Application of Rehmannia glutinosa extracellular vesicles in the preparation of drugs to improve bone damage in breast cancer bone metastasis

Abstract

The present invention discloses the use of Rehmannia glutinosa extracellular vesicles in the preparation of drugs for improving bone damage caused by bone metastasis of breast cancer, and belongs to the field of biomedicine technology. Take the tuberous roots of fresh Rehmannia glutinosa plants, wash them, add PBS buffer, crush them, and filter them through gauze to remove large particle impurities; the filtrate is subjected to high-speed centrifugation to remove insoluble residues, and then ultra-high-speed centrifugation is performed to collect the precipitate, and the precipitate is resuspended and purified with PEG, and the precipitate is collected by centrifugation to obtain the Rehmannia glutinosa extracellular vesicles. Rehmannia glutinosa extracellular vesicles can effectively target bones, promote the proliferation of bone-related cells, and significantly reduce bone damage caused by bone metastasis of breast cancer; bone protection treatment of breast cancer bone metastasis is performed by using Chinese herbal vesicles derived from Rehmannia glutinosa, which provides a safe and effective treatment plan for improving the problem of pathological osteolysis caused by breast cancer bone metastasis, provides new ideas and technical support for the prevention and treatment of diseases related to tumor bone metastasis, and has good prospects for clinical transformation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of Rehmannia glutinosa extracellular vesicles in the preparation of a drug for improving bone damage caused by breast cancer bone metastasis. Background Art

[0002] Bone metastasis is one of the common complications of patients with advanced breast cancer, which seriously affects the quality of life of patients and indirectly shortens life expectancy. Bone metastasis accounts for about 70% of metastatic breast cancer, and about 80% of patients with advanced breast cancer develop bone metastasis. Patients with bone metastasis usually suffer from bone pain, pathological fractures, cachexia and hypercalcemia caused by cancer, which may lead to serious complications such as coma and promote secondary metastasis to organs such as the liver, brain, and lungs. The "Guidelines for the Full-Cycle Planning and Management of Bone Health in Breast Cancer (2023 Edition)" points out that early treatment focuses on preventing bone metastasis and bone loss, while late-stage treatment focuses on preventing and treating bone-related events, and new therapeutic drugs are urgently needed.

[0003] Currently, the main clinical medications for preventing bone loss in breast cancer bone metastasis include bisphosphonates and their derivatives, denosumab (a RANKL antibody), and others. However, these existing bone-related medications may cause adverse reactions such as jaw osteonecrosis, acute phase reactions, and nephrotoxicity. Therefore, the development of novel, highly effective, safe, and readily available drugs for preventing bone loss in breast cancer bone metastasis is urgent.

[0004] In recent years, plant-derived extracellular vesicles (EVs) have demonstrated potential for clinical application over synthetic nanocarriers due to their superior biocompatibility, low immunogenicity, and natural targeting properties. Herbal vesicles, a type of plant-derived vesicle derived from natural Chinese herbs, are multi-component and multifunctional, retaining some of the medicinal properties of the original medicinal materials. They have been reported to exhibit superior bioactivity compared to other plant-derived EVs, demonstrating improved biosafety and potential for cancer treatment.

[0005] Radix rehmanniae is a traditional Chinese herbal medicine. Rehmannia glutinosa The tuberous root of Rehmannia glutinosa, as described in Shennong's Herbal Classic, "treats broken bones and tendons... replenishes bone marrow... and is especially beneficial when fresh." Modern research has revealed that the main components of Rehmannia glutinosa have pharmacological effects such as improving bone metabolism, fighting tumors, enhancing immune regulation, and protecting the liver and kidneys. However, research on Rehmannia glutinosa extracellular vesicles has been limited. Summary of the Invention

[0006] In response to the problems existing in the prior art, the present invention provides the use of Rehmannia glutinosa extracellular vesicles in the preparation of drugs for improving bone damage caused by breast cancer bone metastasis. The herbal vesicles derived from Rehmannia glutinosa perform bone protection treatment on breast cancer bone metastasis, which not only has a good repair effect, but also has the advantages of low production cost and simple operation.

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention discloses the use of Rehmannia glutinosa extracellular vesicles in the preparation of a drug for improving bone damage caused by breast cancer bone metastasis.

[0009] Furthermore, the Rehmannia glutinosa extracellular vesicles are prepared by the following steps:

[0010] (1) Take the tuberous roots of fresh Rehmannia glutinosa plants, wash them, add PBS buffer, crush them, and filter them through gauze to remove large particles of impurities;

[0011] (2) The filtrate of step (1) is subjected to high-speed centrifugation to remove insoluble residues, and then subjected to ultra-high-speed centrifugation to collect the precipitate. The precipitate is resuspended and purified by PEG. After PEG purification, the precipitate is collected by centrifugation to obtain the extracellular vesicles of Rehmannia glutinosa.

[0012] Furthermore, the conditions for high-speed centrifugation in step (2) are: 1000 x g for 10 min, 3000 x g for 20 min, and 10,000 x g for 30 min, and insoluble residues are removed after each centrifugation.

[0013] Furthermore, the ultrahigh-speed centrifugation condition in step (2) is 100,000 x g for 90 min.

[0014] Furthermore, the PEG in step (2) is 50 wt% PEG 6000, and the volume ratio of the liquid after the precipitate is resuspended to the 50 wt% PEG 6000 is 1:1.

[0015] Furthermore, the centrifugal conditions for collecting the precipitate in step (2) are 3000 x g for 10 min.

[0016] Furthermore, the extracellular vesicles of Rehmannia glutinosa are round or saucer-shaped, have a double-layer membrane structure, and are 100-200 nm in size.

[0017] Beneficial effects

[0018] (1) The Rehmannia glutinosa extracellular vesicles (RREVs) of the present invention can effectively target bones, promote the proliferation of bone-related cells, and significantly reduce bone damage caused by breast cancer bone metastasis;

[0019] (2) The present invention provides a non-cellular therapeutic drug with high safety and high efficiency, which uses Chinese herbal vesicles derived from Rehmannia glutinosa to perform bone protection treatment on breast cancer bone metastasis. It not only has a good repair effect, but also has the advantages of low production cost and simple operation.

[0020] (3) The extracellular vesicles of Rehmannia glutinosa of the present invention provide a safe and effective treatment plan for improving the pathological osteolysis caused by breast cancer bone metastasis, provide new ideas and technical support for the prevention and treatment of tumor bone metastasis-related diseases, and have good prospects for clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Characterization diagrams of RREVs, (a) is the transmission electron microscopy image, (b) is the size distribution diagram, and (c) is the potential characterization diagram;

[0022] Figure 2 Laser confocal microscopy of cellular uptake of RREVs;

[0023] Figure 3 This is a diagram representing the effect of RREVs on promoting the proliferation of bone-related cells;

[0024] Figure 4 This is the biological imaging image of the RREVs in vivo experiment;

[0025] Figure 5 This is a fluorescence imaging image of isolated bone;

[0026] Figure 6 These are diagrams representing the mouse iliac artery modeling and bone treatment. (a) is a diagram showing the location of the common iliac artery, (b) is a schematic diagram of the modeling, and (c) is a diagram representing the RREVs bone treatment. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0028] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents and raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents and raw materials used in the present invention are used in accordance with conventional methods in the art or in accordance with the product instructions.

[0029] Example 1

[0030] Isolation and purification of extracellular vesicles from Rehmannia glutinosa:

[0031] (1) Take fresh Rehmannia root, wash it, cut it into pieces, add PBS buffer, crush it with a wall-breaking machine, and filter it with gauze to remove large particles;

[0032] (2) The filtered filtrate was centrifuged at 1000 x g for 10 min, 3000 x g for 20 min, and 10,000 x g for 30 min. Insoluble residues were removed after each centrifugation. The filtrate was then centrifuged at 100,000 x g for 90 min, and the precipitate was collected and resuspended in PBS.

[0033] (3) The resuspended solution from step (2) was mixed with 50 wt% PEG 6000 solution at a volume ratio of 1:1, allowed to stand for 24 h, and centrifuged (3000 x g, 10 min). The precipitate was collected and resuspended again in PBS to obtain a dispersion of Rehmannia glutinosa extracellular vesicles (RREVs) (protein concentration determined by BCA assay: 400 μg / mL), which was stored at -80°C for later use.

[0034] The characterization diagram of RREVs is shown in Figure 2. Figure 1 As shown, the specific description is:

[0035] 10 μL of Rehmannia glutinosa extracellular vesicle dispersion was pipetted onto a copper grid and negatively stained with 2% uranyl acetate for 2 min. The RREVs were observed and photographed using a HITACHI HT 7800 transmission electron microscope. Figure 1 As shown in (a), the isolated RREVs have a typical saucer-like appearance and a disc-shaped structure with concave edges.

[0036] The zeta potential and size distribution of RREVs were evaluated by dynamic light scattering using a nanoparticle size and zeta potential instrument (Zetasier Advance Series, Malvern, UK). The size distribution of RREVs is shown in the figure Figure 1 As shown in (b), the potential representation diagram is as follows Figure 1 As shown in (c), the surface potential of RREVs is -37.0±5.6 mV and the size distribution is 153.0±15.8 nm.

[0037] Example 2

[0038] Cellular uptake experiments

[0039] (1) 5 μL of 10 4 μM Dil working solution, incubate in the dark for 20 min, remove excess dye by ultrafiltration, and obtain Dil-labeled RREVs (red fluorescence);

[0040] (2) After the cells (breast cancer cells 4T1 and MDA-MB-231, bone-related cells OB and BSMC) were cultured and adhered to the wall (20 mm culture dish), 10 μL of Dil-labeled RREVs were added and incubated for 0 h, 6 h, 12 h, and 24 h, respectively, and then rinsed with PBS three times;

[0041] (3) Fix with 4% paraformaldehyde for 15 min and rinse with PBS buffer three times;

[0042] (4) 10 μg / mL Hoechst 33342 cell nuclei (blue fluorescence) staining for 30 min, followed by rinsing with PBS three times;

[0043] (5) Actin-Tracker Green-488 cytoskeleton (green fluorescence) staining for 30 min, followed by rinsing with PBS buffer three times;

[0044] The culture dish was placed under a laser confocal microscope (Zeiss LSM880+Fast Airyscan, 63x oil objective lens) for observation and image capture. The laser confocal micrographs of cells taking up RREVs are shown in Figure 2. Figure 2 shown by Figure 2 It was found that RREVs were internalized in breast cancer cells (4T1 and MDA-MB-231) and bone-related cells (OB and BSMC) in a time-dependent manner.

[0045] Example 3

[0046] Cell proliferation assay

[0047] After incubating bone-related cells (OB and BSMC) with RREVs solutions of different concentrations (0 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, protein concentration determined by BCA) for 6 h, MTT solution was added to each well and incubated for another 4 h. The culture was terminated and the culture supernatant in the well was carefully aspirated and discarded; 150 μL DMSO was added to each well and the cells were shaken for 10 minutes to fully dissolve the crystals; the wavelength of 570 nm was selected, and the light absorbance of each well was measured on an enzyme-linked immunosorbent assay (ELISA) to calculate the cell survival rate. The characterization of the RREVs-promoting bone-related cell proliferation effect is shown in the figure. Figure 3 As shown in the results, it shows that RREVs have a more significant proliferation-promoting effect on bone-related cells.

[0048] Example 4

[0049] In vivo targeting experiments

[0050] (1) Preparation of DiD-labeled RREVs: RREVs were placed in an EP tube. 2.5 μL of DiD (400X) staining solution and 2.5 μL of staining enhancer (400X) were added to every 0.995 mL of the RREV dispersion prepared in Example 1. The mixture was mixed evenly and stained in the dark for 20 min. The excess stain was removed by ultrafiltration.

[0051] The above-mentioned DiD (400X) and staining enhancer (400X) were derived from the DiD kit, Biyuntian, product number C1995S, batch number 111023240507;

[0052] (2) Construction of animal model: Prepare female BALB / c mice and inject them into the tail vein ( iv ) and oral gavage ( ig ) were administered DiD-labeled RREVs; the patients were divided into 4 groups: PBS (blank), RREVs iv Group, RREVs ig The DiD (simple DiD) group was treated with 200 μL of the corresponding solution. Mice in each group were killed at 0 h, 6 h, 12 h, 24 h, and 48 h after administration, and their bones were removed (ex vivo).

[0053] (3) Perform bone bioimaging. The bioimaging images of RREVs in vivo experiments are shown in the following figure: Figure 4 As shown in the figure, a large amount of DiD fluorescence was detected in the abdominal area of ​​mice in both the tail vein administration group and the oral administration group. The fluorescence intensity decreased significantly after 12 hours, while the tail vein administration group still maintained strong fluorescence at 48 hours. In addition, we analyzed the fluorescence of isolated bones. The fluorescence imaging of isolated bones is shown in Figure 2. Figure 5 As shown, RREVs iv The bones of the mice in the WT group had stronger fluorescence intensity, indicating that RREVs have the ability to target bones.

[0054] Example 5

[0055] Figure 1 shows the iliac artery injection model and bone treatment results. Figure 6 As shown, the specific description is:

[0056] (1) Anesthetize the mouse with isoflurane nebulizer inhalation, fix the anesthetized mouse in a cross-legged position with the abdomen facing upward, remove the hair at the surgical site, and disinfect;

[0057] (2) Use scissors to make a vertical skin incision of about 1 cm along the right lower abdomen between the 4th and 5th nipples; use forceps to bluntly separate the muscle and connective tissue (membranous structure); expose the common iliac artery under a 4x magnification (microscope). The location of the common iliac artery is shown in the figure. Figure 6 As shown in (a);

[0058] (3) Gently lift the blood vessel on the forceps and use a 100 μL cell suspension (5 x10 5 Insert a 31 G needle with cells into the artery cavity (blood will enter the needle tip), and slowly inject cells. The modeling diagram is shown in the figure below. Figure 6 As shown in (b);

[0059] (4) Gently pull back the needle, quickly press the arterial incision area with a cotton swab to stop bleeding, disinfect the area locally and suture the skin wound; after surgery, place the mouse on a warming pad to keep warm and observe carefully;

[0060] (5) Iliac artery injection model mice were divided into PBS group, RREVs group and iv Group, RREVs ig The model group was treated with PBS daily; RREVs iv Group, RREVs ig The two groups were treated with RREVs by tail vein injection and oral gavage, and each group was given 200 μL of the corresponding solution every day. After 4 weeks of treatment, the mice were killed by cervical dislocation. The bone tissue of tumor metastasis was removed, placed on a Micro-CT specimen tube, wrapped with paper towels several times and fixed to prevent the specimen from dehydration and displacement during detection. Skyscan1276 Micro-CT was used for layer-by-layer scanning. After the scan was completed, Data viewer software was used for calibration, and then CT-AN software was used to select the region of interest (ROI). Finally, CT vox software was used for three-dimensional reconstruction and analysis. The characterization of RREVs bone treatment is shown in the figure below. Figure 6 As shown in (c), RREVs iv Group, RREVs ig The mice in the PBS group had less bone damage than those in the RREV group, indicating that RREVs have the therapeutic ability of bone protection.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention are included in the scope of protection of the present invention.

Claims

1. Application of extracellular vesicles of Rehmannia glutinosa in the preparation of drugs to improve bone damage caused by breast cancer bone metastasis; The Rehmannia glutinosa extracellular vesicles are prepared by the following steps: (1) Take the tuberous roots of fresh Rehmannia glutinosa plants, wash them, add PBS buffer, crush them, and filter them through gauze to remove large particles of impurities; (2) The filtrate from step (1) is subjected to high-speed centrifugation to remove insoluble residues, and then subjected to ultra-high-speed centrifugation to collect the precipitate. The precipitate is resuspended and purified using PEG, and the precipitate is collected by centrifugation to obtain the extracellular vesicles of Rehmannia glutinosa. The extracellular vesicles of Rehmannia glutinosa are round or saucer-shaped, have a double-layer membrane structure, and are 100-200 nm in size.

2. The use according to claim 1, characterized in that The high-speed centrifugation conditions in step (2) are: 1000 x g for 10 min, 3000 x g for 20 min, and 10,000 x g for 30 min. Insoluble residues were removed after each centrifugation.

3. The use according to claim 1, characterized in that The ultrahigh-speed centrifugation condition in step (2) is 100,000 x g for 90 min.

4. The use according to claim 1, wherein The PEG in step (2) is 50 wt% PEG 6000, and the volume ratio of the liquid after the precipitate is resuspended to the 50 wt% PEG 6000 is 1:

1.

5. The use according to claim 1, characterized in that The centrifugal conditions for collecting the precipitate in step (2) are 3000 x g for 10 min.

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