Physicochemical Mechanism for the Enhanced Ability of Lipid Membrane Penetration of Polyarginine

Abstract

Arginine-rich, cell-penetrating peptides (e.g., Tat-peptide, penetratin, and polyarginine) are used to carry therapeutic molecules such as oligonucleotides, DNA, peptides, and proteins across cell membranes. Two types of processes are being considered to cross the cell membranes: one is an endocytic pathway, and another is an energy-independent, nonendocytic pathway. However, the latter is still not known in detail. Here, we studied the effects of the chain length of polyarginine on its interaction with an anionic phospholipid large unilamellar vesicle (LUV) or a giant vesicle using poly-l-arginine composed of 69 (PLA69), 293 (PLA293), or 554 (PLA554) arginine residues, together with octaarginine (R8). ζ-potential measurements confirmed that polyarginine binds to LUV via electrostatic interactions. Circular dichroism analysis demonstrated that the transition from the random coil to the α-helix structure upon binding to LUV occurred for PLA293 and PLA554, whereas no structural change was observed for PLA69 and R8. Fluorescence studies using membrane probes revealed that the binding of polyarginine to LUV affects the hydration and packing of the membrane interface region, in which the degree of membrane insertion is greater for the longer polyarginine. Isothermal titration calorimetry measurements demonstrated that although the binding affinity (i.e., the Gibbs free energy of binding) per arginine residue is similar among all polyarginines the contribution of enthalpy to the energetics of binding of polyarginine increases with increasing polymer chain length. In addition, confocal laser scanning microscopy showed that all polyarginines penetrate across giant vesicle membranes, and the order of the amount of membrane penetration is R8 ≈ PLA69 < PLA293 ≈ PLA554. These results suggest that the formation of α-helical structure upon lipid binding drives the insertion of polyarginine into the membrane interior, which appears to enhance the membrane penetration of polyarginine.

Cell uptake and cytotoxicity of a novel cyclometalated iridium(III) complex and its octaarginine peptide conjugate

“…S5 in supplementary material. The conjugation of octaarginine peptide has been shown previously [2,31,39,53] to enhance cellular uptake of metal complex dyes without the requirement for membrane permeablization and this is further exemplified here in both cell lines. Dye uptake was measured by live-cell time course analysis and quantified by measuring the dye intensity in each cell type at the same time points after incubation wherein the concentration of each dye applied to the cell medium was identical.…”

Defense peptides: recent developments

“…In de novo designed AMPs, Arg and Lys are popular choices, as they are protonated under physiological conditions. Although both amino acids have comparable charge and Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 7/27/15 1:57 AM hydrophobicity, Lys primary amine interacts less electrostatically with PLs than the Arg guanidium group, which could explain the stronger binding affinity of Arg-containing peptides to lipid membranes than that of Lys-containing counterparts (65)(66)(67).…”

Is There Anybody in There? On The Mechanisms of Wall Crossing of Cell Penetrating Peptides

“…ITC measurements on different CPPs reported binding affinities in the M range that were enthalpically favored and associated with positive heat capacities indicating that electrostatics are important for the interaction [27][28][29][30][31][32]. Using calorimetry, plasmon resonance and fluorescent studies, the presence of negatively charged lipids was shown to be essential or to increase the affinity of TAT [33][34][35], oligoarginine peptides [27]; [29,30,31,35] and also of penetratin [36,[37][38][39][40]; [28]. Plasmon waveguide resonance (PWR) studies have reported an increase of ~20-100 fold in affinity when anionic lipids where included in the membrane [38].…”