Abstract
gH625 constitutes a promising delivery vehicle for the transport of therapeutic biomacromolecules across membrane barriers. We report an application of multivalency to create a complex nanosystem for delivery and to elucidate the mechanism of peptide-lipid bilayer interactions. Multivalency may offer a route to enhance gH625 cellular uptake as demonstrated by results obtained on dimers of gH625 by fluorescence spectroscopy, circular dichroism, and surface plasmon resonance. Moreover, using both phase contrast and light sheet fluorescence microscopy we were able to characterize and visualize for the first time the fusion of giant unilamellar vesicles caused by a membranotropic peptide.
Highlights
The plasma membrane plays numerous functions in maintaining cell survival but it represents one of the major impediments for the delivery of therapeutic agents into cells, the main limitation being the poor passive cell membrane permeability of hydrophilic molecules/drugs
In addition to the remarkable efficiency rates, gH625 is essentially internalized by a non-endocytic pathway avoiding endosomal entrapment[10,11,12,13,14,15,16,17,18,19,20] and is able to penetrate the Blood Brain Barrier both in vitro and in vivo11, 20–22. gH625 is a very good delivery vector and a complete understanding of the uptake mechanism and molecular details of its membrane interactions are of major relevance for optimization of this and similar delivery tools
We have previously demonstrated a strong correlation between the affinity of gH625 for the lipid bilayer and cellular delivery efficiency, which is consistent with its uptake by a physically mediated process[23,24,25,26]; to fully describe the biophysical mechanism involved in the molecular mode of action of gH625, the contribution of electrostatic and hydrophobic forces to the process is an essential piece of the puzzle that needs to be elucidated[7]
Summary
The plasma membrane plays numerous functions in maintaining cell survival but it represents one of the major impediments for the delivery of therapeutic agents into cells, the main limitation being the poor passive cell membrane permeability of hydrophilic molecules/drugs. A possible strategy to overcome the membrane barrier is represented by the use of basic peptide sequences, the so-called cell-penetrating peptides (CPPs), with the ability to autonomously cross biological membranes without the assistance of cell membrane receptors and without cell rupture in a nontoxic and non-immunogenic manner[1] This provides a good strategy to deliver a large variety of cargo molecules for therapy and diagnosis granting a substantial improvement in the cellular uptake. A wider application of CPPs in cellular delivery has been hampered by their entrapment in cellular organelles, which seriously reduces the cargo delivering efficacy; for this reason, a physically driven mechanism appears to be a more efficient process for the delivery of macromolecules of biological relevance To this purpose, one of the main challenges of recent research is the obtainment of novel delivery systems that are able to cross membranes using different internalization mechanisms without being entrapped in endosomes[2]. We have previously demonstrated a strong correlation between the affinity of gH625 for the lipid bilayer and cellular delivery efficiency, which is consistent with its uptake by a physically mediated process[23,24,25,26]; to fully describe the biophysical mechanism involved in the molecular mode of action of gH625, the contribution of electrostatic and hydrophobic forces to the process is an essential piece of the puzzle that needs to be elucidated[7]
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