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Cell-penetrating peptides and bioactive cargoes: Strategies and mechanisms
Stockholm University, Faculty of Science, Department of Neurochemistry.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cell membrane is an impermeable barrier for most macromolecules. Recently discovered cell-penetrating peptides (CPPs) have gained lot of attention because they can cross the membrane, and even more, carry cargoes with them. How CPPs enter cells is still not clear, while the delivery of different cargoes has been convincingly shown. This thesis concentrates on evaluating CPPs as vectors for different biologically relevant cargoes. Proposed internalisation mechanisms are reviewed as well as cargo coupling strategies. Biological activities of antisense oligonucleotides delivered by CPPs have been of particular interest and are explained in greater details.

A new CPP, pIsl, was derived from Islet-1 transcription factor, and compared to archetypical CPPs like penetratin and transportan. All three peptides resided in the headgroup region of lipid bilayers in model membranes. However, penetratin and pIsl did only interact with negatively charged membranes, while transportan did not distinguish negatively charged and neutral membranes. This suggests different translocation pathways for different CPPs. Biotinylated pIsl and penetratin were complexed with avidin, and uptake of avidin into the human melanoma cell line Bowes was observed in both cases. This means that the protein is not unfolded during the translocation process, which is important in delivery of other, biologically active proteins.

Transportan and its analogue TP10 were used for peptide nucleic acid (PNA) antisense oligonucleotide delivery. First, eight human galanin receptor type 1 targeting PNA oligomers were designed, conjugated to transportan and assayed for antisense efficiency. In contrary to avidin-biotinylated peptide conjugate, a covalent bond between PNA oligomers and the transport peptide was necessary for cellular uptake of oligomers. A common problem in antisense technology is inactivity of antisense oligonucleotides due to the secondary structure of the target. Efficiencies of tested galanin receptor type 1 targeting PNA oligomers varied over two orders of magnitude. The most efficient oligomers were targeting coding sequence regions 24-38 and 27-38, and had EC50 values 70 and 80 nM, respectively.

TP10-antisense PNA oligomer conjugates were targeted also to L-type voltage dependent Ca2+ channel subunits CaV1.2 and CaV1.3. Specific down-regulation of respective proteins was demonstrated by immunohistochemistry. Physiological response to the down-regulation of either of Ca2+ channels was studied by alteration of flexor reflex sensitisation. Rats treated with either of the antisense PNA, but not with scrambled PNA lost the action potential windup phenomenon. In conjunction with a variety of drugs, modulating the conductivity and excitability of neuronal membranes, a central role of L-type CaV channels in sensitisation was confirmed. Nevertheless, also N-methyl-D-aspartate and glycine receptors were found to be required.

Finally, delivery of plasmids by TP10 was evaluated. In contrary to many similar CPPs, TP10 was incapable to translocate plasmids to cells. However, addition of TP10 or a TP10-PNA conjugate to polyethyleneimine-condensed plasmids increased the expression of reporter genes.

In summary, different types of cargoes have been delivered by CPPs and different cargo coupling strategies have been used. CPP-mediated antisense oligonucleotide delivery has been used to identify accessible sites in human galanin receptor type 1 mRNA and to determine the role of L-type voltage dependent Ca2+ channels in axon potential windup.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi , 2004. , 75 p.
Keyword [en]
Cell-penetrating peptide, delivery, Peptide nucleic acid
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-308ISBN: 91-7265-986-6 (print)OAI: oai:DiVA.org:su-308DiVA: diva2:192279
Public defence
2004-12-17, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2004-11-25 Created: 2004-11-25 Last updated: 2017-12-01Bibliographically approved
List of papers
1. Cellular internalization of a cargo complex with a novel peptide derived from the third helix of the islet-1 homeodomain: Comparison with the penetratin peptide
Open this publication in new window or tab >>Cellular internalization of a cargo complex with a novel peptide derived from the third helix of the islet-1 homeodomain: Comparison with the penetratin peptide
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2001 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 12, no 6, 911-916 p.Article in journal (Refereed) Published
Abstract [en]

Cellular translocation into a human Bowes melanoma cell line was investigated and compared for penetratin and pIsl, two peptides that correspond to the third helices of the related homeodomains, from the Antennapedia transcription factor of Drosophila and the rat insulin-1 gene enhancer protein, respectively. Both biotinylated peptides internalized into the cells with similar efficacy, yielding an analogous intracellular distribution. When a large cargo protein, 63 kDa avidin, was coupled to either peptide, efficient cellular uptake for both the peptide−protein complexes was observed. The interactions between each peptide and SDS micelles were studied by fluorescence spectroscopy and acrylamide quenching of the intrinsic tryptophan (Trp) fluorescence. Both peptides interacted strongly and almost identically with the membrane mimicking environment. Compared to penetratin, the new transport peptide pIsl has only one Trp residue, which simplifies the interpretation of the fluorescence spectra and in addition has a native Cys residue, which may be used for alternative coupling reactions of cargoes of different character.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-23348 (URN)10.1021/bc0100298 (DOI)
Available from: 2004-09-16 Created: 2004-09-16 Last updated: 2017-12-13Bibliographically approved
2. Interaction and structure induction of cell-penetrating peptides in the presence of phospholipid vesicles
Open this publication in new window or tab >>Interaction and structure induction of cell-penetrating peptides in the presence of phospholipid vesicles
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2001 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1512, no 1, 77-89 p.Article in journal (Refereed) Published
Abstract [en]

Certain short peptides, which are able to translocate across cell membranes with a low lytic activity, can be useful as carriers (vectors) for hydrophilic molecules. We have studied three such cell penetrating peptides: pAntp (‘penetratin’), pIsl and transportan. pAntp and pIsl originate from the third helix of homeodomain proteins (Antennapedia and Isl-1, respectively). Transportan is a synthetic chimera (galanin and mastoparan). The peptides in the presence of various phospholipid vesicles (neutral and charged) and SDS micelles have been characterized by spectroscopic methods (fluorescence, EPR and CD). The dynamics of pAntp were monitored using an N-terminal spin label. In aqueous solution, the CD spectra of the three peptides show secondary structures dominated by random coil. With phospholipid vesicles, neutral as well as negatively charged, transportan gives up to 60% α-helix. pAntp and pIsl bind significantly only to negatively charged vesicles with an induction of around 60% β-sheet-like secondary structure. With all three peptides, SDS micelles stabilize a high degree of α-helical structure. We conclude that the exact nature of any secondary structure induced by the membrane model systems is not directly correlated with the common transport property of these translocating peptides.

Keyword
homeo-peptide, penetratin, transportan, phospholipid vesicle, interaction, secondary structure
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-23347 (URN)10.1016/S0005-2736(01)00304-2 (DOI)000168735500008 ()
Available from: 2004-09-16 Created: 2004-09-16 Last updated: 2017-12-13Bibliographically approved
3. Targeting of antisense PNA oligomers to human galanin receptor type 1 mRNA
Open this publication in new window or tab >>Targeting of antisense PNA oligomers to human galanin receptor type 1 mRNA
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2004 (English)In: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 38, no 5, 316-324 p.Article in journal (Refereed) Published
Abstract [en]

In this work, we have targeted positions 18–38 of the human galanin receptor type 1 (GalR1) mRNA coding sequence with different peptide nucleic acid (PNA) oligomers. This region has previously been shown to be a good antisense region and therefore we aimed to identify the subregions and/or thermodynamic parameters determining the antisense efficacy. Nine different PNA oligomers were conjugated to a cell-penetrating peptide, transportan, to enhance their cellular uptake. Concentration-dependent down-regulation of GalR1 protein expression in human melanoma cell line Bowes was measured by radioligand binding assay. No reduction of GalR1 mRNA level was observed upon PNA treatment, thus, the effect was concluded to be translational arrest. Judging from the EC50 values, antisense PNA oligomers targeting regions 24–38 (EC50 = 70 nM) or 27–38 (EC50 = 80 nM) were the most potent suppressors of protein expression. No parameter predicted by M-fold algorithm was found to correlate with the measured antisense activities. Presence of some subregions was found not to increase antisense efficiency of PNA. Presence of a short unpaired triplet between nucleotides 33 and 35 in the target region was, on the other hand, found to be the most critical for efficient GalR1 down-regulation. Thus, the results are of high impact in designing antisense oligomers. Specific results of this study demonstrate 20-fold more efficient antisense down-regulation of GalR1 as achieved before.

Keyword
antisense, cell penetrating peptide, peptide nucleic acid, M-fold algorithm
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-23481 (URN)10.1016/j.npep.2004.06.005 (DOI)000224748100007 ()
Available from: 2004-11-25 Created: 2004-11-25 Last updated: 2017-12-13Bibliographically approved
4. L-type calcium channels and NMDA receptors: a determinant duo for short term nociceptive plasticity
Open this publication in new window or tab >>L-type calcium channels and NMDA receptors: a determinant duo for short term nociceptive plasticity
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(English)Manuscript (Other academic)
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-23482 (URN)
Available from: 2004-11-25 Created: 2004-11-25 Last updated: 2016-01-29Bibliographically approved
5. 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

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