br Several aptamers have already been developed against a
Several aptamers have already been developed against a wide array of therapeutically relevant targets (Alshaer et al., 2018; Vandghanooni et al., 2018; Zhu et al., 2015). Among these, DNA aptamer AS1411 that targets the nucleolin protein has been extensively used as a cancer-targeting agent (Bates et al., 2017; Vandghanooni et al., 2018c). Nu-cleolin is considered a tumor biomarker as it is overexpressed in the surface of cancer cells, while in non-malignant CB-839 it is found primarily in the nuclei and cytoplasm (Mongelard and Bouvet, 2007). In vivo studies showed that AS1411-linked ligands selectively accumulate in cancer cells following systemic administration, emphasizing the po-tential of using this aptamer as a drug delivery system (Bates et al., 2017). Due to its G-rich nature, AS1411 is able to fold into a G4 structure (Bates et al., 2017). The aptamer structure is however highly polymorphic with at least eight diﬀerent folding patterns detected by size exclusion chromatography (Dailey et al., 2010), complicating the identification of the structure(s) relevant for its biological behavior. More recently, Phan and colleagues reported the NMR structures of a series of AS1411 aptamer derivatives that adopted a single G4 structure in solution with identical antiproliferative activity (Chung et al., 2015; Do et al., 2017). These AS1411 derivatives, namely AT11 and AT11-B0 (a more thermally stable version of AT11) (Do et al., 2017), are po-tentially promising for the development of cancer-targeted systems for the delivery of anticancer ligands.
Herein, using AT11 and AT11-B0 derivatives, we studied aptamer-guided approaches for the delivery of ligands with anticancer potential into HeLa cervical cancer cells. The ligands used were acridine orange (AO) derivatives (C3, C5 and C8) that showed potent inhibitory eﬀect towards HeLa cells (low micromolar IC50 values at 24 h incubation) (Carvalho et al., 2018; Pereira et al., 2017). However, the ligands had a similar antiproliferative eﬀect on non-malignant human fibroblasts (NHDF) and their application in further in vitro or in vivo assays would benefit from a cancer-selective drug delivery system. The ligands were also shown to bind and stabilize a variety of G4 structures such as telomeric and oncogene promoter G4s (Carvalho et al., 2018). Using a supramolecular strategy relying on the G4-binding ability of the AO derivatives, lacking any chemical modifications of both sequences and ligands, the ability of AT11 and AT11-B0 to selectively convey C3, C5 and C8 to HeLa human cervical cancer cells was assessed using an array of spectroscopic and biochemical techniques.
2. Materials and methods
2.1. Acridine orange derivatives and oligonucleotides
Acridine orange ligands 10-(3-(4-iodobenzamide)propyl))-3,6-bis (dimethylamino) acridinium iodide (C3), 10-(5-(4-iodobenzamide) pentyl))-3,6-bis(dimethylamino) acridinium iodide (C5) and 10-(8-(4-iodobenzamide)octyl))-3,6-bis(dimethylamine) acridinium iodide (C8) were synthesized and purified as previously described (Pereira et al., 2017). The ligands were dissolved in DMSO to obtain a 10 mM stock solution and the subsequent dilutions were made with Milli-Q water. AT11 (5′-TGGTGGTGGTTGTTGTGGTGGTGGTGGT-3′), Cy5-AT11, AT11-B0 (5′-TGGTGGTGGTTGGTGGTGGTGGTGGT-3′) and Cy5-AT11-B0 were obtained from Eurogentec (Belgium) and StabVida (Portugal) with HPLC-grade purification. Stock solutions were prepared using Milli-Q water and stored at −20 °C until used. For all the experiments, the oligonucleotides were annealed by heating to 95 °C during 10 min and then cooled down in ice until used. International Journal of Pharmaceutics 568 (2019) 118511
2.2. Thermal diﬀerence spectra (TDS)
Thermal diﬀerence spectra (TDS) experiments were performed using a Thermo ScientificTM EvolutionTM 201 UV–Visible Spectrophotometer (Thermo Fisher Scientific, USA). UV spectra were recorded in the 220–335 nm range with a scan rate of 200 nm/min and 1 nm data intervals, above and below the melting temperature, at 90 °C and 20 °C, respectively. Pre-folded AT11 or AT11-B0 were used at 3 μM concentration in 20 mM phosphate buﬀer containing 65 mM KCl. The TDS spectrum was calculated by subtracting the 20 °C spectrum from the spectrum obtained at 90 °C. The data was normalized relatively to the maximum absorbance.
2.3. Circular dichroism (CD) spectroscopy
CD spectra were acquired in a Jasco J-815 spectropolarimeter equipped with a Peltier temperature control system. AT11 or AT11-B0 oligonucleotides were dissolved in 20 mM potassium phosphate buﬀer containing 65 mM KCl. For titrations experiments, the required amount of acridine orange ligands solution was added to the 1 mm quartz cuvette containing 10 µM of pre-folded AT11 or AT11-B0 solutions. Spectra were recorded through wavelengths ranging from 220 to 340 nm, with a scan speed of 100 nm/min, 1 nm bandwidth, 1 s in-tegration time over 4 averaged accumulations.