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Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides: a comparative study
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-6107-0844
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.

Place, publisher, year, edition, pages
2007. Vol. 407, no 2, 285-292 p.
Keyword [en]
cell-penetrating peptide, cytotoxicity, delivery vector, penetratin, transactivator of transcription (Tat), transport
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:su:diva-24531DOI: 10.1042/BJ20070507OAI: oai:DiVA.org:su-24531DiVA: diva2:197751
Available from: 2007-11-15 Created: 2007-11-07 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Vectorization of oligonucleotides with cell-penetrating peptides: Characterization of uptake mechanisms and cytotoxicity
Open this publication in new window or tab >>Vectorization of oligonucleotides with cell-penetrating peptides: Characterization of uptake mechanisms and cytotoxicity
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
CPP, endocytosis, splice correction, siRNA, PNA, cargo delivery, M918
National Category
Neurosciences
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-7167 (URN)9789171555052 (ISBN)
Public defence
2007-12-07, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2007-11-15 Created: 2007-11-07 Last updated: 2011-03-24Bibliographically approved
2. Cell-penetrating peptides as delivery vectors for oligonucleotides and proteins: Studies on applications and toxicity
Open this publication in new window or tab >>Cell-penetrating peptides as delivery vectors for oligonucleotides and proteins: Studies on applications and toxicity
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cell-penetrating peptides (CPPs) have for a little bit more than a decade been employed as delivery vectors for a wide range of cargoes, ranging from gold particles to entire plasmids. Although CPP are well studied and utilized in numerous publications, our knowledge about CPP mediated transport is still poor. The articles presented in this thesis all consider different aspects of CPP mediated delivery. The first two papers are evaluating and improving already known techniques. In paper I, standard polyethyleneimine (PEI) transfection is improved by conjugating the CPP TP10 to the cationic polymer. In paper II, the same CPP is employed to deliver a dsDNA decoy oligo, resulting in decreased activity of the transcription factor c-Myc. The third paper is a more general overview of the delivery efficiency of well known CPPs and how the delivered cargo influences the CPP mediated toxicity. The study shows that different CPPs are suitable for different cargos and that toxic side effects depend heavily on the cargo and coupling strategy used. In Paper IV, a novel CPP, M918, is evaluated as a delivery vector for a transposon based non-viral gene therapy system. M918 display simultaneous delivery of a plasmid carrying a selection gene and a transposase into cultured cells. This is the first study where two so vastly different molecules as a cationic protein and an anionic plasmid, are simultaneously transported into cells by a peptide vector. The method might be a first step towards a safe peptide based non-viral gene therapy platform. Taken together, the results presented in this thesis might help to improve already existing techniques, increase our understandings about CPP mediated delivery and, at the same time, develop new CPP based delivery systems.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2007. 73 p.
National Category
Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-7067 (URN)978-91-7155-499-4 (ISBN)
Public defence
2007-11-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2007-10-12 Created: 2007-09-11 Last updated: 2017-12-01Bibliographically approved

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