Cytosolic delivery of a p16-peptide oligoarginine conjugate for inhibiting proliferation of MCF7 cells.

One of the major limitations in protein and peptide therapeutics is the requirement of delivery to the cytosol or nucleus of cells. It has recently been shown that a small peptide derived from the p16 protein is able to inhibit cell cycle progression when delivered to the cytosol after conjugation to cell penetrating peptides, however the correlation between delivery efficiency and biological activity has not been made. Additionally, whether or not the biological activity attained was due to membrane transduction has not been established. In this paper, the total internalization, and internalization via endocytosis and transduction of 125I-p16, 125I-p16-C(R)9, and 125I-p16-C(K)9 were determined in MCF7 cultured cell monolayers. The results showed that while p16 and p16-oligopeptide conjugates have similar total internalization, 125I-p16-C(R)9 is predominantly internalized via membrane transduction, while p16 and p16-oligolysine are primarily endocytosed. Therefore, the amount of 125I-p16-C(R)9 delivered to the cytosol is significantly higher than both 125I-p16-C(K)9 and 125I-p16. These results show that biological activity is correlated with membrane transduction efficiency, and not total internalization. Additionally, the biological activity and delivery to the cytosol were not sensitive to endocytic inhibitors, verifying that the biological effect is due to membrane transduction, and not endocytosis.