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Distinct Uptake Routes of Cell-Penetrating Peptide Conjugates
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
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2008 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 19, no 12, 2535-2542 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) are a growing family of peptides that have opened a new avenue in drug delivery, allowing various hydrophilic macromolecules to enter cells. In accordance with most other cationic delivery vectors, CPPs seem to rely mostly on endocytosis for internalization. However, due to conflicting results the exact endocytic pathways for CPP uptake have not yet been resolved. Here, we evaluated the ability of seven CPPs, with different chemical properties, to convey peptide nucleic acids (PNAs) inside cells. Assays based on both splice correction, generating biologically active read-out, and on traditional fluorescence measurements were utilized. The same assays were employed to assess different endocytic pathways and the dependence on extracellular heparan sulfates for internalization. Both highly cationic CPPs (M918, penetratin, and Tat) and amphipathic peptides (transportan, TP10, MAP, and pVEC) were investigated in this study. Conjugate uptake relied on endocytosis for all seven peptides but splice-correcting activity varied greatly for the investigated CPPs. The exact endocytic internalization routes were evaluated through the use of well-known endocytosis inhibitors and tracers. In summary, the different chemical properties of CPPs have little correlation with their ability to efficiently deliver splice-correcting PNA. However, conjugates of polycationic and amphipathic peptides appear to utilize different internalization routes.

Place, publisher, year, edition, pages
2008. Vol. 19, no 12, 2535-2542 p.
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:su:diva-15048DOI: 10.1021/bc800212jISI: 000261767800032PubMedID: 19012426OAI: oai:DiVA.org:su-15048DiVA: diva2:181568
Available from: 2008-11-19 Created: 2008-11-19 Last updated: 2015-04-21Bibliographically approved
In thesis
1. Specificity of antisense oligonucleotide derivatives and cellular delivery by cell-penetrating peptides
Open this publication in new window or tab >>Specificity of antisense oligonucleotide derivatives and cellular delivery by cell-penetrating peptides
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Atypical gene expression has a major influence on the disease profile of several severe human disorders. Oligonucleotide (ON) based therapeutics has opened an avenue for compensating deviant protein expression by acting on biologically important nucleic acids, mainly RNAs. Antisense ONs (asONs) can be designed to target complementary specific RNA sequences and thereby to influence the corresponding protein synthesis. However, cellular uptake of ONs is poor and is, together with the target specificity of the asONs, the major limiting factor for the development of ON based therapeutics.

In this thesis, the mechanisms of well-characterized cell-penetrating peptides (CPPs) are evaluated and CPPs are adapted for cellular ON-delivery. The functionality of ON derivatives in cells is investigated and by optimization of asONs, targeting pre-messenger RNA, high efficiency and specificity is achieved. The optimization of the asONs is based on sequence design and through the choice of nucleic acid analogue composition. It is concluded that asONs, partly composed of locked nucleic acids are attractive for splice-switching applications but these mixmers must be designed with limited number of locked nucleic acid monomers to avoid risk for off-target activity. A protocol allowing for convenient characterization of internalization routes for CPPs is established and utilized. A mechanistic study on cellular CPP uptake and translocation of associated ON cargo reveals the importance of the optimal combination of for example charge and hydrophobicity of CPPs for efficient cellular uptake. Formation of non-covalent CPP:ON complexes and successful cellular delivery is achieved with a stearylated version of the well-recognized CPP, transportan 10.

The results illustrate that CPPs and ON derivatives have the potential to become winning allies in the competition to develop therapeutics regulating specific protein expression patterns involved in the disease profile of severe human disorders.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2009. 64 p.
Keyword
cell-penetrating peptide, splice-switching oligonucleotide, oligonucleotide derivative
National Category
Neurosciences
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-31226 (URN)978-91-7155-950-0 (ISBN)
Public defence
2009-12-22, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Projects
VINNOVA-SAMBIO Multidisciplinary BIO
Note
At the time of doctoral defense, the following papers were unpublished and had s status as follows: Paper 4: Accepted. Peper 5: In press.Available from: 2009-11-30 Created: 2009-11-08 Last updated: 2015-04-21Bibliographically approved

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