Preparation method and application of rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect
By using Rhodiola extracellular vesicle vector to load doxorubicin and modify targeted molecules on it, a targeted drug delivery system was constructed, and the cardiotoxicity and adverse reactions of doxorubicin were solved, and effective targeted treatment of melanoma cells was achieved.
Patent Information
- Application Number
- CN202411795721.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-09
- Publication Date
- 2025-03-11
AI Technical Summary
The existing anti-melanoma drug doxorubicin has serious cardiotoxicity and adverse reactions, making it difficult to selectively distinguish between tumor cells and normal cells.
Rhodiola extracellular vesicles (RELNs) were used as a carrier to load doxorubicin, and the targeted DSPE-PEG-pYEEIE with targeted effect was modified on RELNs to construct a targeted drug delivery system DP-pYEEIE-RELNs-DOX.
It significantly inhibits the proliferation of A375 cells, improves the targeted transport effect of drugs, and reduces the toxic and side effects of drugs, providing a new strategy for treating melanoma.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and in particular, relates to a preparation method and application of a Rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect. Background Art
[0002] Melanoma is a tumor that originates from melanocytes in the skin. It most commonly occurs in the skin, and only about 5% of melanomas occur outside the skin, such as the respiratory mucosa and gastrointestinal mucosa. Although melanoma accounts for only 1% of malignant skin tumors, it has always been the main cause of death from skin cancer due to its highly invasive and high mortality rate. In addition, unlike other tumors, melanoma mainly affects young and middle-aged people, and its incidence rate increases linearly between the ages of 25 and 50, and then decreases. And because of the large population base in my country, the total number of patients cannot be underestimated. Therefore, melanoma is a huge threat that seriously affects the quality of human life, and there is an urgent need to develop new technologies and methods to effectively treat melanoma.
[0003] Extracellular vesicles (Exosome-like nanoparticles, ELNs) are a variety of nano-scale membrane vesicles actively released by different types of cells, which play a role in information and material transfer in intercellular and interspecies communication. Plant derived exosome-like nanoparticles (PELNs) are a kind of membrane vesicles secreted by plant cells, with a lipid bilayer as the basic skeleton, encapsulating various active substances such as proteins and nucleic acids. The size is generally 30 to 200 nm, and it plays an important role in plant growth and development, tissue repair and self-defense. Due to the advantages of nano-scale structure, good safety, abundant sources, high yield and good biocompatibility, PELNs can be used as an efficient carrier material for drug delivery.
[0004] Rhodiola rosea is a perennial herbaceous plant of the genus Rhodiola in the Crassulaceae family. It is mainly distributed in Northeast my country, North China, Northwest China and Southwest China. The earliest record of Rhodiola rosea can be traced back to 1,000 years ago. Li Shizhen listed Rhodiola rosea as a top-grade herbal medicine in his Compendium of Materia Medica.
[0005] Doxorubicin (DOX) is an anthracycline antibiotic discovered from a mutant strain of Streptomyces posse. It is a broad-spectrum, highly effective anticancer drug. Doxorubicin is a non-specific drug for the cell cycle. Its mechanism of action is mainly that it can directly act on DNA, inhibit DNA synthesis, and interfere with RNA transcription, thereby killing or inhibiting tumor cells. However, this effect of preventing cell division cannot selectively distinguish between tumor cells and normal cells. Therefore, like most chemotherapy drugs, DOX has many adverse reactions. In addition, because its affinity with the myocardium is significantly higher than that of other tissues, and it can damage myocardial cells through semiquinone metabolites, it can cause severe dose-dependent cardiotoxicity, which greatly limits its clinical application. Although the cardiotoxicity of DOX can be alleviated to a certain extent by reducing the cumulative dosage, it will also reduce the inhibitory effect on tumors. Therefore, researchers have proposed many solutions, and relying on drug carriers is one of the more feasible strategies.
[0006] Based on this, the present invention provides a preparation method and application of a Rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect. Summary of the invention
[0007] The present invention has found that in addition to the activity of the active ingredients in its homologous plants, RELNs also have certain anti-tumor effects. In addition, RELNs are safe, non-toxic, low-cost and can be mass-produced, and are a natural drug carrier. Therefore, using RELNs to construct a drug delivery system that can improve drug treatment effects and reduce drug toxicity and side effects is one of the effective strategies for treating melanoma.
[0008] DSPE-PEG is a conjugate of lipid polymers. It is a biocompatible amphiphilic material that plays an important role in drug delivery systems. DSPE-PEG modified carriers can prolong blood circulation time and increase the possibility of reaching target sites in the body. In addition, DSPE-PEG provides excellent flexibility for further modifications. It can be easily modified by various bioactive molecules, such as antibodies, peptides or other functional components that can bind to specific receptors on the cell surface. Caffeic acid-pYEEIE is a specific peptide sequence that affects physiological processes such as cell proliferation, differentiation and migration by specifically binding to the SH2 domain. The present invention can guide the entire DSPE-PEG-pYEEIE (hereinafter abbreviated as DP-pYEEIE) structure to accurately locate specific cells or tissues by utilizing the specific binding ability of Caffeicacid-pYEEIE (abbreviated as pYEEIE) to target cell surface or intracellular proteins, thereby achieving application goals such as targeted drug delivery and regulation of specific cell physiological processes.
[0009] The purpose of the present invention is to provide a preparation method and application of a Rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect, which mainly includes the following two aspects: 1. Constructing RELNs drug carriers to provide new ideas for the selection of drug carriers. 2. Constructing a RELNs-loaded DOX targeted drug delivery system DP-pYEEIE-RELNs-DOX, studying the anti-melanoma activity of the drug delivery system, and providing a theoretical solution for the clinical research of medicinal plant vesicle drug delivery systems.
[0010] To achieve the above object, the present invention adopts the following technical solution: A Rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect, wherein the Rhodiola rosea extracellular vesicle targeted drug delivery system is DP-pYEEIE-RELNs-DOX, which uses Rhodiola rosea extracellular vesicles RELNs as carriers, loads the drug doxorubicin DOX, and modifies the Rhodiola rosea extracellular vesicles RELNs with a targeting DP-pYEEIE, thereby constructing the Rhodiola rosea extracellular vesicle targeted drug delivery system DP-pYEEIE-RELNs-DOX; wherein DP-pYEEIE is DSPE-PEG modified with Caffeicacid-pYEEIE.
[0011] Furthermore, the preparation method of Rhodiola rosea extracellular vesicles RELNs includes the following steps: soaking Rhodiola rosea in PBS buffer, crushing, filtering, and collecting the filtrate; pouring the filtrate into a centrifuge tube, balancing it, and centrifuging it at 1000g for 10 min, 3000g for 20 min, and 10000g for 30 min by differential centrifugation to obtain the supernatant, and preliminarily remove large particles, fibers and insoluble matter in the Rhodiola rosea; then centrifuging the obtained supernatant at 150000g for 2.5 h, retaining the precipitate, blowing the bottom of the centrifuge tube with PBS buffer, resuspending the precipitate, obtaining a RELNs solution, and storing it at -80°C.
[0012] Furthermore, the preparation method of DP-pYEEIE includes the following steps: weighing 100 mg of DSPE-PEG2K-NHS and dissolving it in 3 mL of DMF (dimethylformamide), adding polypeptide Caffeicacid-pYEEIE (1.1 eq) and triethylamine (3.0 eq) to dissolve completely, and reacting at room temperature for 12 h; transferring the reaction solution to a dialysis bag (molecular weight cutoff 1000 Da), dialyzing in pure water for 24 h, collecting the dialysate and freeze-drying it to obtain the product DP-pYEEIE.
[0013] Furthermore, the amount of Caffeic acid-pYEEIE is 1.1 times the molar amount of DSPE-PEG2K-NHS; the amount of triethylamine is 3 times the molar amount of DSPE-PEG2K-NHS.
[0014] Furthermore, the preparation method of the Rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect includes the following steps: using a co-incubation method, RELNs solution and DOX are added in a mass ratio of 10:1, and then DP-pYEEIE is added, and the content of the DP-pYEEIE targeting molecule is made to be 10 wt % of the above system, and co-incubated at 37 ° C for 2 h to prepare the targeted drug delivery system DP-pYEEIE-RELNs-DOX, and high performance liquid chromatography is used to evaluate the amount of DOX loaded on RELNs.
[0015] Furthermore, the Rhodiola rosea extracellular vesicle targeted drug delivery system is used in the preparation of anti-melanoma drugs.
[0016] The advantages of the present invention are: DP-pYEEIE-RELNs-DOX can significantly inhibit the proliferation of A375 cells, and it has better cell uptake and the ability to prevent cell division. More interestingly, it was found in the experiment that RELNs also have certain biological activity against melanoma. This system not only has a better targeted transport effect, but also has good drug carrier potential. The present invention proposes for the first time to use RELNs as a targeted transport carrier for drug administration and inhibition of melanoma cell toxicity. This lays the foundation for subsequent animal and clinical experiments, and provides a new idea for the treatment of skin cancer caused by melanoma. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 Schematic diagram of the separation of RELNs and the construction of DP-pYEEIE-RELNs-DOX.
[0018] Figure 2 Isolation and identification of RELNs. (A) Morphological characterization of RELNs by transmission electron microscopy, scale bar is 200 nm. (B) Particle size distribution of RELNs was measured by NTA. (C) UPLC was used to determine the type and content of lipids in RELNs.
[0019] Figure 3 Infrared spectra of DP-pYEEIE, RELNs and DP-pYEEIE-RELNs obtained using an infrared spectrometer.
[0020] Figure 4 All values are expressed as mean ± SD (n = 3).
[0021] Figure 5 Fluorescence images of A375 cells co-incubated with DOX, RELNs-DOX, DP-pYEEIE-RELNs-DOX, RELNs, and DP-pYEEIE-RELNs were obtained using a confocal microscope. Red represents DOX, green represents RELNs, and cell nuclei are represented in blue (DAPI). The scale bar is 50 μm.
[0022] Figure 6 The distribution of cell cycle. A375 cells were treated with DOX, RELNs-DOX, DP-pYEEIE-RELNs-DOX, RELNs and DP-pYEEIE-RELNs, respectively. Flow cytometry was used to analyze and evaluate the cell cycle arrest effect of different drugs on the same cells. DETAILED DESCRIPTION
[0023] In order to make the above features and advantages of the present invention more clearly understood, the following embodiments are given for detailed description. The methods of the present invention are all conventional methods in the art unless otherwise specified.
[0024] 1 Extraction and identification of RELNs 1.1 Extraction Soak 500g of Rhodiola rosea in 1000mL PBS buffer, crush it with a food processor, filter it with gauze, and collect the filtrate. Pour the filtrate into a 50 mL centrifuge tube, balance it, and centrifuge it at 1000g for 10 min, 3000g for 20 min, and 10000g for 30 min by differential centrifugation to remove the large particles, fibers and insoluble matter in Rhodiola rosea. Centrifuge the obtained supernatant at 150000g for 2.5 h, retain the precipitate, blow the bottom of the centrifuge tube with -4℃ precooled PBS buffer, and resuspend the precipitate. The RELNs solution was obtained and stored at -80℃.
[0025] 1.2 Identification 1.2.1 Transmission electron microscopy (TEM) identification of RELNs The RELNs obtained by centrifugation were identified by transmission electron microscopy. The specific experimental process is as follows: Under clean conditions, use tweezers to pick out the copper mesh, use a pipette to take 10 μL of the sample and gently drop it on the copper mesh, then take 10 μL of phosphotungstic acid dye and drop it directly below the sample, use tweezers to pick up the copper mesh with the sample and turn it upside down on the dye, wait for 1 minute, then use clean filter paper to gently absorb the excess liquid, place it in a clean container, and observe the morphology, size and distribution of the sample with a transmission electron microscope.
[0026] 1.2.2 Nanoparticle Tracking Analysis (NTA) of RELNs Nanoparticle analysis of RELNs was performed with the following specific procedures: the obtained RELNs solution was diluted with PBS, 3 to 5 mL was drawn with a syringe, and care was taken to ensure that there were no bubbles in the liquid in the syringe, and the particle size and potential information of RELNs were measured with a nanoparticle tracking analyzer.
[0027] 1.2.3 Determination of protein concentration of RELNs Take an appropriate amount of RELNs, lyse with RIPA lysis buffer, mix well, centrifuge at 10000 g ~ 14000 g for 3 ~ 5 min, take the supernatant for subsequent determination of protein concentration in RELNs. The protein concentration of RELNs was determined using the BCA protein concentration determination kit. First, a standard curve of BSA from 0.025 to 0.5 mg / mL was established, and then the sample was added to a 96-well plate, and the volume of the added sample was recorded. If the sample was less than 20 µL, it was supplemented with diluent, and then 200 µL of BCA working solution was added to each well, incubated in a 37 °C oven for 30 min, and the absorbance was measured at 562 nm. Finally, the sample absorbance was brought into the standard curve to calculate the protein concentration of RELNs.
[0028] 2 Construction of DP-pYEEIE targeting molecule modification of RELNs and DOX loading engineering system The specific synthesis route of the targeting molecule DP-pYEEIE is as follows: weigh 100 mg of DSPE-PEG2K-NHS and dissolve it in 3 mL of DMF (dimethylformamide), add peptide (1.1 eq) and triethylamine (3.0 eq) to dissolve completely, and react at room temperature for 12 h; transfer the reaction solution to a dialysis bag (molecular weight cutoff 1000 Da), dialyze in pure water for 24 h, collect the dialysate and freeze-dry to obtain the product. The specific reaction is as follows: By co-incubation, RELNs solution (protein concentration of 3.88 mg / mL) and DOX were added in a mass ratio of 10:1, and DP-pYEEIE was added, and the content of DP-pYEEIE targeting molecule was 10 wt% of the above system. The system was co-incubated at 37°C for 2 h to prepare the targeted drug delivery system DP-pYEEIE-RELNs-DOX, and the amount of DOX loaded on RELNs was evaluated by high performance liquid chromatography. This part mainly evaluates the ability of RELNs as drug carriers.
[0029] The encapsulation rate is a key quality attribute of RELNs drug carriers. It refers to the percentage of drug content loaded in the lipid bilayer to the total dosage. It can reflect the degree of drug encapsulation and guide the improvement of the preparation process. Calculate the mass of a certain amount of RELNs loaded with DOX, and calculate the encapsulation rate according to the formula: Encapsulation rate = (drug loading / drug dosage) × 100% 3 Infrared spectroscopy analysis The connection between RELNs and DP-pYEEIE was determined by infrared spectroscopy. The operation steps are: mixing the sample with potassium bromide, grinding and pressing into tablets, and detecting with infrared spectroscopy.
[0030] 4 Study on the anti-melanoma effect of DP-pYEEIE-RELNs-DOX in vitro The proliferation inhibitory effect of DP-pYEEIE-RELNs-DOX on human A375 cells was evaluated at the in vitro cell level. Cell viability was determined by CCK-8 method, drug uptake by cells was photographed by laser confocal microscopy, and cell cycle and apoptosis were analyzed by flow cytometry. This part of the study mainly verified the anti-melanoma effect of DP-pYEEIE-RELNs-DOX at the cell level through in vitro cell experiments.
[0031] 4.1 Cell culture After A375 cells and cell culture medium (DMEM medium containing 10% FBS and 1% double antibody) are mixed evenly, they are added to the culture dish. The culture dish is placed in a cell culture incubator at 37°C and 5% CO2 and incubated. The medium is changed every 48 hours. When the cell growth density reaches 80%-90%, the cells are passaged or subjected to subsequent experiments.
[0032] 4.2 Cytotoxicity assay A375 cells in the logarithmic growth phase were seeded into 96-well cell culture plates at a rate of 1000 cells per well (n = 3). After the cells adhered, five groups of drugs, RELNs, DP-pYEEIE-RELNs, DOX, RELNs-DOX, and DP-pYEEIE-RELNs-DOX, were added and cultured for 24 h. Then, CCK-8 solution was added and incubated for 1 h. Its toxicity was evaluated. The absorbance (OD) was measured at a wavelength of 450 nm to analyze the cell survival rate under different conditions.
[0033] 4.3 Cellular uptake assay RELNs were labeled with DiO dye. DiO was added to RELNs, RELNs-DOX, and DP-pYEEIE-RELNs-DOX at a final concentration of 5 μM (dissolved in DMSO). Incubate at 37°C for 30 min and centrifuge at 3500 rpm for 10 min to remove free DiO dye. Three samples labeled with DiO were obtained. A375 cells were seeded in confocal culture dishes, and after the cells adhered, the above five drugs were added and incubated for 24 h. Fix with 1 mL 4% paraformaldehyde for 30 min, and then stain with DAPI for 30 min. Finally, wash with PBS and observe under a confocal laser confocal microscope.
[0034] 4.4 Cell cycle assay The detection of cell cycle was mainly carried out by propidium iodide staining. A375 cells were seeded in 6-well cell culture plates and incubated with the above 5 different drugs for 24 h. The cell culture medium was collected, digested with trypsin, and added to the previously collected culture medium. Centrifuge at 1000 g for 5 min, take the supernatant, and resuspend the cells with 1 mL of pre-cooled PBS buffer. Add 1 mL of pre-cooled 70% ethanol, blow evenly, and fix at 4°C for 12-24 h. Centrifuge at 1000 g for 5 min. Add 1 mL of pre-cooled PBS buffer again, resuspend the cells, and centrifuge the cells again. Add propidium iodide staining solution and RNaseA to each cell sample, incubate in a dark bath at 37°C for 30 min, filter with a cell filter with a pore size of 70 μm, and analyze using flow cytometry.
[0035] 5 Study on the anti-melanoma effect of DP-pYEEIE-RELNs-DOX in vivo 5.1 Establishment of melanoma model mice Male mice aged 6 to 8 weeks were prepared and divided into the following groups: blank group, PBS group, RELNs group, DP-pYEEIE-RELNs group, DOX group, RELNs-DOX group, and DP-pYEEIE-RELNs-DOX group. The hair at the inoculation site of the mice was removed and B16F10 cells were injected subcutaneously. After about ten days, when the tumor volume grew to about 50 mm, the melanoma model mice were established.
[0036] 5.2 Pharmacodynamics of DP-pYEEIE-RELNs-DOX on tumor growth in mice PBS, RELNs, DP-pYEEIE-RELNs, DOX, RELNs-DOX and DP-pYEEIE-RELNs-DOX were injected into the tail vein. During the treatment period, the tumor size and body weight of the mice were recorded once a day and analyzed using GraphPad Prism.
[0037] 5.3 HE staining test Blood was collected from the ophthalmic vein of the mice and stored for blood testing; the mice were dissected, and the heart, liver, spleen, lung, kidney and tumor were removed and fixed in a 4% paraformaldehyde solution; the tissue was removed from the paraformaldehyde, the formaldehyde solution was absorbed with absorbent paper, and the surface of the tissue was trimmed flat with a blade; the trimmed tissue was immersed in 70% (4-12h), 80% (4-12h), 90% (1-2h), and 100% (1-2h) ethanol solutions in turn to replace the formaldehyde solution in the tissue; the tissue was immersed in a xylene solution to replace the ethanol therein, and the immersion was stopped when the edge of the tissue became translucent; an oven containing paraffin was opened in advance to melt the paraffin, the tissue was immersed in the paraffin for 3 h, and then placed in another oven containing paraffin for 1 h to replace the xylene in the tissue. Pour the melted paraffin into the embedding frame, quickly remove the tissue from the paraffin and place it in the embedding frame, with the cut surface facing down, and mark it with label paper. Note that the paraffin should be kept in a melted state during the process; in order to obtain continuous and smooth slices during slicing, pre-cool the wax block containing the tissue sample in a -20°C refrigerator before slicing, fix the wax block on the slicer, and perform continuous slicing; carefully transfer the slices to 40°C warm water for spreading; pick up the slices with a slide, and then place them in an oven for baking. Baking can prevent the slices from falling off the slide during the staining process. After baking, staining can be performed. If the slices are not stained immediately after baking, they can be placed in a slice box after cooling at room temperature and stored in a -20°C refrigerator. They need to be baked again before staining.
[0038] The tissue sections were stained with a hematoxylin and eosin (HE) staining kit. After hematoxylin and eosin staining, the cell nucleus appeared blue and the cytoplasm appeared pink or red, so that the tissue or lesion could be observed. Sample processing: Dewax the paraffin sections in xylene for 5 to 10 minutes, replace with fresh xylene, dewax for 5 to 10 minutes, anhydrous ethanol for 5 minutes, 90% ethanol for 2 minutes, 80% ethanol for 2 minutes, 70% ethanol for 2 minutes, and distilled water for 2 minutes. Hematoxylin and eosin (HE) staining: For the samples treated above, stain with hematoxylin staining solution for 5 to 10 minutes, rinse off the excess staining solution in tap water for about 10 minutes, wash again with distilled water, and stain with eosin staining solution for 30 seconds to 2 minutes. At this time, if direct observation is required, wash twice with 70% ethanol. If direct observation is not required, dehydration, transparency and sealing are still required after washing with 70% ethanol. After staining, obtain pictures of each group of tissues under a microscope.
[0039] 6 Statistical analysis The experimental data were analyzed by one-way analysis of variance or unpaired t-test to determine the differences between groups (Graph PadPrism 9.5). p The results were considered statistically significant when the value was <0.05. All data in this article are greater than or equal to the mean ± standard deviation of three independent experiments.
[0040] 7 Results and discussion 7.1 Isolation and identification of RELNs In this study, RELNs were separated by differential centrifugation and their morphology was characterized by TEM and NTA. Images obtained by transmission electron microscopy showed that RELNs were obviously saucer-shaped and evenly distributed ( Figure 2 The average particle size of RELNs measured by NTA is between 100 and 200 nm ( Figure 2 (B), the morphology and particle size are basically consistent with the characteristics of other types of PELNs in previous studies.
[0041] 7.2 Analysis of lipid and protein content in RELNs UPLC was used to analyze the types and contents of lipids in RELNs ( Figure 2 C). The higher content of RELNs is triglyceride (TG, 33%), diglyceride (DG, 14%), sphingolipid (Cer, 13%, SPH, 10%) and fatty acid (FA, 7%), which together account for 70% of the total lipids of RELNs. Compared with other related studies, the composition and content of PELNs from different sources are significantly different, which also reflects the differences in sources between different plants.
[0042] Meanwhile, the protein concentration of RELNs was determined to be 3.88 mg / mL for subsequent quantification.
[0043] 7.3 Encapsulation efficiency of RELNs DOX was successfully loaded onto RELNs by ultrasound and co-incubation. When co-incubated at 37°C for 2 h, the encapsulation efficiency of the prepared DP-pYEEIE-RELN-DOX reached 35.6%, indicating that RELNs have a certain degree of encapsulation ability for DOX.
[0044] 7.4 Infrared spectroscopy analysis of RELNs, DP-pYEEIE and DP-pYEEIE-RELNs The structures of RELNs, DP-pYEEIE, and DP-pYEEIE-RELNs were analyzed by infrared spectroscopy. Figure 3 ). It can be confirmed that there are multiple characteristic peaks of DP-pYEEIE in DP-pYEEIE-RELNs, and the characteristic peaks of RELNs also exist. -1 The peak at is related to the stretching vibration of the ether chain, which is an important indicator for the successful connection between the phospholipid bilayer and DP-pYEEIE in RELNs.
[0045] 7.5 Evaluation of the cytotoxic effects of different drugs on A375 cells To evaluate the in vitro antitumor effect of DP-pYEEIE-RELNs-DOX, the survival rates of A375 cells treated with different drugs were detected and compared using CCK-8 method ( Figure 4 ). The experimental results showed that DOX, RELNs, DP-pYEEIE-RELNs, RELNs-DOX and DP-pYEEIE-RELNs-DOX had different degrees of inhibitory effects on the proliferation of A375 cells, and the inhibitory effects were concentration-dependent. When the concentration of DOX was greater than 20 ng / mL, DOX, RELNs-DOX and DP-pYEEIE-RELNs-DOX were significantly different from the control group (p<0.001). When the concentration of RELNs was greater than 50 μg / mL, RELNs and DP-pYEEIE-RELNs were significantly different from the control group ( p <0.001). At the same time, it can also indirectly prove that DP-pYEEIE can be successfully modified on RELNs and exert a certain degree of cell proliferation inhibition together with them.
[0046] 7.6 Evaluation of DP-pYEEIE-RELNs-DOX uptake by A375 cells To further demonstrate the effectiveness of the prepared targeted drug delivery system DP-pYEEIE-RELNs-DOX, we used laser confocal microscopy to observe the uptake of different drugs into A375 cells ( Figure 5 ). After treatment with different drugs, red and green fluorescence can be observed on the cells, indicating that these five groups of drugs can be phagocytosed by cells to varying degrees. At the same time, different drugs have different effects on cell phagocytosis. Compared with the DOX group and the RELNs group, especially the DP-pYEEIE-RELNs-DOX group, the red and green fluorescence are the strongest, which is consistent with the previous cytotoxicity test results. RELNs as a drug delivery carrier can increase the uptake of DOX by melanoma cells A375, and the uptake effect of DP-pYEEIE-RELNs-DOX is better.
[0047] 7.7 Cell cycle analysis In order to study the effects of DOX, RELNs, RELNs-DOX, DP-pYEEIE-RELNs and DP-pYEEIE-RELNs-DOX on the cell cycle of A375 cells, thereby inhibiting cell proliferation and achieving cell cycle arrest. A375 cells were treated with DOX, RELNs-DOX, DP-pYEEIE-RELNs-DOX-DOX, RELNs and DP-pYEEIE-RELNs, respectively. Figure 6 ). It can be seen that after treatment with the above five drugs, the proportion of cells in the G1 phase increased significantly (p<0.05), indicating that with the increase in drug concentration, the proliferation rate of cells slowed down, thereby delaying the subsequent S phase, G2 phase and mitosis phase (M phase).
[0048] 8 Conclusion This study investigated the characteristics and activity of RELNs. The engineering of RELNs has been successfully achieved. On this basis, an excellent drug delivery system was constructed. DP-pYEEIE-RELNs-DOX can significantly inhibit the proliferation of A375 cells. It has better cellular uptake and the ability to prevent cell division. More interestingly, it was found in the experiment that RELNs also have certain biological activity against melanoma. This system not only has better targeted transport effects, but also has good potential as a drug carrier. In summary, this study proposed for the first time that RELNs can be used as targeted transport carriers for drug delivery and inhibition of melanoma cytotoxicity. This lays the foundation for subsequent animal and clinical experiments and provides new ideas for the treatment of skin cancer caused by melanoma.
[0049] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. A Rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect, characterized in that: The Rhodiola rosea extracellular vesicle targeted drug delivery system is DP-pYEEIE-RELNs-DOX, which uses Rhodiola rosea extracellular vesicles RELNs as carriers, loads the drug doxorubicin DOX, and modifies the Rhodiola rosea extracellular vesicles RELNs with a targeting DP-pYEEIE, thereby constructing the Rhodiola rosea extracellular vesicle targeted drug delivery system DP-pYEEIE-RELNs-DOX; wherein DP-pYEEIE is DSPE-PEG modified with Caffeic acid-pYEEIE.
2. The Rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect according to claim 1, characterized in that: The preparation method of Rhodiola rosea extracellular vesicles RELNs comprises the following steps: soaking Rhodiola rosea in PBS buffer, breaking, filtering, and collecting filtrate; pouring the filtrate into a centrifuge tube, centrifuging at 1000 g for 10 min, 3000 g for 20 min, and 10000 g for 30 min by differential centrifugation, and then taking the supernatant to preliminarily remove large particles, fibers and insoluble matter in the Rhodiola rosea; centrifuging the obtained supernatant at 150000 g for 2.5 h, retaining the precipitate, blowing the bottom of the centrifuge tube with PBS buffer, resuspending the precipitate, obtaining a RELNs solution, and storing it at -80°C.
3. The Rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect according to claim 1, characterized in that: The preparation method of DP-pYEEIE comprises the following steps: dissolving DSPE-PEG2K-NHS in DMF, adding polypeptide Caffeic acid-pYEEIE and triethylamine, dissolving completely, and reacting at room temperature for 12 hours; transferring the reaction solution to a dialysis bag, dialyzing in pure water for 24 hours, collecting the dialysate and freeze-drying to obtain the product DP-pYEEIE.
4. The Rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect according to claim 3, characterized in that: The amount of DSPE-PEG2K-NHS used was 100 mg, and the amount of DMF used was 3 mL.
5. The Rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect according to claim 3, characterized in that: The amount of caffeic acid-pYEEIE used was 1.1 times the molar amount of DSPE-PEG2K-NHS used.
6. The Rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect according to claim 3, characterized in that: The amount of triethylamine used is 3 times the molar amount of DSPE-PEG2K-NHS.
7. The Rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect according to claim 3, characterized in that: The molecular weight cut-off of the dialysis bag is 1000 Da.
8. The method for preparing the Rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect according to claim 1, characterized in that: The following steps are involved: The RELNs solution was mixed with DOX by the co-incubation method, and then the DP-pYEEIE targeting molecule was added and co-incubated at 37 °C for 2 h to prepare the targeted drug delivery system DP-pYEEIE-RELNs-DOX.
9. The method for preparing the Rhodiola rosea extracellular vesicle targeted drug delivery system with anti-melanoma effect according to claim 8, characterized in that: The mass ratio of RELNs solution to DOX was 10:1, and the amount of DP-pYEEIE was 10% of the total mass of RELNs solution and DOX.
10. Use of the Rhodiola rosea extracellular vesicle targeted drug delivery system according to claim 1 in the preparation of anti-melanoma drugs.