The activity of membrane-active peptides/proteins (MAPs) involves interactions with lipid bilayer regions of cell membranes. For example, antimicrobial peptides, lytic peptides, pore-forming toxins, lipidated peptides, and cell-penetrating peptides are all MAPs. Most MAPs induce damage in cell membranes/lipid bilayers, such as nanopore formation. Various methods have been employed to examine the interactions between MAPs and lipid bilayers, as well as MAP-induced membrane damage. Methods using giant unilamellar vesicles (GUVs) are particularly versatile techniques because they provide useful information regarding both MAP–lipid bilayer interactions and MAP activities such as membrane damage. GUV studies have revealed many aspects of elementary processes of MAP-induced membrane damage and their correlations, thus clarifying the mechanisms of MAPs-induced membrane damage. Here, we focus on GUV-based studies of MAP-induced nanopore formation in lipid bilayers. First, we review the binding of MAPs to the lipid bilayers. Second, we review the rate of MAP-induced nanopore formation and the rate of membrane permeation of fluorescent probes through the nanopores. Third, we review the relationships between several elementary processes involved in MAP-induced nanopore formation (i.e., binding of MAPs, nanopore formation, translocation of MAPs across lipid bilayers) and factors that induce membrane instability to facilitate nanopore formation. The pre-pore model of translocation of MAPs across lipid bilayers is also reviewed. Fourth, we review the effects of membrane tension, membrane potential, and lipid composition on MAP-induced formation of nanopores and their stability. Finally, we describe our perspectives on future GUV-based studies of MAP-induced nanopore formation.

Trp is unique among the amino acids since it is involved in many different types of noncovalent interactions such as electrostatic and hydrophobic ones, but also in π-π, π-cation, π-anion and π-ion pair interactions. In membranotropic peptides and proteins, Trp locates preferentially at the water-membrane interface. In antimicrobial or cell-penetrating peptides (AMPs and CPPs respectively), Trp is well-known for its strong role in the capacity of these peptides to interact and affect the membrane organisation of both bacteria and animal cells at the level of the lipid bilayer. This essential amino acid can however be involved in other types of interactions, not only with lipids, but also with other membrane partners, that are crucial to understand the functional roles of membranotropic peptides. This review is focused on this latter less known role of Trp and describes in details, both in qualitative and quantitative ways: (i) the physico-chemical properties of Trp; (ii) its effect in CPP internalisation; (iii) its importance in AMP activity; (iv) its role in the interaction of AMPs with glycoconjugates or lipids in bacteria membranes and the consequences on the activity of the peptides; (v) its role in the interaction of CPPs with negatively charged polysaccharides or lipids of animal membranes and the consequences on the activity of the peptides. We intend to bring highlights of the physico-chemical properties of Trp and describe its extensive possibilities of interactions, not only at the well-known level of the lipid bilayer, but with other less considered cell membrane components, such as carbohydrates and the extracellular matrix. The focus on these interactions will allow the reader to reevaluate reported studies. Altogether, our review gathers dedicated studies to show how unique are Trp properties, which should be taken into account to design future membranotropic peptides with expected antimicrobial or cell-penetrating activity.

Biophysical studies have a very high impact on the understanding of internalization, molecular mechanisms, interactions, and localization of CPPs and CPP/cargo conjugates in live cells or in vivo. Biophysical studies are often first carried out in test-tube set-ups or in vitro, leading to the complicated in vivo systems. This review describes recent studies of CPP internalization, mechanisms, and localization. The multiple methods in these studies reveal different novel and important aspects and define the rules for CPP mechanisms, hopefully leading to their improved applicability to novel and safe therapies.