Abstract
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 nonaarginine. The molecular basis of the increased activity was elucidated in detail. Loading efficiency and intracellular residence time were assessed by confocal microscopy. Fluorescence correlation spectroscopy was employed 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.
- Fluorescence techniques
- Structure/function/mechanism
- Mechanisms of cell killing/apoptosis
- Pharmacokinetics, metabolism and activation
- Received August 19, 2010.
- Revision received December 30, 2010.
- Accepted December 30, 2010.
- The American Society for Pharmacology and Experimental Therapeutics