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 a D‐chirality CPP as bio‐vector in therapeutic and biosensing applications, but also identify a new expulsion method suggesting a relationship between uptake kinetics, intracellular stability, and export kinetics.

Membrane Internalization Mechanisms and Design Strategies of Arginine-Rich Cell-Penetrating Peptides

“…Isomer is another factor affecting the function of CPPs [ 10 , 64 , 65 , 66 , 67 , 68 ]; a different number of arginine optical isomers in the peptide sequence are proved to affect the uptake efficiency of the delivery peptides. Verdurmen et al [ 69 ] firstly reported the differences in the uptake of L-CPPs (R 9 ) and their D-counterparts (r 9 ); the results demonstrated that cationic L-CPPs (R 9 ) were taken up more efficiently than their D-counterparts (r 9 ) in MC57 fibrosarcoma and HeLa cells but not in Jurkat T leukemia cells (the sequences of the peptides were shown in Table 1 ).…”

Intracellular targets: A multiple cargo transporting molecule

“…The integration of non-natural amino acids, a determined secondary structure, or an increased backbone inflexibility could achieve CPPs stability and protect them against degradation. 18 So, the development of other CPPs that do not present such issues is a goal for the scientific community to apply them in vivo.…”

Supramolecular Chiral Binding Affinity‐Achieved Efficient Synergistic Cancer Therapy

“…It is known direct penetration and endocytosis are the main pathways for the cellular uptake of nanomaterials. [ 34 , 35 ] To clarify if the cellular uptake of WP5⊃D/DL/L‐Arg+DOX+ICG supramolecular nanoparticles is mediated by direct penetration pathway (energy independent), the cellular uptake experiments were performed at 4 °C, with the co‐incubation of WP5⊃D/DL/L‐Arg+DOX+ICG nanoparticles and Hela cells for 24 h. As shown in Figure S17 (Supporting Information), the internalization of WP5⊃D/DL/L‐Arg+DOX+ICG into CT26 cells was completely inhibited at this condition, suggesting the endocytosis‐based cellular uptake of WP5⊃D/DL/L‐Arg+DOX+ICG. To further demonstrate the specific endocytosis pathway, inhibitors, i.e., chlorpromazine (inhibitor of clathrin‐dependent endocytosis), nystatin (inhibitor of caveolin‐dependent endocytosis), and wortmannin (inhibitor of micropinocytosis‐mediated endocytosis) were employed for the pretreatment of CT26 cells.…”