[System] polypeptide self-assembly literature information extraction

[User]
We have synthesized and characterized a self-assembling tripeptide hydrogelator
Ac-l-Phe-l-Phe-l-Ala-NH2. A series of experiments showed that the hydrogel
material could serve as a stabile and biocompatible physical support as it
improves the survival of HEK293T cells in vitro, thus being a promising
biomaterial for use in tissue engineering applications.

Synthesis, characterization and in vitro
biocompatibility assessment of a novel tripeptide
hydrogelator, as a promising scaﬀold for tissue
engineering applications†
We have synthesized and characterized a self-assembling tri-
peptide hydrogelator Ac-L-Phe-L-Phe-L-Ala-NH2. A series of
experiments showed that the hydrogel material could serve as a
stabile and biocompatible physical support as it improves the sur-
vival of HEK293T cells in vitro, thus being a promising biomaterial
for use in tissue engineering applications.
Hydrogel biomaterials, closely mimicking the three-dimen-
sional extracellular matrix (ECM), are considered to be ideal
materials for cell and tissue scaﬀolding applications due to
their biocompatibility, hydrophilic character, mechanical pro-
perties (lower micromechanical modulus) and structural diver-
sity.1 The dynamic nature of the noncovalent hydrogel fibril
network allows the hydrogel material to spontaneously adjust
to the surrounding environment and the cells to migrate
through the matrix compared to covalent hydrogels whose
pores are chemically constrained and relatively inflexible.2
Short self-assembling peptides made from natural amino acids
with alternating hydrophobic and hydrophilic amino acids
undergo spontaneous self-assembly into nanofiber hydrogel
scaﬀolds under physiological conditions.3 The weak non-
covalent interactions such as hydrogen bonding, π–π stacking,
electrostatic and van der Waals interactions are mainly respon-
sible for the self-assembly process. These scaﬀolds consisting
of nanofiber networks surround cells in a manner similar to
the ECM and the process is triggered by the addition of cell
suspensions to the aqueous solutions.4 Ultrashort peptide
hydrogels made of two or three amino acids are especially
suitable for biomedical applications because they are cost-
eﬀective, simple to prepare and accessible to production on a
aLaboratory for Supramolecular Chemistry, Division of Organic Chemistry and
Biochemistry, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia.
E-mail: frkanec@irb.hr
bCroatian Institute for Brain Research, University of Zagreb, School of Medicine,
Šalata 12, 10000 Zagreb, Croatia. E-mail: srecko.gajovic@hiim.hr
† Electronic supplementary information (ESI) available: Synthesis and spectro-
scopic characterization of hydrogel. See DOI: 10.1039/c6bm00287k
large-scale.5 A large number of such scaﬀolds described in the
literature require the use of synthetic protecting groups (such
as Fmoc, naphthalene, etc.) and/or small amounts of organic
solvents (e.g. DMSO) needed for peptide dissolution and self-
assembly.6 However, neither of these approaches are suitable
for biomedical applications because it is advisable to avoid
toxic organic solvents and some aromatic molecules (e.g.
Fmoc) when dealing with cells as it may lead to reduced cell
viability in vitro.7 Continuing the long lasting research of the
IRB group on supramolecular gelation8 we developed a new
type of self-assembling tripeptide hydrogel Ac-L-Phe-L-Phe-L-
Ala-NH2 for use in tissue engineering approaches. Ac-L-Phe-L-
Phe-L-Ala-NH2 self-assembles at physiological pH without the
need for any organic solvent. This tripeptide derivative con-
tains an aromatic diphenylalanine moiety (Phe–Phe) as a mini-
malistic building block to drive the self-assembly of short
peptides into nanostructures and hydrogels of high stability.9
The tripeptide hydrogelator Ac-L-Phe-L-Phe-L-Ala-NH2 was
prepared in five synthetic steps (Scheme S-1†). The starting
Boc-L-Phe-L-Ala-OMe dipeptide was prepared from commer-
cially available reagents by a procedure described in the litera-
ture.10 Boc-deprotection was achieved using TFA11 to give an
unprotected dipeptide L-Phe-L-Ala-OMe. L-Phe-L-Ala-OMe was
then coupled to an activated Boc-protected phenylalanine
succinimide ester to give the Boc-L-Phe-L-Phe-L-Ala-OMe tri-
peptide. After Boc-deprotection, the tripeptide was acetylated
with acetyl chloride to give the acetyl tripeptide methyl ester,
Ac-L-Phe-L-Phe-L-Ala-OMe. In the last step, ammonolysis of the
acetyl tripeptide methyl ester with dry ammonia in methanol
resulted in a final product, Ac-L-Phe-L-Phe-L-Ala-NH2 in 98%
Minimal gelation concentration (MGC) was the lowest con-
centration of the gelator in the solution that could form a
hydrogel. Ac-L-Phe-L-Phe-L-Ala-NH2
minimum gelation concentration of 0.7%, w/v. The tripeptide
hydrogel maintains a stable self-supporting structure under
physiological conditions over weeks at a very low concentration
of 2.64 mg ml
Fig. 1 Chemical structure of hydrogelator and a picture of
hydrogel formed by Ac-L-Phe-L-Phe-L-Ala-NH2 in saline ( pH = 6.12,
2.64 mg ml−1).
properties of immature neurons, thus being suitable for bio-
logical screening in neural tissue engineering.17 HEK293T
cells were encapsulated in vitro within a three-dimensional
network of nanofibers formed by self-assembly of peptide
molecules. The self-assembly was triggered by mixing cell
suspensions in media with a physiological saline solution of
the peptide molecules and the cells survived the growth of the
nanofibers around them. The results of the PicoGreen assay
showed that HEK293T cells proliferate when cultured within
Ac-L-Phe-L-Phe-L-Ala-NH2 hydrogel
cell number was significantly higher on day 5 compared to
day 0 (Fig. 3).
Transmission Electron Microscopy (TEM) images of the
nanofibrous hydrogel network show the presence of a mixture
of fibers and straight ribbons with diameters in the range of
50–500 nm and lengths in the range of micrometers (Fig. 2).
FTIR and NMR investigation pointed toward the cross-β
structure type of hydrogen bonding of the tripeptide in the gel
aggregates.12
Intramolecular hydrogen bonding and non-
covalent interactions between molecules of hydrogelators are
responsible for the formation of the 3D gel network.13
The mechanical properties of the tripeptide hydrogel were
investigated by basic rheological measurements, in order to
determine the suitability of the hydrogel for use in tissue
engineering applications. The storage modulus (G′) is 6 fold
greater than the loss modulus (G″) under constant strain at 2%
(Fig. S-12†) which indicates the viscoelastic properties of the
proposed hydrogel biomaterial. The rheological
confirm that our hydrogel satisfies criteria similar to other
small peptide hydrogels described in the literature.14,15
The tripeptide hydrogel is transparent and therefore suit-
able for microscopy analysis. A series of experiments have
been performed in order to assess in vitro biocompatibility of
the novel material.
Human embryonic kidney cells (HEK293T) have been used
as a reproducible model cell line and were cultured following
established proliferation protocols. These cells were found to
be a simple model cell line used for hydrogel-based biomater-
ial cytocompatibility evaluations.16 In addition, they show
As expected, the total cell number also increased signifi-
cantly in two positive control groups (Matrigel and medium)
and decreased in a negative control group (latex rubber). Sur-
prisingly, the cell number was significantly higher in Ac-L-Phe-
L-Phe-L-Ala-NH2 hydrogel samples compared to Matrigel on the
5th day (549.20 ± 25.17 vs. 296.40 ± 92.11, t-test, p < 0.05). The
novel supramolecular hydrogel was shown to be eﬀective in
enhancing cell proliferation up to 5 days of culturing. In
addition, the obtained results reveal the advantages of the
novel tripeptide hydrogel compared to Matrigel. A small pro-
portion of the cells precipitated during this time at the bottom
of the wells in both Ac-L-Phe-L-Phe-L-Ala-NH2 hydrogel and
Matrigel, but the majority of the cells remained in gels and
formed agglomerates. Matrigel was previously proved to be a
suitable scaﬀolding support for culturing cells. Being a natu-
rally-derived material, Matrigel cannot match requirements for
reproducibility, but this can be overcome by the use of syn-
thetic materials like the Ac-L-Phe-L-Phe-L-Ala-NH2 hydrogel.
There were no significant diﬀerences in the percentage of
live cells between HEK293T cells cultured with Matrigel
(82.7 ± 5.79%), medium (83.78 ± 2.37%) or Ac-L-Phe-L-
Phe-L-Ala-NH2 hydrogel (89.78 ± 1.43%). Morphology of the
HEK293T cells transfected with tubulin-mCherry grown in the
Fig. 2 TEM images of the tripeptide hydrogel network (a) PWK-stained
and (b) Pd-shadowed (saline, c = 2.64 mg ml−1).
Fig. 3 HEK293T cells proliferate well encapsulated within the Ac-L-
Phe-L-Phe-L-Ala-NH2 hydrogel scaﬀold. HEK293T cell number was
determined using Quant-iT™ PicoGreen® dsDNA Kit on day 0 and 5 for
cells grown in the peptide hydrogel scaﬀold, Matrigel, on latex or within
medium only. Results represent mean ± SD of 4 independent experi-
ments performed in triplicate. p < 0.05.
Ac-L-Phe-L-Phe-L-Ala-NH2 hydrogel compared to the cells grown
in medium was observed by using a confocal microscope. The
cells grown in the Ac-L-Phe-L-Phe-L-Ala-NH2 hydrogel
aggregates that are visible in a range of diﬀerent z planes,
while the cells grown in medium adhere to the well surface
and are all visible in the same z planes (Fig. S-13† and Fig. 7).
In addition, the cells were visualised under bright light, and
the cells belonging to one aggregate within the hydrogel could
be clearly distinguished from the cells grown in other aggre-
gates, being the only ones in the clear focus of the microscope,
while the others appeared blurred (Fig. 8).
Fig. 4 HEK293T cells show high viability encapsulated within the Ac-L-
Phe-L-Phe-L-Ala-NH2 hydrogel scaﬀold. Percentage of live cells was
determined using LIVE/DEAD viability/cytotoxicity assay on day 0 and 5
for cells encapsulated within the peptide hydrogel scaﬀold, or grown on
Matrigel, latex or in medium only. Results are shown as percentage of
live cells and represent mean ± SD of 4 independent experiments per-
formed in triplicate.
Fig. 7 HEK293T cells transfected with tubulin-mCherry, grown in Ac-L-
Phe-L-Phe-L-Ala-NH2 hydrogel and observed under a confocal micro-
scope after 72 h. Cells formed aggregates within the hydrogel and are
visible in diﬀerent z planes. Magniﬁcation 100×, scale bar applies to all.
Fig. 5 HEK293T cells in: (A) Ac-L-Phe-L-Phe-L-Ala-NH2, (B) Matrigel, (C)
medium, (D) latex. On the 0 day of the experiment. Live cells are green,
and dead cells are red. Magniﬁcation 10×, scale bar applies to all.
Fig. 8 HEK293T cells grown in: (A) Ac-L-Phe-L-Phe-L-Ala-NH2 hydrogel
and (B) medium and observed 72 h after encapsulation. Bright ﬁeld
microscopy shows that cells form aggregates within the hydrogel, while
cells grown in medium adhered to the surface. Magniﬁcation 20×.

[Assistant] [{"peptide_sequence": "FFA", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Acetylation", "C-terminal_modification": "Amidation", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "7.7", "PH": "6.12", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "FFA", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Acetylation", "C-terminal_modification": "Amidation", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "8.4", "PH": "6.12", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "FFA", "self-assembly_phase": "none", "N-terminal_modification": "Acetylation", "C-terminal_modification": "Amidation", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "6.3", "PH": "6.12", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "FFA", "self-assembly_phase": "none", "N-terminal_modification": "Acetylation", "C-terminal_modification": "Amidation", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.6", "PH": "6.12", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}]


[System] polypeptide self-assembly literature information extraction

[User]
The self-assembly of peptides, specifically dipeptides, offers numerous
advantages for biological applications. We describe an easy, versatile method of
fabricating different types of zwitterionic Phe-Phe dipeptide structures (i.e.,
tubes and vesicles) through solvent-mediated assembly. The stability of the
dipeptide structures is increased by thin polymer coatings of
poly(chloro-p-xylylene), a PPX film. We also investigated protein adsorption
onto PPX-coated peptide tubes and vesicles by varying the thickness of the
polymer film.

Synthesis of Peptide Tubes and Vesicles. Diphenylalanine
(Phe-Phe; Bachem AG, Switzerland) was dissolved in ethanol,
acetone, methanol, water, chloroform, toluene, THF, or benzene
at a concentration of 2 mg/mL at 70 (cid:1)C for 10 min and cooled to
room temperature. To avoid any preaggregation, fresh solutions
were prepared for each experiment. Each peptide solution (50 μL)
was then placed onto cleaned silicon wafers or glass slides and
dried until the solvent evaporated. Subsequently, the dipeptide
tubes and vesicles that were obtained were coated with poly-
(chloro-p-xylylene) (PPX) by using different amounts of dichloro-
di-p-xylylene as a precursor.12-16 The precursor was first sub-
limed at 175 (cid:1)C under a pressure of 0.1-10 Torr. Precursor vapor
was then pyrolyzed at 650-700 (cid:1)C. To control protein adsorp-
tion, PPX-coated samples having different thicknesses were first
immersed into a bovine serum albumin-FITC solution (0.1 mg/
mL in pH 7.4 PBS buffer) overnight at room temperature in a
dark environment. The films were then washed with buffer
solution several times and dried in a stream of air.
Characterization. Scanning electron microscope (SEM)
images were recorded with a Philips XL-40 system after the
samples were coated with gold. To obtain topographical informa-
tion about dipeptide tubes and vesicles, samples were character-
ized by a Nanoscope-E atomic force microscope (AFM) (Veeco
Metrology, CA). Topographical images were collected in ambient
air at room temperature with silicon nitride (SiN) triangular
cantilevers having contact mode tips (DNT-20, Veeco Metrology,
CA). Fluorescence microscopy images were taken using an
Olympus Fluoview 300 confocal laser scanning microscope with
a 50(cid:1) objective. Static contact angle experiments were performed
on an FTA-1000 (Firsttenangstrom Inc., VA) apparatus using
1460DOI:10.1021/la903571yLangmuir2010,26(3),1460–1463PublishedonWeb12/09/2009pubs.acs.org/Langmuir©2009AmericanChemicalSocietyDownloaded via MASSACHUSETTS INST OF TECHNOLOGY on March 21, 2024 at 14:50:55 (UTC).See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.Table 1. Solvent Effect on the Self-Assembly of Phe-Phe
Figure 1. Schematic representation of the self-assembly of Phe-
Phe dipeptides into tubes and vesicles in ethanol and acetone,
respectively.
Figure 2. SEM images of Phe-Phe tubes and vesicles: (a) 2 mg/mL
dipeptide in ethanol vaporized at 25 (cid:1)C, (b) 2 mg/mL dipeptide in
acetone vaporized at 25 (cid:1)C, (c) 2 mg/mL dipeptide in ethanol
vaporized at 80 (cid:1)C, and (d) 2 mg/mL dipeptide in acetone vapor-
ized at 80 (cid:1)C.
10 μL of ultrapure water. ATR-FTIR (Thermo Nicolet IR) data
was also collected with respect to a silicon wafer reference in air
and was recorded at 4 cm-1 resolution.

[Assistant] [{"peptide_sequence": "FF", "self-assembly_phase": "tube", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "10.0"}, {"peptide_sequence": "FF", "self-assembly_phase": "tube", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Methanol", "peptide_concentration(mg/ml)": "2.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "10.0"}, {"peptide_sequence": "FF", "self-assembly_phase": "tube", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Ethanol", "peptide_concentration(mg/ml)": "2.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "10.0"}, {"peptide_sequence": "FF", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Acetone", "peptide_concentration(mg/ml)": "2.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "10.0"}, {"peptide_sequence": "FF", "self-assembly_phase": "none", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 THF", "peptide_concentration(mg/ml)": "2.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "10.0"}, {"peptide_sequence": "FF", "self-assembly_phase": "none", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 THF", "peptide_concentration(mg/ml)": "2.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "10.0"}, {"peptide_sequence": "FF", "self-assembly_phase": "none", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 THF", "peptide_concentration(mg/ml)": "2.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "10.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
© 2017 Wiley Periodicals, Inc.

Dipeptides containing dehydrophenylalanine self-assemble to form
nanotubes or vesicles depending on the N-terminal amino acid.[10]
at 20,000g for 30 min at room temperature. Dilute peptide solutions
(50, 100, and 200 mM) in MeOH were prepared freshly by diluting the
structure of Boc-Cys(Me)-Leu-OMe shows self-assembly into a variety
of superstructures such as hollow rods.[18]
eliminates self-assembly of aliphatic tripeptide methyl esters into orga-
nogels.[19] The solvents that have been used are methanol (MeOH), the
recorded. Thin dried films of peptides were obtained by slowly evapo-
rating the sample solution (20 mL) on the ATR plate. The FTIR spectra
of 64 spectra for concentrated samples and 128 spectra for dilute sam-
ples, at a resolution of 4 cm21, in the wavelength range from 1000 to
4000 cm21.
blanks. From the CD spectrum obtained as a function of wavelength,
the molar ellipticity at any wavelength is given by: [u]M 5 100 3 u/c 3
l; where, [u]M is the molar ellipticity, c is the molar concentration of
peptide, u is the observed ellipticity (degrees), d is the path length (cm),
and the unit of molar ellipticity is deg cm2dmol21.
a linear gradient of 0.1% TFA in H2O to 0.1% TFA in acetonitrile:iso-
propanol (7:3) in 60 min. The molecular masses were confirmed on an
and washed with phosphate buffer (PBS). Aliquots of 1 mL suspension
containing 107 cells were incubated with peptides (10–300 mM) at
378C for 30 min, centrifuged and the absorbance of supernatant was
Peptide solutions (20 mL of (cid:1)3 mM) in 50% aqueous MeOH, were
mixed with 2 lL of different dyes. Rhodamine 6G (15 mL of 0.01 mg/
from MeOH and aqueous MeOH solutions and the Boc group was
essential for forming nanotubes.[21] Silver nanoparticles could be
trapped in the nanostructures formed by the dipeptide.[22] In an effort
varying combinations of Val, Leu and Ile. The solvents for dissolution
were MeOH as well as the structure-promoting solvent TFE[23] and
HFIP.[24,25] Self-assembly of the peptides was also examined from 50%
microscope equipped with Axiocam HRC camera and Axiovision soft-
ware (Carl Zeiss, Germany). To the Boc peptides in 10% MeOH (20 mL
of (cid:1)1.5 mM), ThT solution (2 mL of 10 mM) in 50 mM Tris buffer con-
spots were washed with water to remove excess dye and salt. Filter
with excitation and emission band pass 450–490 and 515–565 nm,
The peptides in 50% MeOH (20 mL of (cid:1)3 mM) were similarly
treated with Cong red (2 mL of 1 mg/mL in water) and Nile red (2 mL of
MeOH/TFE/HFIP, 3 mM in 50% and 1.5 mM in 10% organic-aqueous
mixtures. Long linear rod-like structures (cid:3) 1 mm and (cid:1) 0.3–1 mm diameter,
unbranched. In aqueous solvents, they form prominent hollow tubes. Long
hollow (arrow shows the opening) rounded tubular structures ((cid:1)6 mm inner
and (cid:1)8 mm outer diameter) are observed with 50% aqueous MeOH solu-
rounded edged arms. In 10% aqueous MeOH, the peptide formed hollow
six-sided, 10–20 mm long tubes with open ends on both sides (opening
1D). However, in 50% aqueous MeOH (Figure 1E), long tubes/rods (several
mm long and (cid:1)0.5–1 mm diameter) without any apparent open end were
for 4 in neat MeOH (Figure 1J). In 50% aqueous MeOH (Figure 1K),
hollow long sharp edged tubes ((cid:1)6 mm inner diameter, each side is (cid:1)2
mm wide), with open ends (shown by arrow) were observed. In 10%
aqueous MeOH, shorter (5–10 mm long) sharp edged tubular structures
F I G U R E 1 SEM images of dipeptides in MeOH and aqueous MeOH. A–C, Peptide 1 in MeOH, 50% and 10% aqueous MeOH,
respectively. D–F, Peptide 2 in MeOH, 50% and 10% aqueous MeOH, respectively. G–I, Peptide 3 in MeOH, 50% and 10% aqueous
MeOH, respectively. J–L, Peptide 4 in MeOH, 50% and 10% aqueous MeOH, respectively. Peptide concentration 5 6 mM in MeOH, 3 mM
in 50% aqueous MeOH and 1.5 mM in 10% aqueous MeOH
(Figure 2E) and hollow sharp edged tubes ((cid:1)100 mm long, sides with
(cid:1)10 mm width) were observed in 10% aqueous TFE (Figure 2F). A mix-
structures with tapering ends on both sides were observed from 50%
aqueous TFE (Figure 2H) and branched tubes (3–5 mm diameter, >100
mm long) were observed from 10% aqueous TFE solution of 3 (Figure
(Figure 2J). In 50% aqueous TFE solution, hollow rounded broken tubes
(1–2 mm diameter, 10–20 mm long) are formed (Figure 2K) and from
F I G U R E 2 SEM images of dipeptides in TFE. A–C, Peptide 1 in TFE, 50% and 10% aqueous TFE, respectively. D–F, Peptide 2 in TFE, 50%
and 10% aqueous TFE, respectively. G–I, Peptide 3 in TFE, 50% and 10% aqueous TFE, respectively. J–L, Peptide 4 in TFE, 50% and 10%
aqueous TFE, respectively. The arrows show the openings of the hollow tubular structures. Peptide concentration 5 6 mM in TFE, 3 mM in
50% aqueous TFE and 1.5 mM in 10% aqueous TFE
The structures observed at lower concentrations of the peptides
(50, 100, and 200 mM) were very similar to those observed at higher
F I G U R E 3 SEM images of dipeptides in HFIP and aqueous HFIP. A–C, Peptide 1 in HFIP, 50% and 10% aqueous HFIP, respectively. D–F,
Peptide 2 in HFIP, 50% and 10% aqueous HFIP, respectively. G–I, Peptide 3, in HFIP, 50% and 10% aqueous HFIP, respectively. J–L,
Peptide 4, in HFIP, 50% and 10% aqueous HFIP, respectively. The arrows show the openings of the hollow tubular structures. Peptide
concentration 5 6 mM in HFIP, 3 mM in 50% aqueous HFIP and 1.5 mM in 10% aqueous HFIP
F I G U R E 4 FTIR spectra of dipeptides. A–C, Peptide 1 in neat MeOH, TFE, and HFIP, respectively. D–F, Peptide 2, in MeOH, TFE, and
HFIP, respectively. G–I, Peptide 3, in MeOH, TFE, and HFIP, respectively. J–L, Peptide 4 in MeOH, TFE, and HFIP respectively. Arrows
indicate the presence of (cid:1)1625 cm21 amide I band as a shoulder. Peptide concentration 5 6 mM
From HFIP, 1 (Figure 4C) and 2 (Figure 4F) showed intense amide I
bands at (cid:1)1647 and 1649 cm21 while 3 (Figure 4I) and 4 (Figure 4L)
displayed bands at 1658/1626(sh) cm21 and 1652/1627(sh) cm21,
respectively. Prominent bands at (cid:1)1740 and (cid:1)3313 cm21 correspond-
ing to free methyl ester group and hydrogen bonded NAH group,
respectively, are present though the band at (cid:1)1683 cm21 which is
is absent. In organic solvent-aqueous mixture, all the peptides displayed
amide I bands at 1645/1625 cm21 along with (cid:1)1740 and 1684 cm21.
Studies on peptides in b-sheet conformation, particularly amyloid fibril
forming peptides, indicate bands at 1623 and 1683 cm21 corresponding
to antiparallel b-sheets. The peaks characteristic of cross b-structure
occur at (cid:1)1611–1630 cm21.[28] In model Val peptides in b-sheet con-
formation, bands were observed in the region 1634–1640 cm21.[26] All
the peptides show a prominent band at 1683 cm21. The 1683 cm21
band is assigned to stretching of the hydrogen bonded urethane car-
bonyl group (Boc >C@O).[27] Bands in the region 1645–1650 cm21 are
observed for 1, 2, and 3 in MeOH. A very small shoulder-like band at
(cid:1)1624 is observed for 1 and 4. The small peak at 1624 cm21 is a char-
acteristic amide I absorption peak for >C@O stretching coming from
extended backbone (b-sheet-like)
structures and the peak at
1645 cm21 suggests the presence of random coils within the supramo-
lecular network. Although the bands in the 1645–1650 cm21 have
been taken as an indicator of unordered conformation, we assign it to
disordered b-sheet with weak hydrogen bonds as dipeptides are
unlikely to be organized as a perfect b-sheet. Since the NH and ure-
thane C@O are clearly involved in hydrogen bonding it is an indication
Figure 1D. Monomeric forms of the dipeptides are unlikely to show
CD spectra characteristic of helix, b-sheet or b-turn conformation. The
peptides as a result of self-association. The spectra in MeOH, TFE, and
HFIP show a negative minimum at (cid:1)225–230 nm indicative of
b-structure. In 50% organic solvents (Figure 5B,E,H, respectively) too,
the peptides show prominent negative bands at (cid:1)225 nm and positive
F I G U R E 5 CD spectra of dipeptides. A–C, MeOH, 50% and 10% aqueous MeOH. D–F, TFE 50% and 10% aqueous TFE. G–I, HFIP, 50%
and 10% aqueous HFIP. curve 1- Peptide 1, curve 2- Peptide 2; curve 3- Peptide 3; curve 4- Peptide 4. Peptide concentration 5 6 mM in
neat solvents, 3 mM in 50% and 1.5 mM in 10% organic-aqueous mixtures solvents
chain spacing between chains of neighboring sheets or inter sheet dis-
tance.[30] Amyloid fibrils composed of different proteins have a com-
mon core structure with intense 4.7 Å and a less intense 10–11 Å
reflection which indicates a b-structure. However, the dried dipeptide
samples show more scattering or higher intensity at small angles of the
9.6 and 10.1 Å peaks compared to the (cid:1)4.7 Å indicating that they do
not form cross b-sheets as in amyloid fibrils. The XRD patterns of the
dipeptides indicate that the overall secondary structure present in the
microcrystals correspond to an intermolecular b-sheet arrangement
of 2 from MeOH is different from the other three peptides in MeOH,
it is likely that in all the cases, the peptides are arranged in b-sheet
mitochondria,[33] lipid droplets,[34] exoskeleton in organisms[35] as well
as peptide and protein super structures such as amyloid fibrils,[36]
hydrogels,[37] and peptoid nanosheets.[38] Confocal images of peptides
it is clearly evident that these structures do not pack in the same man-
ner as that of Phe-Phe,[32] Although peptides 1–4 exhibit the presence
of b-sheet like structure, they have lesser intense reflection with
(cid:1)4.6–5.5 Å d-spacing than 9–13.6 Å d-spacing reflection indicating
Information Fig. S2). Nile red stains nanosheets composed of pep-
toids.[38] However, these peptoids are rich in aromatic moieties. Clearly,
phobic enough for a lipophilic dye such as Nile red to bind. ThT binds
to amyloid fibrils which can be imaged by fluorescence microscopy.[39]
F I G U R E 7 The confocal images showing bright field and the corresponding fluorescence images and traces of ROI of peptides treated
with cationic fluorescent dye Rhodamine 6G. A–C, Peptide 1; D–F, Peptide 2; G–I, Peptide 3, J–L, Peptide 4. Peptide concentration was
1 mg/mL ((cid:1)3 mM) in 50% aqueous MeOH solution
erties of the solvents such as dielectric constant and ability to act as
hydrogen bond donor or acceptor.[25,31,40] Both TFE and HFIP have
lower dielectric constants as compared to MeOH.[25] The hydrophobic
superstructures with extensive intermolecular hydrogen bonding. The
positions of the bands suggest b-sheet structures with regions of
tion. Though the CD spectra of peptides in neat TFE and HFIP also
showed a negative band at (cid:1)222 nm as in neat MeOH, the SEM analy-
ized structures are not formed upon rapid drying from HFIP. The four
dipeptides in the monomeric form cannot fold into a b-turn conforma-
peptides are considerably different from the spectra shown in Figure
5.[42] While an assignment based on the CD spectra of proteins in heli-
cal or b-sheet conformation is not possible, the spectra are similar to
those exhibited by amyloid-fibrils forming peptides that show cross
b-conformation. The CD spectra suggest that the peptides self-
associate and are organized in b-conformation and corroborated with
F I G U R E 8 The bright field and the corresponding fluorescence
images of peptides treated with green fluorescent dye ThT are
recorded on Axioplan 2 fluorescence microscope. A and B, Peptide
1; C and D, Peptide 2; E and F, Peptide 3; G and H, Peptide 4.
Peptide concentration was 0.5 mg/mL ((cid:1)1.5 mM) in 10% aqueous
MeOH solution
MeOH. In 50 and 10% organic aqueous mixtures, the dielectric con-
stants of MeOH, TFE, and HFIP are comparable.[25] Hence, drastic dif-
The positions of the peaks in the FTIR spectra indicate extensive
hydrogen bonding, necessarily intermolecular, b-sheet conformation of
F I G U R E 1 0 The X-ray derived nanotube structure where the building blocks of the dipeptide 1 monomers are joined in parallel through
intermolecular hydrogen bonding to form b-sheet structure. Repeats of this dipeptide structure could give rise to the highly ordered struc-
tures observed by SEM. The difference in the XRD patterns from the regular b-sheet forming proteins or b-amyloid fibrils could be due to
formation of the superstructures with greater length along the equatorial axis (c) in the direction of the growing polypeptide chain com-
pared to the inter sheet distance of b-sheets along the H-bonding direction in the meridional axis (a)
different extents showing spectral properties diverse in amplitude and
positions of components into b-sheet secondary structures with hollow
tected aliphatic dipeptides are dependent on the physico-chemical
property of the solvent of dissolution. They tend to adopt ordered b-
conformation although they differ from the classical b-sheet structures
noncovalent interactions to align to form an array which is structurally
like a b-sheet. These strands could further stack as a result of van der
tubes, open-end nanotubes, sharp needle like structures or nanonails at
concentrations as low as 50 mM, in organic solvents and when diluted
apparent sequence or amino acid specificity or a need for the peptide
monomers to adopt b-sheet structure for self-assembly. The crystallites
of peptide aggregates showing b-sheet like structures exhibit XRD pat-
terns, which are distinct from the regular b-sheet forming proteins or
b-amyloid fibrils in their intensities. This could arise due to structures
growing polypeptide chain compared to the inter sheet distance of
b-sheets along the H-bonding direction in the meridional axis as shown

[Assistant] [{"peptide_sequence": "VI", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Methanol", "peptide_concentration(mg/ml)": "2.88", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VI", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 Methanol/H2O", "peptide_concentration(mg/ml)": "1.44", "PH": "8.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VI", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 Methanol/H2O", "peptide_concentration(mg/ml)": "0.72", "PH": "7.5", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VI", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 TFE/H2O", "peptide_concentration(mg/ml)": "1.44", "PH": "5.3", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VI", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 TFE/H2O", "peptide_concentration(mg/ml)": "0.72", "PH": "6.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VI", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 HFP/H2O", "peptide_concentration(mg/ml)": "1.44", "PH": "3.8", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VI", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 HFP/H2O", "peptide_concentration(mg/ml)": "0.72", "PH": "4.5", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VL", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 Methanol/H2O", "peptide_concentration(mg/ml)": "1.44", "PH": "8.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VL", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 Methanol/H2O", "peptide_concentration(mg/ml)": "0.72", "PH": "7.5", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VL", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 TFE/H2O", "peptide_concentration(mg/ml)": "1.44", "PH": "5.3", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VL", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 TFE/H2O", "peptide_concentration(mg/ml)": "0.72", "PH": "6.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VL", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 HFP/H2O", "peptide_concentration(mg/ml)": "1.44", "PH": "3.8", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VL", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 HFP/H2O", "peptide_concentration(mg/ml)": "0.72", "PH": "4.5", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "LV", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Methanol", "peptide_concentration(mg/ml)": "2.88", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "LV", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 Methanol/H2O", "peptide_concentration(mg/ml)": "1.44", "PH": "8.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "LV", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 Methanol/H2O", "peptide_concentration(mg/ml)": "0.72", "PH": "7.5", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "LV", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 TFE", "peptide_concentration(mg/ml)": "2.88", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "LV", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 TFE/H2O", "peptide_concentration(mg/ml)": "1.44", "PH": "5.3", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "LV", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 TFE/H2O", "peptide_concentration(mg/ml)": "0.72", "PH": "6.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "LV", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 HFP/H2O", "peptide_concentration(mg/ml)": "1.44", "PH": "3.8", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "LV", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 HFP/H2O", "peptide_concentration(mg/ml)": "0.72", "PH": "4.5", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VV", "self-assembly_phase": "fiber", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Methanol", "peptide_concentration(mg/ml)": "2.79", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VV", "self-assembly_phase": "fiber", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 Methanol/H2O", "peptide_concentration(mg/ml)": "1.4", "PH": "8.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VV", "self-assembly_phase": "fiber", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 Methanol/H2O", "peptide_concentration(mg/ml)": "0.7", "PH": "7.5", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VV", "self-assembly_phase": "fiber", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 TFE", "peptide_concentration(mg/ml)": "2.79", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VV", "self-assembly_phase": "fiber", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 TFE/H2O", "peptide_concentration(mg/ml)": "1.4", "PH": "5.3", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VV", "self-assembly_phase": "fiber", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 TFE/H2O", "peptide_concentration(mg/ml)": "0.7", "PH": "6.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VV", "self-assembly_phase": "fiber", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "50/50 HFP/H2O", "peptide_concentration(mg/ml)": "1.4", "PH": "3.8", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "VV", "self-assembly_phase": "fiber", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 HFP/H2O", "peptide_concentration(mg/ml)": "0.7", "PH": "4.5", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
BACKGROUND: Conventional cell culture studies have been performed on 2D
surfaces, resulting in flat, extended cell growth. More relevant studies are
desired to better mimic 3D in vivo tissue growth. Such realistic environments
should be the aim of any cell growth study, requiring new methods for culturing
cells in vitro. Cell biology is also tending toward miniaturization for
increased efficiency and specificity. This paper discusses the application of a
self-assembling peptide-derived hydrogel for use as a 3D cell culture scaffold
at the microscale.
RESULTS: Phenylalanine derivative hydrogel formation was seen to occur in
multiple dispersion media. Cells were immobilized in situ within microchambers
designed for cell analysis. Use of the highly biocompatible hydrogel components
and simplistic procedures significantly reduced the cytotoxic effects seen with
alternate 3D culture materials and microstructure loading methods. Cells were
easily immobilized, sustained and removed from microchambers. Differences in
growth morphology were seen in the cultured cells, owing to the 3-dimentional
character of the gel structure. Degradation improved the removal of hydrogel
from the microstructures, permitting reuse of the analysis platforms.
CONCLUSION: Self-assembling diphenylalanine derivative hydrogel provided a
method to dramatically reduce the typical difficulties of microculture
formation. Effective generation of patterned 3D cultures will lead to improved
cell study results by better modeling in vivo growth environments and increasing
efficiency and specificity of cell studies. Use of simplified growth scaffolds
such as peptide-derived hydrogel should be seen as highly advantageous and will
likely become more commonplace in cell culture methodology.

Gel Formation
The self-assembling hydrogel samples were made by first
creating  a  gelling  agent.  Lyophilized  Fmoc-Phe-Phe
dipeptide (Bachem) was weighed and dissolved in dime-
thyl  sulfoxide  (DMSO).  Deviating  from  the  preliminary
methods provided by Mahler et al., DMSO was selected to
replace  1,1,1,3,3,3-hexafluoro-2-propanol  because  it  is
less  detrimental  to  cell  viability.  The  gelling  agent  was
used  in  concentrations  of  25  and  100  mg/ml.  A  small
stock solution was created to run experiments for each day
so  as  to  avoid  any  pre-aggregation  of  dipeptide  deriva-
tives. The gelling agent was applied to the desired disper-
sion medium by pipette addition to a final concentration
of either 5 or 10 mg/ml; the higher concentration corre-
sponded to increased gel stability. The gel was created in
situ  within  the  desired  analysis  structure.  All  structures
were fabricated via silicon etching/masking procedures by
the  Microsystems  Technology  Group  in  the  School  of
Electrical Engineering, Royal Institute of Technology, Swe-
den.  Transparent  glass  cover  slides  were  bonded  to  the
etched surface to create channels and chambers for micro-
scopic observation.
Layer  formation  was  performed  by  repetition  of  the  gel
forming procedure. The initial layer was first generated in
a  generic  mold  and  allowed  to  stabilize.  Upon  stabile
hydrogel formation, the additional dispersion media was
added to the mold, forming a fluid layer above the stable
hydrogel.  An  appropriate  amount  of  gelling  agent  was
directly added to the fluid layer. Layers were only formed
in purified water, but it can be assumed that all successful
dispersion media can also be used in layer formation. We
only examined the use of 25 mg/ml gelling agents for a
final  hydrogel  concentration  of  10  mg/ml.  Diffusion  of
dispersion  medium  in  layers  was  observed  by  adding  a
colored dye in one layer of hydrogel. For general diffusion
observation, a drop of dye was applied to a stable get and
allowed to diffuse into the sample.
Gel Reversing
Mechanical  reversing  was  performed  by  applying  sheer
forces to a stable gel. Large samples were destabilized by
vortex or pipette mixing. Environmental pH changes were
also used to reverse assembled gel stability. NaOH (0.5M)
was  added  to  elevate  the  matrix  solution  above  pH  9.
Enzyme  reversing  was  performed  with  proteinase  K
(Sigma-Aldrich).  Reversing  in  microstructures  was  per-
formed by actively applying a flow of reversing solution:
0.05 mg/ml proteinase K, 1.0 wt% SDS, pH between 9 and
10,  and  temperature  control  at  37°C.  Microchambers
were all flanked by parallel channels (Figure 4) where the
enzyme reversing solution was perfused. Precision pumps
were fitted to drive the solution from a syringe, through
flexible tubing, into the inlets of the parallel microchan-
nels, and out through the outlets. The microchip stage and
perfusion tubing setup is shown in Figure 10.
Detecting Gel Transitions
To confirm gel formation, two protocols were employed.
The first was a macroscopic observation of the gel stabil-
ity. In the presence of a rigid conformation, the hydrogel
should be sufficiently stable to undergo and inversion test
without  conformational  changes.  Inversion  testing  was
performed on 2 ml samples in a 15 ml Falcon Tube. After
appropriate  stabilization  time,  the  sample  was  inverted
and  monitored  for  deformation.  A  sample  undergoing
incomplete gel transition shows deformation in the form
of flow toward the lid (lowest point of the inverted tube).
A complete and stable hydrogel transition was character-
ized by an inversion test resulting in zero visible deforma-
tion.  A  successful
inversion  test  confirmed  stable
macroscopic hydrogel formation.
The second protocol utilized to verify gel stability required
observation  at  the  microscopic  level.  Optically  distin-
guishable foreign material was added to the gel and mon-
itored  by  confocal  microscopy.  The  'impurity'  behavior
was  then  used  to  infer  hydrogel  characteristics.  Fluores-
cent  microspheres  (15  μm  Focal  Check  microspheres,
Molecular  Probes)  were  suspended  in  the  dispersion
medium  of  choice  just  prior  to  addition  of  the  gelling
agent. The microspheres were then induced with 488 nm
laser excitation on a Zeiss LSM 5 Pascal confocal micro-
scope over a 10 minute detection time interval. Confocal
tracking of the microspheres presented the level of stabil-
ity of the hydrogel matrix in the form of sphere immobil-
ity. Immobilization of the suspended microspheres over
an extended time period was a sufficient indication of a
stable hydrogel matrix.
Stable hydrogel samples were monitored for sensitivity to
multiple  reversing  methods.  The  above  gel  transition
methods were utilized to demonstrate reverse transitions
from stable a stable hydrogel matrix to a less stable liquid
solution. If a stable hydrogel sample can be modified such
that it no longer satisfies the discussed requirements for a
stable gel, the sample has reverted from a gel to a liquid
phase  solution.  More  explicitly,  a  sample  that  fails  the
inversion test or shows a dramatic increase in microscopic
movement upon modification is no longer considered a
stable hydrogel.
Cell Inclusion
The cell lines examined in hydrogel cultures include COS-
7, MDCK, and two strains of Astrocytes (CTX TNA2 and
DI TNC1), all obtained from ECACC. Each cell line was
cultured  according  to  the  ECACC  recommendations.
Astrocytes were grown to confluency in a liquid culture of
MEM (Gibco) and the remaining cell lines were grown in
DMEM  (Sigma).  Cells  were  grown  with  an  addition  of
10% fetal bovine serum (Gibco), 1.0% penicillin/strepto-
mycin  (Sigma)  and  1.0%  L-glutamine  (Sigma).  MDCK
cells  were  grown  with  an  additional  treatment  of  1.0%
non-essential  amino  acids  (Sigma).  Incubation  was  per-
formed at 37°C with 5% CO2 and 100% humidity. The
cells  were  harvested  from  the  culturing  petri  dishes  by
trypsinization.  The  harvested  cells  were spun  down  to a
pellet by centrifugation for 5 minutes at 1700 rpm. Cells
were then washed twice before a final spin down. Staining
treatments and washing were performed with a standard
phosphate  buffered  saline  solution  (1×  PBS).  The  final
cell pellet was then immobilized in the peptide derivative
extracellular matrix by first suspending in PBS and adding
the gelling agent to initiate hydrogel self-assembly. A typ-
ical cell number density used was between 5 and 12 mil-
lion cells/ml of final gel volume. Nutrients were supplied
to the immobilized cell suspensions by perfusion as soon
as  the  hydrogel  was  stabilized.  For  long-term  culturing,
the  microstructures  were  removed  from  the  microchip
platform  and  submerged  in  culture  media  under  37°C.
Cells  were  viewed  by  staining  with  1.0  μM  calcein-AM
(Invitrogen). If the cells were to be stained before in situ
culture  formation,  calcein-AM  was  added  to  the  disper-
sion medium. For in situ staining within the microcham-
ber,  calcein-AM  was  added  to  a  perfusion  solution  of
growth medium and perfused through the microchannels.
Cells were maintained in stain solution for 30 minutes to
1 hour at 37°C before confocal observation.

[Assistant] [{"peptide_sequence": "FF", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 DMSO/PBS", "peptide_concentration(mg/ml)": "5.025", "PH": "7.0", "temperature(celsius)": "37.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "FF", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 DMSO/PBS", "peptide_concentration(mg/ml)": "10.05", "PH": "7.0", "temperature(celsius)": "37.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "FF", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "20/80 DMSO/PBS", "peptide_concentration(mg/ml)": "5.025", "PH": "7.0", "temperature(celsius)": "37.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "FF", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "40/60 DMSO/PBS", "peptide_concentration(mg/ml)": "10.05", "PH": "7.0", "temperature(celsius)": "37.0", "heating_cooling": "No", "incubation_period(min)": "nan"}]


[System] polypeptide self-assembly literature information extraction

[User]
Early research has shown that many neurodegenerative diseases are associated
with the absence of a short and natural tripeptide sequence, Lys-Phe-Gly (KFG).
Herein we report results of both experiments and extensive MD simulations of
this tripeptide to understand the self-assembly and morphology as a function of
its concentration. Morphologies of the aggregates formed by the tripeptide at
low concentration (vesicles), and at high concentration (nanotubes) are studied
by several independent 3 μs long Martini coarse-graining MD simulation runs.
Further, prediction from MD at still higher concentrations about the formation
of rectangular blocks, reported for the first time, has been verified through
laboratory experiments. Thus, the computational studies performed are in
agreement with the experimental findings observed in our laboratory and a
complete control over the formation of various nanostructures is achieved simply
by changing the concentration of a short and naturally conserved tripeptide.

Computational Section. Coarse-Grained Molecular Dy-
namics Simulation. Herein we use coarse-grain (CG)
MARTINI V2.1 force ﬁeld37,38 to model the tripeptide, KFG.
This force ﬁeld uses four-to-one mapping where four atoms and
associated hydrogens are represented by one CG bead and three-
to-one mapping for aromatic substituents. Hence one Lys-Phe-
Gly (KFG) molecule is represented by eight beads where Lys is
represented by three, Phe by four, and Gly by one CG bead. The
N-terminal Lys bead has two positive charges and the C-terminal
Gly bead has one negative charge. Since the total charge of each
KFG molecule is +1, we have added CG chloride and CG sodium
ions in requisite amount to electrically neutralize the system
having 1000 molecules of the tripeptide, KFG, and attain the salt
concentration of 0.1 mol/L. CG waters, where four water
molecules correspond to one bead with no charge, are also added
to the system to achieve the concentration of interest. We have
also replaced 0.1 mole fractions of the CG waters with the
“antifreezing” model in order to avoid the unwanted freezing
eﬀect that sometimes occur with MARTINI model.37 Overall it
allows a 4-fold reduction in the number of particles and speeds up
the system by many folds as compared to the all-atom
simulations. All of the molecular dynamics simulation runs
have been carried out with NAMD software.39 The systems are
coupled to external temperature and pressure baths of 310 K and
1 atm respectively using the Langevin coupling methods.40 We
have utilized electrostatic and van der Waals (vdW) interactions
in their shifted forms with a cutoﬀ value of 1.2 nm. The neighbor
list has been updated every 10 steps and the coordinates are saved
every 5 ns for the trajectory monitoring where no coordinates are
constrained during the simulation. All the periodic boxes are
energy minimized using steepest descent integrator at 0 K,
heated to 773 K and cooled to 310 K. System is then equilibrated
at 310 K for 2 ns and simulated for a total of 3 μs at 310 K with a
large time step of 25 fs.41,42
Analysis of Outputs from the MD Simulation. The ﬁnal
trajectories at each concentration have been analyzed using our
in-house-developed codes and NAMD facilities. The various
conformations at diﬀerent time points are monitored to follow
the assembly process and characterized the prevailing structures
Experimental Section. Fourier-Transform Infrared (FT-IR)
Spectroscopy. The infrared (IR) spectra were recorded using a
PerkinElmer Spectrum BX FT-IR spectrometer. The tripeptide
(KFG) suspensions in water were prepared at diﬀerent
concentrations and drop-coated over calcium ﬂuoride cell. It
was air-dried, freeze-dried, and then IR spectra were recorded of
the generated thin ﬁlms.
Scanning Electron Microscopy (SEM). The diﬀerent samples
of the tripeptide suspensions were made by serial dilution from a
concentrated sample. The morphology of the tripeptide, KFG, at
various concentrations has been investigated by scanning
electron microscopy. An aliquot of the suspension (20 μL) is
loaded onto the brass stubs and the samples are kept at room
temperature for 6 h to air-dry. This is followed by freeze-drying
for another 6 h. Finally the samples are used for the SEM study
on a Quanta 200 SEM operated at 5 kV after sputtering 20 nm of
gold over the sample surfaces for proper imaging without
charging eﬀect.
X-ray Diﬀraction (XRD). The aqueous suspension of the
tripeptide at 57 mM concentration was placed on a precleaned
glass plate which, upon air-drying, aﬀorded a thin ﬁlm on the
glass plate. X-ray diﬀraction (XRD) of the ﬁlm was performed
using the reﬂection method with a Phillips X-ray diﬀractometer.
The X-ray beam was generated with a Cu anode and the Cu Kα1
beam of wavelength 1.5418 Å was used for the experiments.
Scans were performed for 2θ values up to 45°.

[Assistant] [{"peptide_sequence": "KFG", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.5", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "KFG", "self-assembly_phase": "tube", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "4.9", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
Hydrogels are promising candidates for biomimetic scaffolds of the extracellular
matrix in tissue engineering applications. However, their use in bone tissue
engineering is limited due to their low mechanical properties. In this study, we
designed and synthesized multicomponent peptide-based hydrogels composed of
fluorenyl-9-methoxycarbonyl diphenylalanine (FmocFF), which contributed to the
rigidity and stability of the hydrogel, and Fmoc-arginine (FmocR), which
mediated high affinity to hydroxyapatite (HAP) due to the arginine moiety. The
new hydrogels composed of nanometric fibril networks were decorated with HAP and
demonstrated high mechanical strength with a storage modulus of up to 29 kPa. In
addition, the hydrogels supported cell adhesion and in vitro cell viability.
These properties suggest using these multicomponent organic-inorganic hydrogels
as functional biomaterials for improved bone regeneration.

commonly implemented for bone regeneration due to their low
mechanical properties compared to those of the natural bone.26
Consequently, the use of synthetic biomaterial hydrogels with
controllable and reproducible properties is appealing for bone
tissue engineering.27
Short peptides can be used as low molecular weight gelators
and form mechanically stable hydrogels through self-assembly
into supramolecular structures,28−34 which typically arise from
noncovalent interactions such as hydrogen-bonding, van der
Waals interactions, π−π stacking, and electrostatic interac-
tions.35−37 Furthermore, the material properties can be tailored
to form multicomponent supramolecular gels composed of (1)
two components that do not form a gel on their own, (2) two
or more gelators that can either coassemble or self-sort into
distinct assemblies, or (3) one or more gelators and one or
more nongelling additives that can aﬀect the assembly process
of the gelator.38−41 Controlling the properties of the resulting
gels is achieved by diﬀerent interactions and by changing the
ratio of the diﬀerent components, enabling modulation of
cellular functionality and tissue morphogenesis.39,42
Fluorenyl-9-methoxycarbonyl (Fmoc)-functionalized pepti-
des are of the most studied low molecular weight gelators. One
notable example is Fmoc-diphenylalanine (FmocFF) that can
form a rigid hydrogel without the need for an additional cross-
linking agent,43 resulting in a biocompatible gel network that
mimics the ECM. The properties of this dipeptide hydrogel can
be easily modiﬁed by changing the amino acid sequence and
the aromatic N-terminus groups.32,44
Incorporation of
inorganic materials such as hydroxyapatite (HAP) into hydrogel
matrices has been shown to support the cellular growth of
human osteoblasts,45 enhance bone mineralization by forming
nucleation sites,46,47 as well as to support angiogenic and
osteogenic activity in collagen scaﬀolds after subcutaneous
ectopic implantation in mice.48 HAP is easily dissolved, forming
a high calcium ion layer and a slightly alkaline environment,
which can eﬀectively promote osteoblast adhesion, prolifer-
ation, and matrix secretion, thus improving the mechanical
properties of the composite material.49
biomineralization.50,51 Speciﬁcally,
the interaction between
3− with both negative and positive charged
Ca2+ and PO4
amino acids glutamate and arginine, respectively, to form HAP
aggregates and later give rise to HAP crystals has been shown
by Cerruti and colleagues.51 In addition, arginine has been
shown to increase bone mineral density by stimulating growth
hormone production.50 Moreover, it is considered to be one of
the most essential amino acids for young developing mammals,
suggesting a role for arginine in tissue development.52 Brasinika
et al. demonstrated enhanced human mesenchymal stem cell
attachment, proliferation, and diﬀerentiation on HAP surfaces
in the presence of collagen and arginine.53 The addition of
arginine can also be used as a strategy to ﬁne-tune the
bioactivity of the HAP crystals, as previously suggested.54,55
In the present study, we designed peptide-based multi-
component hydrogels composed of
two building blocks,
FmocFF and Fmoc-arginine (FmocR). We allowed the
hydrogels to form at diﬀerent ratios of the building blocks to
modulate the hydrogel physical properties. In addition, we
incorporated HAP within the hydrogels to form 3D scaﬀolds
for bone tissue regeneration. We characterized the multi-
component hydrogel’s secondary structure, mechanical proper-
ties, and biocompatibility. An optimum composition of
FmocFF, FmocR, and HAP resulted in a superior rigid and
Materials. Lyophilized Fmoc-Phe-Phe-OH (FmocFF) and Fmoc-
Arg-OH (FmocR) were purchased from Bachem (Budendorf,
Switzerland) and Sigma-Aldrich (Rehovot, Israel), respectively. HAP
nanoparticles were synthesized by a wet precipitation method in an
aqueous system and have been previously shown to be pure HAP
particles with an ellipsoidal shape and an average size of approximately
80 nm without heterogeneous phases.46
Formation of the Hydrogels. FmocFF and FmocR solutions
were prepared separately by dissolving the powders to a concentration
of 100 mg/mL in dimethyl sulfoxide (DMSO) and vortexing until the
solution became transparent. Single peptide solutions were prepared
by adding 50 μL of the aqueous stock solutions to 950 μL of double
distilled water with vortexing. FmocFF:FmocR mixed solutions at a
ﬁnal concentration of 5 mg/mL were prepared by combining the two
peptides stock solutions in DMSO at the desired molar ratios of 3:1
(37.5 and 12.5 μL), 1:1 (25 and 25 μL), and 1:3 (12.5 and 37.5 μL).
The mixed solutions were then diluted in 950 μL of double distilled
water and vortexed. For the HAP-incorporated hydrogels, 1 mg/mL of
HAP was dispersed in 950 μL of DDW by sonication at 0−4 °C to
avoid sample heating. Pure FmocFF and FmocR and their mixed stock
solutions were then added to the HAP aqueous solution with vortex
mixing using the ratios detailed above.
Absorbance Kinetics Analysis. Samples of 150 μL were placed
into a 96-well plate. Absorbances at 400, 500, and 600 nm were
measured every 5 min using a TECAN Inﬁnite M200PRO plate reader
for a total of 24 h.
Transmission Electron Microscopy (TEM). Samples (10 μL) of
FmocFF, FmocR, and hybrids were prepared for TEM analysis
(Electron Microscopy Sciences LTD). The excess liquid was removed
2 min later using cellulose ﬁlter paper. Ten microliters of 2% uranyl
acetate was then deposited on the grid and allowed to adsorb for 2 min
before excess ﬂuid was blotted oﬀ. Prior to analyses, samples were left
to air-dry at ambient conditions. TEM analysis was performed using a
JEM-1400Plus Transmission Electron Microscope (JEM) operating at
EDX Analysis Using High-Resolution Scanning Electron
Microscopy (HR-SEM). Hydrogel samples were placed on glass
slides and left to air-dry at ambient conditions. The samples were then
coated with Cr for conductance and viewed and analyzed using a JSM-
6700 ﬁeld-emission HR-SEM (JEOL, Tokyo, Japan) equipped with a
cold ﬁeld emission gun operating at 10 kV.
Rheological Analysis. Rheological measurements were performed
using an AR-G2 controlled-stress rheometer (TA Instruments, USA).
Time-sweep oscillatory tests in parallel plate geometry were performed
on 250 μL of fresh solution (resulting in a gap size of 0.6 mm) at room
temperature. Oscillatory strain (0.01−100%) and frequency sweeps
(0.01−100 Hz) were conducted to determine the linear viscoelastic
region at which the time sweep oscillatory tests were performed. G′
and G″, the storage and loss moduli, respectively, were obtained at 5
Hz oscillation and 0.5% strain deformation for each sample.
Fluorescence Spectroscopy. The emission spectra of the gels
were recorded using a Horiba JobinYvon FL3-11 ﬂuorimeter (Horiba
JobinYvon, NJ, USA). A quartz cuvette with an optical path length of 1
cm was used. The gels were assembled within the cuvette, and the
spectrum was collected. The experiments were carried out using an
excitation wavelength of 280 and 5 nm excitation and emission slits.
Fourier-Transform Infrared (FTIR) Spectroscopy. FTIR spectra
were collected 4 days after gel preparation using a Nicolet Nexus 470
FTIR spectrometer with a deuterated triglycine sulfate (DTGS)
detector. Hydrogels samples were placed onto disposable KBr IR
sample cards (Sigma-Aldrich, Israel) and vacuum dried. Measurements
were performed using 4 cm−1 resolution and by averaging 2000 scans.
The absorbance maxima values were determined using the OMNIC
analysis program (Nicolet). The obtained transmittance spectra were
smoothed by applying the Savitzky−Golay function to eliminate noise
Figure 1. Hydrogel gelation kinetics. (a) Molecular structure of the two building blocks FmocFF and FmocR. (b) Inverted vials of the single and
hybrid hydrogels and (c) inverted vials of the diﬀerent hydrogels incorporated with HAP: (1) FmocFF, (2) FmocFF:FmocR 3:1, (3)
FmocFF:FmocR 1:1, (4) FmocFF:FmocR 1:3, and (5) FmocR. (d) Absorbance changes of the hydrogels over time (absorbance at 400 nm). (e)
Absorbance changes of the HAP-containing hydrogels over time (absorbance at 400 nm).
Cell Viability Analysis. 3T3 mouse ﬁbroblast cells were purchased
from ATCC and cultured in Dulbecco’s modiﬁed Eagle’s medium
(DMEM) supplemented with 10% fetal calf serum, 100 U mL−1
penicillin, 100 U mL−1 streptomycin, and 2 mmol L−1
L-glutamine (all
from Biological Industries, Israel). The cells were maintained in a Petri
dish at 37 °C in a humidiﬁed atmosphere containing 5% CO2. Gels
were formed in a 24-well plate and repeatedly washed for 3 days with
culture medium to ensure complete removal of excess materials and
solvent followed by UV sterilization. Subsequently, the pH of the ﬁnal
medium over the gels was measured to be 7.4−7.8, as also inferred
from the medium color, which was observed to be the same as that of
the nai ̈ve medium, indicating a suitable pH required for cell culture.
After reaching a conﬂuence of 90%, the cells were separated from the
Petri dish using trypsin A, and 60,000 cells in 0.1 mL of fresh culture
medium were seeded per well on the prewashed gel samples and
incubated for 8 h. A ﬂuorescent live−dead staining assay (Sigma-
Aldrich) containing ﬂuorescein diacetate (6.6 μg/mL) and propidium
iodide (5 μg/mL) was then used to visualize the proportion of viable
versus nonviable cells. The labeled cells were immediately viewed
using a Nikon Eclipse Tiﬂuorescent microscope, and images were
captured by a ZylascMOS camera using Nikon Intensilight C-HGFI
ﬂuorescent lamp.

[Assistant] {"peptide_sequence": "R", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 DMSO/H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "9.7", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}


[System] polypeptide self-assembly literature information extraction

[User]
© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transformation of Dipeptide-Based Organogels into Chiral Crystals by
Cryogenic Treatment
Xingcen Liu+, Jinbo Fei+, Anhe Wang, Wei Cui, Pengli Zhu, and Junbai Li*
Abstract: Controlled molecular assembly is an important
approach for the synthesis of single-component materials with
diverse functions. Unlike traditional heat treatment or solvent
modulation, cryogenic treatment at 77 K enabled the tunable
transition of a self-assembled diphenylalanine organogel into
a hexagonal crystal. Under these conditions, the assembled
molecules undergo an internal rearrangement in the solid state
to form a well-defined chiral crystal structure. Moreover, these
assemblies exhibit enhanced emission. This strategy for the
synthesis of single-component supramolecular assemblies can
create new functions by manipulating phase transitions.
as a breakthrough strategy in nanoscience and nanotechnol-
ogy.[1–3] As it enables the formation of self-assemblies with
desired properties, this bottom-up approach has already
found various applications in biomedicine, electrical engi-
neering, and materials science.[4–7] Peptides contain all of the
molecular information that is needed to rapidly and effi-
ciently form ordered assemblies with various structures,
ranging from nanotubes to fiber bundles, and a series of
conformations, such as a-helices and b-sheets.[8–10] In partic-
ular, short peptides can assemble into diverse aggregate
structures, such as spheres, micelles, vesicles, tubes, rods, and
fibers. Among these peptides, diphenylalanine (FF), the
simplest aromatic dipeptide, has attracted significant interest
because of its favorable features,
including its structural
simplicity, biocompatibility, facile chemical modification, and
the fact that its self-assembly can be tuned particularly
well.[11–15] In our previous work, we constructed various FF-
based microstructures, such as organogels, microcrystals, and
hierarchical mesocrystals, in a controlled manner.[16–19]
Low-molecular-weight gelators (LMWGs) with three-
dimensional open nanostructures have attracted substantial
attention.[20, 21] A wide range of peptides, including FF, exhibit
good LMWG behavior through various intermolecular non-
covalent interactions, such as p–p stacking, hydrogen bond-
ing, electrostatic, and van der Waals interactions.[22, 23] Gen-
erally, gels are unstable systems because of the balance
between the molecular aggregation force and solubilizing
gelator–solvent interactions.[24, 25] Notably, physical gelation is
a spontaneous, thermodynamically and kinetically driven
process.[26] Owing to the weakness of
the non-covalent
interactions exploited to maintain the aforementioned equi-
librium, such metastable physical gels can be converted into
thermodynamically stable crystals. The gel–crystal transitions
in several systems have been observed to be triggered by
various stimuli, such as light irradiation, pH changes, heating/
cooling, a change in ionic strength or solvent, or even
storage.[27–35] For instance, our group has demonstrated that
the structural transition from an organogel to flower-like
microcrystals can be induced in the dipeptide self-assembly
system by using ethanol as a co-solvent.[18]
Herein, we explore cryogenic treatment as an approach to
achieve a tunable transition from a dipeptide-based self-
assembled organogel to a well-ordered crystal (Scheme 1).
Specifically, cryogenic treatment of a FF–toluene gel resulted
in a gel–crystal transition. The transition process was moni-
tored for several cycles as the temperature of the system was
changed from 77 K to room temperature. Interestingly, the
aforementioned transition enables the fabrication of a bio-
molecule-based nanosystem with enhanced emission. Fur-
thermore, this novel and general approach can be used to
facilitate transitions of other FF-based gel systems.
Scheme 1. The transition of a dipeptide-based organogel into a hexago-
nal crystal with enhanced emission under extremely cold conditions
(liquid nitrogen, 77 K).
To gain further insight into how a cryogenic treatment
affects self-assembled organogels, we used scanning electron
microscopy (SEM) to analyze their morphology. As we
reported previously,[17] a transparent gel was rapidly obtained
when a FF/1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) solution
was diluted with toluene. The resulting organogel consisted of
ultrafine fibers (Figure 1 a). Cryogenic treatment for different
periods of time resulted in the appearance of the gels
changing from transparent to opaque. Specifically, after two
treatments, some small fibers appeared, even though the gel
remained transparent (Figure 1 b). When the number of
treatments was increased to five, the morphology clearly
changed to short bundled fibers (Figure 1 c). Meanwhile, the
organogels gradually became opaque after five or more
treatments. As depicted in Figure 1 d, after ten cycles, longer
fibers with few bundles were produced. When the cycle
number was increased to sixteen, the fiber morphology
became homogeneous and dispersive, and the fibers had
lengths of several dozens of micrometers and widths of
approximately 200 nm (Figure 1 e). When the number of
treatments was further increased to as many as 30 cycles, no
obvious morphology changes were observed (see the Sup-
porting Information, Figure S1). The transmission electron
microscopy (TEM) image in Figure 1 f also shows that the
fibers were 200 nm wide, which is in good agreement with the
SEM results.
Figure 1. SEM images of FF–toluene organogels after different num-
bers of cryogenic treatments: a) 0, b) 2, c) 5, d) 10, and e) 16 treat-
ments. The insets show the corresponding photographs. f) TEM image
of an organogel after 16 cryogenic treatment cycles.
The images in Figure 2 a, which were obtained using
a cross-polarized microscope, show that the assembly was not
particularly bright before the treatment. By contrast, after the
treatment, bright fibers were observed (Figure 2 b), which
suggests the presence of fibril bundles, resulting in vibrational
brightness. Specifically, when the macroscopic fibers were
aligned at an angle of 4588 relative to the polarizer, the
anisotropic birefringence was minimized. The birefringence
of these fibers became very strong again when they were
aligned at an angle of 9088 with respect to the polarizer. These
results reveal that the crystalline fibers were aligned after the
cryogenic treatment.
The viscoelastic properties of the gels were measured by
oscillatory rheology experiments. Generally, the viscoelastic
behavior of a gel is determined by two key parameters, the
storage modulus (G’) and the loss modulus (G’’), which
represent the ability of a gel to return to its original geometry
and the tendency of a material to flow under applied stress.[34]
For a gel, G’ is greater than G’’, and G’ is very weakly
dependent on the oscillatory frequency, demonstrating the
dominant elastic behavior of the gel. The results shown in
Figure 3 a confirm the formation of an organogel and its
elastic properties. Importantly, as the cryogenic treatment was
repeated for
the mechanical
strength increased. The higher mechanical strength may
from enhanced molecular interactions in the FF
assembly.
To investigate the molecular interactions during the
transition process,
the system was analyzed by Fourier
transform infrared spectroscopy (FTIR). Figure 3 b shows
the characteristic peaks of an amide I band at 1681 cm@1 and
an amide II absorption band at approximately 1608 cm@1,
corresponding to a hydrogen-bonded b-sheet secondary
structure with antiparallel alignment.[16, 36] When the number
of cryogenic treatments was increased further, the peak at
1608 cm@1 was red-shifted to approximately 1604 cm@1 and
the peak at 1681 cm@1 was
shifted to approximately
1686 cm@1. These shifts may be due to the effect of the
cryogenic treatment on the hydrogen bonding between C=O
and N@H moieties in the FF assembly.[16]
ature, toluene returns to the liquid state much earlier than
HFIP, and the FF–toluene interactions lead to gel aggregation
by stochastic nucleation,[39] that is, the evolution could be
a result of the rebalance of molecular interactions such as p–p
stacking, hydrogen bonding, and electrostatic interactions.[40]
Thus the cryogenic treatment mediates the rebalancing effect,
enabling the transformation of a metastable gel phase into
a thermodynamically stable crystal phase.[34]
Interestingly, during the transition process, the well-
ordered FF crystals exhibited enhanced emission properties.
Specifically, as shown in Figure 4 a, upon excitation at l =
250 nm, the photoluminescence emission peak of the FF/
HFIP solution at l = 285 nm shifted to 295 nm in FF–toluene
gel as the molecules aggregated into gels. As the number of
cryogenic treatments was increased, the emission intensity
increased gradually but significantly (Figure 4 b). This behav-
ior may be due to the decreased vibrational relaxation in the
crystal state.
Figure 4. Photoluminescence emission spectra of a) a solution of FF
in HFIP and a FF–toluene gel and b) FF–toluene gels after different
numbers of cryogenic treatments.
This strategy based on cryogenic treatments can be
extended to achieve the transition of other FF-based gels
into a crystalline state. Figure S3 shows that similar phenom-
ena are observed with FF–CHCl3/isopropanol (3 %), FF–
CH2Cl2/DMF (3 %), FF–benzene, FF–styrene, FF–xylene,
and FF–CCl4/DMF (2 %) systems. SEM images (Figures S4
and S5) illustrate the crystallization process, demonstrating
that the gel–crystal transition also occurs in other gel systems.
In the case of FF–CHCl3/isopropanol (3 %), the transparent
gel became opaque after only two cryogenic treatments,
possibly because the solvent–gelator interactions were much
weaker than those in the FF–toluene system, resulting in
much easier separation of the solvent and FF assembly and
more facile formation of the FF crystal. We also tested two
other organogel systems (cationic diphenylalanine (CDP) and
phenylalanine (F)) and observed similar transitions (Figur-
es S6 and S7). These findings demonstrate the versatility of
our method.
In summary, we have developed a universal approach for
modulating the gel–crystal transition of a dipeptide assembly.
Cryogenic treatment disrupted and reestablished the balance
between the molecular interactions within a newly prepared
gel, resulting in the formation of a dipeptide hexagonal
crystal. Remarkably,
exhibited
enhanced emission, which may be due to the limited vibra-
tional relaxation of the intermolecular bonds in the assembly.
Figure 3. Characterization of FF–toluene organogels after different
numbers of cryogenic treatments (0, 5, 10, and 16): a) Rheological
data, b) FTIR spectra, c) XRD patterns, and d) CD spectra.
Furthermore, the entire evolution process was studied by
X-ray diffraction (XRD; Figure 3 c). Before treatment, the
xerogel exhibited a lamellar structure, with a peak at 2 q =
5.1488 (lamellar thickness = 16.9 c),[17] indicating that the gel
was not crystalline. After five treatments, some weak peaks
appeared, revealing the formation of a small amount of
crystallites. Ten cycles later, more small peaks had arisen, the
intensity of the previously observed peaks had increased, and
the peaks had become sharp, implying crystal formation.
When the number of cycles was increased to sixteen or even
thirty (Figure S1 b), the sharp peaks did not change anymore,
confirming the completion of the gel–crystal transition. After
16 cycles, a hexagonal structure with crystalline properties
was observed.[37, 38] These findings are in good agreement with
the SEM results. Collectively, these results imply that the
cryogenic treatment caused the gel–crystal transition. By
contrast, without the cryogenic treatment, no obvious changes
were observed in the FF–toluene system (see Figure S2).
Furthermore, circular dichroism (CD) spectroscopy was
used to examine the secondary structure of the gels after the
cryogenic treatment (Figure 3 d). Clearly, without treatment,
the gel did not show any CD signals because the gelators did
not form a chiral supramolecular assembly. After more than
five treatments, the CD spectra exhibited a single negative
band with a peak at 278 nm, which was fit to l-Phe-l-Phe.
Moreover, the peak intensities increased with an increasing
number of
cryogenic treatment changes the intermolecular arrangement
in the gel, and that the FF hexagonal crystal is chiral.
For LMWGs, the solvophobic moieties of the molecules
tend to aggregate. Meanwhile, a balance exists between the
solvophobic and solvophilic parts to facilitate the formation
of nanostructures and enable gelator–solvent interactions.
When the gelator is too solvophobic, the gel may crystallize or
precipitate.[28] In our case, FF is easily dissolved in HFIP but it
is insoluble in toluene. Thus, after toluene had been added to
the FF/HFIP solution, a transparent gel was formed imme-
diately. In the temperature range from 77 K to room temper-
This strategy can be generalized to achieve gel–crystal
transitions in other FF-based organogels. These findings will
be useful to gain a deeper understanding of the relationship
between gelation and crystallization in self-assembled sys-
tems and to achieve the controlled assembly and versatile
functionalization of single-component materials.

[Assistant] {"peptide_sequence": "FF", "self-assembly_phase": "organogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "4/96 HFP/Toluene", "peptide_concentration(mg/ml)": "5.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}


[System] polypeptide self-assembly literature information extraction

[User]
DOI: 10.3390/gels3030029
PMCID: PMC6318665
PMID: 30920525

Merck (Milan, Italy). Drugs and chemicals were from Sigma (Milan, Italy). High purity Milli-Q-water
(MQ water) with a resistivity greater than 18 MΩ·cm was obtained from an in-line Millipore
RiOs/Origin system (Merck, Milan, Italy). The 1H-NMR spectra were recorded at 400 MHz, and the
13C-NMR spectra were recorded at 100 MHz on a Varian Innova Instrument (Agilent Technologies,
Milan, Italy) with chemical shift reported as ppm (in dimethyl sulfoxide (DMSO) with tetramethylsilane
as internal standard). ESI-MS spectra were recorded on an Inﬁnity 6120 single quadrupole LC-MS
system (Agilent Technologies, Milan, Italy).
The peptide was synthesised using standard Fmoc solid phase peptide synthesis with HBTU
activation. Brieﬂy, Fmoc-amino acid deprotection was performed in a sintered funnel, with continuous
stirring, in 20% piperidine in N,N-dimethylformamide (DMF) for 20 min (2 × 10 min) until both
bromophenol blue and acetaldehyde/chloranil tests were positive. HBTU activation was performed
with 2.5 equiv. of Fmoc-amino acid, 2.0 equiv. of HBTU, and 2.0 equiv. of HOAt in DMF (4 mL
for every equiv. of resin), with N,N-diisopropyl ethyl amine (DIPEA, 2 mL of a 1 M solution
in DMF for every equiv. of resin). Coupling was performed at room temperature for 1.5 h in a
sintered funnel with continuous stirring, and completeness was monitored by both bromophenol
blue and acetaldehyde/chloranil tests after thorough washes with DMF and DCM (Dichloromethane).
The ﬁnal cleavage was obtained using a mixture of TFA/DCM/TIPS/water (47.5:47.5:2.5:2.5) (TFA:
triﬂuoroacetic acid; TIPS: triisopropyl silane). The crude peptide was too hydrophobic to be
precipitated in cold ether, thus the majority of TFA was evaporated under argon ﬂow, and the
remaining oil was dissolved in a mixture of acetonitrile/water and then puriﬁed by reverse-phase
HPLC (Agilent Technologies, Santa Clara, CA, USA). The HPLC Agilent 1260 Inﬁnity system was
equipped with a preparative gradient pump (1311B), semipreparative C-18 column (Kinetex, 5 microns,
100 Å, 250 mm × 10 mm, Phenomenex, Torrance, CA, USA), autosampler (G1329B), and Photodiode
Array detector (G1315C). The gradient used consisted of acetonitrile (MeCN)/water with 0.1% TFA
with the following program: t = 0–2 min, 25% MeCN; t = 12 min, 50% MeCN; t = 15 min, 95% MeCN;
t = 16 min, 95% MeCN (Retention time tR = 6.8 min). The compound was then freeze-dried to yield the
corresponding peptide as a white ﬂuffy powder. Peptide identity was veriﬁed by ESI-MS, 1H-NMR,
and 13C-NMR [39].
The peptide was dissolved in sodium phosphate buffer (0.1 M pH 11.8), and then diluted with
an equal volume of sodium phosphate at acidic pH (0.1 M pH 5.8) to a ﬁnal peptide concentration
of 10 mM, obtaining a ﬁnal pH of 7.2 ± 0.1. All buffer solutions were ﬁltered (0.2 µm) prior to use.
In the case of the naproxen-containing hydrogels, in a typical protocol naproxene was dissolved
with 200 µL of 1 M NaOH and 150 µL sodium phosphate buffer (0.1 M pH 11.8), then the peptide
DLeu-Phe-Phe was added to achieve a ﬁnal concentration of 10 mM of peptide, with another 150 µL of
sodium phosphate buffer (0.1 M pH 5.8) to obtain a ﬁnal pH of 7.1 ± 0.1. All buffer solutions were
ﬁltered (0.2 µm) prior to use. The ﬁnal concentration of naproxene was 100 mg/mL. In the case of
the ketoprofen-containing hydrogels, ketoprofen was dissolved with 50 µL of 1 M NaOH and 200 µL
of sodium phosphate buffer (0.1 M pH 11.8), then the peptide DLeu-Phe-Phe was added to a ﬁnal
concentration of 10 mM of peptide, with another 250 µL of sodium phosphate buffer (0.1 M pH 5.8)
to obtain a ﬁnal pH of 7.1 ± 0.1. All buffer solutions were ﬁltered (0.2 µm) prior to use. The ﬁnal
concentration of ketoprofen was 25 mg/mL.
Dynamic time sweep rheological analysis was conducted on a Malvern Kinexus Ultra Plus
Rheometer (Alfatest, Milan, Italy) with a 20 mm stainless steel parallel plate geometry. The temperature
was maintained at 25 ◦C using a Peltier temperature controller (Alfatest, Milan, Italy). Samples were
prepared in situ and immediately analysed with a gap of 1.00 mm. Time sweeps were recorded for
1 h, using a frequency of 1.00 Hz and a controlled stress of 2.00 Pa. After 1 h, frequency sweeps were
recorded using a controlled stress of 2.00 Pa, and then stress sweeps were recorded using a frequency
TEM micrographs were acquired on a Jeol, JEM 2100 (Tokyo, Japan) at 100 kV. TEM grids
(copper-grid-supported lacey carbon ﬁlm) were ﬁrst exposed to UV-ozone cleaner (UV-Ozone
Procleaner Plus, BioForce Nanosciences, Ames, IA, USA) for 45 min to make the grid surface more
hydrophilic. Then, 24 h-aged gels were precisely deposited on a TEM grid, dried for 15 min at room
temperature and contrasted by aqueous tungsten phosphate solution (pH 7.4). The average size of the
nanostructures was determined by taking into account at least 100 measurements.
A 0.1 mm quartz cell was used on a Jasco J-815 Spectropolarimeter (Jasco Europe, Cremella, Italy),
with 1 s integrations, one accumulation, and a step size of 1 nm with a bandwidth of 1 nm over a range
of wavelengths from 190 to 280 nm at 25 ◦C unless otherwise stated. Samples were freshly prepared
directly in the CD cell and the spectra immediately recorded.
FT-IR spectra were registered using the KBr-pellet method. The gel was prepared, and after 24 h
it was dried under vacuum. Then, it was mixed with KBr to make the pellet. Spectra were acquired at
1 cm−1 resolution and 128 accumulations on a Perkin Elmer System 2000 (Perkin Elmer, Milan, Italy).
Gels (150 µL) were prepared directly inside the wells of Greiner 96 U Bottom Black Polystyrene
plates (VWR, Milan, Italy). After 1 h, 30 µL of a solution of Thioﬂavin T (22.2 M in 20 mM glycine-NaOH
pH 7.5, ﬁltered with a 0.2 µm ﬁlter) were added into the wells. After 15 min, the ﬂuorescence emission
was analysed using an Inﬁnite M1000 pro (Tecan, Milan, Italy), selecting an excitation wavelength
of 446 nm and an emission wavelength of 490 nm, with a bandwidth of 20 nm. Each condition
was repeated twice in triplicate. The average and standard deviations were calculated and plotted
with Excel.
Hydrogels were prepared as described above (0.500 mL total volume) in an amber glass vial,
and were left to self-assemble overnight. The following morning, 8 mL of phosphate-buffered saline
(PBS) were added on top, ﬁlling the vials. The vials were incubated at 37 ◦C, 60 rpm, and at various
timepoints. Fifty microliter (50 µL) samples were taken and added to 0.200 mL of water and analysed by
reverse-phase HPLC (absorbance at 254 nm, same program as described above for peptide puriﬁcation).
Each experiment was repeated three times. The average and standard deviation values were calculated
and plotted with Excel.
Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/3/3/29/s1.
Figure S1: Amide region of FT-IR spectra of drugs alone (a,b) and included in the peptide hydrogels (c,d).

[Assistant] [{"peptide_sequence": "lFF", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "4.26", "PH": "7.1", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "lFF", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "4.26", "PH": "7.3", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "lFF", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "4.26", "PH": "7.2", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}]