WO2017125080A1 - 富勒烯/金属富勒烯在制备药物中的应用 - Google Patents

富勒烯/金属富勒烯在制备药物中的应用 Download PDF

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WO2017125080A1
WO2017125080A1 PCT/CN2017/071971 CN2017071971W WO2017125080A1 WO 2017125080 A1 WO2017125080 A1 WO 2017125080A1 CN 2017071971 W CN2017071971 W CN 2017071971W WO 2017125080 A1 WO2017125080 A1 WO 2017125080A1
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fullerene
soluble
nano material
oil
micro
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English (en)
French (fr)
Inventor
王春儒
甄明明
张莹
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Beijing Fu Na Kang Biotechnology Co Ltd
Institute of Chemistry CAS
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Beijing Fu Na Kang Biotechnology Co Ltd
Institute of Chemistry CAS
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Priority claimed from CN201610041914.1A external-priority patent/CN105903020B/zh
Priority claimed from CN201610656821.XA external-priority patent/CN106109494A/zh
Application filed by Beijing Fu Na Kang Biotechnology Co Ltd, Institute of Chemistry CAS filed Critical Beijing Fu Na Kang Biotechnology Co Ltd
Priority to EP17741111.3A priority Critical patent/EP3406254A4/en
Priority to JP2018538192A priority patent/JP6775589B2/ja
Priority to US16/072,028 priority patent/US20190038667A1/en
Priority to AU2017209357A priority patent/AU2017209357B2/en
Publication of WO2017125080A1 publication Critical patent/WO2017125080A1/zh
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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
    • A61K33/44—Elemental carbon, e.g. charcoal, carbon black
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • 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
    • 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
    • A61K47/54—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 an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • 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
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention also requires the micro-nano material submitted by the Institute of Chemistry of the Chinese Academy of Sciences and Beijing Funakan Biotechnology Co., Ltd. to the Chinese Patent Office, the application number is CN201610656821.X, and the invention name is "prevention and/or treatment of bone marrow suppression" and its application.
  • the priority of the Chinese Patent Application the entire contents of which is incorporated herein by reference.
  • the invention belongs to the field of biomedicine, and particularly relates to the application of a fullerene micro-nano material and/or a metal fullerene micro-nano material in preparing a medicament for treating myelosuppression.
  • the killing effect of chemotherapy drugs and radiotherapy rays will cause secondary damage to patients.
  • the bone marrow suppression caused by radiotherapy and chemotherapy is initially manifested as a decrease in white blood cells and platelets. In severe cases, red blood cells and hemoglobin are decreased, and even possible. Aplastic anemia, leukemia and other diseases occur. Therefore, one of the key factors for the efficacy of chemoradiotherapy is whether the patient's bone marrow hematopoietic function can be protected during radiotherapy and chemotherapy. Whether bone marrow suppression caused by nursing, radiotherapy and chemotherapy can be alleviated.
  • Fullerenes are another allotrope of carbon other than graphite, diamond and amorphous carbon. Such materials refer to cage structures composed of carbon atoms, including C60, C70, C76, C78, C82, and C84.
  • the interior of the carbon cage of fullerenes is a cavity structure, the internal cavity can be embedded with different atoms, ions or clusters of atoms, called metal fullerenes, such as La@C60, indicating that La is embedded in C60.
  • @ means at, the image expresses the meaning of the inline.
  • Objects of the present invention include the use of a fullerene micro-nano material and/or a metal fullerene micro-nano material for the preparation of a medicament for the treatment of myelosuppression, and the object of the present invention also provides a pharmaceutical composition for treating myelosuppression Things and methods.
  • the fullerene micro-nano material and/or the metal fullerene micro-nano material of the invention maintains the high conjugation of fullerene molecules and the high-efficiency scavenging of free radicals, and can be efficiently and rapidly enriched in the bone marrow, in vivo for Myelosuppression has excellent preventive and therapeutic effects, can effectively reduce the side effects of radiotherapy and chemotherapy drugs on bone marrow and other organs, and treat leukopenia, thrombocytopenia, hemoglobin decline and monocyte decline due to myelosuppression, and is safe. Non-toxic, can be metabolized outside the body.
  • the present invention provides the following technical solutions:
  • the invention also provides a method of treating myelosuppression comprising the step of administering an effective amount of a fullerene micro-nano material and/or a metal fullerene micro-nano material to an organism in need of treatment for myelosuppression.
  • the invention also provides a pharmaceutical composition for treating myelosuppression comprising a fullerene micro-nano material and/or a metal fullerene micro-nano material, further comprising a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent and At least one of the pharmaceutically acceptable excipients.
  • the fullerene micro-nano material and/or metal fullerene micro-nano material comprises at least one active ingredient selected from the group consisting of oil-soluble fuller a composition of an ene micro-nano material, an oil-soluble metal fullerene micro-nano material, the oil-soluble fullerene micro-nano material, and the oil-soluble metal fullerene micro-nano material, a water-soluble fullerene micro-nano material, a water-soluble metal fullerene micro-nano material, a composition of the water-soluble fullerene micro-nano material and the water-soluble metal fullerene micro-nano material, a pharmaceutically acceptable ester of the above six or the above six A pharmaceutically acceptable salt.
  • oil-soluble fuller a composition of an ene micro-nano material, an oil-soluble metal fullerene micro-nano material, the oil-soluble fullerene micro-nano material, and the oil-soluble metal
  • the oil-soluble fullerene micro-nano material is obtained by oil-soluble modification of a fullerene bulk material
  • the oil-soluble metal fullerene micro-nano material is The metal fullerene bulk material is obtained by oil-soluble modification.
  • an effective route for oil-soluble modification is to modify the fullerene bulk material and/or the metal fullerene bulk material by oil-soluble modification by covalent or non-covalent interaction. It is obtained that specific oil-soluble modification methods can be carried out in accordance with the methods disclosed in the prior art.
  • the oil-soluble fullerene micro-nano material and/or the oil-soluble metal fullerene micro-nano material are obtained by coating the fullerene bulk material and/or the metal fullerene bulk material with edible oil.
  • the specific coating can be carried out by ball milling or ultrasonication, that is, the fullerene bulk material and/or the metal fullerene bulk material (both in powder form) and the edible oil are mixed, and the resulting mixture is ultrasonicated.
  • the oil-soluble fullerene micro-nano material and/or the oil-soluble metal fullerene micro-nano material can be prepared by ball milling in a ball mill for a certain period of time.
  • the mixture after ultrasonically or ball milling in a ball mill for a certain period of time, further comprises dissolving the resulting oil-soluble fullerene micro-nano material and/or oil.
  • Step of purifying the metal fullerene micro-nano material the precipitate is sequentially removed by centrifugation, and then the resulting supernatant liquid is filtered to obtain.
  • 0.05-500 mg of fullerene bulk material and/or metal fullerene bulk material is dispersed per 1 ml of the edible oil, the method, method or pharmaceutical composition
  • the disclosure of the scope should be considered as a disclosure of all values in the range, optionally 0.05-1 mg, 0.05-10 mg, 0.05-100 mg, and the like.
  • the mixture is ball milled or sonicated for 30 min to 15 h.
  • concentration of the olefinic bulk material may range from 1200 to 1500 ppm (mg/kg), such as 1500 ppm.
  • the edible oil includes, but is not limited to, at least one of olive oil, linseed oil, sunflower oil, corn germ oil, corn oil, and squalane. .
  • the oil-soluble fullerene micro-nano material and/or the oil-soluble metal fullerene micro-nano material may specifically be an edible oil-coated base metal fullerene.
  • edible oil coated Gd@C82 edible oil coated C60
  • edible oil coated C70 edible oil coated C84
  • edible oil refers to olive oil, linseed oil, sunflower seeds Any of oil, corn germ oil, corn oil and squalane.
  • the oil-soluble fullerene micro-nano material and/or the oil-soluble metal fullerene micro-nano material has all of the following properties: (1) fullerene bulk material and/or metal fullerene The bulk material is coated with the edible oil, and its fat-soluble property makes it easier to enter the cell and can reach the organ through the blood circulation through the blood; (2) The fullerene and/or metal rich in the coated material The olefin retains the integrity of the carbon cage and has a good free radical scavenging effect.
  • the water-soluble fullerene micro-nano material of the present invention is obtained by water-soluble modification of a fullerene bulk material, the water-soluble metal fuller The ene micro-nano material is obtained by water-soluble modification of the metal fullerene bulk material.
  • the water soluble modification can be followed
  • the methods disclosed in the prior art are modified.
  • the method for water-soluble modification comprises any one of the following methods: Method 1, covalently modifying a method for modifying a hydrophilic group on a surface of a bulk material, generally by solid or liquid-liquid reaction under the action of a base Specifically, the fullerene bulk material and/or the metal fullerene bulk material are mixed with hydrogen peroxide and a base (the base is specifically sodium hydroxide or potassium hydroxide) and reacted to obtain a water-soluble fullerene micro-nano A material and/or a water soluble metal fullerene micro/nano material that is modified with a hydroxyl group.
  • the sodium hydroxide and/or potassium hydroxide in the above step may be replaced with ammonia water.
  • Method 2 non-covalent interaction forms a water-soluble carrier with a fullerene bulk material and/or a metal fullerene bulk material by hydrophobic-hydrophobic interaction to form a corresponding water-soluble material.
  • the fullerene bulk material and/or the metal fullerene bulk material is mixed with hydrogen peroxide and a base and reacted to further comprise the following purification step: washing with ethanol, and then Dialysis.
  • the water-soluble fullerene micro-nano material and/or the water-soluble metal fullerene micro-nano material comprises at least one selected from the group consisting of: a carbon cage The full surface is modified with a hydrophilic group, a fullerene having a hydrophilic group, a metal fullerene having a hydrophilic group modified on the outer surface thereof, a fullerene supported by a water-soluble carrier, and a metal fullerene supported by a water-soluble carrier.
  • modified fullerene and/or metal fullerene herein refers to a water-soluble fullerene micro-nano material obtained by modifying or supporting a fullerene bulk material and/or a metal fullerene bulk material. And/or water soluble metal fullerene micro/nano materials.
  • the hydrophilic group includes one or more of a hydroxyl group, a carboxyl group, a thiol group, and an amino group.
  • the water-soluble fullerene micro-nano material and/or the water-soluble metal fullerene micro-nano material may specifically be a water-soluble hydroxylated ruthenium metal fullerene.
  • GFNC water-soluble hydroxylated ruthenium metal fullerene.
  • C60(OH) n water-soluble hydroxylated C60
  • C70(OH) n water-soluble hydroxylated C70
  • the water-soluble carrier is a pharmaceutical carrier commonly used in medicine, including at least one of a liposome, a polymer micelle, and a protein.
  • the polymer micelle is polyglycolide polyethylene glycol (PEG-PLGA), polylysine or chitosan; the protein is albumin or transferrin.
  • the water-soluble fullerene micro-nano material and/or water-soluble metal fullerene micro-nano material has all of the following properties: (1) the surface is a pro Water-based, enabling it to be injected into the living body via the vein and enriched in the bone marrow through blood circulation; (2) The micro-nano material is rigid (ie, not easily deformed), allowing it to pass through the endothelial cell space of the sinusoids of the bone marrow Use the pressure difference inside and outside the blood vessel to quickly enter the bone marrow.
  • the myelosuppression is induced by a drug chemotherapy, a chemical poison or a drug having a myelosuppressive side effect.
  • the drug chemotherapy comprises a drug currently inhibiting bone marrow which is conventionally used in clinical practice, and may be cyclophosphamide (CTX), doxorubicin, cisplatin or paclitaxel;
  • CTX cyclophosphamide
  • the chemical poison may be It is a derivative of benzene or benzene (such as chlorambucil);
  • the drug having myelosuppressive side effects may be chloramphenicol, tetracycline, tababacin or indomethacin.
  • the myelosuppression is induced by tumor radiation therapy or radiation that produces myelosuppressive side effects.
  • the radiation utilized by the radiation therapy may be alpha rays, beta rays, gamma rays, or X-rays; alternatively, the rays that cause bone marrow suppression side effects may be radiation that is inevitably contacted due to related work.
  • the fullerene bulk material comprises one or more cage structures consisting of carbon atoms of the formula C 2m , 30 ⁇ m ⁇ 60, For example; C 60 , C 70 , C 84 , etc.
  • the metal fullerene bulk material comprises M@C 2n , M 2 @C 2n , MA@C 2n , M 3 N@C 2n , M 2 One or more of C 2 @C 2n , M 2 S@C 2n , M 2 O@C 2n and M x A 3-x N@C 2n , wherein: M and A both represent a metal element and M, A is selected from any one of a lanthanide metal element, Sc and Y, 30 ⁇ n ⁇ 60; 0 ⁇ x ⁇ 3.
  • N nitrogen element
  • C carbon element
  • S sulfur element
  • lanthanide metal elements include La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu, Such as: Gd@C82.
  • the water-soluble fullerene micro-nano material and/or the water-soluble metal fullerene micro-nano material have an average particle size ranging from 1 to 500 nm, specifically It is 140-200 nm.
  • the oil-soluble fullerene micro-nano material and/or the water-soluble metal fullerene micro-nano material have an average particle size ranging from 0.5 to 50 nm, specifically It is 0.5-10 nm, 0.5-5 nm, 0.5-2 nm or about 1 nm.
  • the drug or the pharmaceutical composition may be a tablet, a pill, a powder, a lozenge, a sachet, a cachet, an elixir, a suspending agent, Formulations of emulsions, solutions, syrups, aerosols, ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions or sterile packaging powders.
  • the method of preparing an active ingredient into a pharmaceutical or pharmaceutical composition in the present invention can be prepared by a method known to those skilled in the art to provide an immediate release, sustained release or delayed release of the active ingredient after administration to a subject, for example:
  • the active ingredient can be mixed with the carrier, diluted with the carrier or enclosed in a carrier.
  • some examples suitable as carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, resins, Acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methylcellulose, methylparaben And propyl ester, talc, magnesium stearate and liquid paraffin.
  • the medicament or the above pharmaceutical composition in the above application may additionally comprise a lubricant, a wetting agent, an emulsifying and suspending agent, a preservative, a sweetener or a flavoring agent. And other additives.
  • the water-soluble fullerene micro-nano material and/or the water-soluble metal fullerene micro-nano material has a concentration of 0.1 in the preparation. mM-10 mM; the concentration of the oil-soluble fullerene and/or oil-soluble metal fullerene in the formulation is from 300 ppm to 5000 ppm (mg/kg).
  • the fullerene micro-nano material and/or the metal fullerene micro-nano material can be used within 24 hours before the use of the chemotherapeutic drug or the radiation therapy radiation, or can be used during the radiotherapy and chemotherapy process. It can be used continuously for 1-2 weeks after the end of radiotherapy and chemotherapy.
  • the active ingredient is administered at a dose of 1 mg/kg/d to 500 mg/kg/d, optionally 1-100 mg/kg/d, 1-20 mg/kg/d, 1-10 mg/kg/d.
  • the method of administration is injection or oral; optionally, the injection is intraperitoneal or intravenous, and the active ingredient enters the body and directly acts through the blood circulation without permeation.
  • the dosage is small and the curative effect is high; it can also be taken orally, filtered and absorbed by the digestive system, with fewer side effects and remarkable curative effect.
  • the organism is a human or an animal, and the animal may be a mammal such as a mouse, a guinea pig, a rat, a dog, a rabbit, a monkey, or the like.
  • treatment includes its generally accepted meaning, which includes preventing, preventing, inhibiting, ameliorating, and slowing, halting, or reversing the development of a symptom or a desired condition.
  • the invention encompasses both therapeutic and prophylactic administration.
  • an effective dose means that when a biological fullerene micro-nano material and/or a metal fullerene micro-nano material is administered by the method of the present invention, it is sufficient to effectively deliver an active ingredient for preventing myelosuppression. the amount.
  • Those with milder bone marrow suppression can be used in a single dose, and those with severe bone marrow suppression can also be used in multiple doses. Myelosuppression is significantly prevented after single or multiple use.
  • active ingredient refers to a water-soluble fullerene micro-nano material, a water-soluble metal fullerene micro-nano material, a water-soluble fullerene micro-nano material, and a water-soluble metal fullerene micro-nano material.
  • a composition, an oil-soluble fullerene micro-nano material, an oil-soluble metal fullerene micro-nano material, an oil-soluble fullerene micro-nano material, and an oil-soluble metal fullerene micro-nano material, and the above six At least one of a pharmaceutically acceptable ester and a pharmaceutically acceptable salt of the above six.
  • fullerene bulk material refers to fullerenes which are not subjected to oil-soluble modification or water-soluble modification, that is, fullerenes themselves.
  • metal fullerene bulk material means a metal fullerene which is not subjected to oil-soluble modification or water-soluble modification, that is, metal fullerenes themselves.
  • the concentration are all measured by the specific content, concentration, etc. of the corresponding fullerene bulk material or metal fullerene bulk material, for example, the active ingredient is applied at a dose of 1 mg/kg/d-500 mg/kg. /d means that the amount of the corresponding fullerene bulk carbon cage or metal fullerene bulk carbon cage in the active ingredient to be applied per 1 kg of mice per day is from 1 mg to 500 mg.
  • the fullerene micro-nano material and/or the metal fullerene micro-nano material in the invention maintains the high conjugation of fullerene molecules and the high-efficiency scavenging of free radicals.
  • the fullerene micro-nano material and/or the metal fullerene micro-nano material can pass through the blood to circulate through the nanopores between the endothelial cells in the sinusoids of the bone marrow and utilize the intravascular and extravascular pressure differences. Efficiently and rapidly enriched in organs and bone marrow.
  • Fullerene micro-nano materials and/or metal fullerene micro-nano materials have a unit mass content in bone marrow 12 times that of bone, and the residence time in bone marrow is significantly higher than that in other organs. It has good bone marrow targeting.
  • the fullerene micro-nano material and/or the metal fullerene micro-nano material in the invention can effectively scavenge free radicals, has excellent preventive and therapeutic effects on bone marrow suppression in vivo, and can effectively reduce radiation therapy and chemotherapy drugs.
  • the fullerene micro-nano material and/or the metal fullerene micro-nano material of the present invention can be used for the prevention and treatment of myelosuppression caused by chemotherapeutic drugs or radiotherapy rays, and can be used for preparing a medicament for treating myelosuppression.
  • the fullerene micro-nano material and/or the metal fullerene micro-nano material in the invention is simple and rapid to prepare, has no obvious cytotoxicity to living, bone marrow cells and normal cells, is safe and non-toxic; can be metabolized outside the organism, and has good Biocompatibility.
  • the fullerene micro-nano material and/or the metal fullerene micro-nano material in the present invention can also protect the liver Damage to the dirty, kidneys, and spleen.
  • 1A-1D are blood routine indexes of different groups of mice in Example 2 of the present invention.
  • Figure 2 is a graph showing changes in body weight of different groups of mice in Example 2 of the present invention.
  • 3A-3D are related enzyme indexes of different groups of mice in Example 2 of the present invention.
  • 4A-4D are blood routine indexes of different groups of mice in Example 3 of the present invention.
  • Figure 5 is a graph showing changes in body weight of different groups of mice in Example 3 of the present invention.
  • 6A-6D are related enzyme indexes of different groups of mice in Example 3 of the present invention.
  • 7A-7D are blood biochemical indicators of different groups of mice in Example 3 of the present invention.
  • Fig. 8 is a graph showing the particle size distribution of the water-soluble hydroxylated ruthenium metal fullerene prepared in Example 4.
  • Figure 9 is a graph showing the metabolic profile of water-soluble hydroxylated ruthenium metal fullerene in mice by ICP-MS in Example 5.
  • Figure 10 is a graph showing the metabolic distribution of water-soluble hydroxylated ruthenium metal fullerene in mice using the 131I labeling method in Example 6.
  • 11A-F are electron probe spectra of water-soluble hydroxylated ruthenium metal fullerene samples in mouse bone marrow at different time points in Example 7.
  • Figure 12 is a graph showing the organ coefficients of the water-soluble hydroxylated ruthenium metal fullerene group and the control group in Example 8 after 30 days of injection.
  • Figure 13 is a graph showing changes in body weight of a water-soluble hydroxylated ruthenium metal fullerene group and a control group in Example 8.
  • Figure 14 is a blood biochemical test of a water-soluble hydroxylated ruthenium metal fullerene group in Example 8 in the same manner, which is a value after 30 days of injection.
  • Example 15 is a result of an in vitro ESR scavenging free radical experiment of GFNC in Example 9, wherein the solid line is the control group; and the dotted line is the experimental group.
  • Figure 16 is a graph showing the effect of GFNC in scavenging free radicals at the cellular level in Example 10.
  • Figure 17 is a blood routine index of different groups of mice in Example 11.
  • Figure 18 is a graph showing tumor size changes in different groups of mice in Example 12.
  • Figure 19 is a graph showing changes in body weight of different groups of mice in Example 12.
  • Figure 20 is a blood routine index of different groups of mice in Example 13.
  • Figure 21 is a graph showing tumor size changes in different groups of mice in Example 14.
  • Figure 22 is a graph showing changes in body weight of different groups of mice in Example 14.
  • Figure 23 is a blood biochemical index of different groups of mice in Example 14.
  • Figure 24 is a sectional image of H&E staining of pathological sections of tissue of different groups of mice in Example 14.
  • Figure 25 is a graph showing the particle size distribution of the water-soluble hydroxylated hollow fullerene prepared in Example 15.
  • Figure 26 is a graph showing the organ coefficients of the water-soluble hydroxylated hollow fullerene group and the control group in Example 16 after injection for 30 days.
  • Figure 27 is a graph showing changes in body weight of the water-soluble hydroxylated hollow fullerene group and the control group in Example 16.
  • Figure 28 is a blood biochemical test of a water-soluble hydroxylated hollow fullerene group injected into the mice of Example 16 after 30 days of injection.
  • Example 29 is a result of an in vitro ESR scavenging free radical experiment of the water-soluble hollow fullerene in Example 17, wherein the solid line is the control group; and the broken line is the experimental group.
  • Figure 30 is a graph showing the effect of the C60 water-soluble derivative in Example 18 on scavenging free radicals at the cellular level.
  • Figure 31 is a graph showing the blood routine of mice of different groups using the C60 water-soluble derivative in Example 19.
  • Figure 32 is a graph showing the blood routine of mice in different groups using the C60 water-soluble derivative in Example 20.
  • top-to-bottom order of the histogram legends of Figs. 9, 10, 17, 20, and 32 of the present invention are sequentially arranged from left to right in accordance with the histogram.
  • the fullerene solid powders C60, C70 and the metal fullerene solid powder Gd@C82 used in the examples of the present invention were purchased from Xiamen Funa New Material Technology Co., Ltd., and the purity was 99%; the cyclophosphamide used (CTX) ) purchased from Sigma-Aldrich, the product number is C0768; the X-ray therapeutic apparatus used is the product used in the radiotherapy department of Beijing Chaoyang Hospital. Other materials, reagents, instruments and the like used are commercially available unless otherwise specified; other experimental methods are conventional methods unless otherwise specified.
  • the centrifuge is used to repeatedly remove the uncoated solid fullerene solid powder and/or the metal fullerene solid powder by repeated centrifugation at a speed of 10000 r until the solid powder is not centrifuged; The olefin and/or oil coat metal fullerenes.
  • the uncoated uncoated hollow fullerene C60 solid powder is repeatedly centrifuged at 10,000 rpm to prepare a solid powder.
  • Olive oil is coated with hollow fullerenes (hereinafter referred to as Olive-C60).
  • the content of the hollow fullerene C60 solid powder in Olive-C60 was determined to be 1500 ppm (mg/kg), that is, 1500 mg of hollow fullerene C60 solid powder per 1 kg of Olive-C60.
  • Example 2 Effect of Olive-C60 on myelosuppression and related enzyme activities in the first course of living level
  • mice were randomly divided into 5 groups, 6 in each group, corresponding to cyclophosphamide (CTX) + Olive-C60 intragastric administration group, CTX+Olive-C60 abdominal cavity
  • CTX cyclophosphamide
  • the experimental group, the CTX+Olive intragastric administration test group, the CTX experimental group and the blank control group were administered.
  • Cyclophosphamide (CTX)+Olive-C60 intragastric administration group rats were intraperitoneally injected with CTX solution, medicine The dosage was 60 mg/kg of mouse body weight, and the Olive-C60 solution was administered by intragastric administration. The dosage of the drug was 1500 ppm, 100 ⁇ L.
  • Cyclophosphamide (CTX)+Olive-C60 intraperitoneal administration group The rats were intraperitoneally injected with CTX solution, the dosage of the drug was 60 mg/kg of mouse body weight, and the oral administration of Olive-C60 solution was performed at a dose of 1500 ppm and 100 ⁇ L.
  • Cyclophosphamide (CTX)+Olive intragastric administration group rats were intraperitoneally injected with CTX solution, the dosage of the drug was 60 mg/kg mouse body weight, and the pure olive oil Olive was administered with a dosage of 100 ⁇ L.
  • Cyclophosphamide (CTX) experimental group rats were intraperitoneally injected with CTX solution, the dosage of the drug was 60 mg/kg of mouse body weight, and 100 ⁇ L of normal saline was administered to eliminate the difference in the effect of gavage stimulation on mice.
  • the blank control group the drugs injected in the experimental group were replaced with the same volume of physiological saline, and the same volume of physiological saline was injected into the stomach and the abdominal cavity.
  • ICR mice began to inject drugs on the seventh day after the start of the laboratory, and the mice were in stable state.
  • intraperitoneal injection of CTX for 5 consecutive days was intragastric or intraperitoneal.
  • Olive-C60 or Olive the injection was stopped after 5 days and only observation or sampling was performed.
  • FIGS. 1A to 1D The corresponding test results are shown in FIGS. 1A to 1D. From FIGS. 1A to 1D, it is known that the indexes related to bone marrow suppression in the mice in the cyclophosphamide (CTX) experimental group are compared with the blank control group: White blood cells, red blood cells, platelets and hemoglobin have been reduced to varying degrees in mice. The reduction of white blood cells is most obvious, indicating that the bone marrow is damaged and the related indicators are obviously abnormal.
  • CTX cyclophosphamide
  • mice In the CTX+Olive-C60 experimental group, Mice, whether administered intraperitoneally or intragastrically, have a significant increase in white blood cell count compared to the cyclophosphamide (CTX) experimental group due to the scavenging free radical effect and protective effect of C60, while red blood cells and platelets
  • CTX cyclophosphamide
  • the values of hemoglobin are also closer to the normal group, and as time goes on, the relevant indicators are getting closer to the values of normal mice.
  • the body weight of different groups of mice decreased due to the toxic side effects of CTX, but after stopping the drug fight, the weight recovery of the mice in the Olive-C60 experimental group was faster than that of the CTX group. Closer to normal mouse weight levels.
  • Olive-C60 has a significant protective effect on the body's antioxidant system, thereby protecting the further damage caused by chemotherapy.
  • MDA is produced by the oxidation of lipids caused by free radicals.
  • the level of MDA in the body also represents the level of free radicals in the body.
  • the increase in MDA levels indicates that there are too many free radicals in the body that are not cleared in time. It also shows an increase in damage to the cell membrane system.
  • Olive-C60 can reduce the systemic damage of the cell membrane by removing free radicals such as hydroxyl radicals, and protect the body from secondary damage caused by cyclophosphamide and other chemotherapy drugs.
  • Example 3 Effect of Olive-C60 on myelosuppression, related enzyme activity and blood biochemical parameters in the second course of living level
  • mice were randomly divided into 5 groups, 6 in each group, corresponding to cyclophosphamide (CTX) + Olive-C60 intragastric administration group, CTX+Olive-C60 abdominal cavity
  • CTX cyclophosphamide
  • the experimental group, the CTX+Olive intragastric administration test group, the CTX experimental group and the blank control group were administered.
  • Cyclophosphamide (CTX)+Olive-C60 intragastric administration group rats were intraperitoneally injected with CTX solution, the dosage of the drug was 60 mg/kg mouse body weight, and the Olive-C60 solution was administered by intragastric administration. The dosage of the drug was 1500 ppm, 100 ⁇ L.
  • Intraperitoneal administration of cyclophosphamide (CTX)+Olive-C60 intraperitoneal injection of CTX solution, drug The dosage was 60 mg/kg of mouse body weight, and the Olive-C60 solution was intraperitoneally administered at a dose of 1500 ppm and 100 ⁇ L.
  • Cyclophosphamide (CTX)+Olive intragastric administration group rats were intraperitoneally injected with CTX solution, the dosage of the drug was 60 mg/kg mouse body weight, and the pure olive oil Olive was administered with a dosage of 100 ⁇ L.
  • Cyclophosphamide (CTX) experimental group mice were intraperitoneally injected with CTX solution, and the dosage of the drug was 60 mg/kg of mouse body weight. At the same time, 100 ⁇ L of normal saline was administered to eliminate the difference in the effect of gavage stimulation on mice.
  • the blank control group the drugs injected in the experimental group were replaced with the same volume of physiological saline, and the same volume of physiological saline was injected into the stomach and the abdominal cavity.
  • the second course of treatment was started, that is, 21 days of the first course of treatment was taken as the first day of the second course of treatment, and once every day, CTX was intraperitoneally injected for 5 consecutive days.
  • Olive-C60 or Olive was administered intragastrically or intraperitoneally, and administration was stopped after 5 days, and only observation or sampling was performed.
  • MDA is produced by the oxidation of lipids caused by free radicals.
  • the level of MDA in the body also represents the level of free radicals in the body. When the level of MDA is increased, there are too many free radicals in the surface that are not cleared in time. It also shows an increase in damage to the cell membrane system.
  • the MDA level of the cyclophosphamide administration group was higher than that of the normal control group, and the MDA level of the mice administered by intragastric administration was even lower than that of the normal control group, indicating that the olive oil-C60 scavenging free radical effect was remarkable.
  • Olive-C60 can reduce the systemic damage of the cell membrane by removing free radicals such as hydroxyl radicals, and protect the body from secondary damage caused by cyclophosphamide and other chemotherapy drugs.
  • Fig. 7A - Fig. 7D The corresponding test results are shown in Fig. 7A - Fig. 7D. It can be seen from Fig. 7A - Fig. 7D that the levels of alanine aminotransferase, alkaline phosphatase and urinary nitrogen are different in the experimental group after the completion of the multi-treatment course. They are all consistent and return to normal levels. It is worth noting the level of uric acid. The level of uric acid in the serum of mice in the cyclophosphamide group is much lower than that in the normal control group, while the uric acid as a reducing agent is concentrated.
  • the diameter is 140 nm, the particle size distribution is uniform, and the particle size distribution diagram is as shown in FIG. 8.
  • the water-soluble hydroxylated metal fullerene obtained in the present embodiment is referred to as GFNC.
  • mice 4-5 weeks of BALB/c mice were selected, and 10 6 mouse hepatoma cells (H22 cells) (provided by Peking Union Medical Cell Bank) were inoculated with 100 ⁇ l on the right thigh. After 5-7 days of inoculation, the tumor diameter reached 5 mm. Experiment with the left and right;
  • mice 150 ⁇ L of 1 mM water-soluble hydroxylated metal fullerene was intravenously injected, and the mice were sacrificed by cervical dislocation at 15 min, 30 min, 45 min, 1 h, 4 h, 24 h, 7 d, 15 d and 30 d after injection, and immediately dissected and small.
  • the main organs of the mouse (heart, liver, spleen, lung, kidney, intestine, stomach, etc.), mouse brain, tibia and muscle. A part of the organs, brain and muscles were weighed, and the tibia was weighed. Then, the tibia bone marrow of the mouse was washed out with PBS, and the tibia was weighed again to calculate the quality of the bone marrow. After dissolving the above tissues, filtration was performed using a 200 nm filter membrane;
  • Fig. 9 The test results are shown in Fig. 9. It can be seen from Fig. 9 that the water-soluble hydroxylated ruthenium metal fullerene material can have a significant enrichment effect in the bone within 1 h, which is only slightly lower than the concentration of the liver portion. However, with the prolongation of time, the concentration of strontium in the bone can continue at a higher level, and after 30 days, the concentration of the material in the bone is significantly reduced, indicating that it has metabolic properties, which is also metabolizable with fullerenes at the living level. Compatible.
  • the relative content of the water-soluble hydroxylated ruthenium metal fullerene obtained in Example 4 in mouse bone marrow and bone was as shown in Table 1 below. It can be seen from Table 1 that bone and bone marrow were separated and water-soluble hydroxylation was measured. The content of strontium metal fullerenes in mouse bone marrow and bone. After 24 hours of injection, the sputum content in the bone marrow was 5.84 ⁇ 0.58 ng / mg, much higher than the content in the bone (0.45 ⁇ 0.044 ng / mg), further indicating that the material has targeted enrichment in the bone marrow. Characteristics.
  • Bone marrow sputum content (ng/mg) 5 minutes 1.182 ⁇ 0.187 0.098 ⁇ 0.016 15 minutes 1.198 ⁇ 0.051 0.099 ⁇ 0.004 30 minutes 2.652 ⁇ 0.096 0.221 ⁇ 0.008 45 minutes 2.679 ⁇ 0.047 0.223 ⁇ 0.004 1 hour 3.521 ⁇ 0.298 0.293 ⁇ 0.025 4 hours 4.621 ⁇ 0.109 0.385 ⁇ 0.009 24 hours 5.388 ⁇ 0.528 0.449 ⁇ 0.044 7 days 8.185 ⁇ 0.388 0.515 ⁇ 0.032 15 days 5.538 ⁇ 0.512 0.461 ⁇ 0.043 30 days 4.431 ⁇ 0.514 0.369 ⁇ 0.043
  • Example 6 radiolabeling method for measuring the metabolic distribution of GFNC in vivo
  • Animal model 4-5 weeks of BALB/c mice were used, and 10 6 mouse hepatoma cells (H22 cells) were inoculated on the right thigh. After 5-7 days of inoculation, the tumor was drilled to a diameter of about 5 mm.
  • the hydroxyl group present on the surface of the water-soluble hydroxylated ruthenium metal fullerene can be substituted with 131I, and the gamma ray emitted by 131I can be used for high sensitivity to detect its metabolic behavior in the living body.
  • a 37 MBq Na 131I solution (volume ratio of 2:5) was oxidized for 10 min using a chloramine-T aqueous solution (10 mg/mL), and then the sample to be labeled was added, and the reaction was shaken at room temperature for 2 hours.
  • the obtained reaction solution was separated and purified by a Sephadex G-25 gel column to remove inorganic salt ions, thereby obtaining a radioactive iodine-131 labeled sample (radiation purity ⁇ 97%).
  • 50 ⁇ L of the activated solution was injected into the tumor-bearing mice through the tail vein respectively.
  • the mice were sacrificed by cervical dislocation after 1.0 h, 4.0 h, 24 h and 48 h, and the important tissues and organs (heart, liver, spleen, lung, etc.) were dissected.
  • Gamma counts were performed on kidneys, tumors, brain, bone, meat, etc., and the content of the labeled samples in each organ was counted.
  • the test results are shown in Fig. 10. It can be seen from Fig. 10 that the results obtained by the ICP ion concentration test method are similar, and it is confirmed that the water-soluble hydroxylated ruthenium metal fullerene material can have a significant enrichment effect in the bone. At the same time, the mouse bone marrow was punched out from the tibia and the bone marrow and bone quality and the intensity of the gamma count were measured. From Table 2, it can be seen that the water-soluble hydroxylated ruthenium metal fullerenes are mainly enriched in the bone marrow. in.
  • Animal model 4-5 weeks of BALB/c mice were used, and 10 6 mouse hepatoma cells (H22 cells) were inoculated on the right thigh. After 5-7 days of inoculation, the tumor was drilled to a diameter of about 5 mm.
  • mice 4-5 weeks of BALB/c mice were used, and 10 6 mouse hepatoma cells (H22 cells) were inoculated on the right thigh. After 5-7 days of inoculation, when the tumor diameter reached 5 mm, the concentration of 150 ⁇ L was intravenous. 900 ppb of water-soluble hydroxylated metal fullerene.
  • Mouse experiment 150 ⁇ L of water-soluble hydroxylated ruthenium metal fullerene at a concentration of 1 mM was injected intravenously before injection, 0.5 h, 1 h, 4 h and 24 h after injection, and the mice were dissected and the mice were dissected immediately. Both sides of the humerus. Immediately, the mouse bone marrow was washed out with PBS, and evenly spread on the surface of the conductive adhesive, and then the content of the strontium element was detected by an electron probe instrument.
  • Water-soluble hydroxylated ruthenium metal fullerene sample experiment The water-soluble hydroxylated ruthenium metal fullerene sample aqueous solution was evenly applied on the surface of the conductive adhesive, and after drying, it was tested by an electron probe instrument.
  • Water-soluble hydroxylated ruthenium metal fullerene group 150 ⁇ L of water-soluble hydroxylated ruthenium metal fullerene at a concentration of 1 mM was injected intravenously at 15 min, 30 min, 45 min, 1 h, 4 h, 24 h, 7 d, 15 d and 30 d, respectively. Take the eyeballs of the mice and take the blood. Place them in a 5 ml centrifuge tube, centrifuge at 3500 rpm for 15 min, transfer the obtained serum to a 200 ⁇ L centrifuge tube, and measure the blood biochemical indicators, ALT, ALP of the mice in a blood biochemical detector. , AST, BUN, LDH, etc. The change in body weight of the mice was observed within 30 days.
  • Saline group The same treatment as the metal fullerene group was used for comparison, and the water-soluble hydroxylated ruthenium metal fullerene was replaced with physiological saline.
  • the corresponding test results are shown in Fig. 12, Fig. 13 and Fig. 14.
  • the metal fullerenes group and the saline group have no significant difference in the body weight or short-term organ coefficient of the mice, indicating that the material is less toxic and can be Further used in clinical research; it can be seen from Fig. 14 that it is obvious that the metal fullerenes group has no significant decrease in liver and kidney function compared with the saline group, indicating that the toxicity is small, which proves that our materials are safe and non-toxic. of.
  • ESR Electron paramagnetic resonance
  • Fig. 15 The corresponding test results are shown in Fig. 15. It can be seen from Fig. 15 that in the experimental group, when the concentration of GFNC is only 20 ⁇ M, the free radicals generated by ultraviolet light irradiation of hydrogen peroxide can be effectively quenched to protect cells from hydrogen peroxide. The harmful effects of free radicals produced.
  • Example 10 GFNC protects cells from the killing of free radicals by hydrogen peroxide at the cellular level
  • Mouse bone marrow cells (FDC-P1) were selected as the study subjects, and the medium was high glucose DMEM supplemented with the cytokine IL-3.
  • the mouse bone marrow cells were seeded in 96-well plates at a concentration of ca.1 ⁇ 10 4 per well, 8 ⁇ 6 wells, negative control group, 6 wells; 6 wells in the positive control group; 7 in the experimental group A different GFNC concentration, 6 wells per concentration, was repeated three times.
  • the negative control group was not added with hydrogen peroxide and GFNC, only the medium inoculated with mouse bone marrow cells; the positive control group was only added 10 ⁇ L, 30 ⁇ M GFNC, no aqueous solution of hydrogen peroxide; the experimental group was added with a concentration of 100 ⁇ M aqueous solution of hydrogen peroxide in parallel. 20 ⁇ L, 90 ⁇ L of medium, and different concentrations of GFNC (to give final concentrations of 0.5 ⁇ M, 1 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M, and 30 ⁇ M, respectively).
  • the negative control group was loaded in the order of no hydrogen peroxide or GFNC, and was replaced with the same volume of PBS.
  • the order of addition of the positive control group was as follows: hydrogen peroxide was replaced with the same volume of PBS, and the mouse bone marrow cells were incubated for 1 h, after which GFNC was added and incubated for 3 h.
  • the experimental group was loaded in the following order: first added hydrogen peroxide and cell culture medium, and incubated with mouse bone marrow cells. 1h, aspirate the aqueous solution of hydrogen peroxide, wash it three times with PBS buffer, wash away the residual aqueous solution of hydrogen peroxide; add a certain concentration of GFNC, add the cell culture medium, and incubate the cells for 3 hours, then aspirate the medium containing GFNC and buffer with PBS. The solution was washed three times; finally, the cell culture medium was added, and the culture was continued for 24 hours in a cell culture incubator, and then the cell activity was measured with CCK-8.
  • Fig. 16 The corresponding test results are shown in Fig. 16. It can be seen from Fig. 16 that when 20 ⁇ L of a hydrogen peroxide solution having a concentration of 100 ⁇ M is added, the hydrogen peroxide will have a certain killing effect on the mouse bone marrow cells, and the water-soluble hydroxylated ruthenium metal is rich. When the amount of lenene is gradually increased, the activity of the cells is also increased, indicating that at the cellular level, the water-soluble hydroxylated ruthenium metal fullerene has an effect on the scavenging of free radicals and can protect cells from the effects. . At the same time, the positive control group containing only water-soluble hydroxylated ruthenium metal fullerene was added without hydrogen peroxide, and the cell activity was slightly higher than that of the negative control group, indicating that the material was not cytotoxic.
  • mice were randomly divided into 4 groups, 6 in each group, corresponding to blank control group, GFNC experimental group, X-ray irradiation experimental group and X-ray + GFNC experimental group.
  • GFNC GFNC experimental group
  • X-ray irradiation experimental group X-ray + GFNC experimental group.
  • 10 6 mouse hepatoma cells H22 cells
  • the tumor was about 5 mm in diameter, and the experiment was performed.
  • the blank control group the drugs injected in the experimental group were replaced with the same volume of physiological saline, and the same volume of physiological saline was intravenously injected.
  • GFNC experimental group Mice were injected with GFNC aqueous solution (1 mM) in the tail vein at a dose of 0.004 mmol GFNC/kg mouse body weight.
  • mice received 6Gy X-ray irradiation all at once.
  • X-ray + GFNC experimental group The whole body received 6Gy X-ray irradiation once and the GFNC solution was intravenously injected, and the dosage of the drug was 0.004 mmol GFNC/kg mouse body weight.
  • the drug was injected on the seventh day after tumor inoculation.
  • GFNC was intravenously injected for 5 consecutive days, and X-ray irradiation was performed on the first day, followed by the fourth day and the seventh day, respectively.
  • blood was taken from the eyelids of the mice (20 ⁇ l), placed in a 3 ml centrifuge tube, and the blood routine was detected by an automatic blood cell analyzer.
  • the main indicator related to myelosuppression was white blood cells.
  • the indexes related to myelosuppression in the mice in the X-ray irradiation experimental group are: white blood cells, platelets, and hemoglobin in mice.
  • the indexes related to myelosuppression in the mice in the X-ray irradiation experimental group are: white blood cells, platelets, and hemoglobin in mice.
  • the amount of white blood cells, platelets, and hemoglobin was greatly improved compared with the X-ray experimental group due to the protective effect of GFNC.
  • the value of monocytes was closer to the normal group and prolonged with time.
  • the relevant indicators are getting closer and closer to the values of normal mice, indicating that GFNC has a significant protective effect on mouse bone marrow suppression caused by radiotherapy X-rays.
  • Example 12 Effect of GFNC on the therapeutic effect of X-ray radiotherapy on the living body level
  • mice were randomly divided into 4 groups, 6 in each group, corresponding to blank control group, GFNC experimental group, X-ray irradiation experimental group and X-ray + GFNC experimental group.
  • GFNC GFNC experimental group
  • X-ray irradiation experimental group X-ray + GFNC experimental group.
  • 10 6 mouse hepatoma cells H22 cells
  • the tumor was about 5 mm in diameter, and the experiment was performed.
  • Blank control group The drugs injected in the experimental group were replaced with the same volume of physiological saline.
  • X-ray irradiation experimental group The whole body was irradiated with X-rays at a dose of 6 Gy.
  • GFNC Metal fullerene derivative
  • X-ray + GFNC experimental group The whole body was irradiated with X-rays at a dose of 6 Gy, and the GFNC solution was intravenously administered. The dosage of the drug was 0.04 mmol Gd3+/kg of mouse body weight.
  • GFNC was intravenously administered once every 5 days, and the body weight and tumor diameter of the mice were measured every two days.
  • Figs. 18 and 19 The corresponding test results are shown in Figs. 18 and 19. It can be seen from Fig. 18 that the tumor diameter of the mouse in the X-ray + GFNC experimental group is not increased or decreased as compared with the X-ray experimental group, indicating that : GFNC does not affect the therapeutic effect of radiotherapy on mouse tumors, and at the same time can significantly inhibit the myelosuppressive toxicity caused by radiotherapy.
  • mice in the X-ray experimental group have their body weight at the beginning of the fight
  • the recovery function of the mouse itself has a certain degree of recovery, but it is still the lightest group
  • the mice in the X-ray + GFNC experimental group are fighting drugs.
  • its body weight also decreased slightly, but it recovered significantly in the later stage, and it was close to the weight value of normal mice, indicating that GFNC can protect mice from the side effects caused by radiotherapy rays and protect mouse bone marrow from inhibiting toxicity.
  • its toxicity is small and can be further used in clinical research.
  • Example 13 Myelosuppressive protection of water-soluble hydroxylated ruthenium metal fullerenes at the living level
  • mice were randomly divided into 4 groups, 6 in each group, corresponding to blank control group, GFNC experimental group, cyclophosphamide (CTX) experimental group and CTX+GFNC experimental group.
  • CTX cyclophosphamide
  • the blank control group the drugs injected in the experimental group were all replaced with the same volume of physiological saline, and the same volume of physiological saline was injected into the vein and the abdominal cavity.
  • GFNC experimental group Mice were injected with GFNC aqueous solution (1 mM) in the tail vein at a dose of 0.004 mmol GFNC/kg mouse body weight.
  • CTX Cyclophosphamide
  • CTX+GFNC experimental group The rats were intraperitoneally injected with CTX solution, the dosage of the drug was 60 mg/kg mouse body weight, and the GFNC solution was intravenously administered. The dosage of the drug was 0.004 mmol GFNC/kg mouse body weight.
  • the drug was injected.
  • blood was collected from the eyelids of the mice (20 ⁇ l), placed in a 3 ml centrifuge tube, and the blood routine was detected by an automatic blood cell analyzer.
  • the main indicators related to myelosuppression were white blood cell count (WBC), platelet count. (PLT), hemoglobin measurement (HGB), monocyte ratio (MO%).
  • Fig. 20 The corresponding test results are shown in Fig. 20.
  • CTX cyclophosphamide
  • Example 14 Effect of water-soluble hydroxylated ruthenium metal fullerene on the therapeutic effect of chemotherapy drug CTX tumor at the living level and toxicity test
  • mice were randomly divided into 4 groups, 6 in each group, corresponding to blank control group, GFNC experimental group, cyclophosphamide (CTX) experimental group and CTX+GFNC experimental group.
  • CTX cyclophosphamide
  • Blank control group The drugs injected in the experimental group were replaced with the same volume of physiological saline.
  • Cyclophosphamide (CTX) experimental group mice were intraperitoneally injected with CTX solution, and the dosage of the drug was 60 mg/kg of mouse body weight.
  • GFNC Metal fullerene derivative
  • CTX+GFNC experimental group The rats were intraperitoneally injected with CTX solution, the dosage of the drug was 60 mg/kg mouse body weight, and the GFNC solution was intravenously administered. The dosage of the drug was 0.04 mmol Gd3+/kg mouse body weight.
  • the drug was injected.
  • CTX or intravenous GFNC was injected intraperitoneally for 5 consecutive days, and the body weight and tumor diameter of the mice were measured every two days, on the 17th day.
  • mice after the mice were sacrificed by cervical dislocation, the main tissues and organs (heart, liver, spleen, lung, kidney) of the mice were weighed and soaked in a 4% formalin solution. Histopathological sections, H&E staining. At the same time, the blank control group, cyclophosphamide (CTX) experimental group and metal fullerene derivative (GFNC) experimental group were treated the same for comparison.
  • CTX cyclophosphamide
  • GFNC metal fullerene derivative
  • mice in the cyclophosphamide (CTX) experimental group had a significant decrease in body weight at the beginning of the fight, and then the recovery function of the mice themselves had a certain degree of recovery due to the stop of the drug fight.
  • CTX+GFNC experimental group showed a slight decrease in body weight at the beginning of the fight, but recovered significantly in the later stage and was close to the weight value of normal mice, indicating GFNC. It can protect mice from the side effects of chemotherapeutic drugs as much as possible, protect the bone marrow suppression toxicity of mice, and its toxicity is small, which can be further used in clinical research.
  • the diameter is 200 nm, the particle size distribution is uniform, and the particle size distribution diagram is shown in Fig. 25.
  • Water-soluble hydroxylated hollow fullerene group 150 ⁇ L of water-soluble hydroxylated hollow fullerenes at a concentration of 1 mM was injected intravenously. At 30 days after injection, the mice were taken from the eyeballs and blood was taken and placed in a 5 ml centrifuge tube. The serum obtained was centrifuged at 3500 rpm for 15 min, and the obtained serum was transferred to a 200 ⁇ L centrifuge tube, and blood biochemical indexes, ALT, ALP, AST, BUN, LDH, and the like were detected in a blood biochemical detector. At the same time, the changes in body weight of mice within 30 days were observed.
  • Fig. 26 and Fig. 27 show that there is no significant difference between the hollow fullerene group and the saline group in the body weight or short-term organ coefficient of the mouse, indicating that the material It is less toxic and can be further used in clinical research. It can be seen from Fig. 28 that it is obvious that there is no significant decrease in liver and kidney function in the hollow fullerene group compared with the saline group, indicating that its toxicity is small, which proves that we The material is safe and non-toxic.
  • Example 17 the effect of water-soluble hydroxylated hollow fullerenes on scavenging free radicals in vitro
  • ESR Electron paramagnetic resonance
  • Fig. 29 The corresponding test results are shown in Fig. 29.
  • the concentration of the aqueous solution of the hydroxylated hollow fullerene is only 20 ⁇ M, the free radicals generated by the ultraviolet light irradiation of the hydrogen peroxide can be effectively quenched. Protect cells from the damage of free radicals produced by hydrogen peroxide.
  • Example 18 water-soluble hydroxylated hollow fullerene protects cells from free radicals by hydrogen peroxide at the cellular level
  • Mouse bone marrow cells (FDC-P1) were selected as the study subjects, and the medium was high glucose DMEM supplemented with the cytokine IL-3.
  • the mouse bone marrow cells were seeded in 96-well plates at a concentration of ca.1 ⁇ 10 4 per well, 8 ⁇ 6 wells, negative control group, 6 wells; 6 wells in the positive control group; 7 in the experimental group
  • the concentration of aqueous solution of different hydroxylated hollow fullerenes was 6 wells per concentration, and the experiment was repeated three times.
  • the negative control group was no aqueous solution of hydrogen peroxide and hydroxylated hollow fullerene, only the medium inoculated with mouse bone marrow cells; the positive control group was only added 10 ⁇ L, 30 ⁇ M aqueous solution of hydroxylated hollow fullerene, without aqueous solution of hydrogen peroxide.
  • the experimental group was added with 20 ⁇ L of aqueous solution of hydrogen peroxide at a concentration of 100 ⁇ M, 90 ⁇ L of medium, and different concentrations of hydroxylated hollow fullerene aqueous solution (to a final concentration of 0.5 ⁇ M, 1 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M, respectively). And 30 ⁇ M).
  • the negative control group was loaded in the order of no hydrogen peroxide or hydroxylated hollow fullerene solution, and was replaced with the same volume of PBS.
  • the order of the positive control group was as follows: the hydrogen peroxide was replaced with the same volume of PBS, and the mouse bone marrow cells were incubated for 1 h. Then, the hydroxylated hollow fullerene aqueous solution was added and incubated for 3 h.
  • the order of sample loading in the experimental group was as follows: first add hydrogen peroxide and cell culture medium, and incubate with mouse bone marrow cells for 1 hour, aspirate the aqueous solution of hydrogen peroxide, wash it three times with PBS buffer, wash away the residual aqueous solution of hydrogen peroxide; add a certain concentration of hydroxylated hollow rich The aqueous solution of the enol was added to the cell culture medium, and after incubation with the cells for 3 hours, the medium containing the aqueous solution of the hydroxylated hollow fullerene was aspirated and washed three times with PBS buffer; finally, the cell culture medium was added, and the culture was continued in the cell culture incubator. For 24 h, cell viability was then measured using CCK-8.
  • Fig. 30 The corresponding test results are shown in Fig. 30. It can be seen from Fig. 30 that when 20 ⁇ L of a hydrogen peroxide solution having a concentration of 100 ⁇ M is added, hydrogen peroxide can exert a certain killing effect on mouse bone marrow cells, with a hydroxylated hollow fullerene aqueous solution. When the amount of addition is gradually increased, the activity of the cells is also increased, indicating that at the cellular level, the aqueous solution of hydroxylated hollow fullerene has an effect on the scavenging of free radicals and can protect cells from being affected. At the same time, the positive control group without the addition of hydrogen peroxide and only the hydroxylated hollow fullerene aqueous solution was slightly higher than the negative control group, indicating that the material was not cytotoxic.
  • Example 19 Myelosuppressive protection of water-soluble hydroxylated hollow fullerenes at the living level
  • mice were randomly divided into 4 groups, 6 in each group, corresponding to blank control group, water-soluble hydroxylated hollow fullerene experimental group, X-ray irradiation experimental group and X Ray + water-soluble hydroxylated hollow fullerene experimental group.
  • 10 6 mouse hepatoma cells H22 cells were inoculated, and after 5-7 days of inoculation, the tumor was about 5 mm in diameter, and the experiment was performed.
  • the blank control group the drugs injected in the experimental group were all replaced with the same volume of physiological saline, and the same volume of physiological saline was injected into the vein and the abdominal cavity.
  • Water-soluble hydroxylated hollow fullerene experimental group The mice were injected with water-soluble hydroxylated hollow fullerene aqueous solution (1 mM) in the tail vein, and the dosage of the drug was 0.004 mmol of water-soluble hydroxylated hollow fullerene/kg of mouse body weight.
  • X-ray irradiation experimental group The whole body was irradiated with X-rays at a dose of 6 Gy.
  • X-ray + water-soluble hydroxylated hollow fullerene experimental group whole body one-time irradiation X-ray, the irradiation dose is 6Gy, intravenous injection of water-soluble hydroxylated hollow fullerene solution, the dosage of the drug is 0.004mmol water-soluble hydroxyl Hollow fullerene / kg mouse body weight.
  • the drug was injected.
  • the water-soluble hydroxylated hollow fullerenes were intravenously injected for 5 consecutive days, respectively on the fourth day, the seventh day, and the tenth day.
  • blood was collected from the eyelids of the mice (20 ⁇ l), placed in a 3 ml centrifuge tube, and blood routine was detected by an automatic blood cell analyzer.
  • the main indicator related to myelosuppression was white blood cell count (WBC). , platelet count (PLT), hemoglobin measurement (HGB), monocyte ratio (MO%).
  • the indexes related to myelosuppression in the mice in the X-ray experimental group are: white blood cells, platelets, and hemoglobin in mice.
  • Example 20 Myelosuppressive protection of water-soluble hydroxylated hollow fullerenes at the living level
  • mice were randomly divided into 4 groups, 6 in each group, corresponding to blank control group, water-soluble hydroxylated hollow fullerene experimental group, cyclophosphamide (CTX) experimental group. And CTX+ water-soluble hydroxylated hollow fullerene experimental group.
  • 10 6 mouse hepatoma cells H22 cells were inoculated, and after 5-7 days of inoculation, the tumor was about 5 mm in diameter, and the experiment was performed.
  • the blank control group the drugs injected in the experimental group were all replaced with the same volume of physiological saline, and the same volume of physiological saline was injected into the vein and the abdominal cavity.
  • Water-soluble hydroxylated hollow fullerene experimental group The mice were injected with water-soluble hydroxylated hollow fullerene aqueous solution (1 mM) in the tail vein, and the dosage of the drug was 0.004 mmol of water-soluble hydroxylated hollow fullerene/kg of mouse body weight.
  • CTX Cyclophosphamide
  • CTX+ water-soluble hydroxylated hollow fullerene experimental group rats were intraperitoneally injected with CTX solution, the dosage of the drug was 60mg/kg mouse body weight, intravenous injection of water-soluble hydroxylated hollow fullerene solution, the dosage of the drug was 0.004mmol water-soluble hydroxylated hollow Fullerene / kg mouse body weight.
  • the drug was injected.
  • CTX or intravenous injection of water-soluble hydroxylated hollow fullerenes was given for 5 consecutive days, respectively, on the fourth day, the seventh day.
  • blood was taken from the eyelids of the mice (20 ⁇ l), placed in a 3 ml centrifuge tube, and the blood routine was detected by an automatic blood cell analyzer.
  • the main indicator related to myelosuppression was white blood cells.
  • Fig. 32 The corresponding test results are shown in Fig. 32. From Fig. 32, it can be seen that compared with the blank control group, the indexes related to myelosuppression in the mice in the cyclophosphamide (CTX) experimental group are: white blood cells, platelets, and hemoglobin.
  • CTX cyclophosphamide
  • monocytes The value of monocytes is closer to the normal group, and with the prolongation of time, the relevant indicators are getting closer to the values of normal mice, indicating that the water-soluble hydroxylated hollow fullerenes are small for the chemotherapy drug CTX.
  • Murine myelosuppression has a significant protective effect.

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Abstract

富勒烯/金属富勒烯微纳材料在制备药物中的应用，所述药物具有如下性质中的至少一种：1) 治疗骨髓抑制；2）治疗至少一种由于骨髓抑制导致的白细胞、血小板、血红蛋白和单核细胞下降；3）保护骨髓细胞和/或造血细胞；4）能富集于骨髓；5）保护肝、脾和肾；6）清除自由基；7）提升机体的抗氧化系统的功能。

Description

[根据细则37.2由ISA制定的发明名称]　富勒烯/金属富勒烯在制备药物中的应用
交叉引用
本发明要求北京福纳康生物技术有限公司和中国科学院化学研究所向中国专利局提交的、申请号为CN201610041914.1、发明名称为“预防和/或治疗骨髓抑制的富勒烯微纳材料及其应用”的中国专利申请的优先权，该申请的全部内容通过引用结合在本发明中。
本发明还要求中国科学院化学研究所和北京福纳康生物技术有限公司向中国专利局提交的、申请号为CN201610656821.X、发明名称为“预防和/或治疗骨髓抑制的微纳材料及其应用”的中国专利申请的优先权，该申请的全部内容通过引用结合在本发明中。
技术领域
本发明属于生物医药领域，具体涉及一种富勒烯微纳材料和/或金属富勒烯微纳材料在制备治疗骨髓抑制的药物中的应用。
背景技术
近年来恶性肿瘤的发病率越来越高，严重危害着人们的健康。虽然随着科技进步，新兴了多种治疗癌症的方法和手段，但是采用化疗药物治疗以及放疗射线治疗仍然是恶性肿瘤临床常用的治疗方法之一。目前60％-70％的肿瘤病人需要采用化疗药物治疗或放疗射线治疗，一般在肿瘤切除手术后2-4周进行合理的放疗或化疗可以及时杀灭“转移灶”，有效抵制肿瘤的复发和转移。然而，大多数化疗药物以及放疗射线在杀伤癌细胞的同时，亦损伤人体中增生活跃的正常细胞，因此会产生很大的副作用。尤其是对于骨髓细胞、造血干细胞等，化疗药物以及放疗射线的杀伤作用会对病人产生二次伤害，放化疗引起的骨髓抑制最初表现为白细胞、血小板下降，严重时红细胞和血红蛋白均下降，甚至可能发生再生障碍性贫血、白血病等疾病。因此放化疗能否坚持的关键因素之一是患者的骨髓造血功能在放化疗过程中能否得到保 护、放化疗引起的骨髓抑制能否得到缓解。在防治骨髓抑制中，不少的中西医药物虽具有一定的疗效，但由于在人体胃肠道内的吸收率低下，并且易造成胃肠道不良反应，因而限制了该类药物的应用；另一种有效的治疗方法是利用重组人粒细胞集落刺激因子，但因价格昂贵及自身缺陷，目前尚不能广泛使用。因此，寻找能够减弱化疗药物以及放疗射线造成的骨髓抑制毒性的理想药物对于减轻患者病情、进一步治疗肿瘤都具有重要临床意义。
富勒烯是除石墨、金刚石和无定型碳之外碳元素的另一种同素异形体。这类物质指的是由碳原子组成的笼状结构，包括C60、C70、C76、C78、C82和C84等。另外由于富勒烯的碳笼内部为空腔结构，因此其内部空腔可内嵌不同原子、离子或原子簇，称之为金属富勒烯，如La@C60，表示La内嵌在C60的笼状结构中，@表示at，形象的表达了内嵌的含义。
公开于该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
发明内容
本发明的目的包括提供一种富勒烯微纳材料和/或金属富勒烯微纳材料在制备治疗骨髓抑制的药物中的应用，本发明的目的还包括提供一种治疗骨髓抑制的药物组合物及方法。本发明富勒烯微纳材料和/或金属富勒烯微纳材料保持了富勒烯分子的高度共轭性以及高效清除自由基等特点，可高效快速地富集于骨髓中，在体内对于骨髓抑制具有优异的预防和治疗的作用，可以有效降低放疗射线和化疗药物对于骨髓以及其他器官的毒副作用，治疗由于骨髓抑制导致的白细胞下降、血小板下降、血红蛋白下降和单核细胞下降，且安全无毒，可以代谢出生物体外。
为了实现目的，本发明提供了以下技术方案：
一种富勒烯微纳材料和/或金属富勒烯微纳材料在制备药物中的应用，其中所述药物具有如下1)-7)性质中的至少一种：
1)治疗骨髓抑制；2)治疗由于骨髓抑制导致的白细胞下降、血小板下降、 血红蛋白下降和单核细胞下降中的至少一种；3)保护骨髓细胞和/或造血细胞；4)能富集于骨髓中；5)保护肝脏组织、脾脏组织和肾脏组织；6)清除自由基；7)提升机体的抗氧化系统的功能。
本发明还提供了一种治疗骨髓抑制的方法，包括如下步骤：向需要治疗骨髓抑制的生物体内施用有效剂量的富勒烯微纳材料和/或金属富勒烯微纳材料。
本发明还提供了一种治疗骨髓抑制的药物组合物，其包括富勒烯微纳材料和/或金属富勒烯微纳材料，还包括可药用的载体、可药用的稀释剂和可药用的赋形剂中的至少一种。
上述应用、方法或药物组合物在另一种实施方式中，所述富勒烯微纳材料和/或金属富勒烯微纳材料包括至少一种选自下组的有效成分：油溶性富勒烯微纳材料、油溶性金属富勒烯微纳材料、所述油溶性富勒烯微纳材料和所述油溶性金属富勒烯微纳材料的组合物、水溶性富勒烯微纳材料、水溶性金属富勒烯微纳材料、所述水溶性富勒烯微纳材料和所述水溶性金属富勒烯微纳材料的组合物、以上六者的可药用的酯或以上六者的可药用的盐。
上述应用、方法或药物组合物在另一种实施方式中，所述油溶性富勒烯微纳材料由富勒烯本体材料经油溶性修饰得到，所述油溶性金属富勒烯微纳材料由金属富勒烯本体材料经油溶性修饰得到。
上述应用、方法或药物组合物在另一种实施方式中，油溶性修饰的有效途径是通过共价或非共价作用将富勒烯本体材料和/或金属富勒烯本体材料经油溶性修饰得到，具体的油溶性修饰方法均可按照现有技术公开的方法进行。可选的如：用食用油包覆所述富勒烯本体材料和/或金属富勒烯本体材料得到所述油溶性富勒烯微纳材料和/或油溶性金属富勒烯微纳材料，具体包覆的方式可采用球磨或超声的方式，即：将所述富勒烯本体材料和/或金属富勒烯本体材料(均为粉末状)和所述食用油混合，并将所得混合物超声或在球磨机内球磨一定时间，即可制备得到油溶性富勒烯微纳材料和/或油溶性金属富勒烯微纳材料。
上述应用、方法或药物组合物在另一种实施方式中，所述混合物超声或在球磨机内球磨一定时间后，还包括对所得油溶性富勒烯微纳材料和/或油溶 性金属富勒烯微纳材料提纯的步骤：依次离心去除沉淀，然后所得上层液过滤，即得。
上述应用、方法或药物组合物在另一种实施方式中，超声或球磨的混合物中，每1ml所述食用油中分散0.05-500mg富勒烯本体材料和/或金属富勒烯本体材料，该范围的公开应当被视为是范围内所有数值的公开，可选的有0.05-1mg，0.05-10mg，0.05-100mg等。
上述应用、方法或药物组合物在另一种实施方式中，所述混合物经球磨或超声30min-15h。
上述应用、方法或药物组合物在另一种实施方式中，所述油溶性富勒烯微纳材料和/或油溶性金属富勒烯微纳材料中，富勒烯本体材料和/或金属富勒烯本体材料的浓度可为1200～1500ppm(mg/kg)，如1500ppm。
上述应用、方法或药物组合物在另一种实施方式中，所述食用油包括但不限于橄榄油、亚麻籽油、葵花籽油、玉米胚油、玉米油和角鲨烷中的至少一种。
上述应用、方法或药物组合物在另一种实施方式中，所述油溶性富勒烯微纳材料和/或油溶性金属富勒烯微纳材料具体可为食用油包覆钆金属富勒烯(如：食用油包覆Gd@C82)、食用油包覆C60、食用油包覆C70或食用油包覆C84等，其中的“食用油”均指的是橄榄油、亚麻籽油、葵花籽油、玉米胚油、玉米油和角鲨烷中任一种。
由上述叙述可以看出，所述油溶性富勒烯微纳材料和/或油溶性金属富勒烯微纳材料具备下述所有性质：(1)富勒烯本体材料和/或金属富勒烯本体材料被所述食用油包覆，其脂溶性特性，使其更容易进入细胞并能够在生物体内并通过血液循环达到脏器；(2)包覆得到的材料中富勒烯和/或金属富勒烯保持了碳笼的完整性，具有良好的自由基清除效果。
上述应用、方法或药物组合物在另一种实施方式中，本发明所述的水溶性富勒烯微纳材料是由富勒烯本体材料经水溶性修饰得到的，所述水溶性金属富勒烯微纳材料是由金属富勒烯本体材料经水溶性修饰得到。
上述应用、方法或药物组合物在另一种实施方式中，水溶性修饰可按照 现有技术公开的方法进行修饰。可选的所述水溶性修饰的方法包括以下方法中的任一种：方法1，共价修饰使本体材料表面修饰亲水基团的方法一般在碱的作用下通过固液或者液液反应实现，具体为将富勒烯本体材料和/或金属富勒烯本体材料与双氧水和碱(该碱具体为氢氧化钠或者氢氧化钾)混合并进行反应，即可得到水溶性富勒烯微纳材料和/或水溶性金属富勒烯微纳材料，该微纳材料是被羟基修饰的。如果需要获得被氨基修饰的微纳材料，将上述步骤中的氢氧化钠和/或氢氧化钾替换成氨水即可。方法2，非共价作用通过疏水-疏水作用使水溶性载体与富勒烯本体材料和/或金属富勒烯本体材料形成相应的水溶性材料。
上述应用、方法或药物组合物在另一种实施方式中，富勒烯本体材料和/或金属富勒烯本体材料与双氧水和碱混合并进行反应后还包括以下提纯步骤：用乙醇洗涤，然后透析。
上述应用、方法或药物组合物在另一种实施方式中，称取50～300mg的C60或C70或Gd@C82固体，5～30ml 20～40％的双氧水，2～20ml 1M-3M的碱溶液，在50～100℃的条件下混合，至相应C60或C70或Gd@C82固体全部溶解。在此描述中，表现的是各物质之间的添加比例关系，实际应用中并不受50～300mg、5～30ml和2～20ml等具体反应规模的限制，可按照比例进行扩大。
上述应用、方法或药物组合物在另一种实施方式中，所述水溶性富勒烯微纳材料和/或水溶性金属富勒烯微纳材料包括选自下组的至少一种：碳笼外表面修饰有亲水基团的富勒烯、碳笼外表面修饰有亲水基团的金属富勒烯、被水溶性载体负载的富勒烯、被水溶性载体负载的金属富勒烯。这里被修饰、被负载的富勒烯和/或金属富勒烯，就是指将富勒烯本体材料和/或金属富勒烯本体材料经过修饰或负载后所得的水溶性富勒烯微纳材料和/或水溶性金属富勒烯微纳材料。
上述应用、方法或药物组合物在另一种实施方式中，所述亲水基团包括羟基、羧基、巯基和氨基中的一种或多种。
上述应用、方法或药物组合物在另一种实施方式中，所述水溶性富勒烯 微纳材料和/或水溶性金属富勒烯微纳材料具体可为水溶性羟基化钆金属富勒烯(GFNC、Gd@C82(OH)n)，以及水溶性羟基化C60(C60(OH)n)或水溶性羟基化C70(C70(OH)n)等水溶性羟基化富勒烯，
上述应用、方法或药物组合物在另一种实施方式中，所述水溶性载体为医学中常用的药物载体，包括脂质体、聚合物胶束、蛋白质中的至少一种。可选的，所述聚合物胶束为聚乙丙交酯聚乙二醇(PEG-PLGA)、聚赖氨酸或壳聚糖；所述蛋白质为白蛋白或转铁蛋白。
上述应用、方法或药物组合物在另一种实施方式中，所述水溶性富勒烯微纳材料和/或水溶性金属富勒烯微纳材料具备下述所有性质：(1)表面为亲水性，使其能够经由静脉注入到生物体内并通过血液循环富集于骨髓中；(2)微纳材料具有刚性(即不易变形)，使其经血液循环可以通过骨髓血窦的内皮细胞间隙、利用血管内外压力差快速进入骨髓中。
上述的应用、方法或药物组合物在另一种实施方式中，所述骨髓抑制由药物化疗、化学毒物或具有骨髓抑制副作用的药物诱导产生。可选的，所述药物化疗包括目前临床上常规使用的对骨髓具有抑制作用的药物，可为环磷酰胺(CTX)、阿霉素、顺铂或紫杉醇；可选的，所述化学毒物可为苯或苯的衍生物(如苯丁酸氮芥)；可选的，所述具有骨髓抑制副作用的药物可为氯霉素、四环素、他巴哇或消炎痛。
上述的应用、方法或药物组合物在另一种实施方式中，所述骨髓抑制由肿瘤放射疗法或产生骨髓抑制副作用的射线诱导产生。可选的，放射疗法利用的射线可为α射线、β射线、γ射线或X射线；可选的，产生骨髓抑制副作用的射线可为由于从事相关工作不可避免的接触到的放射线。
上述应用、方法或药物组合物在另一种实施方式中，所述富勒烯本体材料包括一种或多种通式为C2m的由碳原子组成的笼状结构，30≤m≤60，例如；C60，C70，C84等。
上述应用、方法或药物组合物在另一种实施方式中，所述金属富勒烯本体材料包括M@C2n、M2@C2n、MA@C2n、M3N@C2n、M2C2@C2n、M2S@C2n、M2O@C2n和MxA3-xN@C2n中的一种或多种，其中：M、A均代 表金属元素且M、A均选自镧系金属元素、Sc和Y中的任意一种，30≤n≤60；0≤x≤3。N代表氮元素，C代表碳元素，S代表硫元素，镧系金属元素包括La、Ce、Pr、Nd、Pm、Sm、Eu、Gd、Tb、Dy、Ho、Er、Tm、Yb和Lu，如：Gd@C82。
上述应用、方法或药物组合物在另一种实施方式中，所述水溶性富勒烯微纳材料和/或水溶性金属富勒烯微纳材料的平均颗粒尺寸范围为1-500nm，具体可为140-200nm。
上述应用、方法或药物组合物在另一种实施方式中，所述油溶性富勒烯微纳材料和/或水溶性金属富勒烯微纳材料的平均颗粒尺寸范围为0.5-50nm，具体可为0.5-10nm，0.5-5nm，0.5-2nm或约1nm。
上述应用中的药物或上述药物组合物在另一种实施方式中，该药物或药物组合物可以是片剂、丸剂、散剂、锭剂、小药囊、扁囊剂、酏剂、悬浮剂、乳剂、溶液剂、糖浆剂、气溶胶、软膏、软和硬明胶胶囊、栓剂、无菌注射溶液或无菌包装粉针剂的制剂。本发明中将有效成分制备成药物或药物组合物的方法可采用本领域普通技术人员公知的方法来制备，使其在施用于受试者后速释、缓释或延迟释放有效成分，例如：有效成分可以与载体混合，用载体稀释或者包封在载体中。
上述应用中的药物或上述药物组合物在另一种实施方式中，适宜作为载体、赋形剂和稀释剂的一些实例包括乳糖、右旋糖、蔗糖、山梨醇、甘露醇、淀粉、树脂、阿拉伯胶、磷酸钙、海藻酸盐、西黄蓍胶、明胶、硅酸钙、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水糖浆(water syrup)、甲基纤维素、尼泊金甲酯和丙酯、滑石粉、硬脂酸镁和液状石蜡。
上述应用中的药物或上述药物组合物在另一种实施方式中，该药物或药物组合物还可以另外包括润滑剂、润湿剂、乳化和悬浮剂、防腐剂、甜味剂或矫味剂等助剂。
上述应用中的药物或上述药物组合物在另一种实施方式中，所述水溶性富勒烯微纳材料和/或水溶性金属富勒烯微纳材料在制剂中的浓度为0.1 mM-10mM；所述油溶性富勒烯和/或油溶性金属富勒烯在制剂中的浓度为300ppm-5000ppm(mg/kg)。
上述方法在另一种实施方式中，富勒烯微纳材料和/或金属富勒烯微纳材料可以在化疗药物或放疗射线使用前的24h内使用，也可以在放化疗过程中使用，也可以在放化疗结束后1-2周内持续使用。
上述方法在另一种实施方式中，所述有效成分的施用剂量为1mg/kg/d-500mg/kg/d，可选的为1-100mg/kg/d，1-20mg/kg/d，1-10mg/kg/d。
上述方法在另一种实施方式中，所述施用的方式为注射或口服；可选的，所述注射为腹腔注射或静脉注射，有效成分进入体内直接经血液循环发挥作用，无需渗透，所用的药剂量小，疗效高；也可为口服摄入，经消化系统过滤吸收，副作用更小，疗效显著。
上述方法在另一种实施方式中，所述生物体为人或动物，动物可以为哺乳动物，如小鼠、豚鼠、大鼠、狗、兔子、猴子等。
本发明所用的术语“治疗”包括其通常被接受的含义，该含义包括阻止、预防、抑制、改善以及减缓、停止或逆转所产生症状或预期病变的发展。照此，本发明涵盖治疗性和预防性的施用。
本发明中所用的术语“有效剂量”是指当通过本发明的方法给予生物体富勒烯微纳材料和/或金属富勒烯微纳材料，足以有效传递用于防护骨髓抑制的活性成分的量。骨髓抑制较轻者可以单剂量使用，骨髓抑制严重者亦可以多剂量使用，单次或多次使用后骨髓抑制现象得到显著防治。
本发明所用的术语“有效成分”指的是水溶性富勒烯微纳材料、水溶性金属富勒烯微纳材料、水溶性富勒烯微纳材料和水溶性金属富勒烯微纳材料的组合物、油溶性富勒烯微纳材料、油溶性金属富勒烯微纳材料、油溶性富勒烯微纳材料和油溶性金属富勒烯微纳材料的组合物，及以上六者的可药用的酯和以上六者的可药用的盐中的至少一种。
本发明所用的术语“富勒烯本体材料”是指没有经过油溶性修饰或水溶性修饰的富勒烯，即富勒烯本身。
本发明所用的术语“金属富勒烯本体材料”是指没有经过油溶性修饰或水溶性修饰的金属富勒烯，即金属富勒烯本身。
为了方便计量，本发明中所有关于水溶性富勒烯微纳材料、水溶性金属富勒烯微纳材料、油溶性富勒烯微纳材料和油溶性金属富勒烯微纳材料的具体含量、浓度等定量的限定均是以其对应的富勒烯本体材料或金属富勒烯本体材料的具体含量、浓度等来衡量的，例如：有效成分的施用剂量为1mg/kg/d-500mg/kg/d是指每1天每1kg的小鼠要施用的有效成分中对应的富勒烯本体碳笼或金属富勒烯本体碳笼的量为1mg-500mg。
与现有技术相比，本发明的有益效果包括：
1、本发明中富勒烯微纳材料和/或金属富勒烯微纳材料保持了富勒烯分子的高度共轭性以及高效清除自由基等特点。
2、本发明中富勒烯微纳材料和/或金属富勒烯微纳材料由于特殊的物理化学性质，其可以通过血液循环穿过骨髓血窦中内皮细胞间的纳米孔隙并利用血管内外压力差高效快速地富集于脏器、骨髓中。富勒烯微纳材料和/或金属富勒烯微纳材料在骨髓中的单位质量含量是骨质中的12倍之多，在骨髓中的停留时间明显高于在其他器官中的时间，说明其具有良好的骨髓靶向性。
3、本发明中富勒烯微纳材料和/或金属富勒烯微纳材料可以高效的清除自由基，在体内对于骨髓抑制具有优异的预防和治疗的作用，可以有效降低放疗射线和化疗药物对于骨髓以及其他器官的毒副作用，治疗由于骨髓抑制导致的白细胞下降、血小板下降、血红蛋白下降和单核细胞下降，且多次疗程后，保护效果仍然比较明显，并且富勒烯微纳材料和/或金属富勒烯微纳材料不影响放疗和化疗药物对于肿瘤的治疗。因此本发明中富勒烯微纳材料和/或金属富勒烯微纳材料可以用于化疗药物或放疗射线等原因引起的骨髓抑制的预防和治疗，可以用于制备治疗骨髓抑制的药物。
4、本发明中富勒烯微纳材料和/或金属富勒烯微纳材料制备简单快速，对活体、骨髓细胞及正常细胞没有明显的细胞毒性，安全无毒；可以代谢出生物体外，具有良好的生物相容性。
5、本发明中富勒烯微纳材料和/或金属富勒烯微纳材料还可以保护肝 脏、肾脏和脾脏等的损伤。
附图说明
图1A-图1D为本发明实施例2中不同组别小鼠血常规指标。
图2为本发明实施例2中不同组别小鼠体重变化曲线。
图3A-图3D为本发明实施例2中不同组别小鼠相关酶指标。
图4A-图4D为本发明实施例3中不同组别小鼠血常规指标。
图5为本发明实施例3中不同组别小鼠体重变化曲线。
图6A-图6D为本发明实施例3中不同组别小鼠相关酶指标。
图7A-图7D为本发明实施例3中不同组别小鼠血生化指标。
图8为实施例4中制备的水溶性羟基化钆金属富勒烯粒径分布图。
图9为实施例5中使用ICP-MS测定水溶性羟基化钆金属富勒烯在小鼠体内代谢分布图。
图10为实施例6中使用131I标记法测定水溶性羟基化钆金属富勒烯在小鼠体内代谢分布图。
图11A-F为实施例7中对水溶性羟基化钆金属富勒烯样品在不同时间点小鼠骨髓中的电子探针谱图。
图12为实施例8中注射水溶性羟基化钆金属富勒烯组和对照组小鼠脏器系数，均为注射30d后的值。
图13为实施例8中注射水溶性羟基化钆金属富勒烯组和对照组小鼠体重变化。
图14为实施例8中注射水溶性羟基化钆金属富勒烯组小鼠血生化测试，均为注射30d后的值。
图15为实施例9中GFNC体外ESR清除自由基实验的结果，其中，实线为对照组；虚线为实验组。
图16为实施例10中GFNC在细胞水平清除自由基实验效果。
图17为实施例11中不同组别小鼠血常规指标。
图18为实施例12中不同组别小鼠肿瘤大小变化曲线。
图19为实施例12中不同组别小鼠体重变化曲线。
图20为实施例13中不同组别小鼠血常规指标。
图21为实施例14中不同组别小鼠肿瘤大小变化曲线。
图22为实施例14中不同组别小鼠体重变化曲线。
图23为实施例14中不同组别小鼠血生化指标。
图24为实施例14中不同组别小鼠组织器官病理切片经H&E染色后的切片图像。
图25为实施例15中制备的水溶性羟基化空心富勒烯粒径分布图。
图26为实施例16中注射水溶性羟基化空心富勒烯组和对照组小鼠脏器系数，均为注射30d后的值。
图27为实施例16中注射水溶性羟基化空心富勒烯组和对照组小鼠体重变化。
图28为实施例16中注射水溶性羟基化空心富勒烯组小鼠血生化测试，均为注射30d后的值。
图29为实施例17中水溶性空心富勒烯体外ESR清除自由基实验的结果，其中，实线为对照组；虚线为实验组。
图30为实施例18中C60水溶性衍生物在细胞水平清除自由基实验效果。
图31为实施例19中使用C60水溶性衍生物不同组别小鼠血常规指标。
图32为实施例20中使用C60水溶性衍生物不同组别小鼠血常规指标。
本发明图9、图10、图17、图20和图32的柱状图图例的从上到下的顺序均是根据柱状图从左到右的顺序依次排列的。
具体实施方式
下面结合附图通过具体实施例对本发明进行说明，但本发明并不局限于下面的实施例中。
本发明实施例中所使用的富勒烯固体粉末C60、C70和金属富勒烯固体粉末Gd@C82均购买于厦门福纳新材料科技有限公司，纯度99％；所使用的环磷酰胺(CTX)购买于Sigma-Aldrich公司，货号为C0768；所使用的X射线治疗仪是北京朝阳医院放疗科所用产品。其它所使用的材料、试剂、仪器等如无特殊说明，均可从商业途径得到；其它实验方法如无特殊说明，均为常规方法。
实施例1、油包覆空心富勒烯和/或油包覆金属富勒烯的制备
1)将富勒烯固体粉末和/或金属富勒烯固体粉末以及橄榄油等食用油以不同比例混合，在球磨机中球磨6-10h；
2)球磨完成后使用离心机，用转速10000r反复离心去除未包覆成功的富勒烯固体粉末和/或金属富勒烯固体粉末，直到无固体粉末被离心下来；即得油包覆空心富勒烯和/或油包覆金属富勒烯。
3)使用紫外-可见光谱仪测试所得油包覆空心富勒烯和/或油包覆金属富勒烯中包覆成功的空心富勒烯固体粉末和/或金属富勒烯固体粉末的具体含量。
在上述方法的一种具体实施例中，将100mg空心富勒烯C60固体粉末和100ml橄榄油混合球磨6h，用转速10000r反复离心去除未包覆成功的空心富勒烯C60固体粉末后，制备得到橄榄油包覆空心富勒烯(以下简称为Olive-C60)。经检测Olive-C60中空心富勒烯C60固体粉末的含量为1500ppm(mg/kg)，即每1kg Olive-C60中有1500mg空心富勒烯C60固体粉末。
实施例2、Olive-C60在活体水平第一疗程中对骨髓抑制和相关酶活性的作用
动物模型：选用4-5周ICR小鼠，将其随机分为5组，每组6只，分别对应环磷酰胺(CTX)+Olive-C60灌胃给药实验组、CTX+Olive-C60腹腔给药实验组、CTX+Olive灌胃给药实验组、CTX实验组和空白对照组。
环磷酰胺(CTX)+Olive-C60灌胃给药实验组：鼠腹腔注射CTX溶液，药 物用量为60mg/kg小鼠体重，灌胃给药Olive-C60溶液，药物用量为1500ppm，100μL。
环磷酰胺(CTX)+Olive-C60腹腔给药实验组：鼠腹腔注射CTX溶液，药物用量为60mg/kg小鼠体重，腹腔给药Olive-C60溶液，药物用量为1500ppm，100μL。
环磷酰胺(CTX)+Olive灌胃给药实验组：鼠腹腔注射CTX溶液，药物用量为60mg/kg小鼠体重，灌胃纯橄榄油Olive，药物用量为100μL。
环磷酰胺(CTX)实验组：鼠腹腔注射CTX溶液，药物用量为60mg/kg小鼠体重，灌胃100μL生理盐水，以消除灌胃刺激对小鼠影响的差别。
空白对照组：实验组所注射的药物均用同体积的生理盐水所代替，灌胃和腹腔均注射同等体积的生理盐水。
4-5周ICR小鼠在实验室开始饲养的第七天、小鼠状态稳定后开始注射药物，作为开始实验的第一天，每天一次，连续5天腹腔注射CTX同时灌胃或腹腔给药Olive-C60或者Olive，5天之后停止注射，只进行观察或取样。
Olive-C60在活体水平第一疗程中对骨髓抑制的作用：分别在第四天、第七天、第十天和第十七天，从小鼠眼眶取血(13μl)，置于3ml离心管中，用血细胞自动分析仪检测血常规，其中和骨髓抑制相关的主要指标为白细胞计数(WBC)、血小板计数(PLT)、红细胞计数(RBC)和血红蛋白测定(HGB)。同时监测小鼠体重变化。
相应的检测结果如图1A-图1D所示，从图1A-图1D可得知：与空白对照组相比，环磷酰胺(CTX)实验组中的小鼠中与骨髓抑制相关的指标：白细胞、红细胞、血小板和血红蛋白在小鼠体内都有着不同程度的减少，其中以白细胞的减少最为明显，说明其骨髓受到损伤，相关指标都有明显的异常；而CTX+Olive-C60实验组中的小鼠，无论是腹腔还是灌胃给药，由于C60的清除自由基效果和保护作用，其白细胞计数水平相较于环磷酰胺(CTX)实验组都有着很大程度的提高，而红细胞、血小板和血红蛋白的数值也都更接近于正常组，并且随着时间的延长，相关指标越来越接近于正常小鼠的值。对于体重的监测在打药初期，由于CTX的毒副作用，不同组别小鼠的体重均有下降，但在停止打药后，Olive-C60实验组的小鼠体重恢复较CTX组更快一些，也 更接近正常小鼠体重水平。
上述数据均表明：Olive-C60对于化疗药物CTX所导致的小鼠骨髓抑制有明显的保护效果，并且不同的给药方式，保护效果都很明显。同时从图2中可以看出，在实验后期，环磷酰胺组小鼠的体重是相对其他组最低的，也说明了Olive-C60对于小鼠整体生存质量的保护和提升作用。
Olive-C60在活体水平第一疗程中对相关酶活性的作用：在实验的第18天，眼眶取全血，离心取血清，测试不同相关酶活性。
相应的检测结果如图3A-图3D所示，从图3A-图3D可得知：在治疗过程中，注射了橄榄油-C60的小鼠体内与抗氧化相关的酶(SOD、CAT和GPx)等的活性均高于仅注射橄榄油的实验组，更是比仅给药环磷酰胺的小鼠酶活性高很多。由于机体内的过氧化物主要由抗氧化系统通过自身的氧化来清除，抗氧化系统对机体免疫力的提高，抗氧化损伤防护具有十分重要的作用，而抗氧化酶的活性降低会导致机体受损加重，因此保护机体的抗氧化系统对于减轻化疗带来的损伤有着十分重要的意义。在本实施例中，可以明显发现，Olive-C60的摄入对于机体的抗氧化系统有着明显的保护效果，从而保护了化疗带来的进一步的伤害。而MDA是由自由基引起的脂质氧化而产生的，体内MDA水平的高低也代表了体内自由基水平的高低，MDA水平升高表明体内有过多的未能及时被清除的自由基，同时也说明了细胞膜系统损伤加剧。在本实施例中，Olive-C60可以通过清除羟基自由基等自由基水平，减少机体细胞膜的系统损伤，保护机体不受环磷酰胺等化疗药物的二次伤害。
实施例3、Olive-C60在活体水平第二疗程中对骨髓抑制、相关酶活性和血生化指标的作用
动物模型：选用4-5周ICR小鼠，将其随机分为5组，每组6只，分别对应环磷酰胺(CTX)+Olive-C60灌胃给药实验组、CTX+Olive-C60腹腔给药实验组、CTX+Olive灌胃给药实验组、CTX实验组和空白对照组。
环磷酰胺(CTX)+Olive-C60灌胃给药实验组：鼠腹腔注射CTX溶液，药物用量为60mg/kg小鼠体重，灌胃给药Olive-C60溶液，药物用量为1500ppm，100μL。
环磷酰胺(CTX)+Olive-C60腹腔给药实验组：鼠腹腔注射CTX溶液，药 物用量为60mg/kg小鼠体重，腹腔给药Olive-C60溶液，药物用量为1500ppm，100μL。
环磷酰胺(CTX)+Olive灌胃给药实验组：鼠腹腔注射CTX溶液，药物用量为60mg/kg小鼠体重，灌胃纯橄榄油Olive，药物用量为100μL。
环磷酰胺(CTX)实验组：小鼠腹腔注射CTX溶液，药物用量为60mg/kg小鼠体重。同时灌胃100μL生理盐水，以消除灌胃刺激对小鼠影响的差别。
空白对照组：实验组所注射的药物均用同体积的生理盐水所代替，灌胃和腹腔均注射同等体积的生理盐水。
于第一疗程实验开始的第20天，小鼠基本恢复后，开始第二疗程实验，即第一疗程的21天作为第二疗程开始的第一天，之后每天一次，连续5天腹腔注射CTX同时灌胃/腹腔给药Olive-C60或者Olive，5天后停止给药，只进行观察或取样。
Olive-C60在活体水平第二疗程中对骨髓抑制的作用：分别在第二疗程的第四天，第七天，第十天和第十七天，从小鼠眼眶取血(13μl)，置于3ml离心管中，用血细胞自动分析仪检测血常规，其中和骨髓抑制相关的主要指标为白细胞计数(WBC)、血小板计数(PLT)、红细胞计数(RBC)和血红蛋白测定(HGB)。同时监测小鼠体重变化。
相应的检测结果如图4A-图4D所示，从图4A-图4D可得知：在第二疗程用药过程中，与空白对照组相比，环磷酰胺(CTX)实验组中的小鼠中与骨髓抑制相关的指标：白细胞、红细胞、血小板和血红蛋白在小鼠体内都有着不同程度的减少，其中以白细胞的减少最为明显，说明其骨髓受到二次损伤，相关指标都有明显的异常；而CTX+Olive-C60实验组中的小鼠，无论是腹腔还是灌胃给药，由于C60的清除自由基效果和保护作用，其白细胞计数水平相较于环磷酰胺(CTX)实验组都有着很大程度的提高，而红细胞、血小板和血红蛋白的数值也都更接近于正常组，并且随着时间的延长，相关指标越来越接近于正常小鼠的值，在第十七天，给药组小鼠的白细胞几乎恢复到正常值，而CTX组小鼠的白细胞仍在一个低于正常值的水平。同时对于小鼠体重的检测可以看出，打药组小鼠体重更接近空白对照组，而CTX组小鼠的体重同其他组别有较大的差距，
上述结果均表明：Olive-C60对于CTX在多疗程用药过程中所导致的小鼠骨髓抑制有明显的保护效果，使得小鼠的造血水平更快的恢复到正常，并且不同的给药方式，保护效果都很明显。同时从图5中可以看出，在实验后期，环磷酰胺组小鼠的体重是相对其他组明显是最低的，也说明了Olive-C60对于小鼠整体生存质量的保护和提升作用。
Olive-C60在活体水平第二疗程中对相关酶活性的作用：在第二疗程实验的第18天，眼眶取全血，离心取血清，测试不同相关酶活性。
相应的检测结果如图6A-图6D所示，从图6A-图6D可得知：在多疗程治疗过程完成后，注射了橄榄油-C60的小鼠体内与抗氧化相关的酶(SOD、CAT和GPx)等的活性也均略高于仅注射橄榄油的实验组，同时高于给药环磷酰胺的小鼠酶活性。同上述，机体内的过氧化物主要由抗氧化系统通过自身的氧化来清除，抗氧化系统对机体免疫力的提高，抗氧化损伤防护具有十分重要的作用，而抗氧化酶的活性降低会导致机体受损加重，因此保护机体的抗氧化系统对于减轻化疗带来的损伤有着十分重要的意义。在本实施例中，可以明显发现，Olive-C60的摄入对于机体的抗氧化系统有着明显的保护效果，从而保护了化疗带来的进一步的伤害。而MDA是由自由基引起的脂质氧化而产生的，体内MDA水平的高低也代表了体内自由基水平的高低，MDA水平升高表面体内有过多的未能及时被清除的自由基，同时也说明了细胞膜系统损伤加剧。在多次疗程之后，环磷酰胺用药组的MDA水平高于正常对照组，而灌胃给药的小鼠MDA水平甚至低于正常对照组，说明橄榄油-C60清除自由基效果显著。在本实施例中，Olive-C60可以通过清除羟基自由基等自由基水平，减少机体细胞膜的系统损伤，保护机体不受环磷酰胺等化疗药物的二次伤害。
Olive-C60在活体水平第二疗程中对血生化指标的作用：在第二疗程实验的第18天，眼眶取全血，离心取血清，测试血生化指标。
相应的检测结果如图7A-图7D所示，从图7A-图7D可得知：在多疗程治疗过程完成后，谷丙转氨酶、碱性磷酸酶和尿氮素的水平，不同实验组几乎都是一致的，都恢复到正常水平，值得注意的是尿酸的水平，在环磷酰胺用药组的小鼠血清中尿酸的水平远低于正常对照组，而作为还原剂的尿酸浓 度降低，会使得机体运动产生大量氧，自由基清除能力下降，其毒性导致肾功能损害，肝损伤等一系列的伤害。而灌胃或者腹腔注射了Olive-C60的小鼠血清内的尿酸含量几乎以正常对照组水平持平，说明Olive-C60很好的保护了机体的清除自由基体系，保护了机体的各脏器和功能。
实施例4、水溶性羟基化钆金属富勒烯的制备
1)将100mg Gd@C82固体粉末加入100ml的单口瓶中，分别加入7ml体积分数为30％的过氧化氢水溶液和3ml 2M的氢氧化钠水溶液，油浴加热到70℃，反应2-5h；
2)反应后使用M.W.＝3500透析袋除去小分子，使用电导率仪监测直至透析完成，浓缩得到的产物即可得到水溶性羟基化金属富勒烯，经DLS测定，其在水溶液中的平均粒径为140nm，粒径分布均一，粒径分布图如图8所示，本实施所得水溶性羟基化金属富勒烯之后简称GFNC。
实施例5、GFNC在活体内代谢分布情况
1)试验前处理：
动物模型：选用4-5周BALB/c小鼠，在右侧大腿上接种106个小鼠肝癌细胞(H22细胞)(北京协和细胞库提供)100μl，接种5-7天后，肿瘤直径达到5mm左右时，进行实验；
2)试验：
静脉注射150μL浓度为1mM的水溶性羟基化钆金属富勒烯，分别在注射后15min、30min，45min，1h、4h、24h、7d、15d和30d后脱颈处死小鼠，并当即解剖取小鼠的主要脏器(心，肝，脾，肺，肾，肠，胃等)、小鼠大脑、胫骨和肌肉。将脏器、大脑和肌肉等部位取一部分称重，将胫骨称重，之后用PBS将小鼠的胫骨骨髓冲洗出来，再次对胫骨称重，算出骨髓的质量。将以上组织消解后，使用200nm滤膜进行过滤；
3)ICP-MS的测定钆离子浓度方法进行测试：
取100ppb浓度的Gd3+标准溶液，配成0、30、50、100、200、300ppb的标准溶液样品，作为ICP-MS测试的标样，分别测得上述消解液中Gd3+的浓度，之后通过换算公式，换算出等质量的不同组织中钆离子的浓度。每组 平行实验有6只小鼠，取平均值。
测试结果如图9所示，从图9可得知：在1h之内水溶性羟基化钆金属富勒烯材料就可以在骨中有明显的富集效果，仅略低于肝脏部位的浓度，但是随着时间的延长，在骨中钆浓度可以持续在较高水平，而在30d之后，骨中材料浓度明显降低，说明其具有可代谢特性，这也与富勒烯在活体水平可代谢是相符合的。
实施例4所得水溶性羟基化钆金属富勒烯在小鼠骨髓和骨质中的相对含量如下表1所示，从表1可得知：将骨质和骨髓分离，分别测定水溶性羟基化钆金属富勒烯在小鼠骨髓和骨质的含量。在注射样品24h后，骨髓中的钆含量为5.84±0.58ng/mg，远远高于在骨质中的含量(0.45±0.044ng/mg)，进一步说明了材料具有在骨髓中靶向富集的特征。
表1、水溶性羟基化钆金属富勒烯在小鼠骨髓和骨质中的相对含量
打药后不同时间点 骨髓钆含量(纳克/毫克) 骨质钆含量(纳克/毫克)
5分钟 1.182±0.187 0.098±0.016
15分钟 1.198±0.051 0.099±0.004
30分钟 2.652±0.096 0.221±0.008
45分钟 2.679±0.047 0.223±0.004
1小时 3.521±0.298 0.293±0.025
4小时 4.621±0.109 0.385±0.009
24小时 5.388±0.528 0.449±0.044
7天 8.185±0.388 0.515±0.032
15天 5.538±0.512 0.461±0.043
30天 4.431±0.514 0.369±0.043
实施例6、放射性标记法测定GFNC活体内代谢分布情况
1)试验前处理：
动物模型：选用4-5周BALB/c小鼠，在右侧大腿上接种106个小鼠肝癌细胞(H22细胞)，接种5-7天后，肿瘤直径达到5mm左右时，进行实验。
2)碘标：
标记条件：底物：～5mg/ml，1ml；pH＝8，H3PO4/NaOH；131I：5.0MBq，20μL；氯铵-T：20μL，3mg/mL；反应温度：30℃；反应时间：3h；终止剂：Na2S2O5；标记率：～80％；放化纯度≥99％。
3)131I放射性标记方法进行测试：
水溶性羟基化钆金属富勒烯表面存在的羟基可以与131I进行置换反应，通过131I发射出的伽马射线，可以用于高灵敏度检测其在生物体内的代谢行为。具体地，首先使用氯胺-T水溶液(10mg/mL)氧化37MBq的Na 131I溶液(体积比位2∶5)10min后，加入待标记样品，常温下震荡反应2h。反应结束后，所得反应液用Sephadex G-25凝胶柱进行分离纯化，去除无机盐离子，即得放射性碘-131标记样品(放化纯度≥97％)。将50μL活化后的溶液分别经尾静脉注射于荷瘤鼠体内，分别在注射1.0h、4.0h、24h和48h后脱颈处死小鼠，解剖取出重要组织器官(心、肝、脾、肺、肾、肿瘤、脑、骨、肉等)进行伽马计数，统计各器官中进行标记的样品含量。
测试结果如图10所示，从图10可得知：和ICP离子浓度测试方法得到的结果类似，佐证了水溶性羟基化钆金属富勒烯材料可以在骨中具有明显的富集效果。同时，将小鼠骨髓从胫骨中冲出，分别测量骨髓和骨质的质量以及伽马计数的强度，从表2中可以看出，水溶性羟基化钆金属富勒烯主要是富集在骨髓中。
表2、实施例4所得水溶性羟基化钆金属富勒烯在小鼠骨髓和骨质中的相对含量
打药后不同时间点 骨髓131I放射强度(ID％/g) 骨质131I放射强度(ID％/g)
1小时 8.25±1.29 0.75±0.07
4小时 9.63±1.12 0.88±0.10
24小时 9.12±0.92 0.83±0.04
48h 4.4±0.688 0.41±0.03
实施例7、电子探针测定GFNC骨髓富集量
1)试验前处理：
动物模型：选用4-5周BALB/c小鼠，在右侧大腿上接种106个小鼠肝癌细胞(H22细胞)，接种5-7天后，肿瘤直径达到5mm左右时，进行实验。
实验组：选用4-5周BALB/c小鼠，在右侧大腿上接种106个小鼠肝癌细胞(H22细胞)，接种5-7天后，肿瘤直径达到5mm左右时，静脉注射150μL浓度为900ppb的水溶性羟基化金属富勒烯。
2)试验：
小鼠实验：静脉注射150μL浓度为1mM的水溶性羟基化钆金属富勒烯，分别在注射前，注射后0.5h、1h、4h和24h，将小鼠脱颈处死，并当即解剖取小鼠双侧胫骨。立即用PBS将小鼠的胫骨骨髓冲洗出来，并且均匀涂抹在导电胶表面，之后用电子探针仪器进行钆元素含量检测。
水溶性羟基化钆金属富勒烯样品实验：将水溶性羟基化钆金属富勒烯样品水溶液均匀涂抹在导电胶表面，待其干燥后，用电子探针仪器进行测试。
相应的测试结果如图11A-F所示，从图11A中可以看出，在2.04A的位置是水溶性羟基化钆金属富勒烯中钆离子在电子探针实验中所处的位置，为接下来对于小鼠骨髓中钆离子含量提供了参考。
从图11B中可以看出，打药前，小鼠的骨髓中几乎检测不到钆离子的存在。在图11C-11F中，我们可以看到在打药后，随着时间的延长，小鼠骨髓中所检测出的钆含量也随着增加，分别为在0.5h时为0.58％(质量百分比，下同)，在1h时为1.33％，在4h时为1.49％，以及在24h时为2.2％。从电子探针的实验也进一步佐证了我们的材料可以快速进入并且富集在小鼠的骨髓中。
实施例8、GFNC在活体水平的毒性研究：
水溶性羟基化钆金属富勒烯组：静脉注射150μL浓度为1mM的水溶性羟基化钆金属富勒烯，分别在注射后15min、30min，45min，1h、4h、24h、7d、15d和30d，取小鼠摘眼球眼眶取血，置于5ml离心管中，于3500r、离心15min，将得到的血清转移到200μL离心管中，在血生化检测仪中检测小鼠的血生化指标，ALT，ALP，AST，BUN，LDH等。观察30d内小鼠体重变化。
生理盐水组：与金属富勒烯组做相同的处理用以对比，用生理盐水代替水溶性羟基化钆金属富勒烯。
相应的测试结果如图12、图13和图14所示，金属富勒烯组和生理盐水组无论在小鼠体重还是短长期脏器系数都无明显区别，说明该材料的毒性较小，可以进一步用于临床研究；从图14可得知：明显看出金属富勒烯组和生理盐水组相比，肝肾功能没有明显降低，说明其毒性较小，佐证了我们的材料是安全无毒的。
实施例9、GFNC在体外清除自由基效果
1)电子顺磁共振(ESR)检测自由基强度实验：
采用紫外诱导产生羟基自由基的方法，对照组为：将50μL质量浓度为37％的双氧水、50μL PBS缓冲液(pH＝7.4)和微量(0.133mM)二甲基吡啶N-氧化物(DMPO、自由基捕获剂)溶液混合，用280nm紫外光照射8min，此时即可产生羟基自由基的信号；实验组为：将50μL质量浓度为37％的双氧水、50μL PBS缓冲液(pH＝7.4)和微量(0.133mM)二甲基吡啶N-氧化物(DMPO、自由基捕获剂)溶液混合，立即加入20μM的水溶性羟基化钆金属富勒烯水溶液10μL，用280nm紫外光照射8min，检测自由基的信号强度。
相应的检测结果如图15所示，从图15可得知：实验组中，在GFNC浓度仅为20μM时，即能有效淬灭由紫外光照射双氧水产生的自由基，保护细胞免受双氧水所产生的自由基的伤害作用。
实施例10、GFNC在细胞水平保护细胞免受双氧水产生自由基的杀伤作用
选用小鼠骨髓细胞(FDC-P1)为所研究对象，其培养基为添加了细胞因子IL-3的高糖DMEM。将小鼠骨髓细胞以每孔ca.1x104个的浓度种在96孔板中，种8×6个孔，阴性对照组，种6个孔；阳性对照组种6个孔；实验组共有7个不同GFNC浓度，每个浓度均种6个孔，实验重复三次。阴性对照组为不加双氧水和GFNC，仅为接种有小鼠骨髓细胞的培养基；阳性对照组为仅加入10μL、30μM的GFNC，不加双氧水溶液；实验组为平行加入浓度为100μM的双氧水溶液20μL、90μL的培养基，以及不同浓度的GFNC(使其最终浓度分别为0.5μM、1μM、2.5μM、5μM、10μM、20μM和30μM)。
阴性对照组的加样顺序为：不加双氧水也不加GFNC，均用相同体积的PBS代替。
阳性对照组的加样顺序为：双氧水用同体积PBS代替，小鼠骨髓细胞孵育1h，之后，加入GFNC，孵育3h。
实验组加样顺序为：先加入双氧水和细胞培养基，和小鼠骨髓细胞孵育 1h，吸出双氧水溶液，用PBS缓冲液洗三次，洗去残留的双氧水溶液；再加入一定浓度的GFNC，同时加入细胞培养基，和细胞孵育3h之后，吸去含有GFNC的培养基，用PBS缓冲液洗三次；最后加入细胞培养基，在细胞培养箱中继续培养24h，然后用CCK-8检测细胞活性。
相应的检测结果如图16所示，从图16可得知：当加入浓度为100μM的双氧水溶液20μL时，双氧水会对小鼠骨髓细胞产生一定的杀伤作用，随着水溶性羟基化钆金属富勒烯的加入量逐渐增加时，细胞的活性也随着有所提升，表明：在细胞水平上，水溶性羟基化钆金属富勒烯对于自由基的清除有效果并且可以保护细胞不受其影响。同时，不添加双氧水，仅仅加入水溶性羟基化钆金属富勒烯的阳性对照组，其细胞活性稍高于阴性对照组，表明材料没有细胞毒性。
实施例11、GFNC在活体水平的骨髓抑制保护
动物模型：选用4-5周ICR小鼠，将其随机分为4组，每组6只，分别对应空白对照组、GFNC实验组、X射线辐照实验组和X射线+GFNC实验组。在小鼠右侧大腿上接种106个小鼠肝癌细胞(H22细胞)，接种5-7天后，肿瘤直径达到5mm左右时，进行实验。
空白对照组：实验组所注射的药物均用同体积的生理盐水所代替，静脉注射同等体积的生理盐水。
GFNC实验组：小鼠尾静脉注射GFNC水溶液(1mM)，药物用量为0.004mmol GFNC/kg小鼠体重。
X射线辐照实验组：小鼠全身一次性接受6Gy X射线辐照。
X射线+GFNC实验组：鼠全身一次性接受6Gy X射线辐照，同时静脉注射GFNC溶液，药物用量为0.004mmol GFNC/kg小鼠体重。
于肿瘤接种后第七天开始注射药物，作为开始实验的第一天，每天一次，连续5天静脉注射GFNC，并在第一天进行X射线辐照，之后分别在第四天，第七天，第十天，第十四天和第十七天，从小鼠眼眶取血(20μl)，置于3ml离心管中，用血细胞自动分析仪检测血常规，其中和骨髓抑制相关的主要指标为白细胞计数(WBC)，血小板计数(PLT)，血红蛋白测定(HGB)，单核细胞 比例(MO％)。
相应的检测结果如图17所示，从图17可得知：与空白对照组相比，X射线辐照实验组中的小鼠中与骨髓抑制相关的指标：白细胞，血小板，血红蛋白在小鼠体内都有着不同程度的减少，其中以白细胞的减少最为明显，其单核细胞出现了异常的增生现象，也说明其骨髓受到损伤，相关指标都有明显的异常；而X射线+GFNC实验组中的小鼠，由于GFNC的保护作用，其白细胞，血小板，血红蛋白的量相较于X射线实验组都有着很大程度的提高，单核细胞的数值更接近于正常组，并且随着时间的延长，相关指标越来越接近于正常小鼠的值，表明：GFNC对于放疗X射线所导致的小鼠骨髓抑制有明显的保护效果。
实施例12、GFNC在活体水平对于X射线放疗肿瘤治疗效果的影响
动物模型：选用4-5周ICR小鼠，将其随机分为4组，每组6只，分别对应空白对照组、GFNC实验组、X射线辐照实验组和X射线+GFNC实验组。在小鼠右侧大腿上接种106个小鼠肝癌细胞(H22细胞)，接种5-7天后，肿瘤直径达到5mm左右时，进行实验。
空白对照组：实验组所注射的药物均用同体积的生理盐水所代替。
X射线辐照实验组：小鼠全身一次性辐照X射线，辐照剂量为6Gy。
金属富勒烯衍生物(GFNC)实验组：小鼠静脉注射GFNC溶液，药物用量为0.04mmol Gd3+/kg小鼠体重。
X射线+GFNC实验组：鼠全身一次性辐照X射线，辐照剂量为6Gy，静脉注射GFNC溶液，药物用量为0.04mmol Gd3+/kg小鼠体重。
于肿瘤接种后第七天开始是注射药物，作为开始实验的第一天，每天一次，连续5天静脉注射GFNC，每两天测量小鼠的体重和肿瘤直径。
相应的检测结果如图18和19所示，从图18可得知：与X射线实验组相比，X射线+GFNC实验组中的小鼠的肿瘤直径不仅没有增大，还变小，表明：GFNC不会影响放疗对于小鼠肿瘤的治疗效果，同时又可以显著的抑制放疗所导致的骨髓抑制毒性，两者之间具有协同作用；从图19可得知：通过对比不同组的小鼠的体重变化，X射线实验组中的小鼠其体重在打药初期有 着明显的下降，之后由于停止打药，而靠着小鼠自身的恢复功能有一定程度的回升，但仍是体重最轻的一组，而X射线+GFNC实验组中的小鼠，虽然在打药初期，其体重也略有下降，但是后期明显恢复，并且接近于正常小鼠的体重值，说明GFNC可以保护小鼠尽可能的免于放疗射线所带来的副作用，保护小鼠骨髓抑制毒性的同时，其本身的毒性较小，可以进一步用于临床研究。
实施例13、水溶性羟基化钆金属富勒烯在活体水平的骨髓抑制保护
动物模型：选用4-5周ICR小鼠，将其随机分为4组，每组6只，分别对应空白对照组、GFNC实验组、环磷酰胺(CTX)实验组和CTX+GFNC实验组。在小鼠右侧大腿上接种106个小鼠肝癌细胞(H22细胞)，接种5-7天后，肿瘤直径达到5mm左右时，进行实验。
空白对照组：实验组所注射的药物均用同体积的生理盐水所代替，静脉和腹腔均注射同等体积的生理盐水。
GFNC实验组：小鼠尾静脉注射GFNC水溶液(1mM)，药物用量为0.004mmol GFNC/kg小鼠体重。
环磷酰胺(CTX)实验组：小鼠腹腔注射CTX溶液，药物用量为60mg/kg小鼠体重
CTX+GFNC实验组：鼠腹腔注射CTX溶液，药物用量为60mg/kg小鼠体重，静脉注射GFNC溶液，药物用量为0.004mmol GFNC/kg小鼠体重。
于肿瘤接种后第七天开始是注射药物，作为开始实验的第一天，每天一次，连续5天腹腔注射CTX或静脉注射GFNC，分别在第四天，第七天，第十天，第十四天和第十七天，从小鼠眼眶取血(20μl)，置于3ml离心管中，用血细胞自动分析仪检测血常规，其中和骨髓抑制相关的主要指标为白细胞计数(WBC)，血小板计数(PLT)，血红蛋白测定(HGB)，单核细胞比例(MO％)。
相应的检测结果如图20所示，从图20可得知：与空白对照组相比，环磷酰胺(CTX)实验组中的小鼠中与骨髓抑制相关的指标：白细胞，血小板，血红蛋白在小鼠体内都有着不同程度的减少，其中以白细胞的减少最为明显，其单核细胞出现了异常的增生现象，也说明其骨髓受到损伤，相关指标都有 明显的异常；而CTX+GFNC实验组中的小鼠，由于GFNC的保护作用，其白细胞，血小板，血红蛋白的量相较于环磷酰胺(CTX)实验组都有着很大程度的提高，单核细胞的数值更接近于正常组，并且随着时间的延长，相关指标越来越接近于正常小鼠的值，表明：GFNC对于化疗药物CTX所导致的小鼠骨髓抑制有明显的保护效果。
实施例14、水溶性羟基化钆金属富勒烯在活体水平对于化疗药物CTX肿瘤治疗效果的影响和毒性实验
动物模型：选用4-5周ICR小鼠，将其随机分为4组，每组6只，分别对应空白对照组、GFNC实验组、环磷酰胺(CTX)实验组和CTX+GFNC实验组。在小鼠右侧大腿上接种106个小鼠肝癌细胞(H22细胞)，接种5-7天后，肿瘤直径达到5mm左右时，进行实验。
空白对照组：实验组所注射的药物均用同体积的生理盐水所代替。
环磷酰胺(CTX)实验组：小鼠腹腔注射CTX溶液，药物用量为60mg/kg小鼠体重。
金属富勒烯衍生物(GFNC)实验组：小鼠静脉注射GFNC溶液，药物用量为0.04mmol Gd3+/kg小鼠体重。
CTX+GFNC实验组：鼠腹腔注射CTX溶液，药物用量为60mg/kg小鼠体重，静脉注射GFNC溶液，药物用量为0.04mmol Gd3+/kg小鼠体重。
于肿瘤接种后第七天开始是注射药物，作为开始实验的第一天，每天一次，连续5天腹腔注射CTX或静脉注射GFNC，每两天测量小鼠的体重和肿瘤直径，在第17天，取小鼠摘眼球眼眶取血，置于5ml离心管中，于3500r，离心15min，将得到的血清转移到200μL离心管中，在血生化检测仪中检测小鼠的血生化指标，ALT，ALP，AST，BUN，LDH，UA，脱颈处死小鼠后，取小鼠主要组织器官(心，肝，脾，肺，肾)称重，泡在4％福尔马林溶液中，之后做组织病理切片，H&E染色。同时空白对照组、环磷酰胺(CTX)实验组和金属富勒烯衍生物(GFNC)实验组做相同的处理用以对比。
相应的检测结果如图21和22所示，从图21可得知：与环磷酰胺(CTX)实验组相比，CTX+GFNC实验组中的小鼠的肿瘤直径不仅没有增大，还变小， 表明：GFNC不会影响化疗药物CTX对于小鼠肿瘤的治疗效果，同时又可以显著的抑制化疗药物所导致的骨髓抑制毒性，两者之间具有协同作用；从图22可得知：通过对比不同组的小鼠的体重变化，环磷酰胺(CTX)实验组中的小鼠其体重在打药初期有着明显的下降，之后由于停止打药，而靠着小鼠自身的恢复功能有一定程度的回升，但仍是体重最轻的一组，而CTX+GFNC实验组中的小鼠，虽然在打药初期，其体重也略有下降，但是后期明显恢复，并且接近于正常小鼠的体重值，说明GFNC可以保护小鼠尽可能的免于化疗药物所带来的副作用，保护小鼠骨髓抑制毒性的同时，其本身的毒性较小，可以进一步用于临床研究。
从图23可得知：CTX+GFNC实验组所测试得到的酶活性相对于CTX实验组有一定的下降或者持平，表明：其对于小鼠体内的活性氧成分有一定的清除效果，同时GFNC毒性较小。
从图24可得知：通过对比组织病理切片图像，CTX+GFNC实验组的主要组织器官并无肿大，坏死或者发炎等现象，和空白对照组几乎无异，而环磷酰胺(CTX)实验组中的小鼠，其组织器官均有一定程度的损伤，而且从肿瘤部位的病理切片亦可看出，空白对照组的肿瘤组织生长很旺盛，肿瘤细胞很致密，而CTX+GFNC实验组和CTX实验组的肿瘤组织细胞出现大量死亡的现象，组织疏松。表明GFNC是安全无毒的并且不影响化疗药物CTX的治疗肿瘤效果。
实施例15、水溶性羟基化空心富勒烯的制备
1)将100mg C60固体粉末加入100ml的单口瓶中，分别加入7ml体积分数为30％的过氧化氢水溶液和3ml 2M的氢氧化钠水溶液，油浴加热到70℃，反应2-5h；
2)反应后使用M.W.＝3500透析袋除去小分子，使用电导率仪监测直至透析完成，浓缩得到的产物即可得到水溶性羟基化金属富勒烯，经DLS测定，其在水溶液中的平均粒径为200nm，粒径分布均一，粒径分布图如图25所示。
实施例16、水溶性羟基化空心富勒烯在活体水平的毒性研究
水溶性羟基化空心富勒烯组：静脉注射150μL浓度为1mM的水溶性羟基化空心富勒烯，在注射后30d，取小鼠摘眼球眼眶取血，置于5ml离心管 中，于3500r、离心15min，将得到的血清转移到200μL离心管中，在血生化检测仪中检测小鼠的血生化指标，ALT，ALP，AST，BUN，LDH等。同时观察30d内小鼠体重变化。
生理盐水组：与空心富勒烯组做相同的处理用以对比，用生理盐水代替水溶性羟基化空心富勒烯。
相应的测试结果如图26、图27和图28所示，图26和图27说明空心富勒烯组和生理盐水组无论在小鼠体重还是短长期脏器系数都无明显区别，说明该材料的毒性较小，可以进一步用于临床研究；从图28可得知：明显看出空心富勒烯组和生理盐水组相比，肝肾功能没有明显降低，说明其毒性较小，佐证了我们的材料是安全无毒的。
实施例17、水溶性羟基化空心富勒烯在体外清除自由基效果
1)电子顺磁共振(ESR)检测自由基强度实验：
采用紫外诱导产生羟基自由基的方法，对照组为：将50μL质量浓度为37％的双氧水、50μL PBS缓冲液(pH＝7.4)和微量(0.133mM)二甲基吡啶N-氧化物(DMPO、自由基捕获剂)溶液混合，用280nm紫外光照射8min，此时即可产生羟基自由基的信号；实验组为：将50μL质量浓度为37％的双氧水、50μL PBS缓冲液(pH＝7.4)和微量(0.133mM)二甲基吡啶N-氧化物(DMPO、自由基捕获剂)溶液混合，立即加入20μM的水溶性羟基化空心富勒烯水溶液10μL，用280nm紫外光照射8min，检测自由基的信号强度。
相应的检测结果如图29所示，从图29可得知：实验组中，在羟基化空心富勒烯水溶液浓度仅为20μM时，即能有效淬灭由紫外光照射双氧水产生的自由基，保护细胞免受双氧水所产生的自由基的伤害作用。
实施例18、水溶性羟基化空心富勒烯在细胞水平保护细胞免受双氧水产生自由基的杀伤作用
选用小鼠骨髓细胞(FDC-P1)为所研究对象，其培养基为添加了细胞因子IL-3的高糖DMEM。将小鼠骨髓细胞以每孔ca.1x104个的浓度种在96孔板中，种8×6个孔，阴性对照组，种6个孔；阳性对照组种6个孔；实验组共有7个不同羟基化空心富勒烯水溶液浓度，每个浓度均种6个孔，实验重复 三次。阴性对照组为不加双氧水和羟基化空心富勒烯水溶液，仅为接种有小鼠骨髓细胞的培养基；阳性对照组为仅加入10μL、30μM的羟基化空心富勒烯水溶液，不加双氧水溶液；实验组为平行加入浓度为100μM的双氧水溶液20μL、90μL的培养基，以及不同浓度的羟基化空心富勒烯水溶液(使其最终浓度分别为0.5μM、1μM、2.5μM、5μM、10μM、20μM和30μM)。
阴性对照组的加样顺序为：不加双氧水也不加羟基化空心富勒烯水溶液，均用相同体积的PBS代替。阳性对照组的加样顺序为：双氧水用同体积PBS代替，小鼠骨髓细胞孵育1h，之后，加入羟基化空心富勒烯水溶液，孵育3h。
实验组加样顺序为：先加入双氧水和细胞培养基，和小鼠骨髓细胞孵育1h，吸出双氧水溶液，用PBS缓冲液洗三次，洗去残留的双氧水溶液；再加入一定浓度的羟基化空心富勒烯水溶液，同时加入细胞培养基，和细胞孵育3h之后，吸去含有羟基化空心富勒烯水溶液的培养基，用PBS缓冲液洗三次；最后加入细胞培养基，在细胞培养箱中继续培养24h，然后用CCK-8检测细胞活性。
相应的检测结果如图30所示，从图30可得知：当加入浓度为100μM的双氧水溶液20μL时，双氧水会对小鼠骨髓细胞产生一定的杀伤作用，随着羟基化空心富勒烯水溶液的加入量逐渐增加时，细胞的活性也随着有所提升，表明：在细胞水平上，羟基化空心富勒烯水溶液对于自由基的清除有效果并且可以保护细胞不受其影响。同时，不添加双氧水，仅仅加入羟基化空心富勒烯水溶液的阳性对照组，其细胞活性稍高于阴性对照组，表明材料没有细胞毒性。
实施例19、水溶性羟基化空心富勒烯在活体水平的骨髓抑制保护
动物模型：选用4-5周ICR小鼠，将其随机分为4组，每组6只，分别对应空白对照组、水溶性羟基化空心富勒烯实验组、X射线辐照实验组和X射线+水溶性羟基化空心富勒烯实验组。在小鼠右侧大腿上接种106个小鼠肝癌细胞(H22细胞)，接种5-7天后，肿瘤直径达到5mm左右时，进行实验。
空白对照组：实验组所注射的药物均用同体积的生理盐水所代替，静脉和腹腔均注射同等体积的生理盐水。
水溶性羟基化空心富勒烯实验组：小鼠尾静脉注射水溶性羟基化空心富勒烯水溶液(1mM)，药物用量为0.004mmol水溶性羟基化空心富勒烯/kg小鼠体重。
X射线辐照实验组：小鼠全身一次性辐照X射线，辐照剂量为6Gy。
X射线+水溶性羟基化空心富勒烯实验组：小鼠全身一次性辐照X射线，辐照剂量为6Gy，静脉注射水溶性羟基化空心富勒烯溶液，药物用量为0.004mmol水溶性羟基化空心富勒烯/kg小鼠体重。
于肿瘤接种后第七天开始是注射药物，作为开始实验的第一天，每天一次，连续5天静脉注射水溶性羟基化空心富勒烯，分别在第四天，第七天，第十天，第十四天和第十七天，从小鼠眼眶取血(20μl)，置于3ml离心管中，用血细胞自动分析仪检测血常规，其中和骨髓抑制相关的主要指标为白细胞计数(WBC)，血小板计数(PLT)，血红蛋白测定(HGB)，单核细胞比例(MO％)。
相应的检测结果如图31所示，从图31可得知：与空白对照组相比，X射线实验组中的小鼠中与骨髓抑制相关的指标：白细胞，血小板，血红蛋白在小鼠体内都有着不同程度的减少，其中以白细胞的减少最为明显，其单核细胞出现了异常的增生现象，也说明其骨髓受到损伤，相关指标都有明显的异常；而X射线+水溶性羟基化空心富勒烯实验组中的小鼠，由于水溶性羟基化空心富勒烯的保护作用，其白细胞，血小板，血红蛋白的量相较于X射线辐照实验组都有着很大程度的提高，单核细胞的数值更接近于正常组，并且随着时间的延长，相关指标越来越接近于正常小鼠的值，表明：水溶性羟基化空心富勒烯对于放疗射线所导致的小鼠骨髓抑制有明显的保护效果。
实施例20、水溶性羟基化空心富勒烯在活体水平的骨髓抑制保护
动物模型：选用4-5周ICR小鼠，将其随机分为4组，每组6只，分别对应空白对照组、水溶性羟基化空心富勒烯实验组、环磷酰胺(CTX)实验组和CTX+水溶性羟基化空心富勒烯实验组。在小鼠右侧大腿上接种106个小鼠肝癌细胞(H22细胞)，接种5-7天后，肿瘤直径达到5mm左右时，进行实验。
空白对照组：实验组所注射的药物均用同体积的生理盐水所代替，静脉和腹腔均注射同等体积的生理盐水。
水溶性羟基化空心富勒烯实验组：小鼠尾静脉注射水溶性羟基化空心富勒烯水溶液(1mM)，药物用量为0.004mmol水溶性羟基化空心富勒烯/kg小鼠体重。
环磷酰胺(CTX)实验组：小鼠腹腔注射CTX溶液，药物用量为60mg/kg小鼠体重
CTX+水溶性羟基化空心富勒烯实验组：鼠腹腔注射CTX溶液，药物用量为60mg/kg小鼠体重，静脉注射水溶性羟基化空心富勒烯溶液，药物用量为0.004mmol水溶性羟基化空心富勒烯/kg小鼠体重。
于肿瘤接种后第七天开始是注射药物，作为开始实验的第一天，每天一次，连续5天腹腔注射CTX或静脉注射水溶性羟基化空心富勒烯，分别在第四天，第七天，第十天，第十四天和第十七天，从小鼠眼眶取血(20μl)，置于3ml离心管中，用血细胞自动分析仪检测血常规，其中和骨髓抑制相关的主要指标为白细胞计数(WBC)，血小板计数(PLT)，血红蛋白测定(HGB)，单核细胞比例(MO％)。
相应的检测结果如图32所示，从图32可得知：与空白对照组相比，环磷酰胺(CTX)实验组中的小鼠中与骨髓抑制相关的指标：白细胞，血小板，血红蛋白在小鼠体内都有着不同程度的减少，其中以白细胞的减少最为明显，其单核细胞出现了异常的增生现象，也说明其骨髓受到损伤，相关指标都有明显的异常；而CTX+水溶性羟基化空心富勒烯实验组中的小鼠，由于水溶性羟基化空心富勒烯的保护作用，其白细胞，血小板，血红蛋白的量相较于环磷酰胺(CTX)实验组都有着很大程度的提高，单核细胞的数值更接近于正常组，并且随着时间的延长，相关指标越来越接近于正常小鼠的值，表明：水溶性羟基化空心富勒烯对于化疗药物CTX所导致的小鼠骨髓抑制有明显的保护效果。

Claims (20)

  1. 一种富勒烯微纳材料和/或金属富勒烯微纳材料在制备药物中的应用，其特征在于，所述药物具有如下1)-7)性质中的至少一种：
    1)治疗骨髓抑制；2)治疗由于骨髓抑制导致的白细胞下降、血小板下降、血红蛋白下降和单核细胞下降中的至少一种；3)保护骨髓细胞和/或造血细胞；4)能富集于骨髓中；5)保护肝脏组织、脾脏组织和肾脏组织；6)清除自由基；7)提升机体的抗氧化系统的功能。
  2. 一种治疗骨髓抑制的方法，其特征在于，包括如下步骤：向需要治疗骨髓抑制的生物体施用有效剂量的富勒烯微纳材料和/或金属富勒烯微纳材料。
  3. 一种治疗骨髓抑制的药物组合物，其特征在于，包括富勒烯微纳材料和/或金属富勒烯微纳材料，还包括可药用的载体、可药用的稀释剂和可药用的赋形剂中的至少一种。
  4. 根据权利要求1所述的应用、权利要求2所述的方法或权利要求3所述的药物组合物，其特征在于，所述富勒烯微纳材料和/或金属富勒烯微纳材料包括至少一种选自下组的有效成分：油溶性富勒烯微纳材料、油溶性金属富勒烯微纳材料、所述油溶性富勒烯微纳材料和所述油溶性金属富勒烯微纳材料的组合物、水溶性富勒烯微纳材料、水溶性金属富勒烯微纳材料、所述水溶性富勒烯微纳材料和所述水溶性金属富勒烯微纳材料的组合物、以上六者的可药用的酯或以上六者的可药用的盐。
  5. 根据权利要求1所述的应用、权利要求2所述的方法或权利要求3所述的药物组合物，其特征在于，所述油溶性富勒烯微纳材料由富勒烯本体材料经油溶性修饰得到，所述油溶性金属富勒烯微纳材料由金属富勒烯本体材料经油溶性修饰得到，所述的水溶性富勒烯微纳材料是由富勒烯本体材料经水溶性修饰得到的，所述水溶性金属富勒烯微纳材料是由金属富勒烯本体材料经水溶性修饰得到。
  6. 根据权利要求5所述的应用、权利要求5所述的方法或权利要求5所 述的药物组合物，其特征在于，所述富勒烯本体材料包括一种或多种通式为C2m的由碳原子组成的笼状结构，30≤m≤60。
  7. 根据权利要求5所述的应用、权利要求5所述的方法或权利要求5所述的药物组合物，其特征在于，所述金属富勒烯本体材料包括M@C2n、M2@C2n、MA@C2n、M3N@C2n、M2C2@C2n、M2S@C2n、M2O@C2n和MxA3-xN@C2n中的一种或多种，其中：M、A均代表金属元素且M、A均选自镧系金属元素、Sc和Y中的任意一种，30≤n≤60；0≤x≤3。N代表氮元素，C代表碳元素，S代表硫元素，镧系金属元素包括La、Ce、Pr、Nd、Pm、Sm、Eu、Gd、Tb、Dy、Ho、Er、Tm、Yb和Lu。
  8. 根据权利要求5所述的应用、权利要求5所述的方法或权利要求5所述的药物组合物，其特征在于，所述油溶性修饰的方法包括用食用油包覆所述富勒烯本体材料和/或金属富勒烯本体材料。
  9. 根据权利要求8所述的应用、权利要求8所述的方法或权利要求8所述的药物组合物，其特征在于，所述食用油包括橄榄油、亚麻籽油、葵花籽油、玉米胚油、玉米油和角鲨烷中的至少一种。
  10. 根据权利要求8所述的应用、权利要求8所述的方法或权利要求8所述的药物组合物，其特征在于，所述油溶性富勒烯微纳材料和/或油溶性金属富勒烯微纳材料为食用油包覆钆金属富勒烯、食用油包覆C60、食用油包覆C70或食用油包覆C84。
  11. 根据权利要求5所述的应用、权利要求5所述的方法或权利要求5所述的药物组合物，其特征在于，所述水溶性修饰的方法包括以下方法的任意一种：在富勒烯本体材料和/或金属富勒烯本体材料的碳笼外表面修饰亲水基团，或使富勒烯本体材料和/或金属富勒烯本体材料被水溶性载体负载。
  12. 根据权利要求12所述的应用、权利要求12所述的方法或权利要求12所述的药物组合物，其特征在于，所述亲水基团包括羟基、氨基和羧基中的至少一种。
  13. 根据权利要求12所述的应用、权利要求12所述的方法或权利要求12所述的药物组合物，其特征在于，所述水溶性载体选自脂质体、聚合物胶 束和蛋白中的至少一种，所述聚合物胶束为聚乙丙交酯聚乙二醇、聚赖氨酸或壳聚糖；所述蛋白为白蛋白或转铁蛋白。
  14. 根据权利要求1所述的应用、权利要求2所述的方法或权利要求3所述的药物组合物，其特征在于，所述骨髓抑制由药物化疗、化学毒物或具有骨髓抑制副作用的药物诱导产生。
  15. 根据权利要求15所述的应用，其特征在于：所述药物化疗采用的药物为环磷酰胺、阿霉素、顺铂或紫杉醇；所述化学毒物为苯或苯的衍生物；所述具有骨髓抑制副作用的药物为氯霉素、四环素或消炎痛。
  16. 根据权利要求1所述的应用、权利要求2所述的方法或权利要求3所述的药物组合物，其特征在于，所述骨髓抑制由肿瘤放射疗法或产生骨髓抑制副作用的射线诱导产生。
  17. 根据权利要求17所述的应用、权利要求17所述的方法或权利要求17所述的药物组合物，其特征在于，肿瘤放射疗法利用的射线为α射线、β射线、γ射线或X射线。
  18. 根据权利要求2所述的方法，其特征在于，所述富勒烯微纳材料和/或金属富勒烯微纳材料在以下阶段的至少一个中向需要治疗骨髓抑制的生物体施用：(1)化疗药物或放疗射线使用前的24h内；(2)在放化疗过程中；(3)在放化疗结束后1-2周内使用。
  19. 根据权利要求2所述的方法，其特征在于，有效成分的施用剂量为1mg/kg/d-500mg/kg/d，可选的为1-100mg/kg/d，1-20mg/kg/d或1-10mg/kg/d。
  20. 根据权利要求2所述的方法，其特征在于，所述生物体为人或动物，动物可以为哺乳动物，如小鼠、豚鼠、大鼠、狗、兔子、猴子。
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