RU2696849C1 - Aptamer cytotoxicity estimation method - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: processes of proliferation and apoptosis in cells after incubation with a sample of the aptamer are compared. That is ensured by measuring the expression ratio of BCL2 and PCNA genes. Aptamer cytotoxicity is judged by a degree greater than 1.EFFECT: invention can be used in pharmacology and medicine to assess cytotoxicity of new synthesized substances, particularly antitumor aptamers.1 cl, 2 tbl, 2 ex

Description

The invention relates to medicine, biotechnology, pharmacology and can be used to assess the cytotoxicity of new synthesized substances, in particular aptamers.

The study of the cytotoxicity of substances in relation to normal cells plays a crucial role in the study of the toxicity of potential antitumor drugs. For further preclinical studies, it is important to determine the ratio of the cytotoxicity of the studied compounds on tumor cells to cytotoxicity on normal cells. New antitumor drugs of an aptameric nature are being developed, and methods for assessing their cytotoxicity are in demand in practice.

Morphological methods are widely used to identify and determine the cytotoxic effect of a substance’s action on a cell, for example, “A method for microscopic assessment of the cytotoxicity of scaffold material components” (1). For this, erythrocytes are isolated from the blood, washed and incubated with components of scaffold materials at 37 ° C for 30 minutes. Then the red blood cells are mixed with autologous blood plasma, a drop is placed on a glass slide and microphotography is carried out with a 100x objective. The cytotoxicity of scaffold components is evaluated by the change in the morphology of red blood cells and their aggregates in autologous blood plasma.

A known method for determining the cytotoxicity of substances by treating at least one cell with a substance, followed by determining the toxicity of the substance by changing the level of intracellular ROS (2). In this method, the change in the intracellular level of ROS is determined by the luminescence of the fluorescent dye DCFH-DA, which can penetrate into the cells and also emit light upon binding to ROS. By changing the intensity of the glow judge the toxicity of the test substance.

The disadvantages of this method is its relatively low sensitivity, which complicates the identification of early signs of toxicity, and the complexity of this method, due to the use of additional reagents, as well as the inability to quantify the level of ROS.

Widely used method, called MTT test. The MTT test method is based on the ability of the colorless tetrazolium salt (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide, MTT)

recover to stained formazan in the presence of mitochondrial enzymes of living cells (3).

MTT test

Dissolve the test substances in DMSO at a concentration of 10-2 M. After 24 hours of incubation, add various concentrations of the test compounds to the HEK293 cell culture by trituration. Perform each concentration in triplicate. Add DMSO solvent in a concentration of not more than 1% to control wells. The final volume of medium in the well should be 200 μl. Place the tablet with the compounds into the CO2 incubator.

After 72 hours of incubation of the HEK293 cell line with experimental substances, add 20 μl of MTT working solution (5 mg / ml) to each well. Incubate for another 2 hours in a CO2 incubator. After 2 hours, remove the plate from the CO2 incubator, replace the medium in each well with a DMSO solution. Shake the tablet gently until the formazan crystals dissolve. Using a plate reader (e.g. Victor, PerkinElmer) determine the optical density of each well at 530 nm, subtract the measured background absorption at 620 nm. The concentration value causing 50% inhibition of cell population growth (IC50) is determined on the basis of dose-dependent curves and / or using software (for example, OriginPro 9.0). Processing of the measurement results is carried out using the WorkOut 2.5 software (DazDag Ltd) installed on the workstation of the Victor3 tablet reader.

The survival of HEK293 cells in the presence of the test substance is calculated by the formula: (OD of test wells - OD of the medium) / (OD of control wells - OD of the environment) × 100%, where OD is the optical density (4).

This method is chosen by us as a prototype. The method is quite reliable and reproducible. The disadvantages of the method include limited information content, since it assesses the state of mitochondrial dehydrogenases. In addition, the disadvantages of this method are its duration and complexity due to the use of additional reagents, as well as the fact that this method allows you to determine only a high degree of toxicity, leading to the loss of living cells. These disadvantages are due to the features of the method. In the study of antitumor substances, in particular, aptamers, it is important to evaluate the cytotoxicity expressed in the inhibition of cell proliferation and activation of apoptosis.

The problem to which the present invention is directed, is to increase the information content of the method. This is done by comparing the processes of proliferation and apoptosis by measuring the expression of BCL2 and PCNA genes in cells exposed to a test sample of aptamer.

The proposed method for solving this problem is as follows. The cell culture is incubated with the test substance. Then total RNA is isolated and purified from the cells. The synthesis of the 1st chain of cDNA by reverse transcriptase is carried out. Quantify the transcript level of the BCL2 and PCNA genes. The PCNA gene is a proliferation marker, and the BCL2 gene is involved in apoptosis. When a toxic agent is exposed to a cell, suppression of proliferation and stimulation of apoptosis are observed. In real-time PCR, the level of gene expression is determined. The cytotoxicity of the test substance is judged by the ratio of BCL2 and PCNA gene expression.

To check the adequacy of the choice of markers, we set up an experiment with the RG-17 aptamer, the cytotoxicity of which was revealed in the MTT test. We incubated the substance in a 4-fold therapeutic dose with a human fibroblast culture. The number of cups was 5. Another 5 cups were control; in them, instead of the preparation, a solvent was added to the medium. The incubation time was 72 hours. In fibroblast culture samples, the level of BCL2 and PCNA gene expression was determined. The results are presented in the table:

The expression value of the PCNA proliferation marker was significantly (p <0.01 according to the Mann-Whitney test) reduced in cell samples incubated with the test substance, compared with the control group of cells. The value of BCL2 gene expression tends to increase in the experimental group compared to the control. The greater the ratio of BCL2 and PCNA gene expression will exceed 1, that is, the stronger the process of cell apoptosis will prevail over the proliferation process, the more pronounced the cytotoxic effect of the test substance.

The invention is illustrated by the following examples of specific performance of the method.

Example 1

A sustained-release aptamer RA-361 at a concentration of 400 μg / ml was added to the incubation medium of a human fibroblast culture. The tests were carried out in 5 parallel samples - Petri dishes, control plates contained a solvent instead of the test substance. After three days of incubation, the cells were removed from the dishes, washed twice in PBS and suspended in 150 μl of this buffer.

Isolation of RNA. In an Eppendorf microtube, cells were lysed with 1 ml TPI reagent MRC for 10 min, while stirring on a rotary mixer. 200 μl of chloroform was added and shaken on a shaker for 15 seconds. The tube was left at room temperature for 10 minutes, and then centrifuged for 15 minutes at 4 ° C and 12000 rpm. The supernatant in a volume of 600 μl was mixed with 700 μl of isopropanol. The tube was left on the table, and after 10 min, RNA was precipitated by centrifugation for 10 min at 12000 rpm and 4 ° C. The RNA pellet was washed with 1 ml of 75% ethanol, then centrifuged under the same conditions, dried in a vacuum centrifugal concentrator and dissolved in 50 μl of DEPC water supplemented with 1 μl of a RNAase inhibitor RiboLock Thermo Scientific. The RNA concentration was determined on a Biowave II spectrophotometer, after which it was treated with DNase I Thermo Scientific DNAse according to the attached protocol. The procedures were followed by precipitation with isopropanol, washing with 75% ethanol, drying the RNA pellet and dissolving in DEPC water with the addition of RiboLock. The concentration of RNA was measured and its degradation was assessed by electrophoresis on a 1% agarose gel.

Reverse transcription reaction

The reverse transcription reaction to obtain cDNA was carried out in a volume of 25 μl. Used ready-made kits of reagents "OT-1" for reverse transcription of SINTOL company. The reverse transcription reaction contained: 2 μg of isolated RNA, 1 μl of Random-6 primer, 10 μl of 2.5x reaction mixture, 1 μl (50 units) of MMLV-RT reverse transcriptase and 0.5 μl of RiboLock. The reaction was carried out for 40 min at 42 ° C, after which the reaction mixture was incubated for 4 min at 92 ° C to inactivate reverse transcriptase. The resulting cDNA-containing reaction mixture was immediately used as a template for PCR.

Real-time PCR

The expression levels of BCL2 and PCNA genes were measured by real-time PCR using the prepared reagent kits “Reagent Kit for PCR-PB in the Presence of SYBR Green I R-402 Dye” from SINTOL company on a CFX96 thermocycler (Bio-Rad Laboratories, USA). The reaction was carried out in a volume of 25 μl according to the set protocol, a mixture of forward and reverse primers at a concentration of 1.5 μM each was added in a volume of 5 μl. Each reaction was set in three parallel samples.

PCR reaction protocol: preliminary heating at 95 ° C for 5 min, 40 main cycles: denaturation at 95 ° C for 10 sec, annealing and elongation: 60 ° C for 30 sec. Normalized expression was calculated using the GUSB, HPRT, and GAPDH reference genes; cDNA of intact human fibroblasts served as a control sample. Primers used in the work:

The ratio of gene expression of BCL2 and PCNA was 2, therefore, we observed a pronounced cytotoxic effect of the test sample of aptamer.

Example 2

The claimed method was determined by the cytotoxicity of the aptamer RA-36. The concentration was 100 mg / ml. The ratio of gene expression of BCL2 and PCNA was 0.8. Therefore, at this dose, the aptamer does not have cytotoxic properties.

Thus, it can be seen from the above examples that the proposed method really significantly increases the information content in comparison with the known methods, in addition, it is easier to implement the known method, selected as a prototype, and has a higher sensitivity. List of references

1. RF patent No. 2653476

2. Igor Pujalty, Isabelle Passagne, Brigitte Brouillaud, Mona Treguer, Etienne Durand, Celine Ohayon-Courtes, Beatrice L'Azou 1, Cytotoxicity and oxidative stress induced by different metallic nanoparticles on human kidney cells ”// Particle and Fiber Toxicology 2011, 8:10, http: //www.particleandfibretoxicology.eom/content/8/l/10

3. Berridge M. V., Herst P. M., Tan A. S. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction // Biotechnology annual review.-2005.-T. 11.-C. 127-152.

4. “Methodology for determining the cytotoxicity of substances with the MTT test on the culture of normal human cells HEK293”, STP-14.621.21.0008.12-2015, Institute of Physiologically Active Substances of the Russian Academy of Sciences.

Claims (1)

A method for assessing the aptamer cytotoxicity, including incubating the aptamer with a cell culture, characterized in that the expression of BCL2 and PCNA genes is measured, and cytotoxicity is judged by the ratio of gene expression levels, the greater the ratio of BCL2 and PCNA gene expression will exceed 1, the more pronounced cytotoxic test substance effect.