Pro-apoptotic peptide amphiphile self-assembled with the assistance of polycations

Article abstract of DOI:10.1246/cl.150399

Herein, we describe how a peptide amphiphile, consisting of a pro-apoptotic peptide and a hydrophobic tail connected by a reduction-responsive cleavable spacer, forms self-assembled nanostructures. In the presence of polycations, this amphiphile exhibits

Full text of DOI:10.1246/cl.150399

Received: April 27, 2015 | Accepted: May 11, 2015 | Web Released: August 5, 2015
CL-150399
Pro-apoptotic Peptide Amphiphile Self-assembled with the Assistance of Polycations
Masato Ikeda,*1,2,3 Maika Kawakami,2 and Yukio Kitade*1,2,3
1Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, Gifu 501-1193
2Department of Biomolecular Science, Graduate School of Engineering, Gifu University, Gifu 501-1193
3United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1193
(E-mail: m_ikeda@gifu-u.ac.jp, ykkitade@gifu-u.ac.jp)
Herein, we describe how a peptide amphiphile, consisting of
a pro-apoptotic peptide and a hydrophobic tail connected by
a reduction-responsive cleavable spacer, forms self-assembled
nanostructures. In the presence of polycations, this amphiphile
exhibits loosely entangled nanofiber morphology with a length
of several hundred nanometers.
With these in mind, we set out to develop a peptide
amphiphile termed AVPI-NP-C12, which would consist of the
AVPI tetrapeptide linked with a hydrophobic dodecyl (C12)
chain through a nitrophenyl (NP)-containing spacer moiety, as
shown Figure 1A. Herein, we describe how, in the presence
of polycations, the peptide amphiphile forms self-assembled
nanostructures with loosely entangled nanofiber morphology
with a length of several hundred nanometers. Moreover, we
expected that the AVPI tetrapeptide could be released from a
self-assembled nanostructure consisting of AVPI-NP-C12 upon
reduction of the nitro group of the spacer based on the
mechanism shown in Figure 1B. Recently, it has been revealed
that the microenvironments around cancers, i.e. the so-called
hypoxic environments, are reductive because of low oxygen
supply and trigger reduction of nitro compounds.8
The synthesis of AVPI-NP-C12 was carried out based upon
a standard solid-phase peptide synthesis of the AVPI tetrapep-
tide, and subsequent coupling with NHS-ester of a NP-C12
derivative (diastereomeric mixture) at the N-terminal amino
group of the peptide (see Supporting Information for details).
The ability of AVPI-NP-C12 to form nanostructures in aqueous
media (50 mM HEPES-KOH at pH 7.6) was evaluated by
measurement of optical transmittance. As shown in Figures 2A
and S6, the optical transmittance (%T) decreased with an
increase in AVPI-NP-C12 concentration in the presence of
cations such as Ca2+ (1 mM) and poly-L-lysine (PLL) (5 mM
monomer unit, PLL4000: Mw = 4000-15000, PLL70000: Mw =
Supramolecular nanostructures constructed through the self-
assembly of peptide amphiphiles have been investigated for
various applications in biomedicine, including drug release,
tissue engineering, and regenerative medicine.1 Nevertheless,
the morphologies of these nanostructures were still limited to
simple spherical, fibrous, or tubular structures. Thus, there is
urgent demand for developing a control strategy to construct
nanostructures with preferred morphologies.2
Recently, the Smac (second mitochondria-derived activator
of caspase) protein3 and an AVPI tetrapeptide,4 located at the
N-terminal of the Smac protein, have been shown to enhance
the intended activity of anticancer drugs through the inhibition
of IAPs (inhibitor of apoptosis proteins).5 The IAPs, which are
over-expressed in several cancer cells, suppress apoptosis at
least in part by inhibition of caspases that are key players in
apoptosis.6 To address the limitation that the pro-apoptotic AVPI
tetrapeptide is not cell permeable, the AVPI peptide was
conjugated to a carrier peptide4 or, alternatively, AVPI mimetic
molecules7 were developed.
Figure 1. (A) Schematic representation showing self-assembly of a pro-apoptotic peptide amphiphile (AVPI-NP-C12) assisted by a
polycation (not to scale) and their chemical structures and (B) plausible mechanism showing the reduction-responsive degradation of AVPI-
NP-C12 to release a pro-apoptotic AVPI peptide.
Chem. Lett. 2015, 44, 1137–1139 | doi:10.1246/cl.150399
© 2015 The Chemical Society of Japan | 1137

Figure 3. Unstained TEM images of AVPI-NP-C12¢cation
nanostructures (cation for (A) Ca2+, (B) PLL4000, and (C)
PLL70000).
Figure 2. (A) Change in optical transmittance (%T) by increas-
ing the concentration of AVPI-NP-C12 in the presence of PLL70000
(5 mM monomer unit) and (B) Plots of %T (600 nm) against
concentration of AVPI-NP-C12 to evaluate CAC. Conditions:
[AVPI-NP-C12] = 0, 10, 20, 50, 100, 200, 300, 400, and 500 ¯M,
[Ca2+] = 1 mM or [PLL (monomer unit)] = 5 mM or [Na+] =
100 mM, 50 mM HEPES-KOH (pH 7.6), 25 °C.
measurement was carried out. The size of the nanostructures
of AVPI-NP-C12 complexed with Ca2+, PLL4000, and PLL70000
were evaluated to be 149 « 93, 159 « 58, and 282 « 144 nm,
respectively (Figure S7). These results indicate that the poly-
cations and divalent cation such as Ca2+ can induce the
assembly of AVPI-NP-C12 to form nanostructures, most
probably owing to electrostatic interactions between a cation
and C-terminal carboxylate anion of AVPI-NP-C12.
To obtain insight into the morphology of the nanostructures,
transmission electron microscopy (TEM) experiments were
carried out. As shown in Figure 3A, ill-defined nanostructures
were observed in the presence of Ca2+. In sharp contrast,
interestingly, dramatically different morphologies were observed
in the presence of PLL. Nanostructures consisting of loosely
entangled nanofibers with a fiber diameter of several tens of
nanometers were observed in the presence of both PLL4000
(Figure 3B) and PLL70000 (Figure 3C). In the case of shorter
PLL4000, fibrous structures wound and packed more closely
compared with PLL70000. Although additional, more detailed
studies are required to elucidate the self-assembled mode of
AVPI-NP-C12 with PLL, we speculate that PLL would define
the growth of the self-assembled structures of AVPI-NP-C12
through electrostatic interactions along the main chain of PLL
and hydrophobic interactions, mainly among dodecyl chains of
AVPI-NP-C12.9
70000-150000). To prevent overgrowth of the assembled
structures to micrometer scale, the concentration of cationic
species was set to be higher than that of AVPI-NP-C12. In
contrast, in the absence of polycations or the presence of the
monovalent cation Na+ (100 mM), almost no decrease in %T
except for that around 270 nm (assignable to an absorption band
of NP group) was observed up to 500 ¯M of AVPI-NP-C12
(Figure S6). From the plot of %T at 600 nm against the
concentration (Figure 2B), apparent critical association concen-
trations (CACs) of AVPI-NP-C12 in the presence of Ca2+
,
PLL4000, and PLL70000 were evaluated to be ca. 100, 100, and
50 ¯M, respectively. We presume that one proline moiety in
the middle of the peptide sequence, which could destabilize
intermolecular hydrogen-bonding interactions to induce self-
assembly, could prevent AVPI-NP-C12 from forming assem-
bled nanostructures without the polycations, at least below the
concentration of 500 ¯M. We also observed no decrease in %T
for the AVPI tetrapeptide without the hydrophobic C12 chain
upon the addition of cations. To assess the size of the
nanostructures above the CACs, dynamic light scattering
1138 | Chem. Lett. 2015, 44, 1137–1139 | doi:10.1246/cl.150399
© 2015 The Chemical Society of Japan

To investigate their reduction-responsive property, AVPI-
NP-C12¢Ca2+ nanostructures were incubated with sodium
dithionite (100 ¯M) in HEPES buffer (pH 7.6) at 37 °C.
Following the process with reverse-phase HPLC (RP-HPLC),
it was revealed that AVPI-NP-C12 decomposed almost com-
pletely within 2 h. Then, to detect the peptide product (AVPI-
tetrapeptide) released from the nanostructures by RP-HPLC, the
reaction mixture was centrifuged and the resultant supernatant
was treated with 2,4,6-trinitrobenzenesulfonic acid10 (TNBS),
which labeled the N-terminal amino group of the peptide to
give TNB-peptide (Figure S8). The presence of released AVPI-
tetrapeptide was confirmed by comparing a HPLC chart of a
control sample prepared from AVPI-tetrapeptide and TNBS as
well as MS spectrum.11 These results support our view that the
reduction of the nitro group of AVPI-NP-C12 can trigger the
cleavage of the linkage between the AVPI-tetrapeptide and the
hydrophobic tail, and eventually release the peptide from the
nanostructures (Figure 1B).
In summary, we demonstrated that a pro-apoptotic peptide
amphiphile bearing a reduction-responsive cleavage site was
capable of forming self-assembled nanostructures, with the
assistance of polycations. Interestingly, nanostructures compris-
ing entanglements of nanofibers were obtained in the presence of
poly-L-lysine. Preliminary experiments also revealed the reduc-
tion stimulus-responsive degradation of the peptide amphiphile
to release a pro-apoptotic peptide. We believe that the nanostruc-
tures created in the present study could find future potential
applications as nanomedicines for encapsulating and delivering
drugs, as well as for sensing reductive environments such as
caused by hypoxia. Exploration of such functions and mod-
ifications of component molecules to improve or modulate
stimuli response is currently underway in our laboratory.
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This work was supported in part by a Grant-in-Aid for
Scientific Research (B) (No. 15H03836) of the Japan Society for
the Promotion of Science and a Grant-in-Aid for Scientific
Research on Innovative Areas “Molecular Robotics” (No.
15H00809) of the Ministry of Education, Culture, Sports,
Science and Technology, Japan. Financial support from the
Takeda Life Science Foundation to M.I. is also acknowledged.
We acknowledge the Division of Instrumental Analysis, Life
Science Research Center, Gifu University for maintenance of the
instruments and kind support.
10 R. B. Freedman, G. K. Radda, Biochem. J. 1968, 108, 383.
TNBS can label amino groups (side chain and N-terminal) of
PLL in addition to AVPI-tetrapeptide, which would hamper
the HPLC analysis of AVPI release. Reduction responsive
property was thus investigated for AVPI-NP-C12¢Ca2+
nanostructures in the present study.
11 MALDI-TOF MS spectrum (negative); Calcd. for [TNB-
¹
AVPI (C25H35N7O11) - H] : m/z = 608.2; Found 608.7
(Figure S8). The overall yield was evaluated to be ca. 5%
from the peak area of HPLC.
Supporting Information is available electronically on J-STAGE.
Chem. Lett. 2015, 44, 1137–1139 | doi:10.1246/cl.150399
© 2015 The Chemical Society of Japan | 1139