Aptamer-based Targeted Delivery of a G-quadruplex Ligand in Cervical Cancer Cells

AS1411 is a G-rich DNA oligonucleotide that functions as an aptamer of the protein nucleolin, found at high levels on the surface of cancer cells but not on the surface of normal cells. Herein, we have studied AS1411 as a supramolecular carrier for the delivery of an acridine-based G-quadruplex ligand, C8, to HeLa cancer cells. Two AS1411 derivatives, LNA-AS1411 and U-AS1411, were also tested, in an attempt to compare AS1411 pharmacological properties. The results showed that AS1411-C8 complexation was made with great binding strength and that it lowered the ligand’s cytotoxicity towards non-malignant cells. This effect was suggested to be due to a decreased internalization of the complexed versus free C8 as shown by flow cytometry. The AS1411 derivatives, despite forming a stable complex with C8, lacked the necessary tumour-selective behaviour. The binding of C8 to AS1411 G-quadruplex structure did not negatively affect the recognition of nucleolin by the aptamer. The AS1411-C8 repressed c-MYC expression at the transcriptional level, possibly due to C8 ability to stabilize the c-MYC promoter G-quadruplexes. Overall, this study demonstrates the usefulness of AS1411 as a supramolecular carrier of the G-quadruplex binder C8 and the potential of using its tumour-selective properties for the delivery of ligands for cancer therapy.

structure is a stable G-quadruplex. Recently, AS1411 was noncovalently bound to porphyrin TMPyP4, in an attempt to increase its delivery to MCF7 breast cancer cells within a photodynamic therapy approach 5 . This strategy was shown to increase the accumulation of TMPyP4 in MCF7 cancer cells when compared to non-malignant cells. Several examples in the literature provide evidence that attaching AS1411 to a wide variety of nanoparticles or other entities is an excellent strategy for delivering these cargoes inside of cancer cells. Moreover, improving AS1411 stability and biological properties, mainly through chemical modification on deoxynucleotides at appropriate positions in AS1411, could also be a strategy to improve the therapeutic function of the aptamer 2 .
Herein, we have evaluated AS1411 and its modified derivatives designated by LNA-AS1411 and U-AS1411 as drug delivery systems to carry an acridine orange-based ligand (C 8 ) 6 to HeLa cervical cancer cells. The ligand C 8 was previously published and shown to bind and stabilize promoter and telomeric G-quadruplexes with high affinity (K D ≈ 10 −7 M). C 8 showed cytotoxic activity against HeLa cells with an IC 50 value around 1 µM at 24 h incubation; however, similar values were observed for non-malignant cells. Therefore, targeted carriers that promote the selective accumulation of C 8 in cancer cells, while preventing toxicity in healthy surrounding cells, are of crucial importance to improve its pharmacological profile as a potential drug for anticancer therapies. To address this goal, we have studied the formation of supramolecular complexes between C 8 and AS1411, as well as with its derivatives LNA-AS1411 and U-AS1411. Herein, we also describe the physical properties and biological activities of the resulting C 8 -oligonucleotide supramolecular complexes.
thermal difference spectra (tDs). Thermal difference spectra (TDS) were collected on a Thermo Scientific TM Evolution TM 201 UV-Visible Spectrophotometer (Thermo Fisher Scientific, USA). Spectra were obtained in the 220-335 nm range (scan rate of 200 nm/min and 1 nm data intervals) above and below the melting temperature, at 90 °C and 20 °C, respectively 8 . DNA was used at 3 μM concentration in 30 mM phosphate buffer (15 mM KH 2 PO 4 , 15 mM K 2 HPO 4 , pH 7.1) containing 100 mM KCl. The TDS spectrum was calculated by subtracting the spectrum at 20 °C from the spectrum at 90 °C. The following UV TDS factors were used for analysis: ΔA 240 /ΔA 295 , ΔA 255 /ΔA 295 , ΔA 275 /ΔA 295 . The data was normalized relative to the maximum absorbance. Circular dichroism. Circular dichroism (CD) experiments was performed on a Jasco J-815 CD spectrometer equipped with a Peltier-type temperature controller (model CDF-426S/15). Spectra acquisition was performed in 1-or 10-mm quartz cuvettes at a DNA concentration of 10 µM in 30 mM potassium phosphate buffer containing 100 mM KCl. Spectral width was set to 220-340 nm, with a scan speed of 100 nm/min, 1 nm bandwidth, 1 s integration time over 4 averaged accumulations. During titrations, the required volume of ligand solution was added to the quartz cell.
For CD melting studies, buffer conditions were adjusted so that the oligos would melt around 50 °C, particularly by using 10 mM lithium cacodylate buffer, pH 7.2, containing 10 mM KCl and 90 mM LiCl. Melting curves were obtained by monitoring a single wavelength between 20 and 110 °C with a heating rate of 2 °C/min. Data were converted into fraction folded (θ) plots, fitted to a Boltzmann distribution using OriginPro 2016 and the melting temperatures (T m ) determined from the two-state transition model. Fluorescence studies. Fluorescence titrations were conducted on a Horiba FluoroMax4 fluorometer (Japan) using a high-precision quartz suprasil cuvette (light path 10 × 4 mm). The fluorescence spectra were obtained between 530-700 nm and averaged over three scans. The excitation wavelength was 498 nm, matching the maximum absorbance of C 8 . To study ligand-aptamer complex formation, previously annealed aptamer sequence was titrated into the cuvette containing a solution of 5 μM C 8 in 30 mM potassium phosphate buffer (pH 7.2) containing 100 mM KCl. Spectra were acquired after a 5 min equilibration period. Fluorescence data was converted into fraction of bound ligand (α) plots, fitted to the saturation binding Hill model (OriginPro 8) and the dissociation constant (K D ) and Hill coefficient (n) values determined.
Fluorescence titrations were also carried with nucleolin protein. In this case, labelled Cy3-AS1411 at 1 μM, was titrated with nucleolin in the absence and presence of C 8 to evaluate the effect of ligand binding in the affinity of the aptamer towards the protein. The fluorescence emission spectra were obtained between 555 and 700 nm and averaged over three scans. The excitation wavelength was 540 nm, matching the maximum absorbance of Cy3.
Cell viability assay. Normal human dermal fibroblasts (NHDF) were grown in RPMI-1640 medium supplemented with 0.01 M HEPES, 0.02 M L-glutamine, 0.001 M sodium pyruvate, 10% fetal bovine serum, and 1% penicillium/streptomycin antibiotic. Human cervical cancer cells (HeLa) were grown in DMEM medium supplemented with 10% fetal bovine serum, and 1% penicillium/streptomycin antibiotic. Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO 2 . For MTT assays, cells were seeded in 48-well plates (5 × 10 3 cells/well) and incubated overnight for cell adhesion. Then, cells were incubated for 7 days with the aptamers at 15 μM, C 8 at 1 μM and the preformed ligand-aptamer complex using the same compound/ DNA concentrations. Complex formation was performed by incubating the aptamers with C 8 for 10 min prior www.nature.com/scientificreports www.nature.com/scientificreports/ to incubation. Wells containing untreated cells were used as control. At the end of incubation, the media was replaced with fresh media containing 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide salt (MTT) and further incubated at 37 °C for 1 or 4 h for HeLa and NHDF, respectively. Finally, the formazan crystals were dissolved in DMSO and absorbance was recorded in a Bio-Rad xMark ™ microplate reader at 570 nm. Cell viability relatively to control was expressed as mean ± SEM from at least three different experiments. The IC50 values of C 8 at 7 days incubation period were determined by incubating the cells with different compound concentrations ranging from 0.01 to 2 μM. All data treatment was performed using GraphPad Prism 6.
Cell membrane damage. The effect of the ligand-aptamer complex on cell membrane integrity was assessed by the lactate dehydrogenase (LDH) assay using Pierce ™ LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific, USA). Cells were seeded in 48-well plates (5 × 10 3 cells/well) and incubated overnight for cell adhesion. Then, cells were incubated for 7 days with the preformed ligand-aptamer complex using the same compound/DNA concentrations used for MTT assay (15 μM aptamer and 1 μM C 8 ). After exposure, 50 μL/well of supernatant were transferred into optically clear 96-well flat-bottom microplates and mixed with 50 μL/well of reaction mixture and incubated for 30 min at room temperature. The absorbance of the samples was then measured at 490 nm using a Bio-Rad xMark ™ microplate reader. The percentage of cytotoxicity is calculated with the following equation 9 : Cells treated with lysis buffer were used as positive control (maximum LDH release). The experiments were performed in at least two different plates for each cell line, using triplicate wells for each condition.
Flow cytometry analysis. Cells were seeded in 12-well plates (5 × 10 5 cells/well) and incubated overnight for cell adhesion. Then, cells were incubated for 24 h with C 8 at 1 μM as control and the preformed ligand-aptamer complex using the same compound/DNA concentrations as used in the MTT assays. After the incubation period, the wells were washed by rinsing with PBS three times. Cells were then trypsinized, resuspended in PBS and analysed in a BD FACSCanto ™ II flow cytometry system (BD Life Sciences, US) to evaluate the uptake of the aptamer-ligand complex. Non-specific colouring and debris were excluded by analysing FSC vs SSC density plot as dead cells and debris have lower forward scatter levels.

RNA isolation and RT-qPCR for gene expression analysis.
To analyse the effect of the ligand-aptamer complex at genetic level, RT-qPCR analysis of c-MYC and nucleolin expression was performed. Cells were seeded in 12-well plates (1 × 10 5 cells/well) and incubated overnight. Following cell adhesion, these were incubated for 7 days with free C 8 at 1 μM and free aptamer at 15 μM as control, and the preformed ligand-aptamer complex using the same compound/DNA concentrations. Total RNA was extracted from the cells using TRIzol reagent (Invitrogen, USA). 500 ng of total RNA were reverse transcribed using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA), according to the kit instructions. For quantitative analysis of gene expression, RT-qPCR amplification of cDNA was performed using SYBR ® Green PCR Master Mix (Thermo Fisher Scientific, USA) on a CFX Connect ™ Real-Time PCR Detection System (Bio-Rad, USA). The relative quantification of gene expression was based on the comparative threshold cycle (C T ) method in which the quantity of transcripts is determined as 2 −(ΔCT target−ΔCT control) , normalized to levels of β-actin and relative to the untreated control cells. The following forward (Fw) and reverse (Rv) primers were used: c-MYC-Fw 5′-TGAGGAGACACCGCCCAC-3′ and c-MYC-Rv 5′-CAACATCGATTTCTTCCTCATCTTC-3′; NCL-Fw 5′-AGGAGGAGGAAGAAGAGGAG-3′ and NCL-Rv 5′-ACAAAGAGATTGAAAGCCGTAG-3′; β-actin-Fw 5′-AAGAGAGGCATCCTCACCCT-3′ and β-actin-Rv 5′-TACATGGCTGGGGTGTTGAA-3′. The RT-qPCR parameters were the following: an initial denaturation at 95 °C for 3 min, followed by 40 cycles with denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, and elongation at 72 °C for 30 s, followed by a dissociation stage at 65 °C 5 s. Each sample was run in triplicate from three different experiments and results are expressed in relative levels to respective controls (means ± SEM).

Fluorescence resonance energy transfer (FRet) melting. Fluorescence resonance energy transfer
(FRET) melting experiments were performed on a CFX Connect ™ Real-Time PCR Detection System (Bio-Rad, USA). AS1411 was purchased with FAM and TAMRA at the 5′ and 3′ ends, respectively. The fluorescence measurements were performed in 10 mM lithium cacodylate buffer containing 10 mM KCl and 90 mM LiCl as previously reported 10 . Labelled-AS1411 was used at 0.2 µM and C 8 at 1 µM. For competition experiments with nucleolin, the protein was previously incubated with AS1411 at 1 μM and 2 µM for 30 min at 37 °C. FAM fluorescence intensity was recorded between 25-95 °C, with a temperature increment of 1 °C/min. The excitation and detection wavelengths were 492 and 516 nm, respectively. Each experimental condition was tested in triplicate in at least two separate plates. The melting temperatures were determined from the normalized curves as the temperature for which the normalized emission was 0.5.

Confocal fluorescence microscopy imaging. HeLa and NHDF cells were cultured in Ibidi 8-well
μ-slides (1 × 10 4 cells/well) overnight at 37 °C in a humidified atmosphere containing 5% CO 2 . After cell adhesion, cells were incubated with 1 μM Cy5-AS1411 in the presence and absence of 0.5 μM C 8 , for 7 days. At the end of each of the 7 days of incubation, the excess of fluorophores was washed off by rinsing with PBS three times. Cell nucleoli were then stained with 1 µM nuclear probe Hoechst 33342 for 15 min. Prior to visualization, excess probe was washed off by rinsing with PBS three times. Images were acquired using a Zeiss AxioObserver LSM 710 microscope with 405, 488 and 564 nm laser excitation for Hoechst 33342, C 8 and Cy5-AS1411, respectively. Appropriate emission bands were selected for the three fluorescent channels, considering the spectral overlap and potential bleed-through between channels. Images were processed with Zeiss ZEN software. The quantitative www.nature.com/scientificreports www.nature.com/scientificreports/ analysis of Cy5-AS1411 internalization in the presence and absence of C 8 was determined as described in literature 11 . In brief, the corrected total cell fluorescence (CTCF) of at least 100 AS1411-positive cells for each experimental condition in three different samples was measured. CTCF can be determined as CTCF = ID − (A × MFB) where the ID, A, and MFB correspond to integrated density, selected cell area, and mean fluorescence of background readings, respectively. statistical analysis. The statistical analysis was performed by using Student's unpaired t test. A p value < 0.05 was considered statistically significant. Data analysis was performed in GraphPad Prism 6 (San Diego, CA, USA).

Results and Discussion
AS1411 aptamer is extensively documented as a potential targeting agent for the development of cancer-selective drug delivery systems. It has been shown before that it can deliver a variety of attached cargoes (both covalently and non-covalently) to various cancer cell types, both in vitro and in vivo 2 . Herein, we non-covalently conjugated ligand C 8 to AS1411, LNA-AS1411 and U-AS1411 in an attempt to improve its biological properties (Table 1). Using a simple supramolecular strategy lacking any linkage conjugation, we tested a ligand delivery system aimed at HeLa cancer cells as schematically depicted in Fig. 1. The use of locked nucleic acids (LNA) or via thymine-to-uracil substitutions may enhance the binding affinity of the aptamer to its target nucleolin 12 . LNA nucleotides comprise a 2′-O, 4′-C-methylene linkage in the sugar moiety, locking it in a N-type conformation mimicking RNA nucleotide. LNAs display enhanced thermal and ribozyme stability, and low cytotoxicity for healthy cells 12 . dT → rU substitutions can contribute to the enhancement of stability due to loss of hydration leading to improved stacking of G-tetrads 13 .
The effect of the modifications on the overall G-quadruplex structure was evaluated by circular dichroism (CD) and UV/Vis spectroscopy, namely by the use of thermal difference spectra (TDS). The CD and TDS spectra of AS1411, LNA-AS1411 and U-AS1411 are presented in Fig. 2. The CD spectra of the three sequences, despite minor changes between them, show the typical CD signature of parallel G-quadruplex topologies ( Fig. 2A), with a positive peak at around 260 nm and a negative peak at around 240 nm. However, as shown previously by Trent and collaborators, AS1411 forms a mixture of at least eight G-quadruplex species in solution and CD spectra could not discriminate among the different species 14 . The TDS spectra confirm the structural polymorphism inherent to the sequence (Fig. 2B). The positive peaks around 243 and 273 nm, and negative peak around 295 nm confirm that a G-quadruplex structure is adopted by the modified AS1411 aptamers 8 Table 1. Nucleotide sequence of the aptamers used in this study and their spectroscopic profile characterization. a Modifications are highlighted in bold, g represents LNA guanine. b "+" and "−" refer to the presence of a positive or negative peak, respectively, at the indicated wavelength. c Ratio between values at two different wavelengths (e.g., 240 and 295 nm for the left column). www.nature.com/scientificreports www.nature.com/scientificreports/ a mixture of G-quadruplex topologies as a factor lower than 2 and higher than 4 is characteristic of antiparallel and parallel topologies, respectively 15 . ΔA 255 /ΔA 295 factor on its turn, was between 1.51 for AS1411 and 3.32 for LNA-AS1411. Likewise, these values demonstrate structural polymorphism as a factor lower than 1.5 and higher than 3.5 is characteristic of antiparallel and parallel topologies, respectively 15 . Finally, ΔA 275 /ΔA 295 factor was 1.84, 2.13 and 2.90 for AS1411, U-AS1411 and LNA-AS1411 respectively. Factors lower than 2 and higher than 3 are characteristic of antiparallel and parallel topologies, respectively 15 . AS1411 value is indicative of an antiparallel topology, despite the inherent polymorphism. This may be due to the fact that some of the possible topologies predicted for AS1411 are antiparallel dimers 14 .
The global analysis of the TDS factors shows that despite the evident structural polymorphism, LNA-AS1411 may fold preferentially into a parallel G-quadruplex as the ΔA 255 /ΔA 295 and ΔA 275 /ΔA 295 values are higher when compared with the other sequences, and closer to the threshold value for a parallel topology. Indeed, LNA guanosines are incompatible with a syn conformation which prevents their assembly into the anti-parallel conformation 16 . It has been proposed that the most stable parallel G-quadruplex is obtained when substituting every guanosine to the LNA except the 5′-terminal one 16 . The observation of the CD spectra shows the loss of a weak negative band at around 280 nm which may be indicative of the structural transitions 17 . Nonetheless, the modifications introduced in AS1411 sequence do not seem to promote the formation of a single G-quadruplex structure nor affect G-quadruplex formation.
The formation of a stable complex between the aptamers and ligand C 8 was assessed and characterized by CD and fluorescence spectroscopy. CD titrations were first performed to assess complex formation and/or structural modification of the G-quadruplex structure upon ligand binding. The results are shown in Fig. 3.
Upon ligand titration, the overall topology was maintained followed by an increase in ellipticity indicating aptamer-ligand association and stabilization of the preformed G-quadruplex structure. A deeper analysis of Fig. 3A,C reveals significant changes in the 270-300 nm region. This may be due to the preferential stabilization of one of the possible topologies of AS1411 and U-AS1411. Such effect is not observed for LNA-AS1411 potentially due to the formation of parallel topologies only.
The binding strength between ligand C 8 and the aptamers was further assessed using fluorimetric titrations. As seen in Fig. 4, upon excitation at 498 nm, ligand C 8 emitted fluorescence as a broad band centred at 568 nm as previously reported 6 . After addition of previously annealed aptamer solution, a ≈10-fold fluorescence enhancement was observed for all sequences denoting a strong interaction between the ligand and the aptamers G4 structure. The fluorescence intensity values were then fitted to a saturation binding model and the apparent dissociation constants (K D ) determined. The K D values for AS1411, LNA-AS1411 and U-1411 were 1.29 × 10 −6 , 1.92 × 10 −6 and 1.37 × 10 −6 M, respectively (SD ≤ 1 × 10 −8 M). These values in the low micromolar range are indicative of moderate to high affinity and stable complex formation between the ligand and the aptamers G4 structure 18 .
The Hill coefficient (n) which can be interpreted as the average number of bound ligands per G4, was 2.8, 2.1 and 2.5 for AS1411, LNA-AS1411 and U-1411, respectively. This suggests a ligand:DNA ratio of 3:1 in the case of AS1411 and U-1411, and 2:1 in the case of LNA-AS1411. Regarding AS1411, as it is assumed to fold into a variety of structures, for instance a dimeric antiparallel G-quadruplex 2 , the 3:1 ratio mas be misestimated. At this point, we have no information on whether ligand C 8 binds preferentially to a specific AS1411 conformation. Additionally, the Hill coefficient is a measure of cooperativity, whereas the n values greater than 1 indicate positive cooperativity meaning that a binding event increases the affinity of additional ligand molecules 6 .
The influence of ligand C 8 on the thermal stability of the aptamers was investigated by thermal denaturation. For that, we performed CD melting experiments and assessed the ligand's effect on the aptamers melting temperature (T m ) (Fig. S1). The ligand clearly increased the T m values of all the aptamers G4 structure, being the effect more prominent on AS1411, as C 8 promoted a ΔT m of 28.4 °C at 2 molar equivalents. U-AS1411 was stabilized for www.nature.com/scientificreports www.nature.com/scientificreports/ 24.5 °C at the same ligand:G4 ratio, while LNA-AS1411 was the least stabilized structure with a ΔT m of 17.5 °C. The ΔT m values agree with the K D values as the AS1411 presented the higher K D and ΔT m while on the other end LNA-AS1411 presented the lowest values. As the G4 folding is necessary for the biological properties of the aptamer, the ligand-induced thermal stability may enhance its nuclease resistance, nucleolin affinity and intracellular trafficking 12,19,20 . The ΔT m values at different ligand:G4 ratios are summarized in Table S1.
The ability of the aptamers AS1411, LNA-AS1411 and U-AS1411 to accumulate C 8 in the cancer cells and to prevent C 8 accumulation in healthy cells was indirectly assessed by the MTT assay. As previously reported, AS1411 has a weak inhibitory effect in HeLa cancer cells and more than 5 days are needed to observe a slight effect on cell viability, even at 10 μM aptamer concentration 21,22 . As such, we tested the effect of the aptamer-ligand complexes for 7 days incubation period at 15 μM aptamer concentration. The free aptamers cytotoxicity towards HeLa and non-malignant NHDF cells is shown in Fig. S2. AS1411 showed reduced cytotoxicity towards HeLa cells (79% mean viability) and nearly no toxicity towards non-malignant cells. The LNA-modified aptamer did not have any effect on the cell viability of both cancer and non-malignant cells. Finally, U-AS1411 presented a similar cytotoxicity for HeLa cells (71% mean viability) as AS1411, however in the case of NHDF cells its cytotoxicity was significantly higher (53% mean viability).
Then, the IC 50 values of C 8 at 7 days incubation time were determined for HeLa and NHDF cells (Fig. S3). The IC 50 was 0.15 and 0.48 µM for HeLa and NHDF, respectively. Based on these values, we decided to use a C 8 concentration of 1 µM which is high enough to elicit significant cell toxicity and still suitable to observe any changes induced by the ligand complexation to the aptamer. After mixing 15 µM aptamer with 1 µM C 8 to allow complex formation, the aptamer-ligand complexes were incubated for 7 days in HeLa and NHDF cells. The results are presented in Fig. 5.
As seen in Fig. 5A, the C 8 toxicity towards non-malignant cells is significantly attenuated upon complexation with AS1411. The lower cytotoxicity of C 8 towards NHDF cells when complexed with AS1411 may be due to the aptamer mechanism involved in selective accumulation in cells. It has been proposed that AS1411 is gradually cleared from normal cells by efflux or exocytosis 24-72 h post-treatment 2 . Additionally, normal cells have increased lysosomal activity in comparison with AS1411-treated cancer cells 2 . Therefore, if the aptamer-ligand complex is capable of sustaining cellular trafficking it may be cleared from normal cells by lysosomal degradation,  www.nature.com/scientificreports www.nature.com/scientificreports/ thus clearing C 8 and reducing its effects. Although AS1411-C 8 has a more pronounced effect on HeLa than on NHDF cells, the cytotoxicity towards HeLa cells is also hindered. In this case, the explanation may be the high affinity of C 8 towards the aptamer which may prevent the ligand to detach from AS1411 once inside the cell. LNA-AS1411 complex on its hand, showed no cytotoxic effect on both cell lines with viability values around 100% (Fig. 5B). Indeed, LNA-containing DNA sequences were already shown to be poorly toxic for HeLa cells 23 . This suggests that the modification of the sugar ring may affect the aptamer cellular behaviour, namely the interaction with its target nucleolin. The apparent viability values above 100% observed for LNA-AS1411 treatment may be due to the compounds influencing the cell's metabolism thus resulting in increased metabolization of MTT 24 (as the MTT assay is a measure of the cellular mitochondrial enzymatic activity) and/or cell growth (in response to stress, a phenomena termed hormesis 25 ) when compared with the control. Opposingly, U-AS1411 complex showed an undesired increased cytotoxicity towards NHDF cells, significantly higher than that observed for HeLa cells (Fig. 5C).
To show the applicability of the system, the MTT experiments were extended to two additional cancer cell lines using AS1411-C 8 complex, namely PC-3 prostate cancer cell line and A549 lung cancer cell lines (Fig. S4).
Similarly, to what was observed for HeLa cells, the aptamer cytotoxicity was improved when complexed with C 8 . In the case of PC-3 cells the complex promoted a viability decrease of about 85% while in the case of A549 cells a milder effect was observed (47% mean viability). These results suggest that this strategy may be employed in other cancer types, particularly prostate cancer because PC-3 cells showed the most promising results. Additionally, the effect of AS1411-C 8 complex on cell membrane integrity was evaluated in the same cancer cell line panel used in the MTT assays. The results shown in Fig. 6 suggest that the AS1411-C 8 complex promoted modest cell membrane damage in the tested cell lines (LDH activity <50%), despite reducing their viability as indicated by the MTT results. Similar effect was already observed for other nanosystems 26 and may be related to a non-apoptotic cell death mechanism 27 . Furthermore, it should be emphasized that while the LDH assay is a measure of cytotoxicity induced by cell membrane damage, the MTT assay provides data on cell viability, specifically the cell's metabolic activity. A correlation between the results is therefore not always attained.
Following the MTT and LDH assays, we employed flow cytometry to assess intracellular accumulation of free and AS1411-complexed C 8 in HeLa cancer cells and NHDF non-malignant cells, based on the intrinsic fluorescence of ligand C 8 . The results are depicted in Fig. 7.
Flow cytometry analysis indicated that the AS1411-C 8 complex is less intracellularly accumulated in both cell types when compared to free C 8 , as observed by the lower fluorescence intensity emitted from the cells, despite an enhanced fluorescence quantum yield of C 8 when complexed to DNA. These results corroborate the lower cytotoxicity observed for the AS1411-C 8 complex when compared with free C 8 .  www.nature.com/scientificreports www.nature.com/scientificreports/  www.nature.com/scientificreports www.nature.com/scientificreports/ Then, we assessed if the reduced cellular uptake of the aptamer-ligand complex could be due to a potential interference with nucleolin binding. For that we employed fluorescence spectroscopy in two different approaches. First, using a solution of fluorescently labelled Cy3-AS1411, we titrated increasing amounts of nucleolin in the presence and absence of C 8 and determined the K D values from the fluorescence intensity changes. The saturation binding plots are shown in Fig. 8. The obtained K D values for the binding interaction between the free AS1411 aptamer and the AS1411-C 8 complex with nucleolin were 8.63 pM and 6.38 pM, respectively. The obtained K D values are lower than those reported in the literature, being potentially overestimated by the use of labelled oligonucleotides or the use of a different peptide than that used in those studies 28,29 . Nonetheless, both values are in the same range, being slightly lower in the case of the aptamer-ligand complex which suggests that the ligand does not negatively affect the recognition of the protein by the aptamer.
Additionally, a competition FRET-melting experiment was conducted to support the fluorescence titration findings. The rationale for the assay design was based on the hypothesis that if the ligand-induced stabilization of AS1411 was similar in the presence and absence of nucleolin, ligand binding would not affect protein recognition. The ligand-induced thermal stabilization of AS1411 was 13.1 °C in the presence of 1 µM C 8 ( Table 2). Upon   www.nature.com/scientificreports www.nature.com/scientificreports/ pre-incubation of AS1411 with equimolar nucleolin, the ΔT m increased to 20.0 °C. Even with an excess of protein (2 molar eq.) the ΔT m was 15.1 °C, reflecting a higher stabilization of the aptamer by C 8 in the presence of nucleolin. This indicates that the ligand does not prevent the recognition of the protein by the aptamer, as suggested by the fluorescence titration experiments, and that it may even enhance the stabilization of AS1411 G-quadruplex structure. This may be due to the ligand providing additional binding sites for the protein interaction (ternary complex), or since the aptamer G-quadruplex structure is necessary for nucleolin binding, the C 8 -induced stabilization of the structure may enhance its binding strength 30 .
To assess the potential effect of the AS1411-C 8 complex on gene expression, RT-qPCR analysis was performed. Oncogene c-MYC and nucleolin mRNA levels were analysed since c-MYC is commonly found upregulated in tumour cells being responsible for cancer cell proliferation 31 , and nucleolin is the AS1411's target. The results are depicted in Fig. 9. Upon treatment with AS1411-C 8 complex, the expression of c-MYC mRNA was downregulated by more than 50% (p < 0.01) which may inhibit cancer cell progression, eventually leading to cell death 31 . The acridine derivative C 8 was already tested for its ability to stabilize the G4 structure formed by c-MYC NHE element of the promoter region 6 . Therefore, it is reasonable to assume that the decrease in the c-MYC mRNA levels may be due to transcription modulation induced by interaction of C 8 and c-MYC G4 structures upon intracellular dissociation from the AS1411-ligand complex 32 . Opposingly, nucleolin mRNA expression was upregulated by 1.5-fold similarly to what was reported for free AS1411 before 33 . This suggests that the biological effects of AS1411 are not negatively affected by its complexation with ligand C 8 . This feedback loop induction may be responsible for increasing the cells sensitivity to AS1411 and/or increasing its intracellular accumulation 33 .
Finally, the subcellular localization of the AS1411-C 8 complex in HeLa and NHDF cells was evaluated by fluorescence confocal microscopy. As seen in Fig. 10, the complex is able to penetrate cell membrane and localizes in the cytoplasm of HeLa cells. The overlapping of C 8 (green) and AS1411 (red) suggests that the complex is still formed intracellularly (yellow) and maintained during cell internalization and trafficking. C 8 can be seen in the nucleoli in a free state which may suggest decomplexation and localization of the nucleolus as described for the ligand 6 . This may be particularly important to explain the downregulation of c-MYC transcriptional level as showed by the RT-qPCR experiments. A modest increase in the uptake of AS1411 can be observed in the presence of C 8 which may correlate with the proposed increased stability of aptamer upon ligand binding, possibly enhancing its association with nucleolin, and the fact that nucleolin is found overexpressed upon treatment with the complex as suggested by RT-qPCR results. The quantitative analysis of the Cy5-AS1411 fluorescence in the absence and presence of C 8 shown in Fig. S5 substantiates an increased uptake of AS1411 in the presence of the ligand.
Similar findings were observed for NHDF cells (Fig. S6). These results disagree with the selective accumulation described above where it has been shown that AS1411 is gradually cleared from normal cells by efflux 24-72 h post-treatment. The explanation may arise from the fact that cells are constantly incubated in media containing the compounds for 7 days straight. To test this hypothesis, we incubated the cells for 6 days in the presence of the aptamer-ligand complex, followed by 1 day in free media. The results shown in Fig. 11 confirm that indeed AS1411 is eliminated from normal cells after one day. C 8 despite being uptaken complexed with AS1411, remains inside the cells, suggesting that the observed differences in cytotoxicity in MTT assays between free C 8 and AS1411-C 8 complex are mostly due to differences in uptake as shown by flow cytometry. A similar experiment was performed in HeLa cells (Fig. S7) and the results suggest that cancer cells are indeed less efficient in eliminating the aptamer in its free or C 8 -complexed state as Cy5-AS1411 red staining is still observed after 1-day incubation in free media. These results agree with the proposed mechanism of cancer-selective accumulation of AS1411 where AS1411-conjugates are able to escape lysosomal vesicles with increased uptake 2,34 . Figure 11. Confocal microscopy images of NHDF cells incubated with Cy5-AS1411 and Cy-AS1411-C 8 complex for 6 days followed by 1-day incubation in fresh media. Cell nuclei are stained with Hoechst 33342 (blue), C 8 emits green fluorescence while Cy5-AS1411 fluorescence is absent. Control cells and C 8 single stain control images are shown on the right. Brightness was adjusted to facilitate analysis. www.nature.com/scientificreports www.nature.com/scientificreports/ Conclusion AS1411 aptamer has been increasingly suggested as an effective targeting system for the selective delivery of a wide range of cargoes to cancer cells. Herein, using a simple supramolecular strategy lacking any chemical modification of the aptamer, we propose a delivery system for conveying a G-quadruplex ligand C 8 into cancer cells in a selective manner. AS1411 unmodified aptamer and two derivatives containing LNA and uracil nucleotides were used. Ligand C 8 was shown to strongly associate with the three aptamers with K D in the 10 −6 M range. Additionally, C 8 is able to thermally stabilize the aptamers structure, possible enhancing its properties. The MTT assays shown that the complexation of C 8 with AS1411 decreases its cytotoxicity to NHDF cells, which was later revealed to be due to differential uptake of free C 8 vs complexed C 8 by flow cytometry analysis. LNA-AS1411 and U-AS1411 complexes shown non-adequate cytotoxic profile, being that the first is non-toxic for HeLa cells and the latter is highly toxic for non-malignant cells. MTT experiments with prostate and lung cancer cells show the potential applicability of the system in other cancer types. Using fluorescence spectroscopy and FRET-melting we propose that ligand C 8 complexation with AS1411 does not affect its recognition of protein nucleolin, which is necessary for its biological effects. RT-qPCR experiments demonstrated that AS1411-C 8 complex is able to downregulate c-MYC expression, which may be attributed to the stabilization of c-MYC G4 structures by the ligand. Finally, confocal microscopy indicated that the complex is efficiently taken up into the cells and that AS1411-C 8 is maintained during cell internalization and trafficking. Moreover, AS1411 uptake is increased in the presence of C 8 which may be due to nucleolin overexpression induced by the complex as suggested by RT-qPCR. This study paves the way for the development of cancer-specific AS1411-based delivery systems for the selective accumulation of G-quadruplex ligands in the target tissues, in a simple non-covalent strategy.