Short self-assembling peptides with a urea bond: A new type of supramolecular peptide hydrogel materials

Article abstract of DOI:10.1002/pep2.24214

There is an increasing need to develop short self-assembling peptides (SAPs) that can form hydrogels for cell engineering and biomedical applications. In this study, we proposed new short self-assembling peptides with a symmetric structure via a urea bond

Full text of DOI:10.1002/pep2.24214

Received: 1 July 2020
Revised: 12 October 2020
Accepted: 2 November 2020
DOI: 10.1002/pep2.24214
F U L L P A P E R
Short self-assembling peptides with a urea bond: A new type
of supramolecular peptide hydrogel materials
Hiroshi Tsutsumi
| Kunifumi Tanaka | Jyh Yea Chia | Hisakazu Mihara
School of Life Science and Technology, Tokyo
Institute of Technology, Yokohama, Kanagawa,
Japan
Abstract
There is an increasing need to develop short self-assembling peptides (SAPs) that can
form hydrogels for cell engineering and biomedical applications. In this study, we pro-
posed new short self-assembling peptides with a symmetric structure via a urea
bond. (FFiO)2 and (FFiK)2 were designed as amphiphilic peptides with a hydrophobic
domain inside of zwitterionic hydrophilic ends and a urea bond embedded in the
hydrophobic domain. The two peptides were synthesized by a liquid-phase method.
The simple structural design of these peptides makes their synthesis on a large scale
possible. Both (FFiO)2 and (FFiK)2 assembled into β-sheet structure and formed sta-
ble hydrogels under physiological pH conditions in a pH-responsive manner. The urea
bond was important for the formation of transparent hydrogels. Since (FFiK)2
exhibited self-assembling properties superior to those of (FFiO)2, (FFiK)2 hydrogels
were used as scaffolds for cell culture. (FFiK)2 hydrogels supported cell proliferation
without significant cytotoxicity. Therefore, (FFiK)2 is a beneficial supramolecular pep-
tide hydrogelator for cell engineering.
Correspondence
Hiroshi Tsutsumi and Hisakazu Mihara, School
of Life Science and Technology, Tokyo
Institute of Technology, 4259 Nagatsuta-cho,
Midori-ku, Yokohama, Kanagawa 226-8501,
Japan.
Email: htsutsum@bio.titech.ac.jp (H. T.) and
Email: hmihara@bio.titech.ac.jp (H. M.)
Funding information
JSPS KAKENHI, MEXT, Japan
K E Y W O R D S
amphiphilic peptide, cell culture, hydrogel, self-assembling peptide, urea bond
1
|
INTRODUCTION
Recently, simple amino acid derivatives and dipeptides have been
recognized as supramolecular hydrogelators.[10–12] The first such
The self-assembly of peptides has received much attention over the
last two decades, because precisely designed peptides can assemble
into various nanostructures such as fibers, sheets and spherical
micelles. In particular, peptide nanofibers that can form hydrogels
have been extensively developed for tissue engineering and biomedi-
cal applications.[1] There are many design approaches for self-
assembling peptides (SAPs) based on amphiphilic β-sheet or α-helical
structures and a peptide amphiphile.[2,3] The first SAP to be designed
was EAK16 (Ac-AEAEAKAKAEAEAKAK-NH2), an amphiphilic 16-mer
β-sheet peptide.[4] The structure-assembling property relationships of
amphiphilic peptides designed based on the EAK16 peptide have been
investigated,[5–7] and shorter SAPs, such as Ac-(FKFE)2-NH2, were dis-
covered.[8,9] Compared to longer peptides, short peptides have advan-
tages in that their synthesis requires fewer steps and their purification
is easier.
example is dibenzoyl-L-cystine, which can form a self-supporting
hydrogel at a 0.1 wt% concentration.[13] After it was reported that a
diphenylalanine (FF) peptide self-assembled into nanotubes in
aqueous medium,[14] FF derivatives with an aromatic cap, such as
Fmoc,[15] at their N-terminus were reported as small molecular
SAPs.[12] In addition, non-natural phenylalanine derivatives, such as
4-nitrophenylalanine, are also used in low-molecular-weight hydroge-
lators.[16] Small molecular SAPs are easier to synthesize than tradi-
tional amphiphilic SAPs. However, special care is required for the
combination of aromatic amino acids with aromatic or hydrophobic
cap groups to obtain transparent hydrogels,[17,18] because strong π-π
stacking interactions often cause SAPs to crystalize.
In this study, we designed new short self-assembling peptides,
(FFiX)2, with a symmetric structure via a urea bond (Figure 1A). Due
to their bolaamphiphilic form, (FFiX)2 was easy to synthesize on a
Pept Sci. 2020;e24214.
wileyonlinelibrary.com/peptidesci
© 2020 Wiley Periodicals LLC
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https://doi.org/10.1002/pep2.24214

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TSUTSUMI ET AL.
FIGURE 1 A, Molecular design of (FFiO)2, (FFiK)2 and Ac-FFiK. B, Schematic illustration of self-assembly of (FFiX)2
large scale, and a hydrophobic/aromatic domain placed inside the
hydrophilic ends suppressed the crystal packing of the peptides. In
addition, the urea bond embedded in the hydrophobic domain is
expected to form a strong hydrogen bond network between peptides
and stabilize the self-assembled structure (Figure 1B). (FFiX)2 peptides
were synthesized by a liquid-phase method, and their self-assembling
properties including hydrogelation were investigated. In addition, pep-
tide hydrogels were applied to cell culture materials.
washed with 1 M HClaq (100 mL × 2), sat NaHCO3aq (100 mL × 1)
and sat NaClaq (100 mL × 1). The organic phase was dried over
MgSO4 and concentrated under reduced pressure. After drying in
vacuo, compound
1 was obtained as a white solid (14.15 g,
26.4 mmol, yield 88%).
1H NMR (400 MHz, CDCl3): δ 7.38-7.32 (6H, m), 7.30-7.25
(4H, m), 7.20-7.14 (6H, m), 6.99-6.96 (4H, m), 5.15-5.04 (4H, m), 4.97
(2H, d, J = 7.6 Hz), 4.83-4.78 (2H, m), 3.07-2.99 (4H, m).
2
|
EXPERIMENTAL SECTION
General
2.2.2
|
Compound 2
2.1
|
Compound 2 was synthesized according to the previous report with
modification.[19] Compound 1 (10.73 g, 20 mmol) was dissolved in
150 mL methanol and 150 mL ethyl acetate with heating at 50 ꢀC.
After cooling to room temperature, to this solution was added cata-
lytic amount of 10% palladium on carbon, and the reaction mixture
was stirred at room temperature under H2 atmosphere. After stirring
for 6 hours, the reaction mixture was filtrated on Celite, and the fil-
trate was concentrated under reduced pressure. The residue was
solidified with excess diethyl ether to give compound 2 as a white
solid (7.06 g, 19.8 mmol, yield 99%).
All chemicals and solvents were of reagent or HPLC grade and were
used without further purification. Amino acid derivatives were pur-
chased from Watanabe Chemical Industries, Ltd (Japan). Other chemi-
cal reagents and solvents were purchased from Fujifilm Wako Pure
Chemical (Japan) and Tokyo Chemical Industry (Japan). NMR mea-
surement was conducted using Agilent UNITY-INOVA-400. MALDI-
TOF MS measurement was conducted using MALDI-8020 (Shimadzu).
1H NMR (400 MHz, DMSO-d6): δ 6.87-6.77 (10H, m), 4.11 (2H, t,
2.2
|
Synthesis of self-assembling peptides
J = 5.6 Hz), 2.74-2.69 (2H, m), 2.59-2.54 (2H, m).
2.2.1
|
Compound 1
2.2.3
|
Compound 3
Compound 1 was synthesized according to the previous report with
modification.[19] To a suspended solution of H-Phe-OBzl hydrochlo-
ride salt (19.26 g, 66 mmol) in 100 mL dry dichloromethane was
added N,N-diisopropylethylamine (DIPEA) (11.5 mL, 66 mmol) and
carbonyldiimidazole (4.86 g, 30 mmol) at ice-water bath. After stirring
at room temperature for 18 hours, the reaction mixture was concen-
trated under reduced pressure. The residue was diluted with 450 mL
ethyl acetate/tetrahydrofuran (2/1) and the organic phase was
Compound 3 was synthesized according to the previous report with
modification.[20] To a solution of Boc-Orn(Z)-OH (9.16 g, 25 mmol) in
75 mL dry dichloromethane was added 4-dimethylaminopyridine
(153 mg, 1.25 mmol), tert-butanol (50 mL, 527 mmol) and Boc2O
(8.6 mL, 37.5 mmol) at ice-water bath. After stirring at ice-water bath
for 30 minutes and at room temperature for 4.0 hours, the reaction
mixture was concentrated under reduced pressure. The residue was

TSUTSUMI ET AL.
3 of 13
purified by silica gel column chromatography (hexane/ethyl ace-
tate = 7/3) to give compound 3a as a colorless clear oil (7.24 g,
17.1 mmol, yield 68%).
washed with 10% aqueous citric acid (150 mL × 2), sat NaHCO3aq
(150 mL × 2) and sat NaClaq (150 mL × 1). The organic phase was
dried over MgSO4 and concentrated under reduced pressure. After
drying in vacuo, compound 5a was obtained as a white amorphous
solid (6.38 g, 11.2 mmol, yield 95%).
1H NMR (400 MHz, CDCl3): δ 7.38-7.18 (10H, m), 5.96 (1H, bs),
5.40 (1H, bs), 5.12-5.05 (3H, m), 4.38-4.34 (1H, m), 4.18-4.09 (1H, m),
3.27-3.10 (3H, m), 3.03-2.98 (1H, m), 1.73-1.55 (2H, m), 1.59-1.51
(1H, m), 1.50-1.47 (2H, m), 1.46 (9H, s), 1.44 (9H, s).
1H NMR (400 MHz, CDCl3): δ 7.38-7.29 (5H, m), 5.09 (2H, s),
5.06 (1H, bs), 4.88 (1H, bs), 4.18-4.15 (1H, m), 3.22 (2H, q, J = 6.4 Hz),
1.85-1.78 (1H, m), 1.67-1.49 (3H, m), 1.46 (9H, s), 1.44 (9H, s).
To a solution of Boc-Lys(Z)-OH (11.4 g, 30 mmol) in 90 mL dry
dichloromethane was added 4-dimethylaminopyridine (184 mg,
1.5 mmol), tert-butanol (60 mL, 632 mmol) and Boc2O (10.3 mL,
45 mmol) at ice-water bath. After stirring at ice-water bath for
30 minutes and at room temperature for 4.0 hours, the reaction mix-
ture was concentrated under reduced pressure. The residue was puri-
fied by silica gel column chromatography (hexane/ethyl acetate = 7/3)
to give compound 3b as a colorless clear oil (9.17 g, 21 mmol,
yield 70%).
To a solution of compound 4b (4.24 g, 14 mmol), Z-Phe-OH
(3.72 g, 12.4 mmol), and HOBt H2O (2.15 g, 14 mmol) in 15 mL dry
dichloromethane was added DIPEA (2.44 mL, 14 mmol) and EDCI HCl
(2.67 g, 14 mmol) at ice-water bath. After stirring at ice-water bath
for 1.0 hour and room temperature for 10 hours, the reaction mixture
was concentrated under reduced pressure. The residue was diluted
with 300 mL ethyl acetate and the organic phase was washed with
10% aqueous citric acid (150 mL × 2), sat NaHCO3aq (150 mL × 2)
and sat NaClaq (150 mL × 1). The organic phase was dried over
MgSO4 and concentrated under reduced pressure. After drying in
1H NMR (400 MHz, CDCl3): δ 7.36-7.29 (5H, m), 5.09 (2H, s),
5.05 (1H, d, J = 6.4 Hz), 4.81 (1H, bs), 4.18-4.13 (1H, m), 3.19 (2H, q,
J = 6.4 Hz), 1.83-1.72 (1H, m), 1.65-1.49 (3H, m), 1.46 (9H, s), 1.43
(9H, s), 1.39-1.25 (2H, m).
vacuo, compound 5b was obtained as
a white solid (8.11 g,
13.9 mmol, yield 99%).
2.2.4
|
Compound 4
1H NMR (400 MHz, CDCl3): 7.38-7.18 (10H, m), 5.68 (1H, bs),
5.40 (1H, bs), 5.09 (2H, s), 5.06 (1H, bs), 4.35-4.30 (1H, m), 4.13-4.06
(1H, m), 3.14-3.10 (3H, m), 3.03-2.98 (1H, m), 1.74-1.66 (1H, m),
1.59-1.50 (1H, m), 1.46 (9H, s), 1.44 (9H, s), 1.38-1.30 (2H, m),
1.28-1.17 (2H, m).
Compound 4 was synthesized according to the previous report with
modification.[21] To a solution of compound 3a (5.5 g, 13 mmol) in
30 mL MeOH was added catalytic amount of 10% palladium on car-
bon, and the mixture was stirred at room temperature under H2 atmo-
sphere. After stirring for 7.0 hours, the reaction mixture was filtrated
on Celite, and the filtrate was concentrated under reduced pressure.
After dry in vacuo, compound 4a was obtained as a white amorphous
solid (3.4 g, 11.8 mmol, yield 91%). Compound 4a was directly used in
the next step.
2.2.6
|
Compound 6
To a solution of compound 5a (1.70 g, 3.0 mmol) in 15 mL methanol
was added catalytic amount of 10% palladium on carbon, and the mix-
ture was stirred at room temperature under N2 atmosphere. After stir-
ring for 5.0 hours, the reaction mixture was filtrated on Celite, and the
filtrate was concentrated under reduced pressure. After drying in
vacuo, compound 6a was obtained as a white amorphous solid
(1.28 g, 2.95 mmol, yield 98%). Compound 6a was directly used in the
next step.
To a solution of compound 3b (6.3 g, 14.4 mmol) in 30 mL MeOH
was added catalytic amount of 10% palladium on carbon, and the mix-
ture was stirred at room temperature under H2 atmosphere. After stir-
ring for 7.0 hours, the reaction mixture was filtrated on Celite, and the
filtrate was concentrated under reduced pressure. After dry in vacuo,
compound 4b was obtained as a white amorphous solid (4.24 g,
14 mmol, yield 97%). Compound 4b was directly used in the
next step.
To a solution of compound 5b (1.75 g, 3.0 mmol) in 15 mL metha-
nol was added catalytic amount of 10% palladium on carbon, and the
mixture was stirred at room temperature under N2 atmosphere. After
stirring for 5.0 hours, the reaction mixture was filtrated on Celite, and
the filtrate was concentrated under reduced pressure. After drying in
vacuo, compound 6b was obtained as a white amorphous solid
(1.34 g, 2.99 mmol, yield 99%). Compound 6b was directly used in the
next step.
2.2.5
|
Compound 5
Compound 5 was synthesized according to the previous report with
modification.[21] To a solution of compound 4a (3.4 g, 11.8 mmol),
Z-Phe-OH (3.21 g, 10.7 mmol), and HOBt H2O (1.81 mg, 11.8 mmol)
in 10 mL dry dichloromethane was added DIPEA (2.05 mL, 11.8 mmol)
and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride
(EDCI HCl) (2.26 g, 11.8 mmol) at ice-water bath. After stirring at ice-
water bath for 1.0 hour and room temperature for 10 hours, the reac-
tion mixture was concentrated under reduced pressure. The residue
was diluted with 300 mL ethyl acetate and the organic phase was
2.2.7
|
Compound 7
To a solution of compound 6a (1.28 g, 2.95 mmol) in 5 mL dry DMF
was added compound (476 mg, 1.34 mmol) and OxymaPure
(417 mg, 2.95 mmol) at room temperature. To this mixture was added
2

4 of 13
TSUTSUMI ET AL.
DIPEA (511 μL, 2.95 mmol) and EDCI HCl (563 mg, 2.95 mmol) at ice-
water bath. After stirring at ice-water bath for 1.0 hour and at room
temperature for 23 hours, the reaction mixture was concentrated
under reduced pressure. The residue was diluted with 400 mL ethyl
acetate/tetrahydrofuran (4/1) and the organic phase was washed with
10% aqueous citric acid (120 mL × 2), sat NaHCO3aq (120 mL × 2)
and sat NaClaq (100 mL × 1). The organic phase was dried over
MgSO4 and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (chloroform/
methanol = from 30/1 to 25/1 [v/v]). After drying in vacuo, com-
1H NMR (400 MHz, DMSO-d6): 8.24-7.86 (8H, m), 7.24-7.10
(20H, m), 6.40 (2H, d, J = 7.2 Hz), 4.42-4.36 (2H, m), 4.26-4.21
(2H, m), 3.81-3.78 (2H, m), 3.11-2.89 (6H, m), 2.84-2.78 (4H, m),
2.67-2.62 (2H, m), 1.77-1.66 (4H, m), 1.57-1.39 (4H, m).
MALDI-TOF MS: m/z obs. 879.21 and 901.22, calcd. 879.44
[M + H]+ and 901.42 [M + Na]+.
Compound 7b (1.22 g, 1.0 mmol) was dissolved in 10 mL
trifluoroacetic acid and the mixture was stirred at room temperature.
After stirring for 1.5 hours, the mixture was concentrated under
reduced pressure. To the residue was added 50 mL diethyl ether, and
the precipitate was collected by centrifugation (3000 rpm, for
10 minutes). The solid was suspended in 50 mL diethyl ether with
sonication and collected by centrifugation (3000 rpm, for 10 minutes).
After dry in vacuo, compound 8b was obtained as a white solid
(1.08 g, 0.95 mmol, yield 95%).
1H NMR (400 MHz, DMSO-d6): 8.19-7.76 (8H, m), 7.23-7.09
(20H, m), 6.48 (2H, d, J = 7.2 Hz), 4.42-4.33 (2H, m), 4.22-4.18
(2H, m), 3.61 (2H, bs), 3.01-2.94 (6H, m), 2.98-2.80 (4H, m), 2.71-2.65
(2H, m), 1.71 (4H, bs), 1.32 (8H, bs).
pound 7a was obtained as
a white solid (1.03 g, 0.87 mmol,
yield 65%).
1H NMR (400 MHz, DMSO-d6): 8.05 (2H, d, J = 8.4 Hz), 7.82 (2H,
t, J = 5.6 Hz), 7.23-7.06 (22H, m), 6.31 (2H, d, J = 7.2 Hz), 4.45-4.39
(2H, m), 4.31-4.26 (2H, m), 3.76-3.71 (2H, m), 3.07-3.00 (2H, m),
2.98-2.77 (8H, m), 2.70-2.65 (2H, m), 1.59-1.39 (8H, m), 1.38
(36H, bs).
MALDI-TOF MS: m/z obs. 1213.90, calcd. 1213.65 [M + Na]+.
To a solution of compound 6b (1.34 g, 2.99 mmol) in 5 mL dry
DMF was added compound 2 (485 mg, 1.36 mmol) and OxymaPure
(424 mg, 2.99 mmol) at room temperature. To this mixture was added
DIPEA (520 μL, 2.99 mmol) and EDCI HCl (572 mg, 2.99 mmol) at ice-
water bath. After stirring at ice-water bath for 1.0 hour and at room
temperature for 23 hours, the reaction mixture was concentrated
under reduced pressure. The residue was diluted with 400 mL ethyl
acetate/tetrahydrofuran (4/1) and the organic phase was washed with
10% aqueous citric acid (120 mL × 2), sat NaHCO3aq (120 mL × 2)
and sat NaClaq (100 mL × 1). The organic phase was dried over
MgSO4 and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (chloroform/
methanol = from 30/1 to 25/1 [v/v]). After drying in vacuo, com-
MALDI-TOF MS: m/z obs. 907.18 and 929.18, calcd. 907.47
[M + H]+ and 929.45 [M + Na]+.
2.3
|
Circular dichroism spectroscopy
(FFiX)2 peptides (TFA salt) were dissolved in 100 μL of an aqueous
40 mM NaOH solution at a concentration of 10 mM. The peptide
solution (100 μL) was mixed with 100 μL of 20 mM aqueous HCl solu-
tion for neutralization, and the neutralized peptide solution (5.0 mM)
was quickly diluted with ultrapure water and 20 mM Tris-HCl buffer
(pH 7.2) to prepare peptide solutions at 100 μM, 200 μM, 300 μM,
500 μM and 1000 μM in 10 mM Tris-HCl buffer (pH 7.2). The diluted
solutions were incubated at 25 ꢀC for 24 hours. Circular dichroism
(CD) spectra were recorded on a JASCO J-1100 spectropolarimeter
using a quartz cell with a 0.1 cm path length at 25 ꢀC.
pound 7b was obtained as
a white solid (1.14 g, 0.94 mmol,
yield 69%).
1H NMR (400 MHz, DMSO-d6): 8.05 (2H, d, J = 8.4 Hz), 7.80 (2H,
t, J = 5.6 Hz), 7.24-7.10 (20H, m), 7.05 (2H, d, J = 8.0 Hz), 6.33 (2H, d,
J = 7.2 Hz), 4.44-4.38 (2H, m), 4.31-4.26 (2H, m), 3.73-3.68 (2H,m),
3.09-3.01 (2H, m), 2.94-2.78 (8H, m), 2.71-2.66 (2H, m), 1.59-1.47
(4H, m), 1.38 (18H, s), 1.37 (18H, s), 1.29-1.11 (8H, m).
Neutralized peptide solution (5.0 mM) was prepared as men-
tioned above and quickly diluted with ultrapure water and 20 mM
Tris-HCl solution (pH 4.2, 7.2 or 10.2) to prepare 300 μM peptide
solution in 10 mM Tris-HCl (pH 4.2, 7.2 or 10.2). The diluted solutions
were incubated at 25 ꢀC for 24 hours.
MALDI-TOF MS: m/z obs. 1241.72, calcd. 1241.68 [M + Na]+.
2.2.8
|
Compound 8
2.4
|
Transmission electron microscopy
Compound 7a (1.19 g, 1.0 mmol) was dissolved in 10 mL
trifluoroacetic acid and the mixture was stirred at room temperature.
After stirring for 1.5 hours, the mixture was concentrated under
reduced pressure. To the residue was added 50 mL diethyl ether, and
the precipitate was collected by centrifugation (3000 rpm, for
10 minutes). The solid was suspended in 50 mL diethyl ether with
sonication and collected by centrifugation (3000 rpm, for 10 minutes).
After dry in vacuo, compound 8a was obtained as a white solid
(1.02 g, 0.92 mmol, yield 92%).
A (FFiO)2 solution at a concentration of 300 μM was prepared in
10 mM Tris-HCl buffer (pH 7.2). (FFiK)2 solutions at a concentration of
300 μM were prepared in 10 mM Tris-HCl solutions (pH 4.2, 7.2 and
10.2). These solutions were incubated at 25 ꢀC for 24 hours. A
collodion-coated copper EM grid was glow discharged for 40 seconds
at 3 mA using an IB2 ion coater. A 10 μL drop of the peptide solution
was applied to the freshly glow discharged grid for 1 minute to adsorb
peptide nanofibers. The excess solution was removed by capillary action

TSUTSUMI ET AL.
5 of 13
with filter paper, and the grid was then washed twice with 10 μL of
ultrapure water. After removal of the ultrapure water, the grid was sta-
ined for 2 minutes with EM Stainer (Nisshin EM) that was diluted
4-fold. The stained grid was blotted with filter paper and allowed to dry
at room temperature. Transmission electron microscopy (TEM) images
were taken with a JEM 1400Plus (JEOL) instrument operated at 80 kV.
parallel plate geometry. Hydrogels (1.0 wt%) of (FFiO)2 and (FFiK)2
were prepared in a tip-cut plastic syringe (20 mL) with 24 hours incu-
bation time. Then, hydrogel samples were transferred to the rheome-
ter stage (20 mm) by slowly extruding. A dynamic frequency sweep
was immediately performed over a range of frequencies from 0.1 to
100 rad s−1 at 0.1% constant strain at 25 ꢀC.
2.5
|
Hydrogelation test
2.7
|
Cell culture
(FFiO)2 (20 mg, 18.0 μmol) or (FFiK)2 (20 mg, 17.6 μmol) was dissolved in
1.0 mL of a NaOH solution (4 equiv.) to prepare a 2.0 wt% peptide solu-
tion. The peptide solution (1.0 mL) was neutralized with 1.0 mL of an HCl
solution (2 equiv.) to prepare a 1.0 wt% peptide solution. The neutralized
peptide solution (1.0 wt%, 0.5 mL) was quickly mixed with 0.5 mL of
20 mM Tris-HCl buffer (pH 7.2) to prepare a 0.5 wt% peptide solution in
10 mM Tris-HCl buffer (pH 7.2). The 0.5 wt% peptide solution was
quickly diluted with 10 mM Tris-HCl buffer (pH 7.2) to prepare peptide
solutions at concentrations of 0.2 wt%, 0.1 wt% and 0.05 wt%. All pep-
tide solutions were incubated at 25 ꢀC for 24 hours with a stir bar.
(FFiO)2 (8 mg, 7.2 μmol) or (FFiK)2 (8 mg, 7.04 μmol) was dis-
solved in 1.0 mL of a NaOH solution (4 equiv.) to prepare a 0.8 wt%
peptide solution. The peptide solution (1.0 mL) was neutralized with
1.0 mL of an HCl solution (2 equiv.) to prepare 0.4 wt% peptide solu-
tion. The neutralized peptide solution (0.4 wt%, 100 μL) was quickly
mixed with 100 μL of sodium citrate/phosphate/borate buffer (each
20 mM, pH = 3.2, 4.2, 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 11.2) or 100 μL of
Dulbecco's modified Eagle medium (DMEM) containing phenol red to
prepare peptide solutions at concentration of 0.2 wt%. All peptide
solutions were incubated at 25 ꢀC for 24 hours.
HepG2 cells were obtained from the RIKEN BRC. The cell line was
tested and authenticated by the RIKEN BRC. HepG2 cells were
maintained in DMEM supplemented with 10% fetal bovine serum
(FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. The cells
were cultured on Petri dishes and grown until reaching 80% conflu-
ence at 37 ꢀC under 5% CO2 condition.
2.8
|
Cell culture on (FFiK)2 hydrogels
An (FFiK)2 peptide solution (2.0 wt%) was prepared as described
above. The (FFiK)2 peptide solution (50 μL) was added to a 96-well
culture plate (EZ-BindShut-II, IWAKI), and mixed with DMEM
(50 μL). Then, the peptide solution was incubated for 15 minutes to
prepare a 1.0 wt% peptide hydrogel. DMEM (200 μL) was added on
the peptide hydrogels and incubated for 15 minutes. After DMEM
was removed, 1 × 103 HepG2 cells in DMEM (100 μL) supplemented
with FBS and antibiotics were added on the hydrogels and cultured
at 37 ꢀC under 5% CO2 for 5 days with the medium change every
2 days. HepG2 cells (1 × 103 cells) were cultured on tissue culture-
treated plate (TCTP) as a control. Live cells and dead cells were sta-
ined with calcein-AM (2.0 μM) and propidium iodide (PI, 4.0 μM),
(FFiO)2 (10 mg, 9.0 μmol) or (FFiK)2 (10 mg, 8.8 μmol) was dis-
solved in 0.5 mL of a NaOH solution (4 equiv.) to prepare a 2.0 wt%
peptide solution. The peptide solution (0.5 mL) was transferred to a
tip-cut plastic syringe (5 mL) and neutralized with 0.5 mL of 20 mM
Tris HCl buffer containing HCl (2 equiv.) to prepare a 1.0 wt% peptide
solution. After 5 minutes of incubation, hydrogels were transferred
onto a ruler for photography.
respectively, after 1,
3 and 5 days cultivation according to
manufacturer's instructions. The cells were observed with a fluores-
cence microscope (Ti-U-PH-1, Nikon). The fluorescence intensity of
live cells stained with calcein was measured with a microplate fluo-
rometer (ARVO MX, PerkinElmer). Measurement was conducted
for three different wells. Statistical significance was indicated
by P < .05.
Nile red (NR) and thioflavin
T
(ThT) were dissolved in
1.0 mM stock solution.
dimethylsulfoxide (DMSO) to prepare
a
(FFiK)2 (5.0 mg, 4.4 μmol) was dissolved in 0.25 mL of a NaOH solu-
tion (4 equiv.) to prepare a 2.0 wt% peptide solution. Stock solutions
of NR or ThT (1.0 μL) were added to the peptide solution (100 μL),
respectively. The fluorophore-loaded peptide solution (2.0 wt%,
40 μL) was injected into 1.5 mL of phosphate-buffered saline (PBS,
pH 7.2) in a quartz cell using a microsyringe (inner needle diameter of
0.15 mm) to fabricate hydrogel fibers. Hydrogel fibers were visualized
by irradiation with UV light (354 nm).
2.9
|
Cell culture on (FFiK)2 hydrogel fibers
An (FFiK)2 peptide solution (2.0 wt%) containing NR (10 μM) was pre-
pared as described above, and 40 μL of the peptide solution was
injected into 1.0 mL of DMEM in
a 24-well culture plate
(EZ-BindShut-II, IWAKI) using a microsyringe (inner needle diameter
of 0.15 mm) to fabricate hydrogel fibers. The hydrogel fibers were
incubated at 37 ꢀC for 5 minutes. After the DMEM was carefully
removed, 2.5 × 103 HepG2 cells in DMEM (100 μL) supplemented
with FBS and antibiotics were added to the well and cultured at 37 ꢀC
under 5% CO2 for 2 days. Live cells were stained with calcein-AM
(2.0 μM) and observed with a fluorescence microscope.
2.6
|
Rheology
Measurements were conducted on a TA Instruments Discovery
Hybrid Rheometer HR 30 operating in oscillatory mode, with a 20 mm

6 of 13
TSUTSUMI ET AL.
3
|
RESULTS AND DISCUSSION
synthesis in the liquid phase. Ac-FFiK, which does not have a urea
unit, was designed and used for comparison (Figure 1A). These pep-
tides were synthesized by liquid phase method according to the
scheme shown in Figure 2 and Figure S1. We have established a syn-
thetic method of (FFiX)2 peptides on a scale larger than that of the
representative solid-phase method. In the case of (FFiK)2, the peptide
could be synthesized on a scale of 20 mmol (approximately 22 g) scale
with high purity. This is one of the advantages of short symmetric
self-assembling peptides that can be synthesized in the liquid phase.
3.1
|
Molecular design and synthesis of short self-
assembling peptides
(FFiX)2 peptides were designed as short self-assembling peptides
composed of six amino acids (Figure 1A). A diphenylalanine (FF) unit
was used as a representative self-assembling motif,[14] and two FF
units were connected via a urea bond that can form strong hydrogen
bonds. A urea unit has been used for precise control of self-assembly
of macrocycle,[22] tunable organogel/hydrogel formation,[23,24] and
functionalized biomaterials.[25] However, there is very few applica-
tions of the urea unit to the design of self-assembling peptide com-
posed of natural amino acids. Two ornithine residues (n = 0, (FFiO)2)
or lysine residues (n = 1, (FFiK)2) were introduced at both ends via an
isopeptide bond to display zwitterion units, because an amino acid
framework can form intermolecular ion pairs.[26,27] Under physiologi-
cal aqueous conditions, (FFiX)2 peptides are expected to spontane-
ously assemble by a hydrophobic interaction between FF units, a
hydrogen bonding network formed due to urea bonds and electro-
static interactions at both ends (Figure 1B). In addition, the (FFiX)2
peptides were designed to have a symmetrical structure for their easy
3.2
|
Secondary structure analysis by CD
spectroscopy
CD spectra were measured to evaluate the secondary structure of
(FFiX)2 peptides in 10 mM Tris-HCl buffer (pH 7.2) (Figure 3). (FFiO)2
at 100 and 200 μM showed a positive maximum around 220 nm and
a negative maximum around 205 nm, indicating a random coil struc-
ture, which is characteristic of phenylalanine-based self-assembling
peptides (Figure 3A).[28] However, 300 μM (FFiO)2 showed a negative
maximum around 215 nm with a shoulder peak at 205 nm. This result
suggests that (FFiO)2 assembled into a β-sheet structure. The peak at
FIGURE 2 Synthesis scheme of (FFiO)2 and (FFiK)2

TSUTSUMI ET AL.
7 of 13
FIGURE 3 Circular dichroism spectra of, A, (FFiO)2 and B, (FFiK)2. The concentration of peptides was varied from 100 μM to 1000 μM at
25 ꢀC in 10 mM Tris-HCl buffer (pH 7.2). Circular dichroism spectra of, C, (FFiO)2 and D, (FFiK)2 at different pH condition. The concentration of
peptides was 300 μM at 25 ꢀC in 10 mM Tris-HCl solution (pH 4.2, 7.2 and 10.2)
215 nm is derived from the hydrogen bonding of amide bonds in
β-sheet structure, and the peak at 205 nm is derived from a π-π inter-
action between phenyl groups of side chains or distorted β-sheet
structure.[9,28] When the peptide concentration increased, the nega-
tive peak shifted from 214 nm to 216 nm (500 μM) and 218 nm
(1000 μM) with the peak intensity increasing. In addition, the shoulder
peak around 205 nm almost disappeared at 1000 μM (FFiO)2. These
results indicate that (FFiO)2 at high concentrations formed many
hydrogen bonds and that a hydrogen bonding network contributed to
the formation of a stable β-sheet structure.
increasing peptide concentration. In addition, at a concentration of
300 μM, (FFiK)2 exhibited a larger CD signal intensity than (FFiO)2,
indicating that (FFiK)2 formed a more stable β-sheet structure than
(FFiO)2. Although (FFiO)2 and (FFiK)2 differ by just one methylene
group, a subtle structural difference might affect the electrostatic
interaction network at the edge of both peptides. At higher concen-
trations of (FFiK)2, the negative peak shifted from 215 nm to 217 nm
(500 μM) and 219 nm (1000 μM), and the peak intensity increased
with the disappearance of the shoulder peak around 205 nm as
observed with (FFiO)2. These results suggest that (FFiK)2 also assem-
bled into a stable β-sheet structure in a concentration dependent
manner similar to (FFiO)2. In addition, IR spectra also supported that
both (FFiO)2 and (FFiK)2 could assemble into β-sheet structure,
because they showed peaks around 1630 cm−1 characteristic with a
β-sheet structure (Figure S2).[29]
In contrast, (FFiK)2 at 100 μM showed negative maxima around
215 nm and 205 nm (Figure 3B). This result suggests that (FFiK)2
could assemble into a β-sheet structure at a lower concentration than
(FFiO)2. When the peptide concentration increased, the CD signal
intensity decreased up to a (FFiK)2 concentration of 300 μM, and the
signal intensity at 215 nm became larger than that at 205 nm. This
result indicates that (FFiK)2 also formed many hydrogen bonds with
Notably, 100 μM of (FFiK)2 assembled into a β-sheet structure,
whereas a similar designed peptide, Ac-(KEFFFFKE)2-NH2, which has

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TSUTSUMI ET AL.
a hydrophobic domain and zwitterionic ends, adopted random coil
structure at 200 μM.[30] Although it is difficult to directly compare the
two peptides, the urea unit of (FFiK)2 might contribute to its superior
self-assembling ability.
intermolecular ion pairs at the edge of peptides contributes to stable
β-sheet formation under physiological pH conditions.
CD spectra of (FFiX)2 peptides were measured under different pH
conditions (pH 4.2, 7.2 and 10.2) to evaluate the contribution of elec-
trostatic interactions at the edge of peptides to self-assembly
(Figure 3C,D). The concentration of both peptides was fixed at 300 μM.
(FFiO)2 and (FFiK)2 showed a positive maximum around 220 nm and a
negative maximum around 195 nm at pH 10.2, indicating that neither
peptide self-assembled into β-sheet structures at pH 10.2. A similar CD
spectrum was reported in the trigonal-(FKFE)2 peptide at pH 11.[31]
Since (FFiO)2 and (FFiK)2 can theoretically have a net charge of −2 at
pH 10.2, electrostatic repulsion between the peptides disrupted the
self-assembly. On the other hand, (FFiO)2 and (FFiK)2 showed a nega-
tive maximum around 210 nm at pH 4.2. This result implies that both
peptides formed β-sheet-like aggregates under acidic condition.
Although (FFiO)2 and (FFiK)2 have a net charge of +2 at pH 4.2, a
hydrogen bond between COOH and NH3+ might contribute to pro-
mote self-assembly over electrostatic repulsion. Both (FFiO)2 and
(FFiK)2 showed a negative maximum around 215 nm at pH 7.2. These
results suggest that (FFiX)2 peptides assemble into a β-sheet structure
in response to pH change. The electrostatic interactions of
3.3
|
Nanostructure analysis by TEM observation
Self-assembled nanostructures of (FFiX)2 peptides were observed by
TEM. The concentration of both peptides in 10 mM Tris-HCl buffer
(pH 7.2) was fixed at 300 μM. Both (FFiO)2 and (FFiK)2 assembled into
nanofibers with an approximately 8 nm width and a more than
500 nm length at pH 7.2 (Figure 4A,B). Because the length of (FFiX)2
peptides in an extended conformation is approximately 3.4 nm, this
result indicates that β-sheets assembled from both peptides stacked
by hydrophobic interactions between phenylalanine residues to form
wider nanofibers. In particular, (FFiK)2 exhibited densely bundled and
networked nanofibers (Figure 4B). This result suggests that (FFiK)2
can form more fibers than (FFiO)2, which is consistent with the supe-
rior self-assembling property of (FFiK)2 compared to (FFiO)2 from the
CD study.
The effect of pH on the self-assembly of (FFiK)2 was investigated
by TEM. The peptide concentration was fixed at 300 μM, and the pep-
tide solutions were incubated at pH 4.2 (Figure 4C) or pH 10.2
(Figure 4D) in a 10 mM Tris solution. Under the acidic condition, (FFiK)2
FIGURE 4 TEM images of, A, (FFiO)2
at pH 7.2, B, (FFiK)2 at pH 7.2, C, (FFiK)2
at pH 4.2 and D, (FFiK)2 at pH 10.2. The
concentration of peptides was 300 μM.
Scale bar is 100 nm

TSUTSUMI ET AL.
9 of 13
was present as long nanofibers with an approximately 8 nm width;
however, no bundled nanofibers were observed. This result does not
contradict the CD results, which indicated that (FFiK)2 could form
β-sheet-like aggregate at pH 4.2. However, detail assembly mode of
(FFiK)2 at pH 4.2 might be different to that at pH 7.2, because the pep-
tide has a net charge of +2 at pH 4.2, and electrostatic repulsion
between peptides could disrupt well-packed β-sheet assembly. Since
(FFiK)2 possibly would form micelle-like nanofibers that did not have
well aligned surface at pH 4.2, nanofibers did not form bundled struc-
tures. In addition, nanofibers would have many positive charge at
pH 4.2, and electrostatic repulsion between fibers might inhibit
networked structures of nanofibers under acidic condition. On the
other hand, (FFiK)2 was present as short fibrils under basic condition.
Since (FFiK)2 showed a random coil structure at pH 10.2 in the CD
experiment, (FFiK)2 could not form a stable nanofiber structure. Based
on the above results, (FFiK)2 self-assembled into highly networked
nanofibrous structures in response to pH.
NaOH solution (4 equiv. to peptide) at 2.0 wt%, because self-
assembly is suppressed under basic condition. The peptide solution
was neutralized with the proper amount of an HCl solution and
quickly diluted with Tris-HCl buffer to prepare 0.5, 0.2, 0.1 and
0.05 wt% solutions. After 24 hours of incubation at 25 ꢀC with a stir
bar, hydrogelation was confirmed by an inverted method (Figure 5A).
(FFiO)2 at concentrations of 0.5, 0.2 and 0.1 wt% formed stable self-
supporting hydrogels. Although the (FFiO)2 hydrogel at 0.5 wt% was
slightly opaque, the 0.2 and 0.1 wt% (FFiO)2 hydrogel were transpar-
ent. At a concentration of 0.05 wt%, a partial gel was observed. In
contrast, (FFiK)2 at all concentration examined formed stable and
transparent self-supporting hydrogels. These results and the results of
CD studies and TEM observation suggest that both (FFiO)2 and
(FFiK)2 self-assembled into networked nanofibers through a β-sheet
structure and formed stable self-supporting hydrogels. In addition,
(FFiK)2 exhibited a lower critical hydrogelation concentration than
(FFiO)2 due to its superior self-assembling ability as demonstrated in
the CD experiment. Hydrogelation of the Ac-FFiK peptide was also
tested at concentrations of 0.2, 0.5, 1.0, 1.5 and 2.0 wt% (Figure S3).
Ac-FFiK did not form hydrogels when at a concentration below 0.5 wt
% and formed opaque hydrogels when at a concentration above
1.0 wt%. This result implies that the symmetric structure of (FFiX)2
with a urea unit is important for transparent hydrogel formation at
low concentrations.
3.4
|
Hydrogelation test of self-assembling
peptides
The hydrogelation properties of (FFiX)2 peptides were investigated in
10 mM Tris-HCl buffer (pH 7.2). First, the peptide was dissolved in a
FIGURE 5 Photographs of (FFiX)2 hydrogels. A, Concentration dependent hydrogelation of (FFiX)2 peptides in 10 mM Tris-HCl buffer
(pH 7.2) with stirring bar. The concentration of peptides was varied from 0.05 wt% to 0.5 wt%. B, pH dependent hydrogelation of (FFiX)2
peptides in sodium citrate/phosphate/borate buffer (each 10 mM, pH 3.2-11.2). DMEM was also tested for hydrogelation of (FFiX)2 peptides.
The concentration of peptides was 0.2 wt%. C, Bulk hydrogels of (FFiX)2 peptides. The concentration of peptides was 1.0 wt%. D, Hydrogel fibers
of (FFiK)2 fabricated by injection of 2.0 wt% (FFiK)2 solution into PBS using microsyringe. Hydrogel fibers were stained with Nile Red (10 μM) or
thioflavin T (10 μM) and visualized under UV light (354 nm) irradiation

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TSUTSUMI ET AL.
Next, pH-responsive hydrogelation of (FFiX)2 peptides was
tested. Peptide solutions at a 0.2 wt% concentration were prepared
using sodium citrate/phosphate/borate buffer (each 20 mM,
pH 3.2-11.2) and incubated at 25 ꢀC for 24 hours (Figure 5B). Both
(FFiO)2 and (FFiK)2 formed stable and transparent hydrogels in the pH
range from 5.2 to 9.2. Electrostatic interactions between peptides
contribute to stabilizing self-assembly, because both ionic peptide ter-
mini maintain a zwitterionic form in this pH range. Under acidic condi-
tions (pH 3.2 and 4.2), both peptides formed partial hydrogels,
indicating that not enough of the peptide nanofiber network was
formed to completely support the solvent. Although (FFiK)2 could
assemble into nanofibers at pH 4.2 as shown in Figure 4C, these
nanofibers did not form bundled or networked structures. On the
other hand, both (FFiO)2 and (FFiK)2 remained as a solution under
basic conditions (pH 10.2 and 11.2). Because (FFiK)2 could not form
long nanofibers at pH 10.2 as shown in Figure 4D, neither peptide
could form hydrogels. Based on these results, both (FFiO)2 and (FFiK)2
can form hydrogels in response to pH, and these peptide hydrogels
are stable under physiological pH conditions. In addition, both (FFiO)2
and (FFiK)2 formed stable and transparent hydrogels when cell culture
medium (DMEM) was used as a solvent (Figure 5B). This result indi-
cates that (FFiX)2 peptides can be used as cell culture materials.
Since solutions of (FFiO)2 and (FFiK)2 at a high concentration rap-
idly formed stable hydrogels in response to pH change, the hydrogels
were cylindrical in shape in the plastic syringe. Basic peptide solutions
(2.0 wt%) were neutralized by the proper amount of an HCl solution,
and incubation for (FFiK)2 for 1 minute and (FFiO)2 for 5 minutes gave
stable hydrogels (1.0 wt% for both peptides) (Figure 5C). These hydro-
gels maintained their structure for at least 1 month under high humid-
ity. In addition, the (FFiK)2 hydrogel was more transparent than the
(FFiO)2 hydrogel. Transparency of the (FFiK)2 hydrogel was quite
higher than that of the (FFiO)2 hydrogel in the range from 400 nm to
700 nm (Figure S4). The detail reason for this difference was not
determined; however, a subtle structural difference between (FFiK)2
and (FFiO)2 might affect the molecular packing in the self-assembled
structures.
FIGURE 6 Rheological analysis of (FFiO)2 and (FFiK)2 hydrogels.
Storage modulus (G0) and loss modulus (G00) measured with frequency
sweeps over 0.1-100 rad s−1, 0.1% constant strain, at 25 ꢀC
fluorescence of Nile red (NR) or thioflavin T under UV light irradiation,
and the fiber width was estimated to be approximately 0.3 mm. This
result indicates that the rapid self-assembly of (FFiK)2 in response to
pH change induced hydrogelation before diffusion of peptide. Nile
red, an environmentally sensitive probe, was used to visualize the
peptide hydrogel fibers, which showed strong fluorescence,
suggesting that Nile red bound to the hydrophobic domain in the
assembled (FFiK)2 nanostructures. In addition, thioflavin T, a fluores-
cent probe used to detect assembled β-sheet structures, also visual-
ized peptide hydrogel fibers with strong fluorescence, indicating that
the hydrogel was composed of a β-sheet assembly of (FFiK)2.
3.5
|
Cell culture on (FFiK)2 hydrogels
Rheological analysis was conducted to determine the storage
modulus (G0) and loss modulus (G00) for (FFiO)2 and (FFiK)2 hydrogels
(1.0 wt%) (Figure 6). The (FFiO)2 hydrogel showed higher G0 (8925
964 Pa) than G00 (1160 358 Pa). The (FFiK)2 hydrogel also showed
higher G0 (4489 866 Pa) than G00 (842 148 Pa). These results sug-
gest that 1.0 wt% solutions of (FFiO)2 and (FFiK)2 meet the technical
definition of a hydrogel.[32] Both (FFiO)2 and (FFiK)2 hydrogels
exhibited large G0 values more than 4000 Pa, while G0/G00 ratios of
(FFiO)2 and (FFiK)2 hydrogels were 7.69 and 5.33, respectively. This
result indicates that (FFiO)2 and (FFiK)2 hydrogels are soft gels to the
extent possible for biological applications.
Since (FFiK)2 exhibited hydrogelation properties superior to those of
(FFiO)2, (FFiK)2 hydrogels were applied as a cell culture scaffold to
confirm its cytocompatibility. The HepG2 cell line, a human hepato-
cyte carcinoma cell line, was used as a cellular model. (FFiK)2 hydro-
gels (1.0 wt%) was prepared from DMEM in a 96-well plate and
HepG2 cells (1 × 103 cells) were cultured on the hydrogel or tissue
culture-treated plate (TCTP) for 5 days. After 1, 3 and 5 days of culti-
vation, live cells and dead cells were stained with calcein-AM (2.0 μM)
and propidium iodide (PI, 4.0 μM), respectively. After 1 day of cultiva-
tion, HepG2 cells adhered to the surface of hydrogels (Figure S5a).
Most HepG2 cells were stained with calcein, and a few cells were sta-
ined with PI (Figure S5b,c). These results indicate that the (FFiK)2
hydrogel exhibits cell compatibility. HepG2 cells proliferated on the
(FFiK)2 hydrogel and formed spheroids after 3 days (Figure S5d,e) and
5 days (Figure 7A,B) of cultivation. During this culture period,
Because the hydrogelation of (FFiK)2 was faster than that of
(FFiO)2, (FFiK)2 was used to fabricate hydrogel fibers. Basic peptide
solutions (2.0 wt%) containing fluorescent dyes were injected into
PBS using a microsyringe (inner diameter = 0.15 mm) to prepare
hydrogel fibers (Figure 5D). The hydrogel fibers were visualized by the

TSUTSUMI ET AL.
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FIGURE 7 A, Bright filed image of HepG2 cells cultured on (FFiK)2 hydrogel after 5 days. B and C, Fluorescence images of HepG2 cells
cultured on (FFiK)2 hydrogel after 5 days. Live cells were stained with Calcein (B) and dead cells were stained with PI (C). D, Fluorescence image
of HepG2 cells on TCTP after 5 days. Live cells were stained with Calcein. Scale bar is 100 μm. E, Fluorescent intensity of Calcein-stained HepG2
cells cultured on (FFiK)2 hydrogels and TCTP. *P < .05
FIGURE 8 A, Photograph of
(FFiK)2 hydrogel fibers stained with
NR (10 μM) in 24-well microplate.
Hydrogel fibers were visualized under
UV light (354 nm) irradiation. B, Bright
filed image of HepG2 cells cultured on
(FFiK)2 hydrogel fibers after 2 days.
C, Fluorescence image of HepG2 cells
cultured on (FFiK)2 hydrogel fibers
after 2 days. D, Fluorescence image of
(FFiK)2 hydrogel fibers stained with
NR (10 μM). Scale bar is 100 μm
significant cytotoxicity was not observed (Figure S5f and Figure 7C).
In addition, HepG2 cells exhibited faster proliferation on the (FFiK)2
hydrogel than on TCTP (Figure 7D). The fluorescence intensity from
live cells on the (FFiK)2 hydrogel was higher than that on TCTP. These
results suggest that the (FFiK)2 hydrogel could promote HepG2 cell
proliferation.

12 of 13
TSUTSUMI ET AL.
Hydrogel fibers of (FFiK)2 were also applied to culture of HepG2
hydrogels, cell culture on hydrogels (1 and 3 days), 1H NMR, and
MALDI-TOF MS of compounds.
cells. Hydrogel fibers stained with NR were prepared by the injection
of a (FFiK)2 solution into DMEM (Figure 8A). HepG2 cells were
suspended with hydrogel fibers and cultured for 2 days. The hydrogel
fibers maintained their structure as prepared under cell culture condi-
tion (37 ꢀC, 5% CO2) (Figure 8D). The HepG2 cells also adhered to the
surface of (FFiK)2 hydrogel fibers and proliferated (Figure 8B,C). This
result shows that the shaped (FFiK)2 hydrogel can be used as a cell
culture scaffold.
ACKNOWLEDGMENTS
This work was supported in part by a JSPS KAKENHI, MEXT, Japan.
The authors thank the Biomaterials Analysis Division, Tokyo Institute
of Technology for TEM observations. The measurements of CD spec-
tra, ATR-FTIR spectra, MALDI-TOF MS were supported by
Open Research Facilities for Life Science and Technology. The
authors also thank Mr Masaki Watanabe and Mr Masayoshi Takano
(TA Instruments Japan, Inc) for rheological analysis of hydrogel
samples.
4
|
CONCLUSION
We designed new short self-assembling peptides, (FFiO)2 and (FFiK)2,
with a symmetric structure via a urea bond. The simple structural
design of the peptides makes their synthesis on a large scale possible.
CD experiments revealed that both (FFiO)2 and (FFiK)2 assembled into
β-sheet structures in a concentration-dependent manner at pH 7.2.
(FFiK)2 exhibited a self-assembling ability superior to that of (FFiO)2,
because (FFiK)2 assembled at a lower concentration than (FFiO)2. In
addition, the self-assembly of (FFiO)2 and (FFiK)2 was dependent on
pH. Electrostatic interactions of ornithine or lysine residues at the
edge of both peptides contributed to their stable assembly in β-sheet
structures under physiological pH condition. TEM observations rev-
ealed that both (FFiO)2 and (FFiK)2 assembled into nanofibers with a
uniform width and length more than 500 nm at pH 7.2. (FFiK)2 was
present as more densely bundled and networked nanofibers than
(FFiO)2 due to its superior self-assembling ability. Nanostructures of
(FFiK)2 were also dependent on pH, as reflecting by the CD results.
Electrostatic interactions at the edge of the peptides were important
for their assembly into highly networked nanofibrous structures.
Hydrogelation experiments revealed that both (FFiO)2 and (FFiK)2
formed transparent hydrogels at pH 7.2. (FFiK)2 exhibited a lower crit-
ical hydrogelation concentration than (FFiO)2, which is consistent with
the CD results. (FFiK)2 formed a more transparent hydrogel than
(FFiO)2; on the other hand, the Ac-FFiK peptide, which does not con-
tain a urea unit, formed an opaque hydrogel at a high concentration.
Not only the lysine residue but also the urea unit were important for
the formation of transparent hydrogels. In addition, hydrogel fibers
were prepared by the injection of a (FFiK)2 solution at a high concen-
tration into PBS or DMEM, because (FFiK)2 exhibited rapid hydroge-
lation. In cell culture experiments, we successfully demonstrated that
the (FFiK)2 hydrogel could be used as a scaffold for the culture of
HepG2 cells without significant cytotoxicity. The (FFiK)2 hydrogel pro-
moted the proliferation of HepG2 cells. The hydrogel fiber of (FFiK)2
also worked as a scaffold for HepG2 cells. Based on these results,
(FFiK)2 is a beneficial supramolecular peptide hydrogelator with cell
compatibility. Further functionalization of (FFiK)2 will produce bioma-
terials useful for cell engineering and biomedical applications, because
(FFiK)2 contains two amino groups that can be conjugated with func-
tional molecules.
CONFLICT OF INTEREST
The authors declare no competing interests.
DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are available in the
supplementary material of this article.
ORCID
Hiroshi Tsutsumi
Hisakazu Mihara
https://orcid.org/0000-0003-3780-7871
https://orcid.org/0000-0003-3549-2036
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