241
control |
50 |
/T |
|
GO-1.57 |
50 |
T |
2.4
2.1
1.8
1.5
1.2
0.9
0.6
0.3
0.0
0 |
10 |
25 |
50 |
75 |
100 |
C / mg∙l−1
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.
242
Table 3.18. Influence of GO-1.57 on parameters of plasma-coagulation haemostasis [116].
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C / mg∙l−1 |
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Test |
Standard |
Control |
10 |
25 |
50 |
75 |
100 |
200 |
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PT/ s |
15–19 |
17.7 |
13.0* |
12.8* |
12.1* |
12.4* |
13.0* |
12.7* |
APTT/ s |
35–45 |
41.5 |
65.0* |
61.9* |
63.3* |
63.9* |
61.1* |
63.8* |
*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.
Parameter |
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Amplitude / % |
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Inductor |
Control |
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C / mg∙l−1 |
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5 |
10 |
25 |
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50 |
75 |
100 |
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78.6 ± |
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66.6 |
± |
58.7 ± |
53.2 |
± |
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ADP |
79.3 ± 5.9 |
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70.1 ± 2.0 |
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42.4 ± 2.1* |
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4.5 |
|
1.9 |
|
2.5* |
2.1* |
*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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243
proportion of reduced radicals is less than 50 %, it is not possible to calculate the IC50. It should also be noted that GO-1.57 exhibits a more pronounced antiradical activity compared to GO, which in turn is associated with the functionalisation of the GO surface. It follows from the data presented in Section 3.5.3.1 that compound 1.57 actively reacts with DPPH (k = (2.43 ± 0.06) 10−3 min−1 at 303.15 K). Thus, we can conclude that compound 1.57 retains its antiradical activity after non-covalent conjugation with GO [117].
50 |
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40 |
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30 |
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A |
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20 |
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10 |
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0 |
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2.5 |
6.25 |
12.5 |
18.75 |
25 |
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C / mg·l-1 |
|
|
Fig. 3.52. Dependence of the fraction of reduced DPPH radicals (% inhibition) on the concentration of GO (light grey) and GO-1.57 (dark grey) in terms of the loading of compound 1.57.
3.8.2.2. Photodynamic properties
Photodynamic properties were evaluated by determining the photodegradation rate constant kdeg (Table 3.20). Radachlorin absorption spectra were recorded in the absence and presence of GO-1.57 (C = 10, 50, 75, and 100 mg l–1) and sodium azide (C = 500 µM).
Fig. 3.53 shows the dependencies in the coordinates ln(A0 / At) – t for the reaction of photodegradation of Radachlorin, Radachlorin in the presence of conjugate GO-1.57 and Radachlorin in the presence of sodium azide.
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244 |
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1.0 |
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0.9 |
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0.8 |
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0.7 |
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) |
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t |
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A |
0.6 |
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/ |
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0 |
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ln((A) |
0.5 |
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0.4 |
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0.3 |
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0.2 |
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0.1 |
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0.0 |
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10 |
20 |
30 |
40 |
50 |
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t / s |
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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.
Agent |
C / mg·l−1 |
kdeg / s−1 |
—— 0.0123 ± 0.0017 10 0.0124 ± 0.0015
GO-1.57 |
25 |
0.0084 ± 0.0007 |
|
50 |
0.0075 ± 0.0022 |
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75 |
0.0071 ± 0.0012 |
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NaN3 |
100 |
0.0042 ± 0.0020 |
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3.8.2.3. Binding of NO-radicals
From the data presented in Fig. 3.54, the GO-1.57 conjugate absorbs NO-radicals, although to a lesser extent than sodium azide and the individual compound 1.57 (Section 3.5.3.4). Therefore, non-covalent conjugation of GO with compound 1.57 reduces its antiradical activity in the reaction of interaction with the NO radical [117].
0.6 |
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0.4 |
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A |
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0.2 |
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0.0 |
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0 |
10 |
25 |
50 |
75 |
100 |
200 |
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C / mg·l−1 |
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Fig. 3.54. Antiradical activity of the conjugate GO-1.57 (light grey) and sodium azide (dark grey) in the NO-radical scavenging reaction.
3.8.3. Genotoxicity
The mean % comet tail DNA content, tail length, and comet tail momentum observed from human PBMCs incubated in the presence of H2O2 (positive control), PBS (negative control), and GO-1.57 are presented in Table 3.21. The amount of DNA damage in the presence of H2O2 is significantly higher than in control cells. As an example, in Fig. 3.55 shows micrographs of DNA comets in the presence of H2O2 (C = 100 μM), PBS, and
GO-1.57 conjugate in the concentration range 1–200 μM. Incubation of human PBMCs with GO-1.57 has been shown to cause dose-dependent damage to DNA integrity (Fig.
3.55).
|
246 |
|
(a) |
(b) |
(c) |
(d) |
(e) |
(f) |
(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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Table 3.21. Effect of GO-1.57 on DNA tail (%), tail length and tail moment.
Amplitude / %
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Negative |
H2O2 |
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Characteristic |
concentration / |
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C / µM |
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control |
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µM |
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0.0 |
100.0 |
10.0 |
25.0 |
50.0 |
75.0 |
100.0 |
|
DNA tail, % |
6.95 ± 0.88 |
86.25 ± 16.88 |
77.61 ± 2.11 |
74.62 ± 2.04 |
74.14 ± 2.04 |
82.50 ± 1.71 |
83.15 ± 2.07 |
|
Tail length / µm |
42.00 ± |
98.69 ± 7.18 |
139.26 ± |
137.90 ± |
151.42 ± |
104.80 ± 6.40 |
146.37 ± 8.62 |
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2.49 |
9.12 |
4.90 |
11.23 |
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Tail moment |
4.12 ± 0.02 |
85.22 ± 1.21 |
108.10 ± |
102.85 ± |
112.27 ± 0.30 |
86.46 ± 0.11 |
111.85 ± 0.17 |
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0.19 |
0.10 |
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247
248
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.
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-158 |
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-160 |
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−1 |
-162 |
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H / mJЧs |
-164 |
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-166 |
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-168 |
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2000 |
4000 |
6000 |
8000 |
10000 |
12000 |
14000 |
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t / s |
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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)
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100 |
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80 |
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/ % |
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Survival |
60 |
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40 |
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20 |
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0 |
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0 |
1.56 |
3.13 |
6.25 |
12.5 |
25 |
50 |
100 |
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C / μM |
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120 |
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100 |
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% |
80 |
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/ |
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Survival |
60 |
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40 |
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20 |
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0 |
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0 |
1.56 |
3.13 |
6.25 |
12.5 |
25 |
50 |
100 |
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C / μM |
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(c) |
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120 |
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100 |
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80 |
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/ % |
60 |
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Survival |
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40 |
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20 |
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0 |
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0 |
1.56 |
3.13 |
6.25 |
12.5 |
25 |
50 |
100 |
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C / μM |
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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.
250
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.
|
100 |
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80 |
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/ % |
60 |
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Survival |
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40 |
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20 |
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0 |
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Control |
GO-1.57 |
Amiloride |
CK-636 |
Dinosor |
Chlorpromazine |
Nystatin |
|
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|
GO-1.57 |
GO-1.57 |
GO-1.57 |
GO-1.57 |
GO-1.57 |
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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