Nucleic acid aptamers can specifically bind to target molecules on the cell membrane that mediate their entrance into the cells. Their small size, high binding affinity, specificity, good biocompatibility, stability and low immunogenicity make them ideal drug delivery systems for cancer therapy. These biopharmaceuticals have potential for the delivery of anticancer compounds to diseased tissues, increasing their effectiveness while mitigating the off-target toxicity towards healthy cells. Herein, we have studied two quadruplex-forming DNA sequences derived from the nucleolin-targeted aptamer AS1411 as supramolecular carriers for the cancer-selective delivery of acridine orange derivatives (C3, C5 and C8) in cervical cancer cells. The devised delivery strategy relied on the non-covalent association of the acridine derivatives and the G-quadruplex (G4) structures. This association is done with a high binding strength, as suggested by the obtained KD values in the 10-6-10-7 M range, leading to the thermal stabilization of the G4 structures, particularly for C8. The stability of the resulting supramolecular conjugates was evaluated in fetal bovine serum, which proved their resistance against serum nucleases up to 48 h. Previous studies showed that the tested acridine orange derivatives were cytotoxic towards cervical cancer cells (HeLa) and non-malignant cells. However, when conjugated to AS1411 derivatives, the cytotoxicity of the free ligands towards non-malignant cells was restrained. Furthermore, conjugated C3 showed an enhanced cytotoxicity against HeLa cancer cells. Confocal microscopy indicated that both G4 sequences appear to colocalize with nucleolin, suggesting their ability to recognize and bind nucleolin on the cell surface. Additionally, the results confirmed the internalization of these delivery systems into HeLa cancer cells and their sustained cell trafficking, although being able to dissociate intracellularly to deliver C8 to the nucleoli. Overall, we showed that AS1411-derived G4s can be used as a potential cancer drug delivery system for cervical cancer.

Centro de Ciências e Tecnologias Nucleares Instituto Superior Técnico Universidade de Lisboa Estrada Nacional 10 2695 066 Bobadela LRS Portugal

CICS UBI Centro de Investigação em Ciências da Saúde Universidade da Beira Interior Av Infante D Henrique 6200 506 Covilhã Portugal

CIMAGO iCBR CIBB Faculdade de Medicina da Universidade de Coimbra Portugal

Institute of Biophysics of the CAS v v i Královopolská 135 612 65 Brno Czech Republic

Instituto Politécnico de Coimbra ESTESC Coimbra Health School Ciências Biomédicas Laboratoriais Portugal

Unidade de Gestão Operacional em Citometria Centro Hospitalar e Universitário de Coimbra Portugal

Université de Bordeaux ARNA Laboratory Inserm U1212 CNRS UMR 5320 IECB F 33600 Pessac France

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