The penetrating properties of the tumor homing peptide LyP-1 in model lipid membranes.

Cell-penetrating peptides (CPPs) have the property to cross the plasma membrane and enhance its permeability. CPPs were successfully used to deliver numerous cargoes such as drugs, proteins, nucleic acids, imaging and radiotherapeutic agents, gold and magnetic nanoparticles, or liposomes inside cells. Although CPPs were intensively investigated over the past 20 years, the exact molecular mechanisms of translocation across membranes are still controversial and vary from passive to active mechanisms. LyP-1 is a cyclic 9-amino-acids homing peptide that specifically binds to p32 receptors overexpressed in tumor cells. tLyP-1 peptide is the linear truncated form of LyP-1 and recognizes neuropilin (NRP) receptors expressed in glioma tumor tissue. Here, we investigate the interaction of the cyclic LyP-1 peptide and linear truncated tLyP-1 peptide with model plasma membrane in order to understand their passive, energy-independent mechanism of uptake. The experiments reveal that internalization of tLyP-1 peptides depends on membrane lipid composition. Inclusion of negatively charged phosphatidylserine (PS) or cone-shaped phosphatidylethanolamine (PE) lipids in the composition of giant unilamellar vesicles facilitates the membrane adsorption and direct penetration but without inducing pore formation in membranes. In contrast, cyclic LyP-1 peptide mostly accumulates on the membrane, with very low internalization, regardless of the lipid composition. Thus, the linear tLyP-1 peptide has enhanced penetrating properties compared with the cyclic LyP-1 peptide. Development of a mutant peptide containing higher number of arginine amino acids and preserving the homing properties of tLyP-1 may be a solution for new permeable peptides that facilitate the internalization in cells and further the endosomal escape as well.

The RH 421 styryl dye induced, pore model-dependent modulation of antimicrobial peptides activity in reconstituted planar membranes.

BACKGROUND: Antimicrobial agents, with different pore-formation mechanisms, may be differently influenced by alteration of the dipolar electric field of a lipid membrane. METHODS: By using electrophysiological measurements on reconstituted lipid membranes, we used alamethicin, melittin and magainin to report on how controlled manipulation of the membrane dipole potential by the styrylpyridinium dye RH 421 affects the kinetic and transport features of peptides within membranes. RESULTS: Our data demonstrate that the increase of the membrane dipole potential caused by RH 421 decreases the activity and single-channel conductance of alamethicin. Surprisingly, we found that RH 421 increases the activity of melittin and magainin, suggesting that RH 421 may contribute via electrostatic repulsions, among others, to an increase in the monolayer spontaneous curvature of the membrane. We propose that RH 421-induced dipole potential and membrane elasticity changes alter the peptide-induced channel dynamics, and the prevalence of one mechanism over the other for particular classes of peptides is dictated by the electrical and mechanical interactions which rule the pore-formation mechanism of such peptides. GENERAL SIGNIFICANCE: These results point to a novel paradigm in which electrical and mechanical effects promoted by chemicals which preferentially alter the electrostatics of the membrane, may be employed to help distinguish among various pore-formation mechanisms of membrane-permeabilizing peptides.

A Protein Nanopore-Based Approach for Bacteria Sensing.

We present herein a first proof of concept demonstrating the potential of a protein nanopore-based technique for real-time detection of selected Gram-negative bacteria (Pseudomonas aeruginosa or Escherichia coli) at a concentration of 1.2 × 108 cfu/mL. The anionic charge on the bacterial outer membrane promotes the electrophoretically driven migration of bacteria towards a single α-hemolysin nanopore isolated in a lipid bilayer, clamped at a negative electric potential, and followed by capture at the nanopore's mouth, which we found to be described according to the classical Kramers' theory. By using a specific antimicrobial peptide as a putative molecular biorecognition element for the bacteria used herein, we suggest that the detection system can combine the natural sensitivity of the nanopore-based sensing techniques with selective biological recognition, in aqueous samples, and highlight the feasibility of the nanopore-based platform to provide portable, sensitive analysis and monitoring of bacterial pathogens.