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    Contents lists available at ScienceDirect
    International Journal of Pharmaceutics
    journal homepage:
    AS1411 derivatives as carriers of G-quadruplex ligands for cervical cancer T cells
    Joana Figueiredoa,1, Jéssica Lopes-Nunesa,1, Josué Carvalhoa,1, Francisca Antunesa, Márcia Ribeiroa, Maria Paula Cabral Campellob, António Paulob, Artur Paivac,d,e, Gilmar F. Salgadof, João A. Queiroza, Jean-Louis Mergnyf,g, Carla Cruza,
    a CICS-UBI – Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal
    b Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Estrada Nacional 10 (km 139,7), 2695-066 Bobadela LRS, Portugal
    c Unidade de Gestão Operacional em Citometria, Centro Hospitalar e Universitário de Coimbra (CHUC), Portugal
    d CIMAGO/iCBR/CIBB, Faculdade de Medicina da Universidade de Coimbra, Portugal
    e Instituto Politécnico de Coimbra, ESTESC-Coimbra Health School, Ciências Biomédicas Laboratoriais, Portugal
    G-quadruplex aptamers
    Drug delivery
    AS1411 derivatives
    Acridine orange ligands
    Cervical cancer 
    Nucleic AZ1495 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 im-munogenicity make them ideal drug delivery systems for cancer therapy. These biopharmaceuticals have po-tential for the delivery of anticancer compounds to diseased tissues, increasing their effectiveness while miti-gating 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-se-lective 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 con-jugated 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 in-tracellularly 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.
    1. Introduction
    Conventional chemotherapeutics suffer from a narrow therapeutic window due to their poor specificity towards tumor/healthy cells, leading to the so called “off-target” side effects (Zhou and Rossi, 2017). There is a need of developing targeted delivery systems to improve drug selective accumulation in cancer cells while increasing its effectiveness.
    Corresponding author.
    E-mail address: (C. Cruz). 1 These authors contributed equally to this work. 
    One approach to tackle such need is the use of cell-specific carriers that guide the delivery of the drug to the cancer cell. Nucleic acid aptamers have emerged as an attractive class of drug delivery systems due to their ability to bind with high affinity to specific ligands, their high chemical flexibility as well as tissue penetration capability (Zhou and Rossi, 2017). They have several advantages such as a relatively small size, flexible structure, inexpensive and reproducible chemical production
    J. Figueiredo, et al.
    when compared to antibodies, ease of chemical modification, high stability and lack of immunogenicity (Zhou and Rossi, 2017). Taking advantage of such properties, nucleic acid aptamers have been used to selectively convey ligands with therapeutic or imaging purposes into cancer cells (Alshaer et al., 2018; Vandghanooni et al., 2018).