Identifying cell culturing parameters that improve endocytic uptake of the HIV-TAT cell penetrating peptide
Delivery tools, including cell-penetrating peptides (CPPs) are often inefficient due to a combination of poor endocytosis and endosomal escape. Herein, the impact of cell culturing techniques on the endocytic uptake of a typical CPP, the TAT peptide (derived from HIV1-TAT), was quantified. Parameters previously found to generally modulate endocytosis such as cell density, washing steps, and cell aging did not affect TAT endocytosis. In contrast, cell dissociation methods, media, temperature, serum starvation, and media composition all contributed to changes in uptake. The combination of these parameters in worst versus best-uptake protocols, led to changes in uptake of more than 13-fold and indicated that small variations in cell culturing techniques have a cumulative effect on CPP uptake. More specifically, modulating cell culture protocols does not result in an increased amount of peptide inside endosomes, rather the number of TMR-TAT containing endosomes increases. Taken together this study highlights how technical aspects of cell culture protocols can be used to improve experimental reproducibility, as well as parameters that can be potentially exploited to improve CPP accumulation in endosomes, and hence increase the possibility of endosomal escape and cytosolic access.