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

Sorting of lyophilisomes by fluorescence-activated cell sorting.

<p>a/b) A representative size distribution of the initial lyophilisome population (a) and sorted lyophilisomes (b) is depicted, showing smaller lyophilisomes after sorting. Note the difference in x and y axes. c) Initial lyophilisome population depicted in a FACS dot plot with forward (size)/FITC-positive lyophilisome (FL1 channel) scatter where gated FITC-positive lyophilisomes were sorted. d) After sorting, the scatter showed merely small lyophilisomes, as large lyophilisomes were removed.</p

Quenching of FITC fluorescent lyophilisomes by trypan blue in the absence of cells.

<p>Results show decreased fluorescence (a) and efficient quenching (b) up to three washings steps of FITC fluorescent lyophilisomes by trypan blue.</p

Structural characteristics of TP10.

<p>Summary of features related to the bipartite character of the hybrid peptide TP10 (positions labeled with CF₃-Bpg are marked in red).</p

Schematic illustration of the conjugation of the cell-penetrating peptide (CPP) TAT to lyophilisomes.

<p>(1) Primary amine groups of lyophilisomes react with Sulfo-GMBS introducing reactive maleimide groups. (2) CPPs (cysteine-functionalized TAT-peptides; C-Ahx-YGRKKRRQRRR) are conjugated to maleimide-conjugated lyophilisomes, resulting in stable CPP-conjugated lyophilisomes. Sulfo-GMBS  =  sulfo-<i>N</i>-[γ-maleimidobutyryloxy]sulfo succinimide ester; Ahx  =  aminohexanoic acid; TAT  =  trans-activating transcriptional activator.</p

Cellular binding and internalization of unmodified lyophilisomes and TAT-conjugated lyophilisomes.

<p>HeLa, OVCAR-3, Caco-2 and SKOV-3 cells incubated with TAT-conjugated and unmodified lyophilisomes resulted in 86±3% and 12±4%, 87±3% and 16±8%, 97±3% and 19±3%, and 95±10% and 67±20% lyophilisome-positive cells, respectively. TAT  =  trans-activating transcriptional activator. *p<0.01 ***p<0.0001.</p

Cellular uptake of TAT-conjugated lyophilisomes as analyzed by transmission electron microscopy.

<p>HeLa cells were incubated with unmodified (a) and TAT-conjugated lyophilisomes (b-d) for 4 h. a) No attachment or uptake was observed using unmodified lyophilisomes. b-d) TAT-conjugated lyophilisomes (white arrows) showed various processes required for effective drug delivery systems, such as attachment (b) and uptake (c). Additionally, signs of degradation of the capsule inside the cell were visualized (black arrows, d). Scale bar represents 1.0 µm. TAT  =  trans-activating transcriptional activator.</p

Internalization of lyophilisomes with and without TAT peptide into HeLa cells.

<p>FACS showed a large number of lyophilisome-positive cells for TAT-conjugated lyophilisomes after 1 h (67±3%) without trypan blue and a cellular uptake of 25±1% with trypan blue. Values for lyophilisomes without TAT peptide were low. When lyophilisomes were incubated for 4 h, TAT-conjugated lyophilisomes conserved the large number of lyophilisome-positive cells (79±8%) with an increased internalization of 59±14%, while unmodified lyophilisomes still showed few lyophilisome-positive cells and little cellular uptake. *p<0.01 **p<0.001. CPP  =  cell penetrating peptide; TAT  =  trans-activating transcriptional activator.</p

<i>D</i>-amino acid “scan” to identify aggregation-prone regions in TP10.

<p>Aggregation of TP10 depends on the position of substitution with the sterically restrictive <b><i>D</i></b><b>-</b>CF₃-Bpg, as monitored by solid-state ¹⁹F-NMR and OCD in oriented DMPC/DMPG (3∶1) at P/L = 1∶50. The boxed spectral regions show the static powder pattern contributions of immobilized molecules with −8 kHz splittings.</p