Exosome composition for reducing side effects of chemotherapy treatment and preparation method of exosome composition

Abstract

The invention provides an exosome composition for reducing side effects of chemotherapy treatment and a preparation method of the exosome composition, and belongs to the technical field of medicines. The preparation method comprises the following steps: preparing a co-crystal from methotrexate, vinblastine and daunorubicin, loading the co-crystal on UiO-66-NH2 grafted with RGD, loading the co-crystal on exosome, embedding the UiO-66-NH2 and the exosome in a compound of acrylic acid, traditional Chinese medicine polysaccharide and pullulan to prepare a slow-release long-half-life exosome system, and uniformly mixing the slow-release long-half-life exosome system with D-ribose, yeast-beta glucan and glutathione to prepare a sustained-release long-half-life exosome. The traditional Chinese medicine polysaccharide is obtained by carrying out boiling water extraction and ethanol precipitation on codonopsis pilosula, angelica sinensis, polygonatum kingianum and dendrobium officinale kimura et migo. The exosome composition for reducing the side effects of chemotherapy is prepared, the drug resistance of tumor cells is reduced, the drug effect is improved, the side effects after chemotherapy are reduced, the effect of targeted slow release of drugs is achieved, and the exosome composition has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an exosome composition for reducing the side effects of chemotherapy and a preparation method thereof. Background Art

[0002] A tumor is a disease caused by the body's inability to regulate localized mutated cells, leading to abnormal proliferation and ultimately the formation of a new organism. Once a tumor forms, it can grow rapidly and uncontrollably, invading and destroying normal tissues and organs. In severe cases, it can metastasize throughout the patient's body, resulting in death.

[0003] Chemotherapy began in the 1840s, first used to alleviate the symptoms of acute lymphoblastic leukemia. With the continued advancement of chemotherapy technology, it was discovered that chemotherapy could also combat solid tumors and provide long-term remission. Later, the clinical therapeutic value of adjuvant chemotherapy after cancer surgery was discovered. By the 1980s and 1990s, after more than a century of exploration and development, new chemotherapy drugs were continuously introduced, further enriching and facilitating the formulation and selection of chemotherapy regimens by physicians, significantly improving the clinical effectiveness of chemotherapy.

[0004] Preoperative chemotherapy for cancer has numerous clinical benefits in treating a variety of tumors; in particular, combined therapy can effectively alleviate symptoms and prolong patients' lives. Therefore, compared with other treatment options, radiotherapy and chemotherapy currently represent a wide range of treatment options, with longer durations of action and more definitive efficacy.

[0005] To date, the medical community continues to research and develop new chemotherapy drugs to further enhance chemotherapy efficacy while mitigating its side effects and improving patient tolerance and compliance. Furthermore, the combined use of chemotherapy drugs with molecularly targeted therapies, as well as new treatment approaches based on personalized precision medicine, are opening up new avenues for cancer chemotherapy. Summary of the Invention

[0006] The purpose of the present invention is to provide an exosome composition and a preparation method for reducing the side effects of chemotherapy treatment, thereby improving the water solubility of chemotherapy drugs, reducing their usage, reducing the drug resistance of tumor cells, synergistically improving the drug efficacy, and reducing side effects after chemotherapy. The UiO-66-NH2 / RGD complex is used for loading, which increases the drug loading capacity and can be well complexed with exosomes to achieve the effect of targeted drug release, thereby reducing drug side effects. The exosomes are embedded in an acidified polysaccharide shell, have good acid resistance, extend the half-life of the exosomes, protect the exosomes from enzymatic hydrolysis and immune attack, and achieve sustained release, which has broad application prospects.

[0007] The technical solution of the present invention is achieved as follows:

[0008] The present invention provides a method for preparing an exosome composition for reducing the side effects of chemotherapy. The exosome composition 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, vinblastine and daunorubicin are prepared into a cocrystal, loaded on UiO-66-NH2 connected with RGD, loaded on exosomes, and embedded in an acrylic acid, traditional Chinese medicine polysaccharide, and pullulan complex to prepare a sustained-release exosome system with a high half-life. The system is evenly mixed with D-ribose, yeast-β-glucan, and glutathione to prepare an exosome composition that reduces the side effects of chemotherapy. The traditional Chinese medicine polysaccharide is a traditional Chinese medicine polysaccharide obtained by boiling water extraction and ethanol precipitation of Codonopsis pilosula, Angelica sinensis, Polygonatum sibiricum, and Dendrobium officinale.

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

[0011] S1. Dissolving methotrexate in an alkaline solution to prepare solution 1; dissolving vinblastine in ethanol to prepare solution 2; and dissolving daunorubicin in water to prepare solution 3; mixing solutions 1, 2, and 3, evaporating them in an open air, filtering, washing, drying, and grinding to obtain a drug eutectic powder;

[0012] S2. Arginine-glycine-aspartic acid RGD was added to water, NHS and EDC were added, stirred for activation, UiO-66-NH2 was added, the reaction was stirred, centrifuged, washed, and dried to obtain a UiO-66-NH2 / RGD complex;

[0013] S3. The UiO-66-NH2 / RGD complex was added to water, NHS and EDC were added, stirred for activation, the drug eutectic powder was added, stirred for reaction, centrifuged, washed, and dried to obtain a drug carrier;

[0014] S4. adding exosomes to water, adding drug carriers, incubating with ultrasound, centrifuging, washing, and drying to prepare an exosome drug-carrying system;

[0015] S5. Codonopsis, Angelica, Polygonatum, Dendrobium candidum were washed, dried, crushed, added to water, heated to boiling for extraction, filtered, the residue was retained, the filtrate was added ethanol for precipitation, filtered, the solid was washed, and dried to obtain a Chinese herbal polysaccharide;

[0016] S6. Add acrylic acid, traditional Chinese medicine polysaccharides, and pullulan to water, add the exosome drug delivery system, then add an initiator, an emulsifier, and N,N'-methylenebisacrylamide, stir and mix thoroughly, add to fish oil, emulsify, cross-link, centrifuge, wash, and dry to produce a sustained-release exosome system with a high half-life;

[0017] S7. Evenly mix D-ribose, yeast-β-glucan, glutathione, and a sustained-release high half-life exosome system 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 5-10wt%, the base is NaOH or KOH, the mass ratio of methotrexate, vinblastine, and daunorubicin is 3-5:1-3:3-6, and the open volatilization time is 7-10d.

[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 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 carrier is 10-12:4-7, the power of the ultrasonic incubation is 300-500W, the temperature is 36-38°C, and the time is 10-12h.

[0022] As a further improvement of the present invention, in step S5, the mass ratio of Codonopsis pilosula, Angelica sinensis, Polygonatum sibiricum and Dendrobium officinale 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, 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 temperature of the cross-linking reaction 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 the 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 heart 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 heart function; it can also significantly improve the symptoms of shortness of breath when moving, frequent palpitations, chest tightness, and qi deficiency.

[0027] Enhance the body's energy and relieve muscle soreness: D-ribose is the starting molecule for synthesizing ATP and an important raw material for muscles to synthesize energy substances. Experiments have confirmed that supplementing D-ribose can improve the body's athletic ability, effectively resist fatigue, and relieve muscle soreness. (Baidu Encyclopedia)

[0028] Yeast-beta glucan (a new resource food) can help treat various cancers, persistent hepatitis B, rheumatic diseases, recurrent oral ulcers, allergies, autoimmune diseases, various infectious diseases, and even the aging process. Yeast-beta glucan can strengthen or restore a weakened or incomplete immune response. It also has anti-radiation and chemical toxin neutralization effects: When ionizing or chemical radiation penetrates biological tissue, the energy from the ions or chemical molecules is directly absorbed by biomacromolecules or, through ion diffusion and the action of free radicals, causes chemical reactions in other biomolecules, causing physical and chemical damage to the human body. Yeast-beta glucan's unique inverted triple helix structure can neutralize toxins in the body, promote hematopoiesis, and enhance the production of blood cells, including granulocytes, monocytes, and erythrocytes, thereby promoting better recovery of human cells from lethal doses of radiation or toxins. (Baidu Encyclopedia)

[0029] Glutathione is a tripeptide containing a gamma-amide bond and a sulfhydryl group. It is composed of glutamic acid, cysteine, and amino acid and is present in nearly every cell in the body. Glutathione helps maintain normal immune system function and has antioxidant and integrative detoxification properties. The sulfhydryl group on cysteine (often abbreviated as G-SH) is its active group and readily binds to certain drugs or toxins, achieving an integrative detoxification effect. Glutathione is not only used in pharmaceuticals but also as a base for functional foods, finding widespread application in anti-aging, immune enhancement, and anti-tumor applications. It also has antioxidant, integrative detoxification, whitening, and spot-lightening properties.

[0030] Baidu Encyclopedia.

[0031] The present invention further protects an exosome composition prepared by the above-mentioned preparation method for reducing 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 for exosomes. MSC exosomes can deliver siRNA to suppress 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 contain a rich array of substances, including RNA, DNA, proteins, antibodies, and small molecule metabolites. These substances can directly activate receptors in target cells and, by transporting macromolecules such as nucleic acids and proteins, act as intercellular messengers, regulating a variety of physiological and pathological processes, such as tumor development and metastasis and the progression of immune diseases. The CD55 and CD59 molecules on their surfaces enhance the stability of exosomes and prolong their circulation. CD47 protects exosomes from clearance by the mononuclear phagocyte system, while membrane proteins such as apoptosis-related gene 2-interacting protein X, tumor susceptibility gene 101, heat shock proteins (HSPs), and tetralipids CD63, CD9, and CD81 contribute to their targeted delivery capabilities. The nanoscale size, fluidity, and targeted nature of exosomes enable them to transcend natural biological barriers, including the dense stroma of cancer cells, enabling efficient, targeted drug delivery.

[0035] However, the problems of short half-life and high content specificity of exosomes will affect the final therapeutic effect. The present invention co-incubates exosomes with drug carriers by ultrasonic co-incubation, and causes vacuoles to appear on the exosome membrane by ultrasonic vibration, allowing the drug to enter, and because the hydrophobic part of the exosome is exposed after ultrasonic treatment, the interaction with the target cell membrane is enhanced. The exosome drug delivery system prepared by the present invention is embedded by acrylic acidized Chinese medicinal polysaccharides and pullulan polysaccharides to form a mutually penetrating network aggregation material shell, which can make the prepared sustained-release high half-life exosome system have good acid resistance, shrink in an acidic environment without releasing, and swell under alkaline conditions so that the contents are slowly released, thereby protecting the exosome drug delivery system from being destroyed by the gastric acid environment, and being able to prolong the half-life of the exosomes, which can protect the exosomes from enzymatic hydrolysis and immune attack, and achieve sustained release.

[0036] Methotrexate is a chemotherapy drug that blocks DNA (deoxyribonucleic acid) assembly and inhibits deoxyribonucleotide synthesis, but it is insoluble in water; vinblastine is a chemotherapy drug that inhibits cell mitosis and tubulin polymerization, but it is also insoluble in water, which greatly limits the biocompatibility of these chemotherapy drugs and requires a very large amount to have a certain effect. Therefore, it is mixed with an aqueous solution of daunorubicin (which disrupts the synthesis process of nucleic acids and hinders RNA (ribonucleic acid) synthesis) and volatilized to form a co-crystal to prepare a water-soluble co-crystal drug, which greatly improves its efficacy and reduces its usage, fundamentally and greatly reduces the side effects of the drug use. Moreover, the synergistic effect of the three chemotherapy drugs with different effects can significantly enhance 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, which can load large doses of chemical drug cocrystal powder. It is coupled with arginine-glycine-aspartic acid RGD, which promotes the adhesion and coupling of drug carriers and exosomes. It can also bind to integrin receptors and specifically bind to tumor neovascularization and integrins overexpressed on the surface of tumor cells, thereby further promoting targeted drug release. In addition, the cyclic RGD peptide segment 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 the present invention are rich in polysaccharides and have good efficacy, as shown in the following:

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

[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 have decreased during radiotherapy and chemotherapy. Codonopsis pilosula can also improve the body's ability to resist stress and enhance its resistance to adverse stimuli such as environmental temperature changes and chemicals.

[0041] Improving Immune Function: Codonopsis polysaccharide can significantly increase the phagocytic function of peritoneal macrophages and improve the carbon particle clearance rate in mice. Codonopsis pilosula water decoction and water decoction alcohol precipitation have similar effects to Codonopsis pilosula polysaccharide, significantly increasing the number of macrophages in mice, enlarging cell volume, increasing pseudopodia, and enhancing phagocytic ability. The activities of DNA, RNA, carbohydrates, ATPase, acid esterase, and succinate dehydrogenase in the cells are also significantly enhanced.

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

[0043] Effect on bacteria: In vitro experiments have shown that meningococci are moderately sensitive to Codonopsis pilosula decoction, while diphtheria, catarrhal diplococci, paracocci, Escherichia coli and human tuberculosis are slightly sensitive.

[0044] Stimulates protein and nucleic acid synthesis: Ginseng total saponins and protein synthesis-promoting factors promote the biosynthesis of protein, DNA, and RNA, increase cytoplasmic ribosomes, and elevate serum protein synthesis rates, albumin, and gamma globulin levels. Therefore, ginseng can promote animal growth, increase weight gain, enhance disease resistance, and promote recovery in patients.

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

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

[0047] Enhance immunity: Angelica sinensis has the effect of promoting specific and nonspecific immune functions. Oral administration of Angelica sinensis decoction can increase the total number of mouse skin cells, significantly increase the phagocytic function of animal peritoneal macrophages, and improve the clearance rate of dyes by the reticuloendothelial system; Angelica sinensis polysaccharide has a significant antagonistic effect on the reduced phagocytic function of macrophages caused by cyclophosphamide.

[0048] Anti-inflammatory and analgesic: Angelica sinensis decoction has a significant inhibitory effect on acute and chronic inflammation caused by three inflammatory agents. Studies have found that the analgesic intensity of Angelica sinensis 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] Anti-tumor: Angelica polysaccharide significantly increases the number of peripheral blood T and B lymphocytes in normal mice, tumor-bearing mice, and X-ray-irradiated tumor-bearing mice. Chinese Materia Medica, 2nd Edition, pp. 988-989.

[0051] Polygonatum sibiricum (ingredients with medicinal and edible properties):

[0052] Cardiovascular effects: Polygonatum sibiricum significantly enhances myocardial contractility in rats and increases perfusion in isolated rabbit hearts. Intravenous injection of this product can counteract acute myocardial ischemia induced by vasopressin. Intraperitoneal injection can improve myocardial oxygen tolerance in mice.

[0053] Agglutination 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 sibiricum polysaccharide can significantly counteract cyclophosphamide-induced leukopenia in peripheral blood of mice and can also increase spleen weight in immunocompromised mice. (Chinese Materia Medica, 2nd Edition, p. 1032).

[0055] Dendrobium officinale (new resource food) nourishes yin and clears heat, nourishes the stomach and produces body fluid, improves eyesight and strengthens the waist.

[0056] [Modern Research] Dendrobium candidum can significantly improve the weakness symptoms of mice with hyperthyroidism and yin deficiency.

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

[0058] Improve immune function: The water decoction of Dendrobium nobile has a significant promoting effect on the phagocytic function of mouse peritoneal phagocytes.

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

[0060] Anti-tumor effects: Dendrobium can prolong the average lifespan of ICR mice implanted with sarcoma 180 cells. Its ethanol extract exhibits significant cytotoxicity against human lung cancer cells, ovarian adenocarcinoma cells, promyelocytic cells, leukemia cells, and other cell lines. Traditional Chinese Medicine, Second Edition, page 1028.

[0061] The present invention prepares an exosome composition for reducing the side effects of chemotherapy treatment, improves the water solubility of chemotherapy drugs, reduces their usage, reduces the drug resistance of tumor cells, synergistically improves drug efficacy, and reduces side effects after chemotherapy. The UiO-66-NH2 / RGD complex is used for loading, which increases the drug loading capacity and can be well complexed with exosomes to achieve the effect of targeted drug release, thereby reducing drug side effects. The exosome composition is embedded in an acidified polysaccharide shell, has good acid resistance, and prolongs the half-life of the exosomes. It can protect the exosomes from enzymatic hydrolysis and immune attack and achieve sustained release, and has broad application prospects. DETAILED DESCRIPTION

[0062] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0063] UiO-66-NH2 was prepared 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). Ultrasonication at 1000 W for 30 minutes was performed to uniformly disperse the mixture. The mixture was then added to a hydrothermal reactor and reacted at 120°C for 24 hours. After cooling, the crude product was separated by centrifugation. The resulting yellow solid was washed and dried to obtain a yellow powder, designated UiO-66-NH2.

[0064] Arginine-glycine-aspartate RGD, CAS number: 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 for reducing the side effects of chemotherapy, comprising the following steps:

[0069] S1. 3 g of methotrexate was dissolved in 100 mL of 5 wt% NaOH solution to prepare solution 1; 1 g of vinblastine was dissolved in 100 mL of ethanol to prepare solution 2; 3 g of daunorubicin was dissolved in 200 mL of water to prepare solution 3; solutions 1, 2, and 3 were mixed, evaporated in an open air for 7 days, filtered, washed, dried, and ground to obtain a drug eutectic powder;

[0070] S2. 2 g of arginine-glycine-aspartic acid RGD was added to 200 mL of water, 0.5 g of NHS and 0.5 g of EDC were added, and the mixture was stirred and activated for 20 min. 7 g of UiO-66-NH2 was added, and the mixture was stirred for 12 h. The mixture was centrifuged, washed, and dried to obtain a UiO-66-NH2 / RGD complex.

[0071] S3. 10 g of UiO-66-NH2 / RGD complex was added to 200 mL of water, 2 g of NHS and 2 g of EDC were added, and the mixture was stirred and activated for 20 min. 3 g of drug eutectic powder was added and stirred for 10 h. The mixture was centrifuged, washed, and dried to obtain a drug carrier.

[0072] S4. Add 10 g of exosomes to 500 mL of water, add 4 g of drug-loaded solids, and incubate with ultrasound at 300 W and 36°C for 10 h. Centrifuge, wash, and dry to obtain an exosome-loaded drug system.

[0073] S5. 5g Codonopsis, 5g Angelica, 3g Polygonatum, 10g Dendrobium officinale were washed, dried, crushed, added to 300mL of water, heated to boiling and extracted for 3h, filtered, the residue was retained, and the filtrate was added with ethanol to an ethanol content of 80wt% and precipitated for 1h, filtered, the solid was washed, and dried to obtain a Chinese herbal polysaccharide;

[0074] S6. Add 3 g of acrylic acid, 5 g of traditional Chinese medicine polysaccharide, and 3 g of pullulan to 200 mL of water, add 6 g of the exosome drug delivery system, then add 0.05 g of potassium persulfate, 0.5 g of Tween-40, and 0.4 g of N,N'-methylenebisacrylamide. Stir and mix for 20 min. Add to 500 mL of fish oil, emulsify at 8000 rpm for 15 min, cross-link at 50°C for 3 h, centrifuge, wash, and dry to produce a sustained-release exosome system with a high half-life.

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

[0076] Example 2

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

[0078] S1. 5 g of methotrexate was dissolved in 100 mL of 10 wt% KOH solution to prepare solution 1; 3 g of vinblastine was dissolved in 100 mL of ethanol to prepare solution 2; 6 g of daunorubicin was dissolved in 200 mL of water to prepare solution 3; solutions 1, 2, and 3 were mixed, evaporated in an open air for 10 days, filtered, washed, dried, and ground to obtain a drug eutectic powder;

[0079] S2. 4 g of arginine-glycine-aspartic acid RGD was added to 200 mL of water, 1 g of NHS and 1 g of EDC were added, and the mixture was stirred and activated for 40 min. 10 g of UiO-66-NH2 was added, and the mixture was stirred for 15 h. The mixture was centrifuged, washed, and dried to obtain a UiO-66-NH2 / RGD complex.

[0080] S3. 15 g of UiO-66-NH2 / RGD complex was added to 200 mL of water, 3 g of NHS and 3 g of EDC were added, and the mixture was stirred and activated for 40 min. 6 g of drug eutectic powder was added and stirred for 14 h. The mixture was centrifuged, washed, and dried to obtain a drug carrier.

[0081] S4. Add 12 g of exosomes to 500 mL of water, add 7 g of drug-loaded solids, and incubate with ultrasound at 500 W and 38°C for 12 h. Centrifuge, wash, and dry to obtain an exosome-loaded drug system.

[0082] S5. 10g Codonopsis, 10g Angelica, 7g Polygonatum, 15g Dendrobium candidum were washed, dried, crushed, added to 300mL of water, heated to boiling and extracted for 5h, filtered, the residue was retained, and the filtrate was added ethanol to an ethanol content of 80wt% system, precipitated for 1h, filtered, the solid was washed, and dried to obtain a Chinese herbal polysaccharide;

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

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

[0085] Example 3

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

[0087] S1. 4 g of methotrexate was dissolved in 100 mL of 7 wt% NaOH solution to prepare solution 1; 2 g of vinblastine was dissolved in 100 mL of ethanol to prepare solution 2; 5 g of daunorubicin was dissolved in 200 mL of water to prepare solution 3; solutions 1, 2, and 3 were mixed, evaporated in an open air for 8 days, filtered, washed, dried, and ground to obtain a drug eutectic powder;

[0088] S2. 3 g of arginine-glycine-aspartic acid RGD was added to 200 mL of water, 0.7 g of NHS and 0.7 g of EDC were added, and the mixture was stirred and activated for 30 min. 8 g of UiO-66-NH2 was added, and the mixture was stirred for 13 h. The mixture was centrifuged, washed, and dried to obtain a UiO-66-NH2 / RGD complex.

[0089] S3. 12 g of UiO-66-NH2 / RGD complex was added to 200 mL of water, 2.5 g of NHS and 2.5 g of EDC were added, and the mixture was stirred and activated for 30 min. 4.5 g of drug eutectic powder was added, and the mixture was stirred for 12 h. The mixture was centrifuged, washed, and dried to obtain a drug carrier.

[0090] S4. Add 11 g of exosomes to 500 mL of water, add 5.5 g of drug-loaded solids, and incubate with ultrasound at 400 W and 37°C for 11 h. Centrifuge, wash, and dry to obtain an exosome-loaded drug system.

[0091] S5. 7g Codonopsis, 7g Angelica, 5g Polygonatum, 12g Dendrobium officinale were washed, dried, crushed, added to 300mL of water, heated to boiling and extracted for 4h, filtered, the residue was retained, and the filtrate was added with ethanol to an ethanol content of 80wt% and precipitated for 1h, filtered, the solid was washed, and dried to obtain a Chinese herbal polysaccharide;

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

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

[0094] Comparative Example 1

[0095] Compared with Example 3, the difference is that methotrexate is not added in step S1.

[0096] The details are as follows:

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

[0098] Comparative Example 2

[0099] Compared with Example 3, the difference is that vinblastine is not added in step S1.

[0100] The details are as follows:

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

[0102] Comparative Example 3

[0103] Compared with Example 3, the difference is that daunorubicin is not added in step S1.

[0104] The details are as follows:

[0105] S1. Dissolve 4 g of methotrexate in 100 mL of 7 wt% NaOH solution to prepare solution 1; dissolve 2 g of vinblastine in 100 mL of ethanol to prepare solution 2; mix solutions 1 and 2, evaporate them open for 8 days, filter, wash, dry, and grind to obtain a drug eutectic powder.

[0106] Comparative Example 4

[0107] Compared with Example 3, the difference is that step S2 is not performed.

[0108] The details are as follows:

[0109] S1. 4 g of methotrexate was dissolved in 100 mL of 7 wt% NaOH solution to prepare solution 1; 2 g of vinblastine was dissolved in 100 mL of ethanol to prepare solution 2; 5 g of daunorubicin was dissolved in 200 mL of water to prepare solution 3; solutions 1, 2, and 3 were mixed, evaporated in an open air for 8 days, filtered, washed, dried, and ground to obtain a drug eutectic powder;

[0110] S2. 12 g of UiO-66-NH2 was added to 200 mL of water, 4.5 g of drug eutectic powder was added, and the reaction was stirred for 12 h, centrifuged, washed, and dried to obtain a drug carrier;

[0111] S3. Add 11 g of exosomes to 500 mL of water, add 5.5 g of drug-loaded solids, and incubate with ultrasound at 400 W and 37°C for 11 h. Centrifuge, wash, and dry to obtain an exosome-loaded drug system.

[0112] S4. 7g Codonopsis, 7g Angelica, 5g Polygonatum, 12g Dendrobium officinale were washed, dried, crushed, added to 300mL of water, heated to boiling and extracted for 4h, filtered, the residue was retained, and the filtrate was added ethanol to an ethanol content of 80wt% and precipitated for 1h, filtered, the solid was washed, and dried to obtain a Chinese herbal polysaccharide;

[0113] S5. Add 5 g of acrylic acid, 7 g of traditional Chinese medicine polysaccharide, and 4 g of pullulan to 200 mL of water, add 7 g of the exosome drug delivery system, then add 0.07 g of sodium persulfate, 0.4 g of Tween-80, 0.3 g of Span-80, and 0.6 g of N,N'-methylenebisacrylamide. Stir and mix for 20 min. Add to 500 mL of fish oil, emulsify at 8000 rpm for 15 min, cross-link at 55°C for 4 h, centrifuge, wash, and dry to prepare a sustained-release exosome system with a high half-life.

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

[0115] Comparative Example 5

[0116] Compared with Example 3, the difference is that no Chinese medicine polysaccharide is added in step S6.

[0117] The details are as follows:

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

[0119] Comparative Example 6

[0120] Compared with Example 3, the difference is that steps S2 and S3 are not performed.

[0121] The details are as follows:

[0122] S1. 4 g of methotrexate was dissolved in 100 mL of 7 wt% NaOH solution to prepare solution 1; 2 g of vinblastine was dissolved in 100 mL of ethanol to prepare solution 2; 5 g of daunorubicin was dissolved in 200 mL of water to prepare solution 3; solutions 1, 2, and 3 were mixed, evaporated in an open air for 8 days, filtered, washed, dried, and ground to obtain a drug eutectic powder;

[0123] S2. Add 11 g of exosomes to 500 mL of water, add 5.5 g of drug eutectic powder, and incubate with ultrasound at 400 W and 37°C for 11 h. Centrifuge, wash, and dry to prepare the exosome drug delivery system.

[0124] S3. 7g Codonopsis, 7g Angelica, 5g Polygonatum, 12g Dendrobium officinale were washed, dried, crushed, added to 300mL of water, heated to boiling and extracted for 4h, filtered, the residue was retained, and the filtrate was added ethanol to an ethanol content of 80wt% system, precipitated for 1h, filtered, the solid was washed, and dried to obtain a Chinese herbal polysaccharide;

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

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

[0127] Comparative Example 7

[0128] Compared with embodiment 3, the difference is that step S6 is not performed.

[0129] The details are as follows:

[0130] S1. 4 g of methotrexate was dissolved in 100 mL of 7 wt% NaOH solution to prepare solution 1; 2 g of vinblastine was dissolved in 100 mL of ethanol to prepare solution 2; 5 g of daunorubicin was dissolved in 200 mL of water to prepare solution 3; solutions 1, 2, and 3 were mixed, evaporated in an open air for 8 days, filtered, washed, dried, and ground to obtain a drug eutectic powder;

[0131] S2. 3 g of arginine-glycine-aspartic acid RGD was added to 200 mL of water, 0.7 g of NHS and 0.7 g of EDC were added, and the mixture was stirred and activated for 30 min. 8 g of UiO-66-NH2 was added, and the mixture was stirred for 13 h. The mixture was centrifuged, washed, and dried to obtain a UiO-66-NH2 / RGD complex.

[0132] S3. 12 g of UiO-66-NH2 / RGD complex was added to 200 mL of water, 2.5 g of NHS and 2.5 g of EDC were added, and the mixture was stirred and activated for 30 min. 4.5 g of drug eutectic powder was added, and the mixture was stirred for 12 h. The mixture was centrifuged, washed, and dried to obtain a drug carrier.

[0133] S4. Add 11 g of exosomes to 500 mL of water, add 5.5 g of drug-loaded solids, and incubate with ultrasound at 400 W and 37°C for 11 h. Centrifuge, wash, and dry to obtain an exosome-loaded drug system.

[0134] S5. 7g Codonopsis, 7g Angelica, 5g Polygonatum, 12g Dendrobium officinale were washed, dried, crushed, added to 300mL of water, heated to boiling and extracted for 4h, filtered, the residue was retained, and the filtrate was added with ethanol to an ethanol content of 80wt% and precipitated for 1h, filtered, the solid was washed, and dried to obtain a Chinese herbal polysaccharide;

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

[0136] Test Example 1

[0137] SPF-grade male BALB / c mice weighing 20-24 g and aged 4-6 weeks were selected. The right axilla was prepared and disinfected with 75% ethanol. 0.2 mL of MFC gastric cancer cell suspension (2.5 × 10 7 A MFC gastric cancer mouse model was established using a 5-fluorouracil (5-FU)-containing exosome (mice / mL) solution. The mice were randomly divided into a model group, a 5-FU group, and groups of Examples 1-3 and Comparative Examples 1-7, with 8 mice in each group. Eight normal mice were also selected as the normal group. Successful modeling was considered when a nodule was visible macroscopically in the right axilla 5-6 days after modeling. Dosing was performed on the 7th day. The groups of Examples 1-3 and Comparative Examples 1-7 were administered 50 mg / kg of the corresponding exosome composition designed to reduce chemotherapy side effects. The 5-FU group was administered 25 mg / kg of 5-FU. The blank and model groups received an equal amount of normal saline. Dosing continued for 9 days, with testing performed on the 10th day.

[0138] 1. Blood index testing

[0139] 2.0 mL of blood was collected from the tail vein of each animal. White blood cells, red blood cells, and platelets were counted 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: * compared with the blank group, P < 0.05; # compared with the model group, P < 0.05.

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

[0144] 2. Organ index

[0145] After anesthetizing mice with sodium pentobarbital, approximately 1 mL of blood was collected from the eyeballs. After standing at room temperature for 1 hour, the blood was centrifuged at 4°C to separate the serum. The liver, kidneys, and thymus were removed, and the organ index was calculated using the formula: organ index = organ mass / body mass. The results are shown in Table 2.

[0146] Table 2

[0147]

[0148] Note: * compared with the blank group, P < 0.05; # compared with the model group, P < 0.05.

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

[0150] 3. Tumor inhibition rate

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

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

[0153] Table 3

[0154]

[0155]

[0156] As can be seen from the above table, the exosome compositions for reducing the side effects of chemotherapy prepared in Examples 1-3 of the present 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 in the scope of protection of the present invention.

Claims

1. A method for preparing an exosome composition for reducing the side effects of chemotherapy, characterized in that: It is prepared from the following raw materials: Codonopsis pilosula, Chinese angelica, Polygonatum sibiricum, Dendrobium officinale, D-ribose, yeast-β-glucan, and glutathione.

2. The preparation method according to claim 1, characterized in that Methotrexate, vinblastine, and daunorubicin were prepared into cocrystals, loaded on UiO-66-NH2 connected with RGD, loaded on exosomes, and embedded in a complex of acrylic acid, traditional Chinese medicine polysaccharides, and pullulan to produce a sustained-release exosome system with a high half-life. This was then evenly mixed with D-ribose, yeast-β-glucan, and glutathione to produce an exosome composition that reduces the side effects of chemotherapy. The traditional Chinese medicine polysaccharides were obtained by boiling water extraction and ethanol precipitation of Codonopsis pilosula, Angelica sinensis, Polygonatum sibiricum, and Dendrobium officinale.

3. The preparation method according to claim 2, characterized in that The following steps are involved: S1. Dissolving methotrexate in an alkaline solution to prepare solution 1; dissolving vinblastine in ethanol to prepare solution 2; and dissolving daunorubicin in water to prepare solution 3; mixing solutions 1, 2, and 3, evaporating them in an open air, filtering, washing, drying, and grinding to obtain a drug eutectic powder; S2. Arginine-glycine-aspartic acid RGD was added to water, NHS and EDC were added, stirred for activation, UiO-66-NH2 was added, the reaction was stirred, centrifuged, washed, and dried to obtain a UiO-66-NH2 / RGD complex; S3. The UiO-66-NH2 / RGD complex was added to water, NHS and EDC were added, stirred for activation, the drug eutectic powder was added, stirred for reaction, centrifuged, washed, and dried to obtain a drug carrier; S4. adding exosomes to water, adding drug carriers, incubating with ultrasound, centrifuging, washing, and drying to prepare an exosome drug-carrying system; S5. Codonopsis, Angelica, Polygonatum, Dendrobium candidum were washed, dried, crushed, added to water, heated to boiling for extraction, filtered, the residue was retained, the filtrate was added ethanol for precipitation, filtered, the solid was washed, and dried to obtain a Chinese herbal polysaccharide; S6. Add acrylic acid, traditional Chinese medicine polysaccharides, and pullulan to water, add the exosome drug delivery system, then add an initiator, an emulsifier, and N,N'-methylenebisacrylamide, stir and mix thoroughly, add to fish oil, emulsify, cross-link, centrifuge, wash, and dry to produce a sustained-release exosome system with a high half-life; S7. Evenly mix D-ribose, yeast-β-glucan, glutathione, and a sustained-release high half-life exosome system to prepare an exosome composition that reduces the side effects of chemotherapy.

4. The preparation method according to claim 3, characterized in that The concentration of the alkaline solution in step S1 is 5-10 wt %, the base is NaOH or KOH, the mass ratio of methotrexate, vinblastine, and daunorubicin is 3-5:1-3:3-6, and the open volatilization time is 7-10 days.

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

6. The preparation method according to claim 3, characterized in that In step S3, the mass ratio of the UiO-66-NH2 / RGD complex, NHS, EDC and chemical eutectic powder is 10-15:2-3:2-3:3-6, the stirring activation time is 20-40 minutes, and the stirring reaction time is 10-14 hours.

7. The preparation method according to claim 3, characterized in that The exosomes in step S4 are mesenchymal stem cell exosomes, the mass ratio of the exosomes to the drug carrier is 10-12:4-7, the power of the ultrasonic incubation is 300-500W, the temperature is 36-38°C, and the time is 10-12h.

8. The preparation method according to claim 3, characterized in that In step S5, the mass ratio of Codonopsis pilosula, Angelica sinensis, Polygonatum sibiricum and Dendrobium officinale is 5-10:5-10:3-7:10-15, and the heating and boiling extraction time is 3-5 hours.

9. The preparation method according to claim 3, characterized in that The mass ratio of acrylic acid, traditional Chinese medicine polysaccharide, pullulan, 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 temperature of the cross-linking reaction is 50-60°C, and the time is 3-5h; 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.

10. An exosome composition for reducing side effects of chemotherapy, obtained by the preparation method according to any one of claims 1 to 9.

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