Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
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.
Show others and affiliations
Number of Authors: 72018 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1860, no 2, p. 491-504Article in journal (Refereed) Published
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 oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular 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. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. 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 monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements 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 peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.

Place, publisher, year, edition, pages
2018. Vol. 1860, no 2, p. 491-504
Keywords [en]
Cell-penetrating peptide, Fluorescence correlation spectroscopy, PepFect14, siRNA, Amphipathic peptide, Peptide aggregates, Plasma membrane
National Category
Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-153640DOI: 10.1016/j.bbamem.2017.09.024ISI: 000424183500029PubMedID: 28962904OAI: oai:DiVA.org:su-153640DiVA, id: diva2:1188852
Available from: 2018-03-08 Created: 2018-03-08 Last updated: 2018-09-24Bibliographically 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. p. 79
Keywords
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: 2018-09-24Bibliographically approved
2. Characterization of nucleic acid delivery with fatty acid modified cell-penetrating peptide nanoparticle formulations
Open this publication in new window or tab >>Characterization of nucleic acid delivery with fatty acid modified cell-penetrating peptide nanoparticle formulations
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Recent advances with techniques used for manipulating gene expression have brought us to an era where various gene therapeutic approaches are becoming common therapeutic tools for many previously incurable diseases. The main factor impeding the wider translation of gene therapy is that the active pharmaceutical ingredients used for interfering with gene expression are based on nucleic acids and synthetic oligonucleotides and such molecules do not readily reach their intracellular targets due to their physicochemical properties and therefore they require delivery vectors to cross the cell membrane. 

Cell-penetrating peptides (CPPs) is one such class of delivery vectors that comprise excellent potential for transporting bioactive cargo molecules across cellular membranes, both in vitro and in vivo conditions. CPPs have shown to be very versatile carriers for various types of bioactive cargo, including different nucleic acids such as plasmids (pDNA), splice-correcting oligonucleotides (SCOs), small interfering RNAs (siRNA) and mRNA, or peptides and proteins or even small molecules.

This thesis focuses on characterizing the delivery of various nucleic acids-based molecules with a variety of novel fatty acid modified CPPs. In order to achieve this we utilize the ability of a family of CPPs called PepFects to non-covalently formulate nucleic acids into nanoparticles. More particularly the aim of the thesis is to find and characterize the key parameters of these peptide/nucleic nanoparticles that would improve their potential applicability as a drug formulation and delivery system for future gene therapies.

By simultaneously characterizing the role of N-terminal fatty acid modification and the peptide/nucleic acid ratio in the nanoparticles we were able to show in Papers I and II that increasing the hydrophobicity and reducing unbound free fraction of the peptide improves delivery efficiency and decreases toxicity of these nanoparticles both in vitro and in vivo.

Based on the findings from Paper I regarding the ability of these amphiphilic peptides to self-associate into supramolecular structures we went deeper in Paper III to study the formation, composition and live cell association of these peptide/nucleic acid complexes at single molecule sensitivity.

And finally in Paper IV we enhanced the specificity of these nanoparticles towards in vivo xenograft tumors by incorporating the capacity to be specifically activated in the tumor microenvironment.

Conclusively, these findings contribute to the field with identifying and characterizing some of the key factors in developing efficient and safe peptide-based delivery vectors for gene modulating therapeutics.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018. p. 86
Keywords
Transfection, gene delivery, oligonucleotide, complexes, non-viral, in vivo
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-160398 (URN)978-91-7797-436-9 (ISBN)978-91-7797-437-6 (ISBN)
Public defence
2018-11-02, Magnélisalen Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, 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: 2018-10-10 Created: 2018-09-21 Last updated: 2018-10-10Bibliographically approved

Open Access in DiVA

No full text in DiVA

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Vasconcelos, LuisLehto, TõnisHällbrink, MattiasLangel, Ülo
By organisation
Department of Neurochemistry
In the same journal
Biochimica et Biophysica Acta - Biomembranes
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 16 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf