Trapped! A Critical Evaluation of Methods for Measuring Total Cellular Uptake versus Cytosolic Localization

Abstract

Biomolecules have many properties that make them promising for intracellular therapeutic applications, but delivery remains a key challenge because large biomolecules cannot easily enter the cytosol. Furthermore, quantification of total intracellular versus cytosolic concentrations remains demanding, and the determination of delivery efficiency is thus not straightforward. In this review, we discuss strategies for delivering biomolecules into the cytosol and briefly summarize the mechanisms of uptake for these systems. We then describe commonly used methods to measure total cellular uptake and, more selectively, cytosolic localization, and discuss the major advantages and drawbacks of each method. We critically evaluate methods of measuring "cell penetration" that do not adequately distinguish total cellular uptake and cytosolic localization, which often lead to inaccurate interpretations of a molecule's cytosolic localization. Finally, we summarize the properties and components of each method, including the main caveats of each, to allow for informed decisions about method selection for specific applications. When applied correctly and interpreted carefully, methods for quantifying cytosolic localization offer valuable insight into the bioactivity of biomolecules and potentially the prospects for their eventual development into therapeutics.

Cytosolic delivery of inhibitory antibodies with cationic lipids

“…When developing intracellular protein delivery technologies, it is imperative to use an assay that only detects cargo proteins that have been successfully delivered to the cytosol. Endosome escape is the major bottleneck for cytosolic delivery, resulting in large false-positive rates for assays that measure total cellular uptake of cargo proteins (22,23). Accordingly, we used a stringent self-assembling splitGFP (24) reporter system in which one half of the splitGFP, the S11 peptide, is fused to pAbBD-ApP, whereas the other half, splitGFP(1-10), is expressed in reporter cells (25)(26)(27)(28).…”

Cellular Uptake and Cytosolic Delivery of a Cyclic Cystine Knot Scaffold

“…30,32 Several groups have reported assays that can measure cytosolic localization without interference from endosomally trapped material. 31,33 One prominent example of an assay that quantitates cytosolic delivery is the chloroalkane penetration assay (CAPA), recently developed by the Kritzer group. 32,34 CAPA allows for quantitative measurement of the cytosolic delivery of peptides in a low-volume, high-throughput format.…”

Status update in the use of cell-penetrating peptides for the delivery of macromolecular therapeutics

“…Regarding the shortcomings in clinical development with the CPPs, similar reviews in the past have repeatedly drawn attention and raised critics about some inadequacies of study designs or interpretation of results [7,40,52,53] as the reason for slow progress in the field. As a positive side, this situation has been improving, which is reflected by the large number of well-designed studies referenced above.…”