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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
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0002-6440-7577
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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: 2017-05-16Bibliographically approved
In thesis
1. Complexes of cell-penetrating peptides with oligonucleotides: Structure, binding and translocation in lipid membranes
Open this publication in new window or tab >>Complexes of cell-penetrating peptides with oligonucleotides: Structure, binding and translocation in lipid membranes
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The fundamental element of life known to man is the gene. The information contained in genes regulates all cellular functions, in health and disease. The ability to selectively alter genes or their transcript intermediates with designed molecular tools, as synthetic oligonucleotides, represents a paradigm shift in human medicine.

The full potential of oligonucleotide therapeutics is however dependent on the development of efficient delivery vectors, due to their intrinsic characteristics, as size, charge and low bioavailability. Cell-penetrating peptides are short sequences of amino acids that are capable of mediating the transport of most types of oligonucleotide therapeutics to the cell interior. It is the interaction of cell-penetrating peptides with oligonucleotides and the transport of their non-covalently formed complexes across the cellular membrane, that constitutes the main subject of this thesis.

In Paper I we studied the effects of different types of oligonucleotide cargo in the capacity of cationic and amphipathic peptides to interact with lipid membranes. We found that indeed the cargo sequesters some of the peptide’s capacity to interact with membranes. In Paper II we revealed the simultaneous interaction of different molecular and supramolecular peptide and peptide/oligonucleotide species in equilibrium, with the cellular membrane. In Paper III we developed a series of peptides with improved affinity for oligonucleotide cargo as well as enhanced endosomal release and consequently better delivery capacity. In Paper IV we investigated the effect of saturated fatty acid modifications to a cationic cell-penetrating peptide. The varying amphipathicity of the peptide correlated with the complex physicochemical properties and with its delivery efficiency.

This thesis contributes to the field with a set of characterized mechanisms and physicochemical properties for the components of the ternary system – cell-penetrating peptide, oligonucleotide and cell membrane – that should be considered for the future development of gene therapy.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2017. 79 p.
Keyword
Cell-penetrating peptide, oligonucleotide, transfection, non-covalent complexes, membrane interaction
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-141881 (URN)978-91-7649-727-2 (ISBN)978-91-7649-728-9 (ISBN)
Public defence
2017-06-16, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2017-05-22 Created: 2017-04-20 Last updated: 2017-05-18Bibliographically approved

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