RU2405772C1 - rac- N-[2,3-DI(TETRADECYLOXY)PROP-1-YL]PYRIDINIUM BROMIDE AS AGENT FOR DELIVERING NUCLEIC ACIDS INTO MAMMAL CELLS - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a novel chemical compound, specifically to rac-N-[2,3-di(tetradecyloxy)prop-1-yl]pyridinium bromide, which can deliver nucleic acids into mammal cells:

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EFFECT: compound has low toxicity and in form of an alcohol solution, the compound can deliver nucleic acids into mammal cells.

1 cl, 5 ex, 4 tbl, 1 dwg

Description

The invention relates to the field of chemistry and medicine, namely to new compounds capable of delivering nucleic acids to mammalian cells.

Gene therapy is designed to treat hereditary, infectious and oncological diseases and is aimed either at correcting a hereditary disease, or at giving the cells new functions. The success of gene therapy depends on the development of effective methods for delivering therapeutic genes to target cells, ensuring their expression and long-term functioning in these cells [1, 2]. Viral vectors are characterized by high efficiency of nucleic acid delivery [1], however, immunogenicity, mutagenicity, toxicity, as well as problems of large-scale production of such viral particles stimulated the search for alternative non-viral transport systems. Currently, cationic lipids, polymers, dendrimers and peptides have been proposed as non-viral vectors [3-5]. Non-viral systems have several advantages, such as low toxicity, non-immunogenicity, as well as the possibility of their easy preparation during controlled processes. Among non-viral transport systems, cationic lipids and liposomes based on them are widely used [3-12]. Lipids protect nucleic acids from degradation and facilitate their transfer through the cell membrane, initiating endocytosis. Cationic lipids are positively charged amphiphilic compounds that consist of three structural domains: hydrophobic, hydrophilic and the spacer group connecting them. The hydrophobic part is most often represented by a residue of cholesterol, diglyceride or long-chain hydrocarbon substituents. As the hydrophilic domain, mono-charged cationic groups obtained by quaternization of aliphatic or heterocyclic bases or residues of polycationic natural or synthetic polyamines are used. The hydrophobic and hydrophilic components of the cationic lipids are usually separated by a spacer and are connected by a carbamoyl, amide, simple or ester bond. The type of binding determines the stability and toxicity of the cationic lipid [3, 13].

The closest to the claimed compound, the prototype, is a cationic lipid, rac-N- [2,3-di (oleyloxy) prop-1-yl] -N, N, N-trimethylammonium chloride (DOTMA) [14]. The structure of this lipid includes the remainder of 1,2-di-O-oleoyl-rac-glycerol, which forms a hydrophobic domain, and a trimethylammonium group, which forms a cationic domain, which binds to phosphate groups of nucleic acids.

The disadvantages of the known compounds are high toxicity and low transfection activity when used as an individual compound.

An object of the invention is to obtain a new effective synthetic compound with high transfection activity, low toxicity and the ability to deliver nucleic acids to mammalian cells.

The stated technical problem is solved by the proposed compound, which is a cationic lipid based on glycerol, the structural formula of which is shown in the drawing.

The inventive compound is obtained by the method described in [15]. The starting compound in the synthesis of the cationic lipid rac-N- [2,3-di (tetradecyloxy) prop-1-yl] pyridinium bromide is 1-bromo-1-deoxy-2,3-di-O-tetradecyl-rac-glycerol, which is obtained by treating 1,2-di-O-tetradecyl-rac-glycerol with an excess of carbon tetrabromide in the presence of triphenylphosphine. Heating of 1-bromo-1-deoxy-2,3-di-O-tetradecyl-rac-glycerol with an excess of pyridine for 43-45 hours at 90 ° C and subsequent recrystallization from diethyl ether give the desired product, rac-N- [2,3-di (tetradecyloxy) prop-1-yl] pyridinium bromide.

For the synthesis of the claimed compounds used solvents and reagents of domestic production. TLC was performed on silica gel (Silicagel 60 F ₂₅₄ , Merck). Compounds were identified using Dragendorf reagent or a solution of Ce (SO ₄ ) ₂ - phosphoformolybdenum acid, followed by heating to 100 ° C. Column chromatography was performed on Kieselgel 60 silica gel (40-63 μm, Merck). ¹ H-NMR spectra (δ, ppm KSSB, Hz) were recorded on a Bruker AMD-400 pulsed Fourier spectrometer with an operating frequency of 400 MHz in CDCl ₃ or a mixture of CDCl ₃ -CD ₃ OD, internal standard Me ₄ Si. Mass spectra were obtained on an LC / MSD Agilent 1100 Series time-of-flight mass spectrometer by electrospray ionization (ESI).

To study the ability of the proposed compound to deliver nucleic acids to mammalian cells, a 25-unit oligodeoxyribonucleotide 5'-dTACAGTGGAATTGTATGCCTATTA-3 ', modified with fluorescein isothiocyanate (FITC) at the 3'-end, plasmid DNA (pEGFP-C2, Germany) was used 21-link double-stranded RNA (siPHK, ICBPM SB RAS) (5'-GCGCCGAGGUGAAGUUCGATT-3 'sense strand sequence, 5'-UCGAACUUCACCUCGGCGCGG-3' antisense strand). FITC-ON was synthesized by the phosphitamide method [16] and purified by ion exchange and reverse phase chromatography. The purity of the oligonucleotide was checked by electrophoresis in 15% PAG under denaturing conditions, visualization of the oligonucleotide in the gel was carried out using Stains-All stain.

The efficiency of penetration of nucleic acids using the claimed compound into mammalian cells in vitro was studied in experiments on transfection of cells with a FITC-labeled oligonucleotide, plasmid DNA encoding a green fluorescent protein (EGFP), and short interfering RNA directed against a matrix RNA gene encoding EGFP.

A comparative analysis of the claimed compounds with well-known and widely used commercial transfection agents, such as Lipofectamine ^® 2000 and Oligofectamine, showed that the proposed compound has the following advantages.

1. The claimed compound has the ability to deliver nucleic acids to mammalian cells, both short and long, which allows it to be considered as a promising agent for transfection.

2. The claimed compound delivers nucleic acids to cells in an individual state in the form of a solution in ethanol and does not require the use of additional auxiliary lipids.

3. The inventive compound has low toxicity to mammalian cells.

4. The inventive compound does not require a complex preparation procedure characteristic of liposomal compositions.

In addition, the claimed compound is stable during storage, both in dry form and in the form of a concentrated alcohol solution.

A search by the sources of scientific, technical and patent literature showed that the compound rac-N- [2,3-di (tetradecyloxy) propyl] pyridinium bromide, which has the ability to deliver nucleic acids to mammalian cells, is not described in known sources.

The invention is illustrated by the following examples.

Example 1. Obtaining rac-N- [2,3-di (tetradecyloxy) prop-1-yl] pyridinium bromide

To a solution of 1,2-di-O-tetradecyl-rac-glycerol (96.5 mg, 0.199 mmol) and triphenylphosphine (109.5 mg, 0.417 mmol) in anhydrous dichloromethane (10 ml) cooled to 0 ° C was added dropwise a solution of tetrabromocarbon (139.6 mg, 0.420 mmol) in anhydrous dichloromethane. After 17 hours, methanol (2 ml) was added to the reaction mixture and stirred for 10 minutes, the solvent was removed in vacuo, the residue was chromatographed on a silica gel column to isolate 1-bromo-1-deoxy-2,3-di-O-tetradecyl-rac- glycerin (108.0 mg, 99%). A mixture of 1-bromo-1-deoxy-2,3-di-O-tetradecyl-rac-glycerol (108 mg, 0.19 mmol) and pyridine (1 ml) was held for 44 hours at 90 ° C. Pyridine was removed in vacuo, the product was recrystallized from anhydrous diethyl ether to give rac-N- [2,3-di (tetradecyloxy) prop-1-yl] pyridinium bromide (82 mg, 66%). Mass spectrum, m / z (I _rel (%)): 546.7 [M-Br] ⁺ (100%). ¹ H NMR spectrum: (δ, ppm, J / Hz): 0.81 (t, 6H, J = 7.0, 2 (CH ₂ ) ₁₁ С Н ₃ ), 1.24 (br s, 44Н, 2 (С H ₂ ) ₁₁ CH ₃ ), 1.27-1.50 (m, 4H, 2 OCH ₂ C H ₂ ), 3.19 and 3.52 (both dt in 1H, J = 6.6 and 9.3, OC H ₂ CH ₂ ), 3.38 (t, 2H, J = 6.7, OC H ₂ CH ₂ ), 3.58 (dd, J = 4.0 and 10.9, OCH H _a ), 3.70 (dd, J = 3.1 and 10.9, OCH H _b ), 3.94-4.00 (m, 1H , CHOC ₁₄ H ₂₉ ), 4.72 (dts, 1H, J = 8.3 and 13.2) and 5.53 (dd, 1H, J = 2.9 and 13.2, CH ₂ N ⁺ ), 7.97 (m, 2H), 8.43 (m, 1H ) and 9.41 (m, 2 N, C ₅ H ₅ N ⁺ ).

Example 2. The study of the effectiveness of transfection into cells of a FITC-labeled oligonucleotide using the inventive cationic lipid

A study of the penetration of a FITC-labeled oligonucleotide into HEK 293 and BHK cells was performed using flow cytometry. Transfection efficiency was evaluated by the number of transfected cells of the total number of cells in the sample. Cells were plated in 24-well plates (2 × 10 ⁵ cells per well for HEK 293 cells and 1 × 10 ⁵ cells per well for BHK cells) and cultured for 24 hours at 37 ° C in an atmosphere containing 5% CO ₂ . Before transfection for experiments in the presence or absence of serum, the medium in the wells was replaced with 200 μl of DMEM medium containing 10% serum or DMEM not containing serum, respectively. A solution of cationic lipid (10 μM) in OptiMEM medium was mixed with a solution of FITC-labeled oligonucleotide (5 μM) in the same medium, the resulting complexes were added to the cells and incubated for 4 hours. After incubation, the cells were washed with phosphate-buffered saline (PBS) (300 μl), 50 μl of trypsin solution was added and incubated for 1-2 min (37 ° C, 5% CO ₂ ). At the end of the incubation, 200 μl of DMEM with 10% serum was added to the wells, the cells were suspended and transferred to tubes. The resulting cell suspension was centrifuged at 1000-1200 rpm, 4 ° C, the medium was taken and washed with 1 ml of PBS. Then the cells were fixed with 500 μl of a 2% solution of formaldehyde in PBS. To study the transfection activity of the cationic lipid, we used the BD FACSAria cell sorter (Becton Dickinson), which measures the fluorescence of cells containing a FITC-labeled oligonucleotide determined at an excitation wavelength of 488 nm (the device uses a coherent sapphire laser (20 mV)).

The results of the penetration into the cells of the FITC-labeled oligonucleotide are shown in table 1.

Table 1 Cells The presence of 10% serum in the growth medium The number of transfected cells,% NEC 293 - 78.6 + 47.2 KSS - 87.8 + 61.9

From the above data it is seen that in the presence of the inventive cationic lipid, the FITC-labeled oligonucleotide effectively (the number of transfected cells exceeds 75%) penetrates into the cells in the absence of serum in the growth medium. The presence of 10% serum in the growth medium reduces the efficiency of transfection, however, the number of transfected cells is approximately 50% or higher.

Example 3. Transfection of HEK 293 cells with plasmid DNA using the inventive cationic lipid

Investigation of the penetration of plasmid DNA into HEK cells. 293 was performed using flow cytometry. Transfection efficiency was evaluated by the number of cells containing green fluorescent protein (EGFP) of the total number of cells in the sample. Cells were plated in 24-well plates (2 × 10 ⁵ cells per well for HEK 293 cells) and cultured for one day at 37 ° C. in an atmosphere containing 5% CO ₂ . Before transfection for experiments in the presence or absence of serum, the medium in the wells was replaced with 200 μl of DMEM medium containing 10% serum or DMEM not containing serum, respectively. A solution of cationic lipid (20 μM) in OptiMEM medium was mixed with a solution of plasmid pEGFP-C2 (0.3 μg / μl) in the same medium, the resulting complexes were added to the cells and incubated for 24 hours. 4 hours after the start of transfection in wells with medium serum-free medium was replaced with DMEM with 10% serum. At the end of the incubation, the cells were processed as described in Example 1. Table 2 presents the results of transfection of pEGFP-C2 plasmid DNA cells using the inventive cationic lipid.

From the above data it is seen that plasmid DNA in the presence of the inventive cationic lipid penetrates into cells in a noticeable amount in the absence of serum in the growth medium.

table 2 Cells The presence of 10% serum in the growth medium The number of transfected cells,% NEC 293 - 22.2 + 2.5

Example 4. Transfection of BHK cells IR-780 short interfering RNA using the inventive cationic lipid

The study of siRNA penetration aimed at suppressing the synthesis of green fluorescent protein (EGFP) was carried out on cells of the BHK line IR-780 stably expressing this protein. As a target, mRNA encoding an EGFP protein was chosen, thus, by reducing the fluorescence of cells, conclusions can be drawn about the efficiency of delivery of short interfering RNA into the cytoplasm. Cells were plated in 24-well plates (0.2 × 10 ⁵ cells per well for IRK-780 BHK cells) and cultured for one day at 37 ° C in an atmosphere containing 5% CO ₂ . Before transfection for experiments in the presence or absence of serum, the medium in the wells was replaced with 200 μl of DMEM medium containing 10% serum or DMEM not containing serum, respectively. A solution of cationic lipid (10 μM) in OptiMEM medium was mixed with a solution of short interfering RNA (50 nM) in the same medium, the resulting complexes were added to the cells and incubated for 72 hours. 4 hours after the start of transfection in wells with serum-free medium was replaced DMEM medium with 10% serum. At the end of the incubation, the cells were processed as described in example 1. Table 3 presents the results of the delivery of short interfering RNA by the claimed cationic lipid.

Table 3 The concentration of lipid, μm The presence of 10% serum in the growth medium Suppression of EGFP Expression,% 3 - 13 + 3.6 5 - 21.7 + 3.9 10 - 41.6 + 2.2

From the above data it is seen that the claimed cationic lipid in an amount of low toxicity to the cells promotes the effective penetration of short interfering RNA into cells in the absence of serum in the growth medium.

Example 5. The effect of the inventive cationic lipid on the cell viability of BHK, BHK IR-780, HeLa, HEK 293

Cells of the BHK lines (Syrian hamster kidney embryonic cells), BHK IR-780 (modified Syrian hamster kidney embryonic cells), HeLa (human cervical carcinoma cells), HEK 293 (human kidney embryonic cells) were cultured in DMEM medium containing 10% - fetal calf serum in an atmosphere of 5% CO ₂ at 37 ° C. Cell viability after incubation with a cationic lipid was determined using the MTT test, which is based on the ability of living cells to convert tetrazole-based compounds (MTT) into brightly colored formazan crystals, which allows spectrophotometric estimation of the number of living cells in the preparation. For this, cells were plated in 96-well plates (0.1 × 10 ⁵ cells per well for HEK 293 cells and 0.03 × 10 ⁵ cells per well for HeLa, BHK, BHK IR-780 cells). After 24 hours, the medium was changed in the wells and a solution of the compound in DMEM medium was added to the cells to a final concentration in the well of 1 to 80 μM. Cells were incubated in the presence of a cationic lipid for another 24 hours under the same conditions. At the end of incubation without changing the medium, MTT solution (5 mg / ml) in phosphate-buffered saline was added to the cells to a concentration of 0.5 mg / ml and incubated for 3 h under the same conditions. The medium was removed, 100 μl of dimethyl sulfoxide, in which the formazan crystals formed in the cells were dissolved, were added to the cells, and the optical density was measured on a multichannel spectrophotometer at wavelengths of 570 and 630 nm, where A ₅₇₀ is the absorption of formazan and A ₆₃₀ is the background of the cells.

From the experimental data, the IC ₅₀ value, the concentration of the compound at which the death of 50% of the cells is observed, was calculated. The IC ₅₀ values of the cationic lipid for the cells are shown in table 4.

From the above data it is seen that the treatment of cells with the claimed cationic lipid causes their effective death only at concentrations of compounds higher than 10 μM, which indicates a low toxicity of the cationic lipid.

Table 4 Cells The presence of 10% serum in the growth medium IC ₅₀ μm NEC 293 + 47 - 55 Hela + eleven - 16 BHK IR-780 + 27 - 16 Bhk + 22 - 21

Thus, the proposed compound, which is a cationic lipid based on glycerol with the structural formula shown in the drawing, has the ability to efficiently deliver nucleic acids to mammalian cells, which allows it to be used as an agent for the delivery of genetic material to cells.

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Claims (1)

rac-N- [2,3-Di (tetradecyloxy) prop-1-yl] pyridinium bromide with the structural formula

as an agent for delivering nucleic acids to mammalian cells.

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