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Cell-penetrating peptides as delivery vectors for oligonucleotides and proteins: Studies on applications and toxicity
Stockholm University, Faculty of Science, Department of Neurochemistry.
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: urn:nbn:se:su:diva-7067ISBN: 978-91-7155-499-4 (print)OAI: oai:DiVA.org:su-7067DiVA: diva2:197584
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
List of papers
1. Evaluation of transportan 10 in PEI mediated plasmid delivery assay
Open this publication in new window or tab >>Evaluation of transportan 10 in PEI mediated plasmid delivery assay
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2005 (English)In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 103, no 2, 511-23 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) are novel high-capacity delivery vectors for different bioactive cargoes. We have evaluated the CPP transportan 10 (TP10) as a delivery vector in different in vitro plasmid delivery assays. Tested methods include: TP10 crosslinked to a plasmid via a peptide nucleic acid (PNA) oligomer, TP10 conjugation with polyethyleneimine (PEI), and addition of unconjugated TP10 to standard PEI transfection assay. We found that without additional DNA condensing agents, TP10 has poor transfection abilities. However, the presence of TP10 increases the transfection efficiency several folds compared to PEI alone. At as low concentrations as 0.6 nM, TP10–PNA constructs were found to enhance plasmid delivery up to 3.7-fold in Neuro-2a cells. Interestingly, the transfection efficiency was most significant at low PEI concentrations, allowing reduced PEI concentration without loss of gene delivery. No increase in cytotoxicity due to TP10 was observed and the uptake mechanism was determined to be endocytosis, as previously reported for PEI mediated transfection. In conclusion, TP10 can enhance PEI mediated transfection at relatively low concentrations and may help to develop future gene delivery systems with reduced toxicity.

Keyword
Transportan 10, Cell-penetrating peptides, Polyethyleneimine, Transfection
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-24463 (URN)10.1016/j.jconrel.2004.12.006 (DOI)
Available from: 2007-10-12 Created: 2007-09-11 Last updated: 2017-12-13Bibliographically approved
2. TP10, a delivery vector for decoy oligonucleotides targeting the Myc protein
Open this publication in new window or tab >>TP10, a delivery vector for decoy oligonucleotides targeting the Myc protein
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2005 (English)In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 110, no 1, 189-201 p.Article in journal (Refereed) Published
Abstract [en]

One approach to investigate gene function, by silencing the activity of certain proteins, is the usage of double stranded decoy oligodeoxynucleotides (ds decoy ODNs). Decoy, in this sense, is ds ODNs bearing the consensus binding sequence for a DNA-binding protein. This can be used in clinical settings to attenuate the effect of overexpressed transcription factors in tumor cells. We here choose to target the oncogenic protein Myc. Since oligonucleotides are poorly internalized to cells, a cell-penetrating peptide, TP10, was coupled to the Myc decoy, using two different strategies. Either TP10 was simply mixed with ds decoy ODNs forming complexes through non-covalent electrostatic interactions, or by having a nona-nucleotide overhang in one of the decoy strands, and adding a complementary PNA sequence coupled to an NLS sequence and TP10, which could hybridize to the Myc decoy.

By using these strategies, uptake was significantly enhanced, especially with the co-incubation approach. Interestingly, various endocytosis inhibitors had no effect on the uptake pattern, suggesting that uptake of these complexes is not mediated via endocytosis. Finally, a decreased proliferative capacity was observed when treating the neuroblastoma cell line N2a with TP10–PNA conjugate hybridized to Myc decoy compared to naked Myc decoy and untreated cells. A dose-dependent decrease in proliferation was also observed in MCF-7 cells, when using both strategies. These results suggest an alternative way to efficiently deliver ds ODNs into cells using the cell-penetrating peptide TP10 and prevent tumor growth by targeting the oncogenic protein Myc.

Keyword
Cell-penetrating peptide, TP10, Cargo delivery, Decoy, Myc, Endocytosis
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-24646 (URN)10.1016/j.jconrel.2005.09.012 (DOI)
Available from: 2008-01-24 Created: 2008-01-11 Last updated: 2017-12-13Bibliographically approved
3. 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.

Keyword
cell-penetrating peptide, cytotoxicity, delivery vector, penetratin, transactivator of transcription (Tat), transport
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-24531 (URN)10.1042/BJ20070507 (DOI)
Available from: 2007-11-15 Created: 2007-11-07 Last updated: 2017-12-13Bibliographically approved
4. Co-transduction of Sleeping Beauty Transposase and Donor Plasmid via a Cell-penetrating Peptide: A simple one-step method
Open this publication in new window or tab >>Co-transduction of Sleeping Beauty Transposase and Donor Plasmid via a Cell-penetrating Peptide: A simple one-step method
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2008 (English)In: International journal of peptide research and therapeutics, ISSN 1573-3904, Vol. 14, no 1, 58-63 p.Article in journal (Refereed) Published
Abstract [en]

Transposable elements have emerged as a promising candidate for human non-viral gene-therapy. The Tc1/mariner transposon Sleeping Beauty is to date one of the most efficient transposons in mammals. Sleeping Beauty transposase has so far mostly been delivered to cells via a DNA source. This might cause spontaneous integration of the transposase gene and cause fatal damage to the affected cell. Hence, it would be advantageous to employ a non-genetic source for the transposase. We here show that a novel Cell-penetrating peptide, M918, has the ability to facilitate cellular delivery of both the transposase Sleeping Beauty as a protein and a transposon donor-plasmid carrying an antibiotic resistance gene in vitro. The technique is a simple and straightforward one-step method that might render a safe and efficient delivery platform for Sleeping Beauty mediated gene therapy.

Keyword
Cell-penetrating peptides, Protein delivery, Transfection, Transposons
National Category
Biological Sciences Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-20685 (URN)10.1007/s10989-007-9114-z (DOI)000253571800009 ()
Available from: 2008-03-04 Created: 2008-03-04 Last updated: 2015-04-21Bibliographically approved

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