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Cell-penetrating peptide based nanocomplexes for oligonucleotide delivery
Stockholm University, Faculty of Science, Department of Neurochemistry. (Ülo Langel)ORCID iD: 0000-0002-4604-6413
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 [en]
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: urn:nbn:se:su:diva-133794ISBN: 978-91-7649-479-0 (print)OAI: oai:DiVA.org:su-133794DiVA: diva2:971282
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
List of papers
1. Rational design of a series of novel amphipathic cell-penetrating peptides
Open this publication in new window or tab >>Rational design of a series of novel amphipathic cell-penetrating peptides
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2014 (English)In: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 464, no 1-2, 111-116 p.Article in journal (Refereed) Published
Abstract [en]

A series of novel, amphipathic cell-penetrating peptides was developed based on a combination of the model amphipathic peptide sequence and modifications based on the strategies developed for PepFect and NickFect peptides. The aim was to study the role of amphipathicity for peptide uptake and to investigate if the modifications developed for PepFect peptides could be used to improve the uptake of another class of cell-penetrating peptides. The peptides were synthesized by solid phase peptide synthesis and characterized by circular dichroism spectroscopy. Non-covalent peptide-plasmid complexes were formed by co-incubation of the peptides and plasmids in water solution. The complexes were characterized by dynamic light scattering and cellular uptake of the complexes was studied in a luciferase-based plasmid transfection assay. A quantitative structure-activity relationship (QSAR) model of cellular uptake was developed using descriptors including hydrogen bonding, peptide charge and positions of nitrogen atoms. The peptides were found to be non-toxic and could efficiently transfect cells with plasmid DNA. Cellular uptake data was correlated to QSAR predictions and the predicted biological effects obtained from the model correlated well with experimental data. The QSAR model could improve the understanding of structural requirements for cell penetration, or could potentially be used to predict more efficient cellpenetrating peptides.

Keyword
Cell-penetrating peptide, Model amphipathic peptide, Plasmid transfection, Structure-activity, QSAR
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-100005 (URN)10.1016/j.ijpharm.2014.01.018 (DOI)000331726000013 ()
Available from: 2014-01-23 Created: 2014-01-23 Last updated: 2017-05-05Bibliographically approved
2. pH-responsive PepFect cell-penetrating peptides
Open this publication in new window or tab >>pH-responsive PepFect cell-penetrating peptides
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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.

Keyword
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:nbn:se:su:diva-128503 (URN)10.1016/j.ijpharm.2016.01.055 (DOI)000370845300004 ()26821060 (PubMedID)
Available from: 2016-04-11 Created: 2016-03-30 Last updated: 2017-04-20Bibliographically approved
3. 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
4. Peptide-Based Delivery of Oligonucleotides Across Blood-Brain Barrier Model
Open this publication in new window or tab >>Peptide-Based Delivery of Oligonucleotides Across Blood-Brain Barrier Model
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2014 (English)In: International Journal of Peptide Research and Therapeutics, ISSN 1573-3904, Vol. 20, no 2, 169-178 p.Article in journal (Refereed) Published
Abstract [en]

Delivery of pharmaceutical agents across a blood–brain barrier (BBB) is a challenge for brain cancer therapy. In this study, an in vitro BBB model was utilized to study the delivery of oligonucleotides across brain endothelial cells targeting to glioma cells in a Transwell™ setup. A series of novel peptides were synthesized by covalent conjugation of cell-penetrating peptides with targeting peptides for delivery of gene-based therapeutics. These peptides were screened for passage across the Transwell™ and we found the most efficient peptide PepFect32 from originating PepFect 14 coupled with the targeting peptide angiopep-2. PepFect32/pDNA nanocomplexes exhibited high transcytosis across the BBB in vitro model and the highest transfection efficiency to glioma cells. In conclusion, PepFect32 revealed the most efficient peptide-based vector for pDNA delivery across in vitro BBB model.

Keyword
Blood–brain barrier model, Cell-penetrating peptide, bEnd.3, Glioma cells, Plasmid transfection, Gene-based therapy
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-100010 (URN)10.1007/s10989-013-9378-4 (DOI)000334420100007 ()
Available from: 2014-01-23 Created: 2014-01-23 Last updated: 2017-05-05Bibliographically approved

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