Kinetic Analysis of Cellular Internalization and Expulsion of
Unstructured D-chirality Cell Penetrating Peptides
Abstract
Most cell penetrating peptides (CPPs) are unstructured and susceptible
to proteolytic degradation. One alternative is to incorporate
D-chirality amino acids into unstructured CPPs to allow for enhanced
uptake and intracellular stability. This work investigates CPP
internalization using a series of time, concentration, temperature, and
energy dependent studies, resulting in a three-fold increase in uptake
and 50-fold increase in stability of D-chirality peptides over
L-chirality counterparts. CPP internalization occurred via a combination
of direct penetration and endocytosis, with a percentage of internalized
CPP expelling from cells in a time-dependent manner. Mechanistic studies
identified that cells exported the intact internalized D-chirality CPPs
via an exocytosis independent pathway, analogous to a direct penetration
method out of the cells. These findings highlight the potential of
D-chirality CPPs as bio-vectors in therapeutic and biosensing
applications, but also identify a new expulsion method suggesting a
relationship between uptake kinetics, intracellular stability, and
export kinetics