Exosome composition for reducing side effects of chemotherapy treatment and method of preparing the same

Abstract

The application provides an exosome composition for reducing side effects of chemotherapy treatment and a preparation method thereof, and belongs to the technical field of medicines. Methotrexate, vinblastine and daunorubicin are prepared into a co-crystal, loaded on UiO-66-NH2 connected with RGD, loaded on exosomes, and embedded in an acrylic acid, traditional Chinese medicine polysaccharide and pullulan compound to obtain a sustained-release high half-life exosome system, which is uniformly mixed with D-ribose, yeast-beta glucan and glutathione to obtain the exosome composition for reducing side effects of chemotherapy treatment. The traditional Chinese medicine polysaccharide is traditional Chinese medicine polysaccharide obtained by boiling water extraction and ethanol precipitation of radix codonopsis, angelica, polygonatum sibiricum and dendrobium officinale. The exosome composition for reducing side effects of chemotherapy treatment is prepared, drug resistance of tumor cells is reduced, drug efficacy is improved, side reactions after chemotherapy are reduced, the effect of targeted slow release of drugs is achieved, and the exosome composition has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an exosome composition for reducing the side effects of chemotherapy and its preparation method. Background Technology

[0002] A tumor is formed when the body loses its ability to regulate locally mutated cells under pathogenic factors, causing these mutated cells to proliferate abnormally and eventually form a new organism. Therefore, once a tumor forms, it grows rapidly and uncontrollably, invading and destroying normal tissues and organs. In severe cases, it can metastasize throughout the body, leading to the patient's death.

[0003] Chemotherapy originated in the 1840s, initially used to alleviate symptoms of acute lymphoblastic leukemia. Later, with further research into chemotherapy techniques, it was discovered clinically that chemotherapy could also provide long-term relief against solid tumors. Subsequently, the clinical value of adjuvant chemotherapy after cancer surgery was also discovered. By the 1980s and 1990s, after more than a century of exploration and development, new chemotherapy drugs were constantly emerging, further enriching and facilitating doctors in formulating and selecting chemotherapy regimens, thus significantly improving the clinical efficacy of chemotherapy.

[0004] Preoperative chemotherapy for cancer has demonstrated significant clinical benefits in the treatment of various tumors; in particular, combined therapy can effectively alleviate symptoms and prolong patient life for many types of cancer. Therefore, compared with other treatment methods, radiotherapy and chemotherapy remain, at present, tumor treatment options with a wide range of applications, long duration of action, and more definite efficacy.

[0005] To date, the medical community continues to research and develop new chemotherapy drugs in order to further improve the efficacy of chemotherapy while reducing its side effects, and to improve patient tolerance and compliance. Meanwhile, the combined use of chemotherapy drugs with molecularly targeted therapies, as well as new treatment methods such as personalized precision medicine, are opening up new directions for cancer chemotherapy. Summary of the Invention

[0006] The purpose of this invention is to propose an exosome composition and its preparation method that reduce the side effects of chemotherapy. This method improves the water solubility of chemotherapeutic drugs, reduces their dosage, decreases drug resistance in tumor cells, synergistically enhances efficacy, and reduces post-chemotherapy side effects. The use of a UiO-66-NH2 / RGD complex for loading increases the drug loading capacity and allows for good complexation with exosomes, achieving targeted drug release and thus reducing side effects. Encapsulated in an acidified polysaccharide shell, it exhibits good acid resistance, prolongs the half-life of exosomes, protects them from enzymatic degradation and immune attack, and enables sustained release, showing broad application prospects.

[0007] The technical solution of this invention is implemented as follows:

[0008] This invention provides a method for preparing an exosome composition that reduces the side effects of chemotherapy, which is prepared from the following raw materials: Codonopsis pilosula, Angelica sinensis, Polygonatum sibiricum, Dendrobium officinale, D-ribose, yeast-β-glucan, and glutathione.

[0009] As a further improvement of the present invention, methotrexate, vincristine, and daunorubicin were prepared into a cocrystal, loaded onto UiO-66-NH2 with RGD, loaded onto exosomes, and embedded in a complex of acrylic acid, traditional Chinese medicine polysaccharide, and pullulan polysaccharide to obtain a sustained-release exosome system with a high half-life. This system was then mixed evenly with D-ribose, yeast-β-glucan, and glutathione to obtain an exosome composition that reduces the side effects of chemotherapy. The traditional Chinese medicine polysaccharide was obtained by extracting Codonopsis pilosula, Angelica sinensis, Polygonatum sibiricum, and Dendrobium officinale with boiling water and precipitating with ethanol.

[0010] As a further improvement to the present invention, the following steps are included:

[0011] S1. Dissolve methotrexate in an alkaline solution to obtain solution 1; dissolve vinblastine in ethanol to obtain solution 2; dissolve daunorubicin in water to obtain solution 3; mix solutions 1, 2, and 3 evenly, allow to evaporate in an open container, filter, wash, dry, and grind to obtain a chemical drug eutectic powder;

[0012] S2. Arginine-glycine-aspartic acid RGD was added to water, NHS and EDC were added, and the mixture was stirred to activate it. UiO-66-NH2 was added, and the mixture was stirred to react. After centrifugation, washing and drying were performed to obtain the UiO-66-NH2 / RGD complex.

[0013] S3. Add the UiO-66-NH2 / RGD complex to water, add NHS and EDC, stir to activate, add drug eutectic powder, stir to react, centrifuge, wash, dry, and obtain the drug carrier;

[0014] S4. Add exosomes to water, add drug carrier, incubate with ultrasound, centrifuge, wash, and dry to obtain exosome drug delivery system;

[0015] S5. Wash, dry, and pulverize Codonopsis pilosula, Angelica sinensis, Polygonatum sibiricum, and Dendrobium officinale. Add water, heat to boiling and extract, filter, keep the residue, add ethanol to the filtrate to precipitate, filter, wash the solid, dry, and obtain the Chinese herbal polysaccharide.

[0016] S6. Add acrylic acid, Chinese herbal polysaccharide, pullulan polysaccharide to water, add exosome drug delivery system, then add initiator, emulsifier, N,N'-methylenebisacrylamide, stir and mix evenly, add to fish oil, emulsify, crosslink reaction, centrifuge, wash, dry, to obtain sustained-release high half-life exosome system;

[0017] S7. Mix D-ribose, yeast-β-glucan, glutathione, and a sustained-release high half-life exosome system evenly to prepare an exosome composition that reduces the side effects of chemotherapy.

[0018] As a further improvement of the present invention, the concentration of the alkaline solution in step S1 is 5-10 wt%, the alkali is NaOH or KOH, the mass ratio of methotrexate, vincristine and daunorubicin is 3-5:1-3:3-6, and the open volatilization time is 7-10 days.

[0019] As a further improvement of the present invention, the mass ratio of arginine-glycine-aspartic acid RGD, NHS, EDC and UiO-66-NH2 in step S2 is 2-4:0.5-1:0.5-1:7-10, the stirring activation time is 20-40 min, and the stirring reaction time is 12-15 h.

[0020] As a further improvement of the present invention, the mass ratio of the UiO-66-NH2 / RGD complex, NHS, EDC and chemical drug eutectic powder in step S3 is 10-15:2-3:2-3:3-6, the stirring activation time is 20-40 min, and the stirring reaction time is 10-14 h.

[0021] As a further improvement of the present invention, the exosomes in step S4 are mesenchymal stem cell exosomes, the mass ratio of the exosomes to the drug-carrying body is 10-12:4-7, the ultrasonic incubation power is 300-500W, the temperature is 36-38℃, and the time is 10-12h.

[0022] As a further improvement of the present invention, the mass ratio of Codonopsis pilosula, Angelica sinensis, Polygonatum sibiricum and Dendrobium officinale in step S5 is 5-10:5-10:3-7:10-15, and the heating and boiling extraction time is 3-5 hours.

[0023] As a further improvement of the present invention, the mass ratio of acrylic acid, traditional Chinese medicine polysaccharide, pullulan polysaccharide, exosome drug delivery system, initiator, emulsifier, and N,N'-methylenebisacrylamide in step S6 is 3-7:5-10:3-5:6-8:0.05-0.1:0.5-1:0.4-0.8. The initiator is selected from at least one of potassium persulfate, ammonium persulfate, and sodium persulfate. The emulsifier is selected from at least one of Span-20, Span-40, Span-60, Span-80, Tween-20, Tween-40, Tween-60, and Tween-80. The crosslinking reaction temperature is 50-60°C, and the time is 3-5 hours.

[0024] As a further improvement of the present invention, the mass ratio of D-ribose, yeast-β-glucan, glutathione, and sustained-release high half-life exosome system in step S7 is 1-3:4-8:2-4:15-20.

[0025] D-ribose (nutritional supplement):

[0026] It can improve myocardial ischemia and enhance cardiac function: Studies have confirmed that oral D-ribose can promote the production of ATP in myocardial cells, ensure the normal function of myocardial cells, and thus significantly improve cardiac function; and can significantly improve symptoms such as shortness of breath, frequent palpitations, chest tightness, and weakness.

[0027] Enhances bodily energy and relieves muscle soreness: D-ribose is the starting molecule for ATP synthesis and an important raw material for muscles to synthesize energy substances; experiments have shown that supplementing with D-ribose can improve the body's athletic ability, effectively resist fatigue, and relieve muscle soreness (from Baidu Encyclopedia).

[0028] Yeast-β-glucan (a new resource food) is beneficial for various cancers or chronic hepatitis B, rheumatic diseases, recurrent oral ulcers, allergies, autoimmune diseases, various infectious diseases, and even the aging process. Yeast-β-glucan can strengthen or restore a weakened or impaired immune response. It also has anti-radiation and chemical toxin neutralization effects: When ionizing radiation or chemical radiation penetrates biological tissues, the energy of the ions or chemical molecules in the radiation is directly absorbed by biological macromolecules, or through ion diffusion and the action of free radicals, it causes other biomolecules to undergo chemical reactions, thereby causing physical and chemical damage to the human body. The unique reverse triple helix structure of yeast-β-glucan can neutralize toxins in the body, promote hematopoietic function, and enhance the production activity of blood cells, including the production of granulocytes, monocytes, and erythrocytes, thereby promoting better recovery of human cells from lethal doses of radiation or toxins. (Source: Baidu Encyclopedia)

[0029] Glutathione is a tripeptide containing a γ-amide bond and a thiol group, composed of glutamic acid, cysteine, and amino acids, and is present in almost every cell of the body. Glutathione helps maintain the normal function of the body's immune system and has antioxidant and detoxification effects. The thiol group on cysteine ​​(its active group) (hence often abbreviated as G-SH) readily binds to certain drugs or toxins, enabling them to achieve detoxification. Glutathione is not only used in pharmaceuticals but also as a base ingredient in functional foods, widely applied in anti-aging, immune enhancement, and anti-tumor activities. It also possesses antioxidant, detoxifying, skin-whitening, and spot-fading properties.

[0030] Baidu Baike (Baidu Encyclopedia)

[0031] The present invention further protects an exosome composition prepared by the above-described preparation method that reduces the side effects of chemotherapy.

[0032] The present invention has the following beneficial effects:

[0033] Mesenchymal stem cells (MSCs) possess many unique advantages and are the most commonly used cell type as exosome donors. MSC exosomes can deliver siRNA to inhibit drug resistance in tumor cells. The miR-379 they carry can reduce COX-2 expression and inhibit cancer cell proliferation; the miR-143-3p they carry can promote apoptosis in pancreatic cancer CFPAC-1 cells and inhibit cell growth, invasion, and migration.

[0034] Exosomes are rich in internal contents, including RNA, DNA, proteins, antibodies, and small molecule metabolites. They can directly activate receptors in target cells and act as intercellular messengers by delivering macromolecules such as nucleic acids and proteins, regulating various physiological and pathological processes, such as tumor development and metastasis, and the progression of immune diseases. The CD55 and CD59 molecules on their surface can increase the stability of in vivo circulation and prolong the in vivo circulation time of exosomes. The CD47 molecule can protect exosomes from the mononuclear macrophage system, thus preventing their elimination. Membrane proteins such as apoptosis-associated gene 2 interactor X, tumor susceptibility gene 101, heat shock protein (HSP), and tetraester proteins CD63, CD9, and CD81 enable them to target and transport drugs. The nanoscale size, fluidity, and targeting ability of exosomes allow them to cross natural biological barriers and overcome the barrier formed by the dense stroma of cancer cells, thereby achieving highly efficient targeted drug delivery.

[0035] However, the short half-life and high content specificity of exosomes can affect the final therapeutic effect. This invention addresses this issue by co-incubating exosomes and drug-carrying bodies with ultrasound. Ultrasonic vibration causes vacuolation in the exosome membrane, allowing the drug to enter. Furthermore, the exposure of the hydrophobic portion of the exosomes after ultrasound treatment enhances their interaction with the target cell membrane. The exosome drug-carrying system prepared by this invention uses acrylic acid-modified traditional Chinese medicine polysaccharides and pullulan polysaccharides to form an interpenetrating network aggregate shell. This results in a sustained-release, high-half-life exosome system with good acid resistance; it contracts without releasing in acidic environments and swells under alkaline conditions, allowing for slow release of the contents. This protects the exosome drug-carrying system from destruction by gastric acid and prolongs the half-life of the exosomes, protecting them from enzymatic degradation and immune attack while achieving sustained release.

[0036] Methotrexate is a chemotherapy drug that inhibits DNA (deoxyribonucleic acid) assembly and deoxyribonucleotide synthesis, but it is insoluble in water. Vincristine is a chemotherapy drug that inhibits cell mitosis and microtubule polymerization, but it is also insoluble in water. This greatly limits the biocompatibility of these chemotherapy drugs, requiring very large doses to achieve a certain effect. Therefore, by mixing them with an aqueous solution of daunorubicin (which inhibits RNA (ribonucleic acid) synthesis by disrupting the nucleic acid synthesis process) to form a co-crystal, a water-soluble co-crystal drug is prepared. This greatly improves the efficacy and reduces the dosage, fundamentally reducing the side effects after drug use. Furthermore, the synergistic effect of the three chemotherapy drugs with different effects can significantly improve the anti-tumor effect and reduce the drug resistance of tumor cells.

[0037] The MOF material UiO-66-NH2 has high porosity and adjustable pore size, allowing for the loading of large doses of drug cocrystal powder. Furthermore, it is coupled with arginine-glycine-aspartic acid RGD, which promotes the adhesion and coupling of drug carriers and exosomes. It can also specifically bind to integrins overexpressed on the surface of tumor angiogenesis and tumor cells by binding to integrin receptors, thereby further promoting targeted drug release. Moreover, the cyclic RGD peptide has higher activity and stability due to its rigid structure, and can resist protease degradation.

[0038] The Codonopsis pilosula, Angelica sinensis, Polygonatum sibiricum, and Dendrobium officinale in this invention are rich in polysaccharides and have good efficacy, as detailed below:

[0039] Codonopsis pilosula (new resource food): replenishes qi and blood, promotes the production of body fluids.

[0040] Modern research has shown that Codonopsis pilosula can increase the number of red blood cells and hemoglobin in the human body, and can also increase the number of white blood cells that decrease during radiotherapy and chemotherapy. Codonopsis pilosula can improve the body's stress resistance and enhance its resistance to adverse stimuli such as changes in environmental temperature and chemicals.

[0041] Enhancing Immune Function: Codonopsis pilosula polysaccharides significantly increase the phagocytic function of peritoneal macrophages and improve the clearance rate of carbon particles in mice. The water decoction and water-decocted alcohol precipitate of Codonopsis pilosula have similar effects to Codonopsis pilosula polysaccharides, significantly increasing the number of macrophages, cell volume, pseudopodia, and phagocytic capacity in mice. The activities of intracellular DNA, RNA, carbohydrates, ATPase, acid esterase, and succinate dehydrogenase are also significantly enhanced.

[0042] Anti-cancer: The combined application of Codonopsis pilosula and cyclophosphamide can prolong the death time and improve the survival rate of tumor-bearing mice.

[0043] Effects on bacteria: In vitro experiments showed that meningococcus was moderately sensitive to Codonopsis pilosula decoction, while diphtheria bacillus, catarrhal diphtheriae, paracolon bacillus, coli, and human tuberculosis bacteria were mildly sensitive.

[0044] Promoting protein and nucleic acid synthesis: Ginseng total saponins and protein synthesis promoting factors can promote the biosynthesis of proteins, DNA, and RNA, increase cytoplasmic ribosomes, and improve serum protein synthesis rate and albumin and gamma globulin content. Therefore, ginseng can promote animal growth, increase body weight, enhance the body's disease resistance, and promote the recovery of patients.

[0045] Angelica sinensis (new resource food): nourishes blood, promotes blood circulation, relieves pain and moistens the intestines.

[0046] [Modern Research] has found that it can promote hematopoiesis. Angelica sinensis can promote the production of hemoglobin and red blood cells, and Angelica sinensis polysaccharides can increase white blood cells and reticulocytes. It has a significant promoting effect on the recovery of red blood cells, hemoglobin, white blood cells and nucleated cells in the femur of anemic mice.

[0047] Enhancing immunity: Angelica sinensis has the effect of promoting specific and non-specific immune functions. Gavage administration of Angelica sinensis decoction can increase the total number of skin cells in mice and significantly increase the phagocytic function of peritoneal macrophages in animals, and improve the clearance speed of dyes by the reticuloendothelial system. Angelica sinensis polysaccharide has a significant antagonistic effect on the decrease in macrophage phagocytic function caused by cyclophosphamide.

[0048] Anti-inflammatory and analgesic: Angelica decoction has a significant inhibitory effect on acute and chronic inflammation caused by three inflammatory agents. Studies have found that the analgesic strength of Angelica is 1.7 times that of sodium acetylsalicylate.

[0049] Anti-radiation: Prophylactic administration of Angelica polysaccharide has a certain radiation protection effect on the hematopoietic tissue of irradiated mice.

[0050] Antitumor effects: Angelica polysaccharides significantly increased the number of peripheral blood T and B lymphocytes in normal mice, tumor mice, and X-ray-irradiated tumor mice. (Chinese Materia Medica, Second Edition, pp. 988-989)

[0051] Polygonatum (a medicinal and edible ingredient):

[0052] Effects on the cardiovascular system: Polygonatum significantly enhances the contractility of rat myocardium and significantly increases the perfusion rate of isolated rabbit hearts. Intravenous injection of this product can counteract acute myocardial ischemia induced by posterior pituitary extract. Intraperitoneal injection can improve the oxygen tolerance of mouse myocardium.

[0053] Aggregation of cancer cells: Polygonatum sibiricum agglutinin 2 has a strong agglutination effect on human uterine cancer cells, human liver cancer cells, and human gastric cancer cell lines.

[0054] Anti-leukopenia: Polygonatum polysaccharides can significantly counteract cyclophosphamide-induced leukopenia in peripheral blood of mice and also increase spleen weight in immunocompromised mice. (Chinese Materia Medica, Second Edition, p. 1032)

[0055] Dendrobium officinale (a new resource food) nourishes yin and clears heat, nourishes the stomach and promotes the production of body fluids, and improves eyesight and strengthens the waist.

[0056] [Modern Research] Nourishes Yin and promotes the production of body fluids. Dendrobium officinale can significantly improve the weakness symptoms of hyperthyroidism and Yin deficiency mice.

[0057] Antipyretic and analgesic: Dendrobium has a certain antipyretic and analgesic effect, but its effect is relatively weaker than that of phenacetin.

[0058] Enhancing immune function: The decoction of Dendrobium nobile has a significant promoting effect on the phagocytic function of peritoneal phagocytes in mice.

[0059] Promotes gastric acid secretion: Dendrobium decoction can promote the secretion of gastric juice and gastric acid, and improve the body's digestive function.

[0060] Antitumor effects: Dendrobium can prolong the average lifespan of ICR mice implanted with sarcoma 180 cells in the peritoneum. Its ethanol extract has significant cytotoxicity against human lung cancer cells, ovarian adenocarcinoma cells, promyelocytic cells, leukemia cells, and other cell lines. (See "Chinese Materia Medica, Second Edition," page 1028).

[0061] This invention prepares an exosome composition that reduces the side effects of chemotherapy. It improves the water solubility of chemotherapeutic drugs, reduces their dosage, decreases the drug resistance of tumor cells, synergistically improves drug efficacy, and reduces post-chemotherapy side effects. The UiO-66-NH2 / RGD complex is used for loading, which increases the drug loading capacity and allows for good complexation with exosomes, achieving targeted drug release and thus reducing drug side effects. The exosomes are embedded in an acidified polysaccharide shell, exhibiting good acid resistance and prolonging the half-life of exosomes. This composition can protect exosomes from enzymatic degradation and immune attack while achieving sustained release, showing broad application prospects. Detailed Implementation

[0062] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0063] The preparation method of UiO-66-NH2 is as follows: Zirconium tetrachloride (0.75 mmol) and 2-aminoterephthalic acid (0.75 mmol) were added to a mixed solvent of N,N-dimethylformamide (38 mL) and acetic acid (2.1 mL), and sonicated at 1000 W for 30 min to disperse it evenly. Then, the mixed solution was added to a hydrothermal reactor and reacted at 120 °C for 24 h. After cooling, the crude product was separated by centrifugation. The obtained yellow solid was washed, dried, and a yellow powder was obtained, which was designated as UiO-66-NH2.

[0064] Arginine-glycine-aspartic acid RGD, CAS No.: 99896-85-2, commercially available.

[0065] Exosomes, mesenchymal stem cell exosomes, commercially available.

[0066] NHS, N-hydroxysuccinimide, EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0067] Example 1

[0068] This embodiment provides a method for preparing an exosome composition that reduces the side effects of chemotherapy, comprising the following steps:

[0069] S1. Dissolve 3g of methotrexate in 100mL of 5wt% NaOH solution to prepare solution 1; dissolve 1g of vincristine in 100mL of ethanol to prepare solution 2; dissolve 3g of daunorubicin in 200mL of water to prepare solution 3; mix solutions 1, 2, and 3 evenly, allow to evaporate in an open environment for 7 days, filter, wash, dry, and grind to obtain chemical drug eutectic powder;

[0070] S2. Add 2g of arginine-glycine-aspartic acid RGD to 200mL of water, add 0.5g of NHS and 0.5g of EDC, stir and activate for 20min, add 7g of UiO-66-NH2, stir and react for 12h, centrifuge, wash, dry, and obtain UiO-66-NH2 / RGD complex.

[0071] S3. Add 10g of UiO-66-NH2 / RGD complex to 200mL of water, add 2g of NHS and 2g of EDC, stir and activate for 20min, add 3g of chemical drug eutectic powder, stir and react for 10h, centrifuge, wash, dry, and obtain drug carrier;

[0072] S4. Add 10g of exosomes to 500mL of water, add 4g of drug carrier, and incubate with ultrasound at 300W power, 36℃ temperature, and 10h time. Centrifuge, wash, and dry to obtain the exosome drug delivery system.

[0073] S5. Wash 5g of Codonopsis pilosula, 5g of Angelica sinensis, 3g of Polygonatum sibiricum and 10g of Dendrobium officinale, dry them, crush them, add them to 300mL of water, heat to boiling and extract for 3h, filter, keep the filter residue, add ethanol to the filtrate until the ethanol content of the system is 80wt%, precipitate for 1h, filter, wash the solid, dry it, and obtain the Chinese herbal polysaccharide.

[0074] S6. Add 3g acrylic acid, 5g Chinese herbal polysaccharide, and 3g pullulan polysaccharide to 200mL of water, add 6g of exosome drug delivery system, then add 0.05g potassium persulfate, 0.5g Tween-40, and 0.4g N,N'-methylenebisacrylamide, stir and mix for 20min, add to 500mL of fish oil, emulsify at 8000r / min for 15min, crosslink at 50℃ for 3h, centrifuge, wash, and dry to obtain a sustained-release exosome system with a high half-life;

[0075] S7. Mix 1g D-ribose, 4g yeast-β-glucan, 2g glutathione, and 15g sustained-release high half-life exosome system for 10 minutes to obtain an exosome composition that reduces the side effects of chemotherapy.

[0076] Example 2

[0077] This embodiment provides a method for preparing an exosome composition that reduces the side effects of chemotherapy, comprising the following steps:

[0078] S1. Dissolve 5g of methotrexate in 100mL of 10wt% KOH solution to prepare solution 1; dissolve 3g of vincristine in 100mL of ethanol to prepare solution 2; dissolve 6g of daunorubicin in 200mL of water to prepare solution 3; mix solutions 1, 2, and 3 evenly, allow to evaporate in an open environment for 10 days, filter, wash, dry, and grind to obtain chemical drug eutectic powder;

[0079] S2. Add 4g of arginine-glycine-aspartic acid RGD to 200mL of water, add 1g of NHS and 1g of EDC, stir and activate for 40min, add 10g of UiO-66-NH2, stir and react for 15h, centrifuge, wash, dry, and obtain UiO-66-NH2 / RGD complex.

[0080] S3. Add 15g of UiO-66-NH2 / RGD complex to 200mL of water, add 3g of NHS and 3g of EDC, stir and activate for 40min, add 6g of chemical eutectic powder, stir and react for 14h, centrifuge, wash, dry, and obtain the drug carrier;

[0081] S4. Add 12g of exosomes to 500mL of water, add 7g of drug carrier, incubate with ultrasound at 500W power, 38℃ temperature for 12h, centrifuge, wash, and dry to obtain the exosome drug delivery system.

[0082] S5. Wash 10g of Codonopsis pilosula, 10g of Angelica sinensis, 7g of Polygonatum sibiricum and 15g of Dendrobium officinale, dry them, crush them, add them to 300mL of water, heat to boiling and extract for 5h, filter, keep the filter residue, add ethanol to the filtrate until the ethanol content of the system is 80wt%, precipitate for 1h, filter, wash the solid, dry it, and obtain the Chinese herbal polysaccharide.

[0083] S6. Add 7g acrylic acid, 10g Chinese herbal polysaccharide, and 5g pullulan polysaccharide to 200mL of water, add 8g of exosome drug delivery system, then add 0.1g ammonium persulfate, 0.5g Tween-60, 0.5g Span 60, and 0.8g N,N'-methylenebisacrylamide, stir and mix for 20min, add to 500mL of fish oil, emulsify at 8000r / min for 15min, crosslink at 60℃ for 5h, centrifuge, wash, and dry to obtain a sustained-release exosome system with a high half-life;

[0084] S7. Mix 3g D-ribose, 8g yeast-β-glucan, 4g glutathione, and 20g sustained-release high half-life exosome system for 10 minutes to obtain an exosome composition that reduces the side effects of chemotherapy.

[0085] Example 3

[0086] This embodiment provides a method for preparing an exosome composition that reduces the side effects of chemotherapy, comprising the following steps:

[0087] S1. Dissolve 4g of methotrexate in 100mL of 7wt% NaOH solution to prepare solution 1; dissolve 2g of vincristine in 100mL of ethanol to prepare solution 2; dissolve 5g of daunorubicin in 200mL of water to prepare solution 3; mix solutions 1, 2, and 3 evenly, allow to evaporate in an open environment for 8 days, filter, wash, dry, and grind to obtain chemical drug eutectic powder;

[0088] S2. Add 3g of arginine-glycine-aspartic acid RGD to 200mL of water, add 0.7g of NHS and 0.7g of EDC, stir and activate for 30min, add 8g of UiO-66-NH2, stir and react for 13h, centrifuge, wash, dry, and obtain UiO-66-NH2 / RGD complex.

[0089] S3. Add 12g of UiO-66-NH2 / RGD complex to 200mL of water, add 2.5g of NHS and 2.5g of EDC, stir and activate for 30min, add 4.5g of chemical drug eutectic powder, stir and react for 12h, centrifuge, wash, dry, and obtain the drug carrier;

[0090] S4. Add 11g of exosomes to 500mL of water, add 5.5g of drug carrier, incubate with ultrasound at 400W power, 37℃ for 11h, centrifuge, wash, and dry to obtain the exosome drug delivery system.

[0091] S5. Wash 7g of Codonopsis pilosula, 7g of Angelica sinensis, 5g of Polygonatum sibiricum and 12g of Dendrobium officinale, dry them, crush them, add them to 300mL of water, heat to boiling and extract for 4h, filter, keep the filter residue, add ethanol to the filtrate until the ethanol content of the system is 80wt%, precipitate for 1h, filter, wash the solid, dry it, and obtain the Chinese herbal polysaccharide.

[0092] S6. Add 5g acrylic acid, 7g Chinese herbal polysaccharide, and 4g pullulan polysaccharide to 200mL of water, add 7g of exosome drug delivery system, then add 0.07g sodium persulfate, 0.4g Tween-80, 0.3g Span-80, and 0.6g N,N'-methylenebisacrylamide, stir and mix for 20min, add to 500mL of fish oil, emulsify at 8000r / min for 15min, crosslink at 55℃ for 4h, centrifuge, wash, and dry to obtain a sustained-release exosome system with a high half-life.

[0093] S7. Mix 2g D-ribose, 6g yeast-β-glucan, 3g glutathione, and 17g sustained-release high half-life exosome system for 10 minutes to obtain an exosome composition that reduces the side effects of chemotherapy.

[0094] Comparative Example 1

[0095] The difference from Example 3 is that methotrexate was not added in step S1.

[0096] Specifically as follows:

[0097] S1. Dissolve 2g of vincristine in 100mL of ethanol to prepare solution 2; dissolve 5g of daunorubicin in 200mL of water to prepare solution 3; mix solutions 2 and 3 evenly, allow to evaporate in an open container for 8 days, filter, wash, dry, grind, and obtain chemical drug eutectic powder.

[0098] Comparative Example 2

[0099] The difference from Example 3 is that vincristine was not added in step S1.

[0100] Specifically as follows:

[0101] S1. Dissolve 4g of methotrexate in 100mL of 7wt% NaOH solution to prepare solution 1; dissolve 5g of daunorubicin in 200mL of water to prepare solution 3; mix solutions 1 and 3 evenly, allow to evaporate in an open environment for 8 days, filter, wash, dry, and grind to obtain chemical drug eutectic powder.

[0102] Comparative Example 3

[0103] The difference from Example 3 is that daunorubicin was not added in step S1.

[0104] Specifically as follows:

[0105] S1. Dissolve 4g of methotrexate in 100mL of 7wt% NaOH solution to obtain solution 1; dissolve 2g of vincristine in 100mL of ethanol to obtain solution 2; mix solution 1 and solution 2 evenly, allow to evaporate in an open environment for 8 days, filter, wash, dry, grind, and obtain chemical drug eutectic powder.

[0106] Comparative Example 4

[0107] The difference from Example 3 is that step S2 was not performed.

[0108] Specifically as follows:

[0109] S1. Dissolve 4g of methotrexate in 100mL of 7wt% NaOH solution to prepare solution 1; dissolve 2g of vincristine in 100mL of ethanol to prepare solution 2; dissolve 5g of daunorubicin in 200mL of water to prepare solution 3; mix solutions 1, 2, and 3 evenly, allow to evaporate in an open environment for 8 days, filter, wash, dry, and grind to obtain chemical drug eutectic powder;

[0110] S2. Add 12g UiO-66-NH2 to 200mL of water, add 4.5g of chemical eutectic powder, stir and react for 12h, centrifuge, wash, dry, and obtain the drug carrier;

[0111] S3. Add 11g of exosomes to 500mL of water, add 5.5g of drug carrier, incubate with ultrasound at 400W power, 37℃ for 11h, centrifuge, wash, and dry to obtain the exosome drug delivery system.

[0112] S4. Wash 7g of Codonopsis pilosula, 7g of Angelica sinensis, 5g of Polygonatum sibiricum and 12g of Dendrobium officinale, dry them, crush them, add them to 300mL of water, heat to boiling and extract for 4h, filter, keep the residue, add ethanol to the filtrate until the ethanol content of the system is 80wt%, precipitate for 1h, filter, wash the solid, dry it, and obtain the Chinese herbal polysaccharide.

[0113] S5. Add 5g acrylic acid, 7g Chinese herbal polysaccharide, and 4g pullulan polysaccharide to 200mL of water, add 7g of exosome drug delivery system, then add 0.07g sodium persulfate, 0.4g Tween-80, 0.3g Span-80, and 0.6g N,N'-methylenebisacrylamide, stir and mix for 20min, add to 500mL of fish oil, emulsify at 8000r / min for 15min, crosslink at 55℃ for 4h, centrifuge, wash, and dry to obtain a sustained-release exosome system with a high half-life.

[0114] S6. Mix 2g D-ribose, 6g yeast-β-glucan, 3g glutathione, and 17g sustained-release high half-life exosome system for 10 minutes to obtain an exosome composition that reduces the side effects of chemotherapy.

[0115] Comparative Example 5

[0116] The difference from Example 3 is that no traditional Chinese medicine polysaccharides were added in step S6.

[0117] Specifically as follows:

[0118] S6. Add 5g acrylic acid and 11g pullulan polysaccharide to 200mL of water, add 7g of exosome drug delivery system, then add 0.07g sodium persulfate, 0.4g Tween-80, 0.3g Span-80 and 0.6g N,N'-methylenebisacrylamide, stir and mix for 20min, add to 500mL of fish oil, emulsify at 8000r / min for 15min, crosslink at 55℃ for 4h, centrifuge, wash and dry to obtain sustained-release high half-life exosome system.

[0119] Comparative Example 6

[0120] The difference from Example 3 is that steps S2 and S3 were not performed.

[0121] Specifically as follows:

[0122] S1. Dissolve 4g of methotrexate in 100mL of 7wt% NaOH solution to prepare solution 1; dissolve 2g of vincristine in 100mL of ethanol to prepare solution 2; dissolve 5g of daunorubicin in 200mL of water to prepare solution 3; mix solutions 1, 2, and 3 evenly, allow to evaporate in an open environment for 8 days, filter, wash, dry, and grind to obtain chemical drug eutectic powder;

[0123] S2. Add 11g of exosomes to 500mL of water, add 5.5g of chemical drug eutectic powder, sonicate at 400W power, 37℃ for 11h, centrifuge, wash, and dry to obtain the exosome drug delivery system.

[0124] S3. Wash 7g of Codonopsis pilosula, 7g of Angelica sinensis, 5g of Polygonatum sibiricum and 12g of Dendrobium officinale, dry them, crush them, add them to 300mL of water, heat to boiling and extract for 4h, filter, keep the filter residue, add ethanol to the filtrate until the ethanol content of the system is 80wt%, precipitate for 1h, filter, wash the solid, dry it, and obtain the Chinese herbal polysaccharide.

[0125] S4. Add 5g acrylic acid, 7g Chinese herbal polysaccharide, and 4g pullulan polysaccharide to 200mL of water, add 7g of exosome drug delivery system, then add 0.07g sodium persulfate, 0.4g Tween-80, 0.3g Span-80, and 0.6g N,N'-methylenebisacrylamide, stir and mix for 20min, add to 500mL of fish oil, emulsify at 8000r / min for 15min, crosslink at 55℃ for 4h, centrifuge, wash, and dry to obtain a sustained-release exosome system with a high half-life.

[0126] S5. Mix 2g D-ribose, 6g yeast-β-glucan, 3g glutathione, and 17g sustained-release high half-life exosome system for 10 minutes to obtain an exosome composition that reduces the side effects of chemotherapy.

[0127] Comparative Example 7

[0128] The difference from Example 3 is that step S6 was not performed.

[0129] Specifically as follows:

[0130] S1. Dissolve 4g of methotrexate in 100mL of 7wt% NaOH solution to prepare solution 1; dissolve 2g of vincristine in 100mL of ethanol to prepare solution 2; dissolve 5g of daunorubicin in 200mL of water to prepare solution 3; mix solutions 1, 2, and 3 evenly, allow to evaporate in an open environment for 8 days, filter, wash, dry, and grind to obtain chemical drug eutectic powder;

[0131] S2. Add 3g of arginine-glycine-aspartic acid RGD to 200mL of water, add 0.7g of NHS and 0.7g of EDC, stir and activate for 30min, add 8g of UiO-66-NH2, stir and react for 13h, centrifuge, wash, dry, and obtain UiO-66-NH2 / RGD complex.

[0132] S3. Add 12g of UiO-66-NH2 / RGD complex to 200mL of water, add 2.5g of NHS and 2.5g of EDC, stir and activate for 30min, add 4.5g of chemical drug eutectic powder, stir and react for 12h, centrifuge, wash, dry, and obtain the drug carrier;

[0133] S4. Add 11g of exosomes to 500mL of water, add 5.5g of drug carrier, incubate with ultrasound at 400W power, 37℃ for 11h, centrifuge, wash, and dry to obtain the exosome drug delivery system.

[0134] S5. Wash 7g of Codonopsis pilosula, 7g of Angelica sinensis, 5g of Polygonatum sibiricum and 12g of Dendrobium officinale, dry them, crush them, add them to 300mL of water, heat to boiling and extract for 4h, filter, keep the filter residue, add ethanol to the filtrate until the ethanol content of the system is 80wt%, precipitate for 1h, filter, wash the solid, dry it, and obtain the Chinese herbal polysaccharide.

[0135] S6. Mix 2g D-ribose, 6g yeast-β-glucan, 3g glutathione, 8.5g traditional Chinese medicine polysaccharide, and 8.5g exosome drug delivery system for 10 minutes to obtain an exosome composition that reduces the side effects of chemotherapy.

[0136] Test Example 1

[0137] SPF-grade male BALB / c mice, weighing 20-24g and 4-6 weeks old, were selected. The skin was prepared under the right axilla, disinfected with 75% ethanol, and then inoculated with 0.2 mL of MFC gastric cancer cell suspension (2.5 × 10⁻⁶). 7 A mouse model of gastric cancer bearing MFC was established using exosomes (e.g., 5-fluorouracil / mL). Mice bearing gastric cancer were randomly divided into a model group, a 5-fluorouracil group, Examples 1-3, and Comparative Examples 1-7, with 8 mice in each group. Eight normal mice were also included in the control group. Successful modeling was indicated by the presence of visible nodules in the right axilla 5-6 days after modeling. Drug administration began on day 7. Examples 1-3 and Comparative Examples 1-7 were administered 50 mg / kg of the corresponding exosome composition for reducing chemotherapy side effects, while the 5-fluorouracil group received 25 mg / kg of 5-fluorouracil. The control and model groups received an equal volume of physiological saline. Administration continued for 9 days, with assessments performed on day 10.

[0138] 1. Blood index testing

[0139] Blood was collected from the tail vein of each animal in a volume of 2.0 mL. The white blood cell, red blood cell, and platelet counts were determined using an automated hematology analyzer. The results are shown in Table 1.

[0140] Table 1

[0141] Group <![CDATA[White blood cell count (10 9 cells / mL)]]> <![CDATA[Red blood cell count (10 12 cells / mL)]]> <![CDATA[Platelet count (10 9 cells / mL)]]> Blank group 16.82±3.46 13.29±6.72 182.25±22.56 Model group 1.03±0.32* 3.35±0.89* 45.72±10.26* 5-Fluorouracil group 2.45±0.64# 4.67±0.67# 52.84±8.94# Example 1 11.25±0.39# 9.05±0.72# 166.56±8.21# Example 2 11.09±0.41# 9.11±0.62# 169.26±9.10# Example 3 11.57±0.33# 9.26±0.59# 170.14±9.04# Comparative Example 1 11.10±0.89 9.02±0.99 162.62±11.56 Comparative Example 2 11.01±0.92 8.95±1.13 163.45±14.24 Comparative Example 3 11.04±0.87 9.00±1.04 163.01±13.25 Comparative Example 4 9.58±0.79 7.89±1.07 142.25±12.85 Comparative Example 5 8.45±0.84 6.89±1.10 126.89±13.42 Comparative Example 6 8.92±0.91 7.02±1.09 130.16±11.78 Comparative Example 7 7.79±0.86 6.38±1.12 114.52±14.54

[0142] Note: * indicates P < 0.05 compared to the control group; # indicates P < 0.05 compared to the model group.

[0143] As shown in the table above, the exosome compositions prepared in Examples 1-3 of this invention for reducing the side effects of chemotherapy significantly improved the bone marrow suppression effect caused by chemotherapy drugs.

[0144] 2. Organ Index

[0145] Mice were anesthetized with sodium pentobarbital, and approximately 1 mL of blood was collected by enucleation. After being left at room temperature for 1 hour, the blood was centrifuged at 4°C to obtain serum. The liver, kidneys, and thymus were harvested, and the organ coefficient was calculated using the formula: Organ Index = Organ Mass / Body Mass. The results are shown in Table 2.

[0146] Table 2

[0147]

[0148] Note: * indicates P < 0.05 compared to the control group; # indicates P < 0.05 compared to the model group.

[0149] As can be seen from the table above, the exosome compositions prepared in Examples 1-3 of this invention for reducing the side effects of chemotherapy have little effect on organs.

[0150] 3. Tumor inhibition rate

[0151] The tumors were removed and weighed, and the tumor weight and tumor inhibition rate of each group were recorded. The results are shown in Table 3.

[0152] Tumor inhibition rate = [(tumor mass in the model group - tumor mass in the treatment group) / tumor mass in the model group] × 100%.

[0153] Table 3

[0154]

[0155]

[0156] As can be seen from the table above, the exosome compositions for reducing the side effects of chemotherapy prepared in Examples 1-3 of this invention have good anti-tumor effects.

[0157] 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, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an exosome composition that reduces the side effects of chemotherapy for gastric cancer, characterized in that, Methotrexate, vincristine, and daunorubicin were prepared into a cocrystal, loaded onto UiO-66-NH2 with RGD, loaded onto exosomes, and embedded in a complex of acrylic acid, traditional Chinese medicine polysaccharides, and pullulan polysaccharides to prepare a sustained-release exosome system with a high half-life. This system was then mixed with D-ribose, yeast-β-glucan, and glutathione to prepare an exosome composition that reduces the side effects of chemotherapy for gastric cancer. The traditional Chinese medicine polysaccharides were obtained by extracting Codonopsis pilosula, Angelica sinensis, Polygonatum sibiricum, and Dendrobium officinale with boiling water and precipitating with ethanol. Includes the following steps: S1. Dissolve methotrexate in an alkaline solution to obtain solution 1; dissolve vinblastine in ethanol to obtain solution 2; dissolve daunorubicin in water to obtain solution 3; mix solutions 1, 2, and 3 evenly, allow to evaporate in an open container, filter, wash, dry, and grind to obtain a chemical drug eutectic powder; the concentration of the alkaline solution is 5-10 wt%, the alkaline is NaOH or KOH, and the mass ratio of methotrexate, vinblastine, and daunorubicin is 3-5:1-3:3-6; S2. Arginine-glycine-aspartic acid RGD was added to water, along with NHS and EDC. The mixture was stirred to activate the solution, and then UiO-66-NH2 was added. The mixture was stirred to react, centrifuged, washed, and dried to obtain the UiO-66-NH2 / RGD complex. The mass ratio of arginine-glycine-aspartic acid RGD, NHS, EDC, and UiO-66-NH2 was 2-4:0.5-1:0.5-1:7-10. S3. The UiO-66-NH2 / RGD complex is added to water, NHS and EDC are added, the mixture is stirred to activate it, the drug eutectic powder is added, the mixture is stirred to react, centrifuged, washed, and dried to obtain the drug carrier; the mass ratio of the UiO-66-NH2 / RGD complex, NHS, EDC and drug eutectic powder is 10-15:2-3:2-3:3-6; S4. Add exosomes to water, add drug carrier, incubate with ultrasound, centrifuge, wash, and dry to obtain an exosome drug delivery system; the exosomes are mesenchymal stem cell exosomes, and the mass ratio of exosomes to drug carrier is 10-12:4-7; S5. Codonopsis pilosula, Angelica sinensis, Polygonatum sibiricum, and Dendrobium officinale are washed, dried, pulverized, added to water, heated to boiling and extracted, filtered, the residue is retained, the filtrate is added to ethanol to precipitate, filtered, the solid is washed and dried to obtain the Chinese herbal polysaccharide; the mass ratio of Codonopsis pilosula, Angelica sinensis, Polygonatum sibiricum, and Dendrobium officinale is 5-10:5-10:3-7:10-15; S6. Add acrylic acid, Chinese herbal polysaccharide, pullulan polysaccharide to water, add to the exosome drug delivery system, then add initiator, emulsifier, N,N'-methylenebisacrylamide, stir and mix evenly, add to fish oil, emulsify, crosslink reaction, centrifuge, wash, dry, to obtain sustained-release high half-life exosome system; S7. Mix D-ribose, yeast-β-glucan, glutathione, and a sustained-release high half-life exosome system evenly to prepare an exosome composition that reduces the side effects of chemotherapy for gastric cancer.

2. The preparation method according to claim 1, characterized in that, The open evaporation time described in step S1 is 7-10 days.

3. The preparation method according to claim 1, characterized in that, The stirring activation time in step S2 is 20-40 min, and the stirring reaction time is 12-15 h.

4. The preparation method according to claim 1, characterized in that, The stirring activation time in step S3 is 20-40 min, and the stirring reaction time is 10-14 h.

5. The preparation method according to claim 1, characterized in that, The ultrasonic incubation in step S4 has a power of 300-500W, a temperature of 36-38℃, and a time of 10-12h.

6. The preparation method according to claim 1, characterized in that, The heating and boiling extraction time in step S5 is 3-5 hours.

7. The preparation method according to claim 1, characterized in that, In step S6, the mass ratio of acrylic acid, traditional Chinese medicine polysaccharide, pullulan polysaccharide, exosome drug delivery system, initiator, emulsifier, and N,N'-methylenebisacrylamide is 3-7:5-10:3-5:6-8:0.05-0.1:0.5-1:0.4-0.

8. The initiator is selected from at least one of potassium persulfate, ammonium persulfate, and sodium persulfate. The emulsifier is selected from at least one of Span-20, Span-40, Span-60, Span-80, Tween-20, Tween-40, Tween-60, and Tween-80. The crosslinking reaction temperature is 50-60℃, and the time is 3-5h. In step S7, the mass ratio of D-ribose, yeast-β-glucan, glutathione, and sustained-release high half-life exosome system is 1-3:4-8:2-4:15-20.

8. An exosome composition for reducing the side effects of chemotherapy for gastric cancer, prepared by the method according to any one of claims 1-7.

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