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Rational modifications of cell-penetrating peptides for drug delivery: Applications in tumor targeting and oligonucleotide delivery
Stockholm University, Faculty of Science, Department of Neurochemistry.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

High molecular weight biomolecules are becoming important in the development of new therapeutics. However, their size and nature creates a major limitation for their application – poor penetration through biological membranes. A new class of peptides, cell-penetrating peptides (CPPs), has shown the capability to transport various macromolecules inside the cells. However, there are at least two limiting factors for successful application of CPPs: the lack of cell-type specificity and restricted bioavailability resulting from endocytic uptake of CPPs and entrapment in endosomal compartments.

This thesis aims at designing delivery vehicles for therapeutic substances. In papers I-III, the CPPs have been rationally modified in order to achieve in vivo selectivity towards cancer cells. The first two papers employ tumor homing peptides as targeting moieties coupled to the N-termini of CPPs. In the third paper, a CPP is C-terminally prolonged with a matrix metalloproteinase 2 (MMP-2) specific cleavage site followed by an inactivating amino acid sequence. In tissues overexpressing MMP-2, i. e. in proximity to cancer, the CPP is activated after proteolytic removal of the inactivating sequence, thus the cargo can be transported inside the cells. In paper IV, several CPPs have been N-terminally modified with a stearyl moiety and applied for the delivery of splice-correcting oligonucleotides. We show that stearyl-TP10 is as effective in oligonucleotide delivery as Lipofectamine™ 2000. Moreover, stearyl-TP10 has preserved efficacy in serum and is not toxic to cells. In conclusion, the rational modifications of CPPs greatly potentiate their application in cargo delivery both in vitro and in vivo.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi , 2009. , 47 p.
Keyword [en]
Cell-penetrating peptide, oligonucleotide, drug delivery, tumor targeting
National Category
Neurosciences
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-8374ISBN: 978-91-7155-792-6 (print)OAI: oai:DiVA.org:su-8374DiVA: diva2:200175
Public defence
2009-01-30, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2009-01-08 Created: 2008-12-08 Last updated: 2010-03-09Bibliographically approved
List of papers
1. Design of a Tumor-Homing Cell-Penetrating Peptide
Open this publication in new window or tab >>Design of a Tumor-Homing Cell-Penetrating Peptide
2008 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 19, no 1, 70-75 p.Article in journal (Refereed) Published
Abstract [en]

Chemotherapy is often limited by toxicity to normal cells. Therefore, an ideal anticancer drug should discriminate between normal tissue and tumors. This would require a target receptor molecule mostly present in tumors. The cyclic peptide cCPGPEGAGC (PEGA) is a homing peptide that has previously been shown to accumulate in breast tumor tissue in mice. PEGA peptide does not cross the plasma membrane per se; however, when attached to the cell-penetrating peptide pVEC, the conjugate is taken up by different breast cancer cells in vitro. Additionally, the homing capacity of the PEGA-pVEC is conserved in vivo, where the conjugate mainly accumulates in blood vessels in breast tumor tissue and, consequently is taken up. Furthermore, we show that the efficacy of the anticancer drug, chlorambucil, is increased more than 4 times when the drug is conjugated to the PEGA-pVEC chimeric peptide. These data demonstrate that combining a homing sequence with a cell-penetrating sequence yields a peptide that combines the desirable properties of the parent peptides. Such peptides may be useful in diagnostics and delivery of therapeutic agents to an intracellular location in a specific tumor target tissue.

Keyword
Cell-penetrating peptide, Homing peptide, Drug delivery, Tumor targeting
National Category
Biochemistry and Molecular Biology Neurosciences
Identifiers
urn:nbn:se:su:diva-25661 (URN)10.1021/bc0701139 (DOI)000252520300012 ()
Available from: 2009-01-08 Created: 2008-12-08 Last updated: 2017-12-13Bibliographically approved
2. Design of a tumor homing cell-penetrating peptide for drug delivery
Open this publication in new window or tab >>Design of a tumor homing cell-penetrating peptide for drug delivery
2009 (English)In: International Journal of Peptide Research and Therapeutics, ISSN 1573-3149, Vol. 15, no 1, 11-15 p.Article in journal (Refereed) Published
Abstract [en]

The major drawbacks with conventional cancer chemotherapy are the lack of satisfactory specificity towards tumor cells and poor antitumor activity. In order to improve these characteristics, chemotherapeutic drugs can be conjugated to targeting moieties e.g. to peptides with the ability to recognize cancer cells. We have previously reported that combining a tumor homing peptide with a cell-penetrating peptide yields a chimeric peptide with tumor cell specificity that can carry cargo molecules inside the cells. In the present study, we have used a linear breast tumor homing peptide, CREKA, in conjunction with a cell-penetrating peptide, pVEC. This new chimeric peptide, CREKA–pVEC, is more convenient to synthesize and moreover it is better in translocating cargo molecules inside cancer cells as compared to previously published PEGA–pVEC peptide. This study demonstrates that CREKA–pVEC is a suitable vehicle for targeted intracellular delivery of a DNA alkylating agent, chlorambucil, as the chlorambucil–peptide conjugate was substantially better at killing cancer cells in vitro than the anticancer drug alone.

Keyword
Cell-penetrating peptide, Homing peptide, Drug delivery, Tumor targeting
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-25662 (URN)10.1007/s10989-008-9156-x (DOI)000265022900002 ()
Available from: 2009-01-08 Created: 2008-12-08 Last updated: 2015-04-21Bibliographically approved
3. Target-activated cell-penetrating peptide
Open this publication in new window or tab >>Target-activated cell-penetrating peptide
Show others...
(English)Manuscript (Other academic)
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-25663 (URN)
Available from: 2009-01-08 Created: 2008-12-08 Last updated: 2016-01-29Bibliographically approved
4. A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy
Open this publication in new window or tab >>A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy
Show others...
2009 (English)In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 134, no 3, 221-227 p.Article in journal (Refereed) Published
Abstract [en]

Aberrations in splicing patterns play a significant role in several diseases, and splice correction, together with other forms of gene regulation, is consequently an emerging therapeutic target. In order to achieve successful oligonucleotide transfection, efficient delivery vectors are generally necessary. In this study we present one such vector, the chemically modified cell-penetrating peptide (CPP) TP10, for efficient delivery of a splice-correcting 2'-OMe RNA oligonucleotide. Utilizing a functional splice correction assay, we assessed the transfection efficiency of non-covalent complexes of oligonucleotides and stearylated or cysteamidated CPPs. Stearylation of the CPPs Arg9 and penetratin, as well as cysteamidation of MPG and TP10, did not improve transfection, whereas the presence of an N-terminal stearyl group on TP10 improved delivery efficiency remarkably compared to the unmodified peptide. The splice correction levels observed with stearyl-TP10 are in fact in parity with the effects seen with the commercially available transfection agent Lipofectamine (TM) 2000. However, the inherent toxicity associated with cationic lipid-based transfections can be completely eliminated when using the stearylated TP10, making this vector highly promising for non-covalent delivery of negatively charged oligonucleotides.

Keyword
Cell-penetrating peptide; Co-incubation, Phosphorothioate 2 '-O-methyl RNA, Splice correction, Stearylation
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-25664 (URN)10.1016/j.jconrel.2008.11.025 (DOI)000264722000010 ()
Available from: 2009-01-08 Created: 2008-12-08 Last updated: 2017-12-13Bibliographically approved

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