Doxorubicin-loaded stevia rebaudiana exosome drug carrier as well as preparation method and application thereof

Abstract

The invention discloses an adriamycin-loaded stevia rebaudiana exosome drug carrier as well as a preparation method and application thereof. The adriamycin loaded on the surface of an extracted and purified stevia rebaudiana exosome is the adriamycin loaded on the surface of the extracted and purified stevia rebaudiana exosome. According to the adriamycin-loaded stevia rebaudiana exosome drug carrier prepared by the invention, active ingredients contained in the stevia rebaudiana exosome have bactericidal and anti-inflammatory effects, so that the inflammatory response of tumor tissues can be relieved or alleviated, and more effective and safer treatment choices are provided. The adriamycin-loaded stevia rebaudiana exosome drug carrier obtained by the invention has high efficiency and targeting property, is beneficial to drug delivery and promotes the treatment effect of liver cancer.

Description

Technical Field

[0001] The present invention relates to a stevia exosome drug carrier loaded with doxorubicin, a preparation method and an application thereof. Background Art

[0002] Nano drug delivery systems show great potential in targeted tumor therapy due to their unique physicochemical and biological properties. Exosomes, as a natural nanoscale vesicle, are secreted by cells and play an important role in intercellular communication. Exosomes are considered to be ideal drug carriers due to their good biocompatibility, targeting and low immunogenicity. Among them, plant-derived exosomes have the advantages of abundant sources, simple preparation and low immunogenicity. In addition, plant-derived exosomes have good oral stability and can maintain integrity in vivo. Hepatocellular carcinoma is a malignant tumor with high morbidity and mortality worldwide. For patients in the middle and late stages, chemotherapy drugs such as doxorubicin are the main treatment methods, but their systemic toxicity and drug tolerance limit the efficacy. Exosome delivery systems can improve the targeting of chemotherapy drugs and reduce systemic toxicity.

[0003] Stevia is a traditional medicinal plant, and its leaves are rich in a variety of bioactive ingredients, such as flavonoids, polyphenols and glycosides, which have antioxidant, anti-inflammatory and anti-tumor activities. At present, there is no research exploring the use of stevia exosomes as drug carriers. In addition, plant-derived exosomes have a similar structure to animal exosomes, are widely available, are cheap, and contain a variety of natural medicinal ingredients. Therefore, the study of exosomes from traditional Chinese medicine plants is extremely important. Summary of the invention

[0004] The purpose of the present invention is to provide a stevia exosome drug carrier loaded with doxorubicin, which has good functions of inhibiting proliferation and inducing apoptosis of liver cancer cells, as well as a preparation method and application thereof.

[0005] The technical solution of the present invention is: A stevia exosome drug carrier loaded with doxorubicin, characterized in that doxorubicin is loaded on the surface of extracted and purified stevia exosomes.

[0006] The stevia exosome drug carrier loaded with doxorubicin has a nearly spherical structure, the average particle size of the stevia exosome is 241.40 nm, and the particle size of the stevia exosome loaded with doxorubicin is 176.16 nm.

[0007] A method for preparing a stevia exosome drug carrier loaded with doxorubicin is characterized by comprising the following steps: S1: preparing stevia exosomes, breaking fresh stevia leaves at high speed to obtain stevia juice, and obtaining stevia exosomes by differential ultracentrifugation; S2: purifying stevia exosomes, separating and purifying exosomes by a sucrose concentration gradient method; S3: loading doxorubicin, incubating the exosome suspension with doxorubicin in a PBS system, and ultrasonically dispersing the mixed system to obtain a homogeneous solution.

[0008] The specific method of S1 is as follows: fresh stevia is collected, washed, and then broken at high speed for 20 minutes to obtain stevia juice; then centrifuged at 3000rpm for 30 minutes, the sediment is removed, the supernatant is collected, and centrifuged at 10000rpm for 30 minutes; the supernatant is collected again and centrifuged at 15000rpm for 1 hour to collect the precipitate stevia exosomes.

[0009] Specific method of S2: Exosomes were purified by centrifugation at 15,000 rpm for 1 hour using 8%, 30%, 45% and 60% sucrose concentration gradient method. The purified exosomes were resuspended in PBS buffer and stored at -80°C.

[0010] The specific method of S3: In the PBS system, the exosome suspension was incubated with doxorubicin at a ratio of 1:3 v / v for 4 h. The mixed system was ultrasonically cycled 6 times, each time for 30 s, each time at 30 W, and the cooling time between cycles was 2 min. Finally, a homogeneous solution of stevia exosomes loaded with doxorubicin was obtained.

[0011] Application of a stevia exosome drug carrier loaded with doxorubicin in the preparation of a drug for treating doxorubicin-resistant hepatocellular carcinoma.

[0012] The stevia exosomes of the present invention contain a variety of bioactive compounds, including polyphenols such as 4-hydroxycinnamic acid, catechol and hydroquinone. These compounds have significant antioxidant and anti-inflammatory properties and can regulate intracellular oxidative stress and inflammatory responses; the stevia exosomes of the present invention are loaded with doxorubicin, which has good functions of inhibiting proliferation and inducing apoptosis of liver cancer cells, thereby achieving the purpose of treating liver cancer.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a stevia exosome drug carrier loaded with doxorubicin, wherein the active ingredients contained in the stevia exosomes have bactericidal and anti-inflammatory effects, can alleviate or reduce the inflammatory response of tumor tissues, and provide a more effective and safe treatment option.

[0014] 2. The stevia exosome drug carrier loaded with doxorubicin prepared in the present invention, as a chemotherapy drug for advanced liver cancer, can be efficiently and targetedly delivered to liver cancer cells and tissues with stevia exosomes as drug carriers, thereby promoting the therapeutic effect of the drug and reducing its toxic effects on other normal tissues.

[0015] 3. The stevia exosome drug carrier loaded with doxorubicin prepared in the present invention has high efficiency and targeting, which is helpful for drug delivery and promotes the therapeutic effect of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0017] Figure 1 The figure is a schematic diagram of the preparation process of the drug carrier of stevia exosomes loaded with doxorubicin according to the present invention; Figure 2 This is a material characterization diagram of the stevia exosome drug carrier loaded with doxorubicin in the present invention; Figure 3 This is a diagram for identifying the active components of stevia exosomes in the present invention; Figure 4 This is a diagram for verifying that liver cancer cells in the present invention take up stevia exosome drug carriers loaded with doxorubicin; Figure 5 The present invention shows the effect of stevia exosomes loaded with doxorubicin on the activity of drug-resistant hepatocellular carcinoma. DETAILED DESCRIPTION

[0018] A method for preparing a stevia exosome drug carrier loaded with doxorubicin includes the preparation of stevia exosomes and the loading of doxorubicin. The specific steps of the preparation method are as follows: S1: Preparation of Stevia Exosomes: The collected stevia leaves were washed and soaked, and homogenized for 20 minutes using a high-speed wall breaking method to obtain stevia leaf juice. The stevia juice was subjected to multi-step centrifugation purification, centrifuged at 3000rpm for 30 minutes to remove the bottom sediment; the supernatant was centrifuged at 10000rpm for 30 minutes, and the supernatant was collected again and centrifuged at 15000rpm for 1 hour to obtain crude exosomes. The purified stevia exosomes were obtained by centrifugation at 15000rpm for 1 hour using 8%, 30%, 45% and 60% sucrose density gradients in sequence.

[0019] S2: Stevia exosomes loaded with doxorubicin.

[0020] The purified stevia exosome suspension was mixed with doxorubicin (1:3), and the mixed system was ultrasonically cycled 6 times, each time for 30 s, each time at 30 W, with a cooling time of 2 minutes between cycles, to finally obtain a homogeneous solution, which was stored at -80°C.

[0021] The present application also provides a drug carrier of stevia exosomes, which is prepared using the above-mentioned preparation method, and its material characterization diagram is as follows Figure 2 shown.

[0022] The present application provides the use of the above-mentioned stevia exosome drug carrier loaded with doxorubicin in drug-resistant hepatocellular carcinoma. The above-mentioned stevia exosome loaded with doxorubicin has good targeting and high efficiency, can help deliver drugs, and promote the therapeutic effect of liver cancer.

[0023] Specifically, the stevia exosomes loaded with doxorubicin are used in drug-resistant hepatocellular carcinoma.

[0024] The following is an explanation of the above content in combination with specific verification experiments: 1. Preparation and characterization of stevia exosome drug carrier loaded with doxorubicin; 1.1 Experimental methods: 1.1.1 Extraction of Stevia Exosomes Stevia leaves were collected, washed and soaked, and homogenized for 20 minutes using a high-speed wall breaking method to obtain stevia leaf juice. The obtained juice was centrifuged at 3000rpm for 30 minutes to remove the bottom sediment; the supernatant was centrifuged at 10000rpm for 30 minutes, and the supernatant was collected again and centrifuged at 15000rpm for 1 hour to obtain crude exosomes. 8%, 30%, 45% and 60% sucrose density gradients were used in sequence, and centrifuged at 15000rpm for 1 hour to obtain purified stevia exosomes.

[0025] 1.1.2 Stevia exosomes loaded with doxorubicin The purified stevia exosome suspension was mixed with doxorubicin (1:3, v / v), and the mixed system was ultrasonically cycled 6 times, each time for 30 s, each time at 30 W, with a cooling time of 2 minutes between cycles, to finally obtain a homogeneous solution, which was stored at -80°C.

[0026] 1.1.3 Morphological characterization The morphological characteristics of blank stevia exosomes and doxorubicin-loaded exosomes were characterized by DLS, TEM and AFM.

[0027] 1.2 Experimental results: The DLS results showed that the average particle size of stevia exosomes was 241.40nm, and the particle size of stevia exosomes loaded with doxorubicin was 176.16nm, which may be the result of stevia exosomes loaded with drugs. The particle size measured by TEM and AFM results was slightly lower than that of DLS results (~50nm). This difference may be due to the slight swelling of exosomes caused by DLS solution. TEM and AFM results showed that stevia exosomes loaded with doxorubicin had a nearly spherical structure, which was conducive to its long-term circulation in the body and enrichment in tumor sites ( Figure 2 ) 2. Identification of active ingredients in stevia exosomes 2.1 Experimental methods: 2.1.1 LC-MS technical analysis To analyze the main active components of stevia exosomes, we transferred the samples to 2 mL centrifuge tubes and added 100 mg of glass beads. Then, 1000 μL of acetonitrile: methanol: H2O mixture (2:2:1, v / v / v) was added and vortexed for 30 seconds. Then, the centrifuge tube containing the sample was placed in the 2 mL adapter attached to the instrument and immersed in liquid nitrogen for rapid freezing for 5 min. After taking out the centrifuge tube and thawing at room temperature, the tube was placed in the 2 mL adapter again, installed in the tissue grinder, and ground at 60 Hz for 2 min. Repeat twice to ensure that the sample was completely lysed. After grinding, centrifuge at 12000 rpm for 10 min at 4 ° C, collect the supernatant, concentrate and dry. Subsequently, accurately add 300 μL of 2-chloro-l-phenylalanine (4 ppm) solution prepared with acetonitrile: 0.1% formic acid (1:9, v / v) to re-dissolve the sample. Filter the supernatant through a 0.22 μm membrane and detect it on the machine.

[0028] 2.2 Experimental results: LC-MS analysis showed that there are a variety of bioactive compounds in stevia exosomes, including polyphenols such as 4-hydroxycinnamic acid, catechol and hydroquinone. These compounds have significant antioxidant and anti-inflammatory properties and play an important role in regulating intracellular oxidative stress and inflammatory responses. It is particularly noteworthy that these polyphenols have the ability to regulate Cu 2+ This may be closely related to its antioxidant mechanism. As an effective antioxidant, catechol can scavenge free radicals and chelate metal ions, thereby alleviating oxidative damage. In addition, the detected amino acid derivatives such as L-2, 4-diaminobutyric acid and acetylcysteine ​​may further enhance the anti-inflammatory and cytoprotective effects of exosomes. On the other hand, the presence of erucic acid suggests that these exosomes may have the potential to regulate lipid metabolism ( Figure 3 ). Therefore, the bioactive components of stevia exosomes themselves may have extensive antioxidant, anti-inflammatory and metal ion regulation capabilities 3: Validation of Stevia exosomes loaded with doxorubicin for liver cancer cell uptake 3.1 Experimental methods: In order to evaluate the cellular targeting and uptake efficiency of stevia exosomes loaded with doxorubicin, we conducted cell uptake experiments using liver cancer cells HepG2 and liver cancer resistant cells HepG2 / ADR. We used DIO labeling and laser confocal microscopy to observe the distribution and content of doxorubicin-loaded exosomes in cells at different time points (2h, 6h, and 12h).

[0029] 3.2 Experimental results: The results showed that as the treatment time increased, the fluorescence intensity of DIO-labeled stevia exosomes loaded with doxorubicin in the cells gradually increased, indicating that MLNP-DOX could be taken up by liver cancer cells ( Figure 4 ). It is worth noting that there was no significant difference in the uptake capacity of HepG2 and HepG2 / ADR cells for MLNP-DOX, indicating that MLNP-DOX can overcome the multidrug resistance problem faced by conventional chemotherapeutic drugs.

[0030] IV: Effect of doxorubicin-loaded stevia exosomes on the activity of drug-resistant hepatocellular carcinoma 4.1 Experimental methods: Cell viability was detected using the CellCounter lite2.0 luminescent cell viability assay kit. First, the cells were digested and seeded into a 96-well plate to reach a cell density of approximately 40%. Then, the cells were treated with different concentrations of MLNP-DOX for 48 h. The CellCounter lite2.0 reagent was added to a volume equal to the cell culture to be tested, equilibrated to room temperature, and the plate was shaken and mixed for 2-5 minutes to fully lyse the cells. Finally, the well plate was placed at room temperature for 10 min and then detected using an enzyme marker.

[0031] 4.2 Experimental Results The results showed that compared with the control group, the MLNP group had no significant effect on the activity of HepG2 cells, indicating that the carrier itself has good biocompatibility. The same concentration of DOX and MLNP-DOX treatment can significantly inhibit the activity of HepG2 cells, and the reduction is about 50%, indicating that DOX and MLNP-DOX can significantly inhibit the growth of liver cancer cells. In DOX-resistant HepG2 / ADR cells, the cell activity of the DOX-treated group only decreased by about 20%, while the cell activity of the MLNP-DOX-treated group decreased to about 25%. This result shows that nanoparticle encapsulation can significantly increase the inhibitory effect of DOX on the growth of liver cancer cells. Through half-inhibitory concentration detection, we found that MLNP-DOX significantly reduced the half-inhibitory amount of DOX, indicating that DOX is encapsulated in stevia exosomes ( Figure 5 ).

[0032] In summary, the present application uses stevia, a traditional medicinal plant, to extract exosomes and load doxorubicin. Stevia leaves are rich in a variety of bioactive ingredients, such as flavonoids, polyphenols and glycosides, which have antioxidant, anti-inflammatory and anti-tumor activities. Similarly, stevia exosomes also contain polyphenols such as 4-hydroxycinnamic acid, catechol and hydroquinone. These compounds have significant antioxidant and anti-inflammatory properties and play an important role in regulating intracellular oxidative stress and inflammatory responses; plant-derived exosomes have good oral stability and can maintain integrity in vivo. At the same time, the exosome delivery system can improve the targeting of chemotherapeutic drugs and reduce systemic toxicity. Stevia exosomes loaded with doxorubicin can be efficiently and targetedly delivered to drug-resistant liver cancer cells, significantly reducing systemic toxicity and drug tolerance, and are an ideal chemotherapeutic drug delivery carrier.

[0033] In summary, the stevia exosome drug carrier loaded with doxorubicin of the present invention has good targeting and high efficiency, can significantly inhibit the proliferation of drug-resistant liver cancer cells, and induce cell apoptosis; at the same time, the plant-derived exosomes can increase the patient's compliance through oral delivery, which is an ideal treatment for mid- and late-stage liver cancer.

Claims

1. A stevia exosome drug carrier loaded with doxorubicin, characterized by: Doxorubicin is loaded on the surface of the extracted and purified stevia exosomes.

2. The stevia exosome drug carrier loaded with doxorubicin according to claim 1, characterized in that: The stevia exosome drug carrier loaded with doxorubicin has a nearly spherical structure, the average particle size of the stevia exosome is 241.40 nm, and the particle size of the stevia exosome loaded with doxorubicin is 176.16 nm.

3. A method for preparing a stevia exosome drug carrier loaded with doxorubicin, characterized in that: The following steps are involved: S1: Preparation of stevia exosomes: fresh stevia leaves were broken at high speed to obtain stevia juice, and stevia exosomes were obtained by differential ultracentrifugation; S2: Purification of stevia exosomes, separation and purification of exosomes using sucrose concentration gradient method; S3: Loading doxorubicin, incubating the exosome suspension with doxorubicin in a PBS system, and ultrasonically dispersing the mixed system to obtain a homogeneous solution.

4. The method for preparing a stevia exosome drug carrier loaded with doxorubicin according to claim 3, characterized in that: The specific method of S1 is as follows: fresh stevia is collected, washed, and then broken at high speed for 20 minutes to obtain stevia juice; then centrifuged at 3000rpm for 30 minutes, the sediment is removed, the supernatant is collected, and centrifuged at 10000rpm for 30 minutes; the supernatant is collected again and centrifuged at 15000rpm for 1 hour to collect the precipitate stevia exosomes.

5. The method for preparing a stevia exosome drug carrier loaded with doxorubicin according to claim 3, characterized in that: Specific method of S2: Exosomes were purified by centrifugation at 15,000 rpm for 1 hour using 8%, 30%, 45% and 60% sucrose concentration gradient method. The purified exosomes were resuspended in PBS buffer and stored at -80°C.

6. The method for preparing a stevia exosome drug carrier loaded with doxorubicin according to claim 3, characterized in that: The specific method of S3: In the PBS system, the exosome suspension was incubated with doxorubicin at a ratio of 1:3 v / v for 4 h. The mixed system was ultrasonically cycled 6 times, each time for 30 s, each time at 30 W, and the cooling time between cycles was 2 min. Finally, a homogeneous solution of stevia exosomes loaded with doxorubicin was obtained.

7. Use of a stevia exosome drug carrier loaded with doxorubicin in the preparation of a drug for treating doxorubicin-resistant hepatocellular carcinoma.

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