Membrane affinity and fluorescent labelling: comparative study of monolayer interaction, cellular uptake and cytotoxicity profile of carboxyfluorescein-conjugated cationic peptides

Abstract

Fluorescent labelling is a common approach to reveal the molecular details of cellular uptake, internalisation, transport, distribution processes in biological systems. The conjugation with a fluorescent moiety might affect relevant physico-chemical and in vitro transport properties of the bioactive component. A representative set of seven cationic peptides-including cell-penetrating peptides as well as antimicrobial peptides and synthetic derivatives-was selected for our comparative study. Membrane affinity of the peptides and their 5(6)-carboxyfluorescein (Cf) derivatives was determined quantitatively and compared applying Langmuir monolayer of zwitterionic (DPPC) and negatively charged (DPPC + DPPG) lipids as cell membrane models. The interaction with neutral lipid layer is mainly governed by the overall hydrophobicity of the molecule which is remarkably increased by Cf-conjugation for the most hydrophobic Magainin, Melittin and Transportan. A significantly enhanced membrane affinity was detected in negatively charged lipid model monolayer for all of the peptides since the combination of electrostatic and hydrophobic interaction is active in that case. The Cf-conjugation improved the penetration ability of Penetratin and Dhvar4 suggesting that both the highly charged character (Z/n) and the increased hydrophobicity by Cf-conjugation present important contribution to membrane interaction. This effect might also responsible for the observed high in vitro internalisation rate of Penetratin and Dhvar4, while according to in vitro studies they did not cause damage of cell membrane. From the experiments with the given seven cationic peptides, it can be concluded that the Cf-conjugation alters the degree of membrane interaction of such peptides which are moderately hydrophobic and highly charged.

Drug Conjugation Induced Modulation of Structural and Membrane Interaction Features of Cationic Cell-Permeable Peptides

“…The other peptides' membrane affinity then gradually increased. This shows agreement with our previous results, where the membrane affinity order of the peptides almost followed their hydrophobicity [37]. Transportan, CM15 and Magainin are shown to be the most hydrophobic ones in the present set, according to their Rt (>12) and H (< 0) values.…”

Cellular Internalization and Inhibition Capacity of New Anti-Glioma Peptide Conjugates: Physicochemical Characterization and Evaluation on Various Monolayer- and 3D-Spheroid-Based in Vitro Platforms

“…However, the ratio for most of the Dau-conjugates with tuftsin derivatives is below 100% at 50 μM (Figure S21). This shows the obvious fact that labeling molecules also have a role in the internalization processes and it can cause a significant difference in the uptake of the same peptide derivative (Dau can also be considered as a labeling molecule) . However, mostly similar conclusions can be drawn if we compare the different conjugates to each other: the uptake rates of Dau-SynB3-conjugates were significantly higher than the uptake rates of most Dau-tuftsin-conjugates; the rate of the tandem conjugates resembled or was slightly lower than the rate of Dau-Aoa-SynB3.…”

Old Polyanionic Drug Suramin Suppresses Detrimental Cytotoxicity of the Host Defense Peptide LL-37

“…As one dye molecule was coupled to each peptide, the fluorescence properties and the cellular uptake values are comparable. Previous investigation on a set of representative peptides demonstrated that the Cf labeling did not modify the tendency compared with those of unlabeled peptides in case of internalization rate as a consequence of altered lipophilicity and charge.…”