Investigating the Insertion Mechanism of Cell-Penetrating Peptide Penetratin into Cell Membranes: Implications for Targeted Drug Delivery

The cell-penetrating peptide (CPP) penetratin (PEN) has garnered attention for its potential to enter tumor cells. However, its translocation mechanism and lack of selectivity remain debated. This study investigated PEN’s insertion into healthy cells (H-) and cancer cells (C-) using micromolar concentrations and various techniques. Raman spectroscopy was used to determine PEN’s location in the lipid bilayer at different lipid-to-peptide ratios. Dynamic light scattering (DLS) and zeta potential analysis were used to measure the lipid–PEN complex’s size and charge. The results showed helical PEN particles directly inserted into C- membranes at a ratio of 110, while aggregated particles stayed on H- surfaces. Raman spectroscopy and scanning electron microscopy confirmed PEN insertion in C- membranes. Zeta potential studies revealed highly negative charges for PEN–C- complexes and neutral charges for PEN–H- complexes at pH 6.8. C- integrity remained unchanged at a ratio of 110. Specific lipid-to-peptide ratios with dipalmitoylphosphatidylserine (DPPS) were crucial for direct insertion. These results provide valuable insights into CPP efficacy for targeted drug delivery in cancer cells, considering membrane composition and lipid-to-peptide ratios.