3 курс / Фармакология / Синтез_и_изучение_свойств_новых_материалов_с_противоопухолевой

Fig. 3.40. Circular dichroism spectra of DNA (C = 6.1 μM) as a function of compound

3.6concentration (C = 3.89–12.3 µM).

3.7.2.3.Study of the binding of compound 3.6 with HSA by spectrophotometric

method

To identify the binding sites of compound 3.6 with HSA, experiments were carried out on the competitive interaction of compound 3.6 with HSA binding sites in the presence of the corresponding binding site markers. To determine the binding constants (Kbin), as well as the stoichiometry of the binding reaction (n), the Scatchard equation was used:

where F0 is fluorescence intensity of HSA in the absence of compound 3.6, F — fluorescence intensity of HSA in the presence of compound 3.6, Q is concentration of compound 3.6, M. Fig. 3.41 shows data on the binding of compound 3.6 to HSA in Hill

coordinates ( lg F0 - F vs lgQ).

F

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The data presented in Table 3.14 allow us to draw the following conclusions: (1) a significant decrease in lgKbin and n is observed in the presence of digitonin; (2) in the presence of ibuprofen, lgKbin and n slightly decrease; (3) an increase in lgKbin and n is observed in the presence of warfarin.

The change in the Gibbs energy (ΔG) of the binding reaction of compound 3.6 with HSA in the temperature range of 298.15–313.15 K was calculated using the equation:

G = H -T S (3.24). Table 3.14. Values of the logarithms of the binding constants (lgKbin) and the stoichiometry of the binding reaction (n) of compound 3.6 with HSA.

Negative values of G (Table 3.15) in the temperature range 298.15–315.15 K indicate thermodynamically favourable binding of compound 3.6 with HSA. The enthalpy change (ΔH) has a negative value (Table 3.15), which is typical for the formation of hydrogen bonds.

Table 3.15. Thermodynamic parameters of the binding of compound 3.6 with HAS. H,

S, G are changes in enthalpy, entropy, and Gibbs energy.

Fig. 3.44. Temperature dependence of the logarithm of the rate constant of the reduction reaction of DPPH with compound 3.6.

The value of the activation energy and the pre-exponential factor for the reduction reaction of DPPH with compound 3.6 are 76.4 ± 12.4 kJ/mol and 23.2 ± 4.9 min−1, respectively.

3.7.3.1. Photodynamic properties of the compound 3.6

The degradation of the photosensitiser was assessed by measuring the rate constants of photodegradation (kdeg), the values of which were determined from the kinetic dependences in the coordinates ln(At / A0) – t. A decrease in kdeg indicates that the compound under study has the properties of a singlet oxygen quencher. In turn, an increase in kdeg indicates that the compound under study has the properties of an inducer of the formation of singlet oxygen. Fig. 3.45 shows experimental data on the photobleaching of Radachlorin in the presence of compound 3.6. Analysis of the dependences obtained indicates that compound 3.6 exhibits antioxidant properties that are dose-dependent. However, the antioxidant properties of compound 3.6 are less pronounced than those of sodium azide [111].

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The obtained experimental data indicate that compound 3.6 has practically no effect on photoinduced haemolysis caused by Radachlorin photobleaching. Thus, under the experimental conditions, compound 3.6 does not have antioxidant properties.

3.7.3.3. Antiradical activity of compound 3.6 with respect to NO-radicals

Fig. 3.47 shows the results of measuring the activity of compound 3.6 and sodium azide in the reaction of interaction with NO-radicals in a model system containing sodium nitroprusside. Over the entire range of concentrations, compound 3.6 does not exhibit antiradical activity [111].

Fig. 3.47. Efficiency of NO-radical capture by compound 3.6 (dark grey) compared to sodium azide (grey). C is the molar concentration of compound 3.6.

3.7.4. Compound 3.6 cytotoxicity

Compound 3.6 showed a pronounced dose-dependent reduction in the survival of human liver adenocarcinoma (SK-HEP-1; Fig. 3.48a) and human glioblastoma (T98G; Fig. 3.48b) cells at a concentration of 50 μM. The cytotoxicity of compound 3.6 is comparable to that of cisplatin and compound 1.57 (Table 3.17) [28].

Fig. 3.49. Compound 3.6 reduces the level of expression of the HIF-1α protein in cell lines HeLa (a) and A549 (b).

Thus, compound 3.6 was shown to decrease HIF-1α stabilisation in HeLa and A549 cell lines. Under hypoxic conditions, lower viability values of HeLa and A549 cells (IC50

= 30.4 ± 3.1 μM and IC50 = 1.8 ± 0.3 μM, respectively) were recorded compared to normoxia.

It has been shown that triazinyltetrazole 3.6 is haemocompatible and cytotoxic with respect to A549, HeLa, PANC-1, SK-HEP-1 and T98G cell lines. A complex of studies on the interaction of compounds 3.6 with DNA allows us to conclude that they have a mixed mechanism of action and have antioxidant activity.

3.8.Biocompatibility of non-covalent conjugate based on GO and compound 1.57

3.8.1.Haemocompatibility study

3.8.1.1.Haemolysis of GO-1.57 conjugate

Fig. 3.50 shows that GO-1.57, when incubated for 1 and 3 h, caused haemolysis over the entire concentration range; the rate of haemolysis was dose and time dependent. It should be noted that nanomaterials are classified as non-haemolytic if the degree of haemolysis does not exceed 5 %.[115].