Application of method for regulating and controlling tumor metabolism and inducing ubiquitous apoptosis through intracellular organelle cascade injury

Abstract

The invention discloses a method and application for regulating and controlling tumor metabolism and inducing pan-apoptosis through intracellular organelle cascade injury. Under a starvation condition, the polyhydroxy fullerene material can force the damage degree of organelles to be greater than the repairable ability of the organelles, so that continuous interactive damage among the organelles is realized, positive feedback superposition of the damage is realized, cancer cells are induced to autonomously execute a pan-apoptosis process, and an efficient and low-toxicity mode is adopted, so that the damage degree of the organelles is reduced. And the development of tumor treatment methods is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for regulating tumor metabolism and inducing pan-apoptosis through cascade damage of intracellular organelles. Background Art

[0002] In the dynamic cytoplasmic environment, eukaryotic cells have many intracellular compartments, such as membrane-bound organelles, non-membrane structures, and the cytoskeleton. First, cytoskeletal components directly exert forces on organelles through actomyosin contraction, cytoskeletal dynamics, or motor protein-based transport. Second, the intertwined transport of organelles in the limited cytoplasmic space promotes physical contact between organelles. At the same time, most organelles are highly dynamic structures, and their rapid remodeling, such as fusion, fission, and tubulation, can work with the cytoskeleton to maintain the normal function of the cell. Organelle homeostasis is very important for maintaining the normal physiological activities of cells.

[0003] The most common organelles in eukaryotic cells include lysosomes, mitochondria, and endoplasmic reticulum. Lysosomes are catabolic organelles that control many cellular processes. They can replenish the cellular storage of amino acids, lipids, sugars, ions, and nucleotides, and provide fuel for cellular anabolic pathways. In addition, abnormal changes in lysosomal abundance are highly associated with many diseases. Mitochondria are important double-membrane-bound organelles and the main site of intracellular oxidative phosphorylation and ATP production. Under normal physiological functions of the body, the mitochondrial life cycle is in a dynamic equilibrium state. Changes in mitochondrial expression and activity are highly correlated with processes such as cell growth and differentiation, information transmission, metabolic needs, and cell apoptosis. As the convergence point of cell death pathways, mitochondria play a key role in apoptosis and non-apoptotic programmed cell death. The endoplasmic reticulum (ER) is the largest endomembrane organelle that controls many cellular functions, including protein folding / misfolding / unfolding, intracellular calcium homeostasis, protein secretion, etc. Although this process is precisely regulated, various external and intracellular factors can destroy protein folding ability and induce ER stress. In tumors, various genetic and transcriptional abnormalities, metabolic alterations, and microenvironmental perturbations (such as oxidative stress, oxygen limitation, nutrient deprivation, and high metabolic demand) enable cancer cells to achieve immune escape and enhance pro-survival mechanisms such as anti-apoptosis, thereby accelerating tumor progression.

[0004] For a long time, among the existing forms of programmed cell death (PCD), pyroptosis, apoptosis and necroptosis have been considered to operate in parallel with little overlap, and their respective complex molecular mechanisms are responsible for the initiation, transduction and execution of cell death, but in fact there is a complex crosstalk between these three pathways, and they can cross-regulate each other. Among them, the new death mode with the characteristics of apoptosis, pyroptosis and necroptosis can be summarized as pan-apoptosis, which has become a way to kill tumor cells, but the way to cause pan-apoptosis still needs to be explored. Summary of the invention

[0005] One of the objectives of the present invention is to provide a polyhydroxyfullerene material.

[0006] Another object of the present invention is to provide the use of the polyhydroxyfullerene material in the preparation of drugs for treating tumors.

[0007] Another object of the present invention is to provide a method for inducing tumor pan-apoptosis.

[0008] To achieve the above objectives, one aspect of the present invention provides a polyhydroxy fullerene material, the structure of the polyhydroxy fullerene material is shown in formula (I):

[0009]

[0010] in,

[0011] F is fullerene;

[0012] T is a compound molecule with a hydroxyl group at the end, and T is covalently bonded to fullerene;

[0013] n is 30-44.

[0014] In one aspect of the present invention, the F and T are combined in the form of a covalent bond through an addition reaction.

[0015] In one aspect of the present invention, the covalent bond is selected from one of a carbon-oxygen single bond, a carbon-carbon single bond or a carbon-nitrogen single bond.

[0016] In one aspect of the present invention, the fullerene is selected from one or more of hollow fullerene and metal fullerene.

[0017] In one aspect of the present invention, the hollow fullerene is selected from C 2n One or more of, wherein 2n is the number of carbon atoms, 30≤n≤60.

[0018] In one aspect of the present invention, the metallofullerene is selected from M@C 2n 、M2@C 2n and MA@C 2n One or more of, wherein M and A both represent metal elements and M, A are independently selected from any one of Sc, Y and lanthanide metal elements; optionally, the lanthanide metal elements include lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, 30≤n≤60.

[0019] In one embodiment, the fullerene is selected from C 60 , C 70 , C 76 , C 78 , C80 One or more of.

[0020] In one embodiment, the fullerene is selected from C 60 , C 70 One or both of.

[0021] In one embodiment, the metallofullerene is Gd@C 82 .

[0022] In one aspect of the present invention, the T is selected from one or more of a hydroxyl group, a terminal hydroxylation-modified saccharide derivative, a terminal hydroxylation-modified amino acid derivative, and a terminal hydroxylation-modified nucleotide derivative.

[0023] In one embodiment, T is hydroxyl.

[0024] In one embodiment, the fullerene is selected from C 60 , C 70 One or two of; the T may be a pure hydroxyl group and / or a terminal hydroxylated modified saccharide derivative.

[0025] In one embodiment, the fullerene is selected from C 60 , C 70 One or two of; the T may be a pure hydroxyl group and / or an amino acid derivative modified with a terminal hydroxylation.

[0026] In one embodiment, the fullerene is selected from C 60 , C 70 One or two of; the T may be a pure hydroxyl group and / or a terminal hydroxylation-modified nucleotide derivative.

[0027] In some embodiments, the average number of terminal hydroxyl groups in the polyhydroxyl fullerene material is 30-44. It is understood that the number of terminal hydroxyl groups is between 30-44, which does not require that the number of hydroxyl groups carried by each fullerene molecule in the fullerene material or the number of terminal hydroxyl groups are all within the range of 30-44. For example, the number of terminal hydroxyl groups carried by fullerene can actually be between 21-41.

[0028] In some embodiments, the average number of terminal hydroxyl groups in the polyhydroxy fullerene material is 38, which helps to improve the phospholipid bilayer membrane penetration effect of the fullerene material.

[0029] In some embodiments, the average number of hydroxyl groups in the polyhydroxyfullerene material is 30-44.

[0030] In some embodiments, the average number of hydroxyl groups in the polyhydroxyfullerene material is 38.

[0031] In some embodiments, the polyhydroxyfullerene material is C60 (OH) 30-44 Preferably, the polyhydroxyfullerene material is C 60 (OH) 38 .

[0032] In some embodiments, the average hydrated particle size of the polyhydroxyfullerene material is between 140-150 nm, preferably 143.6 nm.

[0033] Another aspect of the present invention provides a method for preparing any one of the aforementioned polyhydroxyfullerene materials.

[0034] It is understood that the polyhydroxyl fullerene can be obtained by directly adding hydroxyl groups to the carbon cage of fullerene or by first modifying the carbon cage of fullerene with groups (such as amino groups) that facilitate covalent connection with other molecules to obtain a fullerene precursor, and then connecting the modified groups on the carbon cage that facilitate covalent connection with other molecules with the compound molecules having hydroxyl groups at the end to obtain the fullerene precursor.

[0035] The modification of the carbon cage of fullerene with groups that facilitate covalent connection with other molecules can be carried out by commonly used methods in the art, such as Prato reaction and Bingel reaction.

[0036] In one embodiment, the fullerene may be a fullerene precursor material with a prato monosubstituted group; and T may be a pure hydroxyl group and / or a terminal hydroxylated modified saccharide derivative.

[0037] In one embodiment, the fullerene may be a fullerene precursor material with a prato monosubstituted group; and T may be a pure hydroxyl group and / or an amino acid derivative modified with a terminal hydroxyl group.

[0038] In one embodiment, the fullerene may be a fullerene precursor material with a prato monosubstituted group; and T may be a pure hydroxyl group and / or a terminal hydroxylation-modified nucleotide derivative.

[0039] In one embodiment, the fullerene may be a fullerene precursor material with a bingel monosubstituted group; and T may be a pure hydroxyl group and / or a saccharide derivative modified with a terminal hydroxyl group.

[0040] In one embodiment, the fullerene may be a fullerene precursor material with a bingel monosubstituted group; and T may be a pure hydroxyl group and / or an amino acid derivative modified with a terminal hydroxyl group.

[0041] In one embodiment, the fullerene may be a fullerene precursor material with a bingel monosubstituted group; and T may be a pure hydroxyl group and / or a terminal hydroxylation-modified nucleotide derivative.

[0042] In one embodiment, the method for preparing the polyhydroxy fullerene material comprises the step of adding hydroxyl groups in hydrogen peroxide to the carbon cage of fullerene.

[0043] In one embodiment, the method for preparing the polyhydroxy fullerene material comprises the steps of dissolving the fullerene to be modified in an organic solvent and adding hydroxyl groups in hydrogen peroxide to the carbon cage of the fullerene using a phase transfer catalyst as a medium.

[0044] In one aspect of the present invention, the method for preparing the polyhydroxyfullerene material comprises dissolving the fullerene solid powder to be modified in an organic solvent, adding a phase transfer catalyst and an aqueous hydrogen peroxide solution to the fullerene organic solvent, and obtaining the polyhydroxyfullerene material by constant temperature stirring, recrystallization, washing, and drying.

[0045] In one aspect of the present invention, the phase transfer catalyst is selected from one or more of quaternary ammonium salts, crown ethers, chain polyethylene glycols, cyclic polyethers, amines, and alkaline solutions.

[0046] In one embodiment, the method for preparing the polyhydroxyfullerene material comprises the following steps:

[0047] (1) dissolving fullerene solid powder in an organic solution, adding a hydrogen peroxide solution and TBAH, and reacting them sufficiently;

[0048] (2) cooling, separating the aqueous solution, and adding a poor solvent for recrystallization to obtain a milky white precipitate;

[0049] (3) dissolving the precipitate in deionized water and adding the poor solvent to reprecipitate and wash;

[0050] (4) vacuum drying the precipitate of step (3) at room temperature to obtain polyhydroxyfullerene powder.

[0051] In one embodiment, in the method for preparing the polyhydroxy fullerene material, the fullerene is C 60 .

[0052] In one embodiment, in the method for preparing the polyhydroxyfullerene material, the organic solution is a 1,2,4-trimethylbenzene solution.

[0053] In one aspect of the present invention, in the method for preparing the polyhydroxyfullerene material, the sufficient reaction is placed in air and stirred at 60-90°C for 12-20 hours; preferably stirred at 65-85°C for 15-18 hours; more preferably stirred at 70°C for 16 hours.

[0054] In one embodiment, in the method for preparing the polyhydroxyfullerene material, the washing is performed using an alcohol solvent, preferably methanol.

[0055] In one embodiment, in the method for preparing the polyhydroxyfullerene material, the phase transfer catalyst is tetrabutylammonium hydroxide (TBAH).

[0056] In one embodiment, in the method for preparing the polyhydroxyfullerene material, the mass fraction of the aqueous hydrogen peroxide solution is 30%.

[0057] In one embodiment, in the method for preparing the polyhydroxyfullerene material, the poor solvent is one or more of 2-propanol, ethyl acetate and hexane;

[0058] In one embodiment, in the method for preparing the polyhydroxyfullerene material, the washing is performed 2-4 times, preferably 2 times.

[0059] In one embodiment, the preparation method of the polyhydroxyfullerene material is: 60 The solid powder is dissolved in 1,2,4-trimethylbenzene solution, and then 30% hydrogen peroxide aqueous solution and TBAH are added to the mixed liquid, and the mixture is stirred at 70°C in air for 16 hours until the purple 1,2,4-trimethylbenzene layer becomes a colorless transparent solution; after cooling the mixture, a light yellow aqueous solution is separated, and a mixed solvent of 2-propanol, ethyl acetate and hexane (5:4:5) is added to obtain a milky white precipitate; the precipitate is dissolved in deionized water and re-precipitated by adding a poor solvent as described above, and then washed with methanol; the solid is vacuum dried at room temperature to obtain a polyhydroxyfullerene material (MF) white to yellow powder.

[0060] In one aspect of the present invention, a method for preparing the aforementioned hydroxyl-poor fullerene material (FF) is provided, comprising the steps of dissolving the fullerene to be modified in an organic solvent, and adding hydroxyl groups in hydrogen peroxide to the carbon cage of the fullerene using a phase transfer catalyst as a medium, wherein the phase transfer agent is an alkaline solution; the organic solvent is selected from at least one of the following: toluene, xylene, o-xylene, 1,2,4-trimethylbenzene; preferably, the solvent is toluene.

[0061] In one aspect of the present invention, a method for preparing the aforementioned hydroxyl-poor fullerene material (FF) is provided, wherein C 60 Solid powder 50 mg, in a round-bottom flask, slowly add 5 mL of hydrogen peroxide (30%) (slowly add, avoid splashing the flask wall), slowly stir, evenly disperse, add 15 mL of 15% NaOH solution, stir vigorously after adding (~1000), heat at 70 ° C for about 6 h (aspirate the solution, the solution changes color, less precipitation), recrystallize with a large amount of ethanol, precipitate and wash, dialyze with a dialysis bag (molecular weight 3500), and freeze-dry.

[0062] Another aspect of the present invention provides use of any of the aforementioned polyhydroxyfullerene materials in the preparation of drugs for treating tumors.

[0063] Another aspect of the present invention provides the use of the polyhydroxyfullerene material of formula (I) in the preparation of drugs for treating tumors.

[0064] Another aspect of the present invention provides the use of any of the aforementioned polyhydroxyfullerene materials for treating tumors under starvation conditions.

[0065] In some embodiments, the starvation conditions include: incubation in a medium without glucose, a medium without fetal bovine serum, a medium without pyruvate, a medium without amino acids, a medium without glucose and amino acids, addition of inhibitors of glucose metabolism-related enzymes, addition of small molecules that interfere with cell metabolism such as mTOR inhibitors, knockout of tumor cell glucose transporters, knockout of tumor cell glucose utilization-related enzymes, etc. One or more conditions.

[0066] In one embodiment, the starvation condition is a medium condition without fetal bovine serum.

[0067] In one embodiment, the starvation condition is a starvation condition in which EBSS medium is provided without fetal bovine serum.

[0068] In one embodiment, the tumor treatment is to induce tumor pan-apoptosis, and the tumor pan-apoptosis includes inducing tumor apoptosis, inducing tumor pyroptosis and / or inducing tumor necrosis.

[0069] Another aspect of the present invention provides the use of any of the aforementioned polyhydroxyfullerene materials to induce tumor pan-apoptosis.

[0070] Another aspect of the present invention provides the use of any of the aforementioned polyhydroxyfullerene materials for inducing tumor apoptosis.

[0071] Another aspect of the present invention provides the use of any of the aforementioned polyhydroxyfullerene materials to induce tumor pyroptosis.

[0072] Another aspect of the present invention provides the use of any of the aforementioned polyhydroxyfullerene materials for inducing tumor necrosis.

[0073] In one aspect of the present invention, the tumor is selected from one or more of liver cancer, lung cancer, colorectal cancer, pancreatic cancer, bone cancer, breast cancer, kidney cancer, esophageal cancer, ovarian cancer, oral cancer, laryngeal cancer, bile duct cancer, uterine cancer, nasal cancer, testicular cancer, meningioma, skin cancer, cervical cancer, brain glioma, lymphoma, leukemia or sarcoma.

[0074] In one embodiment, the tumor is selected from one or more of lung cancer, pancreatic cancer, breast cancer, and brain glioma.

[0075] In one embodiment, the tumor is selected from lung cancer.

[0076] Another aspect of the present invention provides a method for regulating tumor metabolism and inducing pan-apoptosis by intracellular organelle cascade damage, which is achieved by co-incubating the above-mentioned polyhydroxyfullerene material with solid tumors or tumor cells, or administering the above-mentioned polyhydroxyfullerene material to a subject in need, specifically as follows:

[0077] (1) Damage to lysosomes, destruction of lysosomal membrane permeability, change of pH value in lysosomes, release of degraded proteins in lysosomes, release of calcium ions, etc.;

[0078] (2) Damage to the endoplasmic reticulum, leading to an increase in misfolded proteins and inducing endoplasmic reticulum stress;

[0079] (3) Damage to mitochondria, destroy mitochondrial membrane potential, lead to oxidative stress, and disrupt electron transfer in the respiratory chain;

[0080] (4) Regulate tumor cell metabolism and inhibit the glycolysis process of cancer cells, leading to the consumption of energy ATP and nutrients;

[0081] (5) Cause apoptosis, pyroptosis and necrosis of tumor cells.

[0082] In a preferred embodiment, the co-incubation of the polyhydroxyfullerene material with solid tumor or tumor cells is performed under starvation conditions.

[0083] The starvation conditions include: incubation in a medium without glucose, a medium without fetal bovine serum, a medium without pyruvate, a medium without amino acids, a medium without glucose and amino acids, addition of glucose metabolism-related enzyme inhibitors, addition of mTOR inhibitors and other small molecules that interfere with cell metabolism, knockout of tumor cell glucose transporters, knockout of tumor cell glucose utilization-related enzymes, and one or more of the following conditions.

[0084] In a preferred embodiment, the polyhydroxyfullerene material needs to be co-administered with an mTOR inhibitor and / or an inhibitor of glucose metabolism-related enzymes to create starvation conditions.

[0085] The present invention provides a new way to induce pan-apoptosis of tumor cells, that is, the permeabilization of lysosomal membrane caused by the combination of polyhydroxyfullerene materials and lysosomal membrane under starvation conditions, destroying lysosomes, disrupting endoplasmic reticulum and mitochondria, causing endoplasmic reticulum stress and mitochondrial damage, and activating the pan-apoptotic process of apoptosis, pyroptosis and necrosis in multiple organelles. By forcing the degree of organelle damage to be greater than the repair capacity of the organelle, continuous mutual damage between organelles is achieved, positive feedback superposition of damage is achieved, and cell death is induced, which is better than the positive drug hydroxychloroquine (CQ). This efficient and low-toxic method is very conducive to the development of new tumor treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 The mass spectra of hydroxyl fullerene materials with different numbers of hydroxyl groups in Example 1 are shown, wherein: Figure 1 1A in the figure is the mass spectrum of FF. Figure 1 1B in FIG. 1 is the mass spectrum of MF.

[0087] Figure 2 The infrared spectra of hydroxyl fullerene materials with different numbers of hydroxyl groups in Example 1 are shown.

[0088] Figure 3 The hydrated particle sizes of the hydroxyfullerene materials with different numbers of hydroxyl groups in Example 1 are shown.

[0089] Figure 4 A schematic diagram showing the simulation of the interaction between hydroxyfullerene materials with different numbers of hydroxyl groups and lysosomal phospholipids in Example 2 is shown.

[0090] Figure 5 The killing effect of the polyhydroxyfullerene material on A549 cells under starvation conditions provided by EBSS in Example 3 is shown.

[0091] Figure 6 The figure shows the effect of hydroxyl fullerene materials with different numbers of hydroxyl groups in Example 4 on the killing effect of A549 cells under starvation conditions provided by EBSS.

[0092] Figure 7 The effect of the polyhydroxyfullerene material in Example 5 on the killing effect of different tumor cell lines under the starvation condition provided by EBSS is shown.

[0093] Figure 8 It shows that different treatment conditions of A549 cells in Example 6 will cause lysosomal damage.

[0094] Fig. 9 It shows that different treatment conditions of A549 cells in Example 7 will cause damage to the endoplasmic reticulum.

[0095] Fig.10The figure shows the damage of mitochondria caused by different treatment conditions of A549 cells in Example 8.

[0096] Fig.11 The figure shows the changes in the metabolic pattern of A549 cells after mitochondria are damaged in Example 9.

[0097] Fig.12 The expression of apoptosis-related proteins in A549 cells before and after different treatments detected by Western blot in Example 10 is shown.

[0098] Fig.13 The expression of pyroptosis-related proteins in A549 cells before and after different treatments detected by Western blot in Example 11 is shown.

[0099] Fig.14 The expression of necrosis-related proteins in A549 cells before and after different treatments detected by Western blot in Example 12 is shown. DETAILED DESCRIPTION

[0100] I. Definitions

[0101] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the relevant terms and laboratory procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0102] As used herein and unless otherwise specified, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0103] In the description herein, reference is made to "one embodiment" or "some implementations", which describe a subset of all possible embodiments, but it is understood that "one embodiment" may be the same subset or a different subset of all possible embodiments and may be combined with each other without conflict.

[0104] As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

[0105] As used herein, the term "fullerene" is a series of spherical cluster molecules composed of an even number of carbon atoms, 12 five-membered rings, and the rest are six-membered rings, and are cage-like structures composed of carbon atoms. Fullerenes include hollow fullerenes and metallofullerenes, and the hollow fullerene is a cage-like structure composed of carbon atoms alone.

[0106] As used herein, the term "metallofullerene" refers to a class of compounds with special structures and properties formed by enclosing various atoms, ions or atomic clusters in the carbon cage structure of fullerene. Such compounds are usually called endohedral fullerenes and are generally represented by M@C 2n It is expressed in the form of 2n, where M represents the metal element and 2n represents the number of carbon atoms.

[0107] The term "Platow reaction" refers to the 1,3-dipolar cycloaddition reaction between fullerene or carbon nanotube and methyleneamine inner salt. This reaction can be used to prepare functionalized fullerene / (single-walled) nanotubes, and is one of the important methods for surface chemical modification of fullerene. A single substitution group usually means that in this reaction, only one substitution group is introduced on the fullerene or carbon nanotube. For example, the heating reflux reaction of sarcosine and paraformaldehyde in toluene can produce methyleneamine inner salt, which undergoes 1,3-dipolar cycloaddition with the 6,6-bond in the fullerene to obtain the corresponding pyrrolidyl-substituted fullerene.

[0108] The term "Bingle reaction" refers to the 60 Fullerene reacts with bromomalonate in the presence of a base (such as sodium hydride, DBU) to form a single 6-6 closed-ring product. This reaction is the main route for the cyclopropanation of fullerenes and is widely used in C 60 Bingel monosubstituted group refers to the reaction in which C 60 Only one substituent is introduced on the fullerene. The Bingel product is called C 60 Methylene derivatives (methanofullerene) are generally stable in various chemical environments. In addition to the Bingel reaction, such compounds can also be synthesized through 1,3-dipolar cycloaddition reactions of diazo compounds, addition reactions with carbenes, and electrochemical methods.

[0109] The term "EBSS medium", Earle's Balanced Salt Solution, is one of the phosphate buffers commonly used in cell isolation or culture. It has the functions of maintaining osmotic pressure, keeping pH stable, and providing simple nutrition, which can meet the basic survival and metabolic needs of tissues, organs or cells in in vitro experiments. Its main components include glucose, NaHCO3, NaCl, KCl and Na2HPO4, and may contain phenol red as a pH indicator. It contains Ca2+ ,Mg 2+ , these ions may affect cell behavior in certain cell culture processes.

[0110] As a low-nutrient culture medium, EBSS can induce cells to enter an autophagic state. Under the conditions of EBSS culture medium, the intracellular ATP level decreases, AMPK activity increases, and mTORC1 is inhibited, leading to upregulation of the expression of autophagy-related genes, thereby initiating the autophagy process; for example, incubating cells in EBSS for a certain period of time (such as 10, 30, 60 minutes or longer) can induce cells to undergo autophagy.

[0111] II. Embodiment

[0112] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0113] The raw materials and equipment used in the specific embodiments of the present disclosure are all known products, which are obtained by purchasing commercially available products.

[0114] Example 1: Preparation of hydroxyl fullerene materials with different hydroxyl numbers

[0115] Preparation of polyhydroxyfullerene materials (MF)

[0116] C 60 The solid powder (0.100 g) was dissolved in 1,2,4-trimethylbenzene solution (7 mL), and 30% H2O2 aqueous solution (5 mL) and TBAH (tetrabutylammonium hydroxide, 40% in water, 500 μL) were added to the mixed liquid, and the mixture was stirred at 70 ° C in air for 16 hours until the purple 1,2,4-trimethylbenzene layer became a colorless transparent solution. After cooling the mixture, a light yellow aqueous solution (about 5 mL) was separated, and a mixed solvent of 2-propanol, ethyl acetate and hexane (5:4:5, 70 mL) was added to obtain a milky white precipitate. The precipitate was dissolved in deionized water and reprecipitated by adding a poor solvent as described above, and then washed three times with methanol. The solid was dried in vacuum at room temperature to obtain a white to yellow powder of polyhydroxyfullerene material (MF).

[0117] The obtained MF was characterized by mass spectrometry.

[0118] Experimental results: Figure 1As shown in the figure, the m / z of MF is concentrated in 1200-1450. Next, the composition of MF will be further determined by combining elemental analysis and thermogravimetric analysis. The C and H contents in MF are 53.19% and 4.54%, respectively. Thermogravimetric analysis infers that the water content in MF is 11.38%. The comprehensive calculation shows that the average molecular formula of MF is C 60 (OH) 38 9H2O.

[0119] Preparation of hydroxyl-poor fullerene materials (FF)

[0120] Weigh C 60 Solid powder 50 mg, in a round-bottom flask, slowly add 5 mL of hydrogen peroxide (30%) (slowly add, avoid splashing the flask wall), slowly stir, evenly disperse, add 15 mL of 15% NaOH solution, stir vigorously after adding (~1000), heat at 70 ° C for about 6 h (aspirate the solution, the solution changes color, less precipitation), recrystallize with a large amount of ethanol, precipitate and wash, dialyze with a dialysis bag (molecular weight 3500), and freeze-dry.

[0121] Experimental results: Figure 1 As shown in the figure, the m / z of FF is concentrated in 800-1200. Next, the composition of FF will be further determined by combining elemental analysis and thermogravimetric analysis. The C and H contents in FF are 41.65% and 2.69% respectively. The water content in MF is 5.16% as inferred by thermogravimetric analysis. The average molecular formula of MF is C 60 (OH) 25 6H2O.

[0122] Depend on Figure 1-3 The results show that MF has more substituted hydroxyl groups; the infrared spectrum of MF has a more obvious hydroxyl absorption peak than FF, and the final hydration particle size characterization shows that the particle size of FF and MF is similar.

[0123] Example 2: Interaction between hydroxyl fullerene materials with different numbers of hydroxyl groups and lysosomal phospholipids

[0124] The molecular dynamics simulation method was used to simulate the structure of the phospholipid bilayer-few hydroxyl fullerene (FF) / phospholipid bilayer-poly hydroxyl fullerene (MF) molecular complex using AMBER software. The molecular dynamics time was >100ns, and the overall average structure of the phospholipid bilayer-FF / phospholipid bilayer-MF molecules was extracted respectively. For the phospholipid bilayer and fullerenes with different hydroxyl numbers, AMBER ff99SB force field and GAFF force field were used respectively. With the phospholipid bilayer-FF / phospholipid bilayer-MF molecules as the center, a 1nm cubic water box was added, the topology and coordinate structure were saved, and then the simulation was performed. It is worth noting that due to the relatively poor structural stability of the phospholipid bilayer itself during the molecular dynamics simulation, the fullerene The phospholipid molecules outside the range are subjected to a heavy atom restraint force of -1500 kcal / mol to prevent the molecules from floating excessively during the dynamic process and to keep the overall phospholipid bilayer structure unchanged; The phospholipid molecules outside the range do not add any restrictive force, thereby obtaining only the changes in the binding mode between the phospholipid molecules around the fullerene molecules and the fullerene.

[0125] The simulation results are as follows Figure 4 As shown, the overall membrane penetration effect of the phospholipid bilayer-FF is not obvious, a more obvious crack appears in the membrane layer of the phospholipid bilayer-MF close to the fullerene, and the phospholipid membrane is obviously broken.

[0126] Example 3: Killing effect of polyhydroxyfullerene materials on A549 cells under EBSS starvation conditions

[0127] Cells: Human non-small cell lung cancer cell line A549.

[0128] Experimental method: CCK8 experimental method was used to detect whether fullerene materials have cytotoxicity. 5 A suspension of human non-small cell lung cancer cells (A549) with a volume of 100 μL / mL was inoculated into a transparent 96-well plate. After 12 hours of adherent culture, the supernatant was aspirated and replaced with 100 μL of polyhydroxyfullerene materials (MF) with different concentrations and EBSS (100 μL) (purchased from beyotime, product number: C0213-500mL); 4 replicate wells per group were cultured for 24 hours. After the experiment, the conditioned medium containing fullerene was aspirated, washed three times with PBS, and 100 μL of CCK-8 solution (diluted with colorless DMEM) was added to each well to test the OD values ​​of different groups. The experiment was repeated three times. Cell activity (%) = OD value of treatment group / OD value of control group.

[0129] Conclusion: If Figure 5As shown, different concentrations of polyhydroxyfullerene materials alone do not cause obvious death of A549 cells, but when EBSS is added, the polyhydroxyfullerene materials are prompted to produce cell killing effects, and have obvious concentration dependence, that is, the higher the concentration of polyhydroxyfullerene materials, the better the killing effect.

[0130] Example 4: Effect of hydroxyl fullerene materials with different numbers of hydroxyl groups on the killing effect of A549 cells

[0131] Cells: Human non-small cell lung cancer cell line A549.

[0132] Experimental method: CCK8 experimental method was used to detect whether fullerene materials have cytotoxicity. 5 A suspension of human non-small cell lung cancer cells (A549) with a volume of 100 μL / mL was inoculated into a transparent 96-well plate. After 12 hours of adherence culture, the supernatant was aspirated and replaced with 100 μL of hydroxyfullerene materials (MF, FF) and EBSS (100 μL) containing different concentrations. Each group had 4 replicate wells and was cultured for 24 hours. After the experiment, the conditioned medium containing fullerene was aspirated, washed three times with PBS, and 100 μL of CCK-8 solution (diluted with colorless DMEM) was added to each well to test the OD values ​​of different groups. The experiment was repeated three times. Cell activity (%) = OD value of treatment group / OD value of control group.

[0133] Conclusion: If Figure 6 As shown, there was no significant toxicity difference between different concentrations of FF and different concentrations of MF in the control group. However, after EBSS treatment, MF had a significant concentration-dependent killing effect compared with FF, and the higher the concentration, the better the killing effect.

[0134] Example 5: Effect of polyhydroxyfullerene materials on the killing effect of different tumor cell lines

[0135] Cells: pancreatic cancer cell PANC-1, human breast cancer cell MCF-7, mouse breast cancer cell 4T1, brain glioma cell U87.

[0136] Experimental method: CCK8 experimental method was used to detect whether fullerene materials have cytotoxicity. 5Cell suspensions of different cell lines (PANC-1, MCF-7, 4T1, U87) with a concentration of 100 μL / well were inoculated into a transparent 96-well plate. After 12 hours of adherent culture, the supernatant was aspirated and replaced with 100 μL of conditioned medium containing different concentrations of polyhydroxyfullerene (MF) and EBSS (100 μL). Each group had 4 replicate wells and was cultured for 24 hours. After the experiment, the conditioned medium containing fullerene was aspirated, washed three times with PBS, and 100 μL of CCK-8 solution (diluted with colorless DMEM) was added to each well to test the OD values ​​of different groups. The experiment was repeated three times. Cell activity (%) = OD value of treatment group / OD value of control group.

[0137] Conclusion: If Figure 7 As shown, compared with the control group, after adding EBSS, the killing effect of MF materials on multiple cell lines was concentration-dependent, and the higher the concentration, the better the killing effect.

[0138] Example 6: EBSS provides polyhydroxyfullerene materials under starvation conditions to cause lysosomal destruction

[0139] Cells: Human non-small cell lung cancer cell line A549.

[0140] Grouping: The blank control group was PBS, and the experimental groups were starvation condition group, 100 μM MF aqueous solution, starvation condition + 100 μM MF aqueous solution, positive drug hydroxychloroquine (CQ, 10 μM), starvation condition + 10 μM CQ aqueous solution.

[0141] Western blot detection of protein levels of LAMP1, LAMP2, Cathepsin B, Cathepsin D and β-actin: A549 cells were cultured in DMEM medium (Gbico, C11995500BT) containing 10% fetal bovine serum (ExCell Bio, 12B173). After 12 hours of normal culture in six-well plates, A549 cells were treated with different doses of the test sample groups for 24 hours, and the treated A549 cells were lysed in RIPA lysis buffer (Beyotime, P0013) containing a protease phosphatase inhibitor cocktail (Beyotime, P1048). Subsequently, the cell lysate was boiled in 5× loading buffer (Beyotime, P0015) for 15 minutes, then dissolved with SDS-polyacrylamide gel (Genscript, M42012), and then transferred to a PVDF membrane. The membranes were incubated overnight at low temperature with antibodies against anti-LAMP1 (proteintech, 21997-1-AP), anti-LAMP2 (proteintech, 27823-1-AP), anti-Cathepsin B (abcam, ab214428), anti-Cathepsin D (abcam, ab75811), and anti-β-actin (Cell signaling technology, 4970), and then incubated with bound secondary antibodies (Cell signaling technology, 7074). All antibodies were diluted in blocking buffer and the antigen-antibody reaction was visualized by enhanced chemiluminescence kit (Absin, abs920).

[0142] Conclusion: If Figure 8 As shown, the bands of lysosomal marker proteins LAMP1 and LAMP2 in A549 cells in the experimental group became significantly lighter, and the bands of lysosomal tissue proteases Cathepsin B and Cathepsin D also became significantly lighter, indicating that polyhydroxyfullerene materials would destroy lysosomes under starvation conditions.

[0143] Example 7: EBSS provides polyhydroxyfullerene materials under starvation conditions to induce endoplasmic reticulum stress

[0144] Cells: Human non-small cell lung cancer cell line A549.

[0145] Grouping: The blank control group was PBS, and the experimental groups were starvation condition group, 100 μM MF aqueous solution, starvation condition + 100 μM MF aqueous solution, positive drug CQ (10 μM), starvation condition + 10 μM CQ aqueous solution.

[0146] Western blot detection of Bid, IRE1, BiP, CHOP and β-actin protein levels: A549 cells were cultured in DMEM medium (Gbico, C11995500BT) containing 10% fetal bovine serum (ExCell Bio, 12B173). After 12 hours of normal culture in six-well plates, A549 cells were treated with different doses of the test sample groups for 24 hours, and the treated A549 cells were lysed in RIPA lysis buffer (Beyotime, P0013) containing a protease phosphatase inhibitor cocktail (Beyotime, P1048). Subsequently, the cell lysate was boiled in 5× loading buffer (Beyotime, P0015) for 15 minutes, then dissolved with SDS-polyacrylamide gel (Genscript, M42012), and then transferred to a PVDF membrane. The membrane was incubated overnight at low temperature with antibodies of anti-Bid (Abclonal, A0210), anti-IRE1 (Bioss, bs-8680R), anti-BiP (Cell signaling technology, 3177), anti-CHOP (Bioss, bs-8875R), and anti-β-actin (Cell signaling technology, 4970), and then incubated with the conjugated secondary antibody (Cell signaling technology, 7074). All antibodies were diluted in blocking buffer, and the antigen-antibody reaction was visualized by enhanced chemiluminescence kit (Absin, abs920).

[0147] Conclusion: If Fig. 9 As shown, compared with the control group, under the premise that the expression level of reference protein β-actin remained unchanged, the expression levels of BiP and IRE1 proteins in the starvation condition + MF group were significantly downregulated, and the expression levels of Bid and CHOP proteins in the starvation condition + MF group were significantly upregulated, indicating that polyhydroxyfullerene materials can induce endoplasmic reticulum stress under starvation conditions.

[0148] Example 8: Damage of polyhydroxyfullerene materials to mitochondria under starvation conditions provided by EBSS

[0149] Cells: Human non-small cell lung cancer cell line A549.

[0150] Grouping: The blank control group was PBS, and the experimental groups were starvation condition group, 100μM MF aqueous solution, starvation condition + 100μM MF aqueous solution, positive drug CQ (10μM), starvation condition + 10μM CQ aqueous solution, and positive reference CCCP (10μM).

[0151] Fluorescence confocal detection of mitochondrial damage: A549 cells were cultured in DMEM medium (Gbico, C11995500BT) containing 10% fetal bovine serum (ExCell Bio, 12B173). After A549 cells were cultured normally in a six-well plate for 12 hours, the cells were treated with different doses of the sample groups to be tested for 24 hours, the culture medium containing the material was aspirated, washed 3 times with PBS, and 1 ml of JC-1 staining working solution was added to each well (JC-1 was diluted by adding 1 ml of JC-1 staining buffer to every 5 μl of JC-1 (200X), beyotime, C2003S), and mixed thoroughly. Incubate at 37°C in a cell culture incubator for 30 minutes. After the incubation at 37°C, the supernatant was discarded and washed twice with JC-1 staining buffer. Then, the cell nucleus was counterstained with Hoechst 33342, and finally 1 ml of colorless DMEM was added to obtain images using a laser scanning confocal microscope (Olympus).

[0152] Conclusion: If Fig.10 As shown, when the mitochondrial membrane potential is normal, the JC-1 polymer exhibits red fluorescence, while when the membrane potential is damaged, the JC-1 monomer exhibits obvious green fluorescence. The green fluorescence of the starvation condition + MF group is equivalent to that of the positive control CCCP, indicating that the mitochondrial membrane potential is significantly decreased and the mitochondria are damaged.

[0153] Example 9: Effects of mitochondrial damage on the metabolic pattern of A549 cells

[0154] Cells: Human non-small cell lung cancer cell line A549.

[0155] Grouping: blank control group (PBS), EBSS provided starvation conditions + 100 μM MF aqueous solution.

[0156] Metabolomics analysis of A549 cells: The treated cells were rinsed with pre-cooled PBS and then collected with a scraper. After centrifugation, the cells were extracted with methanol to obtain proteins and analyzed on a LC-ESI-MS / MS system (Waters ACQUITY H-Class and MS, ) for further analysis. LC-ESI-MS / MS system (Waters ACQUITY H-Class and MS, 6500+ system) for further analysis. For HPLC analysis, an amide column (ACQUITY UPLC BEH, 2.1 ID×100 mm, 1.7 μm, 40° C.) was used. The mobile phase was ultrapure water (phase A) containing 10 mM ammonium acetate with 0.3% ammonia and water containing 90% acetonitrile (phase B), with a flow rate of 0.4 mL / min. In the first 1.2 minutes, the solvent gradient started from 95% of phase B, then decreased to 70% of phase B (8 minutes). 70% of phase B (8 minutes), 50% of phase B (9-11 minutes), and then returned to 95% of phase B (11.1-15 minutes). For mass spectrometry detection, the electrospray ion source (ESI) temperature was 550° C., the ion spray voltage was 5500 V (positive) and -4500 V (negative), and the curtain gas (CUR) was set to 35.0 psi. Qualitative analysis was performed using a standard-based database, and quantitative analysis was performed using triple quadrupole mass spectrometry in multiple reaction monitoring (MRM) mode. Software packages Analyst1.6.3 and MultiQuant 3.0.3 were used to process mass spectrometry data. Metabolomic data were normalized using protein concentrations measured by the BCA method. Methods Metabolomic data were normalized. Unsupervised principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) were performed using R (version 3.5.1). Metabolites with VIP ≥ 1, FC ≥ 2, or FC ≤ 0.5 were identified as significantly up-regulated or down-regulated metabolites. Metabolites with significantly up-regulated or down-regulated metabolites, respectively. Heatmaps of metabolite levels were also visualized using TBtools software.

[0157] Conclusion: If Fig.11 As shown in the figure, compared with the control group, GSEA analysis of glycolysis, MYC, mTORC1 and hypoxia showed a significant negative correlation, indicating that the starvation condition + MF group can significantly inhibit the glycolysis process of cancer cells, leading to energy and nutrient consumption, thereby weakening the resistance mechanism of cancer cells to promote survival.

[0158] Example 10: EBSS provides polyhydroxyfullerene materials to induce apoptosis of A549 cells under starvation conditions

[0159] Cells: Human non-small cell lung cancer cell line A549.

[0160] Grouping: The blank control group was PBS, and the experimental groups were starvation condition group, 100 μM MF aqueous solution, starvation condition + 100 μM MF aqueous solution, positive drug CQ (10 μM), starvation condition + 10 μM CQ aqueous solution.

[0161] Western blot detection of caspase 3, cl caspase 3, Bax, Bcl-2 and β-actin protein levels: A549 cells were cultured in DMEM medium (Gbico, C11995500BT) containing 10% fetal bovine serum (ExCell Bio, 12B173). After A549 cells were cultured normally in six-well plates for 12 hours, they were treated with different doses of the sample groups for 24 hours, and the treated A549 cells were lysed in RIPA lysis buffer (Beyotime, P0013) containing a protease phosphatase inhibitor cocktail (Beyotime, P1048). Subsequently, the cell lysate was boiled in 5× loading buffer (Beyotime, P0015) for 15 minutes, then dissolved with SDS-polyacrylamide gel (Genscript, M42012), and then transferred to a PVDF membrane. The membranes were incubated overnight at low temperature with antibodies against anti-caspase 3 (Cell signaling technology, 9662), anti-cl caspase 3 (Cell signaling technology, 9664), anti-Bax (abcam, ab32503), anti-Bcl-2 (abcam, ab182858), and anti-β-actin (Cell signaling technology, 4970), and then incubated with the conjugated secondary antibody (Cell signaling technology, 7074). All antibodies were diluted in blocking buffer, and the antigen-antibody reaction was visualized by an enhanced chemiluminescence kit (Absin, abs920).

[0162] Conclusion: If Fig.12 As shown in the figure, compared with the control group, under the premise that the expression level of reference protein β-actin remained unchanged, the anti-apoptotic protein Bcl-2 was significantly downregulated in the starvation condition + MF group, and the expression levels of cl-caspase 3 / caspase 3 and pro-apoptotic protein Bax were significantly upregulated in the starvation condition + MF group, indicating that starvation condition + MF can induce cell apoptosis.

[0163] Example 11: EBSS provides polyhydroxyfullerene materials to induce pyroptosis of A549 cells under starvation conditions

[0164] Cells: Human non-small cell lung cancer cell line A549.

[0165] Grouping: The blank control group was PBS, and the experimental groups were starvation condition group, 100 μM MF aqueous solution, starvation condition + 100 μM MF aqueous solution, positive drug CQ (10 μM), starvation condition + 10 μM CQ aqueous solution.

[0166] Western blot detection of caspase 1, GSDMD-N and β-actin protein levels: A549 cells were cultured in DMEM medium (Gbico, C11995500BT) containing 10% fetal bovine serum (ExCell Bio, 12B173). After 12 hours of normal culture in six-well plates, A549 cells were treated with different doses of the sample groups for 24 hours, and the treated A549 cells were lysed in RIPA lysis buffer (Beyotime, P0013) containing a protease phosphatase inhibitor cocktail (Beyotime, P1048). Subsequently, the cell lysate was boiled in 5× loading buffer (Beyotime, P0015) for 15 minutes, then dissolved with SDS-polyacrylamide gel (Genscript, M42012), and then transferred to a PVDF membrane. After overnight incubation at low temperature with antibodies of anti-caspase 1 (Abcam, ab138483), anti-GSDMD-N (Cell signaling technology, 10137), and anti-β-actin (Cell signaling technology, 4970), the membrane was then incubated with the conjugated secondary antibody (Cell signaling technology, 7074), all of which were diluted in blocking buffer, and the antigen-antibody reaction was visualized by an enhanced chemiluminescence kit (Absin, abs920).

[0167] Conclusion: If Fig.13 As shown in the figure, compared with the control group, under the premise that the expression of reference protein β-actin remained unchanged, the expression of caspase 1 and GSDMD-N proteins in the starvation condition + MF group was significantly upregulated, indicating that starvation condition + MF would induce cell pyroptosis. However, the CQ group did not significantly upregulate the expression of GSDMD-N protein, indicating that CQ did not induce cell pyroptosis.

[0168] Example 12: EBSS provides polyhydroxyfullerene materials to induce A549 cell necrosis under starvation conditions

[0169] Cells: Human non-small cell lung cancer cell line A549.

[0170] Grouping: The blank control group was PBS, and the experimental groups were starvation condition group, 100 μM MF aqueous solution, starvation condition + 100 μM MF aqueous solution, positive drug CQ (10 μM), starvation condition + 10 μM CQ aqueous solution.

[0171] Western blot detection of MLKL, p-MLKL, and β-actin protein levels: A549 cells were cultured in DMEM medium (Gbico, C11995500BT) containing 10% fetal bovine serum (ExCell Bio, 12B173). After A549 cells were cultured normally in six-well plates for 12 hours, the cells were treated with different doses of the test sample groups for 24 hours, and the treated A549 cells were lysed in RIPA lysis buffer (Beyotime, P0013) containing a protease phosphatase inhibitor cocktail (Beyotime, P1048). Subsequently, the cell lysate was boiled in 5× loading buffer (Beyotime, P0015) for 15 minutes, then dissolved with SDS-polyacrylamide gel (Genscript, M42012), and then transferred to a PVDF membrane. The membrane was incubated with anti-p-MLKL (Cell signaling technology, 91689), anti-MLKL (Cell signaling technology, bs-14993), anti-β-actin (Cell signaling technology, 4970) antibodies at low temperature overnight, and then incubated with the bound secondary antibody (Cell signaling technology, 7074), all antibodies were diluted in blocking buffer, and the antigen-antibody reaction was visualized by enhanced chemiluminescence kit (Absin, abs920).

[0172] Conclusion: If Fig.14 As shown, compared with the control group, under the premise that the expression level of reference protein β-actin remained unchanged, the expression level of p-MLKL / MLKL protein in the starvation condition + MF group was significantly upregulated, indicating that starvation condition + MF would induce cell necrosis, while the expression level of p-MLKL / MLKL protein in the CQ group was not significantly upregulated, indicating that it did not induce cell necrosis.

[0173] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A polyhydroxyfullerene material, the structure of which is shown in formula (I): in, F is fullerene; T is a compound molecule with a hydroxyl group at the end, and T is covalently bonded to fullerene; n is 30-44; Preferably, F and T are combined in the form of a covalent bond through an addition reaction; Preferably, the covalent bond is selected from one of a carbon-oxygen single bond, a carbon-carbon single bond or a carbon-nitrogen single bond.

2. The polyhydroxyfullerene material according to claim 1, wherein The fullerene is selected from one or more of hollow fullerene and metal fullerene; Preferably, the hollow fullerene is selected from C 2n One or more of, wherein 2n is the number of carbon atoms, 30≤n≤60; Preferably, the metallofullerene is selected from M@C 2n 、M2@C 2n and MA@C 2n One or more of, wherein M and A both represent metal elements and M and A are independently selected from any one of Sc, Y and lanthanide metal elements; optionally, the lanthanide metal elements include lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, 30≤n≤60; More preferably, the fullerene is selected from C 60 , C 70 , C 76 , C 78 , C 80 One or more of; More preferably, the fullerene is selected from C 60 , C 70 One or both of; More preferably, the metallofullerene is Gd@C 82 .

3. The polyhydroxyfullerene material according to any one of claims 1 or 2, wherein: The T is selected from one or more of a hydroxyl group, a terminal hydroxylated modified saccharide derivative, a terminal hydroxylated modified amino acid derivative, and a terminal hydroxylated modified nucleotide derivative; Preferably, T is hydroxy.

4. The polyhydroxyfullerene material according to any one of claims 1 to 3, wherein: The number of hydroxyl groups in the polyhydroxyfullerene material is 30-44; preferably, the average number of hydroxyl groups is 38.

5. The polyhydroxyfullerene material according to any one of claims 1 to 4, wherein: The method for preparing the polyhydroxy fullerene material comprises the steps of adding hydroxyl groups in hydrogen peroxide to the carbon cage of the fullerene; Preferably, the method for preparing the polyhydroxy fullerene material comprises the steps of dissolving the fullerene to be modified in an organic solvent and adding the hydroxyl group in hydrogen peroxide to the carbon cage of the fullerene using a phase transfer catalyst as a medium; Preferably, the phase transfer catalyst is selected from one or more of quaternary ammonium salts, crown ethers, chain polyethylene glycols, cyclic polyethers, amines, and alcohol-water solvents; preferably, the phase transfer catalyst is tetrabutylammonium hydroxide (TBAH).

6. The polyhydroxyfullerene material according to any one of claims 1 to 5, wherein: The preparation method of the polyhydroxyfullerene material comprises the following steps: (1) dissolving fullerene solid powder in an organic solvent, adding a hydrogen peroxide aqueous solution and tetrabutylammonium hydroxide (TBAH) to fully react; (2) cooling, separating the aqueous solution, and adding a poor solvent for recrystallization to obtain a milky white precipitate; (3) dissolving the precipitate in deionized water and adding the poor solvent to reprecipitate and wash; (4) vacuum drying the precipitate from step (3) at room temperature to obtain polyhydroxyfullerene powder; Preferably, in step (1), the fullerene is C 60 ; Preferably, in step (1), the organic solution is a 1,2,4-trimethylbenzene solution; preferably, in step (1), the sufficient reaction is placed in air and stirred at 60-90°C for 12-20 hours; preferably, stirred at 65-85°C for 15-18 hours; more preferably, stirred at 70°C for 16 hours; Preferably, in step (2), the poor solvent is one or more of 2-propanol, ethyl acetate and hexane; Preferably, in step (3), the washing is performed using an alcohol solvent, preferably methanol; Preferably, in step (3), the washing is performed 2-4 times, preferably 3 times.

7. Use of the polyhydroxyfullerene material according to any one of claims 1 to 6 in the preparation of a drug for treating tumors, preferably, the treatment is carried out under starvation conditions.

8. The use according to claim 7, wherein: The starvation conditions include: a medium without glucose, a medium without fetal bovine serum, a medium without pyruvate, a medium without amino acids, a medium without glucose and amino acids, the addition of inhibitors of glucose metabolism-related enzymes, the addition of small molecules that interfere with cell metabolism such as mTOR inhibitors, the knockout of tumor cell glucose transporters, the knockout of tumor cell glucose utilization-related enzymes or one or more of the following conditions; Preferably, the starvation condition is a medium condition without fetal bovine serum; Preferably, EBSS medium is used to provide starvation conditions without fetal bovine serum.

9. The use according to claim 7 or 8, wherein: The tumor treatment is to induce tumor pan-apoptosis, and the tumor pan-apoptosis includes inducing tumor apoptosis, inducing tumor pyroptosis and / or inducing tumor necrosis.

10. The use according to any one of claims 7 to 9, wherein: The tumor is selected from one or more of liver cancer, lung cancer, colorectal cancer, pancreatic cancer, bone cancer, breast cancer, kidney cancer, esophageal cancer, ovarian cancer, oral cancer, laryngeal cancer, bile duct cancer, uterine cancer, nasal cancer, testicular cancer, meningioma, skin cancer, cervical cancer, glioma, lymphoma, leukemia or sarcoma; preferably, selected from one or more of lung cancer, pancreatic cancer, breast cancer and glioma; preferably lung cancer.

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