Cárdenas-Guerra Rosa E, Moreno-Gutierrez David S, Vargas-Dorantes Oscar de J, Espinoza Bertha, Hernandez-Garcia Armando
Laboratorio de Estudios sobre Tripanosomiasis, Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, México.
Laboratory of Biomolecular Engineering and Bionanotechnology, Departamento de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de México, Ciudad de México, México.
Nucleic Acid Ther. 2020 Dec;30(6):392-401. doi: 10.1089/nat.2020.0870. Epub 2020 Sep 9.
, which causes Chagas disease, is one of the most lacerating parasites in terms of health and social impacts. New approaches for its study and treatment are urgently needed since in more than 50 years only two drugs have been approved. Genetic approaches based on antisense oligonucleotides (AONs) are promising; however, to harness their full potential the development of effective carriers is paramount. Here, we report the use of an engineered virus-like protein C-B to transfect AONs into . Using gel electrophoresis, Dynamic Light Scattering, and atomic force microscopy, we found that C-B binds AONs and forms 10-25 nm nanoparticles (NPs), which are very stable when incubated in biological media, only releasing up to 25% of AON. Fluorescence microscopy and qPCR revealed that the NPs successfully delivered AONs into epimastigotes and reduced the expression of a target gene down to 68%. Importantly, the protein did not show cytotoxicity. The combination of high stability and capability to transfect and knock down gene expression without causing cell damage and death makes the protein C-B a promising starting point for the further development of safe and effective carriers to deliver AONs into for biological studies.
克氏锥虫会引发恰加斯病，就健康和社会影响而言，它是最具伤害性的寄生虫之一。由于在50多年里仅有两种药物获批，因此迫切需要针对其研究和治疗的新方法。基于反义寡核苷酸（AONs）的基因方法很有前景；然而，要充分发挥其潜力，开发有效的载体至关重要。在此，我们报告了使用一种工程化病毒样蛋白C-B将AONs转染到克氏锥虫中的情况。通过凝胶电泳、动态光散射和原子力显微镜，我们发现C-B能结合AONs并形成10 - 25纳米的纳米颗粒（NPs），这些纳米颗粒在生物介质中孵育时非常稳定，AON的释放量最高仅为25%。荧光显微镜和定量聚合酶链反应显示，这些纳米颗粒成功地将AONs递送至无鞭毛体中，并将靶基因的表达降低至68%。重要的是，该蛋白未表现出细胞毒性。高稳定性以及在不造成细胞损伤和死亡的情况下转染并敲低基因表达的能力，使得蛋白C-B成为进一步开发安全有效的载体以将AONs递送至克氏锥虫用于生物学研究的一个有前景的起点。
