Fullerene nanomaterials targeting mannose receptor and preparation method thereof

Abstract

The application discloses a fullerene nanomaterial capable of targeting a mannose receptor and a preparation method thereof. The application connects a mannose molecule to a fullerene through an amidation reaction to prepare a fullerene-mannose derivative material with a determined molecular structure. The mannose group targets a mannose receptor overexpressed by a macrophage, so that the fullerene material is efficiently taken by the macrophage, and the superior antioxidant and anti-inflammatory properties of the fullerene are fully exerted in the cell.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis of nanomaterials, specifically relating to a fullerene nanomaterial that can target mannose receptors and its preparation method. Background Technology

[0002] Macrophages, derived from monocytes and precursor cells in the bone marrow, are phagocytic cells whose primary function is to phagocytose cellular debris and pathogens (i.e., phagocytosis and digestion) in the form of fixed or free cells, and to activate lymphocytes or other immune cells to respond to pathogens. Macrophages are immune cells with multiple functions and are important subjects in the study of phagocytosis, cellular immunity, and molecular immunology. Due to their phenotypic and functional diversity, targeting macrophages and regulating their function has become an important therapeutic approach. Currently, most macrophage-targeted therapies utilize receptor-mediated phagocytosis to provide specificity, and the abundant overexpression of receptors on the surface of macrophages facilitates the targeted design of nanomaterials. Among these, the mannose receptor is the most important and highly efficient endocytic lectin receptor, mainly expressed on macrophages, and can specifically recognize mannose glycosyl molecules. As the most promising macrophage targeting group, mannose has many advantages such as non-toxicity, non-immunogenicity, high biocompatibility, and highly efficient endocytosis mediation, and is widely used for targeted modification of drug systems.

[0003] Fullerene (C 60 Carbon is the third allotrope of carbon. Internally, it is a hollow, soccer ball-shaped molecule composed of 60 carbon atoms forming 60 vertices, 32 faces (including 12 regular pentagons and 20 regular hexagons, where the pentagons are not connected to each other but only adjacent to the hexagons), and 30 carbon-carbon double bonds. Each carbon atom is bonded by sp... 2 The hybrid orbitals are connected to the three adjacent carbon atoms, and the remaining p orbitals are in C. 60 The unique conjugated system formed by the outer periphery and inner cavity of the molecule, along with its high electron affinity, endows fullerenes with outstanding antioxidant and anti-inflammatory capabilities. In recent years, the antioxidant and anti-inflammatory effects of fullerenes have been widely used in the treatment of diseases such as type 2 diabetes, tumors, and ulcerative colitis. Summary of the Invention

[0004] The purpose of this invention is to provide a fullerene nanomaterial that can target the mannose receptor on macrophages, possessing a well-defined molecular structure and excellent biomedical potential. This invention involves attaching mannose molecules to fullerenes via amidation, esterification, or condensation reactions to prepare a fullerene-mannose derivative material (C60-Man) with a defined molecular structure. By targeting the mannose receptor overexpressed on macrophages with the mannose group, this fullerene material can be efficiently taken up by macrophages, allowing it to fully exert its superior antioxidant and anti-inflammatory properties within the cell.

[0005] Specifically, in the first aspect, the present invention provides:

[0006] A mannose receptor-targeting fullerene material covalently bound to mannose, as shown in formula (I):

[0007]

[0008] Wherein, F is a fullerene precursor with a terminal group; T is a mannose receptor targeting group with a terminal group;

[0009] The F and T are covalently bonded together through their end groups;

[0010] Wherein, n is selected from an integer from 1 to 12; preferably, n is selected from an integer from 2 to 4.

[0011] Optionally, the covalent bond may be an amide bond, an ester bond, or an anhydride bond;

[0012] The terminal groups forming the amide bond can be carboxyl and amino groups; the terminal groups forming the ester bond can be carboxyl and hydroxyl groups; the terminal groups forming the anhydride bond can be carboxyl and carboxyl groups.

[0013] The end groups that form the covalent bonds can be interchanged at the F and T positions.

[0014] Optionally, the fullerene precursor may be a fullerene precursor material with a carboxyl end group; and T may be an aminolated mannose and / or a mannose derivative.

[0015] Optionally, the fullerene precursor may be a fullerene precursor material with an amino terminal group; and T may be carboxylated mannose and / or mannose derivatives.

[0016] Optionally, the fullerene precursor may be a fullerene precursor material with a carboxyl end group; and T may be hydroxylated mannose and / or mannose derivatives.

[0017] Optionally, the fullerene precursor may be a fullerene precursor material with hydroxyl terminal groups; and T may be carboxylated mannose and / or mannose derivatives.

[0018] Optionally, the fullerene precursor may be a fullerene precursor material with a carboxyl end group; and T may be carboxylated mannose and / or mannose derivatives.

[0019] Optionally, the fullerene material comprises at least one of hollow fullerene and metallofullerene; preferably, the hollow fullerene is selected from C-containing... 2n One or more fullerene molecules, wherein 2n is the number of carbon atoms, 30≤n≤60; preferably, the metallofullerene includes M@C 2n M2@C 2n and MA@C 2n One or more of the following, wherein M and A both represent metallic elements and M and A are independently selected from any one of Sc, Y, and lanthanide elements; optionally, the lanthanide elements include lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium, 30≤n≤60; 0≤x≤3; more preferably, the fullerene is selected from C 60 C 70 C 76 C 78 C 80 C 82 C 84 One or more thereof; more preferably, the fullerene is selected from C. 60 C 70 One or two of them. More preferably, the metallofullerene is Gd@C82.

[0020] The T group is a mannose receptor targeting group, containing a terminal group on one side that binds to the fullerene group. Optionally, T is mannose and / or a mannose derivative; further, the mannose derivative may be one or more of mannoside, mannosamine, and mannan; even further, the mannose derivative may be one or more of amino-D-mannoside, 4-aminophenyl-α-D-mannopyranoside, 4-nitrophenyl-α-D-mannopyranoside, and carbamoyl-D-mannose; preferably, T is selected from amino-D-mannose, carboxy-D-mannose, hydroxy-D-mannose, or carboxy-D-mannose.

[0021] According to one embodiment of the present invention, the mannose receptor-targeted fullerene material covalently bound to fullerene as shown in formula (I) can specifically be C 60 -Man has the following structure:

[0022]

[0023] In a second aspect, the present invention provides a method for preparing a mannose receptor-targeted fullerene material covalently bound to fullerene and mannose as shown in the above formula (I).

[0024] The preparation method provided by the present invention includes the following steps: reacting a fullerene precursor with a terminal group with a mannose receptor targeting group with a terminal group, so that the mannose receptor targeting group is covalently bound to the fullerene, thereby obtaining the fullerene.

[0025] Alternatively, depending on the end group, the reaction may be an amidation reaction, an esterification reaction, or a condensation reaction.

[0026] Optionally, when the reaction is an amidation reaction, the amidation reaction is carried out in the presence of a condensing agent and / or a base;

[0027] The condensing agent is selected from at least one of the following: HBTU, TBTU, HOBt; the alkali is selected from at least one of the following: EDCI, NHS, DIPEA;

[0028] Preferably, the condensing agent is O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), and the base is N,N-diisopropylethylamine (DIPEA).

[0029] Optionally, the amidation reaction is carried out in a solvent selected from at least one of the following: DMF, DMSO, chloroform; preferably, the solvent is DMF.

[0030] Optionally, when the reaction is an esterification reaction, the esterification reaction is carried out in the presence of a dehydrating agent / activator;

[0031] The dehydrating agent is selected from at least one of the following: DCC, DIC; the activator is selected from at least one of the following: EDC, DMAP.

[0032] Optionally, when the reaction is an acid anhydride reaction, the condensation reaction is carried out in the presence of a dehydrating agent;

[0033] The dehydrating agent is selected from at least one of the following: acetic anhydride, phosphorus pentoxide, and sulfuric acid.

[0034] Thirdly, the present invention provides the application of the mannose receptor-targeting fullerene material covalently bound to fullerene of formula (I) in the preparation of products that inhibit macrophage oxidative activity (ROS).

[0035] This invention utilizes the characteristic of macrophages overexpressing mannose receptors on their cell surface to design and synthesize fullerene nanomaterials (C0) covalently bound to mannose targeting groups. 60 -Man), and its physicochemical properties and biotoxicity were characterized.

[0036] The mannose receptor-targeting fullerene material provided by this invention has the following advantages compared with traditional fullerene materials:

[0037] (1) The molecular structure is well-defined and easy to characterize and detect;

[0038] (2) The material preparation is stable and controllable;

[0039] (3) Due to the addition of mannose targeting groups, it can be efficiently taken up by macrophages, giving full play to the anti-inflammatory and antioxidant properties of fullerene itself. Attached Figure Description

[0040] Figure 1 This illustrates the C-type fullerene nanomaterial covalently modified with mannose. 60 -Man's mass spectrum.

[0041] Figure 2 This illustrates the C-type fullerene nanomaterial covalently modified with mannose. 60 - Infrared spectrum of Man.

[0042] Figure 3 This illustrates the C-type fullerene nanomaterial covalently modified with mannose. 60 -Man's hydrated particle size.

[0043] Figure 4 This illustrates the C-type fullerene nanomaterial covalently modified with mannose. 60 -Man's solubility in water, PBS buffer, serum, and culture medium.

[0044] Figure 5 It shows the presence of mannose (C 60 -Man) or mannose-free (C 60 ESR spectra of PEG-modified fullerene nanomaterials.

[0045] Figure 6 The C164 fullerene nanomaterials modified with mannose at different concentrations are shown. 60 -Man's ESR spectrum.

[0046] Figure 7 It shows the presence of mannose (C 60 -Man) or mann-free (C 60 Effects of PEG-modified fullerene nanomaterials on the viability of RAW264.7 macrophage cells.

[0047] Figure 8 It shows the presence of mannose (C 60 Effects of α-Man modified fullerene nanomaterials on the viability of RAW264.7 macrophages, 4T-1 cells, and L02 hepatocytes.

[0048] Figure 9 It shows the presence of mannose (C 60 -Man) or mann-free (C 60The effect of PEG-modified fullerene nanomaterials on the scavenging of reactive oxygen species in LPS-induced RAW264.7 macrophages.

[0049] Figure 10 It shows the presence of mannose (C 60 -Man) or mann-free (C 60 Effects of PEG-modified fullerene nanomaterials on LPS-induced expression of inflammatory factors in RAW264.7 macrophages.

[0050] Figure 11 It shows the presence of mannose (C 60 -Man) or mann-free (C 60 Effects of PEG-modified fullerene nanomaterials on body weight in ob / ob mice.

[0051] Figure 12 It shows the presence of mannose (C 60 -Man) or mann-free (C 60 Effects of PEG-modified fullerene nanomaterials on liver morphology and liver coefficient in ob / ob mice.

[0052] Figure 13 It shows the presence of mannose (C 60 -Man) or mann-free (C 60 Effects of PEG-modified fullerene nanomaterials on liver pathology in ob / ob mice.

[0053] Figure 14 It shows the presence of mannose (C 60 -Man) or mann-free (C 60 Effects of PEG-modified fullerene nanomaterials on lipid accumulation in the liver of ob / ob mice.

[0054] Figure 15 It shows the presence of mannose (C 60 -Man) or mann-free (C 60 Effects of PEG-modified fullerene nanomaterials on liver function in ob / ob mice. Detailed Implementation

[0055] Based on the above description of the present invention, and in accordance with common technical knowledge and conventional methods in the art, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present disclosure.

[0056] I. Definition

[0057] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0058] The term "treatment" includes the prevention, suppression, relief, or elimination of one or more symptoms of the disease, condition, or related disorder being treated.

[0059] The terms “reduction,” “inhibition,” “mitigation,” or “reduction” are used relative to a control. For example, a reduced index in a subject or cell treated with the compound is compared to a response in a subject or cell not treated with the compound.

[0060] II. Examples

[0061] The present invention will be further explained below with reference to specific embodiments. The description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the teachings of this specification. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0063] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0064] Example 1: Preparation of C 60 -(COOH)2 Fullerene Material Precursor

[0065] Synthesis method:

[0066]

[0067] In a 500 mL round-bottom flask, add 29.22 g of 1,3-acetone dicarboxylic acid, 200 mL of dry dichloromethane, 44.47 g of tert-butanol, and 2.00 g of 4-dimethylaminopyridine. Stir and cool to 0 °C in an ice bath, while simultaneously adding 45.59 g of dicyclohexylcarbodiimide over 5 minutes. After holding at 0 °C for another 5 minutes, remove from the ice bath and stir the dark brown reaction mixture at room temperature for 3 hours. Filter to remove the precipitated dicyclohexylurea. Wash the filtrate with two 50 mL aliquots of 0.5 N diluted hydrochloric acid and two 50 mL aliquots of saturated sodium bicarbonate solution. Filter to remove any precipitate formed during the process. Dry the organic solution with anhydrous magnesium sulfate and concentrate in a rotary evaporator. Elute with ethyl acetate and n-hexane using silica gel column chromatography to obtain compound 1.

[0068] 1 g of compound 1 was dissolved in methanol, and 790 mg of benzylglycine hydrochloride and 180 mg of sodium cyanoborohydride were added. The mixture was stirred at room temperature under a nitrogen atmosphere for 48 h, and then acidified with dilute hydrochloric acid. After 30 min, a saturated NaHCO3 solution was added until the mixture became alkaline. The mixture was extracted with dichloromethane and washed with water. The solvent was removed using a rotary evaporator, yielding a colorless oily substance. 1.39 g of the oil was dissolved in a mixture of ethyl acetate (210 mL) and methanol (50 mL) and degassed with nitrogen. Pd / C (10%, 230 mg) was then added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 24 h. The reaction mixture was filtered through diatomaceous earth, and after removing most of the solvent using a rotary evaporator, precipitation was carried out with n-hexane. The precipitate was washed with n-hexane on filter paper and dried to give compound 2.

[0069] 600 mg of compound 2 and 90 mg of paraformaldehyde were added to 660 mL of C60 saturated toluene solution, and the mixture was heated under reflux at 110 °C for 1 h. After cooling, most of the solvent was removed using a rotary evaporator, and the mixture was purified by silica gel column chromatography. Toluene was used as the eluent, and the third component was compound 3.

[0070] 20 mL of trifluoroacetic acid was added to a 60 mL solution of compound 3 (500 mg) in CHCl3. The mixture was stirred at room temperature for 18 hours, the solvent was removed using a rotary evaporator, and the solution was dried to obtain C. 60 -(COOH)2 (compound 4).

[0071] Example 2: Preparation of Man-NH2 precursor

[0072]

[0073] 5 g of d-mannose was dissolved in 200 mL of pyridine, and 39 mL of acetic anhydride was added. The mixture was stirred overnight at room temperature. After removing the solvent, the residue was dissolved in 200 mL of ethyl acetate and washed with 2N HCl, water, saturated sodium bicarbonate, and saturated brine, respectively. The organic layer was dried over anhydrous magnesium sulfate, filtered, and rotary evaporated to give compound 6.

[0074] Approximately 10 g of compound 6 was dissolved in 25 mL of dichloromethane, and 20 mL of 33% HBr / acetic acid was added. The mixture was stirred for 2 h at room temperature under a nitrogen atmosphere. After the reaction was complete, approximately 100 mL of dichloromethane was added, and the mixture was extracted with cold water. The organic layer was neutralized with saturated sodium bicarbonate solution, washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated using a rotary evaporator to obtain compound 7.

[0075] Add 8 ml of tetraethylene glycol (compound 8) and 4 g of sodium hydroxide to a mixture of 23 ml of water and 23 ml of THF. Cool to 0 °C and add 100 ml of a tetrahydrofuran solution of p-toluenesulfonyl chloride (12.35 g). Stir the reaction mixture at 0 °C for 2 h, then at room temperature for 2 h. Pour the mixture into ice water and extract with dichloromethane. Rinse the organic layer with water and dry with anhydrous magnesium sulfate. Remove the solvent by rotary evaporation to give compound 9.

[0076] 22 g of compound 9, 14 g of potassium phthalimide, and 3 g (3 Å) of molecular sieve were added to 160 mL of DMF. The mixture was stirred at 150 °C for 10 hours, then stirred at room temperature for 8 hours. The mixture was filtered through diatomaceous earth and washed with DMF. After removing the solvent by rotary evaporation, compound 10 was purified by silica gel column chromatography (eluting with ethyl acetate) to obtain purified compound 10.

[0077] 2.0 g of compound 7 and 2.4 g of compound 10 were dissolved in 100 mL of dichloromethane. 3 g of molecular sieve (3 Å) was added, and the mixture was stirred for 30 minutes at -20 °C under nitrogen atmosphere in the dark. Approximately 1.9 g of silver trifluoromethanesulfonate was added, and the mixture was stirred overnight at room temperature. The mixture was neutralized with triethylamine, filtered, washed with dichloromethane, and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation, and the crude product was purified by column chromatography to give compound 11.

[0078] 1.7 g of compound 11 was added to 35 mL of methanol, followed by 0.95 mL of 65% hydrazine hydrate. The reaction mixture was stirred under a nitrogen atmosphere at room temperature until precipitation occurred. The solid was filtered off, and the reaction was continued for 1.5 h with 0.1 M sodium methoxide solution. Sodium ions were adsorbed onto a cation exchange resin, and the mixture was filtered and then subjected to column chromatography to obtain Man-NH2 (compound 12).

[0079] Example 3: Preparation of C 60 -Man fullerene materials

[0080]

[0081] Synthesis method: Add C to DMF 60 -(COOH)₂ (11 μmol), Man-NH₂ (34 μmol), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 56 μmol), and N,N-diisopropylethylamine (DIPEA, 56 μmol). After stirring at room temperature for 12 hours, the reaction was complete. The reaction was stopped, and the resulting product was precipitated in anhydrous diethyl ether. After drying to remove the diethyl ether, it was redissolved in water. Dialyzed using a MWCO 3500 dialysis bag for 48 hours, followed by freeze-drying, to obtain the final product C. 60 -Man (yield approximately 80%).

[0082] The obtained C 60 The -Man material was characterized by mass spectrometry, infrared spectroscopy, and hydration particle size.

[0083] Experimental results: such as Figure 1-3 As shown. MALDI-TOF-MS results show that C 60 The molecular weight of the man sample is exactly the same as the theoretical molecular weight, at 1568. It also has high purity, making it suitable for further biological experiments. The infrared spectrum shows obvious absorption peaks for the amide bond and hydroxyl group; C 60 -The hydrated particle size of the Man material is approximately 30 nm.

[0084] Example 4: Preparation of C 60 -PEG fullerene materials

[0085] Add C to DMF 60 -(COOH)₂ (11 μmol), mPEG2000-NH₂ (34 μmol), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 56 μmol), and N,N-diisopropylethylamine (DIPEA, 56 μmol). After stirring at room temperature for 12 hours, the reaction was complete. The reaction was stopped, and the resulting product was precipitated in anhydrous diethyl ether. After drying to remove the diethyl ether, it was redissolved in water. Dialyzed using a MWCO3500 dialysis bag for 48 hours, followed by freeze-drying, to obtain the final product C. 60 -PEG.

[0086] Example 5: C 60 -Man solubility test

[0087] Experimental method: 1 mg C 60 -Man was dissolved in 1 ml of water, 1 ml of PBS buffer, 1 ml of serum, and 1 ml of cell culture medium, respectively.

[0088] Experimental results: such as Figure 4 As shown, material C 60-Man exhibits good solubility in water, PBS buffer, serum, and cell culture medium.

[0089] Example 6: Determination of free radical scavenging ability of fullerene materials with or without mannose modification

[0090] Detection method: The method of generating hydroxyl radicals from hydrogen peroxide using ultraviolet light was employed. 25 μL of C... 60 -Man or C 60 Mix PEG material (240mM) or ultrapure water, 10 μL of free radical scavenger DMPO (200mM), and 25 μL of hydrogen peroxide solution (100mM), irradiate with ultraviolet light for 2 min, and detect free radical signals by electron spin resonance (ESR).

[0091] Conclusion: As attached Figure 5 As shown, compared with the blank control, C 60 -Man or C 60 - PEG materials all have strong hydroxyl radical scavenging ability, and their free radical scavenging ability is roughly the same at the same concentration.

[0092] Example 7: The scavenging effect of fullerene materials modified with different concentrations of mannose on reactive oxygen species.

[0093] Experimental method: 40 μL of DMPO (100 mM) and 20 μL of H2O2 (100 mM) were respectively reacted with 20 μL of deionized water and 20 μL of C 60 -Man aqueous solution (50 μM), 20 μl C 60 -Mand-water solution (100 μM) was mixed. First, the mixture was irradiated with 500 W UV light for 4 minutes, and then the X-band electron paramagnetic resonance (EPR) spectrum of DMPO-OH was recorded in the dark using an EPR spectrometer.

[0094] Conclusion: Figure 6 As shown, C 60 -Man materials have a strong ability to scavenge hydroxyl radicals, and the scavenging efficiency increases with the increase of the material concentration.

[0095] Example 7: Effects of mannose-modified or unmodified fullerene materials on the viability of RAW264.7 macrophage cells

[0096] Cells: Macrophages RAW264.7

[0097] Experimental Methods: The cytotoxicity of fullerene materials was determined using the CCK8 assay. Materials with a density of 5 × 10⁻⁶ were used. 4RAW264.7 cell suspension at 200 μL / well was seeded into clear 96-well plates. After 12 h of adherent culture, the supernatant was aspirated and replaced with 200 μL of material containing different concentrations of fullerene (C). 60 Cells were cultured in conditioned medium containing fullerene (-Man) in six replicates per group for 24 hours. After the experiment, the conditioned medium containing fullerene was aspirated, and the cells were washed three times with PBS. Then, 100 μL of CCK-8 solution (diluted with colorless DMEM) was added to each well, and the OD values ​​of different groups were measured. The experiment was repeated three times. Cell viability (%) = OD value of treatment group / OD value of control group.

[0098] Conclusion: The corresponding test results are as follows Figure 7 As shown, from Figure 7 It can be concluded that: C 60 -Man (left image) and C 60 -PEG (right figure) is not toxic to RAW264.7 cells at concentrations up to 200 μM.

[0099] Example 8: Effects of mannose-modified fullerene materials on different cell viability

[0100] Cells: RAW264.7 macrophages, 4T-1 cells, and L02 hepatocytes.

[0101] Experimental Methods: The cytotoxicity of fullerene materials was determined using the CCK8 assay. Materials with a density of 5 × 10⁻⁶ were used. 4 RAW264.7 cell suspension of / mL was seeded at 200μL per well in a clear 96-well plate. After 12h of adherent culture, the supernatant was aspirated and replaced with 200μL of fullerene material containing different concentrations (C 60 Cells were cultured in conditioned medium containing fullerene (-Man) in six replicates per group for 24 hours. After the experiment, the conditioned medium containing fullerene was aspirated, and the cells were washed three times with PBS. Then, 100 μL of CCK-8 solution (diluted with colorless DMEM) was added to each well, and the OD values ​​of different groups were measured. The experiment was repeated three times. Cell viability (%) = OD value of treatment group / OD value of control group.

[0102] Conclusion: The corresponding test results are as follows Figure 8 As shown, from Figure 8 It can be concluded that: C 60 -Man showed no toxicity to RAW264.7 cells (left), 4T-1 cells (middle), and L02 hepatocytes (right) at concentrations up to 500 mg / L.

[0103] Example 9: The scavenging effect of fullerene materials with or without mannose modification on LPS-induced intracellular reactive oxygen species in RAW264.7 cells.

[0104] Cells: RAW264.7 macrophages.

[0105] Experimental methods and grouping: The normal group consisted of RAW264.7 cells without LPS stimulation; the model group consisted of RAW264.7 cells stimulated with 100 ng / mL LPS for 12 h; C 60 -Man group is 100μM C 60 -Man material and 100 ng / mL LPS were co-incubated with RAW264.7 cells for 12 h; C 60 -PEG group is 100μM C 60 - PEG material and 100 ng / mL LPS were co-incubated with RAW264.7 cells for 12 h. After the experiment, the conditioned medium was discarded, the cells were washed three times with PBS, and then resuspended in 100 μL of DCFH-DA staining solution (diluted 1:1000 in colorless DMEM according to the instructions). After incubation at 37°C in the dark for 30 min, the staining solution was discarded by centrifugation. 300 μL of PBS solution was added to the sample, and the cells were analyzed by flow cytometry as soon as possible.

[0106] Conclusion: Figure 9 As shown, C 60 -Man and C 60 -PEG can eliminate the increase of reactive oxygen species induced by LPS stimulation in RAW264.7 cells, and C 60 -Man is more effective at scavenging reactive oxygen species than C. 60 -The removal effect of PEG.

[0107] Example 10: Effects of mannose-modified or unmodified fullerene materials on LPS-induced expression of inflammatory factors in RAW264.7 cells

[0108] Cells: RAW264.7 macrophages.

[0109] Experimental methods and grouping: The normal group consisted of RAW264.7 cells without LPS stimulation; the model group consisted of RAW264.7 cells stimulated with 100 ng / mL LPS for 12 h; C 60 -Man group is 100μM C 60 -Man material and 100 ng / mL LPS were co-incubated with RAW264.7 cells for 12 h; C 60 -PEG group is 100μM C 60- PEG material and 100 ng / mL LPS were co-incubated with RAW264.7 cells for 12 h. After the experiment, the conditioned medium was discarded, and the cells were washed three times with PBS. RIPA lysis buffer (Beyotime) containing protease inhibitors and phosphatase inhibitors (Beyotime) was added to the cells, and the cells were lysed on ice for 30 min. After high-speed centrifugation, the supernatant was collected and mixed with 5X loading buffer (Beyotime) to prepare a sample protein concentration for loading. The mixture was then boiled for 10 min. SDS-polyacrylamide gel electrophoresis (Genscript) was then performed, followed by transfer to a PVDF membrane. The membrane was incubated overnight at low temperature with antibodies against p-p65 (Abcam), anti-iNOS (Abcam), anti-CD86 (Abcam), and anti-GAPDH (Cell signaling technology). The membrane was then incubated with the bound secondary antibody (Cell signaling technology), and the antigen-antibody reaction was finally visualized using an enhanced chemiluminescence kit (Absin).

[0110] Conclusion: Figure 10 As shown, compared with the model group, C 60 -Man and C 60 -PEG can inhibit the expression of inflammatory factors iNOS, CD86, and p-p65 induced by LPS stimulation in RAW264.7 cells, and C 60 -Man's anti-inflammatory effect is better than C's. 60 - The anti-inflammatory effect of PEG.

[0111] Example 11: Contains mannose (C 60 -Man) or mannose-free (C 60 The therapeutic effect of PEG-modified fullerene nanomaterials on hepatic steatosis in ob / ob mice. Animal model grouping and treatment: SPF grade C57BL / 6J mice (male, weight: 20g±2g, age: 6 weeks) served as normal controls; ob / ob mice (male, weight: 30g±2g, age: 6 weeks) served as ob / ob mice. Mice were randomly divided into four groups of eight mice each, based on body weight. Body weight and food intake were recorded every three days. 200 μL of physiological saline / C was administered intraperitoneally daily. 60 -Man / C 60 -PEG (1mM). Liver samples were collected 19 days after treatment. The liver was photographed, weighed, and fixed. Grouping was as follows:

[0112] a. Blank control group (Control): C57BL / 6J mice were injected with physiological saline daily;

[0113] b. Model group: ob / ob mice were injected with physiological saline;

[0114] c. Treatment group (C 60 -Man): ob / ob mice injected with C 60 -Man;

[0115] d. Material control group (C 60 -PEG): ob / ob mice injected with C 60 -PEG.

[0116] Experimental Results and Analysis:

[0117] 1) Changes in mouse body weight and weight gain during the experiment, as follows: Figure 11 As shown. It can be seen that C 60 -Man can inhibit weight gain in ob / ob mice.

[0118] 2) Changes in liver morphology and liver coefficient: Figure 12 As shown, the livers of ob / ob mice were significantly swollen and white compared to those of normal mice. (C) 60 The liver enlargement in the -Man treatment group was reduced and the liver regained its normal color compared to the model group. Quantitative analysis showed that the ob / ob mice had reduced liver enlargement and normal color compared to the model group. 60 -Man treatment significantly reduced liver quality and decreased liver coefficient ratio. Meanwhile, non-targeted C 60 -PEG treatment is less effective than C 60 -Man's healing effects.

[0119] 3) Liver H&E pathological staining: Mouse livers were fixed in 4% formaldehyde, then embedded in paraffin, sectioned, and stained with H&E. Results are as follows: Figure 13 The results showed that the normal group mice had normal liver structure, orderly liver plate structure, and no fat vacuoles; the model group mice had a large number of white fat vacuoles in their livers, with swollen and deformed cells, and cell nuclei squeezed to one side; C 60 -Man treatment significantly reduced fatty vacuoles in the mouse liver and restored liver plate structure, while non-targeted C 60 -PEG treatment is less effective than C 60 -Man's healing effects.

[0120] 4) Oil Red O staining of liver: Mouse livers were fixed in 4% formaldehyde, then embedded in OCT, frozen sections, and stained with Oil Red O. Results are as follows: Figure 14 As shown, large areas of orange-red lipid droplets can be seen in the livers of the model group mice. C 60 - Following Man treatment, lipid droplets were significantly reduced preferentially around the portal area, C 60 -PEG treatment is less effective than C 60 -Man's healing effects.

[0121] Example 12: Contains mannose (C 60 -Man) or mannose-free (C 60 Effects of PEG-modified fullerene nanomaterials on liver function indicators in ob / ob mice

[0122] Experimental method: As in Example 6, whole blood of mice was collected, and mouse serum was obtained by centrifugation after standing at room temperature for 1 hour. The levels of liver function indicators, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), were detected by an automated blood biochemistry analyzer.

[0123] Experimental results: such as Figure 15 As shown, compared with the normal group, the AST (left figure) and ALT (right figure) levels in the model group were significantly increased. However, after the treatment method in Example 6, the AST and ALT levels in the mice significantly decreased and tended to normalize; indicating that after C 60 -Man treatment significantly improved liver function in mice. 60 -PEG treatment is less effective than C 60 -Man's healing effects.

Claims

1. The application of the mannose receptor-targeting fullerene material covalently bound to fullerene as shown in Formula (I) in the preparation of products containing substances that inhibit the oxidative activity of macrophages; Formula (I) The mannose receptor-targeting fullerene material covalently bound to mannose as shown in formula (I) is C 60 -Man, its structure is as follows: 。 2. A method for preparing a mannose receptor-targeted fullerene material covalently bonded to fullerene and mannose as shown in formula (I) in claim 1, comprising the following steps: reacting a fullerene precursor with an end group with a mannose receptor-targeting group with an end group to covalently bond the mannose receptor-targeting group to the fullerene, thereby obtaining the material.

3. The preparation method according to claim 2, characterized in that: The reaction is an amidation reaction.

4. The preparation method according to claim 3, characterized in that: The amidation reaction is carried out in the presence of a condensing agent and a base; The condensing agent is selected from at least one of the following: HBTU, TBTU, HOBt; the alkali is selected from at least one of the following: EDCI, NHS, DIPEA; The amidation reaction is carried out in a solvent selected from at least one of the following: DMF, DMSO, and chloroform.

5. The preparation method according to claim 4, characterized in that: The condensing agent is HBTU, and the base is N,N-diisopropylethylamine (DIPEA). The solvent is DMF.

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