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

Marco Cavaco,Javier Valle,David Andreu,João D G Correia,Miguel A R B Castanho,Vera Neves,Clara Pérez-Peinado,Rúben D M Silva

doi:10.3389/fbioe.2020.552035

Abstract

The characterization of biologically active peptides relies heavily on the study of their efficacy, toxicity, mechanism of action, cellular uptake, or intracellular location, using both in vitro and in vivo studies. These studies frequently depend on the use of fluorescence-based techniques. Since most peptides are not intrinsically fluorescent, they are conjugated to a fluorophore. The conjugation may interfere with peptide properties, thus biasing the results. The selection of the most suitable fluorophore is highly relevant. Here, a comprehensive study with blood–brain barrier (BBB) peptide shuttles (PepH3 and PepNeg) and antimicrobial peptides (AMPs) (vCPP2319 and Ctn[15-34]), tested as anticancer peptides (ACPs), having different fluorophores, namely 5(6)-carboxyfluorescein (CF), rhodamine B (RhB), quasar 570 (Q570), or tide fluor 3 (TF3) attached is presented. The goal is the evaluation of the impact of the selected fluorophores on peptide performance, applying routinely used techniques to assess cytotoxicity/toxicity, secondary structure, BBB translocation, and cellular internalization. Our results show that some fluorophores significantly modulate peptide activity when compared with unlabeled peptides, being more noticeable in hydrophobic and charged fluorophores. This study highlights the need for a careful experimental design for fluorescently labeled molecules, such as peptides.

Highlights

The development of molecules for biological or biomedical applications relies on their accurate and precise biophysical/biological characterization (La Gatta et al, 2016; Van Norman, 2016)
The necessary data concerning the efficacy, toxicity, mechanism of action, cellular uptake, or intracellular location of such molecules can be gathered using in vitro or in vivo approaches (D’Addio et al, 2016). The collection of this information requires the use of highly sensitive techniques, which usually depend on the use of fluorescent probes (Gonzalez-Vera, 2012; Xu et al, 2018; Gao and Wu, 2019)
Dulbecco’s Modified Eagle Medium (DMEM), DMEM/Ham’s F-12 (DMEM:F12), DMEM:F12 without phenol-red, TrypsinEDTA, attachment factor protein solution (AF), fetal bovine serum (FBS), penicillin-streptomycin (Pen-Strep), and fluorescent dyes Hoechst 33342 were from Life Technologies (Carlsbad, CA, United States)

Summary

Introduction

The development of molecules for biological or biomedical applications relies on their accurate and precise biophysical/biological characterization (La Gatta et al, 2016; Van Norman, 2016). Since most molecules are not intrinsically fluorescent in the visible spectrum range, conjugation to a fluorophore is needed This way, fluorescencebased techniques, such as confocal laser scanning microscopy (CLSM), flow cytometry, and fluorimetry are possible options (Gautam et al, 2015; Radicioni et al, 2015). Despite their extensive use and value, most fluorophores are bulky, rigid, and hydrophobic molecules. Their conjugation to other molecules may alter their physicochemical/biological properties, mainly when dealing with low molecular weight molecules, which may bias the results obtained by a given technique (Toseland, 2013; Sánchez-Rico et al, 2017; Hedegaard et al, 2018)

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