European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical SciencesOct 16, 2025

Tracking how glowing cell-penetrating peptides escape from cellular compartments

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗

Updated Jan 7, 2026

Abstract

The study presents guidelines for assessing cell-penetrating peptide (CPP) endosomal escape.

CPPs can enter cells through endocytosis or direct translocation but often become trapped in endosomes and lysosomes.
Endosomal escape can potentially be enhanced by coupling CPPs with escape-promoting cargos or using permeable small molecules like L-Leucyl-L-Leucine methyl ester (LLOMe).
The effectiveness of assessing CPP endosomal escape is influenced by factors such as the chirality of amino acids, the type of attached fluorophore, and the timing of observations.
Commonly used CPPs for this assessment include R9, TAT, and penetratin.
Guidelines are provided for selecting optimal combinations of amino acid chirality, fluorophore type, and timing based on specific research goals.

Simplified

Cell-penetrating peptides (CPPs) have the capacity to transport cargos into cells either through endocytosis or direct translocation across the plasma membrane. CPPs entering via endocytosis remain generally trapped within endosomes and lysosomes. This entrapment can be alleviated by coupling the CPPs to endosomal escape promoting cargos or by using small molecules that permeate endosomes or lysosomes. L-Leucyl-L-Leucine methyl ester (LLOMe) is an example of such a molecule. The assessment of CPP endosomal escape into the cytosol is not trivial as cytosolic acquisition can also occur via CPP direct translocation across the plasma membrane. Moreover, visualization of CPP cytosolic acquisition often requires the CPP to be tagged with a fluorophore that can affect the behavior of the CPP and that can also be released from the CPP in the degradative environment of endosomes and lysosomes. In the present work, we have explored various parameters that can potentially affect the recording of CPP endosomal escape, such as the chirality of the amino acids making the CPPs, the nature of the fluorophore coupled to the CPPs, and the timing of the recording. For this purpose, we have used three commonly used CPPs: R9 (a synthetic CPP made of 9 arginines), TAT, and penetratin. We have used LLOMe as the endosomal escape promoter. This study presents guidelines for the assessment of CPP endosomal escape. We also provide a framework for selecting appropriate combinations of amino acid chirality, fluorophore type, and assessment timing based on what researchers want to achieve when the CPP-cargo is released from vesicles into the cytosol.

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Competing interests

Declaration of competing interest The authors declare no conflict of interest.

PubMed

Tracking how glowing cell-penetrating peptides escape from cellular compartments

Abstract

Full Text

Funding

Competing interests

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