LAUSR

Crossing the cellular membrane and delivering active pharmaceuticals or biologicals into the cytosol of cells is an essential step in the development of nanomedicines. One of the most important intracellular processes regarding the cellular uptake of biologicals is the endolysosomal pathway. Sophisticated nanocarriers have been developed overcoming a major hurdle, the endosomal entrapment, and delivering their cargo to the required site of action. In parallel, in vitro assays have been established analyzing the performance of these nanocarriers. Among them, the release of the membrane-impermeable dye calcein has become a popular and straightforward method. It is accessible for most researchers worldwide, allows for rapid conclusions about the release potential, and enables the study of release mechanisms. This review is intended to provide an overview and guidance for scientists applying the calcein release assay. It comprises a survey of several applications in the study of endosomal escape, considerations of potential pitfalls, challenges and limitations of the assay, and a brief summary of complementary methods. Based on this review, we hope to encourage further research groups to take advantage of the calcein release assay for their own purposes and help to create a database for more efficient cross-correlations between nanocarriers. This article is protected by copyright. All rights reserved.