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Cell-penetrating peptides targeting glioblastomas for nucleic acid delivery in the blood-brain barrier model
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0003-2265-557X
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glioblastoma multiforme is the most aggressive form of malignant brain tumor with poor prognosis. The efficacy of brain cancer treatment by chemotherapeutics is limited by the blood-brain barrier (BBB) which allows less than 2% of the small molecules and blocks almost all the macromolecules to transport into the brain. Delivery of the large molecules such as proteins and nucleic acids across the BBB is a great challenge for brain-targeted drug delivery. To overcome this obstacle, cell-penetrating peptides (CPPs) were used as vectors for delivery of nucleic acids across the BBB targeting glioblastomas. The CPPs have shown such promising carriers to deliver various cargoes ranging from small molecules to large molecules into the cells. This thesis is focused on the development of glioblastoma-targeting vectors based on modifications of the CPPs and the targeting peptides. The peptide-based vectors were developed to improve the transport of the nucleic acids across the BBB and specifically target glioblastomas.

In this thesis, a series of peptide-based vectors targeting glioblastomas were synthesized and modified with targeting peptides by either covalent conjugation or non-covalent complex formation. The delivery of plasmid DNA (pDNA) in the complex with the peptide-based vectors was studied in the in vitro model of the BBB. The role of receptors expressed on the BBB was investigated. Scavenger receptors class A and B were found to be expressed on the BBB, and they were involved in the delivery of the pDNA across the BBB model. Moreover, various targeting peptides were modified with hexaglutamate to form non-covalent complexes with the CPPs for small interfering RNA (siRNA) delivery to glioblastoma cells. The non-covalent complex of the CPP and the targeting peptide showed greater gene-silencing efficiency than the consecutively covalent conjugation of the CPP and the targeting peptide for siRNA delivery to glioblastoma cells. Lastly, a number of novel, amphipathic peptides were developed based on the model amphipathic peptide. The prediction of the biological effect of the designed peptides using quantitative structure-activity relationship model showed a correlation with the experimental data.

Finally, the CPP-based nucleic acid delivery vectors with homing peptide strategy have a potential for the BBB shuttle and the future use as a glioblastoma-targeted drug carrier in the in vivo studies and the clinical applications.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2017.
Keyword [en]
Blood-brain barrier model, nucleic acid delivery, glioblastomas, cell-penetrating peptides
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-142109ISBN: 978-91-7649-725-8 (print)ISBN: 978-91-7649-726-5 (electronic)OAI: oai:DiVA.org:su-142109DiVA: diva2:1091660
Public defence
2017-06-09, 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: 2017-05-17 Created: 2017-04-27 Last updated: 2017-05-17Bibliographically approved
List of papers
1. 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
2. Role of scavenger receptors in peptide-based delivery of plasmid DNA across a blood-brain barrier model
Open this publication in new window or tab >>Role of scavenger receptors in peptide-based delivery of plasmid DNA across a blood-brain barrier model
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2016 (English)In: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 500, no 1-2, 128-135 p.Article in journal (Refereed) Published
Abstract [en]

Receptor-mediated transcytosis remains a major route for drug delivery across the blood-brain barrier (BBB). PepFect 32 (PF32), a peptide-based vector modified with targeting ligand (Angiopep-2) binding to low-density lipoprotein receptor-related protein-1 (LRP-1), was previously found to be a promising vector for plasmid delivery across an in vitro model of the BBB. Cellular uptake of PF32/plasmid DNA (pDNA) complexes was speculated the internalization via LRP-1 receptor. In this study, we prove that PF32/pDNA nanocomplexes are not only transported into brain endothelial cells via LRP-1 receptor-mediated endocytosis, but also via scavenger receptor class A and B (SCARA3, SCARA5, and SR-BI)-mediated endocytosis. SCARA3, SCARA5, and SR-BI are found to be expressed in the brain endothelial cells. Inhibition of these receptors leads to a reduction of the transfection. In conclusion, this study shows that scavenger receptors also play an essential role in the cellular uptake of the PF32/pDNA nanocomplexes.

Keyword
Blood-brain barrier, bEnd.3, Plasmid transfection, Scavenger receptors, angiopep-2, LRP-1 receptor, Receptor-mediated endocytosis
National Category
Physical Chemistry Biochemistry and Molecular Biology Pharmacology and Toxicology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-128163 (URN)10.1016/j.ijpharm.2016.01.014 (DOI)000370049900013 ()26773601 (PubMedID)
Available from: 2016-03-29 Created: 2016-03-21 Last updated: 2017-05-05Bibliographically approved
3. 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
4. New strategy for siRNA delivery to glioblastoma cells by cell-penetrating peptide
Open this publication in new window or tab >>New strategy for siRNA delivery to glioblastoma cells by cell-penetrating peptide
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(English)Manuscript (preprint) (Other academic)
Keyword
blood-brain barrier, siRNA, cell-penetraing peptides, glioblastoma
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-142101 (URN)
Funder
EU, FP7, Seventh Framework ProgrammeSwedish Cancer SocietySwedish Research Council
Available from: 2017-04-25 Created: 2017-04-25 Last updated: 2017-05-05Bibliographically approved

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