2 курс / Биохимия / Синтез_и_изучение_свойств_новых_материалов_с_противоопухолевой

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Fig. 3.51. Concentration dependence of the degree of photohaemolysis in the presence of

GO-1.57. C is the molar concentration of GO-1.57 in terms of the content of compound 1.57, TGO-1.5750 is the time of photoinduced haemolysis of 50 % in the presence of GO- 1.57, Tcontrol50 is the time of photoinduced haemolysis of 50 % of erythrocytes in the presence of saline.

3.8.1.3. Plasma coagulation haemostasis

As can be seen from the presented data (Table 3.18), GO-1.57 significantly increases APTT in the studied concentration range compared to the control. The individual compound 1.57 in the concentration range of 5–200 μM also exhibited anticoagulant properties and significantly increased the APTT time compared to the control [116]. In turn, it was shown that the GO-1.57 conjugate slightly reduced PT. Thus, the anticoagulant properties of the GO-1.57 conjugate are comparable to the individual compound 1.57.

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Table 3.18. Influence of GO-1.57 on parameters of plasma-coagulation haemostasis [116].

*p < 0.05 compared to control.

3.8.1.4. Platelet aggregation

As can be seen from the data presented in Table 3.19, in tests of ADP-induced platelet aggregation in the presence of GO-1.57 conjugate in the concentration range of 5–

100 mg∙l−1, a statistically significant dose-dependent decrease in platelet aggregation is observed compared to the control.

Table 3.19. Effect of the GO-1.57 conjugate on ADP-induced platelet aggregation.

*p < 0.05 compared to control.

3.8.2. Antioxidant activity of GO-1.57 conjugate

3.8.2.1. Antiradical activity of GO-1.57 conjugate

Fig. 3.52 shows a plot of the fraction of reduced DPPH radicals (% inhibition) vs the concentration of the GO-1.57 conjugate. It can be concluded that the antiradical activity of GO-1.57 increases with increasing concentration (C = 0.25–25.0 mg l−1). Since the

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Fig. 3.53. Kinetic dependence of photodegradation of Radachlorin (■) in the presence of

GO-1.57 (▲ 10 mg∙l−1, ▼ 25 mg∙l−1, 50 mg∙l−1, ● 75 mg∙l−1) compared to sodium azide (◄ 500 µM). A0 and At are the optical densities of Radachlorin solutions at a wavelength of 663.9 nm before and after irradiation.

Table 3.20 shows kdeg values for the photodegradation of Radachlorin in the presence of GO-1.57 and sodium azide. The presented kdeg values indicate that in the presence of GO-1.57, a decrease in Radachlorin kdeg is observed, which indicates the antioxidant properties of the GO-1.57 conjugate.

Table 3.20. Photodegradation constants (kdeg) of Radachlorin in the presence of GO-1.57 and sodium azide. C is a concentration in terms of the content of the compound 1.57.

—— 0.0123 ± 0.0017 10 0.0124 ± 0.0015

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(g)

Fig. 3.55. DNA comets after cell electrophoresis in microgel: (a) — positive control (H2O2), (b) — negative control (PBS), (c–g) — GO-1.57 (in molar concentrations in terms of compound 1.57, C = 10, 25, 50, 75 and 100 μM).

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3.8.4. Study of thermodynamic parameters of GO-1.57 binding to HSA by

calorimetric method

The thermal effects observed during HSA titration with aqueous dispersions of GO- 1.57 (C = 5·10−3 M in terms of compound 1.57 content) (Fig. 3.56) are the heats of mixing of the titrant and titratable substance. A similar result was obtained when HSA was titrated with compound 1.57. It can be concluded that the lack of binding of both compound 1.57 and its GO-1.57 conjugate to HSA indicates that HSA will not perform a transport function in the bloodstream.

Fig. 3.56. Dependence of the thermal effect of the reaction of the interaction of the GO- 1.57 conjugate with HSA on time at 298.15 K.

3.8.5. Cytotoxicity of the GO-1.57 conjugate

Analysis of the obtained data on the cytotoxicity of GO-1.57 shows a dosedependent decrease in the survival of A549 (Fig. 3.57a), PANC-1 (Fig. 3.57b) and HeLa (Fig. 3.57c) cell lines. It follows from the presented data that the maximum cytotoxicity of

GO-1.57 was achieved on the HeLa cell line at IC50 = 2.5 μM. This effect is comparable to doxorubicin (IC50 = 1.5 μM) and more than 4.5 times greater than the cytotoxic effect of the individual compound 1.57 (IC IC50 50 = 11.8 μM) [116,117]. It is important to note

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that GO-1.57 has a significantly lower cytotoxicity against the HEK 293 cell line compared

to doxorubicin, and also compared to compound 1.57 (more than 1.5 times).

(a) (b)

Fig. 3.57. The effect of GO-1.57 on the survival of HeLa cervical tumour cells (a), the effect of GO-1.57 on the survival of A549 human alveolar adenocarcinoma tumour cells (b), the effect of GO-1.57 on the survival of human pancreatic cancer cells PANC-1 (c).

3.8.6. Study of the mechanisms of endocytosis of the GO-1.57 conjugate

The mechanisms of endocytosis have been studied in the presence of the following inhibitors: CK-636 (inhibitor of actin-dependent endocytosis), nystatin (inhibitor of caveolin-dependent endocytosis), dinosor (inhibitor of dynamin-dependent endocytosis), chlorpromazine (inhibitor of clathrin-dependent endocytosis), amiloride (inhibitor of pinocytosis). Endocytosis inhibitors were used at a concentration of C = 10 µM.

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Cytotoxicity data in the presence of inhibitors were compared with controls in the absence of inhibitors.

Analysis of the obtained data shows that in the presence of the endocytosis inhibitor amiloride, the survival of HeLa cells increases with the addition of the GO-1.57 conjugate (Fig. 3.58). Also, the survival of HeLa cells is increased in the presence of chlorpromazine. Therefore, the transport of the GO-1.57 conjugate into cells is possible by two mechanisms: pinocytosis and clathrin-dependent endocytosis.

Fig. 3.58. The effect of endocytosis inhibitors on the survival of the HeLa cell line in the presence of the GO-1.57 conjugate (C = 2.5 μM in terms of an individual cytostatic agent).

Thus, the synthesised conjugate is haemocompatible, has the potential to be used in photodynamic therapy, and has cell-specific cytotoxicity; therefore, GO can be effectively used to develop a system for targeted delivery of compound 1.57 to tumour targets [117].

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