Two-photon-responsive supramolecular hydrogel for controlling materials motion in micrometer space

Article abstract of DOI:10.1002/anie.201404158

Spatiotemporal control of fluidity inside a soft matrix by external stimuli allows real-time manipulation of nano/micromaterials. In this study, we report a two-photon-responsive peptide-based supramolecular hydrogel, the fluidity of which was dramatically controlled with high spatial resolution (10μm×10μm×10μm). The off-on switching of the Brownian motion of nanobeads and chemotaxis of bacteria by two-photon excitation was successfully demonstrated.

Full text of DOI:10.1002/anie.201404158

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Angewandte
Communications
DOI: 10.1002/anie.201404158
Supramolecular Hydrogels
Two-Photon-Responsive Supramolecular Hydrogel for Controlling
Materials Motion in Micrometer Space**
Tatsuyuki Yoshii, Masato Ikeda, and Itaru Hamachi*
Abstract: Spatiotemporal control of fluidity inside a soft
matrix by external stimuli allows real-time manipulation of
nano/micromaterials. In this study, we report a two-photon-
responsive peptide-based supramolecular hydrogel, the fluidity
of which was dramatically controlled with high spatial
resolution (10 mm ꢀ 10 mm ꢀ 10 mm). The off–on switching of
the Brownian motion of nanobeads and chemotaxis of bacteria
by two-photon excitation was successfully demonstrated.
such systems because of its contactless mode and high
spatiotemporal resolution. Indeed, several research groups
have reported photoresponsive supramolecular hydrogels
based on a photoisomerization reaction or photo-click
chemistry.[8] However, these simple systems in which UV
light is used for irradiation, show cytotoxicity without careful
dose adjustment. In regard to biomaterials application, two-
photon responsiveness is superior to one-photon responsive-
ness because of its higher biocompatibility. A few distinctive
chromophores are able to absorb two less energetic photons
simultaneously upon irradiation with intense laser pulses[9] to
generate the same excited state as that with one-photon
excitation. This allowed light with a twofold longer wave-
length to be utilized (typically, near-infrared (NIR) region),
which is more appropriate for fabricating hydrogels with
lower cytotoxicity. As another advantage, three-dimensional
(3D) fabrication inside the gel matrix can be performed with
high spatial resolution by the two-photon process, because the
two-photon excitation event occurs only at a focal point,
which depends on the numerical aperture of the lens, the
wavelength of the light, and the refractive index of the
materials. In this context, it was recently reported that two-
photon-responsive polymer gels,[10] for example, hydrogels
consisting of poly(ethylene glycol) cross-linked with a photo-
labile o-nitrobenzyl group,[10a,b] can be employed as cell
culture matrices for controlling extracellular microenviron-
ment.[10a] Although the inside fluidity was not quantitatively
examined in detail, the polymers remaining after photo-
degradation are presumed to remain in the irradiated space.
By contrast, two-photon-responsive supramolecular hydro-
gels have not yet been developed, despite these being
expected to show a drastic change in fluidity because all the
photogenerated residues are small molecules. We describe
herein the design of a peptide-based supramolecular hydrogel
capable of exhibiting gel–sol transition upon two-photon
excitation, which enables the creation of 3D fluidic micro-
meter-sized spaces inside the gel with high spatial resolution
and with a high fluidity equivalent to an aqueous solution. We
also successfully demonstrated local control over the Brow-
nian motion of nanobeads and the regulation of the chemo-
taxis of living bacteria in an off–on manner, without
inactivation of the biological processes inside the two-
photon-responsive supramolecular hydrogel matrix.
S
upramolecular hydrogels[1] formed by the self-assembly of
small molecules are promising biomaterials for numerous
applications, such as cellular scaffolds,[2] controlled drug
release,[3] and biosensing.[4] Compared to conventional poly-
mer gels, supramolecular hydrogels are anticipated to exhibit
unique functions, such as generating fluidic nanofiber net-
works[5] and dynamic or flexible stimuli-responsive proper-
ties.[6] It is now recognized that precise control of the gel
structure and functions is crucial for the construction of
sophisticated soft biomaterials comprising supramolecular
hydrogels not only to facilitate understanding of the impact of
the surrounding environment on a unique biological function,
but also for manipulating various biological phenomena.[7]
Many stimuli-responsive supramolecular hydrogels[6] have
been developed to date. Light is an attractive stimulus for
[*] T. Yoshii, Prof. Dr. I. Hamachi
Department of Synthetic Chemistry and Biological Chemistry
Graduate School of Engineering, Kyoto University
Katsura, Kyoto 615-8510, (Japan)
E-mail: ihamachi@sbchem.kyoto-u.ac.jp
Dr. M. Ikeda
Department of Biomolecular Science
Graduate School of Engineering, Gifu University
Gifu 501-1193 (Japan)
and
United Graduate School of Drug Discovery and Medical Information
Sciences, Gifu University
Gifu 501-1193 (Japan)
Prof. Dr. I. Hamachi
Japan Science and Technology Agency (JST), CREST
5 Sanbancho, Chiyoda-ku, Tokyo 102-0075 (Japan)
[**] Prof. K. Akiyoshi, Dr. S. Mukai, Dr. H. Aoki (Kyoto University), and
Prof. S. Yokoyama (Kyushu University), Mission of the Center for
Meso-Bio Single-Molecule Imaging (CeMI) are thanked for techni-
cal help in microscopy observations. Dr. Y. Takaoka (Kyoto
We recently established a semirational design strategy for
various stimuli-responsive peptide-based supramolecular
hydrogelators.[11] For example, by incorporating a photosti-
muli-responsive 7-bromo-hydroxycoumarin-4-yl-methoxycar-
bonyl (Bhcmoc) protecting group at the N-terminus of
a dipeptide (FF; F: phenylalanine), a supramolecular gel
was obtained, which showed a gel–sol transition on (one)
University, Hamachi Lab) and Prof. K. Urayama (Kyoto Institute of
Technology) are acknowledged for help with TEM observations and
rheological measurements, respectively. T.Y. acknowledges JSPS
Research Fellowships for Young Scientists. This work was partly
supported by CREST (Japan Science and Technology Agency).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201404158.
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phototriggered cleavage of the Bhcmoc moiety (Figure 1). We
sought to explore two-photon-responsive hydrogels based on
this scaffold. Since the Bhcmoc-FF moiety does not form
a hydrogel at a physiological pH value, probably because of
movement (Brownian motion) of beads with a diameter of
250 nm in the DMACmoc-FF(CF3) gel (0.075 wt%) was
observed (see Figure S4A in the Supporting Information). In
contrast, the Brownian motion of beads having a diameter of
500 nm stopped as a result of entrapment by the entangled gel
fiber mesh (Figure S4B). These results indicate that the
DMACmoc-FF(CF3) hydrogel (0.075 wt%) formed a nano-
mesh with void spaces between 250 nm and 500 nm. The
relationship between the Brownian motion of different sized
beads and gelator concentrations was evaluated in detail (see
Figure S4C in the Supporting Information).
To confirm the photoresponsive nature of the DMAC-
moc-FF(CF3) gel, we firstly irradiated the gel (0.080 wt%)
with one-photon excitation by a stand-alone Hg lamp
(360 nm, 49 mW, 5 min), which caused a macroscopic gel–
sol transition (see Figure S5A in the Supporting Information).
HPLC analysis of the photoirradiated sample revealed
a decrease in the amount of DMACmoc-FF(CF3) and the
concurrent increase in the amount of the cleaved product H-
FF(CF3) in an irradiation time dependent manner (see
Figure S6 in the Supporting Information). These results
indicate that the cleavage of the DMACmoc moiety occurred
by one-photon excitation. The gel to sol transition occurred
after 56% of the gelator had decomposed, which was in good
agreement with the CGC value of this gelator. It is clear that
removal of the DMACmoc group by one-photon (360 nm)
excitation disturbs the delicate balance of the molecular
interactions in the self-assembled nanofiber, which induces
the gel–sol transition. The photoinduced gel-sol transition
process can also be tracked by the disappearance of the
fibrous morphology by CLSM (see Figure S5B in the
Supporting Information).
Figure 1. A) Photoinduced cleavage of a caged-peptide gelator.
B) Schematic representation of the self-assembly of a caged-peptide
gelator to form a supramolecular hydrogel and its photoresponsive
gel–sol transition.
deprotonation of the hydroxy group of Bhcmoc, we decided
to replace the Bhcmoc group at the N-terminus of peptides
with
a
dimethylaminocoumarin-4-yl-methoxycarbonyl[12]
(DMACmoc) group, a typical two-photon absorption dye. A
small library of DMACmoc-modified peptides was con-
structed by varying the peptide sequence (see Figure S1A in
the Supporting Information) and gelator screening was
conducted at pH 7.4. By the tube-inversion method, we
found four gelators that form transparent hydrogels that
should be appropriate for efficient photoabsorption with
minimization of photodiffraction in neutral aqueous condi-
tions (pH 7.4; see Figure S1 in the Supporting Information).
The critical gelation concentrations (CGCs) of each gelator
were evaluated (see Figure S1B in the Supporting Informa-
tion). DMACmoc-FF(CF3) showed the lowest CGC
(0.050 wt%), and was thus mainly used for further studies.
The nanostructure of the dried DMACmoc-FF(CF3) gel
was analyzed by transmission electron microscopy (TEM). A
fibrous structure with diameters of 20–30 nm and lengths of
several micrometers was observed (see Figure S2A in the
Supporting Information). Observation of the DMACmoc-
FF(CF3) gel stained with a cationic fluorescence dye (DEAC-
gua:[4b] 10 mm; see Figure S2B in the Supporting Information)
by confocal laser scanning microscopy (CLSM) clearly
revealed the presence of fiber network in a semiwet state
(Figure S2C). The circular dichroism (CD) spectrum sug-
gested the formation of a b-sheet-like secondary structure and
a chiral arrangement of the aromatic side chains of the
peptide and DMAC chromophore in the fibrous aggre-
gates.[13] The rheological data of the DMACmoc-FF(CF3)
hydrogel showed the typical viscoelastic properties of a hydro-
gel consisting of fiber networks (see Figure S3 in the
Supporting Information).[14] To evaluate the mesh size of the
DMACmoc-FF(CF3) gel, we then encapsulated fluorescent
nanobeads and examined their Brownian motion.[15] Random
We next examined the local gel–sol transition inside the
DMACmoc-FF(CF3) gel upon two-photon excitation (Fig-
ure 2A). A two-photon laser scanning microscope was
utilized both for observing the supramolecular fiber and for
fabricating the supramolecular hydrogel at different powers,
Figure 2. A) Schematic representation of the photofabrication of the
DMACmoc-FF(CF3) gel by two-photon excitation. B) CLSM images of
the DMACmoc-FF(CF3) gel (0.1 wt%) stained with DEAC-gua (10 mm)
before and after two-photon irradiation (740 nm, 2 s). Scale bar:
10 mm. C,D) CLSM images of the x-y and x-z cross-section of the
DMACmoc-FF(CF3) gel fabricated by C) two-photon or D) one-photon
excitation. Scale bar: 10 mm. E) Alphabetical letters patterned by two-
photon excitation. Scale bar: 20 mm.
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typically, 0.4% for the fiber observations and 10% for the gel
fabrications (near-IR (NIR) pulse laser at 740 nm, 3 W,
140 fs). When the DMACmoc-FF(CF3) gel (0.10 wt%)
stained with DEAC-gua (10 mm) was irradiated with the
NIR laser (10%), the entangled fibers disappeared in the
irradiated area after a total irradiation time of only 2 s
(Figure 2B). It is noteworthy that the inside of the DMAC-
moc-FF(CF3) gel was three-dimensionally fabricated by two-
photon excitation with 8 mm spatial resolution in the z-axis
direction (Figure 2C (right)). In contrast, the spatial resolu-
tion in the z-axis by one-photon fabrication (UV laser at
405 nm, 30 mW, 30%, 2 s)) was rather broad, that is 25 mm
(Figure 2D; right). The two-photon fabrication of the
DMACmoc-FF(CF3) gel was apparently three times more
precise than the one-photon fabrication. This difference is
attributed to the difficulty of the one-photon excitation to
perfectly prevent the formation of the excited state in the
space through which the light passes. Furthermore, the
current response time is short (2 s) enough to fabricate
more complex patterns, such as alphabetical letters, by
scanning NIR laser light (Figure 2E).
We subsequently sought to perform a remote photo-
modulation of the Brownian motion of nanobeads inside the
gel matrix through the focal gel-to-sol transition upon two-
photon irradiation. The DMACmoc-FF(CF3) gel encapsulat-
ing 500 nm diameter nanobeads was treated with NIR light
(740 nm, 3 W, 10%, 2 s). Before photoirradiation, the loca-
tions of all the beads at 0 s overlapped well with those after
10 s by CLSM observation (Figure 3A, see also Movie S1 in
the Supporting Information), which indicates that the Brow-
nian motion of the beads stopped through entrapment by the
nanomesh of the gel fibers. After NIR irradiation, the
locations of the beads did not merge in the irradiated area,
whereas the overlap still remained in the unirradiated area,
which revealed that the Brownian motion restarted only on
photoirradiation. To evaluate the speed of the Brownian
motion of the beads inside the gel, the motion of the beads
was traced every 0.49 s and the migration distance was plotted
against time (see Figure S7 in the Supporting Information).
The speed in the irradiated area was determined to be (1.6 Æ
0.6) mmsÀ1. By contrast, the bead speed was approximately
25-fold slower (0.06 Æ 0.04 mmsÀ1) in the unirradiated area
(Figure 3B). Assuming a two-dimensional Brownian motion
according to the Einstein–Smoluchowski relation, the mean-
square displacements were analyzed as a function of time and
using the Stokes–Einstein Equation. This allowed us to
estimate the local viscosity (h) in the irradiated area to be
2.8 ꢀ 10À3 Pas. Remarkably, this is almost comparable to the
viscosity of pure water (8.9 ꢀ 10À4 Pas),[16] which suggests that
highly fluidic focal space was created inside the hydrogel
matrix by the two-photon stimuli. These results indicate that
the fluidity increased (or the viscosity decreased) only in the
irradiated focal space as the fiber disappeared (see above), as
a result of the two-photon responsive gel–sol transition.
Given that the diameter of bacteria (250–1000 nm) is
almost comparable to these beads, it might be reasonable to
expect that a bacteria strain (E. coli RP437[17]) would be
encapsulated in the DMACmoc-FF(CF3) gel (0.075 wt%).
Observation by CLSM confirmed that chemotaxis of the
bacteria stained with SYTO9 did not occur in the DMAC-
moc-FF(CF3) gel (see Figure S8 in the Supporting Informa-
tion), thus indicating that the nanomesh of the DMACmoc-
FF(CF3) gel is strong enough to hinder bacterial chemotaxis.
Interestingly, we observed that bacteria in the area irradiated
with NIR light (740 nm, 3 W, 10%, 2 s) developed a motile
state and moved in the limited microspace (Figure 4, see also
Figure 3. Controlling the local fluidity of the DMACmoc-FF(CF3) gel
(0.075 wt%) by two-photon excitation, evaluated by the Brownian
motion of nanobeads. A) Time-lapse CLSM images of the fluorescent
nanobeads (500 nm). The inset rectangle shown in the “Merge” panel
was irradiated by focused laser light (740 nm). The fluorescence
images were converted into pseudocolors (red: t=0 s, green: t=10 s)
and merged. Scale bar: 5 mm. (see also Movie S1 in the Supporting
Information). B) Comparison of the mean speed of the Brownian
motion of the nanobeads (500 nm) in the area with or without
irradiation by two-photon excitation (740 nm).
Figure 4. 3D spatial control of the chemotaxis of E. coli (RP437).
A) Time-lapse CLSM images of the SYTO9-labeled E. coli in the
DMACmoc-FF(CF3) (0.075 wt%) gel. The inset rectangle was irradiated
with focused laser light (740 nm). The fluorescence images were
converted into pseudocolors (green: t=0 s, red: t=10 s) and merged.
B) Three different tracks of each E. coli in the irradiation area shown in
different colors. Scale bar: 10 mm (see also Movie S2 in the Supporting
Information).
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d) J. P. Schneider, D. J. Pochan, B. Ozbas, K. Rajagopal, L.
Movie S2 in the Supporting Information). The rate of the
bacterial chemotaxis was evaluated to be (6.6 Æ 1.0) mmsÀ1,
which is comparable to the literature value.[18] In sharp
contrast, one-photon irradiation of DMACmoc-FF(CF3) with
UV light (405 nm, 30 mW) did not restore the chemotaxis of
the entrapped bacteria (see Figure S9 in the Supporting
Information), because of its high toxicity, which highlighted
the advantage of two-photon excitation by NIR light for
manipulating living materials.
In summary, we successfully developed the first two-
photon-responsive supramolecular hydrogel which can be
readily fabricated with high spatial resolution by excitation
with two-photon NIR light. As a consequence of the high
biocompatibility of the two-photon NIR fabrication and the
sufficient stiffness of the nanomesh composed of supramolec-
ular fibers, the bacterial chemotaxis was remotely regulated in
an off–on mode. The current supramolecular material is
potentially useful for remote manipulation of not only the
chemotaxis of bacterial cells but also the action of mammalian
cells, such as migration and differentiation or controlled drug
release in deep tissue. Further research towards these goals is
currently in progress.
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Keywords: gels · photochemistry · self-assembly ·
supramolecular chemistry
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