Dioscorea opposita thunb vesicle nano material for breast cancer radiotherapy detoxification, preparation method and application

Abstract

This invention discloses a yam vesicle nanomaterial, its preparation method, and its application for reducing the toxicity of radiotherapy for breast cancer. It belongs to the field of biomedical technology. The method includes adding PBS to the yam at a weight-to-volume ratio of 1:3 for juicing, sieving the yam juice, centrifuging the resulting yam filtrate at low speed to obtain supernatant A, centrifuging supernatant A at medium speed to obtain supernatant B, centrifuging supernatant B at ultra-high speed, resuspending the precipitate in PBS, and obtaining the yam vesicle nanomaterial. In this invention, the yam vesicle nanomaterial uses yam as raw material. The obtained yam vesicle nanomaterial has natural bone marrow targeting properties, requires no additional modification, and can spontaneously accumulate in the bone marrow after intravenous injection, specifically protecting the hematopoietic system. It can also simultaneously prevent the decrease in white blood cells, red blood cells, and platelets caused by radiotherapy, comprehensively alleviating the bone marrow suppression side effects of radiotherapy without affecting the efficacy of tumor radiotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to yam vesicle nanomaterials that can be used to reduce the toxicity of radiotherapy for breast cancer, their preparation methods, and applications. Background Technology

[0002] Cancer is one of the leading causes of death worldwide, posing a serious threat to human health. Statistics from the International Agency for Research on Cancer (IARC) show that approximately 12.7 million new cancer cases are diagnosed globally each year, and 7.6 million people die from cancer. Radiotherapy, as one of the three core methods of cancer treatment, plays an irreplaceable role in comprehensive tumor management. It is estimated that about 50% of cancer patients require radiotherapy during their disease course, and radiotherapy contributes up to 40% to cancer cure rates.

[0003] However, radiotherapy has significant limitations—it lacks tumor specificity, and while killing tumor cells, it inevitably damages surrounding normal tissues. The bone marrow hematopoietic system is extremely sensitive to radiation, and due to its vigorous cell proliferation, it is one of the most common target organs damaged by radiotherapy. Radiation-induced bone marrow suppression mainly manifests as a decrease in all blood cells, including leukocytes, erythrocytes, and platelets. Clinical data shows that approximately 60%-80% of patients receiving radiotherapy experience varying degrees of leukopenia, of which 20%-30% may develop severe bone marrow suppression. Radiation-induced bone marrow suppression not only leads to immune failure, increasing the risk of complications such as infection, bleeding, and severe anemia, but also often forces the interruption or reduction of radiotherapy, directly affecting the efficacy of tumor treatment and patient prognosis.

[0004] Currently, the gold standard for treating myelosuppression is recombinant human granulocyte colony-stimulating factor (G-CSF). However, the mechanism of action of G-CSF mainly focuses on "stimulating" the bone marrow to accelerate the release of mature granulocytes, which is an exogenous "rescue" for increasing the number of blood cells, rather than an endogenous "repair" for the bone marrow hematopoietic microenvironment and stem cell pool. More importantly, G-CSF cannot effectively remove excess reactive oxygen species (ROS) generated during radiotherapy, and long-term use may even exacerbate bone marrow exhaustion. Therefore, developing a prevention and treatment strategy that can efficiently remove ROS, block oxidative stress damage at the molecular level, and comprehensively protect all three hematopoietic cell lines is of great significance.

[0005] In recent years, plant-derived nanovesicles, as a novel type of natural nanoparticle formulation, have attracted much attention in the field of targeted disease therapy due to their unique nanoscale effects, good biocompatibility, and natural capacity to load bioactive molecules. Plant nanovesicles can carry endogenous antioxidant enzymes, polyphenolic compounds, and other active ingredients, possessing strong ROS scavenging capabilities. More importantly, plant nanovesicles from specific sources have natural tissue targeting properties, accumulating in specific organs without additional modification for precise delivery. Yam (Dioscorea opposita), a traditional medicinal and edible plant, is rich in polysaccharides, saponins, allantoin, and other active ingredients, exhibiting significant antioxidant and cell-protective activities. However, there are currently no research reports on the application of yam-derived nanovesicles in radiation-induced bone marrow injury protection, particularly utilizing their ROS scavenging capabilities to fundamentally block oxidative stress and protect the hematopoietic system. To address these issues, we propose yam vesicle nanomaterials for reducing the toxicity of radiotherapy in breast cancer, along with their preparation methods and applications. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing yam vesicle nanomaterials, their preparation methods, and applications for reducing the toxicity of radiotherapy for breast cancer. This solves the problem that radioactive bone marrow suppression in breast cancer radiotherapy not only leads to immune failure in patients, but also increases the risk of complications such as infection, bleeding, and severe anemia, forcing the interruption or reduction of radiotherapy, directly affecting the efficacy of tumor treatment and patient prognosis.

[0007] This invention is achieved by providing a method for preparing yam vesicle nanomaterials for reducing the toxicity of breast cancer radiotherapy and their application. The method for preparing yam vesicle nanomaterials for reducing the toxicity of breast cancer radiotherapy includes: S10, take yam, wash, peel, and cut into small pieces. Add PBS to yam at a ratio of 1:3 (yam weight to PBS volume) and juice to obtain yam juice. Take the yam juice and pass it through a sieve to obtain yam filtrate. S20, take the obtained yam filtrate and centrifuge at low speed to remove large particles, mucoprotein and fiber, discard the precipitate and keep the supernatant to obtain supernatant A; S30, centrifuge the obtained supernatant A at medium speed to remove intact organelles, discard the precipitate and keep the supernatant to obtain supernatant B; S40, the obtained supernatant B is subjected to ultra-high speed centrifugation to precipitate exosome-like nanovesicles, the supernatant is discarded, and the precipitate is resuspended in PBS to obtain yam vesicle nanomaterials.

[0008] Preferably, in step S20, when the yam filtrate is centrifuged at low speed, the yam filtrate is centrifuged under an ice bath at 0-4℃; in step S30, when the supernatant A is centrifuged at medium speed, the supernatant A is centrifuged under an ice bath at 0-4℃; and in step S40, when the supernatant B is centrifuged at ultra-high speed, the supernatant B is centrifuged under an ice bath at 0-4℃.

[0009] Preferably, when centrifuging the yam filtrate at low speed, the low speed centrifugation conditions are 500×g for 10 min, followed by 2000×g for 20 min.

[0010] Preferably, when centrifuging the supernatant A at a medium speed, the medium speed centrifugation is 10000×g for 30 minutes, and it is performed twice.

[0011] Preferably, when the supernatant B is subjected to ultra-high speed centrifugation, the ultra-high speed centrifugation conditions are 100000×g for 120min.

[0012] Preferably, in step S40, the ratio of PBS resuspension is: the precipitate corresponding to every 10 mL of yam filtrate obtained in step S10 is resuspended in 100-200 μL of PBS.

[0013] Preferably, in step S40, the yam vesicle nanomaterials are stored at 4°C for a short period and at -80°C for a long period.

[0014] On the other hand, the present invention also provides yam vesicle nanomaterials that can be used to reduce the toxicity of radiotherapy for breast cancer, which are prepared by the method described above.

[0015] Furthermore, the present invention also provides the application of the aforementioned yam vesicle nanomaterials, which can be used to reduce the toxicity of breast cancer radiotherapy, in the preparation of drugs to prevent radiation-induced bone marrow damage.

[0016] Compared with the prior art, the embodiments of this application have the following main advantages: In this embodiment of the invention, the yam vesicle nanomaterial uses yam as raw material and extracts natural nanovesicles from yam for the first time. The preparation method is simple, requires no organic solvents, is green and environmentally friendly, and can be mass-produced. The obtained yam vesicle nanomaterial has natural bone marrow targeting properties, requires no additional modification, and can spontaneously accumulate in the bone marrow after intravenous injection, specifically protecting the hematopoietic system. It can also prevent the decrease of white blood cells, red blood cells and platelets caused by radiotherapy, comprehensively alleviate the side effects of bone marrow suppression caused by radiotherapy, and does not affect the efficacy of tumor radiotherapy. Attached Figure Description

[0017] Figure 1 Transmission electron microscopy (TEM) images of the yam vesicle nanomaterials prepared in Example 1 are shown.

[0018] Figure 2This diagram illustrates the test results of the radioprotective ability of the yam vesicle nanomaterials prepared according to the embodiments of the present invention to OP9 cells with and without RT.

[0019] Figure 3 A schematic diagram showing the test results of the reactive oxygen species scavenging ability of the yam vesicle nanomaterials prepared in this invention is presented.

[0020] Figure 4 The diagram shows the ability of the yam vesicle nanomaterials prepared in this invention to target OP9 cells.

[0021] Figure 5 The diagram shows the results of targeted enrichment of yam vesicle nanomaterials in bone marrow using in vivo imaging of small animals.

[0022] Figure 6 The graph shows the changes in peripheral blood leukocyte count in mice after radiotherapy in different treatment groups when verifying the use of yam vesicle nanomaterials to prevent radiotherapy-induced decrease in peripheral blood cell counts.

[0023] Figure 7 The graph shows the changes in peripheral blood erythrocyte counts in mice after radiotherapy in different treatment groups when verifying the use of yam vesicle nanomaterials to prevent radiotherapy-induced reduction in peripheral blood cell counts.

[0024] Figure 8 The graph shows the changes in peripheral blood platelet counts in mice after radiotherapy in different treatment groups when verifying the use of yam vesicle nanomaterials to prevent radiotherapy-induced reduction in peripheral blood three lineages. Detailed Implementation

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0026] As described in the background section, radiotherapy-induced myelosuppression not only leads to immune failure in patients, increasing the risk of complications such as infection, bleeding, and severe anemia, but also often forces the interruption or reduction of radiotherapy, directly affecting the efficacy of tumor treatment and patient prognosis. To address these issues, we propose a yam vesicle nanomaterial, its preparation method, and its application for reducing the toxicity of radiotherapy in breast cancer. The yam vesicle nanomaterial uses yam as a raw material and is the first to extract natural nanovesicles from yam. The preparation method is simple, requires no organic solvents, is environmentally friendly, and can be mass-produced. The obtained yam vesicle nanomaterial has natural bone marrow targeting properties, requires no additional modification, and can spontaneously accumulate in the bone marrow after intravenous injection, specifically protecting the hematopoietic system. It can also simultaneously prevent the decrease in white blood cells, red blood cells, and platelets caused by radiotherapy, comprehensively alleviating the side effects of radiotherapy-induced myelosuppression without affecting the efficacy of tumor radiotherapy. Example 1

[0027] This invention provides a method for preparing yam vesicle nanomaterials that can be used to reduce the toxicity of radiotherapy for breast cancer. The method specifically includes: S10, take yam, wash, peel, and cut into small pieces. Add PBS to yam at a ratio of 1:3 (yam weight to PBS volume) and juice to obtain yam juice. Take the yam juice and pass it through a sieve to obtain yam filtrate. S20, take the obtained yam filtrate and centrifuge at low speed to remove large particles, mucoprotein and fiber, discard the precipitate and keep the supernatant to obtain supernatant A; wherein, when centrifuging the yam filtrate at low speed, the low speed centrifugation conditions are 500×g centrifugation for 10min, and then 2000×g centrifugation for 20min.

[0028] S30, the obtained supernatant A is centrifuged at medium speed to remove intact organelles, the precipitate is discarded and the supernatant is retained to obtain supernatant B; when centrifuging supernatant A at medium speed, the centrifugation speed is 10000×g for 30min, and it is performed twice.

[0029] S40, the obtained supernatant B is subjected to ultracentrifugation to precipitate exosome-like nanovesicles. The supernatant is discarded, and the precipitate is resuspended in PBS to obtain yam vesicle nanomaterials. The yam vesicle nanomaterials can be stored for a short period at 4℃ and for a long period at -80℃. When the supernatant B is subjected to ultracentrifugation, the ultracentrifugation conditions are 100000×g for 120 min. The ratio of PBS resuspension is: for every 10 mL of yam filtrate obtained in step S10, the corresponding precipitate is resuspended in 150 μL of PBS.

[0030] In this embodiment of the invention, in step S20, when the yam filtrate is centrifuged at low speed, the yam filtrate is centrifuged under an ice bath at 0-4℃; in step S30, when the supernatant A is centrifuged at medium speed, the supernatant A is centrifuged under an ice bath at 0-4℃; and in step S40, when the supernatant B is centrifuged at ultra-high speed, the supernatant B is centrifuged under an ice bath at 0-4℃.

[0031] In this embodiment of the invention, the yam vesicle nanomaterial uses yam as raw material and extracts natural nanovesicles from yam for the first time. The preparation method is simple, requires no organic solvents, is green and environmentally friendly, and can be mass-produced. The obtained yam vesicle nanomaterial has natural bone marrow targeting properties, requires no additional modification, and can spontaneously accumulate in the bone marrow after intravenous injection, specifically protecting the hematopoietic system. It can also prevent the decrease of white blood cells, red blood cells and platelets caused by radiotherapy, comprehensively alleviate the side effects of bone marrow suppression caused by radiotherapy, and does not affect the efficacy of tumor radiotherapy.

[0032] Performance testing: Transmission electron microscopy (TEM) test: The yam vesicle nanomaterials prepared in Example 1 of this invention were dissolved in anhydrous ethanol to prepare a 1 mg / mL solution. 20 μL of the solution was pipetted onto a copper grid, dried, and used to prepare a sample for transmission electron microscopy (TEM) examination. The sample was then observed using a TEM. Figure 1 A transmission electron microscope (TEM) image of the yam vesicle nanomaterials prepared in Example 1 is shown. From... Figure 1 It can be seen that the yam vesicle nanomaterials have a particle size of approximately 100 nm, and the yam vesicles exhibit a typical spherical or cup-shaped structure. This is a typical morphological feature of plant exosome-like nanovesicles. The vesicles have clear edges and a complete lipid bilayer membrane structure, indicating that the vesicles maintained good structural integrity during the extraction process without rupture or fusion. Therefore, the yam vesicles prepared in this invention have good nanoscale dispersibility and stability, providing a structural basis for subsequent in vivo application as a natural nano-preparation to target bone marrow and clear ROS.

[0033] Cytotoxicity assay: Prepare a 6-well plate, add 1×10 to each well. 5Mouse bone marrow stromal cells (OP9 cells) were cultured for 24 h in a 37°C constant temperature incubator containing 5% CO2. The cells were then treated in two groups: (1) EV group: mouse bone marrow stromal cells were co-incubated with EV materials (yam vesicle nanomaterials) prepared in Example 1 at different concentrations of 0, 5, 10, and 20 μg / mL for 6 h; (2) EV+RT (X-ray irradiation) group: EV materials prepared in Example 1 at different concentrations of 0, 5, 10, and 20 μg / mL were co-incubated with the cells for 6 h, followed by X-ray irradiation at a dose of 8 Gy. After that, the medium was changed and cultured for another 24 h. MTT-containing medium was added to each well and cultured for 3.5 h. The MTT medium was removed, and 70 µL DMSO was added to each well. The cells were shaken on a shaker for 10-15 min to dissolve the purple solid completely. The absorbance value (OD value) at 490 nm was read using an ELISA reader. The number of viable cells was determined based on the measured OD value. Figure 2 This diagram illustrates the results of a radioprotective effect test on OP9 cells using the yam vesicle nanomaterials prepared according to an embodiment of the present invention, with and without RT. Compared with the radiotherapy-only group, the survival rate of OP9 cells gradually increased with the increase of EV material concentration, indicating that the EV material has a significant radioprotective effect on bone marrow stromal cells. Figure 2 It is known that the yam vesicle nanomaterials prepared by this invention can significantly reduce the damage of X-rays to bone marrow stromal cells and have good radioprotective activity.

[0034] ROS cleanup experiment: Prepare confocal cells, adding 2×10⁻⁶ ppm to each well. 5 Mouse bone marrow stromal cells (OP9 cells) were cultured in a 37°C incubator containing 5% CO2 for 24 h. Afterward, each well was treated with a reactive oxygen species (ROS) positive control reagent for 30 min. Subsequently, EV materials prepared in the previous step were co-incubated with mouse bone marrow stromal cells at concentrations of 10 and 40 μg / mL for 6 h respectively. The supernatant culture medium was removed, and DCFH-DA working solution (1 mL / well) was added for staining for 20 min. Intracellular DCF fluorescence was observed using a CLSM with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Figure 3 This diagram illustrates the test results of the reactive oxygen species scavenging ability of the yam vesicle nanomaterials prepared according to the present invention. Figure 3 It can be seen that the green fluorescence gradually weakens over time, indicating that the EV material continuously exerts its ROS scavenging effect in OP9 cells. Among other things, Figure 3 (a) in the image represents the fluorescence microscope image analysis during the reactive oxygen species (ROS) clearance test. Figure 3In (a), EV represents the yam vesicle nanomaterial prepared in Example 1 of the present invention, Rosup represents the ROS positive control reagent, DCFH-DA represents the DCFH-DA working solution, Hoechst represents the blue fluorescent DNA dye, which can be Hoechst 33342 or Hoechst 33258, and Merge represents the image overlay (DCFH-DA (green, ROS signal) and Hoechst (blue, cell nucleus) are displayed together). Figure 3 (b) in the figure represents a quantitative statistical analysis chart of reactive oxygen species clearance capacity. Figure 3 In (b) of this paper, Rosup represents the ROS positive control reagent and Control represents the blank control group. The yam vesicle nanomaterial (EV) prepared in this invention has a significant reactive oxygen species scavenging ability. OP9 cells that produce a large amount of ROS induced by Rosup exhibit strong green fluorescence. However, after adding 10 μg / mL and 40 μg / mL EV, the intensity of green fluorescence decreased, and the decrease was concentration-dependent. The difference in quantitative MFI value was statistically significant (P<0.0001). This result confirms that EV can effectively scavenge excess ROS, block the oxidative stress damage cascade reaction from the source, and reduce DNA damage and apoptosis of hematopoietic stem cells. It provides direct cellular-level evidence for preventing radiation-induced bone marrow suppression and protecting the bone marrow hematopoietic microenvironment, reflecting its core mechanism and significant advantages as a natural nano-antioxidant in the application of breast cancer radiotherapy to reduce toxicity.

[0035] Cellular uptake experiment: EVs were incubated with OP9 cells at concentrations of 0 µg / ml, 10 µg / ml, and 40 µg / ml for 4 hours, and the uptake of nanomaterials by the cells was detected using a fluorescence inverted microscope. Figure 4 The diagram shows the ability of the yam vesicle nanomaterials prepared in this invention to target OP9 cells. The results show that the fluorescence captured by the microscope gradually increases with increasing concentration, indicating that the prepared EVs can be effectively taken up by bone marrow stromal cells in a concentration-dependent manner. Figure 4 In the image (a), the fluorescence image represents the cell uptake experiment. Figure 4In (a) of the study, the Control group (0 μg / mL, blank control group) showed almost no red fluorescence, with only blue cell nuclei visible, indicating that no EVs were taken up by the cells. The 10 μg / mL group showed a small amount of red fluorescence distributed in the cytoplasm. The 20 μg / mL group showed a significant increase in red fluorescence intensity, indicating a more widespread distribution of EVs within the cells. The 40 μg / mL group showed a further increase in red fluorescence intensity, indicating a significantly greater accumulation of EVs within the cells compared to the low and medium concentration groups. Combined with the staining of blue cell nuclei, it was observed that the brightness and coverage of intracellular red fluorescence (EVs) gradually increased with increasing EV concentration, suggesting that the uptake of EVs by OP9 cells increased with increasing concentration. Figure 4 (b) in the figure represents the quantitative analysis results of the average fluorescence intensity (MFI, au) of different EV concentration groups. As can be seen from the figure, MFI increases in a concentration-dependent manner with increasing EV concentration, proving that the uptake efficiency of EV by OP9 cells is positively correlated with concentration.

[0036] Bone marrow targeting of yam vesicle nanomaterials (small animal in vivo imaging): Six-week-old female BALB / c mice were injected via tail vein with DiR-labeled yam vesicle nanomaterials (1 mg / kg), while the control group received an equal volume of PBS. In vivo imaging of the animals was performed 24 h post-injection. Figure 5 The diagram shows the results of in vivo imaging in small animals, revealing the targeted enrichment of yam vesicle nanomaterials in the bone marrow. The results are as follows: Figure 5 As shown, yam vesicles exhibited significant fluorescence enrichment in the bone marrow regions (femur and tibia) 24 hours after injection, reaching a peak at 24 hours. This indicates that yam vesicles possess natural bone marrow targeting capabilities. Figure 5 (a) in the image is a fluorescence image of an isolated organ during a small animal in vivo imaging test, while Figure 5 (b) in the image represents the quantitative fluorescence intensity map for in vivo imaging, which is derived from... Figure 5 As shown in (a), after injecting yam vesicle nanomaterials into the bone marrow region (Femur & tibia, i.e., femur and tibia), strong fluorescence enrichment occurred in the femur and tibia. Figure 5 In this study, "Tumor" represents a tumor, "Heart" represents the heart, "Liver" represents the liver, "Spleen" represents the spleen, "Lung" represents the lungs, "Kidney" represents the kidneys, "Brain" represents the brain, and "Muscle" represents the muscles. The natural bone marrow targeting ability of the yam vesicle nanomaterials of this invention was confirmed at the whole animal level: 24 hours after tail vein injection, in addition to distribution in the liver (due to physiological RES clearance) and spleen, EVs showed significant fluorescent enrichment in the bone marrow regions (femur and tibia), with signal intensity far exceeding that of non-target organs such as tumors, heart, lungs, kidneys, and muscles; thus providing crucial in vivo targeting evidence for the development of EVs as a myeloprotective agent in breast cancer radiotherapy.

[0037] Verification of the use of yam vesicle nanomaterials to prevent radiotherapy-induced decrease in peripheral blood cell lines in mice: Forty 8-week-old male C57 mice were randomly divided into three groups (n=5): NC group (negative control group, PBS, no radiotherapy), RT group (radiotherapy group, PBS + 7.5 Gy whole-body irradiation), and FPA group (radiotherapy combined with yam vesicle treatment group, yam vesicles 1 mg / kg, 7.5 Gy irradiation 24 h later). Blood was collected on day 1 after radiotherapy to detect white blood cell, red blood cell, and platelet counts. Figure 6 The graph shows the changes in peripheral blood leukocyte counts in mice after radiotherapy in different treatment groups when verifying the use of yam vesicle nanomaterials to prevent radiotherapy-induced decrease in peripheral blood cell lines. Figure 7 The graph shows the changes in peripheral blood erythrocyte counts in mice after radiotherapy in different treatment groups when verifying the use of yam vesicle nanomaterials to prevent radiotherapy-induced reduction in peripheral blood cell lines. Figure 8 The graph shows the changes in peripheral blood platelet counts in mice after radiotherapy in different treatment groups when verifying the use of yam vesicle nanomaterials to prevent radiotherapy-induced reduction in peripheral blood cell lines. The results are as follows. Figure 6 (leukocyte), Figure 7 (Red blood cells) Figure 8 (Platelet count) shows that all three cell lines were significantly decreased in the RT group. The decrease in all three cell lines was significantly less pronounced in the FPA group, and the decrease in erythrocytes and platelets was also significantly lower than in the RT group. This indicates that yam vesicle nanomaterials can effectively prevent radiotherapy-induced reduction in all three cell lines.

[0038] This invention represents the first time that natural nanovesicles have been extracted from the medicinal and edible plant *Dioscorea opposita*, pioneering the application of plant-derived exosome-like nanomaterials in the field of radiotherapy protection. Compared with existing technologies that use chemical synthesis of nanocarriers or genetic engineering to prepare protein drugs, this invention employs differential centrifugation, achieving the separation and purification of nanovesicles through only three steps: low-speed, medium-speed, and ultra-high-speed centrifugation. It eliminates the need for organic solvent extraction, surfactants, and complex chemical reactions, making the entire preparation process green, environmentally friendly, simple to operate, and low-cost. This method exhibits good reproducibility and batch stability, facilitating standardized production and quality control, providing a feasible technical route for the industrialization of natural nanomedicines, and significantly reducing production costs and environmental burden.

[0039] Unlike existing nanomedicines that require complex engineering modifications such as antibody modification and ligand conjugation to achieve tissue targeting, the yam vesicles obtained in this invention possess natural bone marrow homing ability. They spontaneously accumulate in the bone marrow region after intravenous injection without any surface modification and are efficiently internalized by bone marrow stromal cells (OP9 cells). This characteristic stems from the natural membrane protein and lipid composition and nanoscale effects on the surface of the yam vesicles, enabling them to evade excessive clearance by the reticuloendothelial system, cross the bone marrow vascular barrier, and specifically interact with cells in the bone marrow microenvironment. This natural targeting not only simplifies the preparation process and avoids the immunogenicity risks introduced by chemical modifications but also ensures efficient drug accumulation in target tissues, providing a structural basis for precise protection of the hematopoietic system.

[0040] Unlike the commonly used clinical strategy of G-CSF, which targets only a single white blood cell lineage and accelerates bone marrow release rather than repairing damage—a "symptomatic treatment" approach—this invention utilizes yam vesicles to efficiently scavenge reactive oxygen species (ROS), addressing the core mechanism of radiation-induced bone marrow damage through oxidative stress. This simultaneously prevents a decrease in white blood cells, red blood cells, and platelets, comprehensively alleviating bone marrow suppression. More importantly, in vivo imaging in small animals shows that yam vesicles do not significantly accumulate in tumor tissue. By protecting normal hematopoietic tissue without affecting the killing effect of radiotherapy on tumor cells, this invention successfully overcomes the critical safety bottleneck of radioprotective agents in protecting tumors, achieving the ideal clinical goal of reducing toxicity without compromising efficacy. It provides a strategy that balances efficacy and safety for radiosensitization and normal tissue protection in solid tumors such as breast cancer.

[0041] In summary, this invention provides yam vesicle nanomaterials, preparation methods, and applications that can be used to reduce the toxicity of radiotherapy for breast cancer. In the embodiments of this invention, the yam vesicle nanomaterials are made from yam, and natural nanovesicles are extracted from yam for the first time. The preparation method is simple, requires no organic solvents, is green and environmentally friendly, and can be mass-produced. The obtained yam vesicle nanomaterials have natural bone marrow targeting properties, require no additional modification, and can spontaneously accumulate in the bone marrow after intravenous injection, specifically protecting the hematopoietic system. They can also prevent the decrease of white blood cells, red blood cells, and platelets caused by radiotherapy, comprehensively alleviate the side effects of bone marrow suppression caused by radiotherapy, and do not affect the efficacy of tumor radiotherapy.

[0042] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0043] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still combine, add, delete, or otherwise adjust the features of the various embodiments of the present invention according to the circumstances without conflict or creative effort, thereby obtaining different technical solutions that do not fundamentally depart from the concept of the present invention. These technical solutions also fall within the scope of protection of the present invention.

Claims

1. A method for preparing yam vesicle nanomaterials that can be used to reduce the toxicity of radiotherapy for breast cancer, characterized in that: The method includes: S10, take yam, wash, peel, and cut into small pieces. Add PBS to yam at a ratio of 1:3 (yam weight to PBS volume) and juice to obtain yam juice. Take the yam juice and pass it through a sieve to obtain yam filtrate. S20, take the obtained yam filtrate and centrifuge at low speed to remove large particles, mucoprotein and fiber, discard the precipitate and keep the supernatant to obtain supernatant A; S30, centrifuge the obtained supernatant A at medium speed to remove intact organelles, discard the precipitate and keep the supernatant to obtain supernatant B; S40, the obtained supernatant B is subjected to ultra-high speed centrifugation to precipitate exosome-like nanovesicles, the supernatant is discarded, and the precipitate is resuspended in PBS to obtain yam vesicle nanomaterials.

2. The method for preparing yam vesicle nanomaterials for reducing the toxicity of breast cancer radiotherapy as described in claim 1, characterized in that: In step S20, when the yam filtrate is centrifuged at low speed, the yam filtrate is centrifuged under an ice bath at 0-4℃. In step S30, when the supernatant A is centrifuged at medium speed, the supernatant A is centrifuged under an ice bath at 0-4℃. In step S40, when the supernatant B is centrifuged at ultra-high speed, the supernatant B is centrifuged under an ice bath at 0-4℃.

3. The method for preparing yam vesicle nanomaterials for reducing the toxicity of breast cancer radiotherapy as described in claim 2, characterized in that: When centrifuging the yam filtrate at low speed, the conditions are 500×g for 10 min, followed by 2000×g for 20 min.

4. The method for preparing yam vesicle nanomaterials for reducing the toxicity of breast cancer radiotherapy as described in claim 2, characterized in that: When centrifuging supernatant A at a medium speed, the centrifugation speed is 10000×g for 30 minutes, and it is repeated twice.

5. The method for preparing yam vesicle nanomaterials for reducing the toxicity of breast cancer radiotherapy as described in claim 4, characterized in that: When supernatant B is subjected to ultra-high speed centrifugation, the ultra-high speed centrifugation conditions are 100000×g for 120min.

6. The method for preparing yam vesicle nanomaterials for reducing the toxicity of breast cancer radiotherapy as described in claim 2, characterized in that: In step S40, the ratio of PBS resuspension is: for every 10 mL of yam filtrate obtained in step S10, the corresponding precipitate is resuspended in 100-200 μL of PBS.

7. The method for preparing yam vesicle nanomaterials for reducing the toxicity of breast cancer radiotherapy as described in claim 6, characterized in that: In step S40, the yam vesicle nanomaterials are stored for a short period at 4°C and for a long period at -80°C.

8. A yam vesicle nanomaterial that can be used to reduce the toxicity of breast cancer radiotherapy, prepared by the method described in any one of claims 1-7.

9. The application of the yam vesicle nanomaterials for reducing the toxicity of breast cancer radiotherapy as described in claim 8 in the preparation of drugs to prevent radiation-induced bone marrow damage.

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