Abstract
Cell-penetrating peptide (CPP) can directly penetrate the cytosol (cytolysis) and is expected to be a potent vector for a drug delivery system (DDS). Although there is general agreement that CPP cytolysis is related to dynamic membrane deformation, a distinctive process has yet to be established. Here, we report the key process and factors controlling CPP cytolysis. To elucidate the task, we have introduced trypsin digestion of adsorbed CPP onto giant unilamellar vesicle (GUV) to quantify the adsorption and internalization (cytolysis) separately. Also, the time-course analysis was introduced for the geometric calculation of adsorption and internalization amount per lipid molecule consisting of GUV. As a result, we found that adsorption and internalization assumed to occur successively by CPP molecule come into contact with membrane lipid. Adsorption is quick to saturate within 10 min, while cytolysis of each CPP on the membrane follows successively. After adsorption is saturated, cytolysis proceeds further linearly by time with a different rate constant that is dependent on the osmotic pressure. We also found that temperature and lipid composition influence cytolysis by modulating lipid mobility. The electrolyte in the outer media is also affected as a chemical mediator to control CPP cytolysis by following the Hoffmeister effect for membrane hydration. These results confirmed the mechanism of cytolysis as temporal and local phase transfer of membrane lipid from Lα to Mesh1, which has punctured bilayer morphologies.
Highlights
Cells are the basic unit of life separated from the environment by a lipid membrane, and life is a system in which molecules come in and out while the system keeps its structures and functions consistent under homeostatic control
The translocation of cell-penetrating peptide (CPP) to giant unilamellar vesicle (GUV) reported in the past were total amounts of CPP trapped by GUV and there is no clarification of adsorption and cytolysis [10,11,12,13,15,16,18]
There is general agreement that CPP cytolysis is related to dynamic membrane deformation, a distinctive process has yet to be established
Summary
Cells are the basic unit of life separated from the environment by a lipid membrane, and life is a system in which molecules come in and out while the system keeps its structures and functions consistent under homeostatic control. There are two known ways of internalization of CPP—one is endocytosis, by which CPP is entrapped in the endosome into the cytosol, and the other is cytolysis as a direct translocation of CPP through the lipid membrane [1,2]. Futaki et al reported that key element of the CPP’s cellular uptake is the richness of cationic amino acids especially arginine in the peptide molecule [2]. They found that the oligomer of Arg, such as R8 and R9, behaves just as the TAT peptide for various cultured cells and proposed the existence of a common internalization mechanism [3]. Wender et al further generalized the basic unit as the number and array of guanidium groups even for the synthetic oligomers such as a peptoid molecule [4]
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