Tracking the Endosomal Escape: A Closer Look at Calcein and Related Reporters

Abstract

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 are developed to overcome a major hurdle, the endosomal entrapment, and delivering their cargo to the required site of action. In parallel, in vitro assays are 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, it is hoped 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.

Enhancing Cytoplasmic Expression of Exogenous mRNA through Dynamic Mechanical Stimulation

“…However, when the calcein released from endosomes into cytoplasm, a diffused calcein signal would be visualized in cytoplasm. [31] The imaging results demonstrate that the oscillation was able to enhance the release of calcein from endosome (Figure 5A&B), in agreement with the colocalization results in Figure 4. All these data collectively confirm the synergistic effect of LNPs-oscillation combination in facilitating endosomal escape, which could be attributed to the heightened kinetic energy of LNPs induced by oscillation that increased their likelihood of close contact with endosomal membrane.…”

Improving Gene Delivery: Synergy between Alkyl Chain Length and Lipoic Acid for PDMAEMA Hydrophobic Copolymers

“…This is advantageous for pH-dependent endosomal release when considering the sponge-effect hypothesis mechanism. [38][39][40][41] On the other hand, a low DoC at physiological pH potentially leads to poor binding and consequently, potential loss of genetic material. This was evident for P0-A and P0-A, which showed inferior performance (Figure S2, Supporting Information).…”

Recent Progress in the Endosomal Escape Mechanism and Chemical Structures of Polycations for Nucleic Acid Delivery

“…Calculating the colocalization ratio between the nucleic acids and typical endosomal protein markers (such as Rab5 or EEA1 (early endosome), Rab7 (late endosome), and LAMP1 (lysosome)), or endosomal staining reagents (e.g., LysoTracker and LysoSensor) provides the nucleic acid location and the endosomal escape ratio of delivery vehicles. [89,90] For example, Gilleron et al formulated fluorescent dye-labeled siRNAs using LNPs. [91] The authors reported that only ∼1-2% siRNAs escape from the endosome.…”