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Spontaneous membrane-translocating peptides: influence of peptide self-aggregation and cargo polarity.

Macchi S, Signore G, Boccardi C, Di Rienzo C, Beltram F, Cardarelli F - Sci Rep (2015)

Bottom Line: We unveil TM9 ability to self-aggregate in a concentration-dependent manner and demonstrate that peptide self-aggregation is a necessary--yet not sufficient--step for effective membrane translocation.These findings are discussed and compared to previous reports.The present results impose a careful rethinking of this class of sequences as direct-translocation vectors suitable for delivery purposes.

View Article: PubMed Central - PubMed

Affiliation: NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12-56127 Pisa, Italy.

ABSTRACT
Peptides that translocate spontaneously across cell membranes could transform the field of drug delivery by enabling the transport of otherwise membrane-impermeant molecules into cells. In this regard, a 9-aminoacid-long motif (representative sequence: PLIYLRLLR, hereafter Translocating Motif 9, TM9) that spontaneously translocates across membranes while carrying a polar dye was recently identified by high-throughput screening. Here we investigate its transport properties by a combination of in cuvette physico-chemical assays, rational mutagenesis, live-cell confocal imaging and fluorescence correlation spectroscopy measurements. We unveil TM9 ability to self-aggregate in a concentration-dependent manner and demonstrate that peptide self-aggregation is a necessary--yet not sufficient--step for effective membrane translocation. Furthermore we show that membrane crossing can occur with apolar payloads while it is completely inhibited by polar ones. These findings are discussed and compared to previous reports. The present results impose a careful rethinking of this class of sequences as direct-translocation vectors suitable for delivery purposes.

No MeSH data available.


Decreasing of pyrene 1:3 ratio at TP2 increasing concentrations, TP2 cell uptake and cell uptake before and after the homogenization of TP2 solution (1% DMSO).(a) Plot of the pyrene 1:3 ratio of free and dye-labeled TP2 peptide. Vertical bars: standard errors. (b) Confocal images of cells treated with increasing concentrations of TP2-ATTO 425 and TP2-TAMRA in free serum medium. Scale bars: 10 μm. (c) Confocal images of cells treated with TP2-ATTO 425 and TP2-TAMRA at a final concentration of 2 μM (peptide added to the cell medium directly from the DMSO stock solution). Scale bars: 10 μm.
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f5: Decreasing of pyrene 1:3 ratio at TP2 increasing concentrations, TP2 cell uptake and cell uptake before and after the homogenization of TP2 solution (1% DMSO).(a) Plot of the pyrene 1:3 ratio of free and dye-labeled TP2 peptide. Vertical bars: standard errors. (b) Confocal images of cells treated with increasing concentrations of TP2-ATTO 425 and TP2-TAMRA in free serum medium. Scale bars: 10 μm. (c) Confocal images of cells treated with TP2-ATTO 425 and TP2-TAMRA at a final concentration of 2 μM (peptide added to the cell medium directly from the DMSO stock solution). Scale bars: 10 μm.

Mentions: At this point, one must check whether the properties unveiled so far for TM9 are shared by its precursor sequence, TP2. To this end, we performed the pyrene 1:3 ratio assay on TP2 dissolved in water at varying concentrations, ranging from 0.5 μM to 100 μM (Fig. 5a). Notably, the calculated I1/I3 parameter varies non-linearly with peptide concentration, as expected for a concentration-dependent aggregation process. In this case, and in contrast to TM9, the characteristic CMC value of the unlabeled peptide (1.0 ± 0.2 μM) increases upon addition of the fluorophore, regardless of fluorophore polarity (CMC = 7.1 ± 0.9 μM in the case of ATTO 425, and CMC = 3.7 ± 0.5 μM in the case of TAMRA, see also Table 1). As expected, DLS measurements confirm the presence of peptide-based aggregates at concentrations above the CMC for both unlabeled and labeled TP2 and yield an estimate of the aggregate size (about 160 nm for TP2 and about 110 nm for both types of dye-labeled TP2). We then investigated cell uptake of the two fluorescent variants of TP2 at concentrations both below and above their respective CMC values (Fig. 5b). Quite surprisingly, no direct translocation was observed for TP2, regardless of the selected concentration and of the chosen fluorescent cargo (polar or apolar). In fact, as shown by confocal images in Fig. 5b, only punctuate fluorescence staining was detected within live cells after peptide administration, suggesting endocytosis as the dominant uptake mechanism for these TP2 adducts.These results do not appear to be consistent with previous reports on TP2714 and this, in turn, suggests the need for a careful comparison of the experimental protocols adopted. For what concerns in vitro cell studies, in previous reports dye-labeled TP2 was added from stock DMSO solutions directly to the cell medium up to the desired final concentration714. Indeed, if we adopt this same procedure, we do obtain direct translocation for both TP2-ATTO 425 and TP2-TAMRA even below the CMC value (see, for instance, the behavior of both TP2-ATTO 425 and TP2-TAMRA at 2 μM reported in Fig. 5c). Unfortunately, although not affecting cell viability (its final concentration in the cell medium is always around 1% or even less, as in the literature), DMSO can influence TP2 solubilization by altering peptide/water miscibility kinetics and lead to the formation of precipitates (see in cuvette absorption measurements reported in Fig. S5). As a consequence, this type of protocol does not appear to be transferable from these in vitro tests to more complex situations like tissues or entire organisms.


Spontaneous membrane-translocating peptides: influence of peptide self-aggregation and cargo polarity.

Macchi S, Signore G, Boccardi C, Di Rienzo C, Beltram F, Cardarelli F - Sci Rep (2015)

Decreasing of pyrene 1:3 ratio at TP2 increasing concentrations, TP2 cell uptake and cell uptake before and after the homogenization of TP2 solution (1% DMSO).(a) Plot of the pyrene 1:3 ratio of free and dye-labeled TP2 peptide. Vertical bars: standard errors. (b) Confocal images of cells treated with increasing concentrations of TP2-ATTO 425 and TP2-TAMRA in free serum medium. Scale bars: 10 μm. (c) Confocal images of cells treated with TP2-ATTO 425 and TP2-TAMRA at a final concentration of 2 μM (peptide added to the cell medium directly from the DMSO stock solution). Scale bars: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4645181&req=5

f5: Decreasing of pyrene 1:3 ratio at TP2 increasing concentrations, TP2 cell uptake and cell uptake before and after the homogenization of TP2 solution (1% DMSO).(a) Plot of the pyrene 1:3 ratio of free and dye-labeled TP2 peptide. Vertical bars: standard errors. (b) Confocal images of cells treated with increasing concentrations of TP2-ATTO 425 and TP2-TAMRA in free serum medium. Scale bars: 10 μm. (c) Confocal images of cells treated with TP2-ATTO 425 and TP2-TAMRA at a final concentration of 2 μM (peptide added to the cell medium directly from the DMSO stock solution). Scale bars: 10 μm.
Mentions: At this point, one must check whether the properties unveiled so far for TM9 are shared by its precursor sequence, TP2. To this end, we performed the pyrene 1:3 ratio assay on TP2 dissolved in water at varying concentrations, ranging from 0.5 μM to 100 μM (Fig. 5a). Notably, the calculated I1/I3 parameter varies non-linearly with peptide concentration, as expected for a concentration-dependent aggregation process. In this case, and in contrast to TM9, the characteristic CMC value of the unlabeled peptide (1.0 ± 0.2 μM) increases upon addition of the fluorophore, regardless of fluorophore polarity (CMC = 7.1 ± 0.9 μM in the case of ATTO 425, and CMC = 3.7 ± 0.5 μM in the case of TAMRA, see also Table 1). As expected, DLS measurements confirm the presence of peptide-based aggregates at concentrations above the CMC for both unlabeled and labeled TP2 and yield an estimate of the aggregate size (about 160 nm for TP2 and about 110 nm for both types of dye-labeled TP2). We then investigated cell uptake of the two fluorescent variants of TP2 at concentrations both below and above their respective CMC values (Fig. 5b). Quite surprisingly, no direct translocation was observed for TP2, regardless of the selected concentration and of the chosen fluorescent cargo (polar or apolar). In fact, as shown by confocal images in Fig. 5b, only punctuate fluorescence staining was detected within live cells after peptide administration, suggesting endocytosis as the dominant uptake mechanism for these TP2 adducts.These results do not appear to be consistent with previous reports on TP2714 and this, in turn, suggests the need for a careful comparison of the experimental protocols adopted. For what concerns in vitro cell studies, in previous reports dye-labeled TP2 was added from stock DMSO solutions directly to the cell medium up to the desired final concentration714. Indeed, if we adopt this same procedure, we do obtain direct translocation for both TP2-ATTO 425 and TP2-TAMRA even below the CMC value (see, for instance, the behavior of both TP2-ATTO 425 and TP2-TAMRA at 2 μM reported in Fig. 5c). Unfortunately, although not affecting cell viability (its final concentration in the cell medium is always around 1% or even less, as in the literature), DMSO can influence TP2 solubilization by altering peptide/water miscibility kinetics and lead to the formation of precipitates (see in cuvette absorption measurements reported in Fig. S5). As a consequence, this type of protocol does not appear to be transferable from these in vitro tests to more complex situations like tissues or entire organisms.

Bottom Line: We unveil TM9 ability to self-aggregate in a concentration-dependent manner and demonstrate that peptide self-aggregation is a necessary--yet not sufficient--step for effective membrane translocation.These findings are discussed and compared to previous reports.The present results impose a careful rethinking of this class of sequences as direct-translocation vectors suitable for delivery purposes.

View Article: PubMed Central - PubMed

Affiliation: NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12-56127 Pisa, Italy.

ABSTRACT
Peptides that translocate spontaneously across cell membranes could transform the field of drug delivery by enabling the transport of otherwise membrane-impermeant molecules into cells. In this regard, a 9-aminoacid-long motif (representative sequence: PLIYLRLLR, hereafter Translocating Motif 9, TM9) that spontaneously translocates across membranes while carrying a polar dye was recently identified by high-throughput screening. Here we investigate its transport properties by a combination of in cuvette physico-chemical assays, rational mutagenesis, live-cell confocal imaging and fluorescence correlation spectroscopy measurements. We unveil TM9 ability to self-aggregate in a concentration-dependent manner and demonstrate that peptide self-aggregation is a necessary--yet not sufficient--step for effective membrane translocation. Furthermore we show that membrane crossing can occur with apolar payloads while it is completely inhibited by polar ones. These findings are discussed and compared to previous reports. The present results impose a careful rethinking of this class of sequences as direct-translocation vectors suitable for delivery purposes.

No MeSH data available.