CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Estrada Nacional 10 (km 139,7), 2695-066 Bobadela LRS, Portugal.
Int J Pharm. 2019 Sep 10;568:118511. doi: 10.1016/j.ijpharm.2019.118511. Epub 2019 Jul 10.
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 (C, C and C) 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 K values in the 10-10 M range, leading to the thermal stabilization of the G4 structures, particularly for C. 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 C 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 C to the nucleoli. Overall, we showed that AS1411-derived G4s can be used as a potential cancer drug delivery system for cervical cancer.
核酸适体可以特异性结合细胞膜上介导其进入细胞的靶分子。它们的体积小、结合亲和力高、特异性强、生物相容性好、稳定性好、免疫原性低，使其成为癌症治疗的理想药物输送系统。这些生物制药具有将抗癌化合物递送到病变组织的潜力，在提高疗效的同时减轻对健康细胞的脱靶毒性。在这里，我们研究了两个源自核仁靶向适体 AS1411 的四链体形成 DNA 序列，作为超分子载体，用于在宫颈癌细胞中选择性递送达那霉素衍生物（C、C 和 C）。设计的递药策略依赖于吖啶橙衍生物与 G-四链体（G4）结构的非共价结合。这种结合具有很强的结合强度，正如在 10-10 M 范围内获得的 K 值所表明的那样，导致 G4 结构的热稳定性增强，特别是对于 C。在胎牛血清中评估了所得超分子缀合物的稳定性，证明它们在 48 小时内抵抗血清核酸酶。先前的研究表明，测试的吖啶橙衍生物对宫颈癌（HeLa）细胞和非恶性细胞具有细胞毒性。然而，当与 AS1411 衍生物缀合时，游离配体对非恶性细胞的细胞毒性受到抑制。此外，缀合的 C 对 HeLa 癌细胞表现出增强的细胞毒性。共焦显微镜表明，两个 G4 序列似乎都与核仁蛋白共定位，表明它们能够识别和结合细胞表面的核仁蛋白。此外，结果证实了这些递药系统能够进入 HeLa 癌细胞并持续进行细胞内转运，尽管能够在细胞内解离以将 C 递送到核仁。总的来说，我们表明，AS1411 衍生的 G4 可以用作宫颈癌的潜在癌症药物输送系统。
