br After days of Cy AT and AT
After 3 days of Cy5-AT11 and -AT11-B0 incubation, AM-251 were able to internalize them in their free (red coloration, Fig. S2) and C8-con-jugated forms.
Interestingly, it was possible to observe that the complex seems to be more internalized by HeLa cells when compared to the free AT11 G4 or AT11-B0 G4. As indicated by the spectroscopic studies, C8 increases the stability of the AT11 or AT11-B0 G4 structures and this eﬀect may lead to a higher uptake as the G4 conformation is involved in their biological activity (Bates et al., 2009). Additionally, the yellow/orange staining in HeLa cytoplasm (Fig. 10) suggests that the complexes
Fig. 12. Flow cytometry analysis of HeLa cells incubated with (A) free C8, (B) AT11 G4-C8 complex and (C) AT11-B0 G4-C8 complex. Cells were incubated for 24 h with the preformed aptamer-ligand complex at a C8 concentration of 1 μM.
Fig. 13. Flow cytometry analysis of NHDF cells incubated with (A) free C8, (B) AT11 G4-C8 complex and (C) AT11-B0 G4-C8 complex. Cells were incubated for 24 h with the preformed aptamer-ligand complex at a C8 concentration of 1 μM.
J. Figueiredo, et al.
remain stable during cell traﬃcking and the green coloration in the nucleoli demonstrates that C8 can be released and reach the same compartment that it attained when administrated alone (Carvalho et al., 2018). Moreover, the complexes promoted changes in the ma-lignant cells morphology, presenting vacuolization and irregular nuclei, which are an evidence of methuosis phenotype (Fig. S3) (Reyes-Reyes et al., 2010).
The internalization of the free AT11 G4 and AT11-B0 G4 (Fig. S4) or AT11 G4-C8 and AT11-B0 G4-C8 (Fig. 11) also occurs in NHDF cells, despite the absence of overexpressed nucleolin at their surface. This eﬀect was already observed in normal cells after AS1411 incubation (Reyes-Reyes et al., 2010). The AT11 G4 and AT11-B0 G4 are taken up by endocytosis being latter expelled by eﬄux, or trapped in lysosomes and degraded (Reyes-Reyes et al., 2010). As the cells were constantly kept in medium with the free AT11 G4 and AT11-B0 G4 or the com-plexes throughout the incubation period, the eﬄux was most likely not observed in the images obtained by confocal microscopy. The Pearson coeﬃcients for NHDF cell line were 0.87 and 0.83 for AT11 G4-C8 and AT11-B0 G4-C8, respectively, suggesting internalization even without overexpression of nucleolin at their surface (Fig. 11).
To support the confocal microscopy data for the internalization of the complexes, flow cytometry experiments were conducted. The re-sults for HeLa and NHDF cells are shown in Figs. 12 and 13, respec-tively. When compared to free C8 (Figs. 12A and 13A), AT11-C8 com-plex seems to be internalized to a similar extent in HeLa cells (Fig. 12B), whereas in NHDF cells the complex seems to be less internalized (Fig. 13B) which may be attributed to the diﬀerential expression of surface nucleolin between cancerous and non-malignant cells, which may lead to increased accumulation of the complex in cancer cells. Regarding AT11-B0-C8 complex, the results suggest less internalization in both cell lines (Figs. 12C and 13C) when compared to free C8. Al-together, these data indicate that the uptake of conjugated-C8 is mediated by the aptamer, most probably involving the interaction with nucleolin at the cell surface.
Following the determination of the subcellular localization of the complexes, MTT studies were performed to determine whether the AT11 G4-ligands (Fig. 14) and AT11-B0 G4-ligands (Fig. 15) could mitigate the cytotoxicity of the free acridine orange derivatives against non-malignant cells. The free AT11 G4 and AT11-B0 G4 were also evaluated in both HeLa and NHDF cell lines (Fig. S5). The free se-quences presented similar toxicity and showed a more pronounced ef-fect on the viability of cancer cells (ca. 60% vs 80% for HeLa and NHDF, respectively). This result agrees with previous studies that report AT11 G4 as being selective for cancer cells (Do et al., 2017).
In the NHDF cell line and at the same dosage, free C3 and AT11 G4-C3 complex presented similar degrees of cytotoxicity, however in the case of HeLa cells the cytotoxicity of the latter was significantly higher. AT11-B0 G4-C3 also presented a higher cytotoxicity against cancer cells but a reduced cytotoxicity against non-malignant cells. AT11 G4-C5 showed similar cytotoxicity in HeLa cells when com-pared with free C5. However, complex formation greatly reduced the toxicity of the C5 ligand towards NHDF cells. Similar eﬀect occurred in the case of AT11-B0 G4-C5, although its cytotoxicity towards the cancer cell line was also reduced (47% of cell viability).
The results obtained for C8 were not so encouraging as those above discussed for the congener C3 and C5. In fact, the C8-complexes induce lower cytotoxicity towards malignant cells than free C8. In the case of AT11-B0 G4 this may be due to decreased internalization of the com-plex when compared to free C8 as suggested by the flow cytometry results. Indeed, when compared to AT11-C8, the AT11-B0-C8 complex showed a highest cytotoxicity reduction in HeLa cells relatively to free C8. By contrast, AT11 G4-C8 and AT11-B0 G4-C8 showed a rather si-milar cytotoxicity against non-malignant NHDF cells, which is quite comparable to the cytotoxicity exhibited by free C8 in the same cell line.