title: TAT peptide and its conjugates: proteolytic stability.
abstract: The proteolytic cleavage of TATp, TATp-PEG(1000)-PE conjugate (TATp-conjugate), and TATp as TATp-conjugate in mixed micelles made of TATp-conjugate and PEG(5000)-PE (2.5% mol of TATp-conjugate, TATp-Mic) were studied by HPLC with fluorescent detection using fluorenylmethyl chloroformate (FMOC) labeling and by MALDI-TOF MS analysis. The cleavage kinetics were analyzed in human blood plasma and in trypsin-containing phosphate buffered saline (PBS), pH 7.4, to simulate the proteolytic activity of human plasma. The trypsinolysis of free TATp, TATp-conjugate, and TATp-Mic revealed that the main initial fragmentation is an endocleavage at the carboxyl terminus resulting in an Arg-Arg (RR) dimer. The trypsinolysis followed pseudo-first-order kinetics. The cleavage of the free TATp was relatively fast with a half-life of a few minutes (t(1/2) ∼ 3.5 min). The TATp-conjugate showed more stability with about a 3-fold increase in half-life (t(1/2) ∼ 10 min). TATp in TATp-Mic was highly protected against proteolysis with an over 100-fold increase in half-life (t(1/2) ∼ 430 min). The shielding of TATp by PEG moieties in the proposed TATp-Mic is of great importance for its potential use as a cell-penetrating moiety for multifunctional "smart" drug delivery systems with detachable PEG.
authors: Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, USA.
authors: Jacob
authors: J
authors: Grunwald
authors: Tomas
authors: T
authors: Rejtar
authors: Rupa
authors: R
authors: Sawant
authors: Zhouxi
authors: Z
authors: Wang
authors: Vladimir P
authors: VP
authors: Torchilin
mesh_terms: D057846:Cell-Penetrating Peptides / Q000097:blood / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008823:Micelles; D010446:Peptide Fragments / Q000097:blood / Q000737:chemistry*; D010714:Phosphatidylethanolamines / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D055550:Protein Stability; D014357:Trypsin / Q000737:chemistry; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000097:blood / Q000737:chemistry*
chemical_list: D057846:Cell-Penetrating Peptides; D008823:Micelles; D010446:Peptide Fragments; D010714:Phosphatidylethanolamines; D054322:tat Gene Products, Human Immunodeficiency Virus; D011092:Polyethylene Glycols; D014357:Trypsin