br The obtained fluorescence data was converted into fraction
The obtained fluorescence data was converted into fraction of li-gand bound (α) plots according to Eq. (2):
I − Iλfree
where I is the fluorescence intensity at 568 nm at each ligand:DNA
J. Figueiredo, et al.
ratio, and Ifree and Ibound are the fluorescence intensity of the free and fully bound ligand, respectively. Data points were then fitted to Hill saturation binding model (OriginPro 8):
where KD is the apparent equilibrium dissociation constant, [DNA] is the concentration of the DNA and n is the Hill constant which describes cooperativity of ligand binding. For the protein binding experiments, data points were fitted to Michaelis-Menten model (OriginPro 8):
2.5. Serum stability assay
2.6. Cell viability assay
Normal human dermal fibroblasts (NHDF) were grown in RPMI medium, supplemented with 10% FBS, 1% streptomycin-penicillin an-tibiotic, 0.01 M HEPES, 0.02 M L-glutamine and 0.001 M sodium pyr-uvate. Additionally, cervical cancer cell line (HeLa) was grown in DMEM medium, supplemented with 10% FBS and 1% streptomycin-penicillin antibiotic. Cell cultures were maintained in a controlled hu-midified SB202190 at 37 °C and 5% CO2. Cells were plated in 48-well culture plates (HeLa or NHDF were seeded at a density of 0.5 × 104 cells/mL or 1 × 104cells/mL, respectively) in 300 µL of medium. In the next day, both cell lines were treated with the pre-formed AT11 or AT11-B0-ligand complexes (2 µM of C3/C5 or 1 µM of C8 with 15 µM of AT11 or AT11-B0 for 10 min) and incubated for 7 days. At the end of incubation, 50 μL of thiazolyl blue tetrazolium bromide (MTT) solution was added to each well and further incubated for 1 h in HeLa or 4 h in NHDF cells. The resulting formazan crystals were solubilized with 250 μL of DMSO and the optical density (OD) was measured at 570 nm. Cell viability was normalized to control condition (cells without li-gands). Statistical analysis was performed by using Student’s unpaired t test. The values of p < 0.05 were considered statistically significant. Data analysis was performed with GraphPad Prism 6 software (San Diego, CA, USA).
2.7. Confocal fluorescence microscopy
To better understand the internalization ability of free AT11 or AT11-B0 and complexes, HeLa and NHDF cells were seeded at a density of 0.3 × 104 and 5 × 104 cells/mL, respectively, in treated 8 well µ-slides (IBIDI, Germany). Cells were grown at 37 °C in 5% CO2 humi-dified atmosphere and treated the day after with the pre-formed com-plex (0.5 µM of acridine orange ligands (C3, C5 or C8) and 1 µM of AT11 and AT11-B0), ligand-only control (0.5 µM) and DNA-only (AT11 and AT11-B0) controls (1 µM). After 3 days of incubation, cells were washed with phosphate-buﬀered saline and nuclei were then stained with 1 µM Hoechst 33,342 nuclear probe for 15 min. Cells were then observed using a Zeiss AxioObserver LSM 710 microscope. International Journal of Pharmaceutics 568 (2019) 118511
For nucleolin immunocytochemistry, HeLa cells were seeded at a density of 1.5 × 104 cells/mL in 8 well µ-slides (IBIDI, Germany) and incubated with free AT11 and AT11-B0, or the ligand complexes for 2 h. Nucleolin antibody (1:100, ref. PA3-16875, Invitrogen) was then added during 2 h and secondary antibody (Alexa Fluor 546®, 1:1000) for 1 h, both at room temperature. The excess fluorophores were washed oﬀ by rinsing with PBS three times and the cells were imaged using a Zeiss AxioObserver LSM 710 microscope.
The degree of complexes colocalization is expressed in Pearson correlation coeﬃcients (rp) values (Manders et al., 1993), which were calculated using the colocalization analysis tool in the ImageJ software.