CN108201540A - Application of Fullerene Structure in Preparation of Drugs for Treating Leukemia - Google Patents

Abstract

The invention discloses an application of a fullerene structure in preparing a medicament for treating leukemia, wherein the fullerene structure comprises at least one selected from the following active ingredients: an oil-soluble fullerene, an oil-soluble endohedral metallofullerene, a composition of said oil-soluble fullerene and said oil-soluble endohedral metallofullerene, a water-soluble fullerene, a water-soluble endohedral metallofullerene, a composition of said water-soluble fullerene and said water-soluble endohedral metallofullerene, a pharmaceutically acceptable ester of the above six or a pharmaceutically acceptable salt of the above six. The active ingredients of the application can be efficiently enriched in bone marrow, the effect is fast, and the biocompatibility is good; and can be used for the prevention of leukemia before the leukemia model matures; when the composition is used after the onset of leukemia, the composition can rapidly prevent and repair damaged hematopoietic function of bone marrow and other organ injuries in leukemia due to the characteristic of targeted enrichment of bone marrow parts of the effective components.

Description

Application of Fullerene Structure in Preparation of Drugs for Treating Leukemia

technical field

The invention relates to the field of medicine, in particular to the application of a fullerene structure in the preparation of medicines for treating leukemia.

Background technique

In recent years, with the change of the environment, the incidence of leukemia is getting higher and higher, seriously endangering people's health. Leukemia is a malignant clonal disease of hematopoietic stem cells. After suffering from leukemia, due to mechanisms such as uncontrolled proliferation of leukemia cells, impaired differentiation, and blocked apoptosis, a large number of proliferation and accumulation in hematopoietic tissues will infiltrate other non-hematopoietic tissues, which will inhibit normal hematopoietic function, and clinically, various degrees of anemia and hemorrhage will occur. , hepatosplenomegaly, bone pain and other symptoms. According to different standards, leukemia can have different classifications. According to the urgency of onset, it can be divided into acute leukemia and chronic leukemia; according to the classification of diseased cell series, leukemia is often divided into lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, etc., among which: diseased cells include myeloid myeloid, Mono, erythroid, megakaryoline and lymphoid T and B cell lines. According to reports, the incidence of leukemia in various regions of my country ranks sixth among various tumors. Although a variety of methods and drugs for the treatment of leukemia have emerged in recent years, such as the introduction of emerging targeted drugs and immunotherapy methods into China, they are still not widely used due to their mediocre effects, drug resistance, and high cost. Therefore, radiotherapy and chemotherapy are still commonly used in the clinical treatment of leukemia. As we all know, while most radiotherapy and chemotherapy kill cancer cells, they also damage the normal cells of the human body, which has relatively large side effects, especially the killing effect on bone marrow cells may cause secondary damage to patients and even aggravate leukemia, which has a serious impact Quality of life of patients and its clinical application. However, bone marrow transplantation, which is recognized as a thorough cure for leukemia, first needs to wait for the bone marrow that matches the patient, and at the same time requires a large amount of funds to ensure that there is still a risk of recurrence after bone marrow transplantation. Many patients suffer from the pain and discomfort caused by transplantation. Fee is unwilling to accept a second transplant.

To sum up, it is possible to find a drug that can treat common leukemia (mainly acute lymphoblastic leukemia, acute myeloid leukemia, acute monocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia), alleviate the patient's condition, and have less side effects , has important clinical research value.

Fullerene is another allotrope of carbon besides graphite, diamond and amorphous carbon. This type of substance refers to a cage-like structure composed of carbon atoms. The most abundant molecule is C60, followed by C70 and C84, followed by relatively small amounts of C76, C78, and C82. In addition, because the carbon cage of fullerene is a cavity structure, different atoms, ions or atomic clusters can be embedded in the inner cavity, which is called an embedded fullerene, such as La@C60, which means that La is embedded in C60 In the cage structure of , @ means at, which vividly expresses the embedded meaning.

The information disclosed in this Background section is only for enhancing the understanding of the general background of the present invention and should not be taken as an acknowledgment or any form of suggestion that the information constitutes the prior art that is already known to those skilled in the art.

Contents of the invention

The purpose of the present invention is to provide the application of a fullerene structure in the preparation of medicines for treating leukemia. Another purpose of the present invention is to provide a pharmaceutical composition and method for treating leukemia. The active ingredient fullerene structure involved in the present invention can be efficiently enriched in the bone marrow, has a fast onset of action, can quickly kill leukemia cells, and prevent and repair damaged bone marrow hematopoietic function and other organ damage in leukemia.

In order to achieve the purpose, the invention provides the following technical solutions:

An application of a fullerene structure in the preparation of a drug for treating leukemia, wherein the fullerene structure includes at least one active ingredient selected from the group consisting of: oil-soluble fullerene, oil-soluble embedded metal fullerene Compositions of alkenes, the oil-soluble fullerene and the oil-soluble metallofullerene, water-soluble fullerene, water-soluble metallofullerene, the water-soluble fullerene The composition of alkenes and the water-soluble metallofullerene embedded in them, the pharmaceutically acceptable esters of the above six or the pharmaceutically acceptable salts of the above six.

The present invention also provides a method for treating leukemia, comprising administering an effective amount of a fullerene structure to a subject suffering from leukemia, the fullerene structure comprising at least one active ingredient selected from the group consisting of: oil-soluble fullerenes, oil-soluble endometallofullerenes, compositions of the oil-soluble fullerenes and the oil-soluble endometallofullerenes, water-soluble fullerenes, water-soluble endometallofullerenes Metallofullerene, the composition of the water-soluble fullerene and the water-soluble intercalated metallofullerene, the pharmaceutically acceptable ester of the above six or the pharmaceutically acceptable salt of the above six.

The present invention also provides a pharmaceutical composition for treating leukemia, comprising at least one fullerene structure selected from the group consisting of oil-soluble fullerenes, oil-soluble embedded metallofullerenes, the oil-soluble Composition of fullerene and said oil-soluble intercalated metallofullerene, water-soluble fullerene, water-soluble intercalated metallofullerene, said water-soluble fullerene and said water-soluble A composition embedded with metallofullerenes, a pharmaceutically acceptable ester of the above six, a pharmaceutically acceptable salt of the above six; the pharmaceutical composition also includes a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent and At least one of the pharmaceutically acceptable excipients.

In another embodiment of the above application, method or pharmaceutical composition, the oil-soluble fullerene is a fullerene whose outer surface of the carbon cage is coated with an oil solution, and the oil-soluble metallofullerene embedded It is an embedded metal fullerene coated with an oil solution on the outer surface of the carbon cage, for example: a fullerene coated with olive oil on the outer surface of the carbon cage, and an embedded metal fullerene coated with sunflower oil on the outer surface of the carbon cage alkene.

In another embodiment of the above application, method or pharmaceutical composition, the oil solution may be a single component oil, or a mixed oil of different oil solutions. Usually vegetable oils, such as olive oil, linseed oil, sunflower oil, corn germ oil, etc., also include animal fats, such as squalane, etc.

In another embodiment of the above-mentioned application, method or pharmaceutical composition, the oil-soluble fullerene is obtained by oil-soluble modification of the fullerene raw material, and the oil-soluble embedded metallofullerene is obtained by It is obtained by modifying the embedded metal fullerene raw material for oil solubility.

In another embodiment of the above application, method or pharmaceutical composition, the oil solubility modification is to disperse at least one of the fullerene raw material and the embedded metal fullerene raw material in the oil solution to obtain Mixed solution; after the mixed solution is subjected to ball milling or ultrasonication, it is successively centrifuged to remove precipitates, and then the obtained supernatant is filtered to obtain the product. The obtained oil-soluble modified liquid.

In another embodiment of the above application, method or pharmaceutical composition, during the oil-soluble modification process, 0.05-500 mg of fullerene raw materials and/or embedded metallofullerene raw materials are dispersed in every 1 ml of oil solution, The disclosure of this range should be regarded as the disclosure of all values within the range, and options include 0.05-1 mg, 0.05-10 mg, 0.05-100 mg, etc.

In another embodiment of the above application, method or pharmaceutical composition, the mixed solution is subjected to ball milling or ultrasonication for 30 min-15 h.

In another embodiment, the above application, method or pharmaceutical composition, the water-soluble fullerene includes at least one fullerene selected from the following group: (1) the outer surface of the carbon cage is modified with a hydrophilic group Fullerenes; (2) Fullerenes supported by water-soluble carriers.

In another embodiment of the above application, method or pharmaceutical composition, the water-soluble intercalated metallofullerene comprises at least one intercalated metallofullerene selected from the following group: (1) the outer surface of the carbon cage An embedded metallofullerene modified with a hydrophilic group; (2) an embedded metallofullerene supported by a water-soluble carrier.

In another embodiment of the above application, method or pharmaceutical composition, the hydrophilic group includes one or more of hydroxyl, carboxyl, sulfhydryl and amino groups. Optionally: the water-soluble embedded metal fullerene is water-soluble hydroxylated Gd@C82, and the water-soluble fullerene is water-soluble hydroxylated C60 or water-soluble hydroxylated C70.

In another embodiment of the above application, method or pharmaceutical composition, the water-soluble carrier is a drug carrier commonly used in medicine, including at least one of liposomes, polymer micelles, and proteins. Optionally, the polymer micelle is polyethylene lactide polyethylene glycol (PEG-PLGA), polylysine or chitosan; the protein is albumin or transferrin.

In another embodiment of the above application, method or pharmaceutical composition, the water-soluble fullerene is obtained by water-soluble modification of the fullerene raw material; the water-soluble embedded metallofullerene It is obtained by water-soluble modification of embedded metal fullerene raw materials.

In another embodiment, the above-mentioned application, method or pharmaceutical composition, the method of water-soluble modification is any of the following methods: (1) The method of surface modification of hydrophilic groups is generally under the action of alkali Realized by solid-liquid or liquid-liquid reaction, specifically, at least one of the fullerene raw material and the embedded metal fullerene raw material is mixed with hydrogen peroxide and alkali and reacted, then washed with ethanol, and then dialyzed to obtain the The corresponding water-soluble hydroxy derivatives of the raw materials. If it is desired to obtain water-soluble aminated derivatives, the sodium hydroxide in the above steps can be replaced by ammonia water. (2) Non-covalent interaction The water-soluble carrier and fullerene raw material or embedded metal fullerene raw material form corresponding water-soluble materials through hydrophobic-hydrophobic interaction.

In another embodiment of the above application, method or pharmaceutical composition, the alkali may specifically be sodium hydroxide or potassium hydroxide.

In another embodiment of the above application, method or pharmaceutical composition, weigh 50-300 mg of C60 or C70 or Gd@C ₈₂ solid, 5-30 ml of 20-40% hydrogen peroxide, and 2-20 ml of 1M-3M alkali solution, mixed at 50-100°C until the corresponding C60 or C70 or Gd@C ₈₂ solids are completely dissolved. In this description, the proportion relationship between the various substances is shown. In practical application, it is not limited by the specific reaction scale of 50-300 mg, 5-30 ml and 2-20 ml, and can be expanded according to the proportion.

In another embodiment of the above application, method or pharmaceutical composition, the fullerene raw material includes one or more cage structures composed of carbon atoms with the general formula _C2m , 30≤m≤60, for example ; C ₆₀ , C ₇₀ , C ₈₄ , etc.

In another embodiment of the above application, method or pharmaceutical composition, the metallofullerene intercalation materials include M@C _2n , M ₂ @C _2n , MA@C _2n , M ₃ N@C _2n , M One or more of ₂ 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 represent metal elements and M , A are all selected from any one of lanthanide metal elements, Sc and Y, 30≤n≤60; 0≤x≤3. N represents nitrogen, C represents carbon, S represents sulfur, and lanthanide metal elements include La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

In another embodiment of the above-mentioned application, method or pharmaceutical composition, the oil-soluble fullerene, the oil-soluble embedded metal fullerene, the oil-soluble fullerene and the oil-soluble embedded metal The composition of fullerene, water-soluble fullerene, water-soluble embedded metal fullerene, the composition of the water-soluble fullerene and the water-soluble embedded metal fullerene, the above six The average particle diameter range of the pharmaceutically acceptable esters of the above six or the pharmaceutically acceptable salts of the above six is 1-1000nm, optionally 10-250nm or 10-150nm.

In another embodiment of the above application, method or pharmaceutical composition, the leukemia includes acute lymphoblastic leukemia, acute myeloid leukemia, acute monocytic leukemia, chronic lymphocytic leukemia, and chronic myeloid leukemia. The etiology of said leukemia can be caused by factors such as virus, chemistry, radiation and heredity.

In another embodiment of the above-mentioned application, method or pharmaceutical composition, the treatment of leukemia includes: 1) making at least one of leukocyte abnormality, platelet abnormality, hemoglobin abnormality and red blood cell abnormality caused by leukemia tend to be normal, such as: Increase the decreased white blood cell count, decreased platelet count, decreased hemoglobin count and decreased red blood cell count caused by leukemia by at least 20%; 2) reduce the leukemic cell content by at least 20%; 3) alleviate the liver and spleen caused by leukemia 4) Relieve the bone pain and fatigue caused by leukemia.

The drug or the above pharmaceutical composition in the above application In another embodiment, the drug or the pharmaceutical composition can be tablet, pill, powder, lozenge, sachet, cachet, elixir, suspension, Preparations as emulsions, solutions, syrups, aerosols, ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions or powders for sterile packaging. In the present invention, the method for preparing the active ingredient into a drug or a pharmaceutical composition can be prepared by methods known to those skilled in the art, so that the active ingredient is released quickly, slowly or delayed after being administered to a subject, for example: The active ingredient can be mixed with the carrier, diluted with the carrier or encapsulated in the carrier.

In another embodiment, the drug in the above application or the above pharmaceutical composition, some examples suitable as carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, resin, Gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methylcellulose, methylparaben and Propyl Ester, Talc, Magnesium Stearate and Liquid Paraffin.

In another embodiment, the medicine or the above-mentioned pharmaceutical composition in the above application, the medicine or the pharmaceutical composition may additionally include a lubricant, a wetting agent, an emulsifying and suspending agent, a preservative, a sweetener or a flavoring agent and other additives.

In another embodiment of the drug in the above application or the above pharmaceutical composition, the concentration of the water-soluble fullerene and/or water-soluble metal fullerene in the preparation is 0.1mM-10mM; The concentration of allerenes and/or oil-soluble metal fullerenes in the preparation is 500ppm-5000ppm (mg/kg).

In another embodiment of the above method, the active ingredient can be administered in a single dose or every other day for patients with mild leukemia, and multiple doses or administered for consecutive days for patients with severe leukemia. Leukemia symptoms can be significantly prevented and treated every other day or continuously.

In another embodiment of the above method, the subject is a leukemia patient or an animal, and the animal can be a mammal, such as a mouse, a rabbit, a monkey, and the like.

In another embodiment of the above method, the method of administration is injection or oral administration; optionally, the injection is intravenous injection, the active ingredient enters the body and directly exerts its effect through blood circulation without penetration, and the dose of medicine used is small , high curative effect; it can also be taken orally, filtered and absorbed through the digestive system, with less side effects and significant curative effect.

The active ingredients in the present invention have all the following properties: (1) the surface is hydrophilic or lipophilic, so that it can enter the living body through intravenous injection or oral administration and be enriched in the bone marrow through blood circulation; (2) The water-soluble active ingredients are rigid (that is, not easy to deform), so that they can pass through the endothelial cell gap of the bone marrow sinus and enter the bone marrow by utilizing the pressure difference between the inside and outside of the blood vessel; (3) The fat-soluble characteristics of the fat-soluble active ingredients make it easier to enter cells, so that it can be injected into the living body through gastric or intraperitoneal injection and reach organs through blood circulation.

The term "treating" as used herein includes its generally accepted meaning, which includes arresting, preventing, inhibiting, ameliorating and slowing down, halting or reversing the development of the resulting symptoms or desired pathological changes. As such, the invention encompasses both therapeutic and prophylactic administration.

The terms "active ingredient", "active ingredient fullerene structure" or "fullerene structure" used in the present invention all refer to oil-soluble fullerenes, oil-soluble embedded metal fullerenes, the oil-soluble The composition of the fullerene and the oil-soluble embedded metal fullerene, the water-soluble fullerene, the water-soluble embedded metal fullerene, the water-soluble fullerene and the water-soluble At least one of the metallofullerene-embedded composition, the pharmaceutically acceptable ester of the above six, or the pharmaceutically acceptable salt of the above six.

The term "effective amount" used in the present invention refers to the amount or dose of the active ingredient that provides the desired effect to the patient being diagnosed or treated by single or multiple administrations to the patient. An effective amount can be determined by the attending diagnostician as a person skilled in the art by known techniques and observations made under similar circumstances. In determining the effective amount or dose of active ingredient to administer, the participating diagnostic physician should consider a variety of factors including, but not limited to: the species of mammal; size, age, and general health; the particular disease involved ; the degree of involvement or severity of the disease; the response of the individual patient; the specific compound administered; the mode of administration; the bioavailability properties of the formulation administered; the selected dosing regimen; the use of concomitant drug therapy; and others related circumstances.

The term "fullerene raw material" used in the present invention refers to fullerenes that have not undergone water-solubility modification or oil-solubility modification, that is, fullerene bulk.

The term "intercalated metallofullerene raw material" used in the present invention refers to the intercalated metallofullerene without water-solubility modification or oil-solubility modification, that is, the intercalated metallofullerene body.

For the convenience of metering, all quantitative limitations such as the specific content and concentration of water-soluble fullerenes, water-soluble metal fullerenes, oil-soluble fullerenes or oil-soluble metal fullerenes in the present invention are based on their corresponding rich It is measured by the specific content and concentration of the fullerene body or the embedded metal fullerene body, for example: the concentration of oil-soluble fullerene in the preparation is 0.1mM-10mM means that oil-soluble modified fullerene can be detected The concentration of the carbon cage of the fullerene body in the preparation is 0.1mM-10mM; another example: the content of the fullerene covered with the oil solution on the outer surface of the carbon cage is 100μM, which refers to the content of the fullerene body carbon cage in the oil solution is 100 μM.

Compared with the prior art, the active ingredient for treating leukemia in the present invention has the following effects:

1) The special physical and chemical properties of the active ingredient and its specific particle size enable it to quickly accumulate in the bone marrow through blood circulation, with fast onset and good biocompatibility; 2) The active ingredient maintains a rich Due to the high conjugation and efficient scavenging of free radicals of the lene molecule, it can be used before the onset of leukemia to prevent leukemia; when used after the onset of leukemia, because the active ingredient has the characteristics of targeted enrichment in the bone marrow, it can quickly Kill leukemia cells, quickly prevent and repair the damaged bone marrow hematopoietic function and other organ damage in leukemia; 3) The active ingredients have no obvious cytotoxicity to bone marrow cells and normal cells, are safe and non-toxic, and can be metabolized out of the body; 4) Effective The preparation of the ingredients is simple and fast, and can be used for industrialization.

Description of drawings

Figure 1 is the effect of water-soluble hydroxylated hollow fullerene in killing leukemia cells at the cellular level in Example 1, wherein Control represents the blank control group, PBS represents the negative control group, and 30 μM, 50 μM, 100 μM and 200 μM represent the 4 levels of the experimental group respectively. concentration group.

Fig. 2 is the effect of the water-soluble gadolinium hydroxylated metallofullerene in Example 2 on killing leukemia cells at the cellular level. Among them, Control represents the blank control group, PBS represents the negative control group, and 30 μM, 50 μM, 100 μM and 200 μM represent the four concentration groups of the experimental group, respectively.

Figure 3 is the effect of water-soluble hydroxylated hollow fullerene on human umbilical vein endothelial cells at the cellular level in Example 3, wherein Control represents the blank control group, and 30 μM, 50 μM, 100 μM, 200 μM and 500 μM represent the five samples of the experimental group respectively. concentration group.

Fig. 4 is the effect of water-soluble gadolinium hydroxylated metallofullerenes on human umbilical vein endothelial cells at the cellular level in Example 4, wherein Control represents the blank control group, and 30 μ M, 50 μ M, 100 μ M, 200 μ M and 500 μ M represent the 50 μ M of the experimental group respectively. concentration group.

Detailed ways

The specific embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

The experimental methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and materials, unless otherwise specified, can be obtained from commercial sources. The medium for leukemia cells used in the following examples is IMDM medium (Iscove's Modified Dulbecco Medium) supplemented with 20% FBS (fetal bovine serum). Both the IMDM medium and FBS were purchased from Beijing Baierdi Biotechnology Co., Ltd. The raw material Gd@C ₈₂ solid powder used in the following examples was purchased from Xiamen Funa New Material Technology Co., Ltd., with a molecular weight of 1141 and a purity of 99.1%. The raw material C60 solid powder used in the following examples was purchased from Xiamen Funa New Material Technology Co., Ltd., with a molecular weight of 720 and a purity of 99%. The CCK-8 test used in the following examples can reflect the number of viable cells, and commercial kits can be used for detection.

Preparation Example

Preparation Example 1

The water-soluble hydroxylated gadolinium metallofullerene is prepared by the following preparation method:

1) Add 300mg of Gd@C ₈₂ solid powder into a 300ml single-mouth bottle, add 20ml of 30% hydrogen peroxide aqueous solution and 10ml of 2M sodium hydroxide aqueous solution respectively, heat the oil bath to 70°C, and react for 6h;

2) After the reaction, use a dialysis bag with MW=3500 to remove small molecules, use a conductivity meter to monitor until the dialysis is completed, and the product obtained by ultrafiltration and centrifugation is water-soluble hydroxylated metal fullerene (Gd@C ₈₂ ) _m (OH) _n , measured by DLS, its average particle size in aqueous solution is 140-150nm, and the particle size distribution is uniform.

Preparation example 2

The water-soluble hydroxylated gadolinium metallofullerene is prepared by the following preparation method:

1) Add 50mg of Gd@C ₈₂ solid powder into a 100ml single-mouth bottle, add 7ml of 30% hydrogen peroxide aqueous solution and 3ml of 2M sodium hydroxide aqueous solution respectively, heat the oil bath to 50°C, and react for 2h.

2) After the reaction, use a dialysis bag with MW=3500 to remove small molecules, use a conductivity meter to monitor until the dialysis is completed, and the product obtained by ultrafiltration and centrifugation is water-soluble hydroxylated metal fullerene (Gd@C ₈₂ ) _m (OH) _n , measured by DLS, its average particle size in aqueous solution is 140-150nm, and the particle size distribution is uniform.

Preparation example 3

The water-soluble hydroxylated hollow fullerene is prepared by the following preparation method:

1) Add 200mg of C60 solid powder into a 200ml single-necked bottle, add 15ml of aqueous hydrogen peroxide solution with a volume fraction of 30% and 6ml of 2M aqueous sodium hydroxide solution, heat the oil bath to 70°C, and react for 5 hours;

2) After the reaction, use a dialysis bag with MW=3500 to remove small molecules, use a conductivity meter to monitor until the dialysis is completed, and the product obtained by ultrafiltration and centrifugation is the water-soluble hydroxylated fullerene (C ₆₀ ) _m (OH) _n , which is tested by DLS Measured, its average particle size in the aqueous solution is 150nm, and the particle size distribution is uniform.

Preparation Example 4

1) Add 50mg of C ₆₀ solid powder into a 100ml single-necked bottle, add 10ml of 30% hydrogen peroxide aqueous solution and 5ml of 2M sodium hydroxide aqueous solution respectively, heat the oil bath to 70°C, and react for 6h.

2) After the reaction, use a dialysis bag with MW=3500 to remove small molecules, use a conductivity meter to monitor until the dialysis is completed, and the product obtained by ultrafiltration and centrifugation is the water-soluble hydroxylated fullerene (C ₆₀ ) _m (OH) _n , which is tested by DLS Measured, its average particle size in the aqueous solution is 150nm, and the particle size distribution is uniform.

As mentioned above, the water-soluble hydroxylated hollow fullerene or water-soluble hydroxylated metal fullerene obtained after dialysis contains more liquid, and it can also be concentrated by ultrafiltration and centrifugation, but no matter whether it is concentrated or not, the raw material metal fullerene Or the water-soluble modification of the metallofullerene embedded in the raw material has been completed, whether it is concentrated or not does not affect its use, and it only needs to adjust the water-soluble fullerene or water-soluble metallofullerene to an appropriate concentration during use.

Preparation Example 5

Oil-coated hollow fullerenes and/or metallofullerenes are prepared by the following preparation method: 1) solid powder of fullerene raw materials and/or metallofullerene raw materials (purchased from Xiamen Funa New Material Technology Co., Ltd. ) and olive oil, linseed oil and other edible oils in different proportions (for example: 200mg of hollow fullerene C60 solid powder and 200ml of olive oil are mixed and ball milled), and ball milled in a ball mill for 6-12h; 2) Use a centrifuge after the reaction, The uncoated fullerene solid was repeatedly centrifuged at a rotational speed of 12000 r until no solid powder was centrifuged. 3) Use a UV-Vis spectrometer to test the specific concentration of the hollow fullerene/metal fullerene coated.

Example 1, the effect of water-soluble hydroxylated hollow fullerene on killing leukemia cells at the cellular level

1) Experimental objects: Kg-1a cells (human acute myeloid leukemia cells) and HL-60 cells (human acute promyelocytic leukemia cells) were selected as the leukemia cells studied.

2) Experimental method: Kg-1a cells and Kg-1a cells were planted in ⁹⁶ -well plates at a concentration of ca.

Blank control group: a total of 6 wells are required for each type of leukemia cells, the leukemia cells are seeded in the wells, the culture medium of leukemia cells is added to the wells, and water-soluble hydroxylated hollow fullerene is not added to the wells;

Experimental group: a total of 6 × 4 wells are required for each type of leukemia cells. Leukemic cells are seeded in the wells, and the culture medium of leukemia cells and the water-soluble hydroxylated hollow fullerene obtained in Preparation Example 3 are added to the wells. The experimental group is provided with 4 different concentrations of water-soluble hydroxylated hollow fullerene (making the final concentrations 30 μM, 50 μM, 100 μM and 200 μM respectively), 6 wells were set for each concentration, and the experiment was repeated three times;

Negative control group: a total of 6 wells are required for each leukemia cell, and the leukemia cells are seeded in the wells, and the culture medium of the leukemia cells and the same volume of PBS solution as the water-soluble hydroxylated hollow fullerene in the experimental group are added to the wells;

After adding samples, each group was incubated at 37°C and 5% CO ₂ for 24 hours, then the culture medium was aspirated and washed three times with PBS buffer; then the cell culture medium was added and incubated at 37°C and 5% CO ₂ The cell culture was continued for 24 hours in the incubator, and then the cell viability was detected with a CCK-8 kit (purchased from Beyontian Biotechnology Co., Ltd.), and the OD value was measured at a wavelength of 450 mM by a microplate reader.

3) Experimental results: as shown in Figure 1 and Table 1:

Table 1

Among them: Control represents the blank control group, PBS represents the negative control group, and 30 μM, 50 μM, 100 μM and 200 μM represent the four concentration groups of the experimental group, respectively.

Can know from Fig. 1 and table 1: along with the adding amount of water-soluble hydroxylated hollow fullerene increases gradually, the viability of two kinds of leukemia cells also declines to some extent, and the leukemia cell viability of negative control group all has risen. The above results show that: at the cellular level, the water-soluble hydroxylated hollow fullerene has a certain effect on the viability and killing of leukemia cells.

Example 2, the effect of water-soluble hydroxylated metal fullerenes on killing leukemia cells at the cellular level

1) Experimental objects: Kg-1a cells (human acute myeloid leukemia cells) and HL-60 cells (human acute promyelocytic leukemia cells) were selected as the leukemia cells studied.

2) Experimental method: Kg-1a cells and Kg-1a cells were planted in ⁹⁶ -well plates at a concentration of ca.

Blank control group: a total of 6 wells are required for each type of leukemia cells, and the leukemia cells are seeded in the wells, the culture medium of leukemia cells is added to the wells, and water-soluble hydroxylated metallofullerenes are not added to the wells;

Experimental group: a total of 6 × 4 wells are required for each type of leukemia cells. Leukemic cells are seeded in the wells, and the culture medium of leukemia cells and the water-soluble gadolinium hydroxylated metallofullerene obtained in Preparation Example 1 are added to the wells. The experimental group is set There are 4 different concentrations of water-soluble hydroxylated gadolinium metallofullerene (making the final concentrations 30 μM, 50 μM, 100 μM and 200 μM respectively), 6 wells are set for each concentration, and the experiment is repeated three times;

Negative control group: a total of 6 wells are required for each type of leukemia cells. Leukemia cells are seeded in the wells, and the culture medium of leukemia cells and PBS solution with the same volume as the water-soluble gadolinium hydroxylated metallofullerene in the experimental group are added to the wells. ;

After adding samples, each group was incubated at 37°C and 5% CO ₂ for 24 hours, then the culture medium was aspirated and washed three times with PBS buffer; then the cell culture medium was added and incubated at 37°C and 5% CO ₂ The cell culture was continued for 24 hours in the incubator, and then the cell viability was detected with a CCK-8 kit (purchased from Beyontian Biotechnology Co., Ltd.), and the OD value was measured at a wavelength of 450 mM by a microplate reader.

3) Experimental results: as shown in Figure 2 and Table 2:

Table 2

Among them: Control represents the blank control group, PBS represents the negative control group, and 30 μM, 50 μM, 100 μM and 200 μM represent the four concentration groups of the experimental group, respectively.

From Figure 2 and Table 2, it can be known that with the addition of water-soluble gadolinium hydroxylated metal fullerenes, the viability of the two types of leukemia cells also decreased, while the viability of leukemia cells in the negative control group Both have risen. The above results show that: at the cellular level, the water-soluble gadolinium hydroxylated metallofullerene has a certain effect on the viability and killing of leukemia cells. And the same concentration of water-soluble gadolinium hydroxylated metal fullerene can kill more leukemia cells, indicating that the effect of metal fullerene on treating leukemia is superior to that of hollow fullerene.

Example 3, the effect of water-soluble hydroxylated hollow fullerene on normal cells

1) Experimental method: replace the Kg-1a cells and HL-60 cells in Example 1 with HUVEC cells (human umbilical vein endothelial cells) normal cells, no negative control group was set, and an additional 500 μM concentration of water-soluble The hollow fullerene groups were selectively hydroxylated so that the final concentration in the wells was 500 μM, and the rest of the steps were exactly the same as in Example 1.

2) Experimental results: as shown in Figure 3 and Table 3:

table 3

Among them: Control represents the blank control group, and 20 μM, 50 μM, 100 μM, 200 μM and 500 μM respectively represent the five concentration groups of the experimental group.

From Figure 3 and Table 3, it can be known that as the addition of water-soluble hydroxylated hollow fullerenes gradually increased, the cell viability of human umbilical vein endothelial cells did not decrease. Vitality has also increased to a certain extent. It shows that: at the cell level, the water-soluble hydroxylated hollow fullerene has no harmful effect on normal human cells.

Embodiment 4, the effect of water-soluble gadolinium hydroxylated metal fullerene on normal cells

1) Experimental method: replace the Kg-1a cells and HL-60 cells in Example 2 with HUVEC cells (human umbilical vein endothelial cells) normal cells, no negative control group was set, and an additional 500 μM concentration of water-soluble Gadolinium hydroxylated metallofullerene groups were used to make the final concentration in the wells 500 μM, and the rest of the steps were exactly the same as in Example 2.

2) Experimental results: as shown in Figure 4 and Table 4,

Table 4

Among them: Control represents the blank control group, and 20 μM, 50 μM, 100 μM, 200 μM and 500 μM respectively represent the five concentration groups of the experimental group.

From Figure 4 and Table 4, it can be known that with the addition of water-soluble gadolinium hydroxylated metal fullerenes, the cell viability of human umbilical vein endothelial cells did not decrease. The activity also increased to a certain extent. It shows that: at the cell level, the water-soluble hydroxylated metal fullerene has no harmful effect on normal human cells.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling others skilled in the art to make and use various exemplary embodiments of the invention, as well as various Choose and change. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims (10)

1. The application of a fullerene structure in the preparation of a medicine for the treatment of leukemia, characterized in that: the fullerene structure includes at least one selected from the group consisting of oil-soluble fullerenes, oil-soluble embedded Metallofullerene, composition of the oil-soluble fullerene and the oil-soluble embedded metalfullerene, water-soluble fullerene, water-soluble embedded metalfullerene, the water-soluble The composition of the fullerene and the water-soluble inner metal fullerene, the pharmaceutically acceptable ester of the above six or the pharmaceutically acceptable salt of the above six. 2. A pharmaceutical composition for treating leukemia, characterized in that it includes at least one active ingredient selected from the group consisting of oil-soluble fullerenes, oil-soluble embedded metallofullerenes, oil-soluble fullerenes Composition of allerene and said oil-soluble endometallofullerene, water-soluble fullerene, water-soluble endometallofullerene, said water-soluble fullerene and said water-soluble endometallofullerene The composition of metallofullerene, the pharmaceutically acceptable ester of the above six substances or the pharmaceutically acceptable salt of the above six substances, also includes a pharmaceutically acceptable carrier, diluent or excipient. 3. application according to claim 1 or the pharmaceutical composition described in claim 2, is characterized in that: described oil-soluble fullerene is the fullerene that the outer surface of carbon cage is coated with oil solution, and described The oil-soluble intercalated metallofullerene is the intercalated metallofullerene whose outer surface of the carbon cage is coated with an oil solution. 4. application according to claim 3 or the pharmaceutical composition described in claim 3, is characterized in that: described oil solution comprises olive oil, linseed oil, sunflower seed oil, corn germ oil, squalane at least one. 5. The application according to claim 1 or the pharmaceutical composition according to claim 2, characterized in that: said water-soluble fullerene comprises at least one fullerene selected from the group consisting of: (1) surface A fullerene modified with a hydrophilic group; (2) a fullerene supported by a water-soluble carrier; the water-soluble embedded metallofullerene includes at least one embedded metallofullerene selected from the following group : (1) embedded metallofullerene with hydrophilic groups on the surface; (2) embedded metallofullerene supported by water-soluble carrier. 6. The application according to claim 5 or the pharmaceutical composition according to claim 5, characterized in that: the hydrophilic group includes one or more of hydroxyl, carboxyl, mercapto and amino; The carrier includes at least one of liposomes, polymer micelles, and proteins; optionally, the polymer micelles are polyethylene lactide polyethylene glycol (PEG-PLGA), polylysine or Chitosan; the protein is albumin or transferrin. 7. The application according to claim 1 or the pharmaceutical composition according to claim 2, characterized in that: said oil-soluble fullerene is obtained by carrying out oil-soluble modification of fullerene raw material; said oil Soluble embedded metallofullerenes are obtained by oil-soluble modification of embedded metallofullerene raw materials; the water-soluble fullerenes are obtained by water-soluble modification of fullerene raw materials; the water-soluble Permanent intercalated metallofullerenes are obtained by water-soluble modification of intercalated metallofullerene raw materials. 8. The application according to claim 1 or the pharmaceutical composition according to claim 2, characterized in that: the fullerene raw material comprises one or more general formulas of cages composed of carbon atoms of _C structure, 30≤m≤60; the embedded metallofullerene materials include M@C _2n , M ₂ @C _2n , MA@C _2n , M ₃ N@C _2n , M ₂ C ₂ @C _2n , M One or more of ₂ S@C _2n , M ₂ O@C _2n and M _x A _3-x N@C _2n , wherein: M and A both represent metal elements and M and A are selected from Sc and Y And any one of the lanthanide metal elements, 30≤n≤60; 0≤x≤3; N represents nitrogen, C represents carbon, S represents sulfur, and lanthanide metal elements include La, Ce, Pr, Nd , Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu. 9. The application according to claim 1 or the pharmaceutical composition according to claim 2, characterized in that: oil-soluble fullerene, oil-soluble embedded metallofullerene, said oil-soluble fullerene and the composition of the oil-soluble metallofullerene, water-soluble fullerene, water-soluble metallofullerene, the water-soluble fullerene and the water-soluble metallofullerene The average particle diameter of the composition of fullerenes, the pharmaceutically acceptable esters of the above six or the pharmaceutically acceptable salts of the above six is 1-1000nm, optionally 10-250nm. 10. The application according to claim 1 or the pharmaceutical composition according to claim 2, wherein said leukemia comprises acute lymphoblastic leukemia, acute myeloid leukemia, acute monocytic leukemia, chronic lymphocytic leukemia, Chronic myelogenous leukemia.