Abstract
Herein, we describe the synthesis of an aptadendrimer by covalent bioconjugation of a gallic acid-triethylene glycol (GATG) dendrimer with the G-quadruplex (G4) AT11 aptamer (a modified version of AS1411) at the surface. We evaluated the loading and interaction of an acridine orange ligand, termed C8, that acts as an anticancer drug and binder/stabilizer of the G4 structure of AT11. Dynamic light scattering experiments demonstrated that the aptadendrimer was approximately 3.1 nm in diameter. Both steady-state and time-resolved fluorescence anisotropy evidenced the interaction between the aptadendrimer and C8. Additionally, we demonstrated that the iodine atom of the C8 ligand acts as an effective intramolecular quencher in solution, while upon complexation with the aptadendrimer, it adopts a more extended conformation. Docking studies support this conclusion. Release experiments show a delivery of C8 after 4 h. The aptadendrimers tend to localize in the cytoplasm of various cell lines studied as demonstrated by confocal microscopy. The internalization of the aptadendrimers is not nucleolin-mediated or by passive diffusion, but via endocytosis. MTT studies with prostate cancer cells and non-malignant cells evidenced high cytotoxicity mainly due to the C8 ligand. The rapid internalization of the aptadendrimers and the fluorescence properties make them attractive for the development of potential nanocarriers.
Keywords:
G-quadruplex aptamers; acridine orange ligands; aptadendrimer; biophysical studies; gallic acid–triethylene glycol dendrimers.
Grants and funding
This work was supported by PESSOA program ref. 5079, project FCT ref. UIDP/00709/2020 ORACLE, “Bolsa de Investigação em Oncologia Dr. Rocha Alves do Núcleo Regional do Centro da Liga Portuguesa Contra o Cancro” and Project CENTRO-01-0145-FEDER-181235, PAPILOMA-Vaginal gel for topical application to precancerous lesions caused by Human Papilloma Virus. Thanks are due to FCT/MCT for the financial support to CICS-UBI (ref. UIDB/00709/2020) research unit, C2TN-IST UID/Multi/04349/2019 research unit, PPBI-Portuguese Platform of BioImaging research unit (POCI-01-0145-FEDER-022122), and to the Portuguese NMR Network (ROTEIRO/0031/2013-PINFRA/22161/2016), through national funds and, where applicable, co-financed by the FEDER through COMPETE 2020, POCI, PORL and PIDDAC. This article/publication is based upon work from COST Action CA 17140 “Cancer Nanomedicine from the Bench to the Bedside” supported by COST (European Cooperation in Science and Technology). The authors acknowledge Project UIDB/04565/2020 of the Research Unit Institute for Bioengineering and Biosciences—iBB, project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB, and PTDC/BIA-BFS/30959/2017 project. This work was supported by the Spanish Ministry of Science and Innovation (RTI2018-102212-B-I00), the Xunta de Galicia (ED431C 2022/21, and Centro Singular de Investigación de Galicia accreditation 2019−2022, ED431G2019/03), Axencia Galega de Innovación (IN845D 2020/09), and the European Union (European Regional Development Fund-ERDF).