3 курс / Фармакология / Синтез_и_изучение_свойств_новых_материалов_с_противоопухолевой
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induced platelet aggregation, an ADP reagent was used to study platelet function (NPO RENAM). Primary (reversible) platelet aggregation was assessed by the response to the addition of a threshold dose of ADP to plasma (C = 10 μM).
When evaluating the aggregation curve, the most informative is the maximum aggregation amplitude (MA) — the maximum increase in the transmittance between the moment the aggregating agent is introduced and the moment the change in the transmittance stops — in % of the light transmission of platelet-poor plasma.
To study the effect of compounds 1.57, 3.6, and GO-1.57 (at loading concentrations of 1.57) on induced platelet aggregation, 270 µl of PRP and 30 µl of test compound solution were mixed in cuvettes at final concentrations of 5, 10, 25, 50, 75, 100 and 200 μM, respectively. The inductor was added to the cuvettes 5 min after the mixture was incubated at 37°C. Aggregation was recorded until the curve reached a plateau.
Plasma coagulation haemostasis
Clotting tests include methods for measuring activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT). These methods make it possible to measure the time interval from the moment of adding a reagent (an activator that starts the coagulation process) to the formation of a fibrin clot in the plasma under study. The effect on plasma-coagulation haemostasis was assessed when it was added to plasma in the APTT, PT, and TT tests.
The principle of the APTT method: the study of the reaction of plasma recalcification under the conditions of standardisation of contact and phospholipid activation of blood coagulation. For this purpose, a contact activator (kaolin) and partial thromboplastin, which is functionally similar to platelet phospholipids, are added to the plasma. The sensitivity of this test to deficiency of plasma coagulation factors (excluding factors VII and XIII) is higher than the plasma recalcification time test, but standard phospholipid activation makes it impossible to detect platelet coagulation deficiency.
The principle of the PT method: determine the clotting time of platelet-poor citrate plasma in the presence of an optimal amount of calcium and an excess of tissue thromboplastin. This is a variant of determining the time of plasma recalcification with the addition of tissue thromboplastin. In combination with factor VII and Ca2+, it directly activates factor X, so that the test results depend on the activity of factor VII, factor X, and
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factors involved in the blood coagulation process at the stages of thrombin and fibrin formation (factors V, II, and I).
The principle of the TT method: the method is based on the ability of thrombin to induce the conversion of fibrinogen into fibrin without the participation of other blood coagulation factors, i.e., it allows you to evaluate the final stage of blood coagulation (fibrinogen and its derivatives, factor XIII activity).
Commercial normal plasma (Technologiya-Standard, Russia) was used for the study. To determine APTT, PT, and TT, we used APTV-TEST, TEHPLASTIN-TEST, and THROMBO-TEST reagent kits from Technologiya-Standard, Russia. The studies were carried out on an APG2-02-P (ECMO) coagulometer. For the study, 50 µl of plasma and 50 µl of a solution (dispersion) of the studied compound were mixed at final concentrations of 5, 10, 25, 50, 75, 100, 200 µM (compounds 1.57, 3.6, and GO-1.57 (with concentrations in terms of loading 1.57)). Next, the solutions (dispersions) were incubated at 37 °C for 60 s, and in accordance with the study protocol, the clotting time was determined on a coagulometer in the APTT, PT, and TT tests.
Study of thermodynamic parameters of DNA and HSA binding by calorimetric
method
The experiments were carried out using a TA Instruments Nano ITC 2G titration microcalorimeter (TA Instruments, USA) equipped with a gold measuring cell with a volume of 1 ml according to the method described in the work [110]. For measurements, an HSA solution (C = 1∙10–4 M) or a DNA solution (C = 5∙10–3 M) was placed in the cell. After equilibrium was established, solutions of compounds 1.57, 3.6, and dispersions of
GO-1.57 (with concentrations in terms of loading 1.57) (C = 1∙10−3 M) were added to the contents of the cell with continuous stirring by successive injections of 10 µl. Phosphate buffered saline (PBS) was used as a medium for the reaction under study. The interval between injections was 40 min, the stirrer rotation speed was 250 rpm. The temperature maintenance accuracy during the experiment was ±0.0003 K.
Study of DNA binding by spectral methods
UV spectra were recorded in the range 200–400 nm on a Beckman Coulter DU 800 spectrometer using quartz cuvettes (l = 1 cm). Circular dichroism (CD) spectra were recorded on a Jasco J-810 spectropolarimeter (l = 0.2 cm). Compliance with the absorption law was checked for compounds 1.57, 3.6, GO-1.57 and DNA working solutions in the
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concentration range (C = 3.6–24.7 μM (for DNA solutions), C = 1–100 μM (for solutions of compounds 1.57, 3.6, and GO-1.57 dispersion (with concentrations in terms of loading
1.57)).Spectral experiments were carried out in physiological saline at pH 7.4.Working solutions were obtained by mixing DNA solutions and solutions of compounds 1.57, 3.6,
GO-1.57 per load of 1.57) at room temperature.
Antioxidant activity
Antiradical activity study
The study of antiradical activity with the stable radical 2,2-diphenyl-1- picrylhydrazyl (DPPH) was carried out on a Thermo Scientific Evolution 300 spectrophotometer. For this, a solution of DPPH in ethanol with a concentration (130 µM), a solution of compounds 1.57, 3.6, and a dispersion of GO-1.57 (with concentrations calculated for loading 1.57) were prepared in PBS buffer with a concentration of 200 µM.
After thermostatting, 1 ml of a DPPH solution and 1 ml of a solution of compounds 1.57,
3.6 and a dispersion of GO-1.57 were placed into a quartz cuvette. A mixture of ethanol and water (1:1) was added to the reference cuvette. To obtain the DPPH reduction kinetic curve, the optical density was recorded on a spectrophotometer at 515 nm, at temperatures of 303.15 K, 308.00 K, 313.00 K, 318.00 K in the dark every minute for 30 min, and also after 6 days after the start of the reaction. The temperature control accuracy was T = 0.1 K.
Photodynamic properties
To study the photodynamic properties, the absorption spectra of the following samples were recorded:
1.Radachlorin.
2.Solution containing Radachlorin and compounds 1.57, 3.6, and GO-1.57 at various concentrations (C = 5–200 μM).
3.Radachlorin solution containing 500 μM sodium azide before and after irradiation with a Laserland LED-2000 red laser (Besram Technology Inc., laser power 55 mW, 659 nm). The effect of compounds 1.57, 3.6 and GO-1.57 on the photobleaching of Radachlorin was evaluated by calculating the photodegradation rate constant kdeg.
Photoinduced haemolysis
Erythrocytes were obtained from citrated blood by centrifugation at 1500 rpm for 10 min, followed by three washings with saline. Next, the cells were stabilised for at least
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24 h at 4 °C in Alsever’s reagent, which is used as an anticoagulant and consists of sodium chloride (0.42 %), citric acid (0.055 %), sodium citrate (0.8 %), and D-glucose (2.05 %).
Before use, erythrocytes were washed three times from Alsever’s reagent with saline, and a standard cell suspension was prepared in PBS (pH 7.4). The optical density of the standard suspension after its 8-fold dilution with a buffer solution was 0.560 ± 0.020 at 800 nm. The measurements were carried out on an SF-2000 spectrophotometer in a cuvette with an optical path length of 5 mm. Registration of the cytolytic activity of compounds 1.57, 3.6 and dispersion of GO-1.57 (with concentrations in terms of loading 1.57) was carried out by recording a decrease in the optical density of the cell suspension at 800 nm at five-second intervals until complete haemolysis [111]. The measurements were carried out in a thermostated cuvette of a spectrophotometer at 37 °C; the concentration of the test sample varied in the range from 5–200 µM.
Antioxidant properties were evaluated using a device for the study of photoinduced cytolysis according to the method published previously [111]. According to this method, an incubation mixture containing 0.1 ml of a standard suspension of erythrocytes, 0.6 ml of PBS (pH 7.4), 0.08 ml of a solution with various contents of compounds 1.57, 3.6, and
GO-1.57 and 0.02 ml of the photosensitiser Radachlorin (0.35 % solution for intravenous administration, the main substance is (7S,8S)-13-vinyl-5-(carboxymethyl)-7-(2- carboxyethyl)-2.8,12,17-tetramethyl-18-ethyl-7H,8H-porphyrin-3-carboxylic acid). An incubation mixture containing physiological saline was used as a control. The resulting incubation mixture, with a total volume of 0.8 ml, was thermostated in the cuvette compartment of the spectrophotometer for three minutes at 37°C with constant stirring, then irradiated with a Laserland LED-2000 red laser (irradiation dose, 3.5 J/cm2). After completion of irradiation, a decrease in the optical density of the solution was recorded at 800 nm.
Study of the binding of NO-radicals
The degree of binding of NO radicals was determined using the Griess–Ilosvay model reaction [55,112]. Sodium nitroprusside at physiological pH is a donor of NOradicals, the interaction of which with oxygen leads to the formation of nitrite anions (NO2−) as a result of a series of transformations. The nitrite anions formed as a result of the reaction are determined using the Griess reagent (a pink-violet colour of the solution is observed). For the experiment, the reaction mixture containing 1 ml of sodium
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nitroprusside (C = 15 μM) and 0.5 ml of an aqueous solution of compounds 1.57, 3.6 and dispersion of GO-1.57 (with concentrations in terms of loading 1.57) (C = 10–200 μM) incubated for 150 min in a thermostatic shaker at 60 °C. Then, 0.5 ml of PBS (pH 7.4) and 0.5 ml of a 1 % solution of the Griess reagent were added to 0.25 ml of the resulting solution. The resulting mixture was incubated for 30 min at room temperature. The resulting diazo compound was determined spectrophotometrically at λ = 540 nm. Similar concentrations of sodium azide were used as controls.
Genotoxicity
The genotoxicity of compounds 1.57, 3.6, and GO-1.57 was evaluated using the DNA comet method based on measuring the effect of test samples on the DNA integrity of human peripheral blood mononuclear cells using alkaline gel electrophoresis[55]. DNA comets were visualised using a Micromed 3 LUM fluorescent microscope. Tail lengths were measured using the CASP software (version 1.2.2). Tail DNA content and tail length were determined experimentally; tail moment was calculated as the percentage of DNA in the tail multiplied by the distance between the centre of the head and the tail [132].
Cytotoxicity
The MTT test (colorimetric test with 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) for compounds 1.57, 3.6 and GO-1.57 was performed on human liver adenocarcinoma cell lines (SK-HEP-1), human glioblastoma (T98G), human alveolar basal epithelium adenocarcinoma (A549), human ovarian teratocarcinoma (PA- 1), human embryonic kidney cell line (HEK293) was used as a control line, doxorubicin was used as a control substance (2, 10, 20, 50, 100, 150, and 200 μM). Cells at a concentration of 5∙103 per well were placed in a 96-well plate and incubated for 12 h in DMEM-F12 medium supplemented with 10 % thermally inactivated fetal bovine serum (FBS), 1% L-glutamine, 50 U∙ml−1 penicillin and 50 µg ml−1 streptomycin. After culturing, fresh DMEM-F12 medium containing various concentrations of test compounds was added to the wells, and the plate was then incubated at 37 °C in a humidified atmosphere of a CO2 incubator in the presence of 20 % O2, 5 % CO2. After 48 h, 0.1 ml of DMEM-F12 and 0.02 ml of the MTT reagent (5 mg ml–1) were added to the wells and the incubation continued for 1 h, after which the supernatant was removed. The formazan crystals formed during MTT reduction by viable cells were dissolved in 0.1 ml of DMSO and the optical density was measured on a BioRad x Marx plate spectrophotometer at λ = 540 nm, subtracting the
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background optical density at λ = 690 nm. For each cell line, the half-maximal inhibition concentration (IC50) was determined. The values obtained were compared with the IC50 of doxorubicin and cisplatin.
Membrane mitochondrial potential
PANC-1 cells were treated with trypsin and washed with PBS containing 10 % FBS. The cells were then resuspended in a mixture of PBS, 5 % FBS, and 50 mM KCl. After cells were incubated with MitoTracker® Orange CMTMRos fluorescent dye (500 nM) at 37 °C for 30 min, they were washed with PBS and plated in a black 96-well plate (80,000 cells per well). The concentration of test compounds (compounds 1.57, 3.6 and GO-1.57) and doxorubicin in the final suspensions was 100 μM. 10 μM FCCP (carbonyl cyanide p- trifluoromethoxyphenylhydrazone) was added to diffuse the proton gradient. Fluorescence measurements were performed using a Varioscan Lux plate reader at excitation/emission wavelengths of 554/576 nm.
Interaction with HSA
The binding of compounds 1.57, 3.6, and GO-1.57 with HSA was performed on a SM 2203 spectrofluorimeter. Emission spectra were recorded in the wavelength range of 310–450 nm and the temperature range of 298.15–31.15 K; excitation wavelength 290 nm.
The HSA concentration was 3 µM, the concentrations of compounds 1.57, 3.6, and GO- 1.57 varied in the range C = 0.3–1.5 µM with a step of 0.3 µM and in the range C = 6.0–
24.0 µM with a step of 3.0 μM. The measurements were carried out in the absence and presence of binding site markers (warfarin, ibuprofen, digitonin with a final concentration of C = 3 μM).
Esterase activity of HSA
To evaluate the effect of compounds 1.57, 3.6, and GO-1.57 on the esterase activity of HSA, the following solutions were prepared: p-nitrophenylacetate in ethanol, HSA, compounds 1.57, 3.6, and GO-1.57 in PBS with pH 7.02. After mixing the solutions, the final concentration of p-nitrophenylacetate was 100 µM, HSA was 3 µM, the concentrations were 1.57, 3.6, and GO-1.57 varied from 0 to 24 µM. The hydrolysis rate of p-nitrophenylacetate was estimated from the formation of the reaction product, p- nitrophenol. The change in optical density was recorded by the spectrophotometric method at a wavelength of 405 nm during the first 10 min from the beginning of the reaction. As a
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result, the kinetic dependences of the hydrolysis reaction of p-nitrophenylacetate with HSA were obtained in the absence and presence of 1.57, 3.6, and GO-1.57.
Endocytosis
The mechanisms of endocytosis were studied using the MTT test to reduce the toxic effects of 1.57, 3.6 and GO-1.57 on HeLa cells in the presence of the following endocytosis inhibitors: CK-636 (an inhibitor of actin-dependent endocytosis); nystatin (an inhibitor of caveolin-dependent endocytosis); dinasor (an inhibitor of dynamin-dependent endocytosis); chlorpromazine (an inhibitor of clathrin-dependent endocytosis); amiloride (pinocytosis inhibitor). Endocytosis inhibitors were used at a concentration of C = 10 µM.
Cytotoxicity data in the presence of inhibitors were compared with controls in the absence of inhibitors.
Immunoblotting
A549 and HeLa cells were seeded in DMEM-F12 culture medium on Petri dishes (d = 10 cm) at a density of 4 106 cells. After 24 h, the culture medium was removed and DMEM-F12 containing 50 μM CoCl2, test samples, and the selective HIF-1α translation inhibitor KC7F2 were added (preincubation was carried out for 8 h). The cells were then homogenised in radioimmunoprecipitation buffer (150 mM NaCl, 0.1 % Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris-HCl solution (pH
8.0) and protease inhibitor cocktail (1:100, Sigma) at +4 °C for 30 min and centrifuged at
12,000g (+4 °C, 20 min). The content of total protein in cell lysates was determined using
Bradford's reagent. HSA was used as a standard The test samples were diluted with
Laemmli’s buffer and electrophoresed in a 12 % polyacrylamide gel in the presence of sodium dodecyl sulphate. Protein transfer from the polyacrylamide gel was carried out using a nitrocellulose membrane with a pore diameter of 0.2 μm (Bio-Rad). Protein transfer was carried out in an electroblotting chamber (Bio-Rad) at a current of 0.3 A for 2 h. antibodies to HIF-1 (Abonova; dilution 1:1000), for 2 hours. After three washes with PBS- T, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase. The chemiluminescent substrate for detecting horseradish peroxidase activity was used according to the manufacturer's instructions (Bio-Rad). The intensity of the chemiluminescence signal was recorded on a Chemidoc instrument (Bio-Rad). To check the equivalence of total protein content, immunoblotting to β-actin was performed using
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monoclonal mouse antibodies against human β-actin (Santa Cruz Biotechnology; dilution 1:6000).
Statistical analysis
Statistical analysis was carried out using the Statistica 8.0 program (StatSoft). The normality of the distribution was tested by the Shapiro-Wilk test. Data are expressed as means ± standard error of the mean. Statistical differences were tested using Student's t- test for independent samples or using analysis of variance followed by Tukey's post-hoc test. Differences were considered statistically significant at p < 0.05.
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MAIN RESULTS AND CONCLUSIONS
1.Synthetic approaches to the production of polynitrogen heterocycles based on 1,3,5- triazines, which are potential cytostatic drugs, have been developed. The obtained compounds were characterised using a set of physicochemical methods of NMR, IR spectroscopy, mass spectrometry, X-ray diffraction analysis, thermogravimetric analysis, X-ray phase analysis and elemental analysis.
2.Compounds 1.57, 3.6, and GO-1.57 were shown to be haemocompatible and exhibit cytotoxic effects on A549, HeLa, PANC-1, SK-HEP-1, and T98G cell lines, comparable or exceeding the effects of such cytotoxic drugs as cisplatin and doxorubicin. A complex of studies on the interaction of compounds 1.57 and 3.6 with DNA allows us to conclude that they have a mixed mechanism of action. Leading compounds have high antioxidant activity and, as a result, can affect the antioxidant-prooxidant balance in tumour cells.
3A comprehensive study of the physicochemical properties of compound 1.57, namely, the temperature and concentration dependences of density, viscosity, sound velocity, and refractive index, has been carried out. The amphiphilicity and high solubility of compound
1.57 in water (up to 43 g/l) were shown. It was found that the synthesised compounds 1.57 and 3.6 are unstable in a weakly acidic medium and hydrolyse with opening of the dioxane ring. The totality of the data obtained confirms the haemocompatibility and indicates the membranotropism of the leader compound.
4.A method has been developed for the synthesis of a non-covalent conjugate based on graphene oxide with a high content of oxygen-containing functional groups (up to 85 %) and compound 1.57 (drug loading is 61 %). The resulting conjugate was characterised using a complex of physicochemical methods.
5.It was found that the GO-1.57 conjugate exhibits antioxidant activity, is haemocompatible, and also has cell-specific cytotoxicity: the maximum cytotoxicity is manifested in relation to the HeLa cell line (IC50 = 2.5 μM), which is comparable to the
action of doxorubicin (IC50 = 1.5 μM) and more than 4.5 times greater than the cytotoxic effect of the individual compound 1.57 (IC50 = 11.8 μM), while GO-1.57 has a significantly lower cytotoxicity against the non-tumour cell line HEK 293 compared to doxorubicin and
compound 1.57.
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