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

Josué Carvalho,Jean-Louis Mergny,Maria Paula Cabral Campello,João A Queiroz,Gilmar F Salgado,Artur Paiva,Carla Cruz,António Paulo

doi:10.1038/s41598-019-44388-9

Abstract

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.

Highlights

AS1411 is a synthetic 26-base DNA aptamer previously discovered by Bates and co-workers as an agent targeting the nucleolin protein with high affinity and specificity, eliciting a potent antiproliferative effect on a variety of cell lines[1,2]
A similar experiment was performed in Human cervical cancer cells (HeLa) cells (Fig. S7) and the results suggest that cancer cells are less efficient in eliminating the aptamer in its free or C8-complexed state as Cy5-AS1411 red staining is still observed after 1-day incubation in free media
The MTT assays shown that the complexation of C8 with AS1411 decreases its cytotoxicity to Normal human dermal fibroblasts (NHDF) cells, which was later revealed to be due to differential uptake of free C8 vs complexed C8 by flow cytometry analysis

Summary

Introduction

AS1411 is a synthetic 26-base DNA aptamer previously discovered by Bates and co-workers as an agent targeting the nucleolin protein with high affinity and specificity, eliciting a potent antiproliferative effect on a variety of cell lines[1,2]. The protein is localized primarily in the nucleus of normal cells, while on cancer cells it is present in the cytoplasm and at the cell surface[3] This property confers a tumour-selective behaviour to AS1411 which targets preferentially the external domain of surface nucleolin of cancer cells. Targeted carriers that promote the selective accumulation of C8 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 C8 and AS1411, as well as with its derivatives LNA-AS1411 and U-AS1411. We describe the physical properties and biological activities of the resulting C8-oligonucleotide supramolecular complexes

Methods

Results

Conclusion