Abstract
The demand for highly efficient macromolecular drugs, used in the treatment of many severe diseases, is continuously increasing. However, the hydrophilic character and large molecular size of these drugs significantly limit their ability to permeate across cellular membranes and thus impede the drugs in reaching their target sites in the body. Cell-penetrating peptides (CPP) have gained attention as promising drug excipients, since they can facilitate drug permeation across cell membranes constituting a major biological barrier. Fluorophores are frequently covalently conjugated to CPPs to improve detection, however, the ensuing change in physico-chemical properties of the CPPs may alter their biological properties. With complementary biophysical techniques, we show that the mode of biomembrane interaction may change considerably upon labeling of the CPP penetratin (PEN) with a fluorophore. Fluorophore-PEN conjugates display altered modes of membrane interaction with increased insertion into the core of model cell membranes thereby exerting membrane-thinning effects. This is in contrast to PEN, which localizes along the head groups of the lipid bilayer, without affecting the thickness of the lipid tails. Particularly high membrane disturbance is observed for the two most hydrophobic PEN conjugates; rhodamine B or 1-pyrene butyric acid, as compared to the four other tested fluorophore-PEN conjugates.
Highlights
The demand for highly efficient macromolecular drugs, used in the treatment of many severe diseases, is continuously increasing
The fluorophores affected the conformation of PEN in solution (e.g. PENRhB) as well as the degree of α-helix formation upon membrane interaction, e.g., the intensity at 222 nm was almost twice for PENPBA and PENMCA in the presence of lipid that of the unmodified PEN
None of the N-terminally conjugated fluorophores induced any decrease in the α-helical folding propensity of PEN, and did not compromise the ability of PEN to adopt a secondary structure upon interaction with the lipid bilayer
Summary
The demand for highly efficient macromolecular drugs, used in the treatment of many severe diseases, is continuously increasing. Fluorophore-PEN conjugates display altered modes of membrane interaction with increased insertion into the core of model cell membranes thereby exerting membrane-thinning effects. This is in contrast to PEN, which localizes along the head groups of the lipid bilayer, without affecting the thickness of the lipid tails. The relatively large molecular size and hydrophilic nature of most therapeutic peptides and proteins constitute challenging factors for achieving sufficient delivery to their target site Their physico-chemical properties limit their ability to permeate across the cell membrane and reach the site of action in the body. We apply a series of biophysical techniques in order to obtain a fundamental understanding of the crucial first step in CPP-membrane interaction by utilizing model phospholipid-based vesicles[5,16,24] and supported lipid bilayers (SLBs)[25,26,27,28]
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