CPL - Chalmers Publication Library
| Utbildning | Forskning | Styrkeområden | Om Chalmers | In English In English Ej inloggad.

Cell-Penetrating Penetratin Peptides - Mechanistic Studies on Uptake Pathways and DNA Delivery Efficiency

Helene Åmand (Institutionen för kemi- och bioteknik, Fysikalisk kemi)
Göteborg : Chalmers University of Technology, 2012. ISBN: 978-91-7385-633-1.- 77 s.
[Doktorsavhandling]

Delivery of gene-targeted drugs is limited by the inherently poor capacity of nucleic acids to overcome the membrane barrier of the cell, and development of vectors that can promote uptake is therefore crucial. Cell penetrating peptides (CPPs) have emerged as promising vector candidates due to their ability to deliver a wide range of macromolecular cargos into cells. Uptake pathways of CPPs have been extensively studied for more than two decades, but we still lack a detailed mechanistic understanding of how CPPs interact with both the cell surface and the cargo to mediate delivery. The work presented in this Thesis has increased our mechanistic insight into how CPPs function by addressing three key steps in CPP-mediated delivery: cell surface binding, stimulated uptake and gene delivery efficiency, using the classical CPP penetratin as a model peptide. The main focus is on the relative importance of the two cationic residues arginine and lysine, in order to rationalize the superior uptake often observed with arginine-rich CPPs. In addition, an important part of this Thesis is the development of a cell-like model system, plasma membrane vesicles (PMVs), for investigating CPP cell surface interactions. By studying both cell surface binding and internalization quantitatively, it is demonstrated that arginines have a greater capacity than lysines to bind to the cell surface and trigger internalization via macropinocytosis. Uptake efficiency is found to be dependent on each peptide’s cell surface affinity, rather than on specific uptake-promoting interactions. However, arginines are also found to be less reliant on cell surface proteoglycans for internalization, and thus more versatile than lysines in promoting uptake via multiple pathways. In addition to promoting uptake, arginines are also demonstrated to be superior to lysines in condensing DNA and mediating gene delivery. However, arginines alone are not sufficient, and hydrophobic residues are found to be necessary to stabilize CPP-DNA interactions. To improve gene delivery further, a strategy based on cysteine modification to allow formation of reversible CPP dimers is assessed. It is demonstrated that functionalization with a single cysteine residue has the capacity to improve stability, enhance endosomal escape and reduce cytotoxicity of CPP-DNA complexes. Altogether, the work presented in this Thesis provides important implications for design of peptide-based gene delivery vectors.

Nyckelord: Cell-penetrating peptide, Penetratin, Uptake, Stimulated endocytosis, Cell surface affinity, Plasma membrane vesicle, Gene delivery, Arginine, Lysine, Cysteine



Den här publikationen ingår i följande styrkeområden:

Läs mer om Chalmers styrkeområden  

Denna post skapades 2012-01-18. Senast ändrad 2013-09-25.
CPL Pubid: 153693

 

Läs direkt!

Lokal fulltext (fritt tillgänglig)


Institutioner (Chalmers)

Institutionen för kemi- och bioteknik, Fysikalisk kemi (2005-2014)

Ämnesområden

Livsvetenskaper
Biofysikalisk kemi

Chalmers infrastruktur

Relaterade publikationer

Inkluderade delarbeten:


Stimulated endocytosis in penetratin uptake: Effect of arginine and lysine


Binding of cell-penetrating penetratin peptides to plasma membrane vesicles correlates directly with cellular uptake


Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation


Examination

Datum: 2012-02-16
Tid: 10:15
Lokal: Kollektorn, Kemivägen 9, Chalmers tekniska högskola
Opponent: Prof. Steven F. Dowdy, University of California, La Jolla, USA

Ingår i serie

Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie