RU2657835C1 - Method for producing a system for delivering an anticancer agent to tumor cells - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to chemical-pharmaceutical industry and is a method for producing a system for delivering an anticancer agent to tumor cells, comprising mixing, in the presence of water, polymer-modified magnetite nanoparticles epitaxially grown on gold nanoparticles, with an organic compound chemically binding to nanoparticles and ensuring the selective penetration of nanoparticles into tumor cells, and an aqueous solution of the anticancer agent, followed by separation of the obtained nanoparticles modified by centrifugation, characterized in that polymer-modified nanoparticles are nanoparticles, obtained by heating to 120 °C in an inert gas atmosphere while stirring a mixture of diphenyl ether, oleic acid, oleylamine and 1,2-hexadecanediol, introducing iron pentacarbonyl into the mixture, maintaining the resultant mixture, followed by the introduction of a solution containing a mixture of aurichlorohydric acid trihydrate and oleylamine in diphenyl ether, pre-aged in an inert gas atmosphere, reheating of the mixture in an inert gas atmosphere from 120 °C to 250–260 °C, maintaining the mixture at 250–260 °C for 25–30 minutes and cooling to room temperature, carried out an inert gas atmosphere, maintaining the mixture at room temperature in the presence of air, adding a monohydric alcohol to the mixture and separating the magnetite nanoparticles by centrifugation, followed by treatment with a solution of a polymer selected from the group consisting of a triblock copolymer consisting of a central block of polypropylene glycol with a polymerization degree of 56 and two polyethylene glycol end blocks with a polymerization degree of 101 each, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol) – 2000] and a triblock copolymer consisting of a central block of polypropylene glycol with a polymerization degree of 30 and two end blocks of polyethylene glycol having a polymerization degree of 78 each, in an organic solvent, then by ultrasound, followed by solvent removal, water injection, ultrasound retreatment and separation of the modified nanoparticles by centrifugation, doxorubicin is used as an anticancer agent, as an organic compound providing the selective penetration of nanoparticles into human benign hyperplastic prostate cells, low molecular weight ligand of the prostatic specific membrane antigen is used, wherein the nanoparticles are first treated with a solution of doxorubicin, then with a solution of a low molecular weight ligand of the prostatic specific membrane antigen.

EFFECT: invention provides increased cytotoxicity of the system for delivering an anticancer agent to human adenocarcinoma cells.

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Description

The invention relates to the field of bionanotechnology and relates to a method for producing a system for delivering an antitumor drug to human malignant tumor cells and can be used for targeted drug delivery.

A known method of obtaining a system for delivering an antitumor drug - doxorubicin to tumor cells, comprising dispersing 5 mg of magnetite nanoparticles with a diameter of 10-15 nanometers (nm) coated with polyvinyl alcohol in 5 ml of an aqueous solution of doxorubicin with a concentration of 0.1 mg / ml, followed by stirring on a rotary shaker at 37 ° C for 26 h, magnetic decantation of modified nanoparticles and their resuspension in water (Kayal S., Ramanujan RV Doxorubicin loaded PVA coated iron oxide nanoparticles for targeted drug delivery // Materials Science and Engineering: C, 2010. V. 30 (3), P. 484-490). This method has such features that coincide with the essential features of the proposed technical solution, such as the use of magnetite nanoparticles coated with a polymer shell, and the treatment of modified nanoparticles with an aqueous solution of doxorubicin.

A known method of obtaining a system for delivering an antitumor drug - paclitaxel to tumor cells, including dispersion in 10 ml of water by ultrasonic treatment for 1 min with 100 mg of 140 nm magnetite nanoparticles coated with glyceryl monooleate, adding paclitaxel solution dropwise to the resulting dispersion acetonitrile with a concentration of 10 mg / ml, stirring the dispersion on a magnetic stirrer overnight and separating the nanoparticles by centrifugation three times at a centrifuge rotor speed of 138 00 rpm for 10 min followed by resuspension of the modified nanoparticles in water (Dilnawaz F., Singh A., Mohanty C. et al. Dual drug loaded superparamagnetic iron oxide nanoparticles for targeted cancer therapy // Biomaterials, 2010. V. 31 (13), P. 3694-3706). This method has such features that coincide with the essential features of the proposed technical solution, such as treatment of magnetite nanoparticles with a polymer solution first, then with ultrasound and separation of drug-modified nanoparticles by centrifugation followed by their resuspension in water.

Closest to the claimed is a known method of obtaining a system for delivering an antitumor drug to tumor cells, comprising mixing in the presence of water polymer-modified (conjugate of dopamine and polyethylene glycol with a terminal carboxyl group) magnetite nanoparticles with an average diameter of 18 nm, epitaxially grown on gold nanoparticles, with water a solution of a mixture of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide, stirring the resulting suspension, separating the excess 1-ethyl-3- (3-d imethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide on a PD-10 chromatographic column washed with a PBS buffer, mixing the suspension with an organic compound (herceptin derivative) that chemically binds to the nanoparticles and ensures selective penetration of the nanoparticles into the intracellular space of the cells of the modified human breast adenocarcinoma, separation by centrifugation followed by the addition of an aqueous solution of an organic ligand that binds the anticancer drug cisplatin derivative by stirring the mixture, removing excess ligand on a PD-10 chromatographic column, mixing with an aqueous solution of the cisplatin derivative, stirring the resulting mixture in the dark overnight, and then separating the obtained modified nanoparticles by centrifugation and chromatography on a PD-10 column (Xu C, Wang B., Sun S. Dumbbell-like Au-Fe ₃ O ₄ Nanoparticles for Target-Specific Platin Delivery // J. Am. Chem. Soc. 2009. V. 131, P. 4216-4217 - prototype).

This method has such features that coincide with the essential features of the proposed technical solution, such as mixing, in the presence of water, polymer-modified magnetite nanoparticles grown epitaxially on gold nanoparticles with an organic compound chemically binding to the nanoparticles and providing selective penetration of the nanoparticles into the intracellular space of human adenocarcinoma cells, and an aqueous solution of an antitumor drug, followed by separation of the obtained modified on nanoparticles by centrifugation.

The disadvantages of this method are its complexity and multi-stage, as well as the fact that the system described in it makes it possible to deliver a relatively small amount of an antitumor drug (17.8% of the mass of the whole nanoparticle, but only from the mass of the gold nanoparticle on which magnetite nanoparticle was grown epitaxially) and the fact that the cytotoxicity of the system (the concentration of half-maximum inhibition of IC ₅₀ , in which 50% of the tumor cells die) quite high (1.76 μg of the preparation / ml of culture medium). If we recalculate the amount of the administered antitumor drug by the mass of the entire magnetite nanoparticle grown epitaxially on the gold nanoparticle, then it will be significantly lower than the above 17.8%.

The objective of the invention is to develop a method for producing a system for delivering an antitumor drug to tumor cells, devoid of the above disadvantages.

The technical result of the invention is to increase the cytotoxicity of the system for delivery of antitumor drug to human adenocarcinoma cells.

Previously, experiments were carried out with various magnetite nanoparticles grown epitaxially on gold nanoparticles, various organic compounds chemically binding to nanoparticles and providing selective penetration of nanoparticles into tumor cells, and various antitumor preparations, which showed that this technical result is achieved when in a method for producing a system for delivering an antitumor drug to tumor cells, comprising mixing in the presence of water of polymer-modified magnetite nanoparticles grown epitaxially on gold nanoparticles, with an organic compound chemically binding to the nanoparticles and providing selective penetration of the nanoparticles into the tumor cells, and an aqueous solution of the antitumor drug, followed by separation of the obtained modified nanoparticles by centrifugation, using nanoparticles as polymer-modified nanoparticles obtained by heating to 120 ° C in an inert gas atmosphere at p stirring a mixture of diphenyl ether, oleic acid, oleylamine and 1,2-hexadecanediol, introducing iron pentacarbonyl into the mixture, keeping the resulting mixture, followed by introducing a solution containing a mixture of hydrochloric acid trihydrate and oleylamine in diphenyl ether, previously stored in an inert gas atmosphere, repeated mixtures in an inert gas atmosphere from 120 ° C to 250 ° -260 ° C, keeping the mixture at 250 ° -260 ° C for 25-30 minutes and cooling it to room temperature, carried out in an inert atmosphere gas, keeping the mixture at room temperature in the presence of air, adding monohydric alcohol to the mixture and separating magnetite nanoparticles by centrifugation, followed by treatment with a polymer solution selected from the group consisting of a triblock copolymer consisting of a central polypropylene glycol block with a polymerization degree of 56 and two end blocks of polyethylene glycol with a degree of polymerization of 101 each (poloxamer 407), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [carboxy (polyethylene glycol) -2000] and triblock copolymer, consisting of a central block of polypropylene glycol with a degree of polymerization of 30 and two end blocks of polyethylene glycol with a degree of polymerization of 78 each (poloxamer 188), in an organic solvent, then ultrasound, followed by removal of the solvent, water, re-treatment with ultrasound and separation of the modified nanoparticles by centrifugation, as the antitumor drug uses doxorubicin, as an organic compound that provides selective penetration of nanoparticles inside human prostate adenocarcinoma cells, use a low molecular weight ligand of a prostatic specific membrane antigen, the nanoparticles being first treated with a solution of doxorubicin, then a solution of a low molecular weight ligand of a prostatic specific membrane antigen.

The proposed method for producing a system for delivering an antitumor drug to tumor cells is new and is not described in the patent and scientific literature. The method of producing magnetite nanoparticles epitaxially grown on gold nanoparticles used in the proposed technical solution is also new and is not described in the patent and scientific literature.

In contrast to the introduction of a pure antitumor drug into the cell culture medium or organism, where this drug substance will penetrate into any type of cell, the proposed method allows to obtain systems for targeted (selective) delivery of the antitumor drug only to cells of a certain type of tumor without penetration of the drug into healthy cells , which reduces the side effects associated with the introduction of drugs for the treatment of malignant neoplasms.

The proposed method can be used for the treatment of cancer - adenocarcinoma of the human prostate gland.

The proposed method uses magnetite nanoparticles with a diameter of 4 ± 1 nm, epitaxially grown on gold nanoparticles with a diameter of 2 ± 1 nm, obtained by heating to 120 ° C in an inert atmosphere with stirring a mixture of diphenyl ether, oleic acid, oleylamine and 1,2-hexadecanediol introducing iron pentacarbonyl into the mixture, keeping the mixture obtained, followed by introducing a solution containing a mixture of hydrochloric acid trihydrate and oleylamine in diphenyl ether, previously incubated in an atmosphere of mercury gas, reheating the resulting mixture from 120 ° C to 250-260 ° C, keeping it at this temperature for 25-30 minutes and then cooling it to room temperature, carried out in an inert gas atmosphere, keeping the mixture in the presence of air, adding into a mixture of monohydric alcohol and the separation of magnetite nanoparticles by centrifugation.

The small diameter of magnetite nanoparticles used in the proposed technical solution facilitates their penetration into tumor cells.

In the proposed method, diphenyl ether is used as a high boiling organic solvent. It was experimentally shown that when using in the proposed method instead of diphenyl ether other high-boiling organic solvents, for example, such as 1-octadecene or dibenzyl ether, magnetite nanoparticles of a significantly larger size (7-14 nm) are formed, which, after their modification, can penetrate into intracellular space, but due to its relatively large size, are not able to pass into the nucleus and / or mitochondria of cells.

In the proposed technical solution, a mixture containing diphenyl ether, oleic acid, oleylamine and 1,2-hexadecanediol is placed in a three-necked round-bottom flask equipped with a reflux condenser, a high-temperature thermometer, an inert gas supply system and a magnetic body for stirring. The amounts of oleic acid, oleylamine, 1,2-hexadecanediol and diphenyl ether introduced may vary, for example, from 1.5-2.5 ml, 1.5-2.5 ml, 2.1-3.1 g and 18-22 ml, respectively. Then, an inert gas current is supplied, an electric heating plate with magnetic stirring function is placed under the flask, the stove is turned on, and the contents of the flask are heated while stirring to 120 ° C in the atmosphere of any inert gas, such as argon, nitrogen, etc. In this case, the heating time can vary and be, for example, 30-90 minutes. Then, liquid iron pentacarbonyl is introduced into the flask in an amount of, for example, 0.25-0.30 ml. It should be noted that this synthesis step must also be carried out in an inert gas atmosphere. Iron pentacarbonyl is administered once, and after its introduction, the reaction mixture is kept in an inert gas atmosphere for a certain time, for example, for 3-5 minutes.

After the reaction mixture is aged, a mixture of hydrochloric acid trihydrate and oleylamine in diphenyl ether is introduced thereinto, previously conditioned in the atmosphere of any inert gas. At the same time, the amounts of added hydrochloric acid trihydrate, oleylamine and diphenyl ether can be different and make up, for example, 38.0-42.0 mg, 0.4-0.6 ml and 4.9-5.1 ml, respectively. When the method is implemented, both thermal decomposition of iron pentacarbonyl occurs with the formation of magnetite nanoparticles, and the recovery of gold hydrochloric acid trihydrate with 1,2-hexadecanediol to form gold nanoparticles. Due to the fact that these processes proceed at different speeds, when implementing the method, 1,2-hexadecanediol and iron pentacarbonyl are first introduced into the reaction mixture, partial thermal decomposition of iron pentacarbonyl is carried out, and only then, for example, 3-5 minutes, into the reaction the system is injected with a solution containing a mixture of hydrochloric acid trihydrate and oleylamine in diphenyl ether. The introduction of 1,2-hexadecanediol, which does not take part in the thermal decomposition of iron pentacarbonyl, at the first stage of synthesis, is due to the fact that 1,2-hexadecanediol is a solid and rather insoluble compound, and it requires a certain time and temperature for complete dissolution in the reaction system . After the introduction of a solution of hydrochloric acid trihydrate and oleylamine, the reaction mixture is reheated from 120 ° C to 250 ° -260 ° C and the heated mixture is kept at this temperature for 25-30 minutes. It should be noted that these synthesis steps are also carried out in the atmosphere of any inert gas.

If it is necessary to obtain more significant amounts of magnetite nanoparticles grown epitaxially on gold nanoparticles, the amount of each of the reagents used should be proportionally increased.

In the proposed method, after keeping the reaction mixture, it is cooled to room temperature in an inert gas atmosphere and kept at room temperature in the presence of air for a certain time, for example, for 30-60 minutes.

If any of the above stages of the synthesis is not carried out at all or carried out under other conditions than indicated above, the technical result of the invention is not achieved.

After the reaction mixture is kept at room temperature, a monohydric alcohol is introduced there, which can be used, for example, methanol, ethanol, isopropanol, etc., which is necessary for the effective precipitation of magnetite nanoparticles grown epitaxially on gold nanoparticles into a precipitate during centrifugation of the obtained mixtures. In this case, the amount of monohydric alcohol introduced can vary and comprise, for example, 150-200% of the volume of the obtained reaction mixture. It is also possible to use a mixture of monohydric alcohols.

The obtained magnetite nanoparticles, epitaxially grown on gold nanoparticles, can be stored at room temperature or at low temperature in a sealed container in a dry form without deterioration of their operational properties for a long time, for example, for a year. In this case, it is advisable to carry out storage in an inert atmosphere in the absence of moisture.

By the method of x-ray phase analysis it was proved that in the proposed method, nanoparticles with a crystalline structure of magnetite, epitaxially grown on gold nanoparticles, are actually formed. The size and size distribution of magnetite nanoparticles grown epitaxially on gold nanoparticles were studied by transmission electron microscopy. It was shown that each spherical magnetite particle having a diameter of 4 ± 1 nm is connected to one spherical gold particle having a diameter of 2 ± 1 nm. The magnetic properties of the obtained nanoparticles were investigated using a VSM-250 vibromagnetometer.

In the proposed technical solution, magnetite nanoparticles grown epitaxially on gold nanoparticles are treated with a polymer solution selected from the group consisting of poloxamer 407 (triblock copolymer consisting of a central hydrophobic block of polypropylene glycol with a degree of polymerization of 56, and two terminal hydrophilic blocks of polyethylene glycol with a degree of polymerization each), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [carboxy (polyethylene glycol) -2000], poloxamer 188 (triblock copolymer consisting of a central hydrophobic th block polypropylene having a degree of polymerization of 30 and two hydrophilic end blocks of polyethylene glycol having a degree of polymerization of 78 each) in an organic solvent.

Each of the above polymers is commercially available. So, poloxamer 407 can be ordered from Sigma-Aldrich (http://www.sigmaaldrich.com/catalog/product/sigma/16758?lang=en&region=RU), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine -N- [carboxy (polyethylene glycol) -2000] can be ordered from Avanti Polar Lipids (http://avantilipids.com/product/880135/), Poloxamer 188 can also be ordered from Sigma-Aldrich (http: // www. sigmaaldrichxom / catalog / product / sigma / p5556? lang = en & region = RU).

The polymers used in the proposed technical solution contain a hydrophobic and hydrophilic component, one of which ensures the stabilization of the used nanoparticles in water, and the other ensures the binding of the nanoparticle to the antitumor drug, doxorubicin. If other known polymers are used as the polymer or the treatment of nanoparticles with the above polymers is not carried out at all, the technical result of the invention is not achieved.

In the proposed method, when processing the above magnetite nanoparticles grown epitaxially on gold nanoparticles with a polymer solution, various organic solvents can be used as a solvent, for example, such as chloroform, dichloromethane, hexane, etc. In this case, the concentration of the polymer in the organic solvent can vary and be, for example, 0.5-2 mg / ml. After mixing magnetite nanoparticles with a solution of the above polymer in an organic solvent, the resulting mixture is treated with ultrasound, while the duration of such processing can vary, for example, 1-5 minutes, the ultrasound power in the bath used for this purpose can also be different and, for example, 300-500 watts. After treating the nanoparticles with a polymer solution, the organic solvent is removed, for example, by drying in air or by using a rotary evaporator. If the solvent is not removed, then the technical result is not achieved. It should be noted that the mass ratio between magnetite nanoparticles grown epitaxially on gold nanoparticles and any of the above polymers can vary and be, for example, 0.2-1. To simplify the method, the treatment of magnetite particles with a solution of the above polymer is advisable to be carried out at room temperature. It was experimentally shown that the above polymers bind to the surface of both magnetite nanoparticles and the surface of gold nanoparticles chemically bonded to them.

Modified magnetite nanoparticles are separated from the excess of the above polymers by introducing water, the amount of which can vary and be, for example, 1-5 ml, repeated treatment with ultrasound, the duration of which can vary and be, for example, 1-5 minutes, followed by centrifugation of the obtained dispersion of modified nanoparticles in water at a rotor speed of a centrifuge of 10,000-14,000 rpm for 5-10 minutes

In the proposed method, an aqueous solution of the well-known antitumor drug, doxorubicin (https: www.macmillan.org.uk/cancerinformation/cancertreatment/treatmenttypes/chemotherapy/individualdrugs/doxorubicin.aspx), is added to the magnetite nanoparticles separated by the wet modified polymer described above), binding magnetite epitaxially grown on gold nanoparticles is not described in the scientific and technical literature. In this case, the mass ratio of the initial unmodified magnetite nanoparticles grown epitaxially on gold nanoparticles and doxorubicin can vary and amount to, for example, 1-3. The concentration of the antitumor drug in water can also vary and make up, for example, 0.1-0.4 mg / ml. After mixing the modified magnetite nanoparticles with a solution of doxorubicin, the resulting mixture is stirred on a magnetic stirrer for 12-24 hours.

Then, an aqueous solution of a low molecular weight ligand of a prostatic specific membrane antigen is introduced into the resulting dispersion, which covalently binds only to the surface of gold nanoparticles, displaces the previously introduced polymer from their surface and ensures selective penetration of nanoparticles into tumor cells of human prostate adenocarcinoma. It should be noted that the production of a low molecular weight ligand of a prostatic specific membrane antigen is described in the scientific and technical literature (Machulkin A.E., Ivanenkov YA, Aladinskaya AV et al. Small-molecule PSMA ligands. Current state, SAR and perspectives // J Drug Target, 2016. V. 24, P. 679-693).

It is necessary to introduce a low molecular weight ligand of a prostatic specific membrane antigen by treating a dispersion of magnetite nanoparticles modified with doxorubicin with its aqueous solution while stirring for 12-24 hours. In this case, the mass ratio of the initial unmodified magnetite nanoparticles grown epitaxially on gold nanoparticles and the low molecular weight specific prostate membrane ligand vary and be, for example, 0.04-0.2. The concentration of a low molecular weight ligand of a prostatic specific membrane antigen in water can also vary and be, for example, 5-10 mg / ml.

Magnetite nanoparticles sequentially modified with doxorubicin and a low molecular weight ligand of a specific prostatic membrane antigen are separated from the excess of the above reagents by centrifuging the resulting dispersion of nanoparticles in water at an ultracentrifuge rotor speed of 10,000-14,000 rpm for 5-10 minutes.

To store magnetite nanoparticles epitaxially grown on gold nanoparticles sequentially modified with a polymer, doxorubicin and a low molecular weight ligand of a prostatic specific membrane antigen, the modified nanoparticles are dispersed in water. The resulting dispersion can be stored both at room temperature and at low temperature in a hermetically sealed container without access to light. Under these conditions, the shelf life of the drug is at least 3 months.

The cytotoxicity of the resulting preparation is determined by the standard MTS test (Heinemann L., Simpson GR, Boxall A., Kottke T., Relph KL, Vile R., Melcher A., Prestwich R., Harrington KJ, Morgan R., Pandha HS Synergistic effects of oncolytic reovirus and docetaxel chemotherapy in prostate cancer // BMC Cancer 2011, V. 11: 221) in the concentration range of 0.5 - 10 μg of doxorubicin / ml of culture medium on cell cultures of human prostate adenocarcinoma LNCP and PC-3, moreover the low molecular weight ligand of the prostatic specific membrane antigen is specific only to LNCaP cells expressing this antigen on voey surface, providing selective penetration into the nanoparticles of this type cells, while cell culture PC-3 was selected as a control because cells of this line do not express a specific prostatic membrane antigen on their surface.

It should be noted that in the proposed technical solution, polymer-modified magnetite nanoparticles grown epitaxially on gold nanoparticles are first treated with an aqueous solution of doxorubicin, then with an aqueous solution of a low molecular weight ligand of a prostatic specific membrane antigen. Moreover, it was experimentally shown that with the reverse sequence of treatments, the cytotoxicity of the proposed system is reduced.

The advantages of the proposed method are illustrated by the following examples.

Example 1

In a three-necked round-bottom flask equipped with a reflux condenser, a high-temperature thermometer, an inert gas supply system and a magnetic body for stirring, 20 ml (126 mmol) of diphenyl ether, 1.9 ml (2 mmol) of oleic acid, 2.0 ml (2 mmol) of oleylamine and 2.6 g (10 mmol) of 1,2-hexadecandiol. Then an argon current is applied, an electric heating plate with magnetic stirring function is placed under the flask, the stove is turned on and the contents of the flask are heated for 30 minutes to 120 ° C with constant stirring, followed by the introduction of 0.28 ml (2 mmol) of iron pentacarbonyl first, then after 3 min, a solution containing a mixture of 40 mg (0.1 mmol) of hydrochloric acid trihydrate and 0.5 ml (1.5 mmol) of oleylamine in 5 ml of diphenyl ether, previously incubated in argon atmosphere. Then the resulting mixture was reheated from 120 ° C to 250 ° C, kept for 25 min at 250 ° C, and then cooled to room temperature, with all stages of the synthesis carried out in an argon atmosphere. The cooled mixture is kept at room temperature in the presence of air for 30 minutes, 45 ml of isopropanol monohydric alcohol are added to the mixture, and magnetite nanoparticles grown epitaxially grown on gold nanoparticles are separated by centrifugation. The separated nanoparticles are dried to constant weight. 45 mg of nanoparticle powder are obtained. The obtained nanoparticles are stored in a hermetically sealed container in an argon atmosphere in the absence of moisture at room temperature. Under these conditions, nanoparticles retain their operational properties throughout the year.

Using transmission electron microscopy, it was shown that the structure of the obtained nanoparticles resembles a dumbbell in which each magnetite nanoparticle having a spherical shape and a diameter of 4 nm is associated with a spherical gold nanoparticle with a diameter of 2 nm. Using a VSM-250 vibromagnetometer, it was shown that the saturation magnetization of the obtained crystals is 55 Am ² / kg.

1 mg of the obtained nanoparticle powder is placed in a round bottom flask, then 2 ml of a triblock copolymer solution consisting of a central hydrophobic block of polypropylene glycol with a degree of polymerization of 56 and two terminal hydrophilic blocks of polyethylene glycol with a degree of polymerization of 101 each, in chloroform with a concentration of 1 mg / are added. l, the flask for 3 min is transferred to an ultrasonic bath with a power of 400 W, after which the flask is placed on a rotary evaporator, and chloroform is removed with it. After removal of chloroform, 2 ml of water is added to the flask, the flask is re-placed for 3 min in an ultrasonic bath with a power of 400 W, then the resulting dispersion is transferred to a plastic tube and centrifuged for 8 min at a rotational speed of the centrifuge rotor of 10000 rpm. Then, 5 ml of an aqueous solution of the antitumor drug, doxorubicin with a concentration of 0.2 mg / ml, is added to a test tube containing separated magnetite nanoparticles epitaxially grown on gold nanoparticles and modified with a polymer. After that, the magnetic body is placed in the tube for stirring, the tube is closed with a lid, suspended on a tripod above the magnetic stirrer, the magnetic stirrer is turned on and the contents of the tube are stirred for 18 hours. Then the tube is opened, 5 ml of an aqueous solution of an organic compound chemically binding to it is introduced into it nanoparticles and providing selective penetration of nanoparticles into the intracellular space of tumor cells (adenocarcinoma of the human prostate gland) of a low molecular weight ligand prostatic th specific membrane antigen at a concentration of 7 mg / ml, and again stirred for 18 hours. The tube was placed in a centrifuge rotor and centrifuged for 8 minutes at a rotor speed of 10,000 rpm. / min. In this case, the nanoparticles are separated from the excess doxorubicin and the low molecular weight ligand of the prostatic specific membrane antigen. The settled nanoparticles are dispersed in 1 ml of water. The resulting dispersion is stored at room temperature in a hermetically sealed container without access of light.

The content of the antitumor drug, doxorubicin in the dispersion of modified nanoparticles, determined by absorption spectroscopy from a calibration curve for a number of standard samples with a known concentration of doxorubicin, is 0.31 mg / ml. The dispersion concentration of the modified nanoparticles, determined by atomic emission spectroscopy from a calibration curve for a number of standard samples with a known concentration of iron and gold, is 0.89 mg / ml. Thus, the content of doxorubicin in the modified nanoparticles, determined by calculation from the data given in the example, is 34.8% by weight of the nanoparticles.

The cytotoxicity of the preparation (1C ₅₀ ) for the LNCaP cell culture specific for the low molecular weight ligand of the prostate specific membrane antigen determined by the MTS test is 1.43 μg of the preparation / ml of culture medium. The cytotoxicity of the drug (IC ₅₀ ) for the control cell culture of PC-3, determined by the MTS test, is 9.74 μg of the drug / ml of culture medium.

Example 2

22 ml (139 mmol) of diphenyl ether, 2.5 ml (7.9 mmol) of oleic acid, 2.5 ml are placed in a three-necked round-bottom flask equipped with a reflux condenser, a high-temperature thermometer, an inert gas supply system and a magnetic body for stirring. (7.5 mmol) oleylamine; and 3.1 g (12 mmol) of 1,2-hexadecandiol. A nitrogen flow is applied, an electric heating plate with magnetic stirring function is placed under the flask, the stove is turned on, and the contents of the flask are heated for 120 minutes to 120 ° C with constant stirring, followed by the initial introduction of 0.30 ml (2.1 mmol) of iron pentacarbonyl, then, after 5 minutes, a solution containing a mixture of 42 mg (0.11 mmol) of hydrochloric acid trihydrate and 0.6 ml (1.8 mmol) of oleylamine in 5.1 ml of diphenyl ether, previously incubated in a nitrogen atmosphere. Then the resulting mixture is reheated from 120 ° C to 260 ° C, kept for 30 min at this temperature, and then cooled to room temperature, and all stages of the synthesis are carried out in a nitrogen atmosphere. The cooled mixture was kept at room temperature in the presence of air for 45 minutes, 35 ml of a monohydric alcohol, methanol, were added to it and magnetite nanoparticles grown epitaxially grown on gold nanoparticles were separated by centrifugation. The separated nanoparticles are dried to constant weight. Get 48 mg of nanoparticle powder. The obtained nanoparticles are stored in a hermetically sealed container in a nitrogen atmosphere in the absence of moisture at room temperature. Under these conditions, nanoparticles retain their operational properties throughout the year.

By transmission electron microscopy, it was shown that the structure of the obtained nanoparticles resembles a dumbbell in which each magnetite nanoparticle having a spherical shape and a diameter of 5 nm is associated with a spherical gold nanoparticle with a diameter of 3 nm. Using the VSM-250 vibromagnetometer, it was shown that the saturation magnetization of the obtained crystals is 59 Am ² / kg.

1 mg of the obtained nanoparticle powder was placed in a round bottom flask, then 2.1 ml of a solution of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [carboxy (polyethylene glycol) -2000] in dichloromethane with a concentration of 2 mg / l was added thereto. , the flask for 1 min is transferred to an ultrasonic bath with a power of 500 W, after which the flask is left open at room temperature, while the dichloromethane is completely evaporated. Then 5 ml of water is added to the flask, the flask is re-placed for 1 min in an ultrasonic bath with a power of 500 W, then the resulting dispersion is transferred to a plastic tube and centrifuged for 10 min at a rotational speed of the centrifuge rotor 12000 rpm. Then, in a test tube containing separated magnetite nanoparticles grown epitaxially on gold nanoparticles and modified with a polymer, 5.1 ml of an aqueous solution of the antitumor drug, doxorubicin with a concentration of 0.4 mg / ml, is added. After that, the magnetic body is placed in the tube for stirring, the tube is closed with a lid, suspended on a tripod above the magnetic stirrer, the magnetic stirrer is turned on and the contents of the tube are stirred for 24 hours. Then the tube is opened, 5.1 ml of an aqueous solution of a low molecular weight prostatic specific ligand is introduced into it membrane antigen with a concentration of 10 mg / ml and re-mixed for 24 hours. After that, the tube is placed in a centrifuge rotor and centrifuged for 10 min at a rotor speed of 12000 rpm./ in. In this case, the nanoparticles are separated from the excess doxorubicin and the low molecular weight ligand of the prostatic specific membrane antigen. The settled nanoparticles are dispersed in 1.1 ml of water. The resulting dispersion is stored at room temperature in a hermetically sealed container without access of light.

The content of the antitumor drug, doxorubicin in the dispersion of modified nanoparticles, determined by absorption spectroscopy from a calibration curve for a number of standard samples with a known concentration of doxorubicin, is 0.47 mg / ml. The dispersion concentration of the modified nanoparticles, determined by atomic emission spectroscopy from a calibration curve for a number of standard samples with a known concentration of iron and gold, is 0.85 mg / ml. Thus, the content of doxorubicin in the modified nanoparticles, determined by calculation from the data given in the example, is 55.3% by weight of the nanoparticles.

The cytotoxicity of the preparation (IC ₅₀ ) for the LNCaP cell culture specific for the low molecular weight ligand of the prostate specific membrane antigen determined by the MTS test is 1.21 μg of the preparation / ml of culture medium. The cytotoxicity of the drug (IC50) for the control cell culture of PC-3, determined by the MTS test, is 9.35 μg of the drug / ml of culture medium.

Example 3

18 ml (113 mmol) of diphenyl ether, 1.5 ml (4.7 mmol) of oleic acid, 1.5 ml are placed in a three-necked round-bottom flask equipped with a reflux condenser, a high-temperature thermometer, an inert gas supply system and a magnetic body for stirring. (4.5 mmol) of oleylamine; and 2.1 g (8 mmol) of 1,2-hexadecandiol. An argon current is applied, an electric heating plate with magnetic stirring function is placed under the flask, the stove is turned on and the contents of the flask are heated for 120 minutes to 120 ° C with constant stirring, followed by the initial introduction of 0.25 ml (1.8 mmol) of iron pentacarbonyl, then, after 4 minutes, a solution containing a mixture of 38 mg (0.09 mmol) of hydrochloric acid trihydrate and 0.4 ml (1.2 mmol) of oleylamine in 4.9 ml of diphenyl ether, previously incubated in an argon atmosphere. Then, the resulting mixture was reheated from 120 ° C to 255 ° C, kept at this temperature for 28 minutes, and then cooled to room temperature, all stages of the synthesis being carried out in an argon atmosphere. The cooled mixture is kept at room temperature in the presence of air for 45 minutes, 40 ml of ethanol - ethanol are added to the mixture and magnetite nanoparticles grown epitaxially grown on gold nanoparticles are separated by centrifugation. The separated nanoparticles are dried to constant weight. 40 mg of nanoparticle powder are obtained. The obtained nanoparticles are stored in a hermetically sealed container in an argon atmosphere in the absence of moisture at room temperature. Under these conditions, nanoparticles retain their operational properties throughout the year.

It was shown by transmission electron microscopy that the structure of the obtained nanoparticles resembles a dumbbell in which each magnetite nanoparticle having a spherical shape and a diameter of 3 nm is associated with a spherical gold nanoparticle with a diameter of 1 nm. Using a VSM-250 vibromagnetometer, it was shown that the saturation magnetization of the obtained crystals is 47 Am ² / kg.

1 mg of the obtained nanoparticle powder is placed in a round bottom flask, then 1.9 ml of a triblock copolymer solution is added thereto, consisting of a central block of polypropylene glycol with a degree of polymerization of 30 and two end blocks of polyethylene glycol with a degree of polymerization of 78 each, in hexane with a concentration of 0.5 mg / l, the flask for 5 min is transferred to an ultrasonic bath with a power of 300 W, after which the flask is placed on a rotary evaporator, and hexane is removed using it. After removing hexane, 1 ml of water is added to the flask, the flask is re-placed for 5 min in an ultrasonic bath with a power of 300 W, then the resulting dispersion is transferred to a plastic tube and centrifuged for 5 min at a rotational speed of the centrifuge rotor of 14,000 rpm. Then, 4.9 ml of an aqueous solution of the antitumor drug, doxorubicin with a concentration of 0.1 mg / ml, is added to a test tube containing separated magnetite nanoparticles epitaxially grown on gold nanoparticles and modified with a polymer. After that, the magnetic body is placed in the tube for stirring, the tube is closed with a lid, suspended on a tripod above the magnetic stirrer, the magnetic stirrer is turned on and the contents of the tube are stirred for 12 hours. Then the tube is opened, 4.9 ml of an aqueous solution of a low molecular weight prostatic specific ligand is introduced into it membrane antigen with a concentration of 5 mg / ml and re-mixed for 12 hours. After that, the tube is placed in a centrifuge rotor and centrifuged for 5 min at a rotor speed of 14000 rpm n In this case, the nanoparticles are separated from the excess doxorubicin and the low molecular weight ligand of the prostatic specific membrane antigen. The settled nanoparticles are dispersed in 0.9 ml of water. The resulting dispersion is stored at room temperature in a hermetically sealed container without access of light.

The content of the antitumor drug, doxorubicin in the dispersion of modified nanoparticles, determined by absorption spectroscopy from a calibration curve for a number of standard samples with a known concentration of doxorubicin, is 0.25 mg / ml. The dispersion concentration of the modified nanoparticles, determined by atomic emission spectroscopy from a calibration curve for a number of standard samples with a known concentration of iron and gold, is 0.93 mg / ml. Thus, the content of doxorubicin in the modified nanoparticles, determined by calculation from the data given in the example, is 26.6% of the mass of the nanoparticles.

The cytotoxicity of the preparation (IC ₅₀ ) for the LNCaP cell culture specific for the low molecular weight ligand of the prostate specific membrane antigen determined by the MTS test is 1.68 μg of the preparation / ml of culture medium. The cytotoxicity of the drug (IC ₅₀ ) for the control cell culture of PC-3, determined by the MTS test, is 9.10 μg of the drug / ml of culture medium.

Thus, from the above examples it is seen that the proposed method compared with the prototype increases the cytotoxicity (reduces by 5-31% IC ₅₀ ) of the system for delivering an antitumor drug to the cells of a malignant tumor - human prostate adenocarcinoma.

Claims (1)

A method of obtaining a system for delivering an antitumor drug to tumor cells, comprising mixing in the presence of water polymer-modified magnetite nanoparticles grown epitaxially on gold nanoparticles, with an organic compound chemically binding to the nanoparticles and providing selective penetration of the nanoparticles into the tumor cells, and an aqueous solution of the antitumor drug with subsequent separation of the obtained modified nanoparticles by centrifugation, characterized in that as polymer-modified nanoparticles use nanoparticles obtained by heating to 120 ° C in an inert gas atmosphere while mixing a mixture of diphenyl ether, oleic acid, oleylamine and 1,2-hexadecanediol, introducing iron pentacarbonyl into the mixture, keeping the mixture obtained, followed by introducing a solution containing the mixture hydrochloric acid trihydrate and oleylamine in diphenyl ether, previously aged in an inert gas atmosphere, reheating the mixture in an inert gas atmosphere from 120 ° C to 2 50 ° -260 ° C, keeping the mixture at 250 ° -260 ° C for 25-30 minutes and cooling it to room temperature, carried out in an inert gas atmosphere, keeping the mixture at room temperature in the presence of air, adding monohydric alcohol to the mixture and separation of magnetite nanoparticles by centrifugation, followed by treatment with a polymer solution selected from the group comprising a triblock copolymer consisting of a central block of polypropylene glycol with a degree of polymerization of 56 and two end blocks of polyethylene glycol with a degree of polymer 101 each, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [carboxy (polyethylene glycol) 2000] and a triblock copolymer consisting of a central polypropylene glycol block with a degree of polymerization of 30 and two end blocks of polyethylene glycol with a degree of polymerization 78 each, in an organic solvent, then ultrasound, followed by removal of the solvent, introduction of water, repeated sonication and centrifugation of the modified nanoparticles, doxorubicin is used as an antitumor preparation, As the organic compound providing selective penetration of nanoparticles into the cells of human prostate adenocarcinoma cells, a low molecular weight ligand of a prostatic specific membrane antigen is used, the nanoparticles being first treated with a solution of doxorubicin, then a solution of a low molecular weight ligand of a prostatic specific membrane antigen.