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
Scavenger receptor-mediated uptake of cell-penetrating peptide nanoparticles with oligonucleotides
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-9912-4887
Show others and affiliations
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.

Place, publisher, year, edition, pages
2012. Vol. 26, no 3, 1172-1180 p.
Keyword [en]
drug delivery, splice correction, cellular uptake
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-75534DOI: 10.1096/fj.11-191536ISI: 000300949300020PubMedID: 22138034OAI: oai:DiVA.org:su-75534DiVA: diva2:517008
Available from: 2012-04-20 Created: 2012-04-20 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Cell-penetrating peptides; chemical modification, mechanism of uptake and formulation development
Open this publication in new window or tab >>Cell-penetrating peptides; chemical modification, mechanism of uptake and formulation development
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Gene therapy holds the promise of revolutionizing the way we treat diseases. By using recombinant DNA and oligonucleotides (ONs), gene functions can be restored, altered or silenced according to the therapeutic need. However, gene therapy approaches require the delivery of large and charged nucleic acid-based molecules to their intracellular targets across the plasma membrane, which is inherently impermeable to such molecules. In this thesis, two chemically modified cell-penetrating peptides (CPPs) that have superior delivery properties for several nucleic acid-based therapeutics are developed. These CPPs can spontaneously form nanoparticles upon non-covalent complexation with the nucleic acid cargo, and the formed nanoparticles mediate efficient cellular transfection. In paper I, we show that an N-terminally stearic acid-modified version of transportan-10 (PF3) can efficiently transfect different cell types with plasmid DNA and mediates efficient gene delivery in-vivo when administrated intra muscularly (i.m.) or intradermaly (i.d.). In paper II, a new peptide with ornithine modification, PF14, is shown to efficiently deliver splice-switching oligonucleotides (SSOs) in different cell models including mdx mouse myotubes; a cell culture model of Duchenne’s muscular dystrophy (DMD). Additionally, we describe a method for incorporating the PF14-SSO nanoparticles into a solid formulation that is active and stable even when stored at elevated temperatures for several weeks. In paper III, we demonstrate the involvement of class-A scavenger receptor subtypes (SCARA3 & SCARA5) in the uptake of PF14-SSO nanoparticles, which possess negative surface charge, and suggest for the first time that some CPP-based systems function through scavenger receptors. In paper IV, the ability of PF14 to deliver siRNA to different cell lines is shown and their stability in simulated gastric acidic conditions is highlighted.

Taken together, these results demonstrate that certain chemical modifications can drastically enhance the activity and stability of CPPs for delivering nucleic acids after spontaneous nanoparticle formation upon non-covalent complexation. Moreover, we show that CPP-based nanoparticles can be formulated into convenient and stable solid formulations that can be suitable for several therapeutic applications. Importantly, the involvement of scavenger receptors in the uptake of such nanoparticles is presented, which could yield novel possibilities to understand and improve the transfection by CPPs and other gene therapy nanoparticles.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University Stockholm University, 2012. 86 p.
Keyword
cell penetrating peptides, gene therapy, scavenger receptors, pharmaceutical formulation, solid dispersion
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-75537 (URN)978-91-7447-464-0 (ISBN)
Public defence
2012-06-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of doctoral defence the following paper was unpublished and had a status as follows: Paper nr 4: SubmittedAvailable from: 2012-05-10 Created: 2012-04-20 Last updated: 2012-04-24Bibliographically approved
2. Cell-penetrating peptides: an uptake mechanism & a new endosomolytic peptide
Open this publication in new window or tab >>Cell-penetrating peptides: an uptake mechanism & a new endosomolytic peptide
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
cell-penetrating peptides
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-94377 (URN)978-91-7447-782-5 (ISBN)
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
3. Cell-penetrating peptides for oligonucleotide delivery: Design and uptake mechanisms
Open this publication in new window or tab >>Cell-penetrating peptides for oligonucleotide delivery: Design and uptake mechanisms
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of oligonucleotides for gene therapy has the potential to efficiently treat a plethora of diseases with minimal side effects. However, the use of oligonucleotides is hampered by the properties of these molecules, which make it essentially impossible for them to permeate the cellular membrane. Therefore, a great deal of research has been focused on developing delivery vectors, which can efficiently and safely deliver oligonucleotides into cells. Cell-penetrating peptides (CPPs) constitute a class of delivery vectors that have received much attention since they were discovered over 20 years ago. CPPs can deliver a wide variety of cargos into cells, such as small molecules, proteins, oligonucleotides and particles, in an efficacious and non-toxic manner.

In this thesis two new CPPs for oligonucleotide delivery were designed. The purpose of the design was to create CPPs, which form stable complexes with oligonucleotides and have endosomolytic properties. The new peptides showed superior potency in intracellular oligonucleotide delivery compared to previously reported CPPs. These results demonstrate that it is possible to drastically improve the efficiency of existing CPPs with relatively simple modifications.

It is well known that CPPs use endocytosis to gain entry into cells, however, why cells endocytose CPPs has never been clearly established. In this thesis we determine that several CPP:oligonucleotide complexes interact with scavenger receptors, and that this interaction leads to endocytosis. The results presented in this thesis provides a deeper understanding of how CPPs function and thereby insights how to improve CPP design.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2014. 62 p.
Keyword
Cell-penetrating peptides, gene therapy, scavenger receptors
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-108344 (URN)978-91-7649-023-5 (ISBN)
Public defence
2014-11-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (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: 2014-11-06 Created: 2014-10-21 Last updated: 2015-04-21Bibliographically approved
4. Cell-penetrating peptides: Uptake mechanism and the role of receptors
Open this publication in new window or tab >>Cell-penetrating peptides: Uptake mechanism and the role of receptors
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:nbn:se:su:diva-120832 (URN)978-91-7649-259-8 (ISBN)
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

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Ezzat, KariemHelmfors, HenrikLindberg, StaffanLangel, Ülo
By organisation
Department of Neurochemistry
In the same journal
The FASEB Journal
Chemical Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 176 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