Conjugation of oligo-His peptides to magnetic γ-Fe2O3@SiO2 core-shell nanoparticles promotes their access to the cytosol

The endosomal entrapment of functional nanoparticles is a severe limitation to their use for biomedical applications. In the case of magnetic nanoparticles (MNPs), this entrapment leads to poor heating efficiency for magnetic hyperthermia and suppresses the possibility to manipulate them in the cytosol. Current strategies to limit their entrapment are based on their functionalization with cell-penetrating peptides in order to promote their translocation directly across the cell membrane or their endosomal escape. However, these strategies suffer from potential release of free peptides in cell and to the best of our knowledge there is currently a lack of effective methods for the cytosolic delivery of MNPs after incubation with cells. Herein, we report the conjugation of fluorescently labelled cationic peptides to γ-Fe2O3@SiO2 core-shell nanoparticles by click chemistry to improve MNP access to the cytosol. We compare the effect of Arg9 and His4 peptides. On one hand, Arg9 is a classical cell-penetrating peptide, able to enter cells by direct translocation and on the other hand, it has been demonstrated that sequences rich in histidine residues promote endosomal escape, most probably by the proton sponge effect. The methodology developed allows to have a high co-localization of the peptides and core-shell nanoparticles in cells and to attest that the grafting onto nanoparticles of peptides rich in histidine promotes NP access to the cytosol. The endosomal escape was confirmed by a calcein leakage assay and by ultrastructural analysis in transmission electron microscopy. No toxicity of the nanoparticles functionalized with peptides was found. We show that our conjugation strategy is compatible with the addition of multiple substrates and can thus be used for the delivery of cytoplasm-targeted therapeutics.

Folding Behaviour and Antibacterial Activity of Ionic Complementary Peptide EAK-16

Aim: A major challenge in the development of new antibiotics is the biocompatibility within biological environment. Ionic complementary peptide (EAK-16) from amyloid protein, have the ability to adopt secondary structure conformation at membrane interfaces. This study aimed to investigate the effect of membrane on EAK-16 peptide folding and their antibacterial applications. Methodology: We studied secondary structural conformation of EAK-16 using circular dichroism (CD) spectroscopy in an aqueous environment and at membrane bilayers interfaces. Initially, the antibacterial efficacy was investigated against both Gram-positive and Gram-negative bacteria. Membrane mimicking models were synthesised with dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS) lipid vesicles using calcein leakage assay. Results: EAK-16 showed transition in secondary structural conformation. In aqueous environment, it was predominantly β-sheets and at membrane interfaces, it was mainly α-helical. EAK-16 peptide was highly active against bacteria (at minimum concentration applied) and membrane leakage was found to be > 60%. This effect was confirmed with both anionic lipids (DMPS) and neutral lipids (DMPC). The helical transition of EAK-16 could be a major factor to disrupt the membrane and bacterial death Conclusion: The secondary structural conformation and calcein leakage data suggest that EAK-16 has potential to kill bacteria by adopting helical tilted conformation and membrane perturbation via lysis. This study revealed structure-function relationship of peptide and lipid bilayers to further investigate the mode of pore formation and mode of action of EAK-16 in membrane perturbation and antibacterial efficacy.

Conformational Changes of Anoplin, W-MreB1–9, and (KFF)3K Peptides near the Membranes

Many peptides interact with biological membranes, but elucidating these interactions is challenging because cellular membranes are complex and peptides are structurally flexible. To contribute to understanding how the membrane-active peptides behave near the membranes, we investigated peptide structural changes in different lipid surroundings. We focused on two antimicrobial peptides, anoplin and W-MreB1–9, and one cell-penetrating peptide, (KFF)3K. Firstly, by using circular dichroism spectroscopy, we determined the secondary structures of these peptides when interacting with micelles, liposomes, E. coli lipopolysaccharides, and live E. coli bacteria. The peptides were disordered in the buffer, but anoplin and W-MreB1–9 displayed lipid-induced helicity. Yet, structural changes of the peptide depended on the composition and concentration of the membranes. Secondly, we quantified the destructive activity of peptides against liposomes by monitoring the release of a fluorescent dye (calcein) from the liposomes treated with peptides. We observed that only for anoplin and W-MreB1–9 calcein leakage from liposomes depended on the peptide concentration. Thirdly, bacterial growth inhibition assays showed that peptide conformational changes, evoked by the lipid environments, do not directly correlate with the antimicrobial activity of the peptides. However, understanding the relation between peptide structural properties, mechanisms of membrane disruption, and their biological activities can guide the design of membrane-active peptides.

Study of the Interaction of a Novel Semi-Synthetic Peptide with Model Lipid Membranes

Most linear peptides directly interact with membranes, but the mechanisms of interaction are far from being completely understood. Here, we present an investigation of the membrane interactions of a designed peptide containing a non-natural, synthetic amino acid. We selected a nonapeptide that is reported to interact with phospholipid membranes, ALYLAIRKR, abbreviated as ALY. We designed a modified peptide (azoALY) by substituting the tyrosine residue of ALY with an antimicrobial azobenzene-bearing amino acid. Both of the peptides were examined for their ability to interact with model membranes, assessing the penetration of phospholipid monolayers, and leakage across the bilayer of large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs). The latter was performed in a microfluidic device in order to study the kinetics of leakage of entrapped calcein from the vesicles at the single vesicle level. Both types of vesicles were prepared from a 9:1 (mol/mol) mixture of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1′-rac-glycerol). Calcein leakage from the vesicles was more pronounced at a low concentration in the case of azoALY than for ALY. Increased vesicle membrane disturbance in the presence of azoALY was also evident from an enzymatic assay with LUVs and entrapped horseradish peroxidase. Molecular dynamics simulations of ALY and azoALY in an anionic POPC/POPG model bilayer showed that ALY peptide only interacts with the lipid head groups. In contrast, azoALY penetrates the hydrophobic core of the bilayers causing a stronger membrane perturbation as compared to ALY, in qualitative agreement with the experimental results from the leakage assays.

Facile Mixing of Phospholipids Promotes Self-Assembly of Low-Molecular-Weight Biodegradable Block Co-Polymers into Functional Vesicular Architectures

In this work, we have used low-molecular-weight (PEG12-b-PCL6, PEG12-b-PCL9 or PEG16-b-PLA38; MW, 1.25–3.45 kDa) biodegradable block co-polymers to construct nano- and micron-scaled hybrid (polymer/lipid) vesicles, by solvent dispersion and electroformation methods, respectively. The hybrid vesicles exhibit physical properties (size, bilayer thickness and small molecule encapsulation) of a vesicular boundary, confirmed by cryogenic transmission electron microscopy, calcein leakage assay and dynamic light scattering. Importantly, we find that these low MW polymers, on their own, do not self-assemble into polymersomes at nano and micron scales. Using giant unilamellar vesicles (GUVs) model, their surface topographies are homogeneous, independent of cholesterol, suggesting more energetically favorable mixing of lipid and polymer. Despite this mixed topography with a bilayer thickness similar to that of a lipid bilayer, variation in surface topology is demonstrated using the interfacial sensitive phospholipase A2 (sPLA2). The biodegradable hybrid vesicles are less sensitive to the phospholipase digestion, reminiscent of PEGylated vesicles, and the degree of sensitivity is polymer-dependent, implying that the nano-scale surface topology can further be tuned by its chemical composition. Our results reveal and emphasize the role of phospholipids in promoting low MW polymers for spontaneous vesicular self-assembly, generating a functional hybrid lipid-polymer interface.

A Molecular Biophysical Approach to Diclofenac Topical Gastrointestinal Damage

Diclofenac (DCF), the most widely consumed non-steroidal anti-inflammatory drug (NSAID) worldwide, is associated with adverse typical effects, including gastrointestinal (GI) complications. The present study aims to better understand the topical toxicity induced by DCF using membrane models that mimic the physiological, biophysical, and chemical environments of GI mucosa segments. For this purpose, phospholipidic model systems that mimic the GI protective lining and lipid models of the inner mitochondrial membrane were used together with a wide set of techniques: derivative spectrophotometry to evaluate drug distribution at the membrane; steady-state and time-resolved fluorescence to predict drug location at the membrane; fluorescence anisotropy, differential scanning calorimetry (DSC), dynamic light scattering (DLS), and calcein leakage studies to evaluate the drug-induced disturbance on membrane microviscosity and permeability; and small- and wide-angle X-ray scattering studies (SAXS and WAXS, respectively), to evaluate the effects of DCF at the membrane structure. Results demonstrated that DCF interacts chemically with the phospholipids of the GI protective barrier in a pH-dependent manner and confirmed the DCF location at the lipid headgroup region, as well as DCF’s higher distribution at mitochondrial membrane contact points where the impairment of biophysical properties is consistent with the uncoupling effects reported for this drug.

Antimicrobial Mechanism of Monocaprylate

ABSTRACTMonoglyceride esters of fatty acids occur naturally and encompass a broad spectrum of antimicrobial activity. Monocaprylate is generally regarded as safe (GRAS) and can function both as an emulsifier and as a preservative in food. However, knowledge about its mode of action is lacking. The aim of this study was therefore to elucidate the mechanism behind monocaprylate's antimicrobial effect. The cause of cell death inEscherichia coli,Staphylococcus xylosus, andZygosaccharomyces bailiiwas investigated by examining monocaprylate's effect on cell structure, membrane integrity, and its interaction with model membranes. Changes in cell structure were visible by atomic force microscopy (AFM), and propidium iodide staining showed membrane disruption, indicating the membrane as a site of action. This indication was confirmed by measuring calcein leakage from membrane vesicles exposed to monocaprylate. AFM imaging of supported lipid bilayers visualized the integration of monocaprylate into the liquid disordered, and not the solid ordered, phase of the membrane. The integration of monocaprylate was confirmed by quartz crystal microbalance measurements, showing an abrupt increase in mass and hydration of the membrane after exposure to monocaprylate above a threshold concentration. We hypothesize that monocaprylate destabilizes membranes by increasing membrane fluidity and the number of phase boundary defects. The sensitivity of cells to monocaprylate will therefore depend on the lipid composition, fluidity, and curvature of the membrane.

Peptide-Lipid Huge Toroidal Pore, a New Antimicrobial Mechanism Mediated by a Lactococcal Bacteriocin, Lacticin Q

ABSTRACT Lacticin Q is a pore-forming bacteriocin produced by Lactococcus lactis QU 5, and its antimicrobial activity is in the nanomolar range. Lacticin Q induced calcein leakage from negatively charged liposomes. However, no morphological changes in the liposomes were observed by light scattering. Concomitantly with the calcein leakage, lacticin Q was found to translocate from the outer to the inner leaflet of the liposomes, after it initially bound to the membrane within 2 s. Lacticin Q also induced lipid flip-flop. These results reveal that the antimicrobial mechanism of lacticin Q can be described by the toroidal pore model. This is the first report of a bacteriocin of gram-positive bacteria that forms a toroidal pore. From liposomes, lacticin Q leaked fluorescence-labeled dextran with a diameter of 4.6 nm. In addition, lacticin Q caused the leakage of small proteins, such as the green fluorescent protein, from live bacterial cells. There are no other reports of antimicrobial peptides that exhibit protein leakage properties. The proposed pore formation model of lacticin Q is as follows: (i) quick binding to outer membrane leaflets; (ii) the formation of at least 4.6-nm pores, causing protein leakage with lipid flip-flop; and (iii) the migration of lacticin Q molecules from the outer to the inner membrane leaflets. Consequently, we termed the novel pore model in the antimicrobial mechanism of lacticin Q a “huge toroidal pore.”