10 research outputs found
Efficient Delivery of Cell Impermeable Phosphopeptides by a Cyclic Peptide Amphiphile Containing Tryptophan and Arginine
Phosphopeptides
are valuable reagent probes for studying protein–protein
and protein–ligand interactions. The cellular delivery of phosphopeptides
is challenging because of the presence of the negatively charged phosphate
group. The cellular uptake of a number of fluorescent-labeled phosphopeptides,
including F′-GpÂYLPÂQTV, F′-NEpÂYTAÂRQ,
F′-AEÂEEIÂYGEÂFEAÂKKÂKK, F′-PEpÂYLGÂLD,
F′-pYÂVNVÂQN-NH<sub>2</sub>, and F′-GpYEEI
(F′ = fluorescein), was evaluated in the presence or absence
of a [WR]<sub>4</sub>, a cyclic peptide containing alternative arginine
(R) and tryptophan (W) residues, in human leukemia cells (CCRF-CEM)
after 2 h incubation using flow cytometry. [WR]<sub>4</sub> improved
significantly the cellular uptake of all phosphopeptides. PEpYLGLD
is a sequence that mimics the pTyr1246 of ErbB2 that is responsible
for binding to the Chk SH2 domain. The cellular uptake of F′-PEpYLGLD
was enhanced dramatically by 27-fold in the presence of [WR]<sub>4</sub> and was found to be time-dependent. Confocal microscopy of a mixture
of F′-PEpÂYLGÂLD and [WR]<sub>4</sub> in live cells
exhibited intracellular localization and significantly higher cellular
uptake compared to that of F′-PEpYLGLD alone. Transmission
electron microscopy (TEM) and isothermal calorimetry (ITC) were used
to study the interaction of PEpÂYLGÂLD and [WR]<sub>4</sub>. TEM results showed that the mixture of PEpÂYLGÂLD and
[WR]<sub>4</sub> formed noncircular nanosized structures with width
and height of 125 and 60 nm, respectively. ITC binding studies confirmed
the interaction between [WR]<sub>4</sub> and PEpÂYLGÂLD.
The binding isotherm curves, derived from sequential binding models,
showed an exothermic interaction driven by entropy. These studies
suggest that amphiphilic peptide [WR]<sub>4</sub> can be used as a
cellular delivery tool of cell-impermeable negatively charged phosphopeptides
Enhanced Cellular Uptake of Short Polyarginine Peptides through Fatty Acylation and Cyclization
Many
of the reported arginine-rich cell-penetrating peptides (CPPs)
for the enhanced delivery of drugs are linear peptides composed of
more than seven arginine residues to retain the cell penetration properties.
Herein, we synthesized a class of nine polyarginine peptides containing
5 and 6 arginines, namely, R<sub>5</sub> and R<sub>6</sub>. We further
explored the effect of acylation with long chain fatty acids (i.e.,
octanoic acid, dodecanoic acid, and hexadecanoic acid) and cyclization
on the cell penetrating properties of the peptides. The fluorescence-labeled
acylated cyclic peptide dodecanoyl-[R<sub>5</sub>] and linear peptide
dodecanoyl-(R<sub>5</sub>) showed approximately 13.7- and 10.2-fold
higher cellular uptake than that of control 5,6-carboxyfluorescein,
respectively. The mechanism of the peptide internalization into cells
was found to be energy-dependent endocytosis. Dodecanoyl-[R<sub>5</sub>] and dodecanoyl-[R<sub>6</sub>] enhanced the intracellular uptake
of a fluorescence-labeled cell-impermeable negatively charged phosphopeptide
(F′-GpYEEI) in human ovarian cancer cells (SK-OV-3) by 3.4-fold
and 5.5-fold, respectively, as shown by flow cytometry. The cellular
uptake of F′-GpYEEI in the presence of hexadecanoyl-[R<sub>5</sub>] was 9.3- and 6.0-fold higher than that in the presence of
octanoyl-[R<sub>5</sub>] and dodecanoyl-[R<sub>5</sub>], respectively.
Dodecanoyl-[R<sub>5</sub>] enhanced the cellular uptake of the phosphopeptide
by 1.4–2.5-fold higher than the corresponding linear peptide
dodecanoyl-(R<sub>5</sub>) and those of representative CPPs, such
as hepta-arginine (CR<sub>7</sub>) and TAT peptide. These results
showed that a combination of acylation by long chain fatty acids and
cyclization on short arginine-containing peptides can improve their
cell-penetrating property, possibly through efficient interaction
of rigid positively charged R and hydrophobic dodecanoyl moiety with
the corresponding residues in the cell membrane phospholipids
Cyclic Peptide–Selenium Nanoparticles as Drug Transporters
A cyclic peptide composed of five
tryptophan, four arginine, and
one cysteine [W<sub>5</sub>R<sub>4</sub>C] was synthesized. The peptide
was evaluated for generating cyclic peptide-capped selenium nanoparticles
(CP–SeNPs) in situ. A physical mixing of the cyclic peptide
with SeO<sub>3</sub><sup>–2</sup> solution in water generated
[W<sub>5</sub>R<sub>4</sub>C]–SeNPs via the combination of
reducing and capping properties of amino acids in the peptide structure.
Transmission electron microscopy (TEM) images showed that [W<sub>5</sub>R<sub>4</sub>C]–SeNPs were in the size range of 110–150
nm. Flow cytometry data revealed that a fluorescence-labeled phosphopeptide
(F′-PEpYLGLD, where F′ = fluorescein) and an anticancer
drug (F′-dasatinib) exhibited approximately 25- and 9-times
higher cellular uptake in the presence of [W<sub>5</sub>R<sub>4</sub>C]–SeNPs than those of F′-PEpYLGLD and dasatinib alone
in human leukemia (CCRF-CEM) cells after 2 h of incubation, respectively.
Confocal microscopy also exhibited higher cellular delivery of F′-PEpYLGLD
and F′-dasatinib in the presence of [W<sub>5</sub>R<sub>4</sub>C]–SeNPs compared to the parent fluorescence-labeled drug
alone in human ovarian adenocarcinoma (SK-OV-3) cells after 2 h of
incubation at 37 °C. The antiproliferative activities of several
anticancer drugs doxorubicin, gemcitabine, clofarabine, etoposide,
camptothecin, irinotecan, epirubicin, fludarabine, dasatinib, and
paclitaxel were improved in the presence of [W<sub>5</sub>R<sub>4</sub>C]–SeNPs (50 μM) by 38%, 49%, 36%, 36%, 31%, 30%, 30%,
28%, 24%, and 17%, respectively, after 48 h incubation in SK-OV-3
cells. The results indicate that CP–SeNPs can be potentially
used as nanosized delivery tools for negatively charged biomolecules
and anticancer drugs
Peptide Amphiphile Containing Arginine and Fatty Acyl Chains as Molecular Transporters
Peptide amphiphiles (PAs) are promising
tools for the intracellular
delivery of numerous drugs. PAs are known to be biodegradable systems.
Here, four PA derivatives containing arginine and lysine conjugated
with fatty acyl groups with different chain lengths, namely, PA1:
R-KÂ(C<sub>14</sub>)-R, PA2: R-KÂ(C<sub>16</sub>)-R, PA3: KÂ(C<sub>14</sub>)-R-KÂ(C<sub>14</sub>), and PA4: KÂ(C<sub>16</sub>)-R-KÂ(C<sub>16</sub>), where C<sub>16</sub> = palmitic acid and C<sub>14</sub> = myristic
acid, were synthesized through Fmoc chemistry. Flow cytometry studies
showed that, among all synthesized PAs, only KÂ(C<sub>16</sub>)-R-KÂ(C<sub>16</sub>), PA4 was able to enhance the cellular uptake of a fluorescence-labeled
anti-HIV drug 2′,3′-dideoxy-3′-thiacythidine
(F′-3TC, F′ = fluorescein) and a biologically important
phosphopeptide (F′-PEpYLGLD) in human leukemia cells (CCRF-CEM)
after 2 h incubation. For example, the cellular uptake of F′-3TC
and F′-PEpYLGLD was enhanced approximately 7.1- and 12.6-fold
in the presence of the PA4 compared to those of the drugs alone. Confocal
microscopy of F′-3TC and F′-PEpYLGLD loaded PA4 in live
cells showed significantly higher intracellular localization than
the drug alone in human ovarian cells (SK-OV-3) after 2 h incubation.
The high-performance liquid chromatography (HPLC) results showed that
loading of Dox by the peptide amphiphile was 56% after 24 h. The loaded
Dox was released (34%) within 48 h intracellularly. The circular dichrosim
(CD) results exhibited that the secondary structure of the peptide
was changed upon interactions with Dox. Mechanistic studies revealed
that endocytosis is the major pathway of the internalization. These
studies suggest that PAs containing the appropriate sequence of amino
acids, chain length, charge, and hydrophobicity can be used as cellular
delivery tools for transporting drugs and biomolecules
Cationic cell-penetrating peptides tested as furin inhibitors.
<p>Cationic cell-penetrating peptides tested as furin inhibitors.</p
Inhibition of furin by the cationic peptides HIV-1 TAT<sub>47-57</sub> and Chariot.
<p>Soluble human furin, pre-incubated for 20 min at room temperature in the presence of (a) HIV-1 TAT (47–57) or (b) Chariot peptide, was tested at the specified concentrations. Furin activity was assessed by measuring the release of the fluorescent mca product from the fluorogenic substrate, pERTKR-mca. Results represent the mean ± S.D., N = 3. *P<0.01; **P<0.05.</p
K<sub>i</sub> values of the synthetic cyclic peptides tested as furin inhibitors.
<p>The data for hexa-D-arginine (D6R) and nona-D-arginine (D9R) are taken from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130417#pone.0130417.ref028" target="_blank">28</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130417#pone.0130417.ref015" target="_blank">15</a>] respectively.</p><p>K<sub>i</sub> values of the synthetic cyclic peptides tested as furin inhibitors.</p
Effect of HIV-1 TAT<sub>47-57</sub> peptide on cell migration of fibrosarcoma HT1080 cells.
<p>Cells were incubated with either 10 μM of peptide or vehicle (water) for 24 h at 37°C and cell migration was measured as described in “Materials and Methods”. Results represent the mean ± S.D., N = 3. **P<0.05.</p
Cyclic polyarginine peptides inhibit cellular convertase activity.
<p>(a) CHO cells were incubated with each compound at 1 μM for 24 h at 37°C, and cell viability was monitored by incubation for 4 h with WST-1. (b) CHO-GRAPfurin cells, expressing secreted alkaline phosphatase tethered to Golgi membranes by a transmembrane domain interrupted by a furin cleavage site, was incubated with 1 μM of each cyclic peptide for 20–24 h at 37 °C. Secreted alkaline phosphatase activity was measured in the medium. Results represent the mean ± S.D., N = 3.</p
Chemical structures of cyclic polyarginine peptides tested as furin inhibitors.
<p>Chemical structures of cyclic polyarginine peptides tested as furin inhibitors.</p