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pH-responsive PepFect cell-penetrating peptides
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-7522-8964
Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
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Number of Authors: 5
2016 (English)In: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 501, no 1-2, 32-38 p.Article in journal (Refereed) Published
Abstract [en]

A series of cell-penetrating PepFect peptide analogues was developed by substitutions of the galanin-derived N-terminal sequence. Histidine modifications were incorporated in order to make the peptides pH-responsive. The peptides were all able to form non-covalent complexes with an oligonucleotide cargo by co-incubation in buffer. The complexes were characterized by dynamic light scattering and circular dichroism, and an assay to evaluate the peptide-cargo affinity was developed. Cellular bioactivity was studied in HeLa cells using a luciferase-based splice correction assay. In addition, the membrane interactions of the peptides in large unilammelar vesicles was studied using a calcein leakage assay. The effects of substitutions were found to be dependent of the non-modified, C-terminal sequence of the peptides; for analogues of PepFect 3 we observed an increase in membrane activity and bioactivity for histidine-containing analogues, whereas the same modifications introduced to PepFect 14 lead to a decreased bioactivity. Peptides modified with a leucine/histidine sequence were found to be pH responsive, complexes formed from these peptides were small at pH 7 and grew under acidic conditions. The most promising of the novel PepFect 3 analogues, PepFect 132 has a significantly higher bioactivity and membrane activity than the parent peptide PepFect 3.

Place, publisher, year, edition, pages
2016. Vol. 501, no 1-2, 32-38 p.
Keyword [en]
Cell-penetrating peptide, Oligonucleotide delivery, Calcein leakage, PepFect, Cellular uptake, pH-responsive
National Category
Chemical Sciences Pharmaceutical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-128503DOI: 10.1016/j.ijpharm.2016.01.055ISI: 000370845300004PubMedID: 26821060OAI: oai:DiVA.org:su-128503DiVA: diva2:918409
Available from: 2016-04-11 Created: 2016-03-30 Last updated: 2017-04-20Bibliographically approved
In thesis
1. Cell-penetrating peptide based nanocomplexes for oligonucleotide delivery
Open this publication in new window or tab >>Cell-penetrating peptide based nanocomplexes for oligonucleotide delivery
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Oligonucleotide-based drugs hold great promise for the treatment of many types of diseases, ranging from genetic disorders to viral infections and cancer. The problem is that efficient delivery across the cell membrane is required for oligonucleotides to have their desired effect. Cell-penetrating peptides (CPPs) provide a solution to this problem. CPPs are capable of transporting cargoes such as drugs or nucleic acids for gene therapy into the cell, either by covalent conjugation to the cargo or by non-covalent complex formation. This thesis is focused on the development of a class of peptides called PepFects, peptides with fatty acid modifications capable of forming nanoparticle-sized complexes with oligonucleotides. These complexes are efficiently internalized by many different cell types and are generally non-toxic and non-immunogenic.

We have developed a number of novel PepFect peptides and a quantitative structure-activity model to predict the biological effect of our peptides. In addition, the involvement of scavenger receptors class A in the endocytic uptake of PepFect complexes as well as other CPPs and polymeric transfection agents was studied. Lastly, we have developed a series of PepFect peptides for delivery across the blood-brain barrier and a model system mimicking the blood-brain barrier in order to evaluate the passage of these peptides.

The general aim of this thesis is to improve the understanding of intracellular delivery of oligonucleotides with PepFect peptides from both a chemical and a biological viewpoint, and further improve the efficacy of this delivery system with the long-term goal of making it useful in clinical settings. 

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2016. 69 p.
Keyword
Cell-penetrating peptides, oligonucleotides, gene therapy, drug delivery, scavenger receptors, blood-brain barrier
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-133794 (URN)978-91-7649-479-0 (ISBN)
Public defence
2016-11-04, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2016-10-12 Created: 2016-09-15 Last updated: 2016-09-26Bibliographically approved
2. 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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