In silico identification and experimental validation of cellular uptake and intracellular labeling by a new cell penetrating peptide derived from CDN1

Bioactive therapeutic molecules are generally impermeable to the cell membrane, hindering their utility and efficacy. A group of peptides called cell-penetrating peptides (CPPs) were found to have the capability of transporting different types of cargo molecules across the cell membrane. Here, we identified a short peptide named P2, which has a higher proportion of basic residues than the CDN1 (cyclin-dependent kinase inhibitor 1) protein it is derived from, and we used bioinformatic analysis and experimental validation to confirm the penetration property of peptide P2. We found that peptide P2 can efficiently enter different cell lines in a concentration-dependent manner. The endocytosis pathway, especially receptor-related endocytosis, may be involved in the process of P2 penetration. Our data also showed that peptide P2 is safe in cultured cell lines and red blood cells. Lastly, peptide P2 can efficiently deliver self-labeling protein HaloTag into cells for imaging. Our study illustrates that peptide P2 is a promising imaging agent delivery vehicle for future applications.