RU2513098C1 - Method of producing water-soluble non-agglomerative fullerene immunostimulating nanoparticle and water-soluble non-agglomerative fullerene immunostimulating nanoparticle - Google Patents

Abstract

FIELD: chemistry.SUBSTANCE: invention relates to a water-soluble non-agglomerative fullerene immunostimulating nanoparticle which consists of a hydrophobic fullerene core which is covalently bonded to a capronyl ligand through a piperazine spacer (1,2-dihydrofullerene-1-(6-piperazin-1-yl)-caproic acid) and a method for production thereof, which involves synthesis of pure 1,2- dihydrofullerene-1-(6-piperazin-1-yl)-caproic acid in four steps, the first of which involves obtaining 4-Boc-1-(5-ethoxycarbonyl-pentyl)-piperazine; the second step involves obtaining 6-(piperazin-1-yl)-caproic acid ethyl ether by dissolving 4-Boc-1-(5-ethoxycarbonyl-pentyl)-piperazine in 2N HCl and evaporating the obtained solution, pH of the precipitate of which is brought to a value of 10.0; extracting with ethyl acetate to dry the organic layer; the third step involves obtaining ethyl ether of 1,2- dihydrofullerene-1-(6-piperazin-1-yl)-caproic acid by mixing and stirring until dissolution of fullerene-Cand toluene with addition, after dissolution, of CpTiCland ethyl ether of 6-(piperazin-1-yl)-caproic acid and subsequent elution with a mixture of hexane and chloroform mixture, and the purified 1,2- dihydrofullerene-1-(6-piperazin-1-yl)-caproic acid in form of the finished product is obtained by dissolving ethyl ether of 1,2-dihydrofullerene-1-(6-piperazin-1-yl)-caproic acid in ethyl alcohol with addition of NaOH solution and heating the mixture while stirring until complete homogenisation. The pH of the reaction mixture is then brought to a neutral value and the precipitated acid is separated by microfiltration on filters for subsequent washing off of nanoparticle residue and evaporation of the drain residue, which consists of the water-soluble non-agglomerative fullerene immunostimulating nanoparticles, consisting of a hydrophobic fullerene core which is covalently bonded to a capronyl ligand through a piperazine spacer.EFFECT: improved method.4 cl, 4 dwg, 1 tbl

Description

The present invention relates to biotechnology, in particular to the use piperazilkapronilovyh C ₆₀ fullerene derivatives as stimulants of the immune response - adjuvants, and may be used in a biological industry at the design and manufacturing of vaccines.

Fullerene C60 - a molecule consisting of 60 carbon atoms (and nothing more), located just like the top of a soccer ball, is the third (after diamond and graphite) allotropic form of carbon, which has extremely low toxicity (Mori T. et al. (2006 ). Preclinical studies on safety of fullerene upon first oral administration and evaluation for no mutagenesis. Toxicology 225, 48-54; Szwarc H, Moussa F. (2011). Toxicity of 60fullerene: confusion in the scientific literature. J. Nanosci. Lett . 1, 61-62).

Fullerene and its derivatives can be used:

- to protect the body from radiation and ultraviolet radiation (Theriot S. A., Casey RC, Moore VC, Mitchell L., Reynolds JO, Burgoyne M., et al. (2010). Dendro [C60] fullerene DF-1 provides radioprotection to radiosensitive mammalian cells. Radiat. Environ. Biophys. 49, 437-445; Andrievsky GV, Bruskov VI, Tykhomyrov AA, Gudkov SV (2009). Peculiarities of the antioxidant and radioprotective effects of hydrated C60 fullerene nanostructures in vitro and in vivo. Free Radic. Biol. Med. 47, 786-793);

- for protection against viruses (Mashino T., Shimotohno K., Ikegami N. .. et al. (2005). Human immunodeficiency virus-reverse transcriptase inhibition and hepatitis C virus RNA-dependent RNA polymerase inhibition activities of fullerene derivatives. Bioorg. Med Chem. Lett. 15, 1107-1109) and bacteria (Lu ZS, Dai T.N., Huang LY, et al. (2010). Photodynamic therapy with a cationic functionalized fullerene rescues mice from fatal wound infections. Nanomedicine 5, 1525-1533);

- for protection against allergies. Thus, in in vivo experiments, the administration of fullerene derivatives inhibits anaphylaxis in mice, and there is no toxic effect;

as an immunity stimulating substance (Xu YY, Zhu JD, Xiang K., Li YK, Sun RH, Ma J., et al. (2011). Synthesis and immunomodulatory acdvity of 60fuUerene-tuftsin conjugates. Biomaterials 32, 9940-9949 );

- as a powerful antioxidant, as it is an active radical scavenger. The antioxidant activity of fullerene is comparable to the action of SkQ class antioxidants (“Skulachev ions”) and is 100-1000 times higher than the action of ordinary antioxidants, such as vitamin E, butylhydroxytoluene, β-carotene;

- as drugs for the fight against cancer (Chen Z., Ma L., Liu Y., Chen C. (2012). Applications of Functionalized Fullerenes in Tumor Theranostics. Theranostics 2, 238-250; Jiao F., Liu Y ., Qu Y. et al. (2010). Studies on anti-tumor and antimetastatic activities of fullerenol in a mouse breast cancer model. Carbon 48, 2231-2243);

- to inhibit angiogenesis, to protect the brain from alcohol, to stimulate nerve growth, to stimulate skin regeneration processes (fullerene is an important component of cosmetic anti-aging agents GRS and CEFINE), to stimulate hair growth and as a medicine with anti-amyloid action.

In addition, fullerene can be used for delivery of various medicinal substances into the cell and non-viral delivery of genetic vectors to the cell nucleus (Montellano A., Da Ros T., Bianco A., Prato M. (2011). Fullerene C (60) as a multifunctional system for drug and gene delivery. Nanoscale 3, 4035-4041; Kuznetsova SA, Oretskaya TS (2010). Nanotransport systems for targeted delivery of nucleic acids to cells. Russian nanotechnology, 5 (No. 9-10), 40- 52).

The mechanisms of interaction of fullerenes with cells are not yet clear, but the results of its application give an idea of a possibly new direction in medicine (Piotrovsky LB (2010). Nanomedicine as part of nanotechnology. Vestnik RAMS 3, 41-46).

From the data of the scientific literature it is known that butyryl and capronyl derivatives of fullerene C60 have pronounced immunostimulating properties (activation of macrophage proliferation, acceleration of phagocytic reactions, increase in the specific production of gamma globulins by lymphoid tissue cells), and the inclusion of nitrogen-containing “tail” in the ligand-forming “tail” heterocycles (pyrrole, pyrrolidine, pyrimidine, oxymethylpurines) can enhance the predicted immunostimulating effect. This conclusion is convincingly substantiated in the analytical review of Devenji & Lakatos (2011).

This conclusion supports the fact that the use of a nitrogen-containing piperazine heterocycle not previously used in the construction of fullerene NPs in combination with capronyl functionalization of the fullerene core can lead to the creation of a nanotechnological product suitable for effective use as an adjuvant in the production of vaccines. This product, first synthesized by our method, meets the specific criteria of nanotechnology imposed on medical nanoparticles by the expert community of RUSNANO and a number of European countries (Nano - Russia, 2011, RUSNANO - Media Strategy, M., 2011).

Among the analogues, a number of experimental studies can be distinguished that did not pose any applied problems, but containing a thorough description of the cellular and molecular mechanisms of the immunostimulating effects of a number of C60 fullerene derivatives. So, Kaidashev et al. described an increase in the functional activity of phagocytic cells under the influence of the C60 nucleus itself (Kaydashev I.P. et al. (2011), Expert. Klin. Farmakol, No. 6: 26-29). The immunogenic properties of fullerene nanocomposites were described by Venterovich et al. (Venterovich N.G. et al. (2011), Immunology, 12 (1); 24-29). Shimanovsky's work analyzed the synergy of the antiviral and immunostimulating effects of amino acid and peptide covalent complexes with fullerene C60 (Shimanovsky N.S. (2009) Internat. Med. J., 1: 131-136).

An analysis of Bakry et al., Which revealed general trends and prospects for the pharmacological and biotechnological use of nanoparticles based on C60 fullerene, is also interesting, indicating, in particular, increasing attention to the prospects for the possible adjuvant use of aliphatic C60 spheres (Bakiy R., et al. (2007 ), Internat. J. Nanomed, 2 (4): 639-649). The mechanism of affinity docking recognition in the trigger-activation system for antibody synthesis in the presence of peptidyl-fullerenes and discussion of the prospects of using this type of NP as adjuvants in the development of vaccines were the subject of research by Xu et al. (Xu Y., et al. (2011), Biomaterialas, 32: 9940-9949).

A few patents (1997-2009) have been identified that protect the rights to synthesis, structure and method of use of a number of water-soluble adducts of fullerene C60 as adjuvants. None of the currently registered patents protects the rights to the class (type, group, family) of compounds, but only to this or that specific structure (RU 2124022 - glycopeptide-C60, RU 2129436 - amino acid and peptide adducts C60, RU 2184566 - amino acid and peptide adducts С60, RU 2184669 - amino acid (serine) adduct С60, RU 2348416 - porphyrin adduct С60 and US 2009/0197951 - oxide-dendrimer-fullerene complex).

For example, it is known to use fullerene derivatives of amino acids and dipeptides of the general formula C ₆₀ -X as adjuvants, where C ₆₀ is the fullerene core, X = NH-CHR-COOH, NH- (CH ₂ ) _n COOH, NH-CHR-CO-NH -CHR-COOH; n = 2 to 6; R is the side radical of a natural amino acid (RU 2129436, A61K 38/05, publ. 04/27/1999). This solution is selected as a prototype for a water-soluble nanoparticle.

And from RU 2213048, СВВ 31/02, С07С 229/06, publ. 09/27/2003, for example, a method for producing water-soluble salts of amino acid derivatives of fullerene of the general formula HC ₆₀ NH (CH ₂ ) _n COOM, where C ₆₀ is a fullerene core, M is an alkali metal, n = 1, 3, 5, including the interaction of fullerene with salt of an amino acid in an organic solvent during heating and subsequent isolation of the target product, while the interaction is carried out in the presence of low molecular weight polyalkylene oxide mol. masses 150-400. This solution is selected as a prototype for a method for producing a water-soluble nanoparticle.

No patents were found that would contain a description of a fullerene nanoadjuvant with an exact indication of the type of nosology for the prevention and / or treatment of which a vaccine containing this nanoadjuvant should be created. Each of the patents found simply offers a non-specific way to increase the activity of cellular and humoral immunity reactions, combining the described nanoadjuvant and antigen in animal experiments, represented by a suspension of cells of killed opportunistic microorganisms (and even their arbitrary mixtures) - such as E. coli, Azotobacter vinilandii , Clostridium pasteurianum, Hehcobacter pilori, Streptococcus carnaei, etc.

The present invention is aimed at achieving a technical result, which consists in creating a new method for producing a water-soluble non-agglomerate fullerene nanoparticle (NP) with immunomodulating properties.

The specified technical result is achieved in that the water-soluble non-agglomerate fullerene immunostimulating nanoparticle consists of a hydrophobic fullerene core covalently linked to the capronyl ligand via a piperazine spacer.

The specified technical result is achieved in that the method for producing a water-soluble non-agglomerate fullerene immunostimulating nanoparticle consists in the synthesis of purified 1,2-dihydrofulleren-1- (6-piperazin-1-yl) -caproic acid in four stages, the first of which 4-Boc -1- (5-ethoxycarbonyl-pentyl) piperazine; in the second step, 6- (piperazin-1-yl) -caproic acid ethyl ester is obtained by dissolving 4-Boc-1- (5-ethoxycarbonyl-pentyl) piperazine in 2H HCl and evaporation of the resulting solution, the pH of the residue of which is adjusted to a value 10.0, extracted with ethyl acetate with drying of the organic layer, in the third step, 1,2-dihydrofulleren-1- (6-piperazin-1-yl) -caproic acid ethyl ester is obtained by mixing and stirring to dissolve fullerene-C ₆₀ and toluene with adding after dissolution of Cp ₂ TiCl ₂ and 6- (piperazin-1-yl) -caproic acid ethyl ester and subsequent elution with a hexane: chloroform mixture and purified 1,2-dihydrofulleren-1- (6-piperazin-1-yl) - caproic acid in the form of a finished product is obtained by dissolving 1,2-dihydrofullerene-1- (6-piperazin-1-ethyl ethyl ester l) -caproic acid in ethyl alcohol with the addition of a NaOH solution and heating the mixture with stirring until complete homogenization. Then the pH of the reaction mixture was adjusted to a neutral pH and the precipitated acid was separated by microfiltration on filters for subsequent washing off of the nanoparticle precipitates and evaporation of the stock precipitate.

These features are significant and are interconnected with the formation of a stable set of essential features sufficient to obtain the desired technical result.

The present invention is illustrated by illustrations, where Fig. 1 shows reaction schemes, structures of intermediates and product.

Figure 1 - scheme for the preparation of 4-Boc-1- (5-ethoxycarbonyl-pentyl) piperazine;

figure 2 - scheme for the preparation of 6- (piperazin-1-yl) -caproic acid ethyl ester;

figure 3 - scheme for the preparation of ethyl ester of 1,2-dihydrofulleren-1- (6-piperazin-1-yl) -caproic acid;

4 is a diagram of the production of 1,2-dihydrofulleren-1- (6-piperazin-1-yl) -caproic acid.

The basis of the method developed by the authors, which is described in stages below, was the functionalization of the fullerene C ₆₀ sphere using the Dies-Alder nucleophilic cycloaddition reaction in a modification of the authors of the present invention.

The target product of the described method is a water-soluble non-agglomerate nanoparticle (0.8-1.2 nm in size), consisting of a hydrophobic fullerene core covalently linked to a capronyl ligand via a piperazine spacer (ligand interface).

This nanotopology of NPs is unique and created by the authors for the first time taking into account the known information about the expressed immunostimulating properties of butyryl and capronyl adducts of fullerene-C _60, on the one hand, and taking into account the predictions about a possible increase in the immunostimulating effect of fullerene NPs with their functionalization with nitrogen-containing heterocycles, with other [Devenji I. & Lakatos S. (2011) A regressive prediction model for fullerene based nanostructures in the immunomimetic approach to vaccination enhancement, pp.207-238, In: Nanopharmaceuticals and Nanodevices for Contemporary Medicine (Waugh RN & Bielka H., Eds.), Szeged University Press: Szeged].

The proposed method consists of four successive stages of the synthesis of intermediates and the target product. The method is shown in the example of obtaining 250 mg of purified 1,2-dihydrofulleren-1- (6-piperazin-1-yl) -caproic acid)

Method Steps

1. Preparation of 4-Boc-1- (5-ethoxycarbonyl-pentyl) piperazine (FIG. 1)

24.3 g (90 mmol) of ethyl 6-iodocaproic acid and 16.79 g of 1-Boc-piperazine were dissolved in 75 ml of THF, 16.95 ml (99 mmol) of diisopropylethylamine were added to the solution. The reaction mixture was boiled for 6 hours. Then, the solvent was evaporated in vacuo, the residue was dissolved in ethyl acetate, the organic layer was washed 3 times with sodium hydrogen carbonate solution and 1 time with water, then dried over magnesium sulfate and the solvent was distilled off. The resulting oil was purified chromatographically on a column of 80 × 400 mm silica gel SL200, the solvent / eluent was ethyl acetate. Yield: 27.6 g of 4-Boc-1- (5-ethoxycarbonyl-pentyl) piperazine (93%).

2. Obtaining 6- (piperazin-1-yl) -caproic acid ethyl ester (figure 2).

26.3 g (80 mmol) of 4-Boc-1- (5-ethoxycarbonyl-pentyl) piperazine was dissolved in 150 ml of 2N HCl and the solution was evaporated on a rotary evaporator at 50 ° C in vacuo. The pH of the residue was adjusted to 10.0 with a 15% sodium hydroxide solution. The product was extracted with ethyl acetate, the organic layer was dried over magnesium sulfate and evaporated. Yield: 12 g of 6- (piperazin-1-yl) -caproic acid ethyl ester (66%).

3. Obtaining ethyl ester of 1,2-dihydrofulleren-1- (6-piperazin-1-yl) -caproic acid (figure 3)

In a glass reactor of 100 ml, set on a magnetic stirrer was charged with 0.056 mMol of fullerene C ₆₀ in 50 mL of toluene was stirred until complete dissolution, was added 0.0112 mMol Cp ₂ TiCl ₂ and 0.056 mmoles of ethyl 6- (piperazin-1-yl) -caproic acid. Stirred at room temperature. The reaction mass was passed through a column filled with L-grade silica gel (100 × 250 mm). The product was isolated by elution with hexane: chloroform (6: 1). Yield: 2.8 g of 1,2-dihydrofulleren-1- (6-piperazin-1-yl) -caproic acid ethyl ester (21%).

4. Obtaining 1,2-dihydrofulleren-1- (6-piperazin-1-yl) -caproic acid (figure 4).

1,2-Dihydrofullerene-1 - (6-piperazin-1-yl) -caproic acid ethyl ester was dissolved in ethanol and a 33% NaOH solution was added, the mixture was heated with stirring until complete homogenization. The reaction mixture was adjusted to neutral pH, the precipitated acid is separated by microfiltration on fiberglass filters Millipore RX7M and then thoroughly washed with a solution of ethanol pretsipitantnym -NaOH (pH 7,0), passing at least 70 ml of it through a 1.0 cm ² surface of the filter. Yield: 62.5 mg of 1,2-dihydrofulleren-1- (6-piperazin-1-yl) -caproic acid (2.3%).

The fiberglass filters containing the final product were thoroughly dried at room temperature (aeration with a hairdryer), after which the nanoparticle sediments (nanoadjuvant) located on them were washed off with a 100-fold volume of demineralized water and evaporated in a rotary evaporator to a volume of 2.5-3.0 ml. Thus obtained portions of the stock solution of the product were combined and used for a qualitative analysis of the composition, identification of the structure, and quantitative determination of NPs.

The synthesis of a nanoadjuvant of the selected structure allowed us to obtain the final product in an amount of 250 mg, which is sufficient to provide the necessary testing according to the program of preclinical testing of vaccine components. The authors have not found a single patent describing the specific structure obtained by the authors of the target product. The piperazine spacer, the immuno-promoter present in the resulting product, is an absolute patent novelty factor.

Quantification of the intermediates and the product in solutions was carried out refractometrically using calibration with the piperazine contribution. IR-VIS differential spectrophotometry in the wavelength ranges of 280-330 / 520-770 nm and 1450-1970 nm was additionally used to accurately determine microgram and milligram quantities of the target product in dilute aqueous solutions. The analysis was performed on a Perkm Elmer Lambda 1050 double-beam scanning spectrophotometer. Diglutamylasparagyl-fullerene and pure piperazine were used as calibration standards. The calculation of the determined concentrations was automated by the LabRun 6A software package and carried out on the HP4100 - 09 analytical module (Hewlett Packard).

The stock solution of the product, adjusted to a concentration of 25 mg / ml, was sterilized by ultrafiltration on Diaflo Y5.0 membranes (Amicon QS400 UF System, 800 p.s.i.) and then poured under sterile conditions (UV-manipulator) into autoclaved hermetically sealed polyalomeric tubes for subsequent use.

LC-MS technology was used to identify the structures of the intermediates and the target product, in which the Shimadzu HPLC 600 TGS System was connected online to the Shimadzu Prizm Analyser JS2200 MALDI mass spectrometer. The chromatographic module included a Buckyclutcher-1 20 × 250 mm column with a stationary phase of 5-PBB (5-pentabromobenzene), the mobile phase - CS2, 16 ml / min, 2000 psi, 20-22 C. Mass spectrometric time-of-flight module included an analyzer based on laser electrospray desorption / ionization of the evaporated HPLC eluate. The structure identification program included the analytical package and data library ERCO 2010 (NC Research Triangle) and was processed in the Shimadzu LMMC 440 XS Analyzer module.

The degree of analytical purity (the presence and content of impurities) was estimated using routine X-ray fluorescence spectrometry of aqueous solutions of the target product in the Finnigan Matt RC708 analytical system.

The size of the obtained nanoparticles was determined using the standard AFM imaging method (NanoFinder AFM Orion - 302).

We also used the method of sedimentation schlieren-optical monitoring with analytical equilibrium ultracentrifugation of the stock solution of NP. A sample of such a solution was applied to a standard Sweberg linear density gradient of cesium chloride (300,000 g, + 4 ° C, 18 hours, Sorvall M77 Analytical Ultracentrifuge). The calibration standards were highly purified preparations of fullerene C60, 0.7 nm, dissolved in toluene, as well as preparations PMC16, 1.8-2.0 nm, and peptidyl-fullerene ASK27, 4.8-5.2 nm, dissolved in water.

The obtained data of a qualitative analysis of the target product (NP - nanoadjuvant piperazine capronyl - fullerene structure) are shown in table 1.

Table 1 Parameter, unit measuring Parameter Value ND preset Installed analytically Like mass, amu 920 920 Amount mg not less than 250 267.7 Chem. purity,% not less than 95 98.4 LF size, nm no more than 2.0 0.8-1.2 *** Fiz. homogeneity,% not less than 90 94.6 *** NOTE: 0.8 nm - according to AFM; 1.2 nm - according to sedimentation analysis.

Based on the data presented in the patents and in the studied scientific literature, we recommend a dose range of 2.5-10 mg / kg for intravenous administration to rats for testing the biological activity and safety of the product in vivo.

Claims (4)

1. A water-soluble non-agglomerate fullerene immunostimulating nanoparticle consisting of a hydrophobic fullerene core covalently linked to a capronyl ligand via a piperazine spacer. 2. The water-soluble non-agglomerate fullerene immunostimulating nanoparticle according to claim 1, characterized in that it has a size of 0.8-1.2 nm. 3. A method of obtaining a water-soluble non-agglomerate fullerene immunostimulating nanoparticle, which consists in the synthesis of purified 1,2-dihydrofullerene-1- (6-piperazin-1-yl) -caproic acid in four stages, the first of which gives 4-Boc-1- ( 5-ethoxycarbonyl-pentyl) piperazine; in the second step, 6- (piperazin-1-yl) -caproic acid ethyl ester is obtained by dissolving 4-Boc-1- (5-ethoxycarbonyl-pentyl) piperazine in 2H HCl and evaporating the obtained the solution, the pH of the residue of which is adjusted to a value of 10.0, is extracted with ethyl acetate with drying organ layer, in the third stage receive ethyl ester of 1,2-dihydrofullerene-1- (6-piperazin-1-yl) -caproic acid by mixing and stirring to dissolve fullerene-C ₆₀ and toluene with the addition after dissolution of Cp ₂ TiCl ₂ and 6- (piperazin-1-yl) -caproic acid ethyl ester and subsequent elution with a hexane: chloroform mixture, and purified 1,2-dihydrofulleren-1- (6-piperazin-1-yl) -caproic acid as a finished product is obtained by dissolving 1,2-dihydrofulleren-1- (6-piperazin-1-yl) -caproic acid ethyl ester in ethyl alcohol with the addition of pouring a NaOH solution and heating the mixture with stirring until complete homogenization, then bring the pH of the reaction mixture to neutral pH and the precipitated acid is separated by microfiltration on filters to subsequently wash off the nanoparticle precipitates and evaporate the stock precipitate. 4. The method according to claim 3, characterized in that 4-Boc-1- (5-ethoxycarbonyl-pentyl) piperazine is prepared by dissolving ethyl ester of 6-Iodocaproic acid and 1-Boc-piperazine in THF with addition of diisopropylethylamine to the solution, followed by boiling the solution and evaporation to obtain a residue, which is dissolved in ethyl acetate and washed with sodium bicarbonate solution and water, and then dried to remove the solvent.

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