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Cell-penetrating peptides: Uptake mechanism and the role of receptors
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-9912-4887
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Genes are the major regulators of biological processes in every living thing. Problems with gene regulation can cause serious problems for the organism; for example, most cancers have some kind of genetic component. Regulation of biological processes using oligonucleotides can potentially be a therapy for any ailment, not just cancer. The problem so far has been that the targets for oligonucleotide-based therapies all reside on the inside of cells, because the cellular plasma membrane is normally impermeable to large and charged molecules (such as oligonucleotides) a delivery method is needed. Cell-penetrating peptides are a class of carrier molecules that are able to induce the cellular membrane into taking them and their cargo molecules into the cells. Understanding how and why cell-penetrating peptides work is one of the first and most important steps towards improving them to the point where they become useful as carriers for oligonucleotide-based therapies. This thesis is comprised of four scientific papers that are steps toward finding an uptake mechanism for cell-penetrating peptides that have been non-covalently complexed with oligonucleotides. In Paper I, we show that the scavenger receptors are responsible for uptake of the cell-penetrating peptide PepFect14 in complex with a short single-stranded oligonucleotide. Paper II expands upon this first finding and shows that the same receptors are key players in the uptake of several other cell-penetrating peptides that have been complexed with either, long double-stranded plasmid DNA or short double-stranded RNA. Paper III improves the luciferase-based assay for short oligonucleotide delivery by increasing the throughput 4-fold and reducing the cost by 95 %. The fourth manuscript uses the assay developed in paper III to investigate the effects on cell-penetrating peptide-mediated delivery by each of the constituents of a 264-member library of ligands for G-protein coupled receptors. We identify three ligands that dose-dependently increase the luciferase expression compared to control cells. These three ligands are one positive-, one negative allosteric modulator of metabotropic glutamate receptor 5 and one antagonist of histamine receptor 3.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2015. , 67 p.
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-120832ISBN: 978-91-7649-259-8 (print)OAI: oai:DiVA.org:su-120832DiVA: diva2:854785
Public defence
2015-11-06, 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 4: Manuscript.

Available from: 2015-10-15 Created: 2015-09-17 Last updated: 2015-10-14Bibliographically 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. A convergent uptake route for peptide- and polymer-based nucleotide delivery systems
Open this publication in new window or tab >>A convergent uptake route for peptide- and polymer-based nucleotide delivery systems
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2015 (English)In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 206, 58-66 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) have been used as vehicles to deliver various cargos into cells and are promising as tools to deliver therapeutic biomolecules such as oligonucleotides both in vitro and in vivo. CPPs are positively charged and it is believed that CPPs deliver their cargo in a receptor-independent manner by interactingwith the negatively charged plasmamembrane and thereby inducing endocytosis. In this study we examine the mechanism of uptake of several different, well known, CPPs that form complexes with oligonucleotides.We show that these CPP:oligonucleotide complexes are negatively charged in transfection-media and their uptake is mediated by class A scavenger receptors (SCARA). These receptors are known to promiscuously bind to, and mediate uptake of poly-anionic macromolecules. Uptake of CPP:oligonucleotide complexes was abolished using pharmacological SCARA inhibitors as well as siRNA-mediated knockdown of SCARA. Additionally, uptake of CPP:oligonucleotide was significantly increased by transiently overexpressing SCARA. Furthermore, SCARA inhibitors also blocked internalization of cationic polymer:oligonucleotide complexes.Our results demonstrate that the previous held belief that CPPs act receptor independently does not hold true for CPP:oligonucleotide complexes, as scavenger receptor class A (SCARA) mediates the uptake of all the examined CPP:oligonucleotide complexes in this study.

Keyword
Cell-penetrating peptides, Oligonucleotide delivery, CPP, Scavenger receptor class A, SCARA, Receptor-mediated endocytosis
National Category
Chemical Sciences Biological Sciences Pharmacology and Toxicology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-108343 (URN)10.1016/j.jconrel.2015.03.009 (DOI)000353361400006 ()
Available from: 2014-10-21 Created: 2014-10-21 Last updated: 2017-12-05Bibliographically approved
3. Optimized luciferase assay for cell-penetrating peptide-mediated delivery of short oligonucleotides
Open this publication in new window or tab >>Optimized luciferase assay for cell-penetrating peptide-mediated delivery of short oligonucleotides
2015 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 484, 136-142 p.Article in journal (Refereed) Published
Abstract [en]

An improved assay for screening for the intracellular delivery efficacy of short oligonucleotides using cell-penetrating peptides is suggested. This assay is an improvement over previous assays that use luciferase reporters for cell-penetrating peptides because it has been scaled up from a 24-well format to a 96-well format and no longer relies on a luciferin reagent that has been commercially sourced. In addition, the homemade luciferin reagent is useful in multiple cell lines and in different assays that rely on altering the expression of luciferase. To establish a new protocol, the composition of the luciferin reagent was optimized for both signal strength and longevity by multiple two-factorial experiments varying the concentrations of adenosine triphosphate, luciferin, coenzyme A, and dithiothreitol. In addition, the optimal conditions with respect to cell number and time of transfection for both short interfering RNA (siRNA) and splice-correcting oligonucleotides (SCOs) are established. Optimal transfection of siRNA and SCOs was achieved using the reverse transfection method where the oligonucleotide complexes are already present in the wells before the cells are plated. Z' scores were 0.73 for the siRNA assay and 0.71 for the SCO assay, indicating that both assays are suitable for high-throughput screening.

Keyword
Cell-penetrating peptide (CPP), Luciferase, Short interfering RNA (siRNA), Splice-correcting oligonucleotide (SCO), High-throughput screening (HTS)
National Category
Chemical Sciences Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-119526 (URN)10.1016/j.ab.2015.05.023 (DOI)000357967500023 ()
Available from: 2015-08-21 Created: 2015-08-17 Last updated: 2017-12-04Bibliographically approved
4. GPCR-ligands influence the short oligonucleotide transfection efficacy of the cell-penetrating peptide; Pepfect14
Open this publication in new window or tab >>GPCR-ligands influence the short oligonucleotide transfection efficacy of the cell-penetrating peptide; Pepfect14
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Cell-penetrating peptides can be used to deliver oligonucleotide-based cargoes into cells. We have previously shown that inhibition or knock-down of scavenger receptor type A results in a decreased oligonucleotide uptake. The remaining question is if the scavenger receptors are the only cell-surface receptors that can affect the uptake. By utilizing an optimized, higher throughput assay, for short oligonucleotide delivery using cell-penetrating peptides, and simultaneously adding a G-protein coupled receptor-ligand library. We show that two allosteric modulators (MPEP and VU 0357121) of metabotropic glutamate receptor type 5 and one histamine H3 receptor antagonist (Ciproxifan) have effects that can increase the transfection efficacy of PepFect in complex with a short single stranded oligonucleotide. Five different estrogen receptor ligands have negative effects on the transfection efficacy.

National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-120831 (URN)
Available from: 2015-09-17 Created: 2015-09-17 Last updated: 2016-01-29Bibliographically approved

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