RU2548971C2 - Method for producing aqueous nanodispersions of fulleren - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention can be used in the chemical industry, cosmetics and medicine in making cosmetic products, therapeutic agents, antioxidants, antimicrobial agents, radioprotective substances, compounds for gene material delivery. An aqueous nanodispersion of fullerene is produced by solving C₆₀ fullerite crystals in N-methylpyrrolidone. The prepared solution is mixed with water and a stabilising agent, which is presented by amino acid, monosaccharide, peptide, polyvinylpyrrolidone or glycerol. That is followed by the dialysis of the prepared mixture. After the dialysis, the solution can be concentrated, e.g. by vacuum vaporisation. The process is safe as uses no toxic solvents.

EFFECT: simplifying the process by eliminating the use of pre-milled fulleren crystals, ultrasonic treatment and heating.

2 cl, 26 dwg, 4 ex

Description

The invention relates to methods for producing stable aqueous solutions (nanodispersions) of fullerene and can be used in a wide range of fields, from electronics to medicine, for example, to create cosmetics, drugs for the treatment of diseases associated with inflammation and oxidative stress (allergy, asthma, dermatitis , ischemic diseases), anticancer drugs, antioxidants, antimicrobial agents, radioprotectors, compounds for the delivery of gene material.

Derivatives of fullerene are known to have various types of biological activity: antitumor, antiviral, antibacterial, antioxidant, neuroprotective, etc. Fullerene Ceo does not exhibit noticeable toxicity and can be completely eliminated from the body within a few days, fullerene and its adducts are able to pass through biological membranes and can be used as an enhancer of drug delivery. Fullerene and its derivatives exhibit antioxidant activity, the ability to inactivate free radicals.

The promising medical use of fullerene is associated with the delivery of nucleic acids, including small interfering RNAs, as universal therapeutic drugs. The introduction of positively charged groups on the surface of fullerene leads to amphiphilic cationic derivatives, which, due to electrostatic interactions, can bind nucleic acids, fold them into a compact structure and ensure their penetration into cells by endocytosis.

The main problem in the use of fullerene in medicine is its almost complete insolubility in aqueous media - 0.02 ng / L. To solve this problem, it is most often used to obtain aqueous nanodispersions of fullerene, the creation of donor-acceptor complexes with hydrophilic compounds, and chemical modification by hanging hydrophilic groups on the fullerene framework.

Most methods for the formation of sufficiently stable aqueous fullerene nanodispersions (NDF) are based on the transfer of fullerene from an organic solution (benzene, toluene, tetrahydrofuran) to the aqueous phase using ultrasound, followed by gradual removal of the organic solvent by vacuum or by blowing with an inert gas (Deguchi Sh., Mukai S.-A. Top-down preparation of dispersions of C ₆₀ nanoparticles in organic solvents Chemistry Letters, 2006; 35 (4): 396-397; Andrievsky GV, Kosevich MV, Vovk O.M., Shelkovsky VS, Vashcenko LA On the production of an aqueous colloidal solution of fullerenes. J. Chem. Soc, 1995, 12, 1281 - 128215). As a rule, the result is a yellow-brown colloidal solution containing hydrated clusters of C ₆₀ molecules (nanocrystals), the size of which depends on the characteristics of the method and varies in the nanometer range (10-1000 nm). The properties of NDF, particle size, stability, pH of coagulation, etc., depend on the method of their preparation. The presence of a negative charge on the surface of the clusters plays an important role in the stabilization of aqueous dispersions of fullerene; therefore, when the pH and ionic strength of the medium change, they easily aggregate. In addition, they cannot be dried, because they lose their ability to dissolve in water. At the same time, the cost of such drugs is quite high due to the small yield of fullerene in solution and the cost of various manipulations, and therefore they are not widely used.

The best-known method for preparing aqueous fullerene dispersions is the solvent replacement method in the organic fullerene solution, where the good solvent for fullerene (benzene, toluene, chlorobenzene) is successively diluted / replaced with a hydrophilic solvent (acetone, tetrahydrofuran) and then with water. The organic solvent is gradually displaced by purging a mixture of inert gases, evaporation and / or intensive ultrasonic treatment to ensure heating of the system (Mchedlov-Petrosyan N.O. Fullerene C ₆₀ solutions: the colloidal aspect. Chemistry, Physics and Technological Sawing, 2010, V. 1, No. 1, 19-37). These organic solvents are toxic and biologically incompatible substances. And, despite the actions in the process of processing dispersions to remove them, aromatic solvents are almost difficult to completely remove from the aqueous dispersion of fullerene, due to their specific (π-stacking) interaction with fullerene molecules (Konarev DV, Litvinov AL, Kovaltvsky A.Yu., Drichko NV, Coppens RN, Lubovskaya RN Molecular complexes of fullerene C ₆₀ with aromatic hydrocarbons: Crystal structures of (TPE) 2C ₆₀ and DPA · C _60. Synthetic Metals, 2003, 133-134, 675-677).

Known methods for producing aqueous dispersions by direct mechanical dispersion of fullerite powder (fullerene crystals) in an aqueous medium, often combined with grinding of fullerene in a mortar and scoring (Labille J., Brant JA, Villieras F., Pelletier M., Wiesner MR, Bottero J .-Y. Affinity of C ₆₀ fullerenes with water. Fullerenes, Nanotubes, and Carbon Nanostructures, 2006, 14 (2-3): 307-14). This method has a rather low efficiency, requires very long mixing (from several days to many months), and the yield of fullerene in the solution is very low. There is an assumption that aerobic conditions, the presence of ozone, lead to the formation of epoxide on the surface of crystalline fullerene nanoparticles, which increases their hydrophilicity and underlies the mechanism of transition of the fullerene crystallite to the nanodispersed state (Murdianti B.S., Damron J.T., Hilburn M .E., Maples RD, Koralege H. RS, Kuriyavar SI, Ausman K. D. C ₆₀ oxide as key component of aqueous C ₆₀ colloidal suspensions. Environ. Sci. Technol., 2012, 46, 7446-7453).

Fullerene can also be converted into solution by boiling fullerite with an aqueous solution of γ-cyclodextrin (CD, cyclic sugars consisting of 6-8 glucose residues) with a predominant molar ratio of C ₆₀ : CD = 1: 2. Fullerene inside the complex is in free and hydrated forms. However, even when using gel chromatography to purify fullerene, it nevertheless slowly decomposes. Thus, the stability of such complexes is insufficient for long-term manipulations and free components will always be in equilibrium in the medium, as a result, such dispersion may turn out to be unstable (Andersson T., Nilsson K., Sundahl M., Westman G., Wennerstrom O. S ₆₀ embedded in γ-cyclodextrin: a water-soluble fullerene. J. Chem. Soc. Chem. Commun., 1992; 604-605; Piotrovsky LB, Kiselev OI Fullerenes in biology. St. Petersburg: Rostock; 2006 g.). Complexes of a similar type also form calixarenes and other “basket-like” compounds.

Of the amphiphilic polymers capable of forming water-soluble complexes with C ₆₀ , the most popular biocompatible polymer is polyvinylpyrrolidone (PVP). Aqueous C ₆₀ / PVP nanodispersions are colored yellow-brown, and the fullerene content in them reaches 0.5-1% depending on the conditions of production and the molecular weight of PVP (Piotrovsky L.B., Kiselev O.I. Fullerenes in biology. St. Petersburg: Rostock; 2006; Yamakoshi YN, Yagami T., Fukuhara K., Sueyoshi S., Miyata N. Solubilization of fullerenes into water with polyvinylpyrrolidone applicable to biological tests. J. Chem. Soc. Chem. Commun., 1994; 517 -518). Such nanodispersions have biological activity comparable to the initial fullerene.

RU 2462474 describes the synthesis of covalent amino acid derivatives of fullerene C ₆₀ , where fullerene and trimethylsilylated amino acids are dissolved in polar aprotic solvents with high dielectric constant: N-methyl-2-pyrrolidone, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, and in the presence of oxygen amino acid adduct. To isolate the final product, the mixture is treated with water to remove trimethylsilyl groups, then the formed trimethylsiloxane is extracted with ethyl acetate, the aqueous layer is dialyzed, removing excess amino acids and acetamide formed during hydrolysis, and the resulting dialysate is lyophilized to isolate the solid substance, fullerene amino acid (adduct). However, in this method, not an aqueous dispersion of free fullerene is obtained, but its derivative, another substance.

The closest known to the described invention is a method for producing aqueous nanodispersions of fullerene (Andrievsky GV, Kosevich M.V., Vovk O.M., Shelkovsky VS, Vashcenko LA On the production of an aqueous colloidal solution of fullerenes. J. Chem. Soc , 1995, 12, 1281-1282), including the preparation of a solution of fullerene in an organic solvent (e.g., toluene), followed by ultrasonic treatment and the transfer of fullerene from the solution to the aqueous medium. After that, the aqueous medium containing fullerenes is additionally filtered through microfilters. Aqueous molecular-colloidal solutions of hydrated fullerenes are obtained in the form of transparent solutions from yellow to brownish-orange, which opalescent in reflected light, indicating their colloidal properties.

The conditions for the implementation of the known method is almost difficult to accurately reproduce, since they depend on the power of the installation, processing time, geometry of the zone exposed, on the temperature of the medium, the cyclical effect, etc. Therefore, the use of ultrasonic treatment for particle crushing, used in all known methods for producing nC ₆₀ , always introduces uncertainty into the protocol of the method, which affects the dispersion efficiency. In addition, some modes can lead to the oxidation of fullerene and the generation of free radicals.

The technical result of the present invention consists in creating a simple effective and soft method for producing an aqueous solution of fullerene C ₆₀ (nanodispersion, nC ₆₀ ), increasing the manufacturability and environmental friendliness of the method for producing aqueous solutions of fullerene C ₆₀ due to the fact that the process does not require preliminary grinding of fullerene crystals, not uses toxic solvents, and also does not apply ultrasonic treatment, nor heating, nor evaporation in vacuum.

The specified technical result is achieved in that the method for producing aqueous fullerene nanodispersions involves dissolving fullerene in the form of C ₆₀ fullerite crystals in N-methylpyrrolidone and diluting the resulting N-methylpyrrolidone-C ₆₀ solution with water, or an aqueous solution of an amino acid, or an aqueous solution of monosaccharide, followed by exhaustive dialysis or diafiltration. If necessary, the resulting solution is concentrated.

The aqueous medium may contain a stabilizing additive. In this case, an amino acid, or a monosaccharide, or a peptide, or glycerin, or a water-soluble polymer, or polyvinylpyrrolidone can be used as a stabilizing additive.

It is preferable to concentrate the solution after dialysis, for example by evaporation in vacuo.

The process is carried out at room temperature. The first stage of the process (dissolution of fullerene in MP) is carried out within a few hours. The time-limiting step of obtaining nC ₆₀ is dialysis, which is carried out over a period of about two days. During dialysis, free amino acids and N-methylpyrrolidone are removed from the solution of fullerene. N-methylpyrrolidone (MP) is a low-toxic biocompatible solvent and is easily removed during dialysis.

To obtain a solution of fullerene as agents, additional natural non-toxic components can be used as stabilizing additives (DM) (L-amino acids, sugars, etc.), which contribute to the dispersion and stabilization of the C ₆₀ dispersion.

A dispersion of nC ₆₀ obtained in the absence of diabetes mellitus (i.e., simply dialysis of a fullerene solution in a MP / water mixture) also forms, however, it is less stable and nanoparticles are rather large (> 0.45 μm), its concentration leads to the formation of a precipitate. As diabetes, peptides and monosaccharides can be used. For example, the tripeptide Arg-Lys-Asp (RKD) is a good stabilizer, the yield of fullerene in the solution is about 100%, as with rhamnose.

In the same way, by mixing a solution of fullerene in N-methylpyrrolidone and a solution of polyvinylpyrrolidone in water followed by dialysis, a complex of fullerene with polyvinylpyrrolidone (C ₆₀ / PVP) is easily obtained. While the known methods for producing such complexes involve the use of toxic solvents, for example, mixing with a solution of C ₆₀ in ortho-dichlorobenzene is carried out with a solution of PVP in chloroform, followed by removal of solvents and dissolving the dry residue in water (RF Patent No. 2255942, IPC C08F 126 / 10, C08F 8/00, A61K 31/785, op. In 2005). Essentially, the described method is conveniently used to obtain complexes of C ₆₀ with any water-soluble polymers and proteins.

Nano-dispersions of nC _{60 are} readily concentrated by evaporation in vacuo. For example, the initial concentration for nC ₆₀ / Gln = 57 μg / ml, and after evaporation on a rotary evaporator in vacuum, it rises to 226 μg / ml (see Fig. 2), i.e. concentration 4 times.

Fullerene molecules, as is known, in the aquatic environment are exclusively in cluster form, forming associates that are unable to pass through the pores of the dialysis membrane, which is convenient for cleaning the dispersion from low molecular weight compounds. As a result of these manipulations, a brownish-yellow solution is formed, which has an electronic spectrum characteristic of nanodispersion of fullerene C ₆₀ : three peaks in the region of 200-220, 260-270 and 335-345 and a weak absorption band between 400 and 500 nm. The concentration of fullerene, measured according to optical density at 340 nm (ε ₃₄₀ = 46450) is 60-140 mg / l, depending on the type of diabetes used (FIGS. 2-11).

A method of obtaining a fullerene dispersion in accordance with the invention is illustrated by drawings and drawings, which show:

in FIG. 1 is a flowchart for preparing an aqueous dispersion of fullerene nC ₆₀ ;

in FIG. 2 is an electronic absorption spectrum (UV-VID) of a C ₆₀ dispersion obtained in the presence of Thr (C ₆₀ / Thr);

in FIG. 3 - electronic absorption spectrum of the dispersion With ₆₀ / Ala;

in FIG. 4 - electronic absorption spectrum of a Ceo / Leu dispersion;

in FIG. 5 - electronic absorption spectrum of the dispersion With ₆₀ / Trp;

in FIG. 6 - electronic absorption spectrum of the dispersion With ₆₀ / Asr;

in FIG. 7 - electronic absorption spectrum of a dispersion of C ₆₀ / Arg;

in FIG. 8 - electronic absorption spectrum of a dispersion of C ₆₀ / His;

in FIG. 9 - electronic absorption spectrum of the dispersion With ₆₀ / Gln;

in FIG. 10 - electronic absorption spectrum of a dispersion of C ₆₀ / Met;

in FIG. 11 is an electronic absorption spectrum of a dispersion of C ₆₀ / L-ramnose;

in FIG. 12 - electronic absorption spectrum of the dispersion With ₆₀ / RKD;

in FIG. 13 shows the electronic absorption spectrum of a C ₆₀ / PVP dispersion;

in FIG. 14 - electronic absorption spectrum of a dispersion of C ₆₀ / -SD (lack of diabetes);

in FIG. 15 - IR-Fourier spectrum of the sample lyophilized dried solution With ₆₀ / Thr;

in FIG. 16 - IR-Fourier spectrum of a sample of lyophilized dried solution of C ₆₀ / L-ramnose;

in FIG. 17 - IR-Fourier spectrum of a sample of lyophilized solution With ₆₀ / His;

in FIG. 18 - IR-Fourier spectrum of a sample of lyophilized dried solution With ₆₀ / Met;

in FIG. 19 - IR-Fourier spectrum of the sample lyophilized solution of C ₆₀ / RKD;

in FIG. 20 — IR-Fourier spectrum of a sample of a lyophilized dried C ₆₀ / PVP solution;

in FIG. 21 - Photo nanodispersion With ₆₀ / Thr;

in FIG. 22 - Photo nanodispersion With ₆₀ / L-ramnose;

in FIG. 23 - Photo of C ₆₀ / His nanodispersion;

in FIG. 24 - Photo nanodispersion With ₆₀ / Met;

in FIG. 25 Photo nanodispersion With ₆₀ / RKD;

in FIG. 26 Photo nanodispersion With ₆₀ / PVP.

Below are examples of obtaining an aqueous dispersion of fullerene in the presence of various diabetes mellitus, including PVP (complex C ₆₀ / PVP).

Example 1

20 mg of crystalline fullerene C ₆₀ (99.9%, SES Research, catalog # 600-9969, USA) was dissolved in 25 ml of N-methylpyrrolidone (MP) (99%, Panreac) by stirring using a magnetic stirrer until completely dissolved 3-4 hours. A dark brown-purple solution of MP is mixed with 20-40 mg of DM in 100 ml of distilled water. As diabetes use L-threonine. The resulting dark red solution was stirred for 1 hour and subjected to exhaustive dialysis using a Spectra / Por, 6-8 kDa membrane tube, Spectrum Labs, Inc. against distilled water (5 l × 4 shifts). The result is a solution, usually brownish yellow (photo in Fig. 21). In FIG. 2a) shows the electronic absorption spectrum (UV-VID spectrum) for this solution (C ₆₀ / Thr), where the initial solution (57 μg C ₆₀ / ml) is diluted 10 times with water; and in FIG. 2b) its UV-VID spectrum is shown after concentration of the initial solution by evaporation to a concentration of 226 μg C ₆₀ / ml (10 times diluted with water for recording the spectrum).

Example 2

A solution of fullerene is prepared analogously to example 1. As a diabetes, take L (+) - ramnose. The process is carried out analogously to example 1. The spectra and type of solution are shown in FIG. 11, 16 and 24.

Example 3

The fullerene solution is prepared analogously to example 1. Arg-Lys-Asp tripeptide (RKD) is taken as DM. The process is carried out as indicated in Example 1. The spectra and type of solution are shown in FIG. 12, 19 and 25.

Example 4

A solution of fullerene is prepared analogously to example 1. As a DM, take PVP. The process is carried out analogously to example 1. The spectra and type of solution are shown in FIG. 13, 19 and 26.

Example 5

A solution of fullerene is prepared analogously to example 1. As a diabetes, glycerol is taken. The process is carried out analogously to example 1.

The IR Fourier spectra (conditions of impaired total internal reflection, FT-IR Spectrometer Alpha, Bruker) of lyophilized nC ₆₀ samples always exhibit narrow absorption peaks characteristic of fullerene, 1182 and 1428 cm ^-1 (CC bond), as well as additional peaks, presumably due to the presence of bound water and OH groups — wide peaks in the region of 3500-3200, 1650-1660, 1000-1070 cm ^-1 (Figs. 12-15). In essence, all UV-VID spectra, as well as IR-Fourier spectra, are of the same type. It can be assumed that the particles in the solution have crystalline packing, which are stabilized by the surface water layer. The dispersions are stable for at least 3 months when stored at 4-8 ° C (maximum observation time). The pH values of the dispersions nC ₆₀ are in the range 5.5-6.5. Lowering the pH to 3.6-3.7 causes flocculation of the nanoparticles, leading to the formation of flocculent precipitates. The process is reversible, the precipitate can be transferred to the solution by increasing the pH.

It should be noted that the particle size of the dispersion decreases with increasing pH (> 9) and diluting the solution with water.

The method in accordance with the invention can be used to obtain aqueous dispersions and other fullerenes (C ₆₀ , C ₇₀ , C ₇₄ , C ₈₀ , etc.), since the main physicochemical properties that determine their behavior in solutions, they have enough are close, therefore, the laws of the formation of nanodispersions will also be similar.

Claims (2)

1. A method of producing aqueous fullerene nanodispersion, comprising dissolving fullerene in the form of C ₆₀ fullerite crystals in N-methylpyrrolidone, mixing the resulting solution with water and a stabilizer, which is used as an amino acid, monosaccharide, peptide, polyvinylpyrrolidone or glycerin, and subsequent dialysis of the resulting mixture. 2. The method according to p. 1, characterized in that after dialysis, the solution is concentrated, for example, by evaporation in vacuo.

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