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Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by Fluorescence Correlation Spectroscopy
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-7522-8964
Karolinska institutet.
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0002-6440-7577
Karolinska institutet.
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2017 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotide cargo is mostly driven by electrostatic and hydrophobic interactions, which may result in polydisperse and polymorphic particles. Here we investigate the coexistence of distinct molecular and supramolecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide’s monomer as well as of peptide aggregates and complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements on peptide/oligonucleotide complexes in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of molecular and supramolecular species, distributed unevenly at the plasma membrane.

Place, publisher, year, edition, pages
2017.
Keyword [en]
Cell-penetrating peptide, Fluorescence correlation spectroscopy, PepFect14, siRNA, amphipathic peptide, peptide aggregates, plasma membrane
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-141095OAI: oai:DiVA.org:su-141095DiVA: diva2:1085821
Funder
Swedish Research Council, 521-2011-2461, 2012-2595Swedish Cancer Society, 2014/259Swedish Foundation for Strategic Research , SBE13-0115Knut and Alice Wallenberg Foundation, 2011.0218EU, FP7, Seventh Framework Programme, GLORIA-602919EU, FP7, Seventh Framework Programme, FP7/2007-2013
Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2017-04-20
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Vasconcelos, LuísLehto, TönisHällbrink, MattiasLangel, Ülo
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