Targeting lipodisks enable selective delivery of anticancer peptides to tumor cells.

Issues concerning non-specificity, degradation and hemolysis severely hamper the development of membranolytic amphiphilic peptides into safe and efficient anticancer agents. To increase the therapeutic potential, we have previously developed a strategy based on formulation of the peptides in biocompatible nanosized lipodisks. Studies using melittin as model peptide show that the proteolytic degradation and hemolytic effect of the peptide are substantially reduced upon loading in lipodisks. Here, we explored the possibilities to increase the specificity and boost the cytotoxicity of melittin to tumor cells by use of targeting lipodisk. We demonstrate that small (~20 nm) EGF-targeted lipodisks can be produced and loaded with substantial amounts of peptide (lipid/peptide molar ratio >7) by means of a simple and straightforward preparation protocol. In vitro cell studies confirm specific binding of the peptide-loaded disks to tumor cells and suggest that cellular internalization of the disks results in a significantly improved cell-killing effect.

Characterizing and Controlling the Loading and Release of Cationic Amphiphilic Peptides onto and from PEG-Stabilized Lipodisks.

Recent studies have identified PEG-stabilized lipid nanodisks (lipodisks) as promising carriers for cationic amphiphilic peptides with antimicrobial and anticancer activity. Using fluorimetric and nanogravimetric methods, we have in this work characterized the parameters describing and controlling the binding of three selected peptides (melittin, LL37, and magainin 2) onto lipodisks. It was found that the affinity of melittin for lipodisks is independent of the disk size and rim charge. On the other hand, the number of binding sites is strongly dependent on both parameters, with the highest loading being obtained for small disks with a negatively charged rim. An optimized composition of the lipodisks was utilized to study the loading of antimicrobial peptides magainin 2 and human LL37. It was observed that although magainin 2 can be loaded in large amounts, it is released very fast upon dilution, which limits future therapeutic applications. In contrast, LL37 can be loaded at relevant concentrations and the formulation is stable. This opens up for applications of LL37-loaded lipodisks as antibiotics and in anticancer treatments.

Label-free characterization of peptide-lipid interactions using immobilized lipodisks.

Lipodisks, planar lipid bilayer structures stabilized by PEG-ylated lipids, were in the present study covalently bound and immobilized onto sensors for quartz crystal microbalance with dissipation monitoring (QCM-D) studies. It is shown that the modified sensors can be used to characterize the interaction of lipodisks with α-helical amphiphilic peptides with an accuracy similar to that obtained with well established fluorimetric approximations. The method presented has the great advantage that it can be used with peptides in their native form even if no fluorescent residues are present. The potential of the method is illustrated by determining the parameters describing the association of melittin, mastoparan X, and mastoparan with immobilized lipodisks. Both thermodynamic and kinetic analyses are possible. The presented method constitutes a useful tool for fundamental studies of peptide-membrane interactions and can also be applied to optimize the design of lipodisks, for example, for sustained release of antimicrobial peptides in therapeutic applications.

Polyethylene glycol-stabilized lipid disks as model membranes in interaction studies based on electrokinetic capillary chromatography and quartz crystal microbalance.

Distearoylphosphatidylcholine (DSPC)/cholesterol/distearoylphosphatidylethanolamine (DSPE)-polyethylene glycol 5000 [PEG(5000)] lipid disks, mimicking biological membranes, were used as pseudostationary phase in partial filling electrokinetic capillary chromatography (EKC) to study interactions between pharmaceuticals and lipid disks. Capillaries were coated either noncovalently with a poly(1-vinylpyrrolidone)-based copolymer or covalently with polyacrylamide to mask the negative charges of the fused-silica capillary wall and to minimize interactions between positively charged pharmaceuticals and capillary wall. Although the noncovalent copolymer coating method was faster, better stability of the covalent polyacrylamide coating at physiological pH 7.4 made it more reliable in partial filling EKC studies. Migration times of pharmaceuticals were proportional to the amount of lipids in the pseudostationary phase, and partition coefficients were successfully determined. Because the capillary coatings almost totally suppressed the electroosmotic flow, it was not practical to use the EKC-based method for partition studies involving large molecules with low mobilities. Hence, the applicability of the biomembrane mimicking lipid disks for interactions studies with large molecules was verified by the quartz crystal microbalance technique. Biotinylated lipid disks were then immobilized on streptavidin-coated sensor chip surface, and interactions with a high-molecular-mass molecule, lysozyme, were studied. Cryo-transmission electron microscopy and asymmetrical flow field-flow fractionation were used to clarify the sizes of lipid disks used.

PEG-stabilized lipid disks as carriers for amphiphilic antimicrobial peptides.

Antimicrobial peptides hold potential as a possible alternative, or complement, to conventional antibiotics but new, safe and efficient means are needed for formulation and administration of the peptides. In this study we have investigated the utility of a novel type of lipid particles, the polyethylene glycol-stabilized lipid disks, as carriers for the model peptide melittin. The structural integrity of the carrier particle when loaded with the peptide was investigated using cryo-transmission electron microscopy. Liposome leakage upon addition of the peptide-lipid disks was monitored as a means to verify the membrane lytic effect of the formulation. The susceptibility of melittin to tryptic digestion was studied and compared in the absence and presence of lipid disks. Finally, the antibacterial effect of the peptide-lipid disk formulation was compared to that of free melittin after both single and repeated exposure to Escherichia coli. The results show that melittin can redistribute from the disk into a new host membrane and that formulation in the disks does not compromise melittin's membrane permeabilizing ability. Further, the peptide was found to be fully protected against degradation when bound to the disks. Time-kill experiments revealed that all the antibacterial effect of melittin administered in free form was gone after a single exposure to E. coli. In contrast, the disk formulation showed significant cell-killing effect also upon a second exposure to bacteria, indicating an extended release of peptide from the lipid disks. These results suggest that the lipid disks constitute a new class of promising carriers for peptide antibiotics.

Effect of α-helical peptides on liposome structure: a comparative study of melittin and alamethicin.

Cryo-transmission electron microscopy was used in combination with turbidity and leakage measurements to explore and compare the membrane perturbing effects of melittin and alamethicin on POPC-based liposomes of varying composition. The results show that the two peptides, despite their differences in physico-chemical properties and proposed mode of action, induce similar structural effects on the liposomes. Importantly, whereas low peptide concentrations leave pure POPC liposomes intact and seemingly unperturbed, POPC liposomes supplemented with 40 mol.% cholesterol change their shape, rupture and fuse in response to the addition of both melittin and alamethicin. In the case of alamethicin, but not melittin, fusion is effectively prevented by inclusion of 10 mol.% POPG in the liposome membranes. By means of a competitive binding assay we furthermore show that alamethicin, in line with earlier findings for melittin, possess high affinity for positively curved lipid surfaces. Moreover, results from the present study show that magainin 2 has a similar preference for curved surfaces.