Molecular assembly composed of a dendrimer template and block polypeptides through stereocomplex formation

Article abstract of DOI:10.1039/c2cc30926b

A 2nd generation polyamideamine (PAMAM) dendrimer bearing right-handed helices to its eight terminals was shown to accommodate eight left-handed helices via stereocomplex formation to generate molecular assemblies of disk structure with 13-14 nm diameter and 6 nm thickness.

Full text of DOI:10.1039/c2cc30926b

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COMMUNICATION
Molecular assembly composed of a dendrimer template and block
polypeptides through stereocomplex formationw
Hisato Matsui, Motoki Ueda, Akira Makino and Shunsaku Kimura*
Received 9th February 2012, Accepted 30th April 2012
DOI: 10.1039/c2cc30926b
A 2nd generation polyamideamine (PAMAM) dendrimer bearing
right-handed helices to its eight terminals was shown to accommodate
eight left-handed helices via stereocomplex formation to generate
molecular assemblies of disk structure with 13–14 nm diameter
and 6 nm thickness.
to self-assemble into planar sheets, which were transformed
into vesicles upon heating.12,13 The driving force of the
molecular assembly process is explained by stereocomplex
formation between right-handed and left-handed helices in
the hydrophobic core region of the sheets due to the convexo-
concave fitting between their surfaces. On the basis of these
results, we introduced the amphiphilic block peptide with the
right-handed helix as a hydrophobic block to eight terminals
of a 2nd generation dendrimer (8RD). We studied here
supramolecular assembly of 8RD with the amphiphilic block
peptide with the left-handed helix as a hydrophobic block due
to stereocomplex formation (Fig. 1a).
Self-assemblies of spherical micelles or vesicles have attracted
much attention in the field of drug delivery systems as
nanocarriers for imaging probes and drugs.1–5 Especially,
nanocarriers in the size range from 10 nm to 100 nm have
been shown to accumulate selectively in solid tumors by
the so-called enhanced permeability and retention (EPR)
effect.6–10 The nanocarriers can leak out of the loosened blood
tube walls in tumor regions and stay there due to the immature
lymphatic draining system. There is, however, a demand for
nanocarrier technology for precise size control and facile
surface modification with biologically active ligands. One
resolution was recently proposed by Tirrell et al. to use a 5th
generation dendrimer bearing a hydrophobic exterior of
alkane chains, which provides an environment to accommodate
biologically active peptides modified with alkane chains.11 But
this method has a drawback of poor stoichiometric control
because formation of the supramolecular structure is based on
hydrophobic interaction and alkyl chain packing. The peptides
of 500 molecules thus finally remained in one dendrimer at a
feed ratio of 20 : 1 peptide : dendrimer. In the present study,
we prepared a molecular assembly using a 2nd generation
dendrimer as a template, and amphiphilic peptides were
assembled into the dendrimer based on stereocomplex
formation between right-handed and left-handed helices.
This strategy improves the stoichiometric control of the
supramolecular assembly.
A hydrophobic dendrimer core was synthesized using 1,3-
diaminopropane as a nucleus and (Boc–NH–(CH2)3)2–N–
CH2COOH as branches (compound 5) (Scheme S1, ESIw). After
synthesizing the 2nd generation dendrimer, 4-pentynoic acid with
an alkyne group was condensed to eight terminals of the 2nd
generation dendrimer (Fig. 1b). On the other hand, 5-azido
pentanoic acid was connected to the C-terminal of the amphiphilic
right-handed block peptide (RP in Fig. 1). The click chemistry was
used for preparation of 8RD (Schemes S2 and S3, ESIw).
The supramolecular assembly was analysed by dynamic light
scattering (DLS) measurements and transmission electron
Helical peptides of a 12-mer alternating sequence of
L-leucine (Leu) or D-leucine (leu) and 2-aminoisobutylic acid
(Aib) were synthesized, and a poly(sarcosine) block was
extended from the N-terminal of the helical peptides. A
mixture of these amphiphilic block polypeptides was shown
Department of Material Chemistry, Graduate School of Engineering,
Kyoto University, Kyoto-Daigaku-Katsura, Nishikyo-ku,
Kyoto, 615-8510, Japan. E-mail: shun@scl.kyoto-u.ac.jp;
Fax: +81 75-383-2401; Tel: +81 75-383-2400
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc30926b
Fig. 1 The diagrams of molecular design: (a) the cartoon-like
diagram of a co-assembling 2nd generation dendrimer template bearing
amphiphilic right-handed block peptides (yellow helix) with amphiphilic
left-handed block peptides (red helix), (b) the schematic diagram of the
2nd generation dendrimer core emphasized on the generation number.
c
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Chem. Commun., 2012, 48, 6181–6183 6181

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microscope (TEM) observations by varying the feed molar
ratios of the amphiphilic left-handed block peptide (LP in
Fig. 1) and 8RD. The molecular assemblies were prepared by
the injection method. An ethanol solution of 8RD and LP at
the specified feed ratio was injected into 1 mL of a 10 mM
Tris buffer solution (pH 7.4, added 0.15 M NaCl). For
example, the molecular assemblies at 8 : 1 LP : 8RD were
prepared by injecting an ethanol solution of 2.50 mL of LP
(0.05 mg mLÀ1) and 1.41 mL of 8RD (0.1 mg mLÀ1) into 1 mL
of the buffer solution.
The hydrodynamic diameters of the self-assemblies have a
minimum value of 42 nm at the feed molar ratio of 8 : 1
LP : 8RD, suggesting that the assembly occurs in a stoichio-
metric manner of 1 : 1 association between the right-handed
helices of 8RD and the left-handed helices of LP. Below the
LP : 8RD feed molar ratio of 8 : 1, lowering the ratio further
resulted in an increase in the hydrodynamic diameter up to a
maximum of 150 nm, the value for pure 8RD. 8RD by itself
therefore could form self-assemblies (sheets of 100 nm), whose
proportion in the total self-assemblies however decreased with
the addition of LP. LP therefore associates with 8RD to
generate smaller self-assemblies (disks of 50 nm). On the other
hand, at the feed molar ratios higher than 8 : 1, the hydro-
dynamic diameters also increased because self-assemblies of
pure LP (hydrodynamic diameter of 200 nm; curved sheets of
200 nm) increased their proportion in the total self-assemblies
(Fig. S1 in ESIw).12 The fractions of the sheets of 100 nm in the
self-assemblies of 200 images shown in the TEM images decreased
in the order 4 : 1 LP : 8RD (163 : 200) > 8 : 1 LP : 8RD
(13 : 200) > 16 : 1 LP : 8RD (8 : 200). The fractions of the
disks of 50 nm shown in the TEM images increased in the
order 4 : 1 LP : 8RD (37 : 200) o 8 : 1 LP : 8RD (187 : 200) o
16 : 1 LP : 8RD (190 : 200). On the other hand, the curved
sheets of 200 nm were not detected with 4 : 1 LP : 8RD
(0 : 200) and 8 : 1 LP : 8RD (0 : 200), but appeared at
16 : 1 LP : 8RD (2 : 200). One molecule of 8RD is therefore
considered to accommodate eight LP molecules probably in a
stoichiometric manner.
Fig. 2 Hydrodynamic diameters of self-assemblies prepared by
varying the feed molar ratios of LP : 8RD. Negative staining TEM
micrographs at the feed molar ratios of (a) 0 : 1, (b) 8 : 1, and
(c) 32 : 1. Each scale bar represents 50 nm.
Fig. 3 Circular dichroism spectra of self-assemblies prepared by
varying the feed molar ratios of LP : 8RD after purification by using
size-exclusion chromatography and a cut-off filter. Each curve shows
0 : 1 (red), 2 : 1 (yellow), 4 : 1 (green), 8 : 1 (sky blue), 16 : 1 (blue),
24 : 1 (purple), 32 : 1 (brown).
The supramolecular assembly of LP with 8RD was analysed
by TEM observations. The TEM micrographs of molecular
assemblies prepared at the feed molar ratios of 0 : 1, 8 : 1, and
32 : 1 (LP : 8RD) are shown in Fig. 2a–c. 8RD self-assembled
into highly curved sheets, and the sizes of self-assemblies
(the average size of 50 nm assemblies in the micrographs)
decreased to 67 nm and 44 nm upon increasing the feed ratios
to 2 : 1 and 4 : 1, respectively (Fig. S2a–d, ESIw). The added
LP therefore acted as an inhibitor for 8RD association for
itself due to LP insertion into 8RD. The self-assemblies were
transformed into small planar sheets at the feed molar ratio of
8 : 1 (Fig. 2b and Fig. S2e, ESIw). These planar sheets
remained as they were at the feed molar ratios of 16 : 1,
24 : 1, and 32 : 1, whilst the amounts of curved sheets
increased (Fig. 2c and Fig. S2f–h, ESIw). The excessively
added LP to more than 8 equivalents of 8RD therefore self-
assembled into curved sheets by themselves. The TEM
observations also support the interpretation of DLS results that
eight molecules of LP and 8RD associate in a stoichiometric way.
Simple calculation of surface areas of 8RD (about 2.6 nm diameter)
and cross-section area of LP (a-helix of about 1.2 nm diameter)
indicates that 8RD has a space to accommodate 10–11 LP
molecules, which is also agreeable with the interpretation.
The self-assemblies were analysed by circular dichroism (CD)
measurements (Fig. 3). The self-assemblies of pure 8RD showed
double minima at 208 and 222 nm indicating a-helical structure.14
The self-assemblies prepared at the feed molar ratios of 2 : 1 and
4 : 1, which were purified by a PD-10 column (packed Sephadex
G-25, GE Healthcare), decreased the intensities of the Cotton
effects upon increasing the amounts of LP, and no signal was
observed with the self-assemblies prepared at the feed molar ratio
of 8 : 1. The self-assemblies prepared at the feed molar ratios of
16 : 1, 24 : 1, and 32 : 1, which were purified by a PD-10 column
and a cut-off filter of 0.1 mm to obtain self-assemblies of several
tens of nm, show no signal at all. These results also support the
interpretation that no more than eight molecules of LP are
inserted into 8RD even in the presence of excess amounts of LP.
The molecular assemblies prepared at the feed molar ratio
of 8 : 1 were sonicated for 10 seconds to obtain relatively
homogeneous disk-shaped assemblies (Fig. 4). The dimensions
c
6182 Chem. Commun., 2012, 48, 6181–6183
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where the yellow and blue regions represent the hydrophobic core
and hydrophilic poly(Sar) chains, respectively.
In the model shown in Fig. 5a, the assembly of eight
molecules of LP and 8RD takes a planar structure on the
whole, which stacks continuously to yield worm-like micelles.
The worm-like shape shown in Fig. 5a however cannot explain
the disk shape observed by TEM. In the other model shown in
Fig. 5b, eight poly(Sar) chains of eight LP molecules and eight
poly(Sar) chains of 8RD are extended to one face of
the dendritic core plane, and the two dendritic core planes
associate face-to-face. This unit may grow in one direction via
contact between plane edges where poly(Sar) chain density is
low. The model shown in Fig. 5b fits to the results of TEM
observations as shown in Fig. 5c.
Fig. 4 Negative staining TEM micrographs of self-assemblies
prepared at the feed molar ratio of 8 : 1 with sonication for 10 seconds.
The right micrograph is the magnified one of the left micrograph. Each
scale bar represents 50 nm.
In summary, eight left-handed helices were incorporated
into a 2nd generation dendrimer bearing eight right-handed
helices at the terminals via stereocomplex formation. This
supramolecular assembly occurred stoichiometrically through
1 : 1 association of the right-handed helix and the left-handed
helix. With this strategy of stereocomplex formation, dendrimers
can be designed as a host platform for accommodating
a defined number of guest molecules with a specified size.
A 3rd generation dendrimer is under research as a template for
sixteen helices.
We gratefully acknowledge the technical help of Prof. J.
Sugiyama, Kyoto University, for TEM measurements.
Notes and references
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Fig. 5 The model structures of self-assemblies prepared at the feed
molar ratio of 8 : 1 (a, b). The yellow and the blue regions represent
the hydrophobic core and the hydrophilic poly(Sar) chains, respec-
tively. (c) The unit model structure for the smallest self-assemblies
prepared at the feed molar ratio of 8 : 1 with 10 seconds sonication.
The noted sizes show the results from DLS and TEM.
were reduced down to 13–14 nm diameter and 6 nm thickness. The
molecular structure of this smallest disk-shape assembly is
discussed under the assumption that the dendritic core with eight
hydrophobic helices at the terminals is hydrophobic with a planar
structure in the assemblies. This planar structure is generally
allowed for 2nd generation dendrimers.15 Consequently, two
structural models are considered, as shown in Fig. 5a and b,
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 6181–6183 6183