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WO2017063328A1 - 一种富勒烯和/或金属富勒烯微纳材料及其应用 - Google Patents

一种富勒烯和/或金属富勒烯微纳材料及其应用 Download PDF

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WO2017063328A1
WO2017063328A1 PCT/CN2016/075831 CN2016075831W WO2017063328A1 WO 2017063328 A1 WO2017063328 A1 WO 2017063328A1 CN 2016075831 W CN2016075831 W CN 2016075831W WO 2017063328 A1 WO2017063328 A1 WO 2017063328A1
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fullerene
metal
bone marrow
micro
nano material
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王春儒
甄明明
张莹
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Beijing Fullcan Bio-Technology Co Ltd
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Beijing Fullcan Bio-Technology Co Ltd
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Priority claimed from CN201510666530.4A external-priority patent/CN105267238A/zh
Priority claimed from CN201510671792.XA external-priority patent/CN105920608B/zh
Priority claimed from CN201610041914.1A external-priority patent/CN105903020B/zh
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    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • 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
    • A61K47/02—Inorganic compounds

Definitions

  • the invention relates to a micro-nano material and its application in bone marrow suppression, and belongs to the field of biomedicine.
  • radiotherapy and chemotherapy can kill metastasis in time, effectively resisting tumor recurrence and metastasis, and the key to whether radiotherapy and chemotherapy can persist. It is the protection of bone marrow hematopoietic cells.
  • many radiotherapy and chemotherapy can cause different degrees of myelosuppression, initially manifested as white blood cells, thrombocytopenia, with increasing doses, red blood cells and hemoglobin are decreased in severe cases, and even aplastic anemia may occur. Therefore, it is of great clinical significance to find an ideal drug that can attenuate the myelosuppressive toxicity caused by chemoradiotherapy drugs without affecting the effect of chemoradiotherapy on tumor inhibition.
  • HGFs hematopoietic growth factors
  • a method for reducing bone marrow suppression induced by chemoradiotherapy drugs can be found to have important clinical research value for reducing the patient's condition.
  • the fullerene and/or metal fullerene micro-nano materials of the present invention have all of the following properties: (1) the surface is hydrophilic, 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 enter the bone marrow through the endothelial cell gap of the sinusoids of the bone marrow.
  • the fullerene and/or metal fullerene micro-nano material of the present invention is obtained by water-soluble modification of fullerene and/or metal fullerene bulk material, said fullerene and/or metal rich
  • the lenidine micro-nano material is a water-soluble derivative of fullerene and/or metal fullerene.
  • the fullerene and/or metal fullerene micro-nano material may have a particle size ranging from 1 to 500 nm, specifically from 140 to 200 nm.
  • the fullerene and/or metal fullerene bulk materials of the present invention include both hollow fullerene C 2n and the like, and also include inlaid metal fullerenes, the main species being M@C 2n , M 2 @C 2n , MA@C 2n , M 3 N@C 2n , M 2 C 2 @C 2n , M 2 S@C 2n , M 2 O@C 2n and M x A 3-x N@C 2n ; where M, A All represent a metal element, and both of M and A are selected from any one of Sc, Y and a lanthanide metal element (La-Lu); 30 ⁇ n ⁇ 60; 0 ⁇ x ⁇ 3.
  • an effective route is to pass a water-insoluble fullerene and/or a metal fuller by covalent or non-covalent interaction.
  • the olefinic bulk material is obtained by water-soluble modification.
  • Commonly used covalent modifications can be prepared by solid-liquid reaction under basic conditions.
  • a hydroxy derivative can be prepared by reacting a fullerene and/or metal fullerene solid powder with H 2 O 2 under basic conditions; passing a fullerene and/or metal fullerene solid powder with H 2 O 2 and ammonia reaction, can prepare amino derivatives, can achieve the purpose of water-soluble modification.
  • the non-covalent bond modification method can form a water-soluble modified fullerene and/or metal fullerene micro-nano material by hydrophobic-hydrophobic interaction using a conventional water-soluble carrier such as a liposome, a polymer micelle, a protein or the like.
  • a conventional water-soluble carrier such as a liposome, a polymer micelle, a protein or the like.
  • the water-soluble modified fullerene and/or metal fullerene micro-nano material may specifically be modified with a hydrophilic functional group hydroxyl group, an amino group and a carboxyl group on the surface of the fullerene and/or metal fullerene bulk material. At least one of, or, by hydrophobic-hydrophobic interaction (non-covalent covalent bond), the fullerene and/or metal fullerene bulk material and a water-soluble carrier (eg, liposome, polymer gel) The bundle, protein, etc.) form the water-soluble fullerene and/or metal fullerene micro-nano material.
  • a water-soluble carrier eg, liposome, polymer gel
  • the water-soluble carrier is a pharmaceutical carrier commonly used in medicine, and may be specifically selected from 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 fullerene and/or metal fullerene micro/nano material may specifically be water-soluble hydroxylated ruthenium metal fullerene (GFNC, Gd@C 82 (OH) n ) or water-soluble hydroxylated hollow fullerene (C 60 (OH) n ).
  • GFNC water-soluble hydroxylated ruthenium metal fullerene
  • C 60 (OH) n ) water-soluble hydroxylated hollow fullerene
  • the water-soluble fullerene and/or metal fullerene micro-nano material of the present invention generally has a size of 1-500 nm in an aqueous solution, and the micro-nano material has a certain rigidity (not easily deformed), so that the micro-nano material passes through the blood.
  • the circulation easily passes through the nanopores between the endothelial cells in the sinusoids of the bone marrow, and can quickly enter the bone marrow using the pressure difference between the blood vessels and the inside and outside.
  • the invention utilizes the special structural properties of the micro-nano material and the bone marrow sinusoidal endothelial cells, can realize enrichment in the bone marrow without further derivatives, and can be metabolized outside the organism.
  • fullerene and/or metal fullerene micro-nano materials of the present invention for the preparation of a drug or drug carrier targeted for enrichment in bone marrow is also within the scope of the present invention.
  • Another object of the present invention is to provide a method for rapidly enriching fullerene and/or metal fullerene micro-nano materials in bone marrow.
  • the method provided by the present invention comprises the steps of: intravenously injecting fullerenes and/or metal fullerene micro-nano materials, through blood circulation, by means of a special structure in the sinusoids of the bone marrow (nanoporosity between endothelial cells), Enter the bone marrow and enrich in the bone marrow.
  • mice were injected intravenously at different time points, and the organs and bone tissues were taken, and the cesium ion concentration and 131 I were determined by ICP-MS.
  • the radiolabeling method tests the metabolic distribution in the body. Two different methods have shown that the drug molecules we obtained can be rapidly enriched in mouse bone, and by further analysis, we found that the water-soluble hydroxylated ruthenium metal fullerene is in the bone mass per unit mass of bone. The quality is 12 times, indicating that it has good bone marrow targeting.
  • the water-soluble fullerene and/or metal fullerene micro-nano material is used as a bone marrow targeting agent, which can be efficiently and rapidly enriched in the bone marrow, has good biocompatibility, and has high-efficiency removal.
  • the ability of free radicals can be further used to protect bone marrow cells.
  • the residence time in the bone marrow is significantly higher than in other organs, and the material can be metabolized in the living body without It has obvious toxic and side effects, so it is a simple and rapid preparation, purification, biocompatibility, rapid enrichment in the bone marrow region, and has a good application prospect as a treatment platform for bone marrow protectants or bone diseases.
  • fullerene and/or metal fullerene micro-nano materials according to the invention in the preparation of a medicament or a pharmaceutical carrier having at least one of the following properties 1) to 5) is also within the scope of protection of the invention: Preventing and/or treating myelosuppression; 2) scavenging free radicals; 3) enriching bone marrow; 4) preventing and/or treating at least one of leukopenia, thrombocytopenia, hemoglobin decline, and monocyte decline due to myelosuppression 5) Protect bone marrow cells and/or hematopoietic cells.
  • the water-soluble fullerene and/or metal fullerene micro-nano material of the present invention generally has a size of 1-500 nm in an aqueous solution, and the micro-nano material has a certain rigidity (not easily deformed), so that the micro-nano material passes through the blood.
  • the circulation easily passes through the nanopores between the endothelial cells in the sinusoids of the bone marrow, and can quickly enter the bone marrow using the pressure difference between the blood vessels and the inside and outside.
  • the myelosuppression may be induced by drug chemotherapy, chemical poisons, X-rays or drugs having myelosuppressive side effects, including drugs currently inhibited by bone marrow which are currently used clinically, such as cyclophosphamide. (CTX), etc.; can also be induced by chemical poisons, such as benzene and its derivatives; it can also be induced by drugs with myelosuppressive side effects, such as chloramphenicol, tetracycline Or indomethacin, etc., or radiation by X-rays.
  • CTX cyclophosphamide.
  • Another object of the present invention is to provide a method for preventing and/or treating myelosuppression using fullerene and/or metal fullerene micro-nano materials.
  • the method of myelosuppression provided by the present invention comprises the steps of: injecting an effective amount of the fullerene and/or metal fullerene micro-nano material into an organism in need of prevention and/or treatment of myelosuppression, said Fuller
  • the olefinic and/or metal fullerene micro-nano material is circulated through the blood through the nanopores between the endothelial cells in the sinusoids of the bone marrow, and can be rapidly enriched in the bone marrow by the intra- and extra-vascular pressure difference, by means of fullerenes and/or metals.
  • Fullerene micro-nano materials are highly effective in scavenging free radicals and prevent and/or treat myelosuppression.
  • the "effective amount" as used in the present invention means an amount of the active ingredient which is sufficient to effectively deliver the active ingredient for preventing myelosuppression when administered to the living body fullerene and/or metal fullerene micro-nanomaterial by the method of the present invention.
  • 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.
  • the fullerene and/or metal fullerene micro/nano material is present in the form of an aqueous solution thereof, and the concentration in vivo is in the range of 0.1 mM to 10 mM.
  • the organism is a mammal, such as a human.
  • the method of the injection may be intravenous injection, directly exerts through the blood circulation, does not require penetration, and the amount of the medicament used is small, and the curative effect is high.
  • the myelosuppression may be induced by a drug such as drug chemotherapy, chemical poison, X-ray or the like or a drug having a myelosuppressive side effect, which includes a drug currently inhibited by bone marrow which is currently used clinically, such as cyclophosphamide (CTX). ), doxorubicin, cisplatin, paclitaxel, etc.; can also be induced by chemical poisons, such as benzene and its derivatives (such as chlorambucil); can also take drugs with myelosuppressive side effects, such as chloramphenicol Or tetracycline, heba or indomethacin.
  • Radiotherapy includes X-rays, gamma rays, and the like currently conventionally used in clinical practice.
  • the chemotherapeutic drug and the X-ray-induced myelosuppression have a protective effect, and the fullerene and/or the metal fullerene micro-nano material can be injected in advance within 24 hours of the use of the chemotherapeutic drug, and used in combination during the chemoradiotherapy process.
  • Efficient and rapid elimination of drugs induces free radicals generated during myelosuppression, which can quickly prevent and repair bone marrow suppression.
  • Example 1 is a particle size distribution diagram of a water-soluble hydroxylated ruthenium metal fullerene prepared in Example 1.
  • Example 2 is a particle size distribution diagram of water-soluble hydroxylated hollow fullerene prepared in Example 2.
  • Fig. 3 is a graph showing the metabolic distribution of water-soluble hydroxylated ruthenium metal fullerene in mice in Example 3 by ICP-MS.
  • 5a-f are electron probe spectra of water-soluble hydroxylated ruthenium metal fullerene samples in Example 4 and mouse bone marrow at different time points.
  • Fig. 6 is a graph showing the organ coefficients of the water-soluble hydroxylated ruthenium metal fullerene group and the control group in Example 5, all after 30 days of injection.
  • Fig. 7 is a graph showing the change in body weight of the water-soluble hydroxylated ruthenium metal fullerene group and the control group in Example 5.
  • Figure 8 is a blood biochemical test of a water-soluble hydroxylated ruthenium metal fullerene group in Example 5, which is a value after 30 days of injection.
  • Figure 9 is a graph showing the organ coefficients of the water-soluble hydroxylated hollow fullerene group and the control group in Example 5, all after 30 days of injection.
  • Figure 10 is a graph showing changes in body weight of a water-soluble hydroxylated hollow fullerene group and a control group in Example 5.
  • Figure 11 is a blood biochemical test of a water-soluble hydroxylated hollow fullerene group injected in Example 5, all after 30 days of injection.
  • Example 12 is a result of an in vitro ESR scavenging free radical experiment of the water-soluble hydroxylated ruthenium metal fullerene in Example 8, wherein the solid line is the control group; and the broken line is the experimental group.
  • Figure 13 is a graph showing the effect of water-soluble hydroxylated ruthenium metal fullerene in the removal of free radicals at the cellular level in Example 8.
  • Example 14 is a result of an in vitro ESR scavenging free radical experiment of the water-soluble hollow fullerene in Example 9, wherein the solid line is the control group; and the broken line is the experimental group.
  • Figure 15 is a graph showing the effect of the C 60 water-soluble derivative in Example 9 on scavenging free radicals at the cellular level.
  • Figure 16 is a blood routine index of different groups of mice in Example 10.
  • Figure 17 is a graph showing tumor size changes in different groups of mice in Example 11.
  • Figure 18 is a graph showing changes in body weight of different groups of mice in Example 11.
  • Figure 19 is a blood biochemical index of different groups of mice in Example 11.
  • Fig. 20 is a sectional image of the pathological section of the tissue of different groups of mice in Example 11 after H&E staining.
  • Figure 21 is a graph showing the blood routine of mice of different groups using the C 60 water-soluble derivative in Example 12.
  • Figure 22 is a blood routine index of different groups of mice in Example 13.
  • Figure 23 is a graph showing tumor size changes of different groups of mice in Example 14.
  • Figure 24 is a graph showing changes in body weight of different groups of mice in Example 14.
  • CTX cyclophosphamide
  • Example 3 Metabolic distribution of water-soluble hydroxylated ruthenium metal fullerenes in vivo
  • 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;
  • Fig. 3 The test results are shown in Fig. 3. It can be seen from Fig. 3 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. 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 in the bone marrow and bone of the mouse in Example 1 is shown in Table 1 below. It can be seen from Table 1 that the bone marrow and the bone marrow are separated, and the water-soluble hydroxylation is measured separately.
  • 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 4 Determination of the metabolic distribution of water-soluble hydroxylated ruthenium metal fullerene in vivo by radiolabeling
  • 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 131 I, and the gamma ray emitted by 131 I can be used for high sensitivity to detect its metabolic behavior in the living body.
  • a 37 MBq Na 131 I solution (volume ratio of 2:5) was oxidized using a chloramine-T aqueous solution (10 mg/mL) for 10 minutes, 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. 4. It can be seen from Fig. 4 that the results obtained by the ICP ion concentration test method are similar, which proves that the water-soluble hydroxylated ruthenium metal fullerene material can have a significant enrichment effect in the bone.
  • 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.
  • Example 5 Determination of bone marrow enrichment by electron probe
  • 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 intravenous injection of 150 ⁇ L of water-soluble hydroxylated ruthenium metal fullerene at a concentration of 1 mM, The mice were sacrificed by cervical dislocation before injection, 0.5 h, 1 h, 4 h and 24 h after injection, and the bilateral tibia of the mice were immediately dissected. 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.
  • Control group The same treatment as the fight group was used for comparison, and the water-soluble hydroxylated ruthenium metal fullerene was replaced by physiological saline.
  • Water-soluble hydroxylated hollow fullerene group 150 ⁇ L of water-soluble hydroxylated hollow fullerene at a concentration of 1 mM was injected intravenously. At 30 days after injection, the mouse eyeballs were taken and blood was taken and placed in a 5 ml centrifuge tube at 3500 rpm. After centrifugation for 15 min, the obtained serum was transferred to a 200 ⁇ L centrifuge tube, and blood biochemical indicators, 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.
  • Control group The same treatment as the fight group was used for comparison, and the water-soluble hydroxylated hollow fullerene was replaced by physiological saline.
  • Example 8 water-soluble hydroxylated ruthenium metal fullerene (GFNC) scavenging free radicals in vitro
  • ESR Electron paramagnetic resonance
  • Fig. 12 The corresponding test results are shown in Fig. 12. It can be seen from Fig. 12 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, and the cells are protected from hydrogen peroxide. The harmful effects of free radicals produced.
  • GFNC Water-soluble hydroxylated ruthenium metal fullerene
  • 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 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 GFNC and add at the same time. After incubated with the cells for 3 h, the medium containing GFNC 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. 13 The corresponding test results are shown in Fig. 13. It can be seen from Fig. 13 that when 20 ⁇ L of a 100 ⁇ M aqueous solution of hydrogen peroxide 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.
  • ESR Electron paramagnetic resonance
  • Fig. 14 The corresponding test results are shown in Fig. 14. It can be seen from Fig. 14 that in the experimental group, when the concentration of the hydroxylated hollow fullerene aqueous solution is only 20 ⁇ M, the free radicals generated by ultraviolet light irradiation of hydrogen peroxide can be effectively quenched. Protect cells from the damage of free radicals produced by hydrogen peroxide.
  • Hydroxylated hollow fullerene aqueous solution protects cells from the free radical scavenging effect of hydrogen peroxide:
  • 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 the experimental group was as follows: first add hydrogen peroxide and cell culture medium, and incubate with mouse bone marrow cells for 1 h. 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 aqueous solution of hydroxylated hollow fullerene, add the cell culture medium, and incubate the cells for 3 hours, then absorb the hydroxylated hollow rich The medium of the aqueous solution of the entrained solution was washed three times with PBS buffer; finally, the cell culture medium was added, and the culture was continued for 24 hours in the cell culture incubator, and then the cell activity was measured with CCK-8.
  • Fig. 15 The corresponding test results are shown in Fig. 15. It can be seen from Fig. 15 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 10 Water-soluble hydroxylated ruthenium metal fullerene at the living level of chemotherapy leads to protection against myelosuppression
  • 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.
  • 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.
  • 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. 16 The corresponding test results are shown in Fig. 16. From Fig. 16, 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: white blood cells, platelets, and hemoglobin There were different degrees of reduction in the body of mice, among which the decrease of white blood cells was the most obvious, and the abnormal growth of monocytes appeared, which also indicated that the bone marrow was damaged, and the related indicators had obvious abnormalities; In the CTX+GFNC experimental group, the amount of white blood cells, platelets, and hemoglobin was significantly improved compared with the cyclophosphamide (CTX) experimental group due to the protective effect of GFNC. The value of monocytes was even higher. Close to the normal group, and with the extension of time, the relevant indicators are getting closer to the value of normal mice, indicating that GFNC has a significant protective effect on mouse bone marrow suppression caused by the chemotherapy drug CTX.
  • CTX
  • Example 11 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 of mouse body weight, and the GFNC solution was intravenously administered. The dosage of the drug was 0.04 mmol Gd 3+ /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
  • Example 12 Water-soluble hydroxylated hollow fullerenes at the living level of chemotherapy lead to protection against myelosuppression
  • 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 mice were injected with GFNC aqueous solution (1 mM) in the tail vein, and the dosage of the drug was 0.004 mmol of water-soluble hydroxylated hollow fullerenes/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. 21 The corresponding test results are shown in Fig. 21. From Fig. 21, 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: white blood cells, platelets, and hemoglobin There were different degrees of reduction in the body of mice, among which the decrease of white blood cells was the most obvious, and the abnormal proliferation of monocytes appeared, which also indicated that the bone marrow was damaged, and the related indexes had obvious abnormalities; while CTX+ water-soluble hydroxylation In the mice in the hollow fullerene experimental group, the amount of white blood cells, platelets and hemoglobin was significantly improved compared with the cyclophosphamide (CTX) experimental group due to the protective effect of water-soluble hydroxylated hollow fullerenes.
  • 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.
  • Example 13 X-ray of water-soluble hydroxylated ruthenium metal fullerene at the living level leads to protection against myelosuppression
  • 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 irradiation + GFNC 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 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.
  • X-ray irradiation experimental group The mouse was intraperitoneally irradiated with XG rays at a dose of 8 Gy.
  • X-ray irradiation group + GFNC experimental group The mouse was intraperitoneally irradiated with XG rays at a dose of 8 Gy, and the GFNC solution was intravenously injected at a dose of 0.004 mmol GFNC/kg of mouse body weight.
  • X-ray irradiation was started on the seventh day after tumor inoculation.
  • GFNC was intravenously administered once every 5 days for the fourth day, seventh day, tenth day, and fourteenth.
  • blood was collected from the eye of the mouse (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 bone marrow suppression were white blood cell count (WBC) and red blood cell count ( RBC), hemoglobin measurement (HGB), monocyte ratio (MO%).
  • the indexes related to myelosuppression in the mice in the X-ray irradiation experimental group are: white blood cells, red blood cells, and hemoglobin in mice.
  • mice in the group due to the protective effect of GFNC, the amount of white blood cells, red blood cells and hemoglobin were greatly improved compared with the X-ray experimental group, and the value of monocytes was further increased. Near the normal group, and with the prolongation of time, the relevant indicators are getting closer to the values of normal mice, indicating that GFNC has a significant protective effect on mouse bone marrow suppression caused by X-ray irradiation.
  • Example 14 Effect of water-soluble hydroxylated ruthenium metal fullerene on the efficacy of X-ray tumor radiotherapy 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, X-ray irradiation experimental group and X-ray irradiation + GFNC 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.
  • Blank control group The drugs injected in the experimental group were replaced with the same volume of physiological saline.
  • mice received X-ray irradiation at a dose of 6 Gy at a time.
  • GFNC Metal fullerene derivative
  • CTX+GFNC experimental group The mouse was intraperitoneally irradiated with 6Gy dose of X-rays, and GFNC solution was injected intravenously. The dosage of the drug was 0.04mmol Gd 3+ /kg mouse body weight.
  • mice changed.
  • the weight of the mice in the X-ray experimental group showed a significant decrease in the initial stage of the fight. After stopping the fight, the recovery function of the mouse itself was somewhat restored, but it was still the lightest.
  • the group while the mice in the X-ray + GFNC experimental group, although the body weight decreased slightly at the beginning of the fight, but significantly recovered in the later stage, and close to the weight value of normal mice, indicating that GFNC can protect the mice It is possible to protect against the side effects of X-ray radiation and protect the bone marrow suppression toxicity of mice. At the same time, it is less toxic and can be further used in clinical research.
  • the fullerene and/or metal fullerene micro-nano materials used in the present invention can be metabolized not only outside the living body but also enriched in the bone marrow through blood circulation after intravenous injection, due to its high efficiency of scavenging free radicals. It has excellent anti-radiotherapy-induced myelosuppression in vivo, does not affect the therapeutic effect of radiotherapy and chemotherapy on tumors, and can effectively reduce the side effects of radiotherapy and chemotherapy on bone marrow and other organs. At the same time, it has no obvious cytotoxicity to bone marrow cells and normal cells, and is safe and non-toxic.

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Abstract

一种富勒烯和/或金属富勒烯微纳材料及其应用。所述富勒烯和/或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，使其可以通过骨髓血窦的内皮细胞间隙进入骨髓中。

Description

[根据细则37.2由ISA制定的发明名称]　一种富勒烯和/或金属富勒烯微纳材料及其应用 技术领域
本发明涉及一种微纳材料及其在骨髓抑制中的应用，属于生物医药领域。
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。虽然近年来新兴了多种治疗癌症的方法手段，但是目前化疗仍然是恶性肿瘤的临床常用的治疗方法之一。众所周知，大多数化疗药物在杀伤癌细胞的同时，亦损伤人体的正常细胞，有较大的副作用，尤其是对于骨髓细胞的杀伤作用可能会对病人产生二次伤害甚至于患上白血病等疾病，严重影响病人的生活质量及其在临床上的应用。目前60％-70％的肿瘤病人需要放化疗，一般手术后2到4周进行合理的放化疗可以及时杀灭“转移灶”，有效抵制肿瘤的复发和转移，而放化疗能否坚持的关键是骨髓造血细胞的保护。然而，许多放化疗均能引起不同程度的骨髓抑制，最初表现为白细胞，血小板下降，随着剂量的增加，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。因此，寻找能够减弱放化疗药物造成的骨髓抑制毒性，又不影响其放化疗抑制肿瘤作用的理想药物具有重要临床意义。在防治骨髓造血抑制中，不少的中西医药物虽有具有一定的疗效，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造血生长因子(HGFs)，但因价格昂贵及自身缺陷，目前尚不能广泛使用。
综上，能够找到一种对于降低放化疗药物诱导的骨髓抑制的方法对于减轻患者病情具有重要临床研究价值。
发明内容
本发明的目的是提供一种微纳材料及其在骨髓抑制中的应用。
本发明所述的富勒烯和/或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)，使其可以通过骨髓血窦的内皮细胞间隙进入骨髓中。
本发明所述的富勒烯和/或金属富勒烯微纳材料是由富勒烯和/或金属富勒烯本体材料经水溶性修饰得到的，所述的富勒烯和/或金属富勒烯微纳材料是富勒烯和/或金属富勒烯水溶性衍生物。
所述的富勒烯和/或金属富勒烯微纳材料的颗粒尺寸范围为1-500nm，具体可为140-200nm。
本发明所述的富勒烯和/或金属富勒烯本体材料既包括空心富勒烯C2n等，亦 包括内嵌金属富勒烯，主要种类有M@C2n、M2@C2n、MA@C2n、M3N@C2n、M2C2@C2n、M2S@C2n、M2O@C2n和MxA3-xN@C2n；其中，M、A均代表金属元素，所述M、A均选自Sc、Y和镧系金属元素(La-Lu)中的任意一种；30≤n≤60；0≤x≤3。
为了获得具有刚性结构的水溶性修饰的富勒烯和/或金属富勒烯微纳材料，有效的途径是通过在共价或非共价作用将非水溶性富勒烯和/或金属富勒烯本体材料经水溶性修饰得到。常用的共价修饰可以通过在碱性条件下的固液反应制备得到。例如，通过富勒烯和/或金属富勒烯固体粉末与H2O2在碱性下反应，即可制备羟基衍生物；通过富勒烯和/或金属富勒烯固体粉末与H2O2和氨反应，可制备氨基衍生物，均能达到水溶性修饰的目的。非共价键修饰方法可以使用常规水溶性载体，如脂质体、聚合物胶束、蛋白等通过疏水-疏水相互作用形成水溶性修饰的富勒烯和/或金属富勒烯微纳材料。上述修饰方法均可按照现有技术公开的方法进行修饰。
所述的水溶性修饰的富勒烯和/或金属富勒烯微纳材料具体可为在所述富勒烯和/或金属富勒烯本体材料表面修饰亲水性官能团羟基、氨基和羧基中的至少一种，或者，通过疏水-疏水相互作用(非共价共价键)使所述富勒烯和/或金属富勒烯本体材料与水溶性载体(如：脂质体、聚合物胶束、蛋白等)形成所述的水溶性富勒烯和/或金属富勒烯微纳材料。
所述水溶性载体为医学中常用的药物载体，具体可选自脂质体、聚合物胶束和蛋白中的至少一种。
其中，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。所述蛋白为白蛋白或转铁蛋白。
所述富勒烯和/或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)或水溶性羟基化空心富勒烯(C60(OH)n)。
本发明所述水溶性富勒烯和/或金属富勒烯微纳材料在水溶液中一般具有1-500nm的尺寸，并且该微纳材料具有一定的刚性(不易变形)，使得微纳材料经血液循环易于穿过骨髓血窦中内皮细胞间的纳米孔隙，能够利用血管内外压力差即可快速地进入骨髓中。
目前已报道的基于富勒烯和/或金属富勒烯相关的亲骨试剂大多需要借助结合磷脂基团靶向骨组织(PCT/US2001/013445；CN 200610011777)，制备流程繁琐，聚集量低，时间久。本发明借助微纳材料与骨髓血窦内皮细胞的特殊结构性质，无需进一步衍生物，即可实现在骨髓中的富集，而且可以代谢出生物体外。
本发明所述的富勒烯和/或金属富勒烯微纳材料在制备靶向富集于骨髓的药物或药物载体中的应用也属于本发明的保护范围。
本发明的目的之二是提供一种将富勒烯和/或金属富勒烯微纳材料快速富集于骨髓中的方法。
本发明所提供的方法，包括如下步骤：通过静脉注射富勒烯和/或金属富勒烯微纳材料，经血液循环，借助骨髓血窦中的特殊结构(内皮细胞间的纳米孔隙)，快速进入骨髓，并富集于骨髓处。
以水溶性钆羟基化金属富勒烯为例，静脉注射小鼠体内，分别于不同时间点脱颈处死小鼠，取其脏器和骨组织，同时使用ICP-MS测定钆离子浓度和131I放射性标记方法测试其体内代谢分布。两种不同的方法均表明我们得到的药物分子可以快速的富集在小鼠骨中，并且通过进一步的分析，我们发现，水溶性羟基化钆金属富勒烯在骨髓中的单位质量含量是骨质的12倍之多，说明其具有良好的骨髓靶向性。
本发明中，以水溶性富勒烯和/或金属富勒烯微纳材料作为骨髓靶向剂，其能高效快速地富集于骨髓中，并且具有良好的生物相容性，同时具有高效清除自由基的能力，可以进一步用于保护骨髓细胞。通过静脉注射进入小鼠体内后，可以在几小时内迅速富集于骨髓中，在骨髓中的停留时间明显高于在在其他器官中的时间，并且材料可以在生物体内代谢出去，对活体无明显的毒副作用，因此是一种制备纯化简单快速，生物相容性好，在骨髓区域可以快速富集，作为一种骨髓保护剂或者骨疾病的治疗平台具有良好的应用前景。
本发明所述的富勒烯和/或金属富勒烯微纳材料在制备具有如下1)-5)中的至少一种性质的药物或药物载体中的应用也属于本发明的保护范围：1)预防和/或治疗骨髓抑制；2)清除自由基；3)骨髓富集；4)预防和/或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；5)保护骨髓细胞和/或造血细胞。
本发明所述水溶性富勒烯和/或金属富勒烯微纳材料在水溶液中一般具有1-500nm的尺寸，并且该微纳材料具有一定的刚性(不易变形)，使得微纳材料经血液循环易于穿过骨髓血窦中内皮细胞间的纳米孔隙，能够利用血管内外压力差即可快速地进入骨髓中。
上述应用中，所述骨髓抑制可由药物化疗、化学毒物、X射线或具有骨髓抑制副作用的药物诱导产生，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，如环磷酰胺(CTX)等；亦可以由于化学毒物诱导产生，如苯及其衍生物等；还可以服用具有骨髓抑制副作用的药物诱导，如氯霉素、四环素 或消炎痛等，亦或是经X射线等放射线辐射。
本发明的另一个目的是提供一种利用富勒烯和/或金属富勒烯微纳材料预防和/或治疗骨髓抑制的方法。
本发明所提供的骨髓抑制方法，包括如下步骤：向需要预防和/或治疗的骨髓抑制的生物体内注射有效剂量的所述富勒烯和/或金属富勒烯微纳材料，所述富勒烯和/或金属富勒烯微纳材料经血液循环穿过骨髓血窦中内皮细胞间的纳米孔隙，利用血管内外压力差即可快速地富集于骨髓中，借助富勒烯和/或金属富勒烯微纳材料高效清除自由基的特性，预防和/或治疗骨髓抑制。
本发明中所述的“有效剂量”是指当通过本发明的方法给予生物体富勒烯和/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。
所述富勒烯和/或金属富勒烯微纳材料是以其水溶液的形式存在，生物体体内使用浓度范围为0.1mM-10mM。
所述生物体为哺乳动物，如：人。
所述注射的方式具体可为静脉注射，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。
所述骨髓抑制可由药物化疗、化学毒物、X射线等放射线或具有骨髓抑制副作用的药物诱导产生，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，如环磷酰胺(CTX)、阿霉素、顺铂、紫杉醇等；亦可以由于化学毒物诱导产生，如苯及其衍生物(如苯丁酸氮芥)等；还可以服用具有骨髓抑制副作用的药物诱导，如氯霉素、四环素、他巴哇或消炎痛等。放疗包括目前临床上常规使用的X射线、伽马射线等。
本发明中对于化疗药物、X射线导致的骨髓抑制具有防护作用，富勒烯和/或金属富勒烯微纳材料可以在化疗药物使用24h内提前注射使用，同时在放化疗过程中配合使用，高效快速清除药物(如化疗药物等)诱导骨髓抑制过程中产生的自由基，可快速防治和修复骨髓抑制现象。
附图说明
图1为实施例1中制备的水溶性羟基化钆金属富勒烯粒径分布图。
图2为实施例2中制备的水溶性羟基化空心富勒烯粒径分布图。
图3为实施例3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4为实施例3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5a-f为实施例4中水溶性羟基化钆金属富勒烯样品和在不同时间点小鼠骨髓中的电子探针谱图。
图6为实施例5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7为实施例5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8为实施例5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9为实施例5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10为实施例5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11为实施例5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12为实施例8中水溶性羟基化钆金属富勒烯体外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13为实施例8中水溶性羟基化钆金属富勒烯在细胞水平清除自由基实验效果。
图14为实施例9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15为实施例9中C60水溶性衍生物在细胞水平清除自由基实验效果。
图16为实施例10中不同组别小鼠血常规指标。
图17为实施例11中不同组别小鼠肿瘤大小变化曲线。
图18为实施例11中不同组别小鼠体重变化曲线。
图19为实施例11中不同组别小鼠血生化指标。
图20为实施例11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21为实施例12中使用C60水溶性衍生物不同组别小鼠血常规指标。
图22为实施例13中不同组别小鼠血常规指标。
图23为实施例14中不同组别小鼠肿瘤大小变化曲线。
图24为实施例14中不同组别小鼠体重变化曲线。
实施发明的最佳方式
下面通过具体实施例对本发明进行说明，但本发明并不局限于此。
下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和材料，如无特殊说明，均可从商业途径获得。
下述各实施例中所用的环磷酰胺(CTX)购买于Sigma-Aldrich公司，货号为C0768。
实施例1、制备水溶性钆金属富勒烯羟基衍生物-水溶性羟基化钆金属富勒烯(简称GFNC)
1)将100mg Gd@C82固体粉末(购买于厦门福纳新材料科技有限公司)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3ml2M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，其相应的测试结果如图1所示，从图1可得知：其在水溶液中的平均粒径为140nm，粒径分布均一。
实施例2、制备水溶性空心富勒烯羟基衍生物-水溶性羟基化金属富勒烯
1)将100mg C60固体粉末(购买于厦门福纳新材料科技有限公司)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的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空心富勒烯，经DLS测定，其相应的测试结果如图2所示，从图2可得知：其在水溶液中的平均粒径为200nm，粒径分布均一。
实施例3、水溶性羟基化钆金属富勒烯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3所示，从图3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1中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4、放射性标记法测定水溶性羟基化钆金属富勒烯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 131 I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4所示，从图4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、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5、电子探针测定骨髓富集量
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5所示，从图5a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5b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5c-5f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6、水溶性羟基化钆金属富勒烯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。
对照组：与打药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6、图7和图8所示，打药组和对照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8可得知：明显看出打药组和对照组相比，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7、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。
对照组：与打药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9、图10和图11所示，打药组和对照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11可得知：明显看出打药组和对照组相比，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8、水溶性羟基化钆金属富勒烯(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12所示，从图12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。
2)水溶性羟基化钆金属富勒烯(GFNC)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13所示，从图13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9、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14所示，从图14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。
2)羟基化空心富勒烯水溶液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15所示，从图15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10、水溶性羟基化钆金属富勒烯在活体水平的化疗导致骨髓抑制的保护
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16所示，从图16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11、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17和18所示，从图17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18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19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20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12、水溶性羟基化空心富勒烯在活体水平的化疗导致骨髓抑制的保护
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GFNC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21所示，从图21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。
实施例13、水溶性羟基化钆金属富勒烯在活体水平的X射线导致骨髓抑制的保护
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射线辐照+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：小鼠腹腔一次性辐照8Gy剂量的X射线。
X射线辐照组+GFNC实验组：小鼠腹腔一次性辐照8Gy剂量的X射线，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是接受X射线辐照，作为开始实验的第一天，每天一次，连续5天静脉注射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)，红细胞计数(RBC)，血红蛋白测定(HGB)，单核细胞比例(MO％)。
相应的检测结果如图22所示，从图22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，红细胞，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射线辐照+GFNC实验组中的小鼠，由于GFNC的保护作用，其白细胞，红细胞，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X射线辐照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X射线肿瘤放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、X射线辐照实验组和X射线辐照+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：小鼠一次性接受6Gy剂量的X射线辐照。
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：小鼠腹腔一次性辐照6Gy剂量的X射线，同时静脉注射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辐照X射线，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23和24所示，从图23可得知：与X射线辐照实验组相比，X射线+GFNC实验组中的小鼠的肿瘤直径并没有增大，表明：GFNC不会影响X射线放射性疗法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24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X射线辐射所带来的副作用，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。
工业应用
本发明中所使用的富勒烯和/或金属富勒烯微纳材料不仅可以代谢出生物体外，而且静脉注射后，通过血液循环富集于骨髓中，由于其高效的清除自由基的效果，在体内具有优异的防治放化疗诱导的骨髓抑制的作用，不影响放化疗对于肿瘤的治疗效果，可以有效降低放化疗对于骨髓以及其他器官的毒副作用。同时，其对骨髓细胞及正常细胞没有明显的细胞毒性，安全无毒。

Claims (14)

  1. 一种富勒烯和/或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，不易变形，使其可以通过骨髓血窦的内皮细胞间隙进入骨髓中。
  2. 如权利要求1所述的富勒烯和/或金属富勒烯微纳材料，其特征在于：所述富勒烯和/或金属富勒烯微纳材料是由富勒烯和/或金属富勒烯本体材料经水溶性修饰得到的；
    所述的富勒烯和/或金属富勒烯微纳材料是富勒烯和/或金属富勒烯水溶性衍生物。
  3. 如权利要求1或2所述的富勒烯和/或金属富勒烯微纳材料，其特征在于：所述的富勒烯和/或金属富勒烯微纳材料的颗粒尺寸范围为1-500nm。
  4. 如权利要求2或3所述的富勒烯和/或金属富勒烯微纳材料，其特征在于：所述富勒烯和/或金属富勒烯本体材料选自空心富勒烯C2n、M@C2n、M2@C2n、MA@C2n、M3N@C2n、M2C2@C2n、M2S@C2n、M2O@C2n和MxA3- xN@C2n中的任一种，其中，M和A均为金属元素，所述M和A均选自Sc、Y和镧系金属元素中的任意一种；30≤n≤60；0≤x≤3。
  5. 如权利要求2-4中任一项所述的富勒烯和/或金属富勒烯微纳材料，其特征在于：所述水溶性修饰的富勒烯和/或金属富勒烯微纳材料为在所述富勒烯和/或金属富勒烯本体材料表面修饰亲水性官能团羟基、氨基和羧基中的至少一种；
    或者，通过疏水-疏水相互作用使所述富勒烯和/或金属富勒烯本体材料与水溶性载体形成所述的水溶性富勒烯和/或金属富勒烯微纳材料。
  6. 如权利要求5所述的富勒烯和/或金属富勒烯微纳材料，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，和/或，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。
  7. 如权利要求1-6中任一项所述的富勒烯和/或金属富勒烯微纳材料，其特征在于：所述的富勒烯和/或金属富勒烯微纳材料为水溶性羟基化钆金属富勒烯或水溶性羟基化空心富勒烯。
  8. 权利要求1-7中任一项所述的富勒烯和/或金属富勒烯微纳材料在制备靶向富集于骨髓的药物或药物载体中的应用。
  9. 权利要求1-7中任一项所述的富勒烯和/或金属富勒烯微纳材料富集于骨髓中的方法，包括如下步骤：通过静脉注射所述富勒烯和/或金属富勒烯微纳材料，经血液循环，借助骨髓血窦中的特殊结构，进入骨髓，并富集于骨髓处。
  10. 权利要求1-7中任一项所述的富勒烯和/或金属富勒烯微纳材料在制备具 有如下1)-5)中至少一种性质的药物或药物载体中的应用：1)预防和/或治疗骨髓抑制；2)清除自由基；3)骨髓富集；4)预防和/或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；5)保护骨髓细胞和/或造血细胞。
  11. 如权利要求10所述的应用，其特征在于：所述骨髓抑制由药物化疗、化学毒物、放射性射线或具有骨髓抑制副作用的药物诱导产生。
  12. 如权利要求11所述的应用，其特征在于：所述药物化疗中的药物为环磷酰胺、阿霉素、顺铂或紫杉醇；所述化学毒物为苯或苯的衍生物；所述放射性射线为X射线或伽马射线；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。
  13. 利用权利要求1-7中任一项所述的富勒烯和/或金属富勒烯微纳材料预防和/或治疗骨髓抑制的方法，包括如下步骤：向需要预防和/或治疗的骨髓抑制的生物体内注射有效剂量的所述富勒烯和/或金属富勒烯微纳材料，所述富勒烯和/或金属富勒烯微纳材料经血液循环穿过骨髓血窦中内皮细胞间的纳米孔隙，利用血管内外压力差即可快速地富集于骨髓中，预防和/或治疗骨髓抑制。
  14. 如权利要求13所述的方法，其特征在于：所述富勒烯和/或金属富勒烯微纳材料是以其水溶液的形式存在，浓度范围为0.1mM-10mM；
    所述生物体为哺乳动物；
    所述注射的方式为静脉注射；
    所述骨髓抑制由药物化疗、化学毒物、放射性射线或具有骨髓抑制副作用的药物诱导产生，所述药物化疗中的药物具体为环磷酰胺、阿霉素、顺铂或紫杉醇；所述化学毒物具体为苯或苯的衍生物；所述放射性射线为X射线或伽马射线；所述具有骨髓抑制副作用的药物具体为氯霉素、四环素或消炎痛。
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