Fluorescence correlation spectroscopy reveals highly efficient cytosolic delivery of certain penta-arg proteins and stapled peptides

Abstract

We used fluorescence correlation spectroscopy (FCS) to accurately and precisely determine the relative efficiencies with which three families of "cell-penetrating peptides" traffic to the cytosol of mammalian cells. We find that certain molecules containing a "penta-arg" motif reach the cytosol, intact, with efficiencies greater than 50%. This value is at least 10-fold higher than that observed for the widely studied cationic sequence derived from HIV Tat or polyarginine Arg8, and equals that of hydrocarbon-stapled peptides that are active in cells and animals. Moreover, we show that the efficiency with which stapled peptides reach the cytosol, as determined by FCS, correlates directly with their efficacy in cell-based assays. We expect that these findings and the associated technology will aid the design of peptides, proteins, and peptide mimetics that predictably and efficiently reach the interior of mammalian cells.

Figure 1

(A) FCS workflow to determine the cytosolic concentration of a rhodamine-tagged molecule. (B) Three families of cell-penetrating molecules studied using FCS in this report.

Figure 2

Total cell uptake as measured by flow cytometry. (A, B) Flow cytometry histograms and (C–E) bar plots illustrating the relative uptake of the indicated Rho-tagged molecule (500 nM) after 30 min incubation with HeLa cells. Values of F₅₉₀/cell reflect the total cell-associated fluorescence, including fractions associated with the cell surface, endosomal compartments, as well as molecules that have reached the cytosol.

Figure 3

Quantifying cytosolic delivery efficiencies of rhodamine-peptide conjugates using fluorescence correlation spectroscopy (FCS). (A) Representative x,z-section fluorescence intensity scans of single HeLa cells treated for 30 min with the indicated Rho-tagged peptide alongside the corresponding FCS trace displaying the diffusion time (τ_D), anomalous coefficient (a), and counts per molecule (cpm, Hz) associated with that individual measurement. Cell outlines are shown as dotted lines. (B) Intracellular concentrations of rhodamine-peptide conjugates extrapolated from respective autocorrelation fits. The value shown represents the average concentration of between 8 and 50 cells. (C) Cytosolic delivery efficiency of respective rhodamine-peptide conjugates determined by dividing the intracellular concentration by treatment concentration. Error bars represent standard error of the mean.

Figure 4

Biochemical analysis of HeLa cell cytosol after incubation with ZF 5.3^R and 5.3^R. (A) HeLa cells were treated with 1 μM of either ZF 5.3^R and 5.3^R for 30 min before homogenization in an isotonic sucrose solution and ultracentrifugation at 100g for 30 min. (B, C) Samples of the supernatant after ultracentrifugation were injected onto a Shimadzu UFLC-XR UPLC alongside authentic samples and resolved on an Agilent Poroshell 120 SB-C18 column at 580 nm.

Figure 5

FCS analysis of intracellular concentrations achieved in HeLa cells by hydrocarbon-stapled peptide inhibitors of p53-HDM2 complexation. (A) Sequences of hydrocarbon-stapled peptides analyzed herein. (B) Representative x,z-section fluorescence intensity scans of single HeLa cells treated for 30 min with the indicated Rho-tagged molecule under standard culture conditions, and corresponding intracellular FCS traces displaying diffusion time (τ_D), anomalous coefficient (a), and counts per molecule (cpm, Hz) values. Cell outlines are shown as dotted lines. (C) Intracellular concentrations of Rho-tagged molecules extrapolated from respective autocorrelation fits (solid bars) and corresponding cytosolic delivery efficiencies (hatched bars). Error bars represent standard error of the mean.