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Cell-penetrating peptides: an uptake mechanism & a new endosomolytic peptide
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-9912-4887
2013 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Peptide-based drugs have slowly begun migrating from laboratories into pharmacies and now there are several on the market. However, currently only one gene based therapy that is relies on a viral delivery vector has been approved. The long-term goal of our research is to leverage the cell-penetrating peptide (CPP) technology into a potent, safe and simple delivery vector for oligonucleotide (ON) based therapies.

Cell-penetrating peptides have been actively researched for more than 20 years, and many CPPs have been discovered. However, it is not fully understood how the peptides are able to enter cells. In this thesis we present a novel receptor for CPP:ON complexes. Pharmacological inhibition and siRNA knockdown of the class A scavenger receptors (SCARAs) demonstrate that these receptors are the main pathway by which CPP:ON complexes are taken up. As the intracellular fate of particles taken up by (receptor mediated) endocytosis is entrapment in endosomes this thesis also presents a new peptide for ON delivery that has endosomolytic properties. Additionally this new peptide (PepFect 15) is also taken up via receptor-mediated endocytosis by the SCARAs. 

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2013.
Keyword [en]
cell-penetrating peptides
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-94377ISBN: 978-91-7447-782-5 (print)OAI: oai:DiVA.org:su-94377DiVA: diva2:653423
Presentation
2013-10-24, Heilbronnsalen, Department of Neurochemistry, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2013-10-04 Created: 2013-10-04 Last updated: 2015-03-09Bibliographically approved
List of papers
1. Scavenger receptor-mediated uptake of cell-penetrating peptide nanoparticles with oligonucleotides
Open this publication in new window or tab >>Scavenger receptor-mediated uptake of cell-penetrating peptide nanoparticles with oligonucleotides
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2012 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26, no 3, 1172-1180 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) are shortcationic peptides that penetrate cells by interacting withthe negatively charged plasma membrane; however, thedetailed uptake mechanism is not clear. In contrary to theconventional mode of action of CPPs, we show here thata CPP, PepFect14 (PF14), forms negatively charged nanocomplexeswith oligonucleotides and their uptake is mediatedby class-A scavenger receptors (SCARAs). Specificinhibitory ligands of SCARAs, such as fucoidin, polyinosinicacid, and dextran sulfate, totally inhibit the activityof PF14-oligonucleotide nanocomplexes in the HeLapLuc705 splice-correction cell model, while nonspecific,chemically related molecules do not. Furthermore, RNAinterference (RNAi) knockdown of SCARA subtypes(SCARA3 and SCARA5) that are expressed in this cell lineled to a significant reduction of the activity to <50%. Inline with this, immunostaining shows prevalent colocalizationof the nanocomplexes with the receptors, and electronmicroscopy images show no binding or internalizationof the nanocomplexes in the presence of theinhibitory ligands. Interestingly, naked oligonucleotidesalso colocalize with SCARAs when used at high concentrations.These results demonstrate the involvement ofSCARA3 and SCARA5 in the uptake of PF14-oligonucleotidenanocomplexes and suggest for the first time thatsome CPP-based systems function through scavenger receptors,which could yield novel possibilities to understandand improve the transfection by CPPs.

Keyword
drug delivery, splice correction, cellular uptake
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-75534 (URN)10.1096/fj.11-191536 (DOI)000300949300020 ()22138034 (PubMedID)
Available from: 2012-04-20 Created: 2012-04-20 Last updated: 2017-12-07Bibliographically approved
2. PepFect15, a novel endosomolytic cell-penetrating peptide for oligonucleotide delivery via scavenger receptors
Open this publication in new window or tab >>PepFect15, a novel endosomolytic cell-penetrating peptide for oligonucleotide delivery via scavenger receptors
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2012 (English)In: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 441, no 1-2, 242-247 p.Article in journal (Refereed) Published
Abstract [en]

Gene-regulatory biomolecules such as splice-correcting oligonucleotides and anti-microRNA oligonucleotides are important tools in the struggle to understand and treat genetic disorders caused by defective gene expression or aberrant splicing. However, oligonucleotides generally suffer from low bioavailability, hence requiring efficient and non-toxic delivery vectors to reach their targets. Cell-penetrating peptides constitute a promising category of carrier molecules for intracellular delivery of bioactive cargo. In this study we present a novel cell-penetrating peptide, PepFect15, comprising the previously reported PepFect14 peptide modified with endosomolytic trifluoromethylquinoline moieties to facilitate endosomal escape. Pepfect15 efficiently delivers both splice-correcting oligonucleotides and anti-microRNA oligonucleotides into cells through a non-covalent complexation strategy. To our knowledge this is the first work that describes peptide-mediated anti-microRNA delivery. The peptide and its cargo form stable, negatively charged nanoparticles that are taken up by cells largely through scavenger receptor type A mediated endocytosis.

Keyword
Cell-penetrating peptide, Drug delivery, Splice correction, MicroRNA, Endosomal escape, Scavenger receptor
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-84942 (URN)10.1016/j.ijpharm.2012.11.037 (DOI)000314054200028 ()23200958 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer SocietySwedish Foundation for Strategic Research
Available from: 2013-01-03 Created: 2013-01-03 Last updated: 2017-12-06Bibliographically approved

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