Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal.
FEBS J. 2014 Jan;281(1):191-215. doi: 10.1111/febs.12587. Epub 2013 Nov 28.
Cell penetrating peptides (CPPs) can be used as drug delivery systems for different therapeutic molecules. In this work two novel CPPs, pepR and pepM, designed from two domains of the dengue virus (DENV) capsid protein, were studied for their ability to deliver nucleic acids into cells as non-covalently bound cargo. Translocation studies were performed by confocal microscopy in HepG2, BHK and HEK cell lineages, astrocytes and peripheral blood mononuclear cells. Combined studies in HepG2 cells, astrocytes and BHK cells, at 4 and 37 °C or using specific endocytosis inhibitors, revealed that pepR and pepM use distinct internalization routes: pepM translocates lipid membranes directly, while pepR uses an endocytic pathway. To confirm these results, a methodology was developed to monitor the translocation kinetics of both peptides by real-time flow cytometry. Kinetic constants were determined, and the amount of nucleic acids delivered was estimated. Additional studies were performed in order to understand the molecular bases of the peptide-mediated translocation. Peptide-nucleic acid and peptide-lipid membrane interactions were studied quantitatively based on the intrinsic fluorescence of the peptides. pepR and pepM bound ssDNA to the same extent. Partition studies revealed that both peptides bind preferentially to anionic lipid membranes, adopting an α-helical conformation. However, fluorescence quenching studies suggest that pepM is deeply inserted into the lipid bilayer, in contrast with pepR. Moreover, only pepM is able to promote the fusion and aggregation of vesicles composed of zwitterionic lipids. Altogether, the results show that DENV capsid protein derived peptides serve as good templates for novel CPP-based nucleic acid delivery strategies, defining different routes for cell entry.
细胞穿透肽 (CPP) 可作为不同治疗分子的药物传递系统。在这项工作中，研究了两种新型 CPP pepR 和 pepM，它们是从登革热病毒 (DENV) 衣壳蛋白的两个结构域设计而来，用于将核酸作为非共价结合的货物递送到细胞中。通过共聚焦显微镜在 HepG2、BHK 和 HEK 细胞系、星形胶质细胞和外周血单核细胞中进行转位研究。在 HepG2 细胞、星形胶质细胞和 BHK 细胞中进行的联合研究，在 4 和 37°C 下或使用特定的内吞作用抑制剂，表明 pepR 和 pepM 使用不同的内化途径： pepM 直接穿过脂质膜，而 pepR 使用内吞途径。为了证实这些结果，开发了一种方法来通过实时流动细胞术监测这两种肽的转位动力学。确定了动力学常数，并估计了递送到的核酸量。进行了额外的研究以了解肽介导的转位的分子基础。基于肽的固有荧光，定量研究了肽-核酸和肽-脂质膜相互作用。pepR 和 pepM 与 ssDNA 的结合程度相同。分配研究表明，两种肽都优先与阴离子脂质膜结合，采用α-螺旋构象。然而，荧光猝灭研究表明 pepM 深深地插入脂质双层中，与 pepR 相反。此外，只有 pepM 能够促进由两性离子脂质组成的囊泡的融合和聚集。总之，这些结果表明 DENV 衣壳蛋白衍生肽可作为新型 CPP 基于核酸传递策略的良好模板，定义了不同的细胞进入途径。
