Cell-Penetrating Penetratin Peptides - Mechanistic Studies on Uptake Pathways and DNA Delivery Efficiency
Doktorsavhandling, 2012

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.

Plasma membrane vesicle

Lysine

Stimulated endocytosis

Gene delivery

Cell surface affinity

Penetratin

Cell-penetrating peptide

Cysteine

Uptake

Arginine

Kollektorn, Kemivägen 9, Chalmers tekniska högskola
Opponent: Prof. Steven F. Dowdy, University of California, La Jolla, USA

Författare

Helene Åmand

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

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

Biochimica et Biophysica Acta - Biomembranes,; Vol. 1808(2011)p. 1860-1867

Artikel i vetenskaplig tidskrift

Stimulated endocytosis in penetratin uptake: Effect of arginine and lysine

Biochemical and Biophysical Research Communications,; Vol. 371(2008)p. 621-625

Artikel i vetenskaplig tidskrift

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

Biochemical and Biophysical Research Communications,; Vol. 418(2012)p. 469-474

Artikel i vetenskaplig tidskrift

Genetiska sjukdomar orsakas av fel i vår arvsmassa. Genom att föra in nya, friska gener skulle dessa kunna bli botade, vilket kallas genterapi. Ett av de största hindren för utvecklingen av genterapi är att DNA måste ta sig över cellens skyddsbarriär; cellmembranet. För att DNA, som är en stor och negativt laddad molekyl, ska kunna ta sig över membranet på ett effektivt sätt behövs en vektor – en slags bärarmolekyl. Denna avhandling handlar om en sorts molekyler som skulle kunna fungera just som vektorer för DNA: Cell-penetrerande peptider. Dessa är korta aminosyrasekvenser (d.v.s. små proteiner) som har visat sig ha den sällsynta förmågan att ta sig över membranbarriären och in i levande celler. Forskningen som presenteras i denna avhandling syftar till att förstå hur cell-penetrerande peptider fungerar genom att studera hur den kemiska strukturen hos peptiden påverkar dess förmåga att ta sig in i cellerna och leverera DNA. Jag visar att genom att byta ut en aminosyra (lysin) mot en annan (arginin), kan en peptid öka sin förmåga att binda till cellmembranet och stimulera upptag genom endocytos – en av cellens naturliga mekanismer för att ta upp molekyler. Dessutom visar jag att peptiden med högre upptagseffektivitet också är bättre på att binda till och mediera transport av DNA in i celler. Min forskning bidrar därmed till värdefulla insikter kring hur cell-penetrerande peptider ska kunna utvecklas och designas för att bli effektiva vektorer för genterapi.

Ämneskategorier

Fysikalisk kemi

Fundament

Grundläggande vetenskaper

Styrkeområden

Livsvetenskaper och teknik

ISBN

978-91-7385-633-1

Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie

Kollektorn, Kemivägen 9, Chalmers tekniska högskola

Opponent: Prof. Steven F. Dowdy, University of California, La Jolla, USA