Cytosolic Delivery of Macromolecules in Live Human Cells Using the Combined Endosomal Escape Activities of a Small Molecule and Cell Penetrating Peptides

Abstract

Ineffective cellular delivery is a common problem in numerous biological applications. Developing delivery reagents that work robustly in a variety of experimental settings remains a challenge. Herein, we report how peptides derived from the prototypical cell penetrating peptide TAT can be used in combination with a small molecule, UNC7938, to deliver macromolecules into the cytosol of cells by a simple co-incubation protocol. We establish successful delivery of peptides, DNA plasmids, and a single-chain variable fragment antibody. We also demonstrate that delivery works in hard-to-transfect mammalian cells and under conditions typically inhibitory to cell-penetrating peptides. Mechanistically, UNC7938 destabilizes the membrane of endosomes. This, in turn, enhances the endosome-leakage activity of cell-penetrating peptides and facilitates the endosomal escape of macromolecules initially internalized by mammalian cells via endocytosis. This combined selective membrane-destabilization represents a new chemical space for delivery tools and provides a novel solution to the problem of endosomal entrapment that often limits the effectiveness of reagent-based delivery approaches.

Figure 1.

UNC7938 enhances cytosolic penetration of the CPP D-dfTAT. A) Structures of the peptide D-dfTAT and of the small molecule UNC7938. Lower case letters in the peptide sequence refer to D-amino acids. dfTAT has the identical structure but is synthesized with L-amino acids instead. B) Fluorescence microscopy images of HeLa cells treated with D-dfTAT and UNC7938 for 30 min. Images are overlays of fluorescence images pseudo-colored red for TMR and blue for Hoechst 33342. An overlay with Lysotracker Green, pseudo-colored green, is also shown for the condition showing a punctate peptide distribution. Nucleoli are highlighted with white arrows in zoomed-in images. C) HeLa cells were incubated with D-dfTAT and UNC7938 for 30 min, washed, incubated with SYTOX green and Hoechst 33342, and imaged. The cells displaying D-dfTAT positive nucleolar staining while excluding SYTOX green were counted as alive and positive for cell penetration by the peptide. The total number of cells present was established by counting all Hoechst 33342 stained nuclei. The data represented are the means of biological triplicates with corresponding standard deviations (>500 cells counted per experiment). D) Quantification of cell viability 1 and 24 h after incubation with D-dfTAT and UNC7938 (30 min incubation), as measured by a SYTOX Green exclusion assay (SEA) and MTT assay. The data represented correspond to the mean of biological triplicates (>500 cells counted per experiment). E) Effect of UNC7938 on D-dfTAT cell penetration in the F11 and neuro-2a cell lines.

Figure 2.

UNC7938 enhances the cytosolic entry of D-dfTAT by facilitating endosomal leakage. A) Hela cells were incubated with combinations of D-dfTAT, UNC7938, and bafilomycin for 1h. Cells were then washed and incubated for an additional 30 min. Cells were imaged after the first and second incubation period and cytosolic penetration of D-dfTAT was scored. B) Total peptide uptake of D-dfTAT (1 μM) with or without UNC7938 after 30 in incubation. C) Comparison of the effect of UNC7938 and chloroquine on D-dfTAT cytosolic penetration. D) Calcein-encapsulated LUVs were treated with D-dfTAT with or without UNC7938, or vehicle (0.1% DMSO). E) Binding of UNC7938 to multi-lamellar vesicles. The label nd (not detected) denotes conditions where no binding was detected. F) HeLa cells transfected with EGFP-Chmp1 and CFP-Lamp1 were treated with vehicle (DMSO), LLOME, or UNC7938. The number of EGFP-Chmp1 puncta present in imaged cells was counted and the total number of pixels displaying a punctate distribution quantified. Representative 100X images are overlay of EGFP-Chmp1 and Hoechst (scale bar: 10 μm). Magnified images show partial co-localization of Chmp1 puncta with Lamp1 positive endosomal organelles (scale bar: 2 μm). In all panels, the data reported corresponds to the mean of biological triplicates (>500 cells per experiment). The label ns corresponds to p>0.05, * corresponds to p≤0.05, ** corresponds to p≤0.01 by a two-tailed t-test.

Figure 3.

UNC7938 rescues the cytosolic penetration of D-dfTAT under conditions were CPP alone fail. A) Total uptake of D-dfTAT (5 μM) in HeLa and COS7 cells, with or without UNC7938 after 30 min incubation (ns = not significant by a two-tailed t-test). B) Uptake of TMR-r9 (5 μM) in HeLa, COS7, and HEK293T, as assessed by flow cytometry. Incubation were performed for 30 min, with or without UNC7938 (10 μM). C) Effect of UNC7938 on D-dfTAT penetration in COS7 cells. COS7 cells were treated for 30 min with D-dfTAT (5 μM) and with UNC7938. D) Effect of UNC7938 on D-dfTAT penetration in HEK293T cells. HEK293T cells were incubated for 30 min with D-dfTAT in the absence or presence of UNC7938 (5 μM). E) Effect of FBS on D-dfTAT (5 μM) penetration in HeLa cells, in the presence or absence of UNC7938 (10 μM). F) Delivery of DEAC-k5 in Hela cells with and without FBS. Cells were incubated with DEAC-k5 (10 μM), D-dfTAT (2 μM) and UNC7938 (10 μM) in L15 media with or without 10 % FBS. Representative 20X fluorescence images, pseudo-colored cyan for DEAC and overlaid with bright field images, are shown. A 100X fluorescence image of highlighting the subcellular localization of DEAC-k5 obtained after delivery with D-dfTAT and UNC7938 is provided. The cell penetration of DEAC-k5 is assessed by counting the number of cells displaying nucleolar staining of the peptide. In all panels, the data represented correspond to the mean of biological triplicates (>500 cells counted per experiment). Scale bars 20X: 50 μm, 100X: 10 μm.

Figure 4.

A dfTAT/UNC7938 cocktail mediates the transfection of DNA into cells. A) Gel mobility shift assay. The mTurquoise-H2A plasmid (280 ng) was mixed with dfTAT (3 μM) and UNC7938 (10 μM) and loaded in an agarose gel. Samples were then separated by electrophoresis and DNA was visualized by staining with ethidium bromide. B) Representative images of Jurkat cells incubated with dfTAT, the Gwiz-GFP DNA plasmid, and UNC7938. Images are overlay of bright field images and of dfTAT fluorescence, pseudocolored red. Scale bar 100X: 10 μm. C) Transfection experiments in Hela cells with the mTurquoise-H2A plasmid. Representative images of HeLa (20X) transfected with either lipofectamine®2000 or dfTAT/UNC7938. Images are overlays of fluorescence images from the mTurquoise signal and bright field images. Scale bars 20X: 50 μm. Histograms represent the percentage of cells transfected 24h after incubation with the reagents, as monitored by fluorescence microscopy. The data represented are the average of biological triplicates and the corresponding standard deviation. D) Transfection experiments in Jurkat T lymphocytes with Gwiz-GFP. Data were acquired as in C, with the exception that Jurkat are suspension cells. Live cell imaging was performed after letting cells settle at the bottom of a culture dish. Scale bars 100X: 10 μm.

Figure 5.

A dfTAT/UNC7938 cocktail delivers a cyclic peptide and a scFv antibody into the cytosol of live human cells. A) Delivery of fluorescein-phalloidin in COS7 cells. Cells were incubated with fluorescein-phalloidin (10 μM, fluorescein is represented by a green disc) and D-dfTAT (2 μM), UNC7938 (10 μM), or both. Representative 100X overlay fluorescence images, pseudo-colored green for the fluorescein signal and blue for Hoechst 33342, are shown. A magnified image displaying the cytoskeletal staining of phalloidin upon cell entry is provided. Scale bars 100X: 10 μm. B) Delivery of anti-NM II scFV SF9 into MCH58 human skin fibroblasts. SF9 was either incubated with fixed and permeabilized cells or with live cells, in the absence or presence of dfTAT and UNC7938. Images are overlay fluorescence images pseudocolored green for SF9, red for dfTAT and blue for Hoechst. Two different representative images of SF9 delivered into live cells are provided. Magnified images are shown and their contrast is adjusted to emphasize the staining of fiber-like structures. Scale bars 100X: 10 μm.