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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.


Cell uptake and diffusion kinetics of direct translocating TM9-ATTO 425 in GAGs rich and deficient cells.(a,c,e) diffusion of TM9-ATTO 425 solutions 12 μM in CHO-K1, 10 μM in PgsA-745 and 10 μM in GAGs cleaved CHO-K1, respectively. A NZ-dependent uptake is clearly detectable in the former case, while a homogeneous diffusion of peptide is shown for GAG-deficient and GAG-cleaved cells. The colored arrows indicate the ROIs used to evaluate peptide diffusion. (b,d,f) plot of the average fluorescence (AU) with respect to time (s) in the selected ROIs. In the case of CHO-K1 a different trend is shown with respect to the location within the cell, while, a linear increase occurs in all the locations within PgsA-745 and GAG-cleaved cells. Scale bars: 10 μm.
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f3: Cell uptake and diffusion kinetics of direct translocating TM9-ATTO 425 in GAGs rich and deficient cells.(a,c,e) diffusion of TM9-ATTO 425 solutions 12 μM in CHO-K1, 10 μM in PgsA-745 and 10 μM in GAGs cleaved CHO-K1, respectively. A NZ-dependent uptake is clearly detectable in the former case, while a homogeneous diffusion of peptide is shown for GAG-deficient and GAG-cleaved cells. The colored arrows indicate the ROIs used to evaluate peptide diffusion. (b,d,f) plot of the average fluorescence (AU) with respect to time (s) in the selected ROIs. In the case of CHO-K1 a different trend is shown with respect to the location within the cell, while, a linear increase occurs in all the locations within PgsA-745 and GAG-cleaved cells. Scale bars: 10 μm.

Mentions: In order to get further insight on peptide translocation into cells we incubated CHO-K1 cells in serum-free medium, added the selected concentration of TM9-ATTO 425 (see also the Experimental Procedures section) and performed a time-lapse, real-time imaging of peptide internalization process, this time without replacing the cell medium (Supporting Information, Movie 1). This latter procedure, made possible by the intrinsic spectral properties of ATTO 425 (as discussed above), allowed us to image the otherwise elusive process of peptide entry into cells. Interestingly, in fact, at concentrations above the CMC, few micrometric zones (typically one or two per cell) in which the fluorescence signal suddenly increases (i.e. the peptide locally concentrates) are detected on the plasma membrane shortly after peptide administration. Then fluorescence rapidly diffuses into the rest of the cell (Fig. 3a). Accordingly, if the average fluorescence intensity is measured in time at different locations within the cell (e.g. arrows in Fig. 3a) a quantitative picture of the timing of peptide intracellular spreading can be achieved. As reported in the plot of Fig. 3b, a nearly-constant intensity value is obtained if the ROI is placed at the peptide entry point, while a time lag of fluorescence intensity is measured if the ROI is far from the entry point (with time lag proportional to the distance from the entry point). Overall, about 50% of the cells are interested by the translocation process. Of note, in a number of reports similar translocation processes originating from spatially-restricted sites of the plasma membrane (also known as “Nucleation Zones”, NZs) were related to peptide binding to selected extracellular-matrix components and subsequent translocation into the intracellular medium17222324. Here we test the hypothesis that TM9 uptake and translocation might be regulated also at the extracellular level. In this regard, a classical molecular target is represented by glycosaminoglycans (GAGs) that, together with proteins, constitute the “glycocalyx” that covers cell surface. To test the possible role of GAGs we performed a confocal microscopy experiment using PgsA-745 cells. This cell line, derived from CHO-K1 cells, does not produce GAGs, as it does not express xylosyltransferase (a required enzyme in GAG synthesis)2526. We administered TM9-ATTO 425 at 10 μM to PgsA-745 and performed a time-lapse acquisition (see also Supporting Information, Movie 2). We observed that, differently from wild type CHO-K1 cells, the peptide enters nearly 100% of the cells by a translocation process that is homogeneously distributed throughout the plasmatic membrane (i.e. no NZs are detected, Fig. 3c). This is confirmed by the spatial analysis of peptide uptake in different locations within the cell (i.e. a linear increase of fluorescence in all the selected ROIs is detected, with no time lag, Fig. 3d). As a last control experiment, we analyzed peptide entry into CHO-K1 cells in which the GAGs were enzymatically digested by chondroitinase ABC (ChABC, Fig. S4). Upon administration of TM9-ATTO 425 at 10 μM concentration in serum-free medium, we monitored internalization by real-time imaging (Supporting Information, Movie 3). We detected homogeneous spreading of the fluorescence signal in 100% of the cells (Fig. 3e); fluorescence increased steadily during the observation time, with no time lag (Fig. 3f). Collectively, these results point at a significant role of integer GAGs in re-directing the peptide entry process towards spatially-restricted sites of the plasma membrane or NZs. Based on the present data and on the well-recognized role of GAGs as endocytosis effectors272829, we cannot exclude that NZs are sites of GAG absence (or marked spatial inhomogeneity) rather than GAG accumulation.


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)

Cell uptake and diffusion kinetics of direct translocating TM9-ATTO 425 in GAGs rich and deficient cells.(a,c,e) diffusion of TM9-ATTO 425 solutions 12 μM in CHO-K1, 10 μM in PgsA-745 and 10 μM in GAGs cleaved CHO-K1, respectively. A NZ-dependent uptake is clearly detectable in the former case, while a homogeneous diffusion of peptide is shown for GAG-deficient and GAG-cleaved cells. The colored arrows indicate the ROIs used to evaluate peptide diffusion. (b,d,f) plot of the average fluorescence (AU) with respect to time (s) in the selected ROIs. In the case of CHO-K1 a different trend is shown with respect to the location within the cell, while, a linear increase occurs in all the locations within PgsA-745 and GAG-cleaved cells. Scale bars: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: Cell uptake and diffusion kinetics of direct translocating TM9-ATTO 425 in GAGs rich and deficient cells.(a,c,e) diffusion of TM9-ATTO 425 solutions 12 μM in CHO-K1, 10 μM in PgsA-745 and 10 μM in GAGs cleaved CHO-K1, respectively. A NZ-dependent uptake is clearly detectable in the former case, while a homogeneous diffusion of peptide is shown for GAG-deficient and GAG-cleaved cells. The colored arrows indicate the ROIs used to evaluate peptide diffusion. (b,d,f) plot of the average fluorescence (AU) with respect to time (s) in the selected ROIs. In the case of CHO-K1 a different trend is shown with respect to the location within the cell, while, a linear increase occurs in all the locations within PgsA-745 and GAG-cleaved cells. Scale bars: 10 μm.
Mentions: In order to get further insight on peptide translocation into cells we incubated CHO-K1 cells in serum-free medium, added the selected concentration of TM9-ATTO 425 (see also the Experimental Procedures section) and performed a time-lapse, real-time imaging of peptide internalization process, this time without replacing the cell medium (Supporting Information, Movie 1). This latter procedure, made possible by the intrinsic spectral properties of ATTO 425 (as discussed above), allowed us to image the otherwise elusive process of peptide entry into cells. Interestingly, in fact, at concentrations above the CMC, few micrometric zones (typically one or two per cell) in which the fluorescence signal suddenly increases (i.e. the peptide locally concentrates) are detected on the plasma membrane shortly after peptide administration. Then fluorescence rapidly diffuses into the rest of the cell (Fig. 3a). Accordingly, if the average fluorescence intensity is measured in time at different locations within the cell (e.g. arrows in Fig. 3a) a quantitative picture of the timing of peptide intracellular spreading can be achieved. As reported in the plot of Fig. 3b, a nearly-constant intensity value is obtained if the ROI is placed at the peptide entry point, while a time lag of fluorescence intensity is measured if the ROI is far from the entry point (with time lag proportional to the distance from the entry point). Overall, about 50% of the cells are interested by the translocation process. Of note, in a number of reports similar translocation processes originating from spatially-restricted sites of the plasma membrane (also known as “Nucleation Zones”, NZs) were related to peptide binding to selected extracellular-matrix components and subsequent translocation into the intracellular medium17222324. Here we test the hypothesis that TM9 uptake and translocation might be regulated also at the extracellular level. In this regard, a classical molecular target is represented by glycosaminoglycans (GAGs) that, together with proteins, constitute the “glycocalyx” that covers cell surface. To test the possible role of GAGs we performed a confocal microscopy experiment using PgsA-745 cells. This cell line, derived from CHO-K1 cells, does not produce GAGs, as it does not express xylosyltransferase (a required enzyme in GAG synthesis)2526. We administered TM9-ATTO 425 at 10 μM to PgsA-745 and performed a time-lapse acquisition (see also Supporting Information, Movie 2). We observed that, differently from wild type CHO-K1 cells, the peptide enters nearly 100% of the cells by a translocation process that is homogeneously distributed throughout the plasmatic membrane (i.e. no NZs are detected, Fig. 3c). This is confirmed by the spatial analysis of peptide uptake in different locations within the cell (i.e. a linear increase of fluorescence in all the selected ROIs is detected, with no time lag, Fig. 3d). As a last control experiment, we analyzed peptide entry into CHO-K1 cells in which the GAGs were enzymatically digested by chondroitinase ABC (ChABC, Fig. S4). Upon administration of TM9-ATTO 425 at 10 μM concentration in serum-free medium, we monitored internalization by real-time imaging (Supporting Information, Movie 3). We detected homogeneous spreading of the fluorescence signal in 100% of the cells (Fig. 3e); fluorescence increased steadily during the observation time, with no time lag (Fig. 3f). Collectively, these results point at a significant role of integer GAGs in re-directing the peptide entry process towards spatially-restricted sites of the plasma membrane or NZs. Based on the present data and on the well-recognized role of GAGs as endocytosis effectors272829, we cannot exclude that NZs are sites of GAG absence (or marked spatial inhomogeneity) rather than GAG accumulation.

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.