A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos

Abstract

Cell-penetrating peptides (CPPs) are capable of transporting molecules to which they are tethered across cellular membranes. Unsurprisingly, CPPs have attracted attention for their potential drug delivery applications, but several technical hurdles remain to be overcome. Chief among them is the so-called ‘endosomal escape problem,’ i.e. the propensity of CPP-cargo molecules to be endocytosed but remain entrapped in endosomes rather than reaching the cytosol. Previously, a CPP fused to calmodulin that bound calmodulin binding site-containing cargos was shown to efficiently deliver cargos to the cytoplasm, effectively overcoming the endosomal escape problem. The CPP-adaptor, “TAT-CaM,” evinces delivery at nM concentrations and more rapidly than we had previously been able to measure. To better understand the kinetics and mechanism of CPP-adaptor-mediated cargo delivery, a real-time cell penetrating assay was developed in which a flow chamber containing cultured cells was installed on the stage of a confocal microscope to allow for observation ab initio. Also examined in this study was an improved CPP-adaptor that utilizes naked mole rat (Heterocephalus glaber) calmodulin in place of human and results in superior internalization, likely due to its lesser net negative charge. Adaptor-cargo complexes were delivered into the flow chamber and fluorescence intensity in the midpoint of baby hamster kidney cells was measured as a function of time. Delivery of 400 nM cargo was observed within seven minutes and fluorescence continued to increase linearly as a function of time. Cargo-only control experiments showed that the minimal uptake which occurred independently of the CPP-adaptor resulted in punctate localization consistent with endosomal entrapment. A distance analysis was performed for cell-penetration experiments in which CPP-adaptor-delivered cargo showing wider dispersions throughout cells as compared to an analogous covalently-bound CPP-cargo. Small molecule endocytosis inhibitors did not have significant effects upon delivery. The real-time assay is an improvement upon static endpoint assays and should be informative in a broad array of applications.

A reversible cell penetrating peptide-cargo linkage allows dissection of cell penetrating peptide- and cargo-dependent effects on internalization and identifies new functionalities of putative endolytic peptides

“…All proteins were assayed by biolayer interferometry to assure high affinity (low nm-to-high pm) binding between adaptors and CBS-cargos in the presence of calcium and fast dissociation upon its removal with EDTA. All evinced kinetics and affinities highly similar to CaM with a natural binding partner ( McMurry et al, 2011 ) and within the ranges observed for previously reported adaptor-cargo pairings ( Salerno et al, 2016 ; Ngwa et al, 2017 ; Gentry et al, 2021 ). Table 1 lists all adaptors and cargos used in this study and lists the plasmids from which they were expressed.…”

“…The resultant adaptor, TAT-NMR-CaM, has a lesser net negative charge (−5) and evinced exactly the behavior we predicted, delivering cargos that had resisted efficient TAT-CaM-mediated delivery, e.g. polyA binding protein PAB-1 ( Gentry et al, 2021 ).…”

“…Plasmids used in this study were previously described ( Salerno et al, 2016 ; Ngwa et al, 2017 ; Gentry et al, 2021 ) or constructed as described in Supplementary Figure S1 from parent vectors pET19b and pET22b (EMD Millipore, United States), pMalc-5x (New England Biolabs, United States) or pCal-N-FLAG (Agilent Technologies, United States). For example, pJM161, encoding TAT-naked mole rat calmodulin (TAT-NMR-CaM) consisted of an E. coli -optimized synthetic gene (GeneScript, Piscataway, NJ, United States) cloned into NdeI and BamHI sites in pET19b with an in-frame stop codon prior to the BamHI site.…”

“…For example, pJM161, encoding TAT-naked mole rat calmodulin (TAT-NMR-CaM) consisted of an E. coli -optimized synthetic gene (GeneScript, Piscataway, NJ, United States) cloned into NdeI and BamHI sites in pET19b with an in-frame stop codon prior to the BamHI site. The encoded TAT-NMR-CaM protein consists of the TAT peptide sequence (YGRKKRRQRRR) N-terminally fused to Heterocephalus glaber (naked mole rat) calmodulin (GenBank: EHB02604.1) ( Gentry et al, 2021 ). A vector-encoded 10xHis tag containing an enterokinase cleavage site and several spacer residues is N-terminal to TAT.…”

Utilization of a cell-penetrating peptide-adaptor for delivery of human papillomavirus protein E2 into cervical cancer cells to arrest cell growth and promote cell death

“…However, during endosomal trafficking, calcium efflux results in cargo dissociation from TAT-CaM and subsequent release to the cytoplasm of living mammalian cells. Delivery is rapid, tunable and efficient and a wide variety of cargos can be delivered into living cells (22)(23)(24) Using the TAT-CaM adaptor system, the hypothesis that bioactive CBS-E2 protein delivered directly into cervical cancer cells would inhibit cellular proliferation and/or cell death was tested. Following delivery, E2 showed distinct cell-cycle dependent subcellular localization patterns and was found in both the cytoplasm and the nucleus.…”