Fluorophore labeling of a cell-penetrating peptide significantly alters the mode and degree of biomembrane interaction

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.

Endosomal Escape and Cytosolic Penetration of Macromolecules Mediated by Synthetic Delivery Agents

“…These fluorophores, by contributing some hydrophobicity, may themselves be involved in some aspects of the membrane disruption process. This has been clearly demonstrated in the context of monomeric CPPs, where the fluorophore used for peptide labeling impacts cell viability, peptide uptake, and CPP binding to bilayers (39,(66)(67)(68). Similarly, fluorophores also impact multivalent systems such as 3TAT, as suggested by the fact that a non-labeled 3TAT construct is noticeably less prone to inducing membrane leakage that an analog labeled with tetramethylrhodamine (the relationship 3TAT>2TAT>1TAT in regard to membrane penetration is however true when comparing labeled or unlabeled analogs) (39).…”

Membrane Active Peptides and Their Biophysical Characterization

“…The choice of the fluorescent probe is therefore critical and must not be disruptive. Fluorescent probes can also be used to monitor leakage induced by membrane active peptides [ 81 , 234 , 261 ].…”

To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models

“…Despite their extensive use and value, most fluorophores are bulky, rigid, and hydrophobic molecules. Hence, their conjugation to other molecules may alter their physicochemical/biological properties, mainly when dealing with low molecular weight molecules, which may ultimately bias the results obtained by a given technique (Toseland, 2013;Sánchez-Rico et al, 2017;Hedegaard et al, 2018).…”

“…The assumption that unlabeled-and labeled-molecules have the same properties often relies on wish much more than evidence (Zhao et al, 2016). The advent of studies reporting differences between fluorophores raised awareness to the importance of their selection (Fischer et al, 2002;Toseland, 2013;Knutson et al, 2016;Zhao et al, 2016;Birch et al, 2017;Hedegaard et al, 2018). In addition, some studies also report the impact of the molecule on the properties of the fluorophore (Toseland, 2013;Szabó et al, 2018).…”