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Cell-penetrating peptides: Uptake, stability and biological activity
Stockholm University, Faculty of Science, Department of Neurochemistry. (Ülo Langel)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cell-penetrating peptides (CPPs) have emerged as a group of remarkable delivery vectors for various hydrophilic macromolecules, otherwise excluded from cells due to the protective plasma membrane. Unbiased conclusions regarding e.g. uptake mechanism, intracellular distribution and cargo delivery efficacy is complicated by the use of different methodological parameters by different laboratories. The first paper in this thesis introduced unifying protocols enabling comparison of results from different research groups. One of these methods, HPLC, was used in paper II to investigate CPP uptake and degradation in yeasts. Both parameters varied depending on peptide and yeast species; however pVEC emerged as a promising delivery vector in yeast since it internalized into both species tested without concomitant degradation. Protein mimicry was another investigated phenomenon and in paper III a 22-mer peptide from the p14Arf protein (Arf (1-22)) was found to be sufficient for retaining its function as a tumor suppressor. This peptide comprised a combination of apoptogenic property and CPP in one unity, thus providing opportunity to conjugate cytotoxic agents boosting the tumoricidal activity. Surprisingly, a partially inverted control peptide to Arf (1-22), called M918, was found to be an extraordinary CPP. In paper IV, it was shown to be superior to well-established CPPs in delivery of both peptide nucleic acids and proteins. Albeit the promising results these two peptides displayed, their utility in vivo, as with all peptides, is hampered by rapid degradation. With the aim of improving their stability, Arf (1-22) and M918 were synthesized with D-amino acids in the reverse order, a modification called retro-inverso (RI) isomerization. Their cell-penetrating ability was retained, but the treated cells displayed unexpected morphological alterations indicative of apoptosis. The presented results demonstrate the versatility of CPPs, functioning as vectors in both yeast and mammalian cells and as protein mimicking peptides with biological activity. Their potential as drug delivery agents is obvious; however, peptide degradation is an issue that requires further improvements before clinical success is in reach.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2011. , 99 p.
National Category
Natural Sciences
Research subject
Neurochemistry and Neurotoxicology
Identifiers
URN: urn:nbn:se:su:diva-55664ISBN: 978-91-7447-269-1OAI: oai:DiVA.org:su-55664DiVA: diva2:405976
Public defence
2011-05-06, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 5: In press.Available from: 2011-04-14 Created: 2011-03-24 Last updated: 2011-03-24Bibliographically approved
List of papers
1. Studying the uptake of cell-penetrating peptides
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2006 (English)In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 1, no 2, 1001-1005 p.Article in journal (Refereed) Published
Abstract [en]

More than a decade ago, it was discovered that cationic peptides could traverse the cellular plasma membrane without specific transporter proteins or membrane damage. Subsequently, it was found that these peptides, known as cell-penetrating peptides (CPPs), were also capable of delivering cargos into cells, hence the great potential of these vectors was acknowledged. Today, many different research groups are working with CPPs, which necessitates efforts to develop unified assays enabling the comparison of data. Here we contribute three protocols for evaluation of CPPs which, if used in conjunction, provide complementary data about the amount and mechanism of uptake (fluorometric analysis and confocal microscopy, respectively), as well as the extent of degradation (HPLC analysis of cell lysates). All three protocols are based on the use of fluorescently labeled peptides and can be performed on the same workday.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-20766 (URN)10.1038/nprot.2006.174 (DOI)17406337 (PubMedID)
Available from: 2008-01-14 Created: 2008-01-14 Last updated: 2015-04-21Bibliographically approved
2. Uptake of cell-penetrating peptides in yeasts
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2005 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 579, no 23, 5217-5222 p.Article in journal (Refereed) Published
Abstract [en]

The uptake of different cell-penetrating peptides (CPPs) in two yeast species, Saccharomyces cerevisiae and Candida albicans, was studied using fluorescence HPLC-analyses of cell content. Comparison of the ability of penetratin, pVEC and (KFF)(3)K to traverse the yeast cell envelope shows that the cellular uptake of the peptides varies widely. Moreover, the intracellular degradation of the CPPs studied varies from complete stability to complete degradation. We show that intracellular degradation into membrane impermeable products can significantly contribute to the fluorescence signal. pVEC displayed highest internalizing capacity, and considering its stability in both yeast species, it is an attractive candidate for further studies.

Keyword
cell-penetrating peptides, internalization, degradation, Saccharomyces cerevisiae, Candida albicans
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-55654 (URN)10.1016/j.febslet.2005.07.099 (DOI)000232194300020 ()
Available from: 2011-03-24 Created: 2011-03-23 Last updated: 2015-04-21Bibliographically approved
3. Characterization of a novel cytotoxic cell-penetrating peptide derived from p14ARF protein
Open this publication in new window or tab >>Characterization of a novel cytotoxic cell-penetrating peptide derived from p14ARF protein
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2008 (English)In: Molecular Therapy, ISSN 1525-0016, Vol. 16, no 1, 115-123 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24648 (URN)000251821000020 ()
Note
Part of urn:nbn:se:su:diva-7287Available from: 2008-01-24 Created: 2008-01-11 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.

Keyword
Pep tides, Nucleic acids, Proteins, Cell membranes, Endocytosis
National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-24649 (URN)10.1038/sj.mt.6300255 (DOI)000249778000016 ()
Available from: 2008-01-24 Created: 2008-01-11 Last updated: 2015-04-21Bibliographically approved
5. Retro-inversion of certain cell-penetrating peptides causes severe cellular toxicity
Open this publication in new window or tab >>Retro-inversion of certain cell-penetrating peptides causes severe cellular toxicity
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2011 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, Vol. 1808, no 6, 1544-1551 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) are a promising group of delivery vectors for various therapeutic agents but their application is often hampered by poor stability in the presence of serum. Different strategies to improve peptide stability have been exploited, one of them being "retro-inversion" (RI) of natural peptides. With this approach the stability of CPPs has been increased, thereby making them more efficient transporters. Several RI-CPPs were here assessed and compared to the corresponding parent peptides in different cell-lines. Surprisingly, treatment of cells with these peptides induced trypsin insensitivity and rapid severe toxicity in contrast to l-peptides. This was measured as reduced metabolic activity and condensed cell nuclei, in parity with the apoptosis inducing agent staurosporine. Furthermore, effects on mitochondrial network, focal adhesions, actin cytoskeleton and caspase-3 activation were analyzed and adverse effects were evident at 20μM peptide concentration within 4h while parent l-peptides had negligible effects. To our knowledge this is the first time RI peptides are reported to cause cellular toxicity, displayed by decreased metabolic activity, morphological changes and induction of apoptosis. Considering the wide range of research areas that involves the use of RI-peptides, this finding is of major importance and needs to be taken under consideration in applications of RI-peptides.

Keyword
Cell-penetrating peptide, Retro-inverso, Cytotoxicity, Hydrophobicity, Apoptosis
National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-52312 (URN)10.1016/j.bbamem.2010.10.019 (DOI)000290705700014 ()21070744 (PubMedID)
Note

authorCount :7

Available from: 2011-01-13 Created: 2011-01-13 Last updated: 2015-04-21Bibliographically approved

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