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Cell-penetrating peptides in cargo delivery: In vitro studies on uptake and in vivo studies on tumor targeting
Stockholm University, Faculty of Science, Department of Neurochemistry.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The capability of cell-penetrating peptides (CPPs) to transport cargos over different cellular membranes both in vitro and in vivo have drawn major attention in the past decade. Three main topics on the application of CPPs have been studied in this thesis.

First, several well-known CPPs, with fluorescein as a cargo, were shown to translocate into Nicotiana tabacum cultivar SR-1 protoplasts. By coupling different cargos to CPP it might be possible to effectively transport them inside the plant protoplasts. The translocation of CPPs into plant protoplasts might open up a new method for transformation of plant cells.

Next, the cell-penetrating ability of the novel peptide YTA2 was characterized and it was established that chemical coupling between YTA2 and the protein cargo is not needed for the transport of the cargo over the cellular membrane in vitro. The delivery of proteins into cells by mere coincubation with CPPs is an improvement, since the chemical coupling between the CPP and the cargo molecule, which adds time-consuming synthesis and purification steps, can be omitted.

Finally, by conjugating each of the two breast tumor homing peptides, the cyclic cCPGPEGAGC (PEGA) or the linear CREKA peptide, to the CPP pVEC, two cell-penetrating peptides with homing properties were obtained. Both, PEGA-pVEC and CREKA-pVEC were taken up by different breast cancer cells in vitro. Moreover, the homing capacity of the PEGA-pVEC and CREKA-pVEC was conserved in vivo, where the conjugates mainly accumulated in blood vessels in breast tumor tissue and were subsequently translocated into cells. Conjugating the anti-cancer drug chlorambucil to PEGA-pVEC or CREKA-pVEC markedly improved its efficiency. Furthermore, systemic treatment of tumor-bearing mice with chlorambucil-CREKA-pVEC significantly reduced tumor growth compared to control groups. These tumor-homing CPPs might improve both diagnosis and treatment of breast cancer tumors, by conjugation to therapeutic agents.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi , 2008. , 67 p.
National Category
Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-7263ISBN: 978-91-7155-554-0 (print)OAI: oai:DiVA.org:su-7263DiVA: diva2:197942
Public defence
2008-02-01, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2015-03-23Bibliographically approved
List of papers
1. Internalisation of cell-penetrating peptides into tobacco protoplasts
Open this publication in new window or tab >>Internalisation of cell-penetrating peptides into tobacco protoplasts
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2005 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, Vol. 1669, no 2, 101-107 p.Article in journal (Refereed) Published
Abstract [en]

Cells are protected from the surrounding environment by plasma membrane which is impenetrable for most hydrophilic molecules. In the last 10 years cell-penetrating peptides (CPPs) have been discovered and developed. CPPs enter mammalian cells and carry cargo molecules over the plasma membrane with a molecular weight several times their own. Known transformation methods for plant cells have relatively low efficiency and require improvement. The possibility to use CPPs as potential delivery vectors for internalisation in plant cells has been studied in the present work. We analyse and compare the uptake of the fluorescein-labeled CPPs, transportan, TP10, penetratin and pVEC in Bowes human melanoma cells and Nicotiana tabacum cultivar (cv.) SR-1 protoplasts (plant cells without cell wall). We study the internalisation efficiency of CPPs with fluorescence microscopy, spectrofluorometry and fluorescence-activated cell sorter (FACS). All methods indicate, for the first time, that these CPPs can internalise into N. tabacum cv. SR-1 protoplasts. Transportan has the highest uptake efficacy among the studied peptides, both in mammalian cells and plant protoplast. The internalisation of CPPs by plant protoplasts may open up a new effective method for transfection in plants.

Keyword
Cell-penetrating peptide, Plant protoplast, Membrane penetration, Transportan, TP10, Penetratin, pVEC
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-24617 (URN)10.1016/j.bbamem.2005.01.006 (DOI)
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2015-04-21Bibliographically approved
2. Protein delivery by the cell-penetrating peptide YTA2
Open this publication in new window or tab >>Protein delivery by the cell-penetrating peptide YTA2
2007 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 18, no 1, 170-174 p.Article in journal (Refereed) Published
Abstract [en]

In most cases, the transport of cell-penetrating peptide (CPP) with a cargo molecule over the plasma membrane requires a cross-linking of the cargo molecule to the peptide. Lately, a method of cargo delivery, coincubation with CPP, has been applied. We have studied uptake and toxicity of the CPP, YTA2, in the Bowes human melanoma cell line and human MDA-MB-231 breast cancer cell line and compared the results with known cell-penetrating peptides. The results show that fluoresceinyl YTA2 is taken up by the Bowes cells with 3.23 nmol/mg protein and shows low membrane toxicity to the cells with an EC50 of 60 μM. Furthermore, we show that YTA2 is capable of delivering cargo proteins, such as β-galactosidase and tetramethyl rhodamine iso-thiocyanate (TRITC) labeled streptavidin into cells by coincubation. The delivery of TRITC-labeled streptavidin was quantified to 42.4 pmol streptavidin/mg protein. The delivery of proteins into the cells by mere coincubation is an advantage, since the chemical coupling between the CPP and the cargo molecule, which adds time-consuming synthesis and purification steps, can be omitted. In addition, the flexibility in CPP cargo delivery is increased.

National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-20830 (URN)10.1021/bc060266g (DOI)000243503400022 ()
Available from: 2008-01-14 Created: 2008-01-14 Last updated: 2015-04-21Bibliographically approved
3. 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: 2015-04-21Bibliographically approved
4. Inhibition of tumor growth by chlorambucil conjugated to a new tumor targeting peptide vector
Open this publication in new window or tab >>Inhibition of tumor growth by chlorambucil conjugated to a new tumor targeting peptide vector
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(English)Manuscript (Other academic)
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-24620 (URN)
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2016-01-29Bibliographically approved

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