Live-Cell Raman Imaging Elucidates Intracellular Distribution of Macrocyclic Peptides Designed as HIV Protease Inhibitors.

Macrocyclic peptides have garnered increasing attention due to their therapeutic potential. However, the low cell membrane permeability of macrocyclic peptides limits their access to intracellular targets. This study aimed to apply live-cell Raman imaging to detect intracellular macrocyclic peptides as a potential therapeutic modality. Alkyne-tags with specific Raman bands were attached to several types of macrocyclic peptides originally designed as HIV protease inhibitors. Membrane permeability was measured by using the traditional penetration test. Variations in permeability were observed among the compounds, with the alkyne group showing a minimal effect on the results. Macrocyclic peptides with an alkyne-tag, which exhibited no permeability in the penetration test, were added to the live cells and evaluated using Raman imaging. Two-dimensional (2D) and three-dimensional (3D) Raman imaging detected the macrocyclic peptides in the intracellular region. However, the alkyne signal was not observed in cells treated with the macrocyclic peptides, showing cell penetration in the penetration test. These results are consistent with the observed pharmacological activities and suggest that the traditional penetration test may be insufficient for effective drug design of macrocyclic peptides. We conclude that combining emerging techniques, such as Raman imaging, with traditional methods can provide a more detailed evaluation of the permeability of macrocyclic peptides.