Membrane affinity and fluorescent labelling: comparative study of monolayer interaction, cellular uptake and cytotoxicity profile of carboxyfluorescein-conjugated cationic peptides
- 195 Downloads
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.
KeywordsFluorescent labelling Membrane affinity Cell-penetrating peptides Lipid monolayer Penetration Cellular uptake
Valuable assistance of Mrs. I. Hórvölgyi and H. Szatmári in Langmuir experiments is acknowledged. This work was financially supported by National Research Development and Innovation Office, Hungary (OTKA 104275, 115431, 124077) and VEKOP-2.3.2-16-2017-00014 European Union and the State of Hungary, co-financed by the European Regional Development Fund. K. Horváti was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. G. Gyulai was supported by the Hungarian Academy of Sciences Postdoctoral Research Program.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
This article does not contain any studies with human participants or animals performed by any of the authors.
- Bellet-Amalric E, Blaudez D, Desbat B, Graner F, Gauthier F, Renault A (2000) Interaction of the third helix of Antennapedia homeodomain and a phospholipid monolayer, studied by ellipsometry and PM-IRRAS at the air–water interface. Biochim Biophys Acta 1467:131–143. https://doi.org/10.1016/S0005-2736(00)00218-2 CrossRefPubMedGoogle Scholar
- Castanho MARB (ed) (2009) Membrane-active peptides: methods and results on structure and function. IUL biotechnology series. International University Line, La Jolla. ISBN 978-0972077453Google Scholar
- Christiaens B, Symoens S, Verheyden S, Engelborghs Y, Joliot A, Prochiantz A, Vandekerckhove J, Rosseneu M, Vanloo B, Vanderheyden S (2002) Tryptophan fluorescence study of the interaction of penetratin peptides with model membranes. Eur J Biochem 269:2918–2926. https://doi.org/10.1046/j.1432-1033.2002.02963.x CrossRefPubMedGoogle Scholar
- Flasinski M, Hac-Wydro K, Wydro P, Dynarowicz-Łatka P (2014) Influence of platelet-activating factor, lyso-platelet-activating factor and edelfosine on Langmuir monolayers imitating plasma membranes of cell lines differing in susceptibility to anti-cancer treatment: the effect of plasmalogen level. J R Soc Interface 11:20131103. https://doi.org/10.1098/rsif.2013.1103 CrossRefPubMedPubMedCentralGoogle Scholar
- Hedegaard SF, Derbas MS, Lind TK, Kasimova MR, Christensen MV, Michaelsen MH, Campbell RA, Jorgensen L, Franzyk H, Cárdenas M, Nielsen HM (2018) Fluorophore labeling of a cell-penetrating peptide significantly alters the mode and degree of biomembrane interaction. Sci Rep 8(1):6327. https://doi.org/10.1038/s41598-018-24154-z CrossRefPubMedPubMedCentralGoogle Scholar
- Hegedüs R, Manea M, Orbán E, Szabó I, Kiss É, Sipos É, Halmos G, Mező G (2012) Enhanced cellular uptake and in vitro antitumor activity of short-chain fatty acid acylated daunorubicin-GnRH-III bioconjugates. Eur J Med Chem 56:155–165. https://doi.org/10.1016/j.ejmech.2012.08.014 CrossRefPubMedGoogle Scholar
- Hill K, Pénzes CB, Schnöller D, Horváti K, Bősze S, Hudecz F, Keszthelyi T, Kiss É (2010) Characterization of the membrane affinity of an isoniazid peptide-conjugate by tensiometry, atomic force microscopy and sum-frequency vibrational spectroscopy, using a phospholipid Langmuir monolayer model. Phys Chem Chem Phys 12:11498–11506. https://doi.org/10.1039/C002737E CrossRefPubMedGoogle Scholar
- Horváti K, Bacsa B, Kiss E, Gyulai G, Fodor K, Balka G, Rusvai M, Szabó E, Hudecz F, Bősze S (2014) Nanoparticle encapsulated lipopeptide conjugate of antitubercular drug isoniazid: in vitro intracellular activity and in vivo efficacy in a Guinea pig model of tuberculosis. Bioconjug Chem 12:2260–2268. https://doi.org/10.1021/bc500476x CrossRefGoogle Scholar
- Horváti K, Bacsa B, Mlinkó T, Szabó N, Hudecz F, Zsila F, Bősze S (2017) Comparative analysis of internalization, haemolytic, cytotoxic and antibacterial effect of membrane-active cationic peptides: aspects of experimental setup. Amino Acids 49:1053–1067. https://doi.org/10.1007/s00726-017-2402-9 CrossRefPubMedGoogle Scholar
- Horváti K, Gyulai G, Csámpai A, Rohonczy J, Kiss É, Bősze S (2018) Surface layer modification of poly(d,l-lactic-co-glycolic acid) nanoparticles with targeting peptide: a convenient synthetic route for pluronic F127-tuftsin conjugate. Bioconjug Chem 5:1495–1499. https://doi.org/10.1021/acs.bioconjchem.8b00156 CrossRefGoogle Scholar
- Illien F, Rodriguez N, Amoura M, Joliot A, Pallerla M, Cribier S, Burlina F, Sagan S (2016) Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry. Sci Rep 6:36938. https://doi.org/10.1038/srep36938 CrossRefPubMedPubMedCentralGoogle Scholar
- Kapus A, Grinstein S, Wasan S, Kandasamy R, Orlowski J (1994) Functional characterization of three isoforms of the Na+/H+ exchanger stably expressed in Chinese hamster ovary cells. ATP dependence, osmotic sensitivity, and role in cell proliferation. J Biol Chem 269:23544–23552 (PMID: 8089122) PubMedGoogle Scholar
- Kiss É, Heine ET, Hill K, He Y-C, Keusgen N, Pénzes CB, Schnöller D, Gyulai G, Mendrek A, Keul H, Moeller M (2012) Membrane affinity and antimicrobial properties of polyelectrolytes with different hydrophobicity. Macromol Biosci 12:1181–1189. https://doi.org/10.1002/mabi.201200078 CrossRefPubMedGoogle Scholar
- PEP-FOLD server, Paris Diderot University (FR) (2018) De novo peptide structure prediction [Internet]. http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD. Accessed 10 June 2018
- Zhang Lab, University of Michigan (2018) I-TASSER Protein Structure and Function Predictions [Internet]. https://zhanglab.ccmb.med.umich.edu/I-TASSER/. Accessed 10 June 2018