RU2617059C2 - Method for production of amphiphilic block copolymers n, n-dimethylaminoethyl methacrylate for nucleic acid delivery to living cells - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to macromolecular chemistry and medicine, namely to versions of method for creation of pseudo-living radical polymerization of amphiphilic block copolymers for eukaryotic cells transfection, comprising a cationic block - poly (N, N-dimethylaminoethyl methacrylate) and a hydrophobic block - poly (oligopropylene glycol acrylate). According to the first version, the method includes preparation of an azoic isobutyric acid dinitrile (AAD) solution and a reversible chain transfer agent, selected from cyano-isopentanoic acid dithiobenzoate and S,'S-bis-(2-methyl-isobutyrate)trithiocarbonate, in N,N-dimethylaminoethyl methacrylate, solution degassing, polymerization to obtain a polymeric reversible chain transfer agent such as poly(N, N-dimethylaminoethyl methacrylate) containing a dithiobenzoate or a trithiocarbonate group, its dilution in a oligopropylene glycol acrylate/benzene mixture, and polymerization followed by isolation, purification and drying of the resultant block copolymer. According to the second version, the process includes preparation of AAD solution and reversible chain transfer agent in the oligopropylene glycol acrylate/benzene mixture, solution degassing, polymerization to obtain a polymeric reversible chain transfer agent such as poly(oligopropylene glycol acrylate) containing a dithiobenzoate or a trithiocarbonate group, its dilution with AAD mixed with N,N-dimethylaminoethyl methacrylate/benzene, polymerization followed by isolation, purification and drying of the resultant block copolymer.

EFFECT: high transfection activity.

3 cl, 3 ex, 6 dwg

Description

The invention relates to the field of chemistry of macromolecular compounds, molecular biology and medicine and can be used for gene therapy of hereditary, oncological and infectious diseases. The use of this invention is possible for the treatment of viral diseases for which virus-specific antisense oligonucleotides, ribozymes, DNAzymes or siRNAs have been developed that are capable of effectively inhibiting the reproduction of viruses. The invention can also be used to deliver nucleic acids to invitro eukaryotic cells to produce transgenic eukaryotic lines.

State of the art

Nucleic acids encoding various genes, antisense oligonucleotides, ribozymes, aptamers, and small interfering RNAs could be ideal in the selectivity of action of drug substances. However, nucleic acids practically do not penetrate into living cells due to the high density of the negative charge of the same name with respect to the charge of the cell membrane. Therefore, the creation of carriers capable of reversibly binding nucleic acids, delivering them to the cell membrane and releasing them in the cytosol or nucleus could significantly expand the arsenal of drugs to combat various diseases of both infectious and metabolic etiology.

In the last two decades of gene therapy, as a method of treating viral infections, significant attention has been paid. The efforts of researchers are currently focused on creating an effective transfecting agent for gene therapy, which compacts and protects genetic material from serum nucleases and will facilitate its penetration into target cells [1].

Non-viral carriers, such as cationic lipids, dendrimers and peptides, are all capable of compacting and binding nucleic acids (NKs), facilitating their penetration into cells. However, unlike viral analogues, which have developed methods to overcome cellular barriers and immune response mechanisms in the body, non-viral NK carriers show much less transfection ability, since they are retained by numerous extra- and intracellular protective barriers. However, the biocompatibility and the possibility of synthesizing these compounds make them very attractive for use in gene therapy. As a result, over the past ten years, numerous studies have been devoted to the production of cationic compounds, which can form complexes with NK and overcome cell barriers [1-2].

The first barrier to overcome the polyplex on the way to the target cell is blood and extracellular medium. That is why it is necessary to select such physicochemical parameters of the complex in order to increase its stability, avoid non-specific interactions and the possibility of an immune response. First, as part of a polyplex, DNA must be protected from the action of extracellular nucleases. Secondly, negatively charged serum proteins (albumin, fibrinogen, immunoglobulins, etc.), as well as extracellular matrix proteins (collagens) are able to adsorb on the surface of charged polyplexes, which leads to a change in the surface charge of polyplexes, leading to an increase in the size of complexes and to their aggregation.

The next step in the delivery of genetic material to target cells is their interaction with the plasma membrane and cell uptake. The binding of polyplexes to cells occurs as a result of electrostatic interaction with a negatively charged (due to negatively charged surface membrane proteins - proteoglycans) plasma membrane. In most cases, such polyplexes are absorbed by non-specific endocytosis. For effective penetration through the plasma membrane of the cell, the optimal size of the DNA-polycation complex is 70-90 nm.

One of the most important stages of the transport path of polyplexes is their exit from endosomes. Endosomes are vesicles with a protein-lipid membrane, the function of which is to transport proteins to certain parts of the cell. Due to the operation of proton pumps, the pH in them decreases (pH 6.5). Further transport can follow either the recirculation path with the release of absorbed molecules into the extramembrane space, or the political path, when the medium is further acidified in the late endosomes, and the macromolecules enter the lysosomes. In lysosomes, the contents are acidified to pH 5, and the absorbed molecules are degraded by hydrolytic enzymes that are active at low pH. Degradation products are removed from the cell by exocytosis or transferred to the cytoplasm, where they are used as building material [3].

After leaving lysosomes, polyplexes end up in the cytosol, after which the complex dissociates into free polycation and DNA. It is believed that this occurs due to competition for cationic groups between the phosphate groups of DNA and low molecular weight compounds and anions of the cytoplasm. In some cases, the dissociation of the complex occurs, apparently, in the nucleus. The main barrier to plasmid DNA in the cell nucleus is the double nuclear membrane. Only small molecules (<40 kD, ~ 10 nm) can pass through the nuclear pore complex by passive diffusion. Since the free plasmid DNA freed after dissociation of the complex does not have a nuclear localization signal, a very small part of plasmid DNA will pass into the nucleus (not more than 0.1-0.001%). In addition, it was found that about 50% of the injected DNA degrades in the cytosol within 1-2 hours after injection. In tumor cells, this problem is solved by active proliferation: DNA easily penetrates the nuclei of daughter cells during the mitotic cycle, when the nuclear membrane dissolves.

When small interfering RNA is delivered to the cell, the problem of nuclear transport is not at all worth it, because the phenomenon of RNA interference occurs in the cytosol. However, in this case, it is especially important that the oligonucleotide is freed from the carrier polymer and is able to bind to RISC proteins.

Thus, to ensure efficient delivery of DNA to cells, polycations must reversibly interact with DNA, forming stable positively charged complexes of small size at physiological pH and ionic strength. These complexes must be resistant to the presence of macromolecular components of biological fluids and, finally, the DNA molecules included in the complex must be protected from biodegradation.

Currently, a number of approaches based on the use of polycations for the delivery of DNA to eukaryotic cells have been patented. So, one of the first patents regarding the use of polycations for transfection of living cells belongs to the company Rhone-PoulencRore. It proposes the use of polythenyleneimine (PEI) with a molecular weight of up to 50 kDa for transfection of eukaryotic cells [4]. Around the same time, it was also proposed to use polyethyleneimine modified with hydrocarbon radicals for DNA delivery [5, 6].

The high transfection reactivity of PEI is largely due to the fact that this polymer is a weak polybase, and at pH 7, i.e. in the environment surrounding the cell, it is protonated only by 10-30%. When it enters endosomes and lysosomes, the pH of which is about 5-5.5, this polymer is protonated, which leads to an increase in endosomal pH. This activates the endosomal H + -ATPase, which begins to pump protons into the internal environment of the endosomes. As a result, this can lead to osmotic swelling and lysis of the endosomal membrane with the release of the polymer and DNA complex into the cytosol [7].

Further designs involving PEI were improved. In particular, it was proposed to use the same polymer with grafted polyethylene glycol chains carrying the TAT oligopeptide (peptide sequence of the envelope protein of type 1 human immunodeficiency virus) at the opposite end, and this polymer is effective for delivering genetic material to the lungs [8]. Polyethyleneimine can also enhance transfection into compositions with proteins that cause DNA compaction, for example, with histones [9]. Later it was proposed to use PEI at the stage of complex formation with DNA at low pH (3.5–4.5), which contributes to more efficient nucleic acid compaction [10]. It was also proposed to use biodegradable PEI analogues containing disulfide bonds that are prone to cleavage under reducing conditions, including in a living cell [11]. PEI modified with aromatic amino acid residues was proposed to be used for delivery of small interfering RNAs [12].

There are a number of patents in which poly-L-lysine (PL) is used as the polycationic base for transfecting cells. This polymer, as well as a number of carbochain polymers with primary amino groups (polyallylamine, poly (aminoethyl methacrylate, etc.), were proposed for compacting DNA [13]. Although this polypeptide itself is ineffective for DNA delivery, it can serve as a convenient platform for creating modular constructs containing peptide sequences that facilitate the penetration of nucleic acids into the cell (for example, imidazole groups) [14], as well as receptor-recognizing molecules (carbohydrates that can bind to membrane lectins, hormones s, RGD-peptides, etc.) [15].

There are also a number of methods for delivering nucleic acids to cells based on the use of the natural chitosan polysaccharide and its derivatives. Thus, it was shown that fractionation of partially depolymerized chitosan preparations by molecular weights with the separation of a fraction containing about 18 units (spread from 15 to 21 units) leads to a three-fold increase in transfection efficiency [16]. Modification of chitosanamidazoleacetic acid with N-ethyl-N'-isopropylcarbodiimide leads to an increase in its transfection activity by almost 2 orders of magnitude [17].

Closest to the patented method is a method for delivering DNA to cells using cationic polymer particles consisting of polyacrylates, polymethacrylates, including poly (N, N-dimethylaminoethyl methacrylate), polyalkylcyanoacrylates, preferably polybutylcyanoacrylates, polyarylamides, polylactates, polyglycolates , polysaccharides, albumin, polystyrene, polyvinyl ethers, polyacrolein, polyglutaric aldehyde and its derivatives, their copolymers and mixtures [18]. The polymer carrier claimed in this patent is solid particles of polybutyl cyanoacrylate coated with cationic polymers and Tween 80 surfactants to stabilize the particles in an aqueous solution.

The carrier for the delivery of DNA patented in this application is a block copolymer with a narrow molecular weight distribution. This compound contains hydrophobic and hydrophilic blocks, i.e. has an amphiphilic structure. In an aqueous solution, this block copolymer forms micelles, the size of which depends heavily on the temperature of the solution.

Another analogue of the proposed approach is a series of patents for the production of lipopoliamines, in which it is proposed to use polyethyleneimine modified with hydrophobic radicals for DNA delivery [6, 19]. It was shown that such a modification increases the transfection activity of polyamine, however, its cytotoxicity significantly increases.

In the present invention, the task is to create a polycationic carrier capable of destabilizing endosomal membranes not only due to the effect of the proton sponge, but also due to the structure of its hydrophobic block. For this, polycationic carriers with a block structure were synthesized and contained weak polyamine poly (N, N-dimethylaminoethyl methacrylate) (PDMAEMA) in their cationic block and hydrophobic poly (oligopropylene glycol acrylate) (POPGA). For the synthesis of block copolymers, radical polymerization was used under conditions of reversible chain transfer (OPC polymerization), which ensured a narrow molecular weight distribution.

The features distinguishing the present invention from analogues are:

- cationic carrier for transfection has a block structure;

- the cationic block is a weak polyamine containing secondary or tertiary amino groups;

- the hydrophobic block is a branched or brush-type polymer or oligomer containing hydrophobic polyalkylene oxides or other flexible chain polymers characterized by a lower critical dissolution temperature (NCTR);

- the block copolymer has a narrow molecular weight distribution;

- the block copolymer is capable of forming micelles in an aqueous solution;

- micelle formation of the block copolymer is temperature dependent;

- the block copolymer interacts with nucleic acids in a water-salt buffer solution, causing their compaction;

- the structure and size of complexes of block copolymers with nucleic acids depend on temperature;

- transfection activity of carriers exceeds the activity of polyethyleneimine (25 kDa) and is comparable with the activity of the commercial drug Lipofectamine 2000 (Invitrogen, USA).

Disclosure of invention

The essence of the invention lies in the fact that the method of producing amphiphilic block copolymers for transfection of eukaryotic cells, involves the use of pseudo-living methods of radical polymerization, and in the structure of all synthesized block copolymers there is a cationic block of weakly polyamine-poly (N, N-dimethylaminoethyl methacrylate) and a hydrophobic block representing a polymer or oligomer of a branched or brush structure - poly (oligopropylene glycol acrylate), which includes, at the first stage, the preparation of a polymer agent fraternal chain transfer based on N, N-dimethylaminoethyl methacrylate by preparation of a solution containing 0.01-0.001 mol / L dinitrile azoisobutyric acid and 0.1-0.01 mol / L solution of dithiobenzoate (cyano-isopentanoic acid dithiobenzoate) or tritiocarbonate (S, S'-bis ( methyl 2-isobutyrate) tritiocarbonate) reversible chain transfer agent in N, N-dimethylaminoethyl methacrylate, attaching it to a vacuum unit and carefully removing traces of oxygen, sealing the resulting degassed solution into an ampoule and heating at 80 ° C for 24-48 hours ow, resulting in the formation of a polymer of N, N-dimethylaminoethyl methacrylate containing a trithiocarbonate or dithiobenzoate group, in the second step the polymer obtained (0.1-0.01 mol / l) and dinitrilazoisobutyric acid (0.01-0.001 mol / l) is dissolved in a mixture of oligopropylene glycol acrylate / benzene 3: 7) and polymerization is carried out at 80 ° C for 24-48 hours, the resulting block copolymer is isolated from the solution by evaporation of the free monomer and purified by dialysis against distilled water, followed by freeze drying in vacuum.

Preferably, polymeric poly (oligopropylene glycol acrylate) polymers containing a dithiobenzoate or trithiocarbonate group that are prepared using two types of reversible chain transfer agents — cyano-isopentanoic dithiobenzoate and S, S'-bis (methyl-2- isobutyrate) trithiocarbonate.

In particular cases of the implementation of the method, the synthesized block copolymers interact with nucleic acids in a water-salt buffer solution, causing their significant compaction, and in cultures of eukaryotic cells have high transfection activity in combination with various types of nucleic acids, including plasmid DNA and small interfering RNA

или

, способные участвовать в реакции роста цепи вплоть до следующего акта взаимодействия с ОПЦ-агентом (фиг. 1), вследствие чего радикальная полимеризация приобретает черты живых анионных процессов [21, 22, 23].The inventive method for the preparation (synthesis) of cationic block copolymers of poly (N, N-dimethylaminoethyl methacrylate) (PDMAEMA) and poly (oligopropylene glycol acrylate) POPHA is based on the phenomenon of radical polymerization under conditions of reversible chain transfer [20]. This approach is based on the fact that the thiocarbonyl compounds ZC (= S) SR (OPC agents) reversibly react with a growth radical, forming intermediates Int1 and Int2, capable of being cleaved by the radical mechanism; during these reactions, macroradicals are regenerated in the system

or

capable of participating in the chain growth reaction up to the next act of interaction with the OPC agent (Fig. 1), as a result of which radical polymerization takes on the features of living anion processes [21, 22, 23].

This approach to the synthesis of polymers has two significant advantages in terms of polymer design in general and the creation of polymer carriers for biologically active compounds in particular.

First, when it is used, the molecular weight of the polymer increases linearly as the monomer is consumed in the reaction mixture, i.e. with increasing conversions. In this case, the resulting polymer, as a rule, has a narrower molecular weight distribution than the polymer obtained by classical radical polymerization.

Secondly, this approach opens up broad prospects for obtaining block copolymers with a controlled sequence and block length. The fact is that most macromolecules formed under the conditions of OPC polymerization contain groups of the OPC agent. Therefore, if such a polymer ZC (= S) SA _{n is} cleaned of unspent monomer and reacted with another monomer B in the presence of an initiator, then the block B _m will grow precisely on the chain A _n , as shown in FIG. 2.

In the aspect of biomedical applications of polymers obtained using OPC polymerization, the issue of toxicity of the OPC agent that is part of the polymer is relevant. This issue was studied in the literature, and the results show that the toxicity level of polymers obtained by the OPC polymerization method strongly depends on the type of cells and the OPC agent used [24, 25]. Thus, cells of the macrophage line Raw264.7 are more sensitive to polymers obtained by OPC polymerization than the fibroblastoid line NIH3T3 or ovary cells of the Chinese hamster CHO. [Mistake! Unknown key argument., Error! Unknown key argument.].

In the present invention, PDMAEMA and POPHA polymers, which were obtained using two types of reversible chain transfer agents, S, S'-bis (methyl-2-isobutyrate) tritiocarbonate (MIBTC) and cyano-isopentane dithiobenzoate, were used as reagent for chain reversal in this invention. acids (CCB) (Fig. 3).

S, S'-bis- (methyl-2-isobutyrate) trithiocarbonate (MIBTC) and cyano-isopentanoic acid dithiobenzoate (CCB) were used as reverse chain transfer agents in the present invention.

MIBTK is a symmetric tritiocarbonate OPC agent and contains two identical labile C-S bonds, which ensures the growth of the polymer chain in two ends, making it possible to easily obtain triblock copolymers of the ABA type. In this case, the trityocarbonate group-S-C (= S) -S- remains in the center of the polymer chain (Fig. 4a).

The dithiobenzoate OPC agent (CIB) contains only one labile C-S bond; therefore, in its presence, the remainder of the OPC agent Ph-C (= S) S-, which ensures the implementation of the pseudo-living mechanism, is at the end of the chain. In this case, the addition of the second monomer to the polymer OPC agent results in the formation of an AB type diblock copolymer containing a dithiobenzoate group at the end of the chain (Fig. 4b).

Effective control of OPC polymerization is possible only if the equilibrium in the reaction of the formation of intermediate 1 (Fig. 2) is shifted to the right. In this case, upon fragmentation of the intermediate, a macromolecule is formed containing a dithioether group and capable of performing the functions of a polymer OPC agent. Naturally, the ratio of the rate constants of the direct and reverse reaction of the formation of intermediate 1, k _ad / k _-ad , depends on the nature of the OPC agent and the monomer.

The block copolymers based on DMAEMA and OPGA obtained by OPC polymerization have a polydispersity index of 1.2-1.9, preferably 1.5. Moreover, the degree of polymerization of PDMAEMA varies from 100 to 300, preferably about 200. The degree of polymerization of the POPA unit can vary from 20 to 100, preferably 40-80, while in the POPA monomer the degree of polymerization of propylene glycol should vary from 5 to 10 units, preferably - Must be 7 links.

PDMAEMA and POPGA block copolymers are amphiphilic in nature, therefore, they are prone to aggregation in an aqueous solution with the formation of micelles. Under physiological conditions, the critical micelle concentration of block copolymers varies from 1 to 10 μg / ml, preferably 2.5 μg / ml, which is an order of magnitude lower than the CMC of most polymeric surfactants.

The structure and stability of micelles depend on the concentration of low molecular weight electrolyte, pH and temperature of the solution, and with increasing temperature, the average hydrodynamic radius of the micelles increases from 15 to 300 nm. An increase in ionic strength above 0.15 M or a pH above 8.5 leads to a decrease in micelle size.

The obtained block copolymers interact with DNA in an aqueous solution to form complexes with an average hydrodynamic radius of 100-150 nm, which is more than an order of magnitude smaller than the hydrodynamic radius of a free nucleic acid molecule. Nevertheless, the size of the complexes strongly depends on the temperature at which they are prepared, and the smallest complexes (about 70 nm) are formed by mixing solutions of nucleic acids and block copolymers heated to a temperature of + 37 ° C. The complexes of a slightly larger, but similar in order of magnitude, size (about 100 nm) are formed at + 4 ° C. It is strongly not recommended to prepare complexes at room temperature, since under these conditions extremely polydisperse particles of complexes with an average hydrodynamic radius exceeding 300 nm are formed, which may be greater than the thickness of small blood capillaries, which means that the use of such complexes in vivo can lead to embolization.

The PDMAEMA and POPGA block copolymers exhibit significantly lower cytotoxicity than the PDMAEMA and PEI homopolymers used as an analog in the invention. The highest non-toxic concentration (NNK) of block copolymers for ovarian cancer cells of the Chinese hamster CHO ranged from 20 to 50 μg / ml depending on the composition, while the NMA of the PDMAEMA homopolymer was 12 μg / ml and PEI was 4 μg / ml Thus, the PDMAEMA and POPGA block copolymers exhibit markedly lower cytotoxicity than the previously used analogues.

The obtained block copolymers exhibit high ability to transfect eukaryotic plasmid DNA cells. In this case, the most efficient transfection of plasmid DNA is demonstrated by the complexes of plasmid DNA with triblock copolymers of POPGA-block-PDMAEMA-block-POPGA with the composition of the complex corresponding to the molar ratio of polymer amine groups to phosphate groups of nucleic acid, N: P = 7.5, and with diblockE copolymers of PDM -Block-POPGA with a ratio of N: P = 15. The transfection efficiency of plasmid DNA using POPGA-block-PDMAEMA-block-POPGA (at a ratio of N: P = 7.5) was 3 times higher than that of the commercially available Lipofectamine 2000 and PEI.

The same block copolymers showed a high ability to deliver small interfering RNAs into cells. Two different systems were used in the invention to evaluate the effectiveness of suppressing the target gene. Firstly, a CHO-luc cell culture stably transfected with the firefly luciferase gene. The complexes of synthetic RNA directed to the luciferase gene and the triblock copolymer POPGA-block-PDMAEMA-block-POPGA inhibit luciferase activity by 89 and 93% at a ratio of N: P = 7.5 and 15, respectively, which is 7 and 10% more than that of the Lipofectamine 2000 reference preparation. PDMAEMA-block-POPHA diblock copolymers are most effective at a ratio of N: P = 15 and cause a suppression of luciferase activity of 80-90%, which is close to the effect exerted by Lipofectamine 2000.

The implementation of the invention

Example 1

Synthesis of block copolymers based on reversible chain transfer reagents of poly (N, N-dimethylaminoethyl methacrylate) -dithiobenzoate or poly (N, N-dimethylaminoethyl methacrylate) -tritiocarbonate.

For the synthesis of polymeric agents for reversible chain transfer based on N, N-dimethylaminoethyl methacrylate, a solution of azoisobutyric acid dinitrile was prepared in a concentration of 0.01 to 0.001 mol / L in freshly distilled dimethylaminoethyl methacrylate. Cyanisopentanoic acid dithiobenzoate or S, S'-bis- (methyl-2-isobutyrate) trithiocarbonate was added to the resulting solution to a final concentration of 0.1 to 0.01 mol / L. The reaction mixture was poured into an ampoule, connected to a vacuum unit, and degassed by repeating freeze-thaw cycles to a residual pressure of 5 × 10 ^-3 mm Hg, then the ampoule was sealed. The sample was polymerized at 80 ° C for 24-48 h. The polymer was isolated by freeze drying from benzene in a vacuum unit. The sample was characterized by gel permeation chromatography. As a result, depending on the OPC agent used, poly (N, N-dimethylaminoethylmethacrylate) -dithiobenzoate (PDMAEMA-B) or poly (N, N-dimethylaminoethylmethacrylate) -tritiocarbonate (PDMAEMA-TK) was obtained.

To synthesize the PDMAEMA-block-POPGA diblock copolymer based on the monofunctional OPC agent of cyan-isopentanoic acid dithiobenzoate, a solution of azoisobutyric acid dinitrile (0.01-0.001 mol / L) in an oligopropylene glycol acrylate / benzene mixture (3: 7 by volume) was prepared it to the polymer OPC agent (PDMAEMA-B, 0.1-0.01 mol / l). The resulting mixture was poured into an ampoule and degassed in a vacuum unit, the ampoule was sealed and placed in a thermostat heated to 80 ° C for 24-48 hours. After polymerization, the ampoule was opened, the contents were dissolved in excess benzene, and then the solvent was distilled off on a rotary evaporator. The resulting dry residue was dissolved in water, dialyzed, and the polymer was isolated by freeze drying from an aqueous solution in a vacuum unit. The block copolymer was characterized by gel permeation chromatography.

Example 2

Synthesis of block copolymers based on polymeric agents for reversible chain transfer of poly (oligopropylene glycol acrylate) -dithiobenzoate (POPGA-B) or poly (oligopropyl anglycol acrylate) -tritiocarbonate (POPG A-TK).

For the synthesis of reversible chain transfer reagents based on poly (oligopropylene glycol acrylate), a solution of azoisobutyric acid dinitrile (0.01-0.001 mol / L) in an oligopolypropylene glycol acrylate / benzene mixture (1: 1 by volume) was prepared. Cyanisopentanoic acid dithiobenzoate or S, S'-bis- (methyl-2-isobutyrate) trithiocarbonate was added to the resulting solution to a final concentration of 0.01-0.1 mol / L. The reaction mixture was poured into an ampoule, connected to a vacuum unit, and degassed by repeating freeze-thaw cycles to a residual pressure of 5 × 10 ^-3 mm Hg, then the ampoule was sealed. The sample was polymerized at 80 ° C for 4-24 hours. The polymer was isolated by freeze drying from benzene in a vacuum unit. Molecular weight characteristics of the sample were determined by gel permeation chromatography. As a result, depending on the OPC agent used, poly (oligopropylene glycol acrylate) -dithiobenzoate (POPGA-B) or poly (oligopropylene glycol acrylate) -tritiocarbonate (POPGA-TK) was obtained.

For the synthesis of the triblock copolymer POPGA-block-PDMAEMA-block-POPGA, a solution of POPGA-TK (0.01-0.001 mol / L) and dinitrile azoisobutyric acid (0.1-0.01 mol / L) in a mixture of dimethylaminoethyl methacrylate / benzene was prepared (4: 1 by volume). The resulting mixture was poured into an ampoule and degassed at a vacuum stop, the ampoule was sealed and placed in a thermostat, heated to 80 ° C for 4-24 h. After polymerization, the ampoule was opened, the contents were dissolved in excess benzene, and then the solvent was distilled off on a rotor. The resulting dry residue was dissolved in water, dialyzed, and the polymer was isolated by freeze drying from an aqueous solution in a vacuum unit. The block copolymer was characterized by gel permeation chromatography. In a similar manner, a PDMAEMA-block-POPGA-block-PDMAEMA block copolymer with an inverted sequence of blocks was obtained using PDMAEMA-TK as a polymer agent for reversible chain transfer.

Example 3

Assessment of the transfection activity of complexes of amphiphilic block copolymers with plasmid DNA and small interfering RNAs (siRNAs).

To assess the transfection activity of complexes of amphiphilic block copolymers with plasmid DNA and siRNA, Chinese hamster ovary CHO and CHO-luc cells were used (CHO cells stably transfected with the firefly luciferase gene), respectively.

CHO cell culture was treated with complexes containing plasmid DNA pCMVluc (Promega, USA) and preparations of synthesized cationic amphiphilic block copolymers: POPGA-block-PDMAEMA-block-POPGA, PDMAEMA-block-POPGA-block-PDMAEMA, PDMAEMA-block-POPGA, POPGA-block-PDMAEMA, as well as PDMAEMA homopolymer in various ratios N: P = 7.5 and 15. Transfection efficiency was evaluated by luciferase activity 24 hours after treatment with complexes using a commercial Luciferase assay system reagent kit (Promega, USA) in a tablet analyzer. Lipofectamine 2000, a commercial transfection agent (Invitrogene, USA) and 25 kD branched polyethyleneimine (PEI) (Sigma, USA), were used as comparison preparations. The negative control, which was a preparation of naked plasmid DNA or siRNA, was also included in the experiments. DNA complexes with copolymers for transfection were prepared in N: P ratios of 7.5 and 15. The highest efficiency of plasmid DNA transfection was demonstrated by DMAEMA-OPGA copolymers in a N: P ratio of 7.5, as well as POPGA-block-PDMAEMA and POPGA-block -PDMAEMA-block-POPGA in the ratio of 15 (Fig. 5a, 5b), both in comparison with other synthesized copolymers and with the starting blocks for copolymers.

In the ratio N: P - 15, the efficiency of transfection using the POPGA-block-PDMAEMA-block-POPGA and POPGA-block-PDMAEMA complexes was 83 and 53 times higher than in experiments with the PDMAEMA homopolymer and not lower than the effectiveness of the comparison drugs (PEI and Lipofectamine 2000). The efficiency of transfection of plasmid DNA using PDMAEMA-block-POPGA (at a ratio of N: P of 7.5) was 64 times higher than that of PDMAEMA and was not inferior in comparison to comparison drugs. With an increase in the N: P ratio to 15, the efficiency of transfection of the PDMAEMA-block-POPGA preparation decreased (2.4 times compared with the efficiency at N: P = 7.5). This is probably due to an increase in the toxic effect of the studied copolymers with an increase in their concentration.

Complexes for siRNA transfection were prepared in ratios N: P = 7.5 and 15. In this series of experiments, a CHO-luc cell culture stably transfected with the firefly luciferase gene was used. Complexes for transfection were prepared from synthetic siRNAs directed to the luciferase gene and amphiphilic block copolymers. The results obtained in experiments with block copolymers were presented as luciferase activity in cells treated with complexes based on specific (directed to the luciferase gene) and nonspecific siRNA (directed to the P gene of respiratory syncytial virus). The results were expressed as the percentage of residual luciferase activity in cells treated with specific siRNA compared with non-specific (Fig. 6a, 6b). As in experiments with plasmid DNA, the drug POPGA-block-PDMAEMA-block-POPGA demonstrated high efficiency of miRNA transfection, which suppressed luciferase activity by 78% at a N: P ratio of 7.5. Block copolymers of PDMAEMA-block-POPGA showed equally high transfection efficiency at a ratio of N: P of 7.5 and 15, and ensured the suppression of luciferase activity by 89%. This is 7% more compared to Lipofectamine 2000.

Thus, the preparations of amphiphilic cationic copolymers based on PDMAEMA and POPGA showed high transfection activity both during transfection of plasmid DNA cell culture and siRNA, comparable or even superior to the efficiency achieved using comparison preparations.

In the ratio N: P - 15, the efficiency of transfection using the POPGA-block-PDMAEMA-block-POPGA and POPGA-block-PDMAEMA complexes was 83 and 53 times higher than in experiments with the PDMAEMA homopolymer and not lower than the effectiveness of the comparison drugs (PEI and Lipofectamine 2000). The efficiency of transfection of plasmid DNA using PDMAEMA-block-POPGA (at a ratio of N: P - 7.5) was 64 times higher than that of PDMAEMA and was not inferior in comparison to the comparison drugs. With an increase in the N: P ratio to 15, the efficiency of transfection of the PDMAEMA-block-POPGA preparation decreased (2.4 times compared with the efficiency at N: P = 7.5). This is probably due to an increase in the toxicity of the studied copolymers.

Complexes for RNA transfection were prepared in ratios N: P = 7.5 and 15. In this series of experiments, we used a CHO-luc cell culture stably transfected with the firefly luciferase gene. Complexes for transfection were prepared from synthetic siRNAs directed to the luciferase gene and amphiphilic block copolymers. The results obtained in experiments with block copolymers were presented as luciferase activity in cells treated with complexes based on specific (directed to the luciferase gene) and nonspecific siRNA (directed to the P gene of respiratory syncytial virus). The results were expressed as the percentage of residual luciferase activity in cells treated with specific siRNA compared with non-specific (figa, 66). As in experiments with plasmid DNA, POPGA-block-PDMAEMA-block-POPGA demonstrated high efficiency of RNA transfection, which suppressed luciferase activity by 78% at a N: P ratio of 7.5. PDMAEMA-block-POPGA block copolymers showed the same high transfection efficiency at N: P ratios of 7.5 and 15, and ensured 89% suppression of luciferase activity. This is 7% more compared to Lipofectamine 2000.

Thus, the preparations of amphiphilic cationic copolymers based on PDMAEMA and POPGA showed high transfection activity both during transfection of plasmid DNA cell culture and siRNA, comparable or even superior to the efficiency achieved using comparison preparations.

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Description of drawings

FIG. 1. Scheme of processes occurring during pseudo-living radical polymerization with reversible chain transfer

и

- радикалы роста; М - мономер; Z и R - алкильные или арильные заместители в молекуле ОПЦ-агента.

and

- growth radicals; M is a monomer; Z and R are alkyl or aryl substituents in the OPC agent molecule.

FIG. 2. Obtaining block copolymers using OPC polymerization.

If the polymer ZC (= S) SA _{n is} cleaned of unspent monomer and reacted with another monomer B in the presence of an initiator, then the block B _m will grow precisely on the chain A _n .

FIG. 3. The formulas of the OPC agents used in the invention are -S, S'-bis- (methyl-2-isobutyrate) trithiocarbonate (MIBTC) and cyano-isopentanoic acid dithiobenzoate (CCB).

FIG. 4. Scheme of OPC polymerization in the presence of (a) trithiocarbonates and (b) dithiobenzoates

FIG. 5. The results of transfection of CHO cells with copolymer preparations in combination with plasmid DNA pCMVluc in the ratio N: P: (a) 7.5, (b) 15.

POPGA - poly (oligopropylene glycol acrylate); PDMAEMA - poly (N, N-dimethylaminoethyl methacrylate); PEI 25 kD - branched polyethyleneimine, 25 kD.

FIG. 6. Results of transfection of CHO-luc cells with copolymer preparations in complex with siRNA to the firefly luciferase gene (siRNA-luc) and nonspecific siRNA to the R gene of the respiratory syncytial virus (siRNA-RSV).

POPGA - poly (oligopropylene glycol acrylate); PDMAEMA - poly (N, N-dimethylaminoethyl methacrylate).

Claims (3)

1. A method of producing amphiphilic block copolymers for transfection of eukaryotic cells by pseudo-living radical polymerization, said block copolymers comprising a cationic block, such as poly (N, N-dimethylaminoethyl methacrylate), and a hydrophobic block, such as poly (oligopropylene glycol acrylate), characterized in that at the first stage involves the preparation of a solution containing 0.01-0.001 mol / l of dinitrile azoisobutyric acid and 0.1-0.01 mol / l of a reversible chain transfer agent selected from cyano-isopentanoic acid dithiobenzoate and S, S'-bis- (methyl-2-isobutyrate) trithiocarbonate, in N, N-dimethylaminoethyl methacrylate, placing said solution in an ampoule, attaching the ampoule to a vacuum unit, thoroughly removing traces of oxygen by degassing, sealing the degassed solution into the ampoule and holding polymerizing at 80 ° C for 24-48 hours to obtain a reversible chain transfer polymer agent, such as a poly (N, N-dimethylaminoethyl methacrylate) containing a dithiobenzoate or trithiocarbonate group; and in the second stage, it involves dissolving the polymer agent of the reversible chain transfer obtained in the first stage at a concentration of 0.1-0.01 mol / l in an oligopropylene glycol acrylate / benzene mixture with a volume ratio of oligopropylene glycol acrylate and benzene equal to 3: 7, respectively, and polymerization at 80 ° C for 24-48 hours, isolating the obtained block copolymer from the solution by evaporation of the free monomer and purifying it by dialysis against distilled water, followed by freeze drying in vacuum. 2. A method of producing amphiphilic block copolymers for transfection of eukaryotic cells by pseudo-living radical polymerization, said block copolymers comprising a cationic block such as poly (N, N-dimethylaminoethyl methacrylate) and a hydrophobic block such as poly (oligopropylene glycol acrylate), characterized in that at the first stage involves the preparation of a solution containing 0.01-0.001 mol / l of dinitrile azoisobutyric acid and 0.1-0.01 mol / l of a reversible chain transfer agent selected from cyano-isopentanoic acid dithiobenzoate and S, S'-bis- (methyl-2-isobutyrate) trithiocarbonate, in a mixture of oligopropylene glycol acrylate / benzene with a volume ratio of oligopropylene glycol acrylate and benzene equal to 1: 1, respectively, placing said solution in an ampoule, attaching an ampoule to a vacuum unit, thoroughly removing traces oxygen by degassing, sealing the degassed solution into a vial and polymerizing at 80 ° C for 4-24 hours to obtain a polymeric reversible chain transfer agent such as poly (oligopropylene glycol acrylate) containing tiobenzoatnuyu or tritiokarbonatnuyu group; and in the second stage, it involves dissolving the polymer agent of the reversible chain transfer obtained in the first stage at a concentration of 0.01-0.001 mol / L and 0.1-0.01 mol / L of dinitrile azoisobutyric acid in a mixture of N, N-dimethylaminoethyl methacrylate / benzene with a volumetric the ratio of N, N-dimethylaminoethyl methacrylate and benzene equal to 4: 1, respectively, and polymerization at 80 ° C for 4-24 hours, isolation of the obtained block copolymer from the solution by evaporation of the free monomer and purification by dialysis against distilled water followed by lyophilization nym drying in vacuo. 3. The method according to any one of paragraphs. 1 or 2, characterized in that the synthesized block copolymers interact with nucleic acids in a water-salt buffer solution, causing their significant compaction, and possess high transfection activity in eukaryotic cell cultures in combination with various types of nucleic acids, including plasmid DNA and small interfering RNAs.

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