The most commonly studied of the cell-penetrating peptides (CPP) is "penetratin" (pAntp), which functions as a carrier (vector), even for large hydrophilic (cargo) molecules. pAntp originates from the third helix of the Antennapedia homeodomain protein. The peptide is known to interact with negatively charged phospholipid vesicles, which leads to induction of secondary structure. In the present study, circular dichroism (CD) spectroscopy has been used to characterize the different secondary structures induced upon interaction with small unilamellar vesicles (SUVs) from mixtures of zwitterionic 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG). The interaction was monitored using an electron paramagnetic resonance (EPR) spin probe attached to the peptide, and the intrinsic fluorophore (tryptophan). We measured the secondary structure as a function of surface charge density, total lipid-to-peptide (L/P) molar ratio, and salt concentration, for completely bound peptide. With vesicles from POPG/POPC in a molar ratio below 30:70, at a high L/P, the peptide adopts a mainly helical conformation. Increasing the charge density, at the same L/P, promotes a higher degree of beta-structure. At a fixed charge density, reducing the L/P also results in an alpha-->beta structure conversion. Hence, low membrane surface charge density and low pAntp concentration both favor a mainly helical conformation, while high charge density and pAntp concentration promote a dominating beta-structure. We conclude that pAntp, when residing at the surface of a membrane, is chameleon-like in terms of its induced structure.

Certain short peptides, which are able to translocate across cell membranes with a low lytic activity, can be useful as carriers (vectors) for hydrophilic molecules. We have studied three such cell penetrating peptides: pAntp ('penetratin'), pIsl and transportan. pAntp and pIsl originate from the third helix of homeodomain proteins (Antennapedia and Isl-1, respectively). Transportan is a synthetic chimera (galanin and mastoparan). The peptides in the presence of various phospholipid vesicles (neutral and charged) and SDS micelles have been characterized by spectroscopic methods (fluorescence, EPR and CD). The dynamics of pAntp were monitored using an N-terminal spin label. In aqueous solution, the CD spectra of the three peptides show secondary structures dominated by random coil. With phospholipid vesicles, neutral as well as negatively charged, transportan gives up to 60% alpha-helix. pAntp and pIsl bind significantly only to negatively charged vesicles with an induction of around 60% beta-sheet-like secondary structure. With all three peptides, SDS micelles stabilize a high degree of alpha-helical structure. We conclude that the exact nature of any secondary structure induced by the membrane model systems is not directly correlated with the common transport property of these translocating peptides.

The practical application of nanoparticles (NPs) as chemotherapeutic drug delivery systems is often hampered by issues such as poor circulation stability and targeting inefficiency. Here, we have utilized a simple approach to prepare biocompatible and biodegradable pHresponsive hybrid NPs that overcome these issues. The NPs consist of a drug-loaded polylactic-co-glycolic acid (PLGA) core covalently 'wrapped' with a crosslinked bovine serum albumin (BSA) shell designed to minimize interactions with serum proteins and macrophages that inhibit target recognition. The shell is functionalized with the acidity-triggered rational membrane (ATRAM) peptide to facilitate internalization specifically into cancer cells within the acidic tumor microenvironment. Following uptake, the unique intracellular conditions of cancer cells degrade the NPs, thereby releasing the chemotherapeutic cargo. The drugloaded NPs showed potent anticancer activity in vitro and in vivo while exhibiting no toxicity to healthy tissue. Our results demonstrate that the ATRAM-BSA-PLGA NPs are a promising targeted cancer drug delivery platform.

Islet amyloid polypeptide (IAPP) is a peptide hormone cosecreted with insulin by pancreatic β-cells. In type II diabetes, IAPP aggregates in a process that is associated with β-cell dysfunction and loss of β-cell mass. The relationship between IAPP's conformational landscape and its capacity to mediate cell death remains poorly understood. We have addressed these unknowns by comparing the cytotoxic effects of sequence variants with differing α-helical and amyloid propensities. IAPP was previously shown to oligomerize cooperatively on binding to lipid bilayers. Here, comparable transitions are evident in cell culture and are associated with a change in subcellular localization to the mitochondria under toxic conditions. Notably, we find that this toxic gain of function maps to IAPP's capacity to adopt aggregated membrane-bound α-helical, and not β-sheet, states. Our findings suggest that upon α-helical mediated oligomerization, IAPP acquires cell-penetrating peptide (CPP) properties, facilitating access to the mitochondrial compartment, resulting in its dysfunction.

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