WO2023236700A1 - 一种功能化纳米硒水溶胶于抗肿瘤方面的应用 - Google Patents

一种功能化纳米硒水溶胶于抗肿瘤方面的应用 Download PDF

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WO2023236700A1
WO2023236700A1 PCT/CN2023/092664 CN2023092664W WO2023236700A1 WO 2023236700 A1 WO2023236700 A1 WO 2023236700A1 CN 2023092664 W CN2023092664 W CN 2023092664W WO 2023236700 A1 WO2023236700 A1 WO 2023236700A1
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selenium
cordyceps mycelium
polysaccharide protein
selenite
concentration
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French (fr)
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黄家兴
董晓莉
陆家谦
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香港理工大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/066—Clavicipitaceae
    • A61K36/068—Cordyceps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the preparation and functionalization of Cordyceps mycelium Cs4 water-soluble polysaccharide protein nano-selenium (Cs4-SeNPs) hydrosol and its application in anti-tumor aspects.
  • Cs4-SeNPs Cordyceps mycelium Cs4 water-soluble polysaccharide protein nano-selenium
  • Selenium is one of the essential trace elements for human and animal health and has a wide range of physiological functions (e.g. anti-tumor, antioxidant, anti-aging, improving immunity, protecting and repairing cells, etc.). More than 40 countries around the world are in selenium-deficient areas, and many provinces in my country are in selenium-deficient or low-selenium zones. These areas have high incidence rates of tumors, liver diseases, cardiovascular diseases, etc., and require selenium supplementation. However, as a nutritional supplement or cancer preventive agent, the range of beneficial and toxic doses of selenium is extremely narrow, which can easily cause selenium poisoning and seriously limits the potential of selenium in disease prevention and treatment. Selenium's toxicity depends on its chemical form. In recent years, nano-selenium obtained by using nanotechnology has become a new research hotspot and a new favorite in selenium supplementation due to its high biocompatibility, low toxicity and significant biological activity.
  • physiological functions e.g. anti-tumor,
  • the preparation method of nano-elemental selenium usually adopts the reduction method, using oxygen-containing salts or oxides of selenium through various reducing agents to obtain elemental selenium, and at the same time using modifiers or regulators to modify and regulate the particle size and morphology, thereby obtaining An ideal product.
  • various existing methods for preparing nano-elemental selenium use various regulators or modifiers to regulate or modify the morphology, without considering its application needs.
  • none of these modulators or modifiers It is a biologically active material and cannot effectively improve the biological activity of nano-elemental selenium, especially the anti-tumor activity, that is, the obtained product is not functionalized nano-elemental selenium.
  • Cs4 water-soluble polysaccharide protein of Cordyceps mycelium that is, the Cs4 water-soluble polysaccharide protein of Cordyceps mycelium, which has antioxidant, anti-tumor and bone growth-promoting activities, was introduced into the liquid nano-selenium system.
  • the Cs4 polysaccharide protein functionalized nano-selenium (Cs4-SeNPs) was prepared, which not only improved the bioavailability and biological activity of selenium, but also reduced the cytotoxicity of selenium and verified its significant activity in promoting bone growth in vitro and in vivo.
  • Cs4-SeNPs have great potential for promoting bone development in children and adolescents and preventing and treating osteoporosis, bone loss, fractures and other disease states in middle age. See the authorized patent "A nano-selenium hydrosol and its preparation method and application” (patent application number: ZL201911215358.5).
  • Cancer has become one of the major diseases affecting health and lifespan in humans and animals such as companion animals.
  • AVMA American Veterinary Medical Association
  • cancer causes almost 50% of deaths in companion animals over the age of 10.
  • Common cancers in companion animals include: skin cancer, testicular cancer, abdominal cancer, and bone cancer. Cancers common in companion animals are also common in humans, such as lymphoma, melanoma and osteosarcoma.
  • the present invention provides the use of a complex comprising Cordyceps mycelium Cs4 polysaccharide protein functionalized nano-selenium hydrosol in the preparation of a formulation for treating tumors in a subject in need thereof.
  • the subject is a human.
  • the subject is a companion animal.
  • companion animals include cats and dogs.
  • the tumors include osteosarcoma and malignant melanoma.
  • the selenium is from selenium dioxide or selenite.
  • the selenite salt is sodium selenite.
  • the formulation prepared from the complex of the present invention may be an injection or a spray.
  • the preparation method of the Cordyceps mycelium Cs4 polysaccharide protein functionalized nano-selenium hydrosol includes the following steps:
  • step (b) Add selenium dioxide solution or selenite solution to the Cordyceps mycelium Cs4 polysaccharide protein solution obtained in step (a), and mix evenly to obtain mixed solution A;
  • the molar ratio of vitamin C to selenium dioxide or selenite is 5:1.
  • the concentration of Cordyceps mycelium Cs4 polysaccharide protein is 60-6000mg ⁇ L -1
  • the concentration of vitamin C is 1.0-100mmol ⁇ L -1
  • selenium dioxide or selenious acid The concentration of salt is 0.2 to 20 mmol ⁇ L -1 .
  • the concentration of Cordyceps mycelium Cs4 polysaccharide protein is 300 to 1200 mg ⁇ L -1
  • the concentration of vitamin C is 10.0 to 100 mmol ⁇ L -1
  • selenium dioxide or selenious acid The concentration of salt is 2.0 to 20 mmol ⁇ L -1 .
  • the present invention provides a preparation comprising Cordyceps mycelium Cs4 polysaccharide protein functionalized nano-selenium hydrosol.
  • the formulation is an injection or spray.
  • Figure 1 Anti-proliferative effects of Cs4-SeNPs, PTR-SeNPs and Chitosan-SeNPs on OSCA-8 and CGMD-5 cells.
  • Figure 2 DNA flow cytometry analysis of Cs4-SeNPs on OSCA-8 and CGMD-5 Effects of changes in cell cycle distribution.
  • Figure 3 Explore the ability of Cs4-SeNPs to evert phosphatidylserine on OSCA-8 and CGMD-5 cell membranes.
  • ⁇ Cs4-SeNPs can be used in various dosage forms such as injection and spray.
  • ⁇ Cordyceps mycelium Cs4 polysaccharide protein is rich in hydrophilic hydroxyl (-OH) and amino (-NH) groups, which improves the water solubility of nano-selenium, enhances the affinity between selenium and cancer cells, and improves tumor cells' sensitivity to nano-selenium.
  • the intake of selenium can achieve the overall therapeutic effect of reducing medication dosage, increasing efficacy, and reducing toxic and side effects.
  • Cordyceps sinensis is a precious medicinal fungus that has been used as a tonic and therapeutic traditional Chinese medicine for more than 700 years in China. Since wild Cordyceps sinensis is very rare and expensive, a Cordyceps mycelium (Cs4) successfully isolated for the fourth time by the Chinese Academy of Sciences has made a huge contribution to commercial production and scientific research in the past 40 years. Pharmacological and clinical studies have shown that polysaccharide protein is one of the main bioactive components of Cs4 and has a wide range of health-promoting and therapeutic effects, such as immune regulation, anti-tumor and bone protection.
  • Cordyceps mycelium Cs4 polysaccharide protein contains at least one of water-soluble polysaccharides, polysaccharide proteins and proteins.
  • the total sugar content is about 41.5% and the protein content is about 23.6%.
  • the polysaccharide part is composed of glucose, Mainly mannose and galactose, with small amounts of mannose, arabinose and xylose.
  • the present invention uses the Cordyceps mycelium Cs4 polysaccharide protein with broad biological activity as the functionalization factor of nano-selenium to obtain a functionalized nano-selenium hydrosol with anti-cancer effect.
  • the present invention provides the use of a complex comprising Cordyceps mycelium Cs4 polysaccharide protein nano-selenium hydrosol in the preparation of a preparation for treating tumors in a subject in need.
  • subjects in need thereof may include various animals, such as mammals.
  • Formulations containing one or more of the compounds disclosed herein can be administered to mammals using a variety of routes. Suitable routes of administration for the methods of treating cancer as disclosed herein include both local and systemic administration.
  • the subject is a companion animal.
  • Companion animals have a well-known meaning in the art, such as common cats and dogs.
  • the complexes of the invention prepare formulations for the treatment of cancer, including common forms of lung, brain, central nervous system, breast, colon, leukemia, myeloma, prostate, and ovarian cancer.
  • Sarcomas are malignant tumors derived from connective tissue or stromal cells.
  • Blastomas are common malignant tumors that resemble immature or embryonic tissue.
  • Lymphoma and leukemia are malignant tumors derived from hematopoietic (blood-forming) cells.
  • the preparation prepared from the drug complex of the present invention is used to treat osteosarcoma and malignant melanoma.
  • the selenium is from selenium dioxide or selenite.
  • Selenite includes sodium selenite, potassium selenite, barium selenite, zinc selenite, etc.
  • formulations prepared from the complexes of the present invention may be produced using any of a variety of processes including, but not limited to, conventional mixing, dissolving, granulating, coating, emulsifying, encapsulating, and lyophilizing.
  • Preparations can be in any of the following forms, including but not limited to hydrosols, sterile solutions, suspensions, emulsions, lyophilizers, tablets, pills, pellets, capsules, powders, and syrups , elixir, or any other dosage form suitable for administration.
  • Preferred dosage forms are injections or sprays.
  • Formulations prepared from the complexes disclosed herein may optionally include, but are not limited to, other pharmaceutically acceptable components (or pharmaceutical ingredients), including, but not limited to, buffers, preservatives, tonicity adjusters, salts, antioxidants, osmolytes, Temperature regulators, physiological substances, pharmacological substances, fillers, emulsifiers, wetting agents, sweeteners or flavoring agents, etc.
  • buffers include, but are not limited to, acetate buffer, borate buffer, citrate buffer, phosphate buffer, neutral buffered saline, and phosphate buffered saline.
  • the present invention provides a preparation comprising Cordyceps mycelium Cs4 polysaccharide protein functionalized nano-selenium hydrosol.
  • the preparation method of the Cordyceps mycelium Cs4 polysaccharide protein functionalized nano-selenium hydrosol includes the following steps:
  • step (b) Add selenium dioxide solution or selenite solution to the Cordyceps mycelium Cs4 polysaccharide protein solution obtained in step (a), and mix evenly to obtain mixed solution A;
  • the Cordyceps mycelium Cs4 polysaccharide protein can be prepared by the following method: adding water to the Cordyceps mycelium Cs4 according to the weight ratio of the Cordyceps mycelium Cs4 to water is 1:20, Heat to 95 ⁇ 100°C, extract the filtrate after 2 hours; repeat the extraction of the filter residue once for 2 hours according to the previous steps, combine the filtrate obtained from the two extractions, dialyze for 24 hours, and obtain the molecular weight cutoff of 8,000. Described Cordyceps mycelium Cs4 polysaccharide protein.
  • the molar ratio of vitamin C to the selenium dioxide or selenite is 5:1.
  • Cordyceps mycelium Cs4 polysaccharide protein has a special chemical structure.
  • the polysaccharide part has a polyhydroxyl structure, so it has a strong physical adsorption effect on nano-selenium, preventing further aggregation and precipitation of nano-selenium, and effectively modifying the surface of nano-selenium. Play a good role in particle size control and stabilization.
  • the Cordyceps mycelium Cs4 polysaccharide protein is rich in hydrophilic hydroxyl (-OH) and amino (-NH) groups, which improves the water solubility of nano-selenium and enhances its affinity with tumor cells, greatly improving The uptake of nano-selenium by tumor cells can achieve the overall therapeutic effect of reducing drug dosage, increasing efficacy, and reducing toxic and side effects.
  • the new nano-selenium prepared by the method of the present invention can be stored in the form of hydrosol in the liquid phase for a long time, has good dispersion and stability, and can be used in various dosage forms such as injection and spray.
  • the cordyceps mycelium Cs4 polysaccharide protein solution, selenium dioxide solution or selenite solution and vitamin C solution used in the present invention all use water as the solvent.
  • step (d) whether the reaction of mixed solution B is complete can be judged based on the color of the product. For example, when the red color in the product no longer deepens, it means that the reaction is complete.
  • the vitamin C solution can be added dropwise into mixed solution A.
  • vitamin C is reduced vitamin C.
  • the concentration of the Cordyceps mycelium Cs4 polysaccharide protein is 60 to 6000 mg ⁇ L -1
  • the concentration of selenium dioxide or selenite is 0.2 to 20mmol ⁇ L -1
  • the concentration of vitamin C is 1.0 ⁇ 100mmol ⁇ L -1 .
  • Technicians can calculate the concentration volume required for the above-mentioned raw materials based on the new nano-selenium hydrosol required at different concentrations.
  • the molar ratio of vitamin C to the selenium dioxide or selenite is 5:1.
  • the concentration of Cordyceps mycelium Cs4 polysaccharide protein is 300 to 1200 mg ⁇ L -1
  • the concentration of vitamin C is 10.0 to 100 mmol ⁇ L -1
  • selenium dioxide or selenium dioxide The concentration of selenate is 2.0 to 20 mmol ⁇ L -1 .
  • novel nano-selenium hydrosol of the present invention can be stored in sol form at 2 to 10°C, and has a storage period of 30 to 60 days.
  • Cs4-SeNPs Cs4-SeNPs, tiger milk mushroom polysaccharide protein functionalized nano-selenium (PTR-SeNPs) and chitosan-functionalized nano-selenium (Chitosan-SeNPs) hydrosol were prepared.
  • the half cell inhibitory concentration (IC 50 ) commonly used in this field is used to evaluate the ability of drugs to inhibit tumor proliferation.
  • the anti-proliferative effects of different types of nano-selenium on canine osteosarcoma (OSCA-8) cancer cells and canine oral malignant melanoma (CMGD-5) were evaluated by MTS cell proliferation assay (BMG Labtech, Clariostar).
  • MTS is an MTT (thiazolyl blue) analogue.
  • MTS thiazolyl blue
  • Cs4-SeNPs (3-40 ⁇ M) induction by DNA flow cytometry OSCA-8 and CMGD-5 cell cycle arrest and apoptosis abilities.
  • Cs4-SeNPs can cause the percentage of apoptotic peak cells (Sub-G1) to increase in a dose-effect manner (OSCA-8: from 7.0% in the control group to 90.7%; CMGD-5: from (from 5.3% in the control group to 53.0%), indicating that Cs4-SeNPs can effectively induce apoptosis of OSCA-8 and CMGD-5 cells, and the effect is concentration-dependent.
  • the cell membrane will change during apoptosis.
  • Phosphatidylserine (PS) of normal cells is located on the inside of the cell membrane.
  • PS Phosphatidylserine
  • the PS of the cell membrane will flip from the inside to the outside.
  • Cs4-SeNPs (3-40 ⁇ M) to induce apoptosis in OSCA-8 and CMGD-5 cells was explored using the Annexin-V kit, as shown in Figure 3A-H.
  • Annexin-V fluorescent probe specifically binds to PS and produces green fluorescence, and the intensity of green fluorescence is proportional to the degree of PS eversion.
  • OSCA-8 canine osteosarcoma
  • CMGD-5 canine oral malignant melanoma
  • Cs4-SeNPs can exert a significantly stronger anti-proliferative effect on OSCA-8 and CMGD-5 cells.
  • Further mechanism studies showed that Cs4-SeNPs achieved anti-tumor activity mainly by inducing apoptosis of OSCA-8 and CMGD-5 cells (including the increase of sub-G1 cell population and phosphatidylserine eversion).

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Abstract

本发明提供了虫草菌丝体Cs4多糖蛋白功能化纳米硒水溶胶的制备和抗癌应用。

Description

一种功能化纳米硒水溶胶于抗肿瘤方面的应用 技术领域
本发明涉及虫草菌丝体Cs4水溶性多糖蛋白纳米硒(Cs4-SeNPs)水溶胶的制备、功能化以及其于抗肿瘤方面的应用。
背景技术
硒是人及动物健康必需的微量元素之一，具有广泛的生理功能(e.g.抗肿瘤、抗氧化、抗衰老、提高机体免疫力、保护修复细胞等)。全球约有40多个国家处于缺硒地区，我国多个省份处于缺硒或低硒地带，这些地区是肿瘤、肝病、心血管疾病等发病率高的地区，需要进行补硒。然而作为营养补充剂或癌症预防剂，硒的有益剂量和毒性剂量范围却是极其狭窄，容易造成硒中毒，严重限制了硒在疾病防治方面的潜力。硒的毒性依赖于其化学形式。近年，利用纳米技术获得的纳米硒，因其生物兼容性高、毒性低和生物活性显著而成为新的研究热点和补硒新宠。
纳米单质硒的制备方法通常采用还原法，利用硒的含氧酸盐或氧化物通过各种还原剂获得单质硒，并同时使用修饰剂或调控剂对粒径和形貌进行修饰调控，从而获得理想产物。然而，现有的各种纳米单质硒制备方法均是通过各种不同的调控剂或修饰剂对形貌进行调控或者修饰，并没有考虑其应用需要。此外，这些调控剂或修饰剂均不 是具有生物活性的材料，不能有效的提高纳米单质硒的生物活性，尤其是抗肿瘤活性，即所得产物并不是功能化纳米单质硒。
我们利用虫草菌丝体Cs4水溶性多糖蛋白成功将纳米硒功能化，即把具有抗氧化、抗肿瘤及促进骨骼生长等活性的虫草菌丝体Cs4水溶性多糖蛋白引入液相纳米硒体系中，制备出Cs4多糖蛋白功能化纳米硒(Cs4-SeNPs)，不但提高了硒的生物利用度和生物活性，而且降低了硒的细胞毒性并验证了其显著的体内外促进骨骼生长活性。由此可见，Cs4-SeNPs用于儿童青少年期促进骨发育以及中年期防治骨质疏松症、骨流失、骨折等疾病状态拥有巨大的潜力。参见已授权专利“一种纳米硒水溶胶及其制备方法与应用”(专利申请号：ZL201911215358.5)。
癌症，在人和动物例如陪伴动物中已经成为影响健康和寿命的主要疾病之一。
根据American Veterinary Medical Association(美国兽医协会)(AVMA)，在10岁以上的陪伴动物中，癌症几乎造成50％的死亡。陪伴动物常见的癌症包括：皮肤癌，睾丸癌，腹部癌和骨癌。常见于陪伴动物身上的癌症也常见于人类，如淋巴瘤，黑色素瘤和骨肉瘤。
随着我国城市化进程的加快以及空巢老人、空巢青年等群体数量的迅速增加，养陪伴动物的人群近年也急速增长。随着生活水平及消费力的提高，人们对陪伴动物的健康越来越关注，陪伴动物保健品市场得以高速发展，潜力巨大。因陪伴动物患癌的机会跟人类相约，但陪伴动物肿瘤治疗多数只是采用人的药品，事实证明特异性不高且价钱昂贵。
本领域需要可方便用于预防和治疗人和动物例如陪伴动物肿瘤的廉价纳米硒药品或者保健品。
发明内容
在一个方面，本发明提供了包含虫草菌丝体Cs4多糖蛋白功能化纳米硒水溶胶的复合物在制备治疗有需要的受试者中肿瘤的制剂中的用途。
在一个实施方案中，受试者为人。
在一个实施方案中，受试者为陪伴动物。
在一个实施方案中，陪伴动物包括猫和犬。
在一个实施方案中，肿瘤包括骨肉瘤及恶性黑色素瘤。
在一个实施方案中，硒来自二氧化硒或亚硒酸盐。
在一个实施方案中，亚硒酸盐为亚硒酸钠。
在一个实施方案中，本发明的复合物制备的制剂可为注射剂或喷雾剂。
在一个实施方案中，虫草菌丝体Cs4多糖蛋白功能化纳米硒水溶胶的制备方法包括以下步骤：
(a)通过水提虫草菌丝体Cs4得到虫草菌丝体Cs4多糖蛋白溶液；
(b)向步骤(a)得到的虫草菌丝体Cs4多糖蛋白溶液中加入二氧化硒溶液或亚硒酸盐溶液，混合均匀，得到混合溶液A；
(c)向混合溶液A中滴加还原性维生素C溶液，混匀，得到混合溶液B；
(d)待混合溶液B反应完全，经过透析得到虫草菌丝体Cs4多糖蛋白功能化纳米硒水溶胶。
在一个实施方案中，于混合溶液B中，维生素C与二氧化硒或亚硒酸盐的摩尔比为5∶1。
在一个实施方案中，于混合溶液B中，虫草菌丝体Cs4多糖蛋白的浓度为60～6000mg·L-1，维生素C的浓度为1.0～100mmol·L- 1，二氧化硒或亚硒酸盐的浓度为0.2～20mmol·L-1。
在一个实施方案中，于混合溶液B中，虫草菌丝体Cs4多糖蛋白的浓度为300～1200mg·L-1，维生素C的浓度为10.0～100mmol·L-1，二氧化硒或亚硒酸盐的浓度为2.0～20mmol·L-1。
在第二方面，本发明提供了一种制剂，其包括虫草菌丝体Cs4多糖蛋白功能化纳米硒水溶胶。
在一个实施方案中，所述制剂为注射剂或喷雾剂。
附图说明
图1：Cs4-SeNPs，PTR-SeNPs以及Chitosan-SeNPs对OSCA-8及CGMD-5细胞的抗增殖作用。
图2：DNA流式细胞术分析Cs4-SeNPs对OSCA-8及CGMD-5 细胞周期分布变化影响。
图3：探讨Cs4-SeNPs对OSCA-8及CGMD-5细胞膜磷脂酰丝氨酸外翻能力。
本发明的优点
·Cs4-SeNPs可作注射、喷雾等多种剂型应用。
·虫草菌丝体Cs4多糖蛋白富含亲水性的羟基(-OH)和氨基(-NH)基团，提高了纳米硒的水溶性，增强了硒与癌细胞的亲和力，提高肿瘤细胞对纳米硒的摄取量，达到减少用药剂量、提增疗效、减少毒副作用的整体治疗效果。
具体实施方式
冬虫夏草(Cordyceps sinensis)是一种珍贵的药用真菌，在中国作为滋补及治疗中药已有700多年的历史。由于野生冬虫夏草非常稀少且价格昂贵，因此中国科学院于第4次成功分离的一种虫草菌丝体(Cs4)，对过去40年的商业生产和科学研究有非常巨大的贡献。药理学和临床研究表明，多糖蛋白是Cs4的主要生物活性成分之一，具有广泛的健康促进和治疗作用，如免疫调节、抗肿瘤以及骨保护等。虫草菌丝体Cs4多糖蛋白含有水溶性多糖、多糖蛋白及蛋白质中的至少一种物质，例如总糖含量约为41.5％，蛋白质含量约为23.6％的水溶性多糖蛋白，其中多糖部分以葡萄糖、甘露糖和半乳糖为主，含有少量甘露糖、阿拉伯糖和木糖。
现有的各种纳米单质硒制备方法均是通过各种不同的调控剂或 修饰剂对形貌进行调控或者修饰，得到理想粒径和形貌的产物，但所得产物并不是功能化纳米单质硒。为解决这问题，本发明以具有广泛生物活性的虫草菌丝体Cs4多糖蛋白作为纳米硒的功能化因子，获得一种具有抗癌作用的虫草菌丝体Cs4多糖蛋白功能化纳米硒水溶胶。
在第一个方面，本发明提供了包含虫草菌丝体Cs4多糖蛋白纳米硒水溶胶的复合物在制备治疗有需要的受试者中肿瘤的制剂中的用途。
在一个实施方案中，有需要的受试者可以包括各种动物，例如哺乳动物。包含一种或多种本文公开的制剂可使用各种途径被施用于哺乳动物。适于治疗如本文公开的癌症的方法的施用途径包括局部施用和全身施用两者。
在一个实施方案中，受试者为陪伴动物。陪伴动物在本领域有公知的含义，例如常见的猫和犬。
在一个实施方案中，本发明的复合物制备的制剂用于治疗癌症，包括肺癌、脑癌、中枢神经系统癌症、乳腺癌、结肠癌、白血病、骨髓瘤、前列腺癌和卵巢癌的常见形式。肉瘤是来源于结缔组织或间质细胞的恶性肿瘤。胚细胞瘤是常见的恶性肿瘤，其类似于未成熟组织或胚胎组织。淋巴瘤和白血病是来源于造血(血液形成)细胞的恶性肿瘤。在一个优选的实施方案中，本发明的药复合物制备的制剂用于治疗骨肉瘤及恶性黑色素瘤。
在一个实施方案中，硒来自二氧化硒或亚硒酸盐。亚硒酸盐包括亚硒酸钠、亚硒酸钾、亚硒酸钡、亚硒酸锌等。
在一个实施方案中，本发明的复合物制备的制剂可使用下述各种过程中任一种来生产，包括但不限于常规混合、溶解、制粒、包衣、乳化、封装和冻干。制剂可采用下述各种形式的任一种，包括但不限于水溶胶、无菌溶液、混悬剂、乳剂、冻干剂、片剂、滴丸剂、微丸剂、胶囊剂、粉剂、糖浆剂、酏剂或适于施用的任何其它剂型。优选的剂型为注射剂或喷雾剂。
本文公开复合物制备的制剂可任选地包括但不限于其它药学上可接受的组分(或药学组分)，包括但不限于缓冲剂、防腐剂、张力调节剂、盐、抗氧化剂、渗透度调节剂、生理物质、药理物质、填充剂、乳化剂、润湿剂、甜味剂或调味剂等。调节pH的各种缓冲液和方法可用于制备本文公开的制剂。这种缓冲液包括但不限于醋酸盐缓冲液，硼酸盐缓冲液，柠檬酸盐缓冲液，磷酸盐缓冲液，中性缓冲盐水以及磷酸盐缓冲盐水。
在第二个方面，本发明提供了一种制剂，其包括虫草菌丝体Cs4多糖蛋白功能化纳米硒水溶胶。
在一个实施方案中，虫草菌丝体Cs4多糖蛋白功能化纳米硒水溶胶的制备方法包括以下步骤：
(a)通过水提虫草菌丝体Cs4得到虫草菌丝体Cs4多糖蛋白溶液；
(b)向步骤(a)得到的虫草菌丝体Cs4多糖蛋白溶液中加入二氧化硒溶液或亚硒酸盐溶液，混合均匀，得到混合溶液A；
(c)向混合溶液A中滴加还原性维生素C溶液，混匀，得到混合溶液B；
(d)待混合溶液B反应完全，经过透析得到虫草菌丝体Cs4多糖蛋白功能化纳米硒水溶胶。
上述步骤(a)中，虫草菌丝体Cs4多糖蛋白可采用下述方法制备得到：按照虫草菌丝体Cs4与水的重量比为1∶20的比例，向虫草菌丝体Cs4中加入水，加热至95～100℃，提取2小时后取滤液；按照前面的步骤将滤渣重复提取一次，重复提取的时间为2小时，合并两次提取所得滤液，透析24小时，截留分子量为8,000，得到所述虫草菌丝体Cs4多糖蛋白。
上述步骤(c)中，所述的混合溶液B中，维生素C与所述的二氧化硒或亚硒酸盐的摩尔比为5∶1。
虫草菌丝体Cs4多糖蛋白在化学结构上具有特殊性，多糖部分具有多羟基结构，因而对纳米硒具有很强的物理吸附作用，避免纳米硒进一步聚集沉淀，有效的对纳米硒表面进行修饰，发挥良好的粒径调控和稳定作用。此外，虫草菌丝体Cs4多糖蛋白富含亲水性的羟基(-OH)和氨基(-NH)基团，提高了纳米硒的水溶性，增强其与肿瘤细胞之间的亲和力，大大提高了肿瘤细胞对纳米硒的摄取量，达到减少用药剂量、提增疗效、减少毒副作用的整体治疗效果。此外，采用本发明方法制得的新型纳米硒，可在液相中以水溶胶的形式长时间保存，分散性和稳定性好，可作注射、喷雾等多种剂型应用。
一个实施方案中，本发明采用的虫草菌丝体Cs4多糖蛋白溶液、二氧化硒溶液或亚硒酸盐溶液以及维生素C溶液均是以水作为溶剂。上述步骤(d)中，混合溶液B是否反应完全可依据产物的颜色进行判断，例如，待产物中出现的红色不再加深即表明反应完全。另外，向 混合溶液A中加入维生素C溶液时，可采用将维生素C溶液滴加到混合溶液A中的方式。上述步骤(c)中，维生素C为还原型维生素C。
作为本发明所述的纳米硒水溶胶制备方法，于混合溶液B中，虫草菌丝体Cs4多糖蛋白的浓度为60～6000mg·L-1，二氧化硒或亚硒酸盐的浓度为0.2～20mmol·L-1，维生素C的浓度为1.0～100mmol·L- 1。技术人员可根据不同浓度需要的新型纳米硒水溶胶，计算出上述的原料所需采用的浓度体积。
在一个实施方案中，于混合溶液B中，维生素C与所述的二氧化硒或亚硒酸盐的摩尔比为5∶1。
在一个优选的实施方案中，于混合溶液B中，虫草菌丝体Cs4多糖蛋白的浓度为300～1200mg·L-1，维生素C的浓度为10.0～100mmol·L-1，二氧化硒或亚硒酸盐的浓度为2.0～20mmol·L-1。
本发明所述的新型纳米硒水溶胶可在2～10℃下以溶胶形态保存，保存期为30～60天。
实施例
本实施例中所用的材料和试剂均商业可得，且所用到的方法是本领域熟知的。
实施例1
本实施例制备了Cs4-SeNPs、虎奶菇多糖蛋白功能化纳米硒(PTR-SeNPs)以及壳聚糖功能化纳米硒(Chitosan-SeNPs)水溶胶。具体方法 为：常温常压下(15～35℃，1标准大气压)，取质量浓度为2.5g·L- 1的虫草菌丝体Cs4多糖蛋白溶液6.0mL、虎奶菇多糖蛋白溶液6.0mL、壳聚糖溶液3.0mL分别加入已装有10mL双蒸水的25mL容量瓶中，然后加入浓度为0.025mol·L-1的亚硒酸钠溶液2.0mL，轻轻摇匀使之混合充分，然后再滴加浓度为0.1mol·L-1的维生素C溶液2.5mL，边滴加边轻轻摇匀，滴加完毕，加水定容至25mL，待红色不再加深，即得到3种功能化纳米硒水溶胶。产物经过24小时透析(截留分子量8,000)后经硝化ICP方法测定当中的硒含量(浓度约为1.0～1.5mmol/L)。
实施例2
采用本领域常用的细胞半数抑制浓度(IC50)用来评估药物抑制肿瘤增殖的能力。通过MTS细胞增殖检测法(BMG Labtech，Clariostar)评估不同种类纳米硒对犬骨肉瘤(OSCA-8)癌细胞及犬口腔恶性黑色素瘤(CMGD-5)之抗增殖作用。MTS是一种MTT(噻唑蓝)类似物，在偶联剂存在下，被活细胞内的脱氢酶类转化成液态可溶的甲臢化合物，这种甲臢化合物在490nm的吸收值可以直接在96孔板上测量，颜色的深浅和细胞数量呈线性关系，以此来反映细胞的增殖情况。结果如图1A-F所示，跟单独的虫草菌丝体Cs4多糖蛋白、虎奶菇多糖蛋白及壳聚糖截然不同，3种功能化纳米硒对OSCA-8及CMGD-5细胞的增殖具有不同程度的抑制作用，而Cs4-SeNPs4能发挥显著最强的抗增殖作用(OSCA-8：IC50＝3.82μM；CMGD-5：IC50＝3.28μM)。
实施例3
进一步通过DNA流式细胞术分析Cs4-SeNPs(3-40μM)诱导 OSCA-8及CMGD-5细胞周期阻滞及细胞凋亡的能力。如图2A-H所示，Cs4-SeNPs能引起凋亡峰细胞(Sub-G1)的百分数呈剂量效应地增加，(OSCA-8：由对照组7.0％上升到90.7％；CMGD-5：由对照组5.3％上升到53.0％)，说明Cs4-SeNPs能有效诱导OSCA-8及CMGD-5细胞凋亡，效果呈浓度依赖性。
实施例4
细胞凋亡时细胞膜会发生改变，正常细胞的磷脂酰丝氨酸(Phosphatidylserine，PS)位于细胞膜的内侧，在凋亡的早期，细胞膜的PS会从内侧翻转到外侧。通过Annexin-V试剂盒探讨Cs4-SeNPs(3-40μM)诱导OSCA-8及CMGD-5细胞凋亡的能力见附图3A-H。Annexin-V荧光探针与PS特异结合并产生绿色荧光，而绿色荧光强度与外翻PS程度成正比。结果显示，Cs4-SeNPs引起的犬癌细胞PS外翻百分数呈剂量效应地增多增强(OSCA-8：由对照组4.80％上升到25.6％；CMGD-5：由对照组7.40％上升到22.1％)。结果表明，Cs4-SeNPs能有效诱导OSCA-8及CMGD-5细胞早期凋亡，效果呈浓度依赖性。
综上，本发明人在实验中发现Cs4-SeNPs能有效抑制犬骨肉瘤(OSCA-8；IC50＝3.82μM)及犬口腔恶性黑色素瘤(CMGD-5；IC50＝3.28μM)的增殖。另外，与其他不同种类的纳米硒比较，我们发现Cs4-SeNPs对OSCA-8及CMGD-5细胞能发挥显著更强的抗增殖作用。进一步的机理研究表明，Cs4-SeNPs主要是通过诱导OSCA-8及CMGD-5细胞凋亡来实现抗肿瘤活性(包括sub-G1细胞群增多和磷脂酰丝氨酸外翻)。
以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (14)

  1. 包含虫草菌丝体Cs4多糖蛋白功能化纳米硒水溶胶的复合物在制备治疗有需要的受试者中肿瘤的制剂中的用途。
  2. 根据权利要求1的用途，其中受试者为人。
  3. 根据权利要求1的用途，其中受试者为陪伴动物。
  4. 根据权利要求3的用途，其中陪伴动物包括猫和犬。
  5. 根据权利要求1-4中任一项的用途，其中肿瘤包括骨肉瘤及恶性黑色素瘤。
  6. 根据权利要求1-4中任一项的用途，其中硒来自二氧化硒或亚硒酸盐。
  7. 根据权利要求6的用途，其中亚硒酸盐为亚硒酸钠。
  8. 根据权利要求1-4中任一项的用途，其中虫草菌丝体Cs4多糖蛋白通过水提虫草菌丝体Cs4得到。
  9. 根据权利要求1-4中任一项的用途，其中虫草菌丝体Cs4多糖蛋白功能化纳米硒水溶胶的制备方法包括以下步骤：
    (a)通过水提虫草菌丝体Cs4得到虫草菌丝体Cs4多糖蛋白溶液；
    (b)向步骤(a)得到的虫草菌丝体Cs4多糖蛋白溶液中加入二氧化硒溶液或亚硒酸盐溶液，混合均匀，得到混合溶液A；
    (c)向混合溶液A滴加还原性维生素C溶液，混匀，得到混合溶液B；
    (d)待混合溶液B反应完全，经过透析得到虫草菌丝体Cs4多糖蛋白功能化纳米硒水溶胶。
  10. 根据权利要求9的用途，于混合溶液B中，虫草菌丝体Cs4多糖蛋白的浓度为60～6000mg·L-1，还原性维生素C的浓度为1.0～100mmol·L-1，二氧化硒或亚硒酸盐的浓度为0.2～20mmol·L-1。
  11. 根据权利要求9的用途，于混合溶液B中，虫草菌丝体Cs4多糖蛋白的浓度为300～1200mg·L-1，维生素C的浓度为10.0～100 mmol·L-1，二氧化硒或亚硒酸盐的浓度为2.0～20mmol·L-1。
  12. 根据权利要求9的用途，于混合溶液B中，还原性维生素C与二氧化硒或亚硒酸盐的摩尔比为5∶1。
  13. 一种用于治疗肿瘤的制剂，其包括虫草菌丝体Cs4多糖蛋白功能化纳米硒水溶胶。
  14. 根据权利要求13的制剂，其中所述制剂为注射剂或喷雾剂。
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