Enzyme Instructed Self-assembly of Naphthalimide-dipeptide: Spontaneous Transformation from Nanosphere to Nanotubular Structures that Induces Hydrogelation

Article abstract of DOI:10.1002/asia.202000575

Understanding the structure-morphology relationships of self-assembled nanostructures is crucial for developing materials with the desired chemical and biological functions. Here, phosphate-based naphthalimide (NI) derivatives have been developed for the

Full text of DOI:10.1002/asia.202000575

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Enzyme Instructed Self-assembly of Naphthalimide-dipeptide:
Spontaneous Transformation from Nanosphere to Nanotubular
Structures that Induces Hydrogelation
Authors: Rajan Deepan Chakravarthy, Mohiuddin Mohammed, and
Hsin-Chieh Lin
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10.1002/asia.202000575
Chemistry - An Asian Journal
Enzyme Instructed Self-assembly of Naphthalimide-
dipeptide: Spontaneous Transformation from Nanosphere to
Nanotubular Structures that Induces Hydrogelation
Rajan Deepan Chakravarthy, Mohiuddin Mohammed and Hsin-Chieh Lin*
Department of Materials Science and Engineering, National Chiao Tung University, Hsinchu,
Taiwan, Republic of China 300
E-mail: hclin45@nctu.edu.tw
Graphical abstract:
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Abstract: Understanding the structure-morphology relationships of the self-assembled
nanostructures is very crucial for developing the materials with the desired chemical and
biological functions. Here, phosphate-based naphthalimide (NI) derivatives have been
developed for the first time to study the enzyme instructed self-assembly process. Self-
assembly of simple amino acid derivative NI-Yp resulted in non-specific amorphous
aggregates in the presence of alkaline phosphatase enzyme. On the other hand, NI-
FYp dipeptide forms spherical nanoparticles in the aqueous condition which slowly
transformed into partially unzipped nanotubular structures during the enzymatic catalytic
process through multiple stages which subsequently resulted in hydrogelation. The self-
assembly is driven by the formation of β-sheet type structures stabilized by offset
aromatic stacking of NI core and hydrogen bonding interactions which is confirmed with
PXRD, Congo-red staining and molecular mechanical calculations. We propose a
mechanism for the self-assembly process based on TEM and spectroscopic data. The
nanotubular structures of NI-FYp precursor exhibited higher cytotoxicity to human
breast cancer cells and human cervical cancer cells when compared to the nanofiber
structures of the similar Fmoc-derivative. Overall this study provides a new
understanding of the supramolecular self-assembly of small molecular weight
hydrogelators.
Keywords: Supramolecular chemistry • Naphthalimide • Peptide • Enzyme catalysis •
Hydrogel
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Molecular self-assembly is a critical process in living systems to forming
macromolecular assemblies including the construction of lipid bilayers, protein
quaternary complex and DNA double helix.1,2 In this context, small peptides especially
aromatic peptides have particularly been proven to be a versatile class of organic
building blocks for self‐assembled materials.3-6 It has been very well established that at
the nano‐to‐mesoscale, the architectures obtained from peptide units exhibit higher
stability with a large variety of morphologies, which include nanotubes, nanoribbons,
and nanowires.7 In addition to that small peptide-based self-assemblies provide
significant advantages including biological compatibility, straightforward synthesis and
functionalization.8 These peptides offer greater flexibility to design and control the
supramolecular architectures because of their characteristic self-assembling building
blocks and highly stable architecture. The self-assembly of peptide molecules depends
on the structural and dynamical properties of their building blocks that are
predominantly governed by their amino acid sequences.9 The morphologies of these
nano-assemblies are crucial for specific applications, e.g., nanoparticles are useful for
drug delivery while nanotubes and nanofibers are used for biosensor fabrication and
tissue engineering, respectively.10 Design of peptide-based small molecules to
controlling the morphology of self-assembling nanostructures is highly challenging.11-21
For example, Stupp and coworkers reported the self-assembly of long chain terapeptide
amphiphiles that formed a giant nanobelts structures.11,12 Ulijn and coworkers have
systematically studied the morphological transformations of Fmoc-Yp and Fmoc-FYp
materials under enzymatic dephosphorylation method.13,14 To our knowledge, there are
currently only a few examples of the nanostructure transformation of low molecular
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peptide assemblies available. However, recent findings and innovations in the field have
created plenty of interest and generous space for further improvements.
Naphthalimide (NI)-derivatives has received increasing attention in recent years due
to its unique photophysical property and photostability.22,23 Napthalimides with a variety
of functionalization have been investigated as aggregation-induced emission (AIE)
luminogens. Recent reviews article summarizes the continuing efforts in this area.24 The
significance of NI for various biological applications has also been very well
documented.25 More recently NI has been implemented as an aromatic core for creating
self-assembled materials due to its strong π-stacking propensity. For example, Tao Yi
and coworkers have studied gelation behaviors of NI compounds and they found that
materials displayed high fluorescence quantum yields in both solution and solid-state.26
Recently we reported a NI-based supramolecular hydrogelator, which exhibits AIE
phenomenon.27-29 Particularly, we have shown the self-assembly of NI-F that results in
the formation of AIE-active microfibers which facilitate the high contrast visualization of
3D live cell imaging.27 In this work, our focus is to study the self-assembly of phosphate-
based NI derivatives.
Enzyme instructed self-assembly (EISA) of small molecules has become an attractive
approach in anticancer drug developments, molecular imaging, and nanotechnology.30
Recent studies revealed that EISA-based supramolecular assemblies are not only
capable of inhibiting undruggable protein targets of various cancers but also exhibited
activities to prevent acquired drug resistance. Hence, EISA is being considered as a
reliable complementary anticancer therapy to the ligand-receptor approach.30-32 Few
attempts have also been made to monitor the EISA process in biological milieu by
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conjugating peptides with suitable fluorescent dye molecules.33 However, the enzymatic
hydrogelation with NI based system was never established. NI is an AIE chromophore
which is weakly emissive in molecularly dissolved state and highly emissive under self-
assembled condition. Integration of such AIE chromophore would be beneficial for
selective imaging and killing of tumor cells. In the present work, we develop NI based
low molecular weight compounds and examine their self-assembly process under EISA
condition. The mechanism of the self-assembly was also systematically investigated
using various spectroscopic and microscopic techniques.
Figure 1. The chemical structures of Fmoc and NI based compounds used for this
study.
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Results and Discussion
The supramolecular hydrogelation process of amino acids or short peptide
hydrogelators typically requires an aromatic hydrophobic group. NI aromatic unit
possess strong hydrophobicity and aromatic π-π stacking system. NI group also
having two-dimensional arrangement of p-orbitals, which result in stronger London
dispersion forces between molecules compared to the commonly used aromatic
capping agents like naphthalene or Fmoc motif. It is anticipated that functionalizing the
simple amino-acids or dipeptides with NI-chromophore would facilitate the self-
assembly of the molecules in water. Thus, in this work, we have designed and
synthesized two new phospho precursors NI-Yp and NI-FYp with the covalently
attached NI core at N-terminus for studying the EISA based hydrogelation
process. The synthetic procedure for the preparation of new NI compounds and
the complete characterization data are provided in supporting information. We
also synthesized the Fmoc-Yp and Fmoc-FYp based on the literature protocol for
comparing the hydrogelation ability with NI materials.13,14 The chemical structures of
various Fmoc and NI building blocks are depicted in Figure 1. The EISA process was
examined for four different phosphate compounds and the results are shown in
Table 1 and Figure S1.
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Table 1. Physical properties of amino acid and peptide amphiphiles under various
conditions
cLog P[a]
value of
gelator
Morphology
based on
TEM
ALP
S.No
Precursor
Gelator
Result
(Unit)[b]
1
2
3
4
Fmoc-Yp
NI-Yp
Fmoc-Y
NI-Y
4.2
2.8
5.4
-
50 U
Hydrogel
Nanofibers
Amorphous
particles
50-200 U Precipitate
Fmoc-FYp Fmoc-FY
50 U
-
Hydrogel
Micelles
Nanofibers
Spherical
nanoparticles
NI-FYp
NI-FYp
-
Partially
unzipped
5
NI-FY
4.0
50 U
Hydrogel
nanotubes[c]
[a] The values of cLogP values were calculated with ChemDraw [b] Amount of ALP unit
added in 0.2 mL of the peptide solution dissolved in Tris-buffer at 1 wt%. [c] Sheet type
morphological features has also been noticed (see supporting information)
Self-assembly of amino acid-based compounds:
We begin our study to the self-assembly of NI-Yp material. The newly designed
NI-Yp system containing hydrophilic O-phosphate side chain and carboxylic acid
group and the hydrophobic NI chromophore. Hydrophobicity is among the factors
that influence the self-assembly of the low-molecular weight (LMW) compounds
in aqueous condition. The calculated partition coefficient clogP value is the most
commonly used measure of hydrophobicity/lipophilicity. The cLogP values of NI-
Yp and the corresponding dephosphorylated compound NI-Y were found to be
1.5 and 2.8 respectively indicating that our NI-Y material is hydrophobic in nature.
Several examples of low molecular weight hydrogelators (LMWHs) are known in
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the literature with the cLogP values

2.0.34,35 This led us to infer that NI-Yp
molecules would have hydrogelation ability under EISA mechanism. The results
however, go against our hypothesis. Hydrogelation was not obtained for the NI-
Yp molecule irrespective of the substrate (1-3 wt%) and ALP concentrations (50-
200 U). On the other hand, the Fmoc-Yp (1 wt%) fragment exhibited pronounced
hydrogel formation under EISA condition. The morphology of the enzyme-
catalyzed Fmoc-Yp and NI-Yp samples was determined by transmission electron
microscope (TEM). Self-assembly of Fmoc-Yp in presence of ALP resulted in the
formation of nanofibers which is consistent with the literature. The TEM image of
the Fmoc-Y hydrogelator revealed a highly entangled and long continuous
nanofiber network, with average diameters of 12.6 ± 1.6 nm.36 However, NI-Y
molecule exhibited non-specific aggregates under the similar catalytic condition
(Figures 2a and 2b). We acknowledge that NI-Y will produce hydrogelation at
high acidic pH, but neutral or basic pH seems to be the most suitable condition
for ALP catalytic hydrogelation process. These findings lead us to conclude that
“hydrogelation propensity” is not confined to a single hydrophobic effect, at least
in the case of simple amino acids.
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Figure 2. Negatively stained TEM images of ALP treated (a) Fmoc-Yp, (b) NI-Yp, (c)
Fmoc-FYp and (d) NI-FYp.
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Figure 3. EISA based self-assembly of NI-FYp (1 wt%, at 37 °C) in Tris buffer. (a) TEM
image of NI-FYp nanoparticles in Tris-buffer (10 mM). (b) Particle size distribution
histogram of NI-FYp (10 mM) nanoparticles by TEM and DLS measurements. TEM
images of (c) NI-FYp treated with 10 U of ALP (d-f) NI-FYp with 20 U of ALP and (g-i)
NI-FYp with 40 U of ALP.
Self-assembly of dipeptide-based compounds:
Next, we set out to investigate the EISA process for dipeptide hydrogelator. Again
Fmoc-FYp is the smallest dipeptide core established for EISA process, we
prepared a similar dipeptide molecule with NI based N-capping agent. The
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calculated cLogP values of Fmoc-FY and NI-FY were 5.4 and 4.0 respectively.
Both the molecules resulted in stable hydrogel under the ALP condition. Self-
assembly of Fmoc-FYp resulted in the formation of nanofibers which is
consistent with the literature (Figure 2c).37 The TEM image of the hydrogelator
Fmoc-FY hydrogelator revealed a highly entangled and long continuous
nanofiber network, with average diameters of 7.0 ± 0.9 nm. The TEM image of
enzymatically dephosphorylated NI-FY exhibited nanotubular morphology under
the ALP condition (Figures 2d and S3). More interestingly, the width of the
nanotubular structure is extended to about 75 nm.
Figure 4. EISA based self-assembly of NI-FYp (1 wt%, at 37 °C) in Tris buffer. (a-b)
SEM images of NI-FYp sample treated with 20 U of ALP and (c) SEM cross-section
image of FIB milled nanotubular structure.
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Supramolecular hydrogels of many reported peptide molecules self-assembled
into nanofibrillar morphology with the smallest possible width size, which is much
less than 50 nm. 27 Few examples of higher-order self-assembled aggregates are
also known in the literature.19,38,39 Understanding the self-assembly mechanism
for the formation of such large aggregates is of significant importance but
remains challenging. In this work, we conducted a systematic investigation of the
NI-FYp system to elucidate the possible mechanism of nanotubular formation.
We have done a series of TEM analyses for various samples and the results are
summarized in Figure 3 and Table S1. NI-FYp at 10mM concentration in water
displayed spherical particles in TEM analysis. Such particles also exhibited
Tyndall-effect when the solution was irradiated with red-laser light (Figure S2).
The particle size and size distribution were determined with dynamic light
scattering (DLS) and the value was found to be 50 nm (Figure 3b). The statistical
analysis of TEM data shows that the size of the nanoparticles was 27.1 ± 4.6 nm
(Figure 3a). The nanoparticles NI-FYp self-assembled into a weak hydrogel or
viscous solution in presence of 10 U ALP. Increasing the concentration of ALP
from 10 to 20 U resulted in a stable hydrogel which could withstand the vial
inversion test. Stable hydrogels have also been produced with the ALP range of
20-50 U. Further increasing the concentration of ALP led to the formation of
immediate precipitation. In all cases, the self-assembly was noticed within 20
min after the addition of ALP. TEM data were collected and analyzed for the
samples at various concentrations of ALP and the results are summarized in
Figure 3. The viscous solution with 10U of ALP forms highly ordered nanosheet
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aggregates with average diameters of nm 27.3 ± 6.9 nm. Interesting results were
obtained as in the case of the NI-FYp sample with 20 and 40U of ALP addition.
Nanosheet type structures were obtained as the major morphological feature for
the NI-FYp (1 wt%) in aqueous tris-buffer. In some portions of the TEM grid the
nanosheets were grooved/curved inwards and rolled-up to form nanotubes. The
coexistence of a few twisted nanofibers was also observed with all these
conditions (see ESI). Evidently, as depicted in TEM micrographs, these
nanostructures are largely entangled, leading to the formation of self-supporting
hydrogel networks. In the case of 100U ALP addition, spherical particles were
predominantly observed (Figure S5). As we previously described the hydrogels of
NI-FY at pH 7.0 exhibited nanofibrillar morphological features.29 However, the
EISA based self-assembly generated nanotubular peptide assembly.
To
understand this self-assembly behavior, we have done a series of experiments
with NI-FY under various pH conditions. The concentration-dependent self-
assembly behavior of NI-FY was examined and the results are shown in figure S6
and S7. Well-defined twisted nano-fibrils were observed at the low peptide
concentrations (0.5-1.0 wt%) under neutral pH condition. Increasing the
concentration of NI-FY resulted in the formation of thicker and rigid nanosheet
type structure without any rolled-up morphological features. Further, the higher
concentration of peptide (> 2 wt%) destabilizes the gel-phase and accelerates the
formation of solid precipitates. The differences in gelation structures are likely
due to the influence of salt on the hydrogelation process.40 We then characterized
the self-assembly of NI-FY (1 wt%) at acidic pH value (6.3) and the TEM data
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showed the coexistence of nanofibers and nanosheet morphologies (Figure S8).
From these results, it is evident that nanosheet structure formation has
proceeded through the association of multiple nanofibrils. We propose a similar
mechanism for catalytic hydrogelation of NI-FYp. However, it is not clear how the
large spherical particles formed at the higher ALP concentrations.
NI-FYp samples with lower ALP concentration (20 U) produced highly ordered
nanostructures. Samples with 40U and 50 U of ALP showed only minor changes
in the morphological features, so we decided to perform further experimental
analysis with 20U of ALP. We have recorded the SEM data for the NI-FYp (1 wt%)
sample treated with ALP (20 U). The SEM data revealed the nanotubular
structures formed through the association of multiple nanofibrils which is similar
to that of TEM results (Figure 4). Focused Ion Beam Scanning Electron
Microscopes (FIB-SEM) technique was used for site-specific milling and imaging
of the nanomaterials (Figures 4c and S9). Groove type nanostructures were
observed for the self-assembled NI-FY peptides which is consistent with the TEM
data. The width of the resulting nanostructure is about 100 nm. The findings
indicate that the hydrogelation of NI-FYp proceed through a hierarchical self-
assembly mechanism in which nanofiber formed in the first stage of the self-
assembly process. Such fibers served as building blocks for the formation of
nanosheet morphology which subsequently rolled-up into nanotubular structures.
The self-assembly mechanism of EISA triggered NI-FYp molecule is similar to
that of the model proposed for the growth of carbon nanotube.41 However, the NI-
dipeptide nanotubes predominantly existed as partially unzipped structures and
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the completely matured nanotube formation was not noticed in any experimental
conditions. It could be possible that self-assembly of higher peptide analogue
would form stable nanotube structures. Further research is needed on NI-based
systems to precisely understand such phenomena. We captured the various
stages of the self-assembly process with the aid of TEM and SEM and provide
conclusive evidences for the molecular mechanism. Nanotube formation is also
possible for uncapped diphenylalanine derivatives, but for the N-capped peptide
amphiphiles, such mechanism is rare. For example, Gazit et.al proposed a similar
mechanism for diphenylalanine molecule and the inter-molecular packing of such
molecules is stabilized through strong attractive electrostatic interactions between
a free amine and carboxylic acid groups along with the hydrophobic interactions
of aromatic side chains.42 However, since the NI-FY molecule possesses only
carboxylic acid, such interactions are less possible. Thus the molecular skeleton
of NI-FY would have different self-assembling mechanism compare to the
diphenylalanine derivatives. Overall, the findings indicate that the EISA based
self-assembly of NI-FYp molecule proceeds through a hierarchal mechanism with
the formation of tubular structures (Figure 5).
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Figure 5. The proposed model for the formation of nanotubular structures that
induces hydrogelation.
Figure 6. (a) 31P-NMR spectra (in D2O solvent) for following the EISA based self-
assembly of NI-FYp sample (1 wt%) (i) without ALP, (ii) with 20 U of ALP measured at 5
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min and (iii) 20 min respectively. (b) HPLC profile for monitoring the ALP catalysis of NI-
FYp at different time interval i) without ALP, (ii) with 20 U of ALP measured at 5 min (iii)
10 min and (iv) 15 min respectively. (c) Time-sweep rheological data of NI-FYp treated
with 20 U of ALP for the measurement of gelling point (GP).
Gelation kinetics and rheological studies:
To gain insight into the kinetics of the biocatalytic hydrogelation process we conducted
a series of experiments. The hydrogelation process was monitored by 31P-NMR spectra
and the data is presented in Figure 6a. The singlet peak at 0.21 ppm is attributed to
the signal of NI-FYp. For the NMR analysis, the NI-FYp sample (1 wt%, 50mM
Tris in D2O) was subjected to the catalytic reaction with ALP (20U). As a result of
dephosphorylation, a new peak for the naked phosphate by-product was noticed
at 2.66 ppm. The signal intensity of NI-FYp decreases with the time and major
conversion of NI-FYp to NI-FY was noticed within 20 minutes of the reaction time
suggesting rapid dephosphorylation process. To further examine the enzymatic
catalysis process, we decided to use High-performance liquid chromatography
(HPLC) and the results are shown in Figure 6b. The HPLC trace exhibited a signal
for NI-FYp phosphate precursor at retention time (Rt) of 23.2 min. Catalytic
dephosphorylation of NI-FYp (1 wt%) with 20U of ALP resulted in a new signal at
Rt value of 26.5 min confirming the formation of NI-FY. The ALP-catalyzed
dephosphorylation reaction was completed within 15 min. Such fast hydrogelation
kinetics might provide higher rigidity to hydrogel samples through the formation of
dense cross-linking networks of the nanostructures.43 We also carried out the
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rheological analysis to understand the mechanical stability and gelation kinetics
of the NI-FYp material in the presence of ALP. Dynamic frequency sweep and
time sweep rheological data were measured and the results are shown in Figures
6c and S10. The storage modulus G' (elastic response) and loss modulus G''
(viscous behavior) of the NI-FYp sample (treated with 20 U ALP) are independent
over the entire frequency range of 0.1-100 Hz which indicate that the hydrogel is
viscoelastic in nature. The average G' of the hydrogel at 1 wt% was found to be
97 Pa. The time-dependent rheological data exhibited very low complex modulus
initially and the value quickly increased to more than 30 Pa within 250 sec
indicating the self-assembly and gelling point (GP) of the sample.
Figure 7. (a) Emission spectra of NI-FYp treated with various unit of ALP and (b)
Time dependent emission spectra of NI-FYp treated with 20 U of ALP.
To understand the supramolecular interactions that are responsible for the self-
assembly and hydrogelation process, a series of fluorescence emission spectra
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was measured with the excitation at 340 nm (Figure 7 and S11). The emission
spectra of NI-FYp molecule (1 wt%) in the presence of 20U of ALP showed the
fluorescence intensity change in the naphthalimide environment upon the
transformation from nanosphere to nanotubular structures. The significant
decrease of PL intensity was noticed with the higher concentration of ALP
indicating the self-assembly of NI-dipeptide. Time-dependent emission study of
NI-FYp sample (1 wt%) with ALP (20U) also showed the change in NI emission
intensity indicating the aggregation of NI-chromophore. Overall, the results
indicating spontaneous self-assembly and hydrogelation of NI-dipeptide.
Spectroscopic and mechanistic studies:
To gain more insights into the intermolecular interactions that are responsible for
the self-assembly and hydrogelation process, we carried out UV-Vis absorption,
and fluorescence emission measurements.28 Compound NI-FY exhibited a strong
absorption band at around 340 nm which can be attributed to the electronic transition of
NI chromophore (Figure S11). Emission spectra of the NI-FY dipeptide in aqueous
buffer is shown in Figure 8. The concentration-dependent emission spectra of NI-FY
were measured with the excitation at 340 nm. The emission signals of NI-FY appeared
as two peaks at 381 and 394 nm respectively. The emission maximum of NI-FY was
slightly red-shifted about 6 nm at a higher concentration which is probably due to the
presence of strong aromatic interactions. Besides we also carried out the aggregation
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study of NI-FY in DMSO:water mixture and the results are summarized in in Figures 8b
and 8c. To explore whether hydrogelator NI-FY has AIE or AIEE characteristics in the
assemblies, the PL spectra of NI-FY (at 500 μM) were measured in DMSO solvent with
different volume fractions of water (fw). Note that NI-FY was completely dissolved and
weakly luminescent in DMSO solvent. The emission is intensified with the gradual
addition of water into DMSO. When the water fraction fw  60 vol%, a strong blue
emission was visually observed under UV irradiation. Upon aggregation at fw = 80 vol%,
the NI-FY molecule showed a 5.9-fold fluorescent enhancement when compared to that
in DMSO. All these results indicate that aggregation has proceeded through strong
aromatic intermolecular interactions with AIE characteristics.
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Figure 8. (a) Normalized emission spectra of NI-FY at various concentrations in Tris
buffer. (b) Emission spectra of NI-FY (500 M) in DMSO:H2O mixtures with increasing
water fractions (ex = 340 nm) and (c) Plot of emission intensity of NI-FY (500 M)
versus the water fractions (fw). The inset of the figure shows the images of NI-FY in
DMSO-water mixtures with water fraction of 0% and 80 % under UV light.
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14000
12000
10000
8000
6000
4000
2000
0
d = 4.9 Å
d = 4.4 Å
d = 3.9 Å
d = 4.5 Å
d = 3.3 Å
d = 3.5 Å
10
15
20
25
30
35
2(degree)
Figure 9. Powder XRD pattern of freeze-dried NI-FYp hydrogel with 20U of ALP.
The powder X-ray diffraction (PXRD) pattern of this freeze-dried hydrogel sample
showed strong reflections at 4.9, 4.4 and 3.9 Å, indicating the formation of highly
ordered structure of the gel samples (Figures 9 and S12). Moreover, the
reflections were characteristic of a

-sheet-type structure.44,45 A minor reflection
” type packing orientations.46 The reflection
at 4.5 Å is most likely due to “cross-

at 3.5 Å and 3.3 Å are presumably due to aromatic stacking of NI-core and amino
acid side chain.27-29 The congo-red staining of NI-FYp hydrogel showed apple-
green birefringence with polarized light indicating the existence of
structures on the hydrogel (Figure S13).
-sheet type
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We also used molecular mechanical (MM) calculations with Dreiding Force Field
to evaluate noncovalent interactions of the nanostructures of NI-FY. We develop
an oligomer of ten-molecule model to study the secondary interactions of the
basic fibril unit. According to the simulations, the one-dimensional -sheet-type
packing of NI-FY is likely to have hydrogen bonding ( 1.8 Å ) and offset aromatic
stacking of NI-core with H-centroid distance of around 3.3 Å which is in agreement
with the XRD value. (see Figures 10 and S14). The stacking is also stabilized by
the hydrogen bonding of the phenolic side chain. These findings conclude that
the smallest hydrogel unit is composed of
-sheet-type aggregation along the
long axis. During the self-assembly process, two or more such oligomeric units
undergo structural transformation stabilized by Phe-Phe interactions and
hydrogen bonding, thereby generating matured fibers with a hydrophobic inner
core and solvent-exposed carboxyl groups.47,48 The matured fibers subsequently
self-assembled through solvent-fiber interactions into nanosheet type structures,
which further rolled-up into the tubular morphology.
Figure 10. Optimized packing model for NI-FY molecules. Possible molecular
interactions that are responsible for hydrogelation; (a) Parallel β-Sheet type interactions
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via hydrogen boding of amino-acid backbone (b) Aromatic-stacking of NI core and (c)
Hydrogen bonding between phenolic side chain with NI carbonyl group of the
neighboring molecule (O−H···O═C). Dimer is derived from the optimized one-
dimensional packing model of NI-FY (see supporting information)
MTT assay for cell toxicity test:
Recently, EISA has been established as a suitable process for selectively
killing cancer cells. In order to investigate the cytotoxicity of NI-FYp and Fmoc-
FYp hydrogelators two different types of cancer cell lines were selected; i) human
breast cancer cells (MCF7) and ii) human cervical cancer cells (HeLa). The cell
viability assay49,50 was performed using MTT, the MCF7 and HeLa cells were
treated with various concentrations (10 to 200 µM) of NI-FYp and Fmoc-FYp
over a period of 3 days. The viability rate of MCF7 cells treated with Fmoc-FYp
was found to be 81% after 3 days’ exposure at a high concentration of 200 µM
whereas the percentage of cell viability value of Fmoc-FYp treated HeLa cells
was decreased to 61.4% at 200µM concentration. The NI-FYp treated MCF7
cells displayed cell survival ratio over 51.2% at 200 µM concentration at day 3, In
contrast, a large diminution in cell viability was observed with HeLa cells when
treated with NI-FYp at 200 µM concentration, only 32.3% cells are viable. Overall,
the highest cytotoxic activity was observed with NI-FYp (Figure 11). Clearly, the
findings indicate that the structure-morphology of the self-assembled material has
a significant effect on the cytotoxicity of cancer cells
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10.1002/asia.202000575
Chemistry - An Asian Journal
Figure 11. Viability ratios of the HeLa and MCF-7 cells incubated with (a-b) NI-FYp and
(c-d) Fmoc-FYp.
Conclusion
In summary, we have demonstrated the first example of phosphate-based
NI/dipeptide conjugate that forms supramolecular hydrogels under the EISA
process. The self-assembly of NI-FYp has proceeded through multiple stages.
The nanofibril structures formed at the initial stage laterally associate to generate
nanosheet morphology that further rolled-up into partially unzipped nanotubular
structures. The spectroscopic and computational model suggest that the
nanostructure of the EISA triggered NI-FYp hydrogel is composed of
-sheet-
type structure connected by intra- and intermolecular of hydrogen bonding and
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10.1002/asia.202000575
Chemistry - An Asian Journal
aromatic interactions. The phenylalanine side chain provides a possible aromatic
interaction for stabilizing the cross- -structure of the peptide aggregates, but the

future research with other NI/dipeptide conjugates is needed to determine the
significance of such interactions. The self-assembled nanostructures of NI-FY
dipeptide have emission characteristics and exhibited higher cytotoxicity to
human breast cancer cells (MCF7) and human cervical cancer cells (HeLa), thus
making it a potential material for the applications in cancer therapy. We are
currently exploring the influences of other parameters that modulate the
structures and biological functions of NI-capped hydrogelators.
Acknowledgements
This work was supported by the Ministry of Science and Technology of the Republic
of China, Taiwan (MOST grant: 108-2113-M-009-024-).
References
1. G. M. Whitesides, B. Grzybowski, Science, 2002, 295, 2418-2421.
2. Y. Lin, C. Mao, Front. Mater. Sci., 2011, 5, 247-265.
3. R. Mecham, The Extracellular Matrix: An Overview, Springer, Berlin, Heidelberg, 2011.
4. H.-C. Lin and B. Xu, Applications of Supramolecular Chemistry, ed. H. J. Schneider, CRC,
Boca Raton, FL, 2012, ch. 13
5. M. Zelzer, R. V. Ulijn, Chem. Soc. Rev., 2010, 39, 3351-3357.
6. N. Singh, M. Kumar, J. F. Miravet, R. V. Ulijn, B. Escuder, Chem. Eur. J. 2017, 23, 981-993.
7. B. B. Gerbelli, S. V. Vassiliades, J. E. U. Rojas, J. N. B. D. Pelin, R. S. N. Mancini, W. S. G.
Pereira, A. M. Aguilar, M. Venanzi, F. Cavalieri, F. Giuntini, W. A. Alves, Macromol. Chem.
Phys., 2019, 220, 1900085.
8. M. Konda, B. Kauffmann, D.B. Rasale, A. K. Das Org. Biomol. Chem., 2016, 14, 4089-4102.
This article is protected by copyright. All rights reserved.

10.1002/asia.202000575
Chemistry - An Asian Journal
9. M. De Zotti, B. Muzzi, E. Gatto, B. Di Napoli, C. Mazzuca, A. Palleschi, E. Placidi, F.
Formaggio, C. Toniolo, M. Venanzi, J. Phys. Chem. B, 2018, 122, 6305-6313.
10. J. J. Panda, V. S. Chauhan, Polym. Chem., 2014, 5, 4418-4436.
11. E. T. Pashuck, S. I. Stupp, J. Am. Chem. Soc., 2010, 132, 8819-8821.
12. H. Cui, T. Muraoka, A. G. Cheetham, S. I. Stupp, Nano Lett., 2009, 9, 945-951.
13. J. W. Sadownik, J. Leckie, R. V. Ulijn, Chem. Commun., 2011, 47, 728-730.
14. K. Thornton, Y. M. Abul-Haija, N. Hodsonc, R. V. Ulijn Soft Matter, 2013, 9, 9430–9439.
15. W. Wang, Y. Chau, Soft Matter, 2009, 5, 4893–4898.
16. M. Ueda, A. Makino, T. Imai, J. Sugiyama, S. Kimura, Chem. Commun., 2011, 47, 3204-
3206.
17. H. Shao, J. R. Parquette, Angew. Chem. Int. Ed., 2009, 48, 2525-2528.
18. S. Y. Qin, Y. F. Chu, L. Tao, S. S. Xu, Z. Y. Li, R. X. Zhuo, X. Z. Zhang, Soft Matter, 2011,
7, 8635–8641.
19. C. Tang, A. M. Smith, R. F. Collins, R. V. Ulijn and A. Saiani, Langmuir, 2009, 25, 9447-
9453.
20. C. Diaferia, G. Morelli and A. Accardo, J. Mater. Chem. B, 2019, 7, 5142-5155.
21. J. Raeburn, G. Pont, L. Chen, Y. Cesbron, R. Lévy and D. J. Adams, Soft Matter, 2012, 8,
1168-1174.
22. D. Jacquemin, E. A. Perpète, G. Scalmani, I. Ciofini, C. Peltier, C. Adamo, Chem. Phys.,
2010, 372, 61-66.
23. S. Huang, R. Han, Q. Zhuang, L. Du, H. Jia, Y. Liu, Y. Liu, Biosens. Bioelectron., 2015, 71,
313–321.
24. P. Gopikrishna, N. Meher, P. K. Iyer, ACS Appl. Mater. Interfaces, 2018, 10, 12081-12111.
25. S. Banerjee, E. B. Veale, C. M. Phelan, S. A. Murphy, G. M. Tocci, L. J. Gillespie, D. O.
Frimannsson, J. M. Kelly, T. Gunnlaugsson, Chem. Soc. Rev., 2013, 42, 1601-1618.
26. X. Cao, L. Meng, Z. Li, Y. Mao, H. Lan, L. Chen, Y. Fan, T. Yi, Langmuir, 2014, 30, 11753-
11760.
27. S.-M. Hsu, F.-Y. Wu, H. Cheng, Y.-T. Huang, Y.-R. Hsieh, D. T.-H. Tseng, M.-Y. Yeh, S.-C.
Hung, H.-C. Lin, Adv. Healthcare Mater., 2016, 5, 2406-2412.
28. M. Y. Yeh, C. T. Huang, T. S. Lai, F. Y. Chen, N. T. Chu, D. T. H. Tseng, S. C. Hung, H. C.
Lin, Langmuir, 2016, 32, 7630- 7638.
29. S.-M. Hsu, R. D. Chakravarthy, H. Cheng, F.-Y. Wu, T.-S. Lai, H.-C. Lin, New J. Chem.,
2018, 42, 4443-4449.
30. J. Zhou, X. W. Du, N. Yamagata, B. Xu, J. Am. Chem. Soc., 2016, 138, 3813- 3823.
This article is protected by copyright. All rights reserved.

10.1002/asia.202000575
Chemistry - An Asian Journal
31. Z. Yang, G. Liang, B. Xu, Acc. Chem. Res., 2008, 41, 315-326.
32. Q. Yao, Z. Huang, D. Liu, J. Chen, Y. Gao, Adv. Mater., 2019, 31, e1804814.
33. J. Zhou, X. Du, C. Berciu, H. He, J. Shi, D. Nicastro, B. Xu, Chem., 2016, 1, 246-263.
34. L. S. Birchall, S. Roy, V. Jayawarna, M. Hughes, E. Irvine, G. T. Okorogheye, N. Saudi, E.
De Santis, T. Tuttle, A. A. Edwards, R. V. Ulijn, Chem. Sci., 2011, 2, 1349-1355.
35. C. A. Angulo-Pachón, S. Díaz-Oltra, J. J. Ojeda-Flores, E. Falomir, F. Galindo, J. F. Miravet,
ChemNanoMat, 2018, 4, 769-771.
36. Z. Yang, H. Gu, D. Fu, P. Gao, J. K. Lam, B. Xu, Adv. Mater., 2004, 16, 1440-1444.
37. J. K. Sahoo, S. Roy, N. Javid, K. Duncan, L. Aitken, R. V. Ulijn, Nanoscale, 2017, 9, 12330-
12334.
38. Y. Lin, M. Penna, M. R. Thomas, J. P. Wojciechowski, V. Leonardo, Y. Wang, E. T.
Pashuck, I. Yarovsky and M. M. Stevens, ACS Nano, 2019, 13, 1900-1909.
39. W. Liyanage, W. W. Brennessel and B. L. Nilsson, Langmuir, 2015, 31, 9933-9942.
40. S. Roy, N. Javid, J. Sefcik, P. J. Halling and R. V. Ulijn, Langmuir, 2012, 28, 16664-16670.
41. C. Gao, Z. Guo, J.-H. Liu, X.-J. Huang, Nanoscale, 2012, 4, 1948-1963.
42. E. Gazit, Chem. Soc. Rev., 2007, 36, 1263-1269.
43. C. X. Chen, Y. F. Gu, L. Deng, S. Y. Han, X. Sun, Y. C. Chen, J. R. Lu, H. Xu, ACS Appl.
Mater. Interfaces, 2014, 6, 14360-14368.
44. O. Rathore, D. Y. Sogah, J. Am. Chem. Soc., 2001, 123, 5231–5239.
45. M. Sunde, L. C. Serpell, M. Bartlam, P. E. Fraser, M. B. Pepys, C. C. F. Blake, J. Mol. Biol.,
1997, 273, 729–739.
46. H. Shao, T. Nguyen, N. C. Romano, D. A. Modarelli and J. R. Parquette, J. Am. Chem.
Soc., 2009, 131, 16374-16376.
47. J. K. Wychowaniec, M. Iliut, M. Zhou, J. Moffat, M. A. Elsawy, W. A. Pinheiro, J. A. Hoyland,
A. F. Miller, A. Vijayaraghavan and A. Saiani, Biomacromolecules, 2018, 19, 2731-2741.
48. I. Ramos Sasselli, P. J. Halling, R. V. Ulijn and T. Tuttle, ACS Nano, 2016, 10, 2661-2668
49. K. Reichenbacher, H. I. Suss and J. Hulliger, Chem. Soc. Rev., 2005, 34, 22.
50. J. L. Guan, Cell Migration: Developmental Methods and Protocols, Humana Press, 2005.
This article is protected by copyright. All rights reserved.