Coupling to polymeric scaffolds stabilizes biofunctional peptides for intracellular applications.
Here, we demonstrate that coupling to N-hydroxypropyl methacrylamide (HPMA) copolymer greatly enhances the activity of apoptosis-inducing peptides inside cells. Peptides corresponding to the BH3 domain of Bid were coupled to a thioester-activated HPMA (28.5 kDa) via native chemical ligation in a simple one-pot synthesis. Peptides and polymer conjugates were introduced into cells either by electroporation or by conjugation to the cell-penetrating peptide nona-arginine. The molecular basis of the increased activity is elucidated in detail. Loading efficiency and intracellular residence time were assessed by confocal microscopy. Fluorescence correlation spectroscopy was used as a separation-free analytical technique to determine proteolytic degradation in crude cell lysates. HPMA conjugation strongly increased the half-life of the peptides in crude cell lysates and inside cells, revealing proteolytic protection as the basis for higher activity.
Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Ivo R
IR
Ruttekolk
Alokta
A
Chakrabarti
Martin
M
Richter
Falk
F
Duchardt
Heike
H
Glauner
Wouter P R
WP
Verdurmen
Jörg
J
Rademann
Roland
R
Brock
D017209:Apoptosis / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D019169:Jurkat Cells; D008689:Methacrylates / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D011108:Polymers / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D055550:Protein Stability; D017434:Protein Structure, Tertiary / Q000502:physiology
D008689:Methacrylates; D009842:Oligopeptides; D010455:Peptides; D011108:Polymers; C491427:nonaarginine; C032802:hydroxypropyl methacrylate