Deciphering Structural Determinants Distinguishing Active from Inactive Cell-Penetrating Peptides for Cytosolic mRNA Delivery

The formation of noncovalent complexes by mixing of positively charged polymers with negatively charged oligonucleotides (ONs) is a widely explored concept in nanomedicine to achieve cellular delivery of ONs. Uptake of ON complexes occurs through endocytosis, which then requires release of ON from endosomes. As one type of polymer, cell-penetrating peptides (CPPs) are being used which are peptides of about 8–30 amino acids in length. However, only a few CPPs yield effective cytosolic ON delivery and activity. Several strategies have been devised to increase cellular uptake and enhance endosomal release, among which an increase of osmotic pressure through the so-called proton sponge effect, disruption of membrane integrity through membrane activity, and disulfide-mediated polymerization. Here, we address the relevance of these concepts for mRNA delivery by incorporating structural features into the human lactoferrin-derived CPP, which shows uptake but not delivery. The incorporation of histidines was explored to address osmotic pressure and structural motifs of the delivery-active CPP PepFect14 (PF14) to address membrane disturbance, and finally, the impact of polymerization was explored. Whereas oligomerization increased the stability of polyplexes against heparin-induced decomplexation, neither this approach nor the incorporation of histidine residues to promote a proton-sponge effect yielded activity. Also, the replacement of arginine residues with lysine or ornithine residues, as in PF14, was without effect, even though all polyplexes showed cellular uptake. Ultimately, sufficient activity could only be achieved by transferring amphipathic sequence motifs from PF14 into the hLF context with some benefit of oligomerization demonstrating overarching principles of delivery for CPPs, lipid nanoparticles, and other types of delivery polymers

Identification of Short Hydrophobic Cell-Penetrating Peptides for Cytosolic Peptide Delivery by Rational Design

Cell-penetrating peptides (CPPs) enhance the cellular uptake of membrane-impermeable molecules. Most CPPs are highly cationic, potentially increasing the risk of toxic side effects and leading to accumulation in organs such as the liver. As a consequence, there is an unmet need for less cationic CPPs. However, design principles for effective CPPs are still missing. Here, we demonstrate a design principle based on a classification of peptides according to accumulated side-chain polarity and hydrophobicity. We show that in comparison to randomly selected peptides, CPPs cover a distinct parameter space. We designed peptides of only six to nine amino acids with a maximum of three positive charges covering this property space. All peptides were tested for cellular uptake and subcellular distribution. Following an initial round of screening we enriched the collection with short and hydrophobic peptides and introduced d-amino acid substitutions and lactam bridges which increased cell uptake, in particular for long-term incubation. Using a GFP complementation assay, for the most active peptides we demonstrate cytosolic delivery of a biologically active cargo peptide

The Intracellular Pharmacokinetics of Terminally Capped Peptides

With significant progress in delivery technologies, peptides and peptidomimetics are receiving increasing attention as potential therapeutics also for intracellular applications. However, analyses of the intracellular behavior of peptides are a challenge; therefore, knowledge on the intracellular pharmacokinetics of peptides is limited. So far, most research has focused on peptide degradation in the context of antigen processing, rather than on peptide stability. Here, we studied the structure–activity relationship of peptides with respect to intracellular residence time and proteolytic breakdown. The peptides comprised a collection of interaction motifs of SH2 and SH3 domains with different charge but that were of similar size and carried an N-terminal fluorescein moiety. First, we show that electroporation is a highly powerful technique to introduce peptides with different charge and hydrophobicity in uniform yields. Remarkably, the peptides differed strongly in retention of intracellular fluorescence with half-lives ranging from only 1 to more than 10 h. Residence times were greatly increased for retro-inverso peptides, demonstrating that rapid loss of fluorescence is a function of peptide degradation rather than the physicochemical characteristics of the peptide. Differences in proteolytic sensitivity were further confirmed using fluorescence correlation spectroscopy as a separation-free analytical technique to follow degradation in crude cell lysates and also in intact cells. The results provide a straightforward analytical access to a better understanding of the principles of peptide stability inside cells and will therefore greatly assist the development of bioactive peptides

Exploration of the Design Principles of a Cell-Penetrating Bicylic Peptide Scaffold

Cell-penetrating peptides (CPPs) possess the capacity to induce cell entry of themselves and attached molecular cargo, either by endocytosis or by direct translocation. Conformational constraints have been described as one means to increase the activity of CPPs, especially for direct crossing of the plasma membrane. Here, we explored the structure–activity relationship of bicyclic peptides for cell entry. These peptides may be considered minimal analogues of naturally occurring oligocyclic peptide toxins and are a promising scaffold for the design of bioactive molecules. Increasing numbers of arginine residues that are primarily contributing to cell-penetrating activity were introduced either into the cycles, or as stretches outside the cycles, at both ends or at one end only. In addition, we probed for the impact of negatively charged residues on activity for both patterns of arginine substitution. Uptake was investigated in HeLa cells by flow cytometry and confocal microscopy. Overall, uptake efficiency showed a positive correlation with the number of arginine residues. The subcellular distribution was indicative of endocytic uptake. One linear stretch of arginines coupled outside the bicycle was as effective in promoting uptake as substituting the same number of arginines inside the bicycles. However, the internally substituted analogues were more sensitive to the presence of negatively charged residues. For a given bicyclic peptide, uptake was more effective than for the linear counterpart. Introduction of histidine and tryptophans further increased uptake efficiency to comparable levels as that of nonaarginine despite the larger size of the bicyclic backbone. The results demonstrate that both arginine clustering and spatial constraints are uptake-promoting structural principles, an observation that gives freedom in the introduction of cell-penetrating capacity to structurally constrained scaffolds

Measured cluster numbers and cell sizes.

<p>Measured cluster numbers and cell sizes.</p

Quantification of the effect of CD28 expression on cell surface spreading and tyrosine phosphorylation.

<p>The original images of the experiment of Fig. 2 were quantified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079277#pone.0079277.s009" target="_blank">Macro S1</a>) and the values were normalized to the mean value of the measured property within that image. Normalized values of experiments with inverted stamp and overlay configurations were pooled. The graphs show the mean ± SEM. <i>A-C</i>) Cells stimulated with stripes containing αCD3 and stripes containing αCD28. (<i>n</i> = 10 images from two separate samples in which stamp and overlay stimuli were reversed (Fig. 2<i>B & C</i>) in total counting 1010 CD28 low and 127 CD28 high cells). <i>D-F</i>) Cells stimulated with stripes containing αCD3 and stripes containing unspecific IgG2a only. (<i>n</i> = 10 images from two separate samples in which stamp and overlay stimuli were reversed (Fig. 2<i>D & E</i>) in total counting 921 CD28 low and 97 CD28 high cells). <i>G-I</i>) Cells stimulated with stripes containing unspecific IgG2a only and stripes containing αCD28. (<i>n</i> = 10 images from two separate samples in which stamp and overlay stimuli were reversed (Fig. 2<i>F & G</i>) in total counting 1006 CD28 low and 165 CD28 high cells). <i>A, D & G</i>) The background-corrected, αphosphotyrosine intensity per surface area. Corrected model p-values were determined by two-way factorial ANOVAs in which no interaction terms were included. <i>B, E & H</i>) The contact surface area per cell. Two-sample T-tests were used to generate the p-values. C, <i>F & I</i>) The integrated, background-corrected, αphosphotyrosine intensity per cell (Two-sample T-tests).</p

Image processing of phosphoPLCγ1 signals and cluster formation.

<p>Overview of the image processing protocol as described in Materials and Methods and used for the analysis of the experiments described in Fig. 4. In order to resolve clusters in print, an enlarged segment of a microscopy image labeled with αphospho-PLCγ1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079277#pone.0079277.s003" target="_blank">Fig. S3</a>) is shown as an example. Image processing and quantification was done on a per image basis. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079277#pone.0079277.s010" target="_blank">Macro S2</a> describes the full procedure utilized to analyze the images. In short, the pPLCγ1 signal was thresholded to generate a binary mask of all cells. This image was inverted to generate a mask of the background signal. The CFSE image was thresholded and was used in combination with the mask of all cells to generate a mask of CFSE labeled cells and a mask of unlabeled cells. The image of the printed stripes was thresholded to generate a mask of the printed structures and inversed to also generate a mask of the overlaid areas. Combining the masks of the printed structures and overlaid areas with the masks of the cells formed the masks of the CFSE labeled cells on stamped stripes, the CFSE labeled cells on overlaid structures, the unlabeled cells on stamped stripes and the unlabeled cells on overlaid structures. These four masks were used to measure the surface areas the cells covered on both surfaces. Combining the stripe and overlay masks with the background mask enabled the measurement of surface areas not covered by cells. The last six generated masks were, in turn, applied to the original pPLCγ1 image and from the resulting images the total pPLCγ1 signal per condition could be determined. Together with the total surface areas of the specific condition, the signal intensity per µm² was calculated. Surface specific background corrections were applied. In addition, a binary cluster mask was generated from the pPLCγ1 image. This mask was segmented using the four masks of cells on surfaces creating four new masks. From these masks cluster numbers were counted and by applying them to the original pPLCγ1 image cluster intensities could be determined. Finally, the cell numbers per image were determined by eye using the original transmission images and the cell masks. The various colors correspond to the graphs in Fig. 6 and indicate which masks and images are required to produce the particular data.</p

Protocol for microcontact printing.

<p>A microstructured silicon master is used as a template for the generation of PDMS stamps. The stamp is coated with antibodies, including a fluorescently labeled indifferent antibody for visualization of stamped features. Stamping transfers a monolayer of antibodies to a clean microscope slide. The areas in between stamped patterns are coated by incubation (‘overlay’) with a second antibody solution. Finally, the surface is blocked with BSA.</p

Effect of SHP2 depletion on IL2 expression.

<p>SHP2 KD and wt Jurkat E6.1 T cells were stimulated with PMA + ionomycin (+), αCD3 & αCD28, αCD3 alone, αCD28 alone or were left unstimulated (–) for 22 h. IL2 in the supernatants was quantified by sandwich ELISAs. Given are the absorption values ± SEM. The p-values are from a full factorial two-way ANOVA and represent the significance of the overall corrected model (corr m), the effect of CD28 expression (CD28 expr), the effect of the stimulus and the interaction factor (int fact) between stimuli and CD28 expression. For all conditions <i>n</i> = 3 samples, all from a single experiment representative of four independent experiments.</p

Detection of the stimulus dependence of total tyrosine phosphorylation (<i>B</i>) and phosphoY783 PLCγ1 (<i>C</i>) in Jurkat cells and SHP2 KD cells.

<p><i>A</i>) For the side-by-side analysis of signaling in Wt and SHP2 KD Jurkat E6.1 T cells, one of the lines was labeled with the cell tracer CFSE. After overnight serum starvation the cells are pooled and incubated on micropatterned, stimulating surfaces for 10 min. Subsequently, the cells are fixed with 3% PFA, permeabilized and immunolabeled for the detection of signaling clusters. <i>B & C</i>) In the top panels, SHP2 KD cells are CFSE labeled and in the bottom panels, wt cells are labeled. Panels from left to right: transmission images; CFSE; immunofluorescence; overlay of the stamped pattern (blue) and the immunolabel (grayscale). In the overlay panels the contrast and brightness for both channels were adjusted proportionally for clarity. 12.5 µg/ml αCD3 + 12.5 µg/ml αCD28 coated stamps were used to generate a striped pattern which was overlaid with 5 µg/ml αCD3. CFSE channels were recorded with saturated signals to facilitate image processing. Scale bars 20 µm.</p