Change search
ReferencesLink to record
Permanent link

Direct link
Vectorization of oligonucleotides with cell-penetrating peptides: Characterization of uptake mechanisms and cytotoxicity
Stockholm University, Faculty of Science, Department of Neurochemistry.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The hydrophobic plasma membrane constitutes an indispensable barrier for cells in living animals. Albeit being pivotal for the maintenance of cells, the inability to cross the plasma membrane is still one of the major obstacles to overcome in order to progress current drug development. A group of substances, with restricted access to the interior of cells, which has shown great promise for future clinical use is oligonucleotides that are exploited to interfere with gene expression. Short interfering RNAs that are utilized to confer gene silencing and splice correcting oligonucleotides, applied for the manipulation of splicing patterns, are two classes of oligonucleotides that have been explored in this thesis.

Cell-penetrating peptides (CPPs) are a class of peptides that has gained increasing focus in last years. This ensues as a result of their remarkable ability to convey various, otherwise impermeable, macromolecules across the plasma membrane of cells in a relatively non-toxic fashion. This thesis aims at further characterizing well-established, and newly designed, CPPs in terms of toxicity, delivery efficacy, and internalization mechanism.

Our results demonstrate that different CPPs display different toxic profiles and that cargo conjugation alters the toxicity and uptake levels. Furthermore, we confirm the involvement of endocytosis in translocation of CPPs, and in particular the importance of macropinocytosis. All tested peptides facilitate the delivery of splice correcting oligonucleotides with varying efficacy, the newly designed CPP, M918, being the most potent. Finally we conclude that by promoting endosomolysis, by exploring new CPPs with improved endosomolytic properties, the biological response increases significantly. In conclusion, we believe that these results will facilitate the development of new CPPs with improved delivery properties that could be used for transportation of oligonucleotides in clinical settings.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi , 2007. , 86 p.
Keyword [en]
CPP, endocytosis, splice correction, siRNA, PNA, cargo delivery, M918
National Category
Research subject
Neurochemistry and Molecular Neurobiology
URN: urn:nbn:se:su:diva-7167ISBN: 9789171555052OAI: diva2:197756
Public defence
2007-12-07, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00 (English)
Available from: 2007-11-15 Created: 2007-11-07 Last updated: 2011-03-24Bibliographically approved
List of papers
1. Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides: a comparative study
Open this publication in new window or tab >>Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides: a comparative study
2007 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 407, no 2, 285-292 p.Article in journal (Refereed) Published
Abstract [en]

The use of CPPs (cell-penetrating peptides) as delivery vectors for bioactive molecules has been an emerging field since 1994 when the first CPP, penetratin, was discovered. Since then, several CPPs, including the widely used Tat (transactivator of transcription) peptide, have been developed and utilized to translocate a wide range of compounds across the plasma membrane of cells both in vivo and in vitro. Although the field has emerged as a possible future candidate for drug delivery, little attention has been given to the potential toxic side effects that these peptides might exhibit in cargo delivery. Also, no comprehensive study has been performed to evaluate the relative efficacy of single CPPs to convey different cargos. Therefore we selected three of the major CPPs, penetratin, Tat and transportan 10, and evaluated their ability to deliver commonly used cargos, including fluoresceinyl moiety, double-stranded DNA and proteins (i.e. avidin and streptavidin), and studied their effect on membrane integrity and cell viability. Our results demonstrate the unfeasibility to use the translocation efficacy of fluorescein moiety as a gauge for CPP efficiency, since the delivery properties are dependent on the cargo used. Furthermore, and no less importantly, the toxicity of CPPs depends heavily on peptide concentration, cargo molecule and coupling strategy.

cell-penetrating peptide, cytotoxicity, delivery vector, penetratin, transactivator of transcription (Tat), transport
National Category
urn:nbn:se:su:diva-24531 (URN)10.1042/BJ20070507 (DOI)
Available from: 2007-11-15 Created: 2007-11-07 Last updated: 2015-06-01Bibliographically approved
2. Induction of splice correction by cell-penetrating peptide nucleic acids
Open this publication in new window or tab >>Induction of splice correction by cell-penetrating peptide nucleic acids
2006 (English)In: Journal of Gene Medicine, ISSN 1099-498X, E-ISSN 1521-2254, Vol. 8, no 10, 1262-1273 p.Article in journal (Refereed) Published
Abstract [en]


Directing splicing using oligonucleotides constitutes a promising therapeutic tool for a variety of diseases such as β-thalassemia, cystic fibrosis, and certain cancers. The rationale is to block aberrant splice sites, thus directing the splicing of the pre-mRNA towards the desired protein product. One of the difficulties in this setup is the poor bioavailability of oligonucleotides, as the most frequently used transfection agents are unsuitable for in vivo use. Here we present splice-correcting peptide nucleic acids (PNAs), tethered to a variety of cell-penetrating peptides (CPPs), evaluating their mechanism of uptake and ability to correct aberrant splicing.


HeLa cells stably expressing luciferase containing an aberrant splice site were used. A previously described PNA sequence, capable of correcting the aberrant splicing, was conjugated to the CPPs, Tat, penetratin and transportan, via a disulfide bridge. The ability of the CPP-PNA conjugates to correct splicing was measured, and membrane disturbance and cell viability were evaluated using LDH leakage and WST-1 assays. Lysosomotropic agents, inhibition of endocytosis at 4 °C and confocal microscopy were used to investigate the importance of endocytosis in the uptake of the cell-penetrating PNAs.


All the three CPPs were able to promote PNA translocation across the plasma membrane and induce splice correction. Transportan (TP) was the most potent vector and significantly restored splicing in a concentration-dependent manner. Interestingly, TP also rendered a concentration-dependent splice correction in serum, in contrast to Tat and penetratin. Addition of the lysosomotrophic agent chloroquine increases the splice correction efficacy of the CPP-PNA conjugates up to 4-fold, which together with experiments at 4 °C and the visual information from confocal microscopy, indicate that the mechanism of uptake responsible for internalization of CPP-PNA conjugates is mainly endocytic. Finally, co-localization studies with dextran further indicate that conjugates, at least in the case of TP, internalize via endocytosis and in particular macropinocytosis.


These data demonstrate that CPPs can be used for the delivery of splice-correcting PNAs, with potential to be used as a therapeutic approach for regulating splicing in a variety of diseases. Transportan presents itself as the overall most suitable vector in this study, generating the most efficient conjugates for splice correction.

antisense, cell-penetrating peptide, endocytosis, PNA, splice correction
National Category
Biological Sciences Chemical Sciences
urn:nbn:se:su:diva-23008 (URN)10.1002/jgm.950 (DOI)
Available from: 2006-10-31 Created: 2006-10-31 Last updated: 2015-04-21Bibliographically approved
3. Assessing the delivery efficacy and internalization route of cell-penetrating peptides
Open this publication in new window or tab >>Assessing the delivery efficacy and internalization route of cell-penetrating peptides
2007 (English)In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 2, no 8, 2043-2047 p.Article in journal (Refereed) Published
Abstract [en]

Developing efficient delivery vectors for bioactive molecules is of great importance within both traditional and novel drug development, such as oligonucleotide (ON)-based therapeutics. To address delivery efficiency using cell-penetrating peptides (CPPs), we here present a protocol based on splice correction utilizing both neutral and anionic antisense ONs, either covalently conjugated via a disulfide bridge or non-covalently complexed, respectively, that generates positive readout in the form of luciferase expression. The decisive advantage of using splice correction for evaluation of CPPs is that the ON induces a biological response in contrast to traditionally used methods, for example, fluorescently labeled peptides. An emerging number of studies emphasize the role of endocytosis in translocation of CPPs, and this protocol is also utilized to determine the relative contribution of different endocytic pathways in the uptake of CPPs, which provides valuable information for future design of novel, more potent CPPs for bioactive cargoes.

National Category
urn:nbn:se:su:diva-20686 (URN)10.1038/nprot.2007.302 (DOI)000253139400024 ()17703217 (PubMedID)
Available from: 2007-11-28 Created: 2007-11-28 Last updated: 2015-04-21Bibliographically approved
4. A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids
Open this publication in new window or tab >>A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids
2007 (English)In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 15, no 10, 1820-1826 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) have attracted increasing attention in the past decade as a result of their high potential to convey various, otherwise impermeable, bioactive agents across cellular plasma membranes. Albeit different CPPs have proven potent in delivery of different cargoes, there is generally a correlation between high efficacy and cytotoxicity for these peptides. Hence, it is of great importance to find new, non-toxic CPPs with more widespread delivery properties. We present a novel CPP, M918, that efficiently translocates various cells in a non-toxic fashion. In line with most other CPPs, the peptide is internalized mainly via endocytosis, and in particular macropinocytosis, but independent of glycosaminoglycans on the cell surface. In addition, in a splice correction assay using antisense peptide nucleic acid (PNA) conjugated via a disulphide bridge to M918 (M918-PNA), we observed a dose-dependent increase in correct splicing, exceeding the effect of other CPPs. Our data demonstrate that M918 is a novel CPP that can be used to translocate different cargoes inside various cells efficiently.

Pep tides, Nucleic acids, Proteins, Cell membranes, Endocytosis
National Category
Chemical Sciences
urn:nbn:se:su:diva-24649 (URN)10.1038/ (DOI)000249778000016 ()
Available from: 2008-01-24 Created: 2008-01-11 Last updated: 2015-04-21Bibliographically approved
5. Delivery of short interfering RNA using endosomolytic cell-penetrating peptides
Open this publication in new window or tab >>Delivery of short interfering RNA using endosomolytic cell-penetrating peptides
Show others...
2007 In: FASEB Journal, ISSN 0892-6638, Vol. 21, no 11, 2664-2671 p.Article in journal (Refereed) Published
urn:nbn:se:su:diva-24535 (URN)000249237500008 ()
Part of urn:nbn:se:su:diva-7167Available from: 2007-11-15 Created: 2007-11-07Bibliographically approved

Open Access in DiVA

fulltext(735 kB)744 downloads
File information
File name FULLTEXT01.pdfFile size 735 kBChecksum MD5
Type fulltextMimetype application/pdf

By organisation
Department of Neurochemistry

Search outside of DiVA

GoogleGoogle Scholar
Total: 744 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 454 hits
ReferencesLink to record
Permanent link

Direct link