CN109096118B - Aminofullerene derivative and preparation method and application thereof - Google Patents
Aminofullerene derivative and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an amino fullerene derivative and a preparation method thereof. The derivative has good water solubility, positive charges on the surface, high free radical capture efficiency and good free radical removal effect, and is suitable for phagocytosis of cells; and has no cytotoxicity in a certain concentration range, but has good cell protection effect. The preparation method has high yield and simple and convenient purification treatment method.
Description
Technical Field
The invention belongs to the field of biological medicine materials. Specifically, the invention relates to an amino fullerene derivative, a preparation method thereof and application thereof in the biomedical field, and more specifically relates to application of the amino fullerene derivative in scavenging free radicals and protecting cells from free radical damage.
Background
Free radicals are groups whose outer orbitals contain unpaired electrons and are highly chemically active.
Free radicals are inevitably produced in organisms, and during normal life, low levels of free radicals are necessary to sustain life. Free radicals are continuously generated and continuously eliminated in the body, and the free radicals are in dynamic equilibrium, so that the free radicals and the free radicals are maintained at a normal physiological level. The research shows that: a certain concentration of free radicals is one of the necessary conditions for the body to perform normal vital activities, but too much free radicals are harmful to the body. It can attack DNA directly, causing its permanent damage; unsaturated fatty acids that can attack biological membranes cause lipid peroxidation; can attack proteins to cause changes in their structure and conformation, resulting in peptide chain breakage, polymerization and cross-linking. All these biomacromolecule structural and functional alterations necessarily lead to a disturbance of cell function, which leads to many diseases such as coronary heart disease, aging, brain injury, inflammation, lung injury, eye tissue injury, atherosclerosis, neurodegenerative diseases, rheumatoid arthritis and other autoimmune diseases.
Free radicals in the human body originate mainly from two aspects, namely exogenous free radicals including atmospheric pollutants (NO)2) Ionizing radiation (ultraviolet), certain drugs (antineoplastic drugs, antibiotics, antipyretics and analgesics), various trace elements (lead, aluminum and the like), alcohol, caused by high-pressure oxygen poisoning; the second is endogenous free radicals, which are produced primarily by some enzymatic or non-enzymatic reactions.
The now recognized biologically antioxidant substances or exogenous natural free radical scavengers mainly consist of: vitamins (vitamin E, vitamin C and carotene), flavonoids (flavones, mushrooms and polysaccharides), lignins, alkaloids, trace elements, gold or palladium nanoparticles and cerium oxide nanoparticles, which have the advantages of single scavenging free radical, low free radical capture efficiency, large dosage and side effects.
Fullerenes are clusters of atoms with closed structures consisting of different numbers of carbon atoms. The fullerene has the advantages of good stability, high strength, large specific surface area and the like, and shows excellent performance in the application of biomedicine, energy storage, hydrogen storage, catalysis and the like. And the electron-deficient polyene structure of the fullerene is easy to generate addition reaction with free radicals, is called as a free radical sponge, and can protect cells from being damaged by the free radicals. However, the fullerene can only be dissolved in partial organic solvent and can not be dissolved in water, so that the fullerene can not be directly applied to a biological system.
A variety of water-soluble fullerene derivatives have been developed, and there are two main types of methods for preparing water-soluble fullerene derivatives: coating fullerene molecules by a polymer; or water-soluble groups such as amino and carboxyl are bonded to the fullerene carbon cage through chemical reaction. However, the polymer coating method has potential danger of fullerene leakage, and the coated particles are large and difficult to phagocytize by cells after entering into the body, so that the application of the water-soluble fullerene derivative prepared by the method in the body is limited. The chemical bond and the method mostly need to dissolve fullerene in an organic solvent for reaction, the solubility of the fullerene in the organic solvent is limited, so that large-scale mass production is difficult, and the process of later purifying and removing the organic solvent is complicated and the removal efficiency is low. In addition, the existing chemical bonding method is a solid-liquid method, a water-soluble group is a bond and is arranged on a nano cluster formed by gathering fullerene molecules, the obtained fullerene derivative is a nano particle, the agglomeration is easy, the water solubility is still poor, and a freeze-dried product is insoluble in water. In addition, in the water-soluble fullerene derivative, the polyhydroxy of the fullerol destroys the integrity of a carbon cage, so that the effect of scavenging free radicals is reduced compared with the fullerene derivative, and the fullerol is not suitable for further derivatization, thereby limiting other applications of the fullerol in the biomedical field. The preparation process of the carboxylic acid fullerene derivative is complicated, the number of bonded carboxylic acids is small, and the water solubility is poor.
Therefore, how to modify fullerene to prepare water-soluble fullerene derivatives while maintaining or even improving the radical scavenging ability thereof to meet the requirements of biomedical applications still remains a technical problem to be solved.
In addition, how to improve the preparation method of the water-soluble fullerene derivative can conveniently control the number of the modification groups bonded on the obtained water-soluble fullerene derivative by adjusting the reaction temperature or the reaction time, and can improve the water solubility, the particle size and the dispersibility of the prepared water-soluble fullerene derivative so as to solve the problems that the water-soluble fullerene derivative prepared at present is easy to agglomerate, has low water solubility and the like.
Disclosure of Invention
The invention provides a novel method for preparing an amino fullerene derivative, and the amino fullerene derivative prepared by the method has high water solubility, small particle size, difficult agglomeration and high activity of removing free radicals.
One aspect of the present invention provides an aminofullerene derivative.
According to the invention, the amino fullerene derivative has the molecular formula of C2n(organic amine)mWherein, C2nN is more than or equal to 30 and less than or equal to 60, the organic amine is liquid at the temperature of 10-40 ℃, m is more than or equal to 5 and less than or equal to 15, and the organic amine molecules are bonded on the surface of the fullerene.
According to the invention, 30. ltoreq. n.ltoreq.45 is preferred, for example n is 30, 35, 38, 39, 42 etc.
According to the invention, the fullerene is preferably C60、C70、C76、C78Or C84. In one embodiment of the present invention, the fullerene is C60Or C70。
According to the invention, the organic amine is preferably at least one of ethylenediamine, propylenediamine, propanolamine and diethanolamine. In one embodiment of the invention, the organic amine is ethylenediamine.
The second aspect of the invention is to provide a preparation method of the amino fullerene derivative.
According to the invention, the preparation method comprises the following steps:
(a) dissolving fullerene in an organic solvent, adding organic amine into the organic solvent for reaction, and collecting precipitate after the precipitate appears;
(b) adding the precipitate obtained in the step (a) into organic amine for reaction;
(c) drying the reaction solution obtained in the step (b), and dissolving the dried residual solid with water to obtain a corresponding aqueous solution.
According to the invention, the organic amine is an organic amine which is liquid at 10 ℃ to 40 ℃. In one embodiment of the present invention, the organic amine is at least one of ethylenediamine, propylenediamine, propanolamine, and diethanolamine. In one embodiment of the invention, the organic amine is ethylenediamine.
According to the invention, the fullerene has the molecular formula C2nN is 30. ltoreq. n.ltoreq.60, preferably 30. ltoreq. n.ltoreq.45. In one embodiment of the present invention, the fullerene is C60、C70、C76、C78Or C84. In one embodiment of the present invention, the fullerene is C60Or C70。
In one embodiment of the invention, the aminofullerene derivative has the formula C2n(organic amine)mWherein, C2nN is more than or equal to 30 and less than or equal to 60, the organic amine is liquid at the temperature of 10-40 ℃, m is more than or equal to 5 and less than or equal to 15, and the organic amine molecules are bonded on the surface of the fullerene.
Preferably 30. ltoreq. n.ltoreq.45, for example n is 30, 35, 38, 39, 42 etc.
Preferably, the fullerene is C60、C70、C76、C78Or C84. In one embodiment of the present invention, the fullerene is C60Or C70。
Preferably, the organic amine is at least one of ethylenediamine, propylenediamine, propanolamine and diethanolamine. In one embodiment of the invention, the organic amine is ethylenediamine.
According to the present invention, the organic solvent in step (a) is an organic solvent capable of dissolving fullerene, including but not limited to: at least one of ortho-xylene, toluene, xylene, carbon disulfide, and chlorobenzene. In one embodiment of the present invention, the organic solvent is ortho-xylene.
The organic solvent capable of dissolving the fullerene is adopted to dissolve the fullerene, so that liquid organic amine is favorably reacted with the fullerene dissolved in the organic solvent, and the organic amine is bonded to a single fullerene molecule rather than the nanocluster of the fullerene molecule in the reaction in the step (a) in a higher probability and proportion, so that the polarity of the single fullerene molecule bonded with the organic amine is favorably enhanced, and the single fullerene molecule is easily separated out from the organic solvent. Namely, the precipitate formed from the organic solvent is mainly a single fullerene molecule bonded with organic amine, and the proportion of the fullerene molecule nanocluster bonded with organic amine is smaller, so that the controllability of the preparation method provided by the invention is established, and the characteristic that the water solubility of the prepared derivative can be improved by the preparation method provided by the invention is established.
According to the present invention, the method of dissolving fullerene in the organic solvent in the step (a) may employ various methods known in the art, including but not limited to: ultrasonic, stirring or shaking, etc.
According to the present invention, the mass ratio of the fullerene to the organic amine in the step (a) may be 1: (100-300), preferably 1 (150-200), for example, the mass ratio of the fullerene to the organic amine is 1:180, 1:160, 1:170, 1:190, and the like.
According to the present invention, the reaction time in step (a) may be several minutes to several hours, and may be adjusted according to the temperature of the reaction. For example, if the reaction is carried out at room temperature, the reaction system is stirred to react for 1 hour; alternatively, the reaction was carried out under heating, stirring and refluxing conditions for a dozen minutes. According to the present invention, the temperature for heating and stirring may be determined according to the boiling point of the organic amine. According to the invention, the stirring rate can be adjusted as appropriate, for example 1000 rpm/min.
According to the invention, the mass ratio of the fullerene to the organic amine in the step (b) can be 1 (500-1500), preferably 1 (800-1200), more preferably 1: (900-1100), for example, the mass ratio of the fullerene to the organic amine is 1:1080, 1:1000, 1:900, or the like.
According to the present invention, the reaction time in the step (b) may be several hours to several tens of hours, and may be adjusted according to the reaction temperature, for example, 2 to 24 hours. According to the embodiment of the present invention, the mixed solution may be stirred at room temperature for 24 hours; alternatively, the reaction was carried out under heating, stirring and refluxing conditions for 8 hours. According to the present invention, the temperature for heating and stirring may be determined according to the boiling point of the organic amine. According to the invention, the stirring rate can be adjusted as appropriate, for example 1000 rpm/min.
According to the invention, in the reaction of step (b), organic amine molecules are further bonded to fullerene molecules to form fullerene molecules bonded with a plurality of organic amine molecules, the polarity of the fullerene molecules is further increased, so that the fullerene molecules can be completely dissolved in the organic amine, and finally a clear and transparent solution can be obtained after the reaction.
According to the invention, the reaction of steps (a) and (b) is preferably carried out under an inert gas atmosphere, for example under a nitrogen, helium or argon atmosphere.
According to the present invention, the drying treatment in step (c) may employ various drying means known in the art, including but not limited to: rotary evaporator, freeze drying, etc. In one embodiment of the invention, the organic amine is evaporated off using a rotary evaporator.
According to the present invention, when the remaining solid after drying is dissolved in water in the step (c), if there is any insoluble matter, the insoluble matter can be accelerated by adjusting the pH of the solution to 4 to 6. In one embodiment of the invention, the pH value of the solution is adjusted by adding an acid solution, wherein the acid solution is 0.5-1.5 mol/L. The acid may be various inorganic and organic acids known in the art, such as hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, and the like.
According to the invention, in step (c) if an acid solution is added, there remains an insoluble material, which is typically a fullerene molecule to which no organic amine is bonded or a fullerene molecule to which only a small amount of organic amine is bonded. The insolubles can be removed by conventional filtration means. Through the step (c), the ratio of fullerene molecules bonded with a plurality of organic amine molecules in the product can be further improved.
According to the invention, optionally, a purification operation is added after said step (c).
According to the invention, said purification operation comprises:
step (d): filtering the aqueous solution obtained in the step (c), adjusting the pH value of the aqueous solution obtained by filtering to be 4-6, and dialyzing until the conductivity of the aqueous solution is less than 1 mu s/cm.
Optionally, further comprising step (e): subjecting the dialyzed solution of step (d) to anion exchange treatment. If an acid solution is added in the step (c) when the dried remaining solid is dissolved, the amino fullerene acid salt can be converted into the amino fullerene by an anion exchange treatment operation.
Optionally, further comprising step (f): dialyzing the anion exchange treated solution of step (e) until the conductivity of the solution is less than 1 μ s/cm.
According to the invention, said step (d) enables dialysis to remove unreacted organic amine.
According to the invention, said step (f) enables dialysis to remove residual inorganic substances.
In one embodiment of the present invention, the dialysis treatment may be performed using a dialysis bag, for example, having a cut-off molecular weight of 3500.
In one embodiment of the present invention, the dialysis treatment is performed by using ultrapure water having an electric conductivity of not less than 18.2 M.OMEGA.cm.
The third aspect of the present invention provides the amidofullerene derivative prepared by the preparation method of the second aspect of the present invention.
Preferably, the amino fullerene derivative has a molecular formula of C2n(organic amine)mWherein, C2nN is more than or equal to 30 and less than or equal to 60, the organic amine is liquid at the temperature of 10-40 ℃, m is more than or equal to 5 and less than or equal to 15, and the organic amine molecules are bonded on the surface of the fullerene.
Preferably 30. ltoreq. n.ltoreq.45, for example n is 30, 35, 38, 39, 42 etc.
Preferably, the fullerene is C60、C70、C76、C78Or C84. In one embodiment of the present invention, the fullerene is C60Or C70。
Preferably, the organic amine is at least one of ethylenediamine, propylenediamine, propanolamine and diethanolamine. In one embodiment of the invention, the organic amine is ethylenediamine.
The fourth aspect of the invention provides the application of the amino fullerene derivative in scavenging free radicals.
The free radical includes at least one of singlet oxygen radical, hydroxyl free radical, hydrogen peroxide free radical, superoxide anion free radical, etc.
According to the invention, said use may be as a therapeutic or prophylactic agent for use in the human or animal body; it may also be the application of non-therapeutic or prophylactic drugs, for example, in certain chemical reaction conditions, in order to avoid the occurrence of free radicals and interference in the reaction system, the amino fullerene derivative is added in advance to the reaction system.
In the embodiment of the present invention, in the research of scavenging hydroxyl radicals by the amidofullerene derivative, the concentration of the amidofullerene derivative is as follows: 20. 50, 100, 200 and 400 mu M, and the concentration of the hydroxyl radical scavenger is 50 mu M, the effect of scavenging free radicals is obvious, and the effect of scavenging free radicals can reach 90 percent at 400 mu M.
The fifth aspect of the invention provides the application of the amino fullerene derivative in preparing a free radical scavenger.
The free radical includes at least one of singlet oxygen radical, hydroxyl free radical, hydrogen peroxide free radical, superoxide anion free radical, etc.
The free radical scavenger is used to protect cells against free radical damage.
In the embodiment of the invention, in the cytotoxicity research of the amino fullerene derivative, the concentration of the used amino fullerene derivative is in the range of 0-400 mu M, the amino fullerene derivative has no cytotoxicity, and the cell activity is higher than that of a control group without the addition of the amino fullerene derivative.
In the embodiment of the invention, in the cell antioxidant protection experiment of the amino fullerene derivative, the concentration of the used amino fullerene derivative is in the range of 0-100 mu M, and the cell antioxidant protection effect is obvious at 2.5 mu M.
The invention has the advantages that:
1. the preparation method of the amino fullerene derivative provided by the invention can conveniently control the number of amino groups in the obtained amino fullerene derivative by adjusting the reaction temperature or the reaction time; the subsequent purification treatment is simple and convenient to operate, residual organic amine and inorganic impurities can be completely removed only by adopting acidification treatment, dialysis purification, anion exchange and the like, and the purification effect is good.
2. The preparation method of the amino fullerene derivative can prepare the amino fullerene derivative with improved water solubility, and the amino fullerene derivative can be completely re-dissolved in ultrapure water after freeze drying; the particle size is small, the surface of the particle is positively charged, the dispersibility is good, the aggregation is not easy to occur, the particle is suitable for phagocytosis, the free radical capturing efficiency is high, and the effect of removing the free radicals is good.
3. Compared with carboxylic acid fullerene derivatives, amino in the amino fullerene derivatives can further generate bonding reaction with biomolecules such as DNA and amino acid, so that the application of the amino fullerene derivatives in the biomedical field can be further enriched.
4. The prepared amino fullerene derivative has no cytotoxicity in a certain concentration range, and has good cell protection and free radical damage resistance effects.
Drawings
FIG. 1 shows the IR absorption spectra of aminofullerene derivatives prepared in example 1 and example 2 of the present invention;
FIG. 2 shows hydrated particle sizes and Zeta potentials of amino fullerene derivatives prepared in example 1 and example 2 of the present invention;
FIG. 3 shows the effect of the Aminoperfluorene derivatives of the present invention prepared in example 1 and example 2 on scavenging hydroxyl radicals using ESR;
FIG. 4 shows the cytotoxicity of aminofullerene derivatives prepared in example 1 and example 2 of the present invention using HUVEC cells;
FIG. 5 shows that HUVEC cells are used to test the effect of the aminofullerene derivatives prepared in examples 1 and 2 of the present invention on protecting cells against free radical damage.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: preparation C60(EDA)6
(a) Weighing 5 with analytical balance0mg solid fullerene C60(purity: 99%, Xiamen new materials science and technology Co., Ltd.) was dissolved in 25mL of o-xylene solution, ultrasonic dispersion was carried out for 30min, 10mL of ethylenediamine (analytical reagent, Chinese medicine reagent) was measured in a measuring cylinder, 250mL of conical flask with stopper was added with the o-xylene solution and ethylenediamine, and then magnetons were added, and the mixture was stirred for 1h with a magnetic stirrer (temperature: room temperature, rotation speed: 1000r/min, N2As protective gas), standing and collecting the precipitate.
(b) Adding 50mL of ethylenediamine (analytically pure, Chinese medicine reagent) into the collected precipitate, stirring and reacting for 24h (temperature: room temperature, rotating speed: 1000r/min, N)2As a shielding gas).
(c) The solution obtained in step (b) was added to a 250ml round-bottom flask and the reaction solution was dried by rotary evaporation using a rotary evaporator (model: IKA RV10basic) (temperature: 60 ℃ C., rotation speed: 80 r/min). Adding ultrapure water to dissolve the solid matter obtained after drying, if a small amount of insoluble matter exists, adding a dilute hydrochloric acid solution (concentration: 1mol/L) into the round-bottom flask, and shaking the flask to dissolve the evaporated matter on the inner wall in the dilute hydrochloric acid to obtain a reddish brown clear solution.
(d) And (c) neutralizing the solution obtained in the step (c) by using NaOH aqueous solution (the concentration is 10mol/L), and detecting the pH with a pH test paper to be weakly acidic (the pH is about 4-6) so as to ensure that the excessive ethylenediamine exists in a chloride form and can be sufficiently removed in the subsequent dialysis step. Putting the neutralized solution into a dialysis bag (with a cut-off molecular weight of 3500), and dialyzing in ultrapure water until the electric conductivity of the ultrapure water is less than 1 μ s/cm.
(e) And (3) carrying out anion exchange treatment on the dialyzed solution to completely convert chloride ions into hydroxide ions, thereby converting the amino fullerene hydrochloride into the amino fullerene.
(f) And dialyzing the solution after the anion exchange treatment again until the conductivity of the ultrapure water is less than 1 mu s/cm, thus obtaining the water-soluble amino fullerene derivative.
The reddish brown solution was dropped onto a silver mirror and dried naturally for infrared spectroscopy (IR) testing.
As shown in FIG. 1, the infrared characteristic absorption (-NH) of the sample at around 3300nm2Stretching vibration absorption peak of (C-C) and C ═ C) demonstrates that ethylenediamine is bonded to the carbon cage of fullerene, while changes in the absorption at the 800-.
The sample solution is directly used for Dynamic Light Scattering (DLS) test, as shown in figure 2, the hydrated particle size is about 120nm, the surface of the sample solution is positively charged, and the sample solution is suitable for being captured and phagocytized by cells.
The samples were freeze dried for C, H, N Elemental Analysis (EA) and 2 sites were randomly selected from the samples, the results are shown in Table 1, and the chemical composition C was determined from the N: C ratio60(EDA)6。
Table 1: c60(EDA)6Results of elemental analysis
By adjusting the reaction time of the ethylenediamine and the fullerene in the step (a), the quantity of the bonded ethylenediamine can be regulated and controlled, and C is obtained60(EDA)m，m＝5～15。
Example 2: preparation C70(EDA)8
The reaction conditions and procedure were as in example 1, except that 50mg of fullerene C was weighed70Solid (purity: 99%, Xiamen New Material science and technology Co., Ltd.) substituted for C60. Finally obtaining a catalyst containing C70(EDA)8A clear reddish brown solution of (a).
The obtained product was subjected to component detection using the same test methods and conditions as in example 1.
As shown in FIG. 1, the infrared characteristic absorption (-NH) of the sample at around 3300nm2Stretching vibration absorption peak of (C-C) and C ═ C) proves that ethylenediamine is bonded to the carbon cage of fullerene, and also that ethylenediamine has been bonded to the carbon cage as a result of a change in the infrared characteristic absorption (stretching vibration peak of C-C and C ═ C on the carbon cage) at 800 to 1500 nm.
The sample solution is directly used for Dynamic Light Scattering (DLS) test, as shown in figure 2, the hydrated particle size is about 140nm, the surface of the sample solution is positively charged, and the sample solution is suitable for being captured and phagocytized by cells.
The samples were freeze dried for C, H, N Elemental Analysis (EA), 2 sites were randomly selected from the samples, the results are shown in Table 2, and the chemical composition C was determined from the N: C ratio70(EDA)8。
Table 2: c70(EDA)8Results of elemental analysis
By adjusting the reaction time of the ethylenediamine and the fullerene, the quantity of the bonded ethylenediamine can be regulated and controlled to obtain C70(EDA)m，m＝5～15。
The sample can be quantified by the content of C on fullerene, and the prepared C is purified by ultrapure water60(EDA)6And C70(EDA)8Water-soluble aminofullerene derivative solutions of different concentrations were prepared and used in the following examples.
Example 3: c60(EDA)6、C70(EDA)8Scavenging test for hydroxy radical
Induction of H by Ultraviolet (UV)2O2Hydroxyl free radicals are generated, and DMPO is taken as a free radical capture agent, so that DMPO is quickly combined with the free hydroxyl free radicals to generate DMPO-OH which can be detected by EPR.
Solution preparation: 100 μ L of DMPO (100mM) and 50 μ L of H2O2A blank control was prepared by mixing (100mM) and 50. mu.L of ultrapure water.
Test C60(EDA)6、C70(EDA)8When the free radicals are eliminated, the concentrations of the added samples in the test system are respectively as follows: 20. 50, 100, 200, 400 μ M.
The test results are shown in FIG. 3. C60(EDA)6、C70(EDA)8At a concentration of 50. mu.M, there is a significant effect of scavenging free radicals, when C is present60(EDA)6、C70(EDA)8Are respectively provided withAt 400 mu M, the effect of clearing free hydroxyl can reach 90 percent, and the dose-effect relationship is obvious. For ease of illustration, only 50 μ M and 400 μ M results are presented in FIG. 3.
Example 4: cytotoxicity test
Testing of C prepared in examples 1, 2 Using Human Umbilical Vein Endothelial Cells (HUVEC)60(EDA)6、C70(EDA)8Cytotoxicity of (a):
HUVEC cells (Chinese microbial strain net) were subcultured in DMEM medium containing 15% calf serum, trypsinized, and then cultured at 5X 104Perml cell density seed 96-well plate (4 wells are used as 1 experimental group, the first group is used as a control group), 200. mu.L of cell dispersion liquid is added into each well, Phosphate Buffer Solution (PBS) is added into the peripheral circle of wells, and the wells are incubated in an incubator for 24 hours; the medium was replaced with 180. mu.L of fresh medium, 20. mu.L of C was added at concentrations of 100, 200 and 400. mu. mol/L, respectively60(EDA)6Solutions or C70(EDA)8Incubating the solution for 3 hours in the dark; the cells were incubated for 120min with 90. mu.L of colorless medium and 10. mu.L of CCK-8 (cell viability detector) until the color became bright orange, and the cell viability was measured with a microplate reader.
The results of cell viability are shown in FIG. 4, with addition of C60(EDA)6Or C70(EDA)8The activity of the cells in the experimental group is higher than that in the control group, and the dose-effect relationship is shown, namely, the cells are added with the addition of C60(EDA)6Or C70(EDA)8The concentration of (a) is increased, and the cell activity is increased. As can be seen, C60(EDA)6、C70(EDA)8The sample did not show any cytotoxicity and the cell activity was increased compared to the control group, indicating that C60(EDA)6、C70(EDA)8Has good cell protection effect.
Example 5: cell antioxidation experiment
Testing of C prepared in examples 1, 2 Using Human Umbilical Vein Endothelial Cells (HUVEC)60(EDA)6、C70(EDA)8Cell antioxidant assay of (1):
HUVEC cells (Chinese microbial strain net) were subcultured in DMEM medium containing 15% calf serum, trypsinized, and then cultured at 5X 104Perml cell density seed 96-well plate (8 wells for 1 experimental group, first group for control group, second group for H2O2Control group), 200. mu.L of cell dispersion was added to each well, and Phosphate Buffered Saline (PBS) was added to the peripheral wells to incubate for 24 hours; the medium was replaced with 90. mu.L of fresh medium, 10. mu.L of C was added at a concentration of 2.5, 5, 10, 20, 40, 80. mu. mol/L, respectively60(EDA)6、C70(EDA)8Incubating the solution for 3 hours in the dark; the cells were incubated for 1 hour with 100. mu.L hydrogen peroxide (3mM), for 24 hours with 200. mu.L cell culture medium, and for 120min with 90. mu.L colorless medium and 10. mu.L CCK-8 (cell activity detector) until the color changed to bright orange, and then the cell activity was measured with a microplate reader.
The results of cell viability are shown in FIG. 5, using H2O2The activity of the treated cells is reduced, and C is used60(EDA)6、C70(EDA)8The degree of decrease in cell activity was suppressed in the pretreated cell sample, indicating that C60(EDA)6、C70(EDA)8Has certain protection effect on hydrogen peroxide damaged cells.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (12)
1. A preparation method of an amino fullerene derivative is characterized by comprising the following steps:
(a) dissolving fullerene in an organic solvent, adding organic amine into the organic solvent for reaction, and collecting precipitate after the precipitate appears; the mass ratio of the fullerene to the organic amine in the step (a) is 1: (100-300);
(b) adding the precipitate obtained in the step (a) into organic amine for reaction; the mass ratio of the fullerene to the organic amine in the step (b) is 1 (500-1500);
(c) drying the reaction solution obtained in the step (b), and dissolving the dried residual solid with water to obtain a corresponding aqueous solution;
(d) filtering the aqueous solution obtained in the step (c), adjusting the pH value of the aqueous solution to 4-6, and dialyzing until the conductivity of the aqueous solution is less than 1 mu s/cm;
optionally, further comprising step (e): subjecting the dialyzed solution of step (d) to anion exchange treatment;
optionally, further comprising step (f): dialyzing the solution after the anion exchange treatment in the step (e) until the conductivity of the solution is less than 1 mu s/cm;
the organic amine is liquid at 10-40 ℃;
the molecular formula of the fullerene is C2n，30≤n≤60；
The molecular formula of the amino fullerene derivative is C2n(organic amine)mWherein, C2nRepresents fullerene, m is more than or equal to 5 and less than or equal to 15, and organic amine molecules are bonded on the surface of the fullerene.
2. The method according to claim 1, wherein the organic amine is at least one of ethylenediamine, propylenediamine, propanolamine, and diethanolamine.
3. The method of claim 1, wherein 30. ltoreq. n.ltoreq.45.
4. The method of claim 3, wherein the fullerene is C60、C70、C76、C78Or C84。
5. The method according to any one of claims 1 to 4, wherein the mass ratio of the fullerene to the organic amine in the step (a) is 1 (150 to 200).
6. The production method according to any one of claims 1 to 4, wherein the organic solvent in the step (a) is at least one of toluene, xylene, carbon disulfide and chlorobenzene.
7. The method of claim 6, wherein the xylene is ortho-xylene.
8. The method according to any one of claims 1 to 4, wherein the mass ratio of the fullerene to the organic amine in the step (b) is 1 (800-1200).
9. The method of claim 8, wherein the mass ratio of fullerene to organic amine in step (b) is 1: (900-1100).
10. A process according to any one of claims 1 to 4, wherein, when the remaining solid after drying is dissolved in water in step (c), if any insoluble matter remains, the dissolution of the insoluble matter is promoted by adjusting the pH of the solution to 4 to 6.
11. The method according to claim 10, wherein the pH of the solution is adjusted by adding an acid solution, wherein the acid solution is 0.5 to 1.5 mol/L.
12. The method of claim 11, wherein the acid is hydrochloric acid, sulfuric acid, acetic acid, or phosphoric acid.
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| US5688486A (en) * | 1992-02-11 | 1997-11-18 | Nycomed Salutar, Inc. | Use of fullerenes in diagnostic and/or therapeutic agents |
| CN1906149A (en) * | 2003-12-15 | 2007-01-31 | Nano-C公司 | Higher fullerenes useful as radical scavengers |
| CN107555418A (en) * | 2016-06-30 | 2018-01-09 | 中国科学院化学研究所 | Amino fullerene and preparation method thereof |
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| US5688486A (en) * | 1992-02-11 | 1997-11-18 | Nycomed Salutar, Inc. | Use of fullerenes in diagnostic and/or therapeutic agents |
| CN1906149A (en) * | 2003-12-15 | 2007-01-31 | Nano-C公司 | Higher fullerenes useful as radical scavengers |
| CN107555418A (en) * | 2016-06-30 | 2018-01-09 | 中国科学院化学研究所 | Amino fullerene and preparation method thereof |
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