Freulon I, Roche A C, Monsigny M, Mayer R
Centre de Biophysique Moléculaire, CNRS and Université d'Orléans, UPR 4301, Glycobiologie, Vectorologie et trafic intracellulaire, Rue Charles Sadron, F-45071 Orléans Cedex 2, France.
Biochem J. 2001 Mar 15;354(Pt 3):671-9. doi: 10.1042/0264-6021:3540671.
The use of antisense oligonucleotides as putative therapeutic agents is limited by their poor delivery into the cytosol and/or the nucleus because they are not able to efficiently cross lipid bilayers. To circumvent this pitfall, anionic amphipathic peptides derived from the influenza virus fusogenic peptide have been used to destabilize membranes in an acidic environment. In this paper, we compare the ability of a monomeric and a dimeric peptide to introduce oligonucleotides into the cytosol and nuclei of several types of cultured cells. Cells incubated at pH 6.2 or at a slightly lower pH in the presence of the monomeric peptide but not the dimeric peptide were efficiently permeabilized. The location of fluorescent derivatives of peptides and of oligonucleotides was assessed by confocal microscopy. Both the peptides and oligonucleotides remained entrapped in vesicular compartments at neutral pH; at acidic pH, oligonucleotides in the presence of the monomeric peptide were mainly in the nucleus, while in the presence of the dimeric peptide they co-localized with the peptide into vesicles. The data are interpreted on the basis of the spectroscopic behaviour of monomeric and dimeric peptides in relation to the environmental pH.
反义寡核苷酸作为潜在治疗剂的应用受到其向细胞质和/或细胞核递送效率低下的限制，因为它们无法有效穿过脂质双层。为了克服这一缺陷，源自流感病毒融合肽的阴离子两亲性肽已被用于在酸性环境中破坏膜的稳定性。在本文中，我们比较了单体肽和二聚体肽将寡核苷酸导入几种培养细胞的细胞质和细胞核的能力。在pH 6.2或略低pH下，在单体肽存在但二聚体肽不存在的情况下孵育的细胞被有效通透化。通过共聚焦显微镜评估肽和寡核苷酸荧光衍生物的位置。在中性pH下，肽和寡核苷酸都被困在囊泡区室中；在酸性pH下，在单体肽存在下寡核苷酸主要位于细胞核中，而在二聚体肽存在下它们与肽共定位于囊泡中。根据单体肽和二聚体肽相对于环境pH的光谱行为对数据进行了解释。
