Biofunctional supramolecular hydrogels fabricated from a short self-assembling peptide modified with bioactive sequences for the 3D culture of breast cancer MCF-7 cells

Article abstract of DOI:10.1016/j.bmc.2021.116345

Self-assembling peptides are a type of molecule with promise as scaffold materials for cancer cell engineering. We have reported a short self-assembling peptide, (FFiK)₂, that had a symmetric structure connected via a urea bond. In this study, we functionalized (FFiK)₂ by conjugation with various bioactive sequences for the 3D culture of cancer cells. Four sequences, RGDS and PHSRN derived from fibronectin and AG73 and C16 derived from laminin, were selected as bioactive sequences to promote cell adhesion, proliferation or migration. (FFiK)₂, and its derivatives could co-assemble into supramolecular nanofibers displaying bioactive sequences and form hydrogels. MCF-7 cells were encapsulated in functionalized peptide hydrogels without significant cytotoxicity. Encapsulated MCF-7 cells proliferated under 3D culture conditions. MCF-7 cells proliferated with spheroid formation in hydrogels that displayed RGDS or PHSRN sequences, which will be able to be applied to drug screening targeting cancer stem cells. On the other hand, since MCF-7 cells migrated in a 3D hydrogel that displayed AG73, we could construct the metastatic model of breast cancer cells, which is helpful for the elucidation of breast cancer cells and drug screening against cancer cells under metastatic state. Therefore, functionalized (FFiK)₂ hydrogels with various bioactive sequences can be used to regulate cancer cell function for tumor engineering and drug screening.

Full text of DOI:10.1016/j.bmc.2021.116345

Bioorg. Med. Chem. 46 (2021) 116345
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Biofunctional supramolecular hydrogels fabricated from a short
self-assembling peptide modified with bioactive sequences for the 3D
culture of breast cancer MCF-7 cells
Jyh Yea Chia, Takayuki Miki, Hisakazu Mihara, Hiroshi Tsutsumi*
School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho 4259-B40, Midori-ku, Yokohama 226-8501, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Self-assembling peptides are a type of molecule with promise as scaffold materials for cancer cell engineering. We
have reported a short self-assembling peptide, (FFiK)2, that had a symmetric structure connected via a urea bond.
In this study, we functionalized (FFiK)2 by conjugation with various bioactive sequences for the 3D culture of
cancer cells. Four sequences, RGDS and PHSRN derived from fibronectin and AG73 and C16 derived from
laminin, were selected as bioactive sequences to promote cell adhesion, proliferation or migration. (FFiK)2, and
its derivatives could co-assemble into supramolecular nanofibers displaying bioactive sequences and form
hydrogels. MCF-7 cells were encapsulated in functionalized peptide hydrogels without significant cytotoxicity.
Encapsulated MCF-7 cells proliferated under 3D culture conditions. MCF-7 cells proliferated with spheroid
formation in hydrogels that displayed RGDS or PHSRN sequences, which will be able to be applied to drug
screening targeting cancer stem cells. On the other hand, since MCF-7 cells migrated in a 3D hydrogel that
displayed AG73, we could construct the metastatic model of breast cancer cells, which is helpful for the eluci-
dation of breast cancer cells and drug screening against cancer cells under metastatic state. Therefore, func-
tionalized (FFiK)2 hydrogels with various bioactive sequences can be used to regulate cancer cell function for
tumor engineering and drug screening.
Self-assembling peptide
Bioactive sequences
Hydrogel
Cancer cell
3D culture
1. Introduction
glycol (PEG)2 have been used to construct tumor tissue models.
Although these materials can create 3D scaffolds for cell growth, most of
Tissue-engineered cancer models that mimic the in vivo tumor mi-
croenvironments have contributed not only to understanding the dy-
namic tumor development and progression but also to studies on cancer
therapy.1,2 Tissue engineering using functional scaffolds for cancer cell
culture can contribute to the advancement of cancer biology research.3
Most tumors in the human body exist within three-dimensional (3D)
hydrogel milieus composed of complexed biomolecules, including
extracellular matrix proteins and polysaccharides.1 Conventional two-
dimensional (2D) culture often poorly presents tumor conditions such
as cell-cell interactions or signalling between cells residing within the
outer proliferative regions and cells situated inside tumors.4,5 On the
other hand, 3D cancer cell culture systems have recently greatly pro-
moted the understanding of the microenvironments that characterize
tumor tissues, including cell-cell interactions and cell-ECM interactions.
Various 3D biomimetic scaffolds from natural materials such as Matri-
gel,6 alginate,7 and collagen8 and synthetic materials such as polyethene
them lack mechanical or biochemical properties that promote cancer
development. Molecular self-assembly is one of powerful approaches in
the construction of functional materials for cancer tissue engineering. In
particular, self-assembling peptides that can form hydrogels have
attracted much attention for the construction of 3D cancer microenvi-
ronments, particularly EAK 169 and RADA-16.10 Self-assembling pep-
tides with either α-helix or β-sheet structure design can assemble into
nanofibrous structures via noncovalent interactions and form hydrogels
that can encapsulate cancer cells for 3D culture.11,12
Breast cancer is a leading cause of death. In previous studies, 3D
breast cancer models have been developed with various biomimetic
materials, such as PEG-fibrinogen,2 PEG-heparin,13,14 collagen,15 chi-
tosan16 and Matrigel,17 for the investigation of tumorigenic phenomena,
for studies of tumor-associated cell population behaviour, and for anti-
cancer drug testing. However, some of these materials, especially
Matrigel, showed some structural weakness, which allowed only thin-
* Corresponding author.
E-mail address: htsutsum@bio.titech.ac.jp (H. Tsutsumi).
https://doi.org/10.1016/j.bmc.2021.116345
Received 1 June 2021; Received in revised form 27 July 2021; Accepted 30 July 2021
Available online 11 August 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

J.Y. Chia et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116345
layer and short-term analysis (<4 days).18 Although a self-assembling
peptide hydrogel was also used for the encapsulation and 3D culture
of breast cancer cells, biological activity was not installed into the self-
assembling peptide materials. 19
relationship between bioactive sequences and cell behaviour in hydro-
gels was investigated to determine the proper bioactive sequences for
the control of spheroid formation and metastatic activity of MCF-7 cells.
This work could contribute to tumor engineering for spheroid formation
related to cancer cell stemness and metastatic model of breast cancer
cells toward drug screening targeting breast cancer cells.
Previously, we reported that a self-assembling peptide E1Y9 with an
amphiphilic β-sheet structure could form the hydrogel as a cell culture
scaffold.20 E1Y9 peptide could be functionalized with bioactive se-
quences to enhance its cell adhesion activity and applied as a drug
releasing carrier.21,22 Recently, we reported a short self-assembling
peptide, (FFiK)2, that can fabricate a stable hydrogel in response to pH
change (Fig. 1a).23 (FFiK)2 has a symmetric structure composed of two
diphenylalanine (FF) units interconnected by a urea bond at the center
and a lysine residue at each terminal via isopeptide bonds. Hydrophobic
interactions between FF units and hydrogen-bonding networks of urea
and amide bonds acted as the driving force for self-assembly, and elec-
trostatic interactions at the zwitter ionic termini controlled the self-
assembly in response to pH change. (FFiK)2 assembles into supramo-
lecular nanofibers at physiological pH and forms a stable and trans-
parent hydrogel at final peptide concentrations as low as 0.5 wt%. The
(FFiK)2 hydrogel is a biocompatible material due to non-cytotoxic
property. Since (FFiK)2 is easy to synthesize on a large scale compared
to other self-assembling peptides such as RADA16 and E1Y9 due to its
simple and symmetric structure, we consider that (FFiK)2 is a promising
candidate material for the construction of larger tumor tissue models.
However, (FFiK)2 showed weak cell adhesion activity for cell culture.
Therefore, functionalization of (FFiK)2 is required for the 3D culture of
cancer cells.
2. Materials and methods
2.1. General
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 chemical
reagents and solvents were purchased from Fujifilm Wako Pure Chem-
ical (Japan) and Tokyo Chemical Industry (Japan). NMR measurements
were conducted using an Agilent UNITY-INOVA-400. ESI MS measure-
ments were conducted using an LCMS-2010 (Shimadzu).
2.2. Synthesis of (FFiK)2 derivative
(FFiK)2 was synthesized according to a previous report.23 The syn-
thesis scheme of (FFiK)2 using the liquid phase synthesis method was
shown in Fig. S1. A single Boc group was removed from fully protected
(FFiK)2 and the partially deprotected (FFiK)2 was used for conjugation
with various bioactive sequences. The detailed experimental procedures
are described in the supplementary data (Fig. S2-S3).
In this study, (FFiK)2 was conjugated with various bioactive se-
quences derived from extracellular matrix (ECM) proteins to improve
the cell adhesion and growth activities for the 3D culture of cancer cells.
The RGDS and PHSRN sequences derived from fibronectin are known to
promote cell adhesion, while the AG73 and C16 sequences derived from
laminin are known to enhance cancer cell adhesion and proliferation.
These bioactive sequences were conjugated with (FFiK)2 to produce
tailored artificial ECMs. The self-assembling properties of (FFiK)2 de-
rivatives were investigated including the hydrogelation property. The
contribution of installed bioactive sequences to adhesion and prolifer-
ation of MCF-7, a breast cancer cell line, for functionalized hydrogels
was evaluated. In addition, (FFiK)2 hydrogels that displayed bioactive
sequences were applied to the 3D culture of MCF-7 cells. The
2.3. Solid phase peptide synthesis and conjugation of (FFiK)2 with
bioactive sequences
All bioactive sequences were synthesized using a solid-phase method
based on standard Fmoc chemistry.24 The syntheses were carried out on
TentaGel S RAM resin (HiPep Laboratories, Japan) to yield C-terminal
amides. Fmoc-protected amino acid (3 eq. for amino group on resin) was
activated by 3 eq. of HOBt⋅H2O and 3 eq. of HBTU in 2 mL of NMP with
6 eq. of DIEA. In the syntheses of biotinylated peptides, Fmoc-Lys(Bio-
tin)–OH was used. After elongation, the N-termini of all the bioactive
sequences were bromoacetylated using preactivated bromoacetic acid
(20 eq.) by 10 eq. of N,N′-diisopropylcarbodiimide at room temperature
(a)
(b)
Fig. 1. Structure of (FFiK)2 and peptide sequences of (FFiK)2 derivatives (a) Chemical structure of (FFiK)2. (b) Peptide sequences of (FFiK)2 derivatives. The bioactive
sequences derived from natural ECMs, RGDS, PHSRN, AG73 and C16, were conjugated with (FFiK)2 peptide at N-terminus via a GGG linker, respectively.
2

J.Y. Chia et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116345
for 30 min in 2 mL of NMP. Bromoactylated peptides were conjugated
with (FFiK)2 partially deprotected by the removal of a single Boc group
(2 eq.) in 2 mL of NMP supplemented with potassium iodide (1 eq) and
DIEA (1 eq.) at room temperature for 24 h. Cleavage of peptides and
deprotection of side chains were performed with a mixture of tri-
fluoroacetic acid (TFA) (95% v/v), triisopropylsilane (2.5% v/v) and
water (2.5% v/v) for 1.5 h at room temperature. The crude peptides
were then precipitated in cold diethyl ether, collected by centrifugation
and dried under vacuum. All peptide purification was performed by
reversed-phase HPLC on a COSMOSIL 5C18-ARII packed column (10 ×
250 mm) with a linear gradient of acetonitrile containing 0.08% (v/v)
TFA and ultrapure water containing 0.1% (v/v) TFA at a flow rate of 3
mL/min. The purified peptide was lyophilized to obtain peptides as
powder. The products were identified by electrospray ionization mass
spectrometry (ESI-MS). The ESI-MS results are shown in Table S1
(supplementary data).
2.6. Hydrogelation analysis
Mixtures of (FFiK)2 and (FFiK)2-bioactive sequences (molar ratio =
4/1) were dissolved in ultrapure water containing 4 eq. of NaOH to
prepare a total 1.0 wt% peptide solution. A 100 µL aliquot of peptide
solution was mixed with 100 µL of RPMI-1640 without phenol red to
prepare a 0.5 wt% solution. After incubation at room temperature for 30
min, hydrogelation was confirmed by inverting the glass vial.
2.7. Cell culture
MCF-7 cells were obtained from the RIKEN BRC. The cell line was
tested and authenticated by the RIKEN BRC. MCF-7 cells were main-
tained in RPMI-1640 medium 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%
confluence at 37 ◦C under 5% CO2.
2.4. Attenuated total reflection infrared (ATR-IR) spectroscopy
2.8. Cell adhesion on hydrogels
(FFiK)2, (FFiK)2-RGDS, (FFiK)2-PHSRN, (FFiK)2-AG73 and (FFiK)2-
C16 were dissolved in ultrapure water with the addition of 4 eq. of 1 M
NaOH solution to prepare 1 mM solutions, respectively. Then, 2 eq. of
HClaq was added to the peptide solutions to neutralize the peptide so-
lution and incubated at room temperature for 48 h prior to ATR-IR
measurement. A 10 µL of each peptide solution was mounted on the
reflective surface of an FT-IR instrument (IRPrestige-21 with DuraSampl
IR-II, Shimadzu) and air-dried to form a peptide film. ATR-IR spectra
were obtained from 1300 cmꢀ 1 to 1800 cmꢀ 1 with the interferograms
(FFiK)2/(FFiK)2-bioactive sequences (molar ratio = 4/1) were dis-
solved in sterile water with 4 eq. of NaOH to prepare 1.0 wt% peptide
solutions. Aliquots of the peptide solutions (150 μL) were placed in 24-
well nontreated plate (Falcon) and mixed with 150 μL of culture medium
(RPMI-1640 supplemented with 1% penicillin- streptomycin) to prepare
0.5 wt% hydrogels. After incubation at 37 ◦C under 5% CO2 for 30 min,
1.0 × 105 MCF-7 cells in serum-free RPMI 1640 (300
μL) were added on
top of the hydrogels. The cells were incubated at 37 ◦C under 5% CO2,
and the number of adhered polygonal-shaped cells and non-adhered
round-shaped cells were counted under a microscope (Ti-U-PH-1,
Nikon) after 6 h of incubation. The average percentages of adhered
cells/total cells were calculated using 3 different views from the same
well. Statistical significance was accepted at p < 0.05.
coadded 100 times and Fourier transformed at a resolution of 4 cmꢀ 1
.
2.5. Transmission electron microscopy (TEM)
(FFiK)2, (FFiK)2-RGDS, (FFiK)2-PHSRN, (FFiK)2-AG73 and (FFiK)2-
C16 were dissolved in ultrapure water with the addition of 4 eq. of 1 M
NaOH solution to prepare 1 mM peptide solutions, respectively. The
peptide solutions were further diluted to 100 µM with ultrapure water
containing 2 eq. of HCl to neutralize the peptide solution. The diluted
solutions were incubated at room temperature for 48 h prior to TEM grid
coating. A collodion-coated copper EM grid was glow discharged for 40
sec at 3 mA using an IB2 ion coater. A 10 µL of peptide solution was
mounted on the collodion-coated copper EM grid for 30 sec to adsorb the
peptide nanofibers. The excess solution was removed by capillary action
with filter paper, and the grid was washed by adding a drop of ultrapure
water (10 µL). After removal of the ultrapure water, the grids were
stained twice with 10 µL of NanoW staining solution (2% solution in
water at pH 6.8) (Nanoprobes) for 1 min each time. The stained grids
were blotted with filter papers and allowed to dry at room temperature
overnight. TEM images were taken with a JEM 1400Plus (JEOL) that
was operated at 80 kV.
2.9. Cell proliferation on/in hydrogels
sfGFP-MCF-7 cells (5.0 × 103) were cultured on hydrogels prepared
in a 96-well tissue culture plate for 7 days with medium changes every
two days. Cell proliferation was evaluated using a Cell Counting Kit-8
(CCK-8) colorimetric assay (Dojindo) at 1, 3, 5 and 7 days according
to the manufacturer’s instructions. The absorbance at 450 nm was
measured on a microplate reader (ARVO MX, PerkinElmer) using a 450
nm filter.
The sfGFP-MCF-7 cells (5.0 × 103) encapsulated within hydrogels
were cultured in a 96-well tissue culture plate for 10 days with medium
changes every two days. Cell proliferation was evaluated in the same
manner described above.
In 2D and 3D cell culture experiments, three wells were used for each
condition. Error bars represent standard deviations (SD) of the means of
three independent experimental values. Statistical significance was
accepted at p < 0.05.
Mixed solutions of (FFiK)2 and biotin-modified (FFiK)2 derivatives
(10 wt%) were prepared as 100 μM solutions in ultrapure water by the
same procedure described above. After 48 h of incubation, 10 µL of
peptide solution was mounted on the collodion-coated copper EM grid
for 30 sec to adsorb the peptide nanofibers. The excess solution was
removed by capillary action with filter paper, and the grid was washed
by adding a drop of ultrapure water (10 µL). After removal of the ul-
trapure water, 10 µL of the streptavidin-gold nanoparticle (10 nm)
conjugates (streptavidin-GNP) (Cytodiagnostics) was mounted on the
collodion-coated copper EM grid for 1 min. After removal of excess
streptavidin-GNP by capillary action with filter paper, the grid was
washed by adding 10 µL of ultrapure water. Then, the sample was
stained with NanoW by the same procedurer described above, and TEM
images were taken.
2.10. 3D cell culture in hydrogels
The MCF-7 cell line stably expressing sfGFP (sfGFP-MCF-7) was
established and used for the 3D cell culture experiment. The detailed
procedure for the establishment of sfGFP-MCF-7 is described in the
supplementary data. (FFiK)2/(FFiK)2-bioactive sequences (molar ratio
= 4/1) were dissolved in sterile water with 4 eq. of NaOH to prepare 1.0
wt% peptide solutions and incubated at 4 ◦C for 24 h prior to hydrogel
preparation. Aliquots of the peptide solutions (50 µL) were placed in 96-
well glass bottom plates (Iwaki) and mixed with RPMI-1640 supple-
mented with 1% penicillin-streptomycin containing 5.0 × 103 sf-GFP-
MCF-7 cells to prepare 0.5 wt% of hydrogels. The hydrogels were
incubated at 37 ◦C under 5% CO2 for 30 min. After hydrogelation, 100
µL of RPMI-1640 supplemented with 10% FBS and 1% penicillin-
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J.Y. Chia et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116345
streptomycin was added to the hydrogels. The cells were incubated at
37 ◦C under 5% CO2 with medium changes every two days and observed
under a laser scanning confocal microscope (LSM780, Carl Zeiss) on
days 1, 3, 5, 7 and 10.
these two sequences were reported to promote cancer cell adhesion and
enhance tumor growth and tumor cell metastasis.28,29 (FFiK)2 was
conjugated with RGDS, PHSRN, AG73 or C16 at the primary amine of
(FFiK)2 via a GGG linker to produce (FFiK)2-RGDS, (FFiK)2-PHSRN,
(FFiK)2-AG73 and (FFiK)2-C16, respectively (Fig. 1b, Fig. S4). The
partially deprotected (FFiK)2 precursor (Fig. S2) was successfully
allowed to react with bioactive sequences that had a bromoacetyl group
at the N-terminus on the solid support. After cleavage from the solid
support and HPLC purification, (FFiK)2 derivatives were obtained in
approximately 39–68% yields on solid phase peptide synthesis
(Table S1).
2.11. Immunofluorescence staining
MCF-7 cells (1 × 105) were cultured in hydrogels for 10 days. The
MCF-7 cell clumps were collected from the hydrogel by gentle resus-
pension using a pipette and centrifuged at 1100g. The supernatant was
removed, and the cells were fixed with 4% paraformaldehyde for 1 h in a
microcentrifuge tube and further permeabilized with 0.5% Triton-X 100
in PBS for 10 min at room temperature. Cell clumps were washed with
PBS for three times by gentle resuspension and centrifugation. Cell
clumps were blocked with 0.5% BSA for 1 h at room temperature in
microcentrifuge tubes and centrifuged at 1100g to remove the blocking
reagent. The primary antibody (rabbit anti-E-cadherin EP700Y inter-
cellular junction marker, ab40772, 1:500 dilution, Abcam) was incu-
bated for 1 h at room temperature with shaking, and followed by the
secondary antibody [goat anti-rabbit IgG H&L (Alexa Fluor ® 488),
ab205718, 1:200 dilution, Abcam] for 1 h at room temperature with
shaking. The cells were washed three times for 5 min each in 0.l% Tween
20 in PBS, then incubated with Alexa-633-phalloidin (Phalloidin-iFluor
633, ab176758, Abcam) and DAPI for 30 min and washed three times
with PBS. Finally, the cells were transferred to 96-well glass bottom
plates (Iwaki) and observed under a confocal microscope.
3.2. Structure analysis of (FFiK)2 derivatives in self-assembly
To demonstrate the potential of all (FFiK)2 derivatives in β-sheet
formation, attenuated total reflection infrared (ATR-IR) spectra were
measured. The (FFiK)2 and (FFiK)2 derivatives showed predominant
peaks around 1620–1640 cmꢀ 1 that were assigned to a β-sheet struc-
ture30 (Fig. 2). This result suggests that all (FFiK)2 derivatives could
assemble into a β-sheet structure. The shoulder peaks around 1670 cmꢀ 1
of (FFiK)2-PHSRN and (FFiK)2-AG73 might come from a random struc-
ture of bioactive sequences conjugated with (FFiK)2.
Self-assembled nanostructures of (FFiK)2 and (FFiK)2 derivatives
were evaluated by TEM observation. (FFiK)2 (Fig. 3a) and (FFiK)2-RGDS
(Fig. 3b) formed nanofibers with widths of approximately 8 nm and 10
nm, respectively. This result suggests that the RGDS sequence does not
alter the self-assembling property of (FFiK)2. On the other hand, (FFiK)2-
PHSRN, (FFiK)2-AG73 and (FFiK)2-C16 did not form uniform nanofiber
structures like (FFiK)2 (Fig. S5). (FFiK)2-PHSRN, (FFiK)2-AG73 and
(FFiK)2-C16 have + 1, +4 and +2 net charges, respectively; on the other
hand, (FFiK)2 and (FFiK)2-RGDS are neutral. Electrostatic repulsion due
to the conjugated sequences might affect the self-assembly of (FFiK)2
derivatives.
2.12. Western blotting analysis of E-cadherin
After cell culture in peptide hydrogels, the cells were lysed with 100
µL of RIPA lysis buffer [50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% (w/
v) Nonidet P40 substitute, 0.5% (w/v) sodium deoxycholate, 0.1% (w/
v) SDS)] supplemented with a cocktail of protease inhibitor (Sigma-
Aldrich) for 1 h with rotation at 4 ◦C. Cell lysates were centrifuged at
17,000g for 15 min at 4 ◦C. The supernatant was harvested to obtain the
whole cell proteins, and the protein concentration was determined by a
BCA protein concentration kit (Takara Bio). 30 µg of protein from each
sample was mixed with Laemmli sample loading buffer for SDS-PAGE.
After transfer to PVDF membranes (Immobilon®-P, Merck) using a
semidry transfer cell device (Bio-Rad), the blots were incubated with
blocking buffer (5% skim milk) for 1 h at room temperature. Blots were
incubated with primary antibodies against E-cadherin (rabbit anti-E-
cadherin EP700Y intercellular junction marker, ab40772, 1:20,000
dilution, Abcam) and GAPDH [(D16H11)XP® rabbit mAB, 1:5000, Cell
Signalling Technology] overnight at 4 ◦C and further incubated with
secondary antibody (anti-rabbit IgG, HRP linked antibody #7074, Cell
Signalling Technology) for 1 h at room temperature. The blot was
visualized by SuperSignal West Femto Maximum Sensitivity Substrate
(Thermo Scientific) according to the manufacturer’s instruction and
observed by LuminoGraph I (Atto). Quantitative image analysis was
performed with ImageJ.
Next, we tried to display bioactive sequences on assembled nano-
fibers by the co-assembly of (FFiK)2 derivatives with a parent (FFiK)2.
For this purpose, 10 mol% of (FFiK)2-RGDS, (FFiK)2-AG73, or their
3. Results and discussion
3.1. Design of (FFiK)2 functionalized with bioactive sequences
The (FFiK)2 peptide (Fig. 1a) was synthesized according to our pre-
vious report.23 To enhance the biological properties of (FFiK)2, we
conjugated (FFiK)2 with various bioactive sequences derived from nat-
ural ECM proteins. The sequence RGDS derived from fibronectin was
chosen to enhance the cell adhesion of a (FFiK)2 hydrogel, because RGDS
has been reported to promote cell adhesion when it is displayed on the
surface of the materials.25,26 The sequence PHSRN, which enhances cell
adhesion synergistically with RGDS26,27 was chosen as another bioactive
sequence derived from fibronectin. Both AG73 (RKRLQVQLSIRT) and
C16 (KAFDITYVRLKF) were chosen from laminin proteins, because
Fig. 2. ATR-IR spectra of (FFiK)2, (FFiK)2-RGDS, (FFiK)2-PHSRN and (FFiK)2-
AG73 after incubation over 48 h. All spectra were measured from air-dried
peptide films.
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Bioorganic & Medicinal Chemistry 46 (2021) 116345
(a)
(b)
(c)
(d)
(f)
(e)
Fig. 3. TEM images of (a) (FFiK)2, (b) (FFiK)2-RGDS, (c) 10% (FFiK)2-RGDS, (d) 10% (FFiK)2-RGDS-Biotin, (e) 10% (FFiK)2-AG73 and (f) 10% (FFiK)2-AG73-Biotin in
the presence of 10 nm streptavidin-conjugated gold nanoparticles. All samples were prepared in 100 µM and negatively stained using NanoW. Scale bar is 100 nm.
Red arrow showed the position of 10 nm streptavidin-conjugated gold nanoparticles attached on the biotinylated peptide nanofibers.
biotinylated derivatives was mixed with (FFiK)2. Both (FFiK)2/(FFiK)2-
RGDS (10 nm width) and (FFiK)2/(FFiK)2-AG73 (13 nm width) showed
long nanofibers with uniform width (Fig. 3c, e). This result indicates that
the presence of (FFiK)2-derivatives does not disturb the self-assembly of
(FFiK)2 peptides. To confirm whether bioactive sequences are displayed
on nanofibers, co-assembled nanofibers of (FFiK)2/(FFiK)2-RGDS-Biotin
and (FFiK)2/(FFiK)2-AG73-Biotin were labelled by streptavidin-gold
nanoparticles (GNPs). Fig. 3d and f clearly show that GNPs were
observed along with co-assembled nanofibers of (FFiK)2/(FFiK)2-RGDS-
Biotin and (FFiK)2/(FFiK)2-AG73-Biotin, respectively. There were no
GNPs on co-assembled nanofibers of (FFiK)2/(FFiK)2-RGDS (Fig.3c) and
(FFiK)2/(FFiK)2-AG73 (Fig. 3e). These results suggest that (FFiK)2 de-
rivatives can co-assemble into nanofibers with the parent (FFiK)2 and
that bioactive sequences are displayed on the assembled nanofibers.
Since not only the short sequence RGDS but also the long sequence AG73
was successfully displayed on nanofibers, various bioactive sequences
can be used by the co-assembly method. Since highly cationic AG73
sequence of 12 residues could be displayed on self-assembled nano-
fibers, we considerd that the shorter PHSRN and less cationic C16 with
the same amino acid length can be displayed on self-assembled
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Bioorganic & Medicinal Chemistry 46 (2021) 116345
nanofibers in the same co-assembly manner.
3.3. Hydrogelation test
Hydrogelation tests of (FFiK)2 and (FFiK)2 derivatives were carried
out to evaluate the macroscopic structure of the (FFiK)2 and (FFiK)2
derivatives. (FFiK)2 derivatives and the parent (FFiK)2 were mixed at a
molar ratio of 1:4 to prepare a 0.5 wt% solution in cell culture medium.
All peptide mixtures formed self-supporting hydrogels within 30 min of
incubation at room temperature (Fig. 4). This result suggests that
(FFiK)2-conjugates with bioactive sequences of different lengths (4 to 12
residues) can form stable hydrogels by co-assembly with the parent
(FFiK)2. (FFiK)2, (FFiK)2-RGDS and (FFiK)2-PHSRN gave transparent
hydrogels, while (FFiK)2-AG73 and (FFiK)2-C16 formed slightly opaque
hydrogels. This result indicates that the short sequences of (FFiK)2-RGDS
and (FFiK)2-PHSRN disperse homogenously in hydrogels. On the other
hand, the long sequences of (FFiK)2-AG73 and (FFiK)2-C16 might un-
dergo partial aggregation in hydrogels due to their amphiphilic struc-
tures. However, these results show that (FFiK)2 and (FFiK)2 derivatives
can co-assemble into networked nanofibers in the presence of high water
content and form stable hydrogels. The parent framework of (FFiK)2 has
stable self-assembling ability enough to display long bioactive sequences
of more than 10 residues even though (FFiK)2 is a short peptide of 6
residues.
3.4. Cell adhesion on 2D hydrogels
Cell adhesion assays were performed to investigate the effect of
bioactive sequences. The peptide hydrogels (0.5 wt%, molar ratio of
(FFiK)2/(FFiK)2 derivatives = 4/1) were prepared in a 24-well micro-
plate, and MCF-7 cells were cultured on the hydrogels for 6 h. The
adherent percentages of MCF-7 cells were evaluated under a microscope
(Fig. 5, Fig. S6). The hydrogels of (FFiK)2, (FFiK)2/(FFiK)2-RGDS and
(FFiK)2/(FFiK)2-PHSRN showed smooth surfaces, and MCF-7 cells
adhered to the surface (Fig. S6a-c). On the other hand, the hydrogels of
(FFiK)2/(FFiK)2-AG73 and (FFiK)2/(FFiK)2-C16 showed rough surfaces
(Fig. S6d-e), probably due to partial aggregation of the bioactive se-
quences. The (FFiK)2-AG73 hydrogel exhibited the highest cell adhesion
activity (59%) among all hydrogels. The hydrogels functionalized with
RGDS and C16 showed similar cell adhesion ability (38% and 44%,
respectively). The (FFiK)2-PHSRN hydrogel showed lower cell adhesion
ability (26%) than the (FFiK)2-RGDS hydrogel. In contrast, the (FFiK)2
hydrogel showed only 10% cell adhesion activity. These results suggest
that the bioactive sequences successfully improved the cell adhesion
activity of the (FFiK)2 hydrogel.
Fig. 5. Cell adhesion on (FFiK)2/(FFiK)2-derivative mixed hydrogels. The
number of adhered cells on the hydrogel were counted and divided by the total
number of cells and the average percentages were calculated using 3 different
views. Error bar represent standard deviations (SD) of the means of three in-
dependent experimental value. Statistical significance was accepted at *p
< 0.05.
Many of integrins expressed on the surfaces of cancer cells can
recognize the tripeptide motif of RGD contained in the ECM proteins,
and the interaction between integrins and the RGD motif promotes the
adhesion and progression of cancer cells. 31 Therefore, installation of the
RGDS sequence into (FFiK)2 could improve the adhesion of MCF-7 cells.
At first, the PHSRN motif was identified as a synergystic sequence that
enhanced cell adhesion together with RGD sequence.32 The PHSRN
Fig. 4. Photographs of self-supporting hydrogels of (FFiK)2 and (FFiK)2 derivatives. (a) (FFiK)2 (b) 20% (FFiK)2-RGDS (c) 20% (FFiK)2-PHSRN (d) 20% (FFiK)2-AG73
(e) 20% (FFiK)2-C16. The total concentration of peptides was 0.5 wt%.
6

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Bioorganic & Medicinal Chemistry 46 (2021) 116345
peptide could promote cell adhesion, but its activity was weaker than
the RGD peptide. Thereafter, the PHSRN sequence was reported to
mediate cell adhesion through a common mechanism as the RGD motif
but binds competitively to the integrin receptors with lower affinity
rather than RGD.33 These reports support that (FFiK)2-PHSRN hydrogel
exhibited higher cell adhesion than the (FFiK)2 hydrogel but lower cell
adhesion than (FFiK)2-RGDS hydrogel. On the other hand, several types
of breast cancer cells, including MCF-7 cells, strongly adhered to
microplates coated with the AG73 peptide.34 This report supports the
highly improved adhesion activity of the (FFiK)2-AG73 hydrogel. It was
reported that C16 could bind to β1 integrin and promote the adhesion
and migration of breast cancer cells through the regulation of GPNMB
expression35; therefore, the (FFiK)2-C16 hydrogel showed enhanced cell
adhesion.
This result implies that the improvement of the cell adhesion alone may
be insufficient for the cell proliferation.
The proliferation of sfGFP-MCF-7 cells was also evaluated in 3D
hydrogels (Fig. 6b). After 1 day of cultivation, the (FFiK)2-C16 hydrogel
exhibited the highest cell number among the hydrogels. However, sig-
nificant cell proliferation was not observed in the (FFiK)2-C16 hydrogel
over 7 days of cultivation. The C16 peptide was reported to stimulate
proteolytic activity, which eventually leads to cell apoptosis.36,37 The
combination of the increased number of dead cells observed with pro-
longation of the culture period and the proteolytic activity stimulated by
the C16 peptide contributed to the low proliferation rate detected in
sfGFP-MCF-7 cells (Fig. S8q-t). On the other hand, MCF-7 cells contin-
uously proliferated in other hydrogels, and the (FFiK)2-RGDS, (FFiK)2-
PHSRN and (FFiK)2-AG73 hydrogels exhibited higher cell proliferation
than the (FFiK)2 hydrogel at day 7. This result indicates that RGDS,
PHSRN and AG73 effectively promote cell proliferation in 3D hydrogels.
Interestingly, the cell proliferation rates in these three hydrogels were
different. In the 3D (FFiK)2-AG73 hydrogel, the cell proliferation rate
was slower than in the (FFiK)2-RGDS and (FFiK)2-PHSRN hydrogels,
although the cell proliferation on the 2D (FFiK)2-AG73 hydrogel was
faster than that of the (FFiK)2-RGDS and (FFiK)2-PHSRN hydrogels
(Fig. 6a). This difference might come from the 3D environment inside
the hydrogels.
3.5. Cell proliferation on hydrogels (2D) and in hydrogels (3D)
Before the cell proliferation assay, the MCF-7 cell line stably
expressing superfolder GFP (sfGFP-MCF-7) was established (Supporting
data). In the cell proliferation assay, sfGFP-MCF-7 cell proliferation on
hydrogels was monitored for up to 7 days by a WST assay using a CCK-8
kit (Fig. 6a). Fig. 6 shows the absorbance at 450 nm, which is related to
the number of live cells. After 1 day of cultivation, the (FFiK)2-AG73
hydrogel exhibited the highest cell numbers among all the tested con-
ditions. In addition, the (FFiK)2-AG73 hydrogel maintained the highest
cell numbers over 7 days of cultivation. These results suggest that the
AG73 sequence is the most effective in not only cell adhesion but also
cell proliferation. The (FFiK)2-C16 hydrogel exhibited the second
highest cell proliferation activity among the hydrogels. This result re-
flects the cell adhesion activity, as shown in Fig. 6. In contrast to cell
adhesion, the (FFiK)2-RGDS and (FFiK)2-PHSRN hydrogels did not
markedly promote cell proliferation compared to the (FFiK)2 hydrogel.
3.6. Cell imaging in 3D hydrogels
The sfGFP-MCF-7 cells were encapsulated within 0.5 wt% hydrogels
of (FFiK)2/(FFiK)2 derivatives (molar ratio = 4/1) under well-dispersed
conditions (Fig. S7). Encapsulated cells were cultured in hydrogels for 7
days (Fig. 7 and Fig. S8). Dead cells were stained by propidium iodide
(PI). In the (FFiK)2 hydrogel, MCF-7 cells proliferated with the spheroid
formation (Fig. 7a). The size of spheroids was around 87 μm (Fig. 7f). A
few cells were stained with PI over 7 days of cultivation. These results
show that the (FFiK)2 hydrogel enables the 3D culture of MCF-7 cells
without significant cytotoxicity. Since the (FFiK)2 hydrogel does not
have positive adhesion activity for MCF-7 cells as demonstrated in the
cell adhesion experiment, strong cell-cell interactions might cause the
spheroid formation. In the case of the (FFiK)2-RGDS hydrogel, MCF-7
cells formed more compact spheroids than the (FFiK)2 hydrogel, with
spheroid size of around 62 μm (Fig. 7b and f). The interaction between
MCF-7 cells and the RGDS sequence displayed on peptide nanofibers
might induce the compact spheroid formation. In the case of the (FFiK)2-
PHSRN hydrogel, similar spheroid formation was observed (Fig. 7c and
f). MCF-7 cells also formed spheroids when cultured in the (FFiK)2-C16
hydrogel (Fig. 7e and f), however, many dead cells were observed after
7 days of cultivation. This result is consistent with the cell proliferation
results in the (FFiK)2-C16 hydrogel. C16 stimulates cell receptor β1
integrin and increases invadopodia activity to promote cell adhesion.38
This implies that invadopodium activity enhanced the cell adhesion
activity but simultaneously induced cell apoptosis.
In contrast, MCF-7 cells proliferated in the (FFiK)2-AG73 hydrogel
without significant spheroid formation (Fig. 7d and Fig. S8 m-p).
Puchalapalli et al. reported the direct binding relationship of AG73 se-
quences to syndecan in breast cancer cells, which promotes filopodium
formation and supports tumor cell adhesion and invasion.34 Therefore,
syndecan and integrin signalling downstream of AG73 might regulate
changes in cancer cell behaviour, promoting migration and metastasis
activity. The increased number of dead cells observed (Fig. 7d) with a
prolonged culture period might be due to the aggregation of conjugated
peptides (opaque gel) formed in AG73 and the induction of apoptosis in
encapsulated cells. This might cause the slower cell proliferation rate
observed in the 3D (FFiK)2-AG73 (Fig. 6b).
Fig. 6. Proliferation of sfGFP-MCF-7 cells over 7 days culture. Viable cells of 1,
3, 5 and 7 days cultured (a) on 2D hydrogel and (b) in 3D hydrogel. Error bar
represent standard deviations (SD) of the means of three independent experi-
mental value. Statistical significance was accepted at *p < 0.05.
3.7. Immunostaining of MCF-7 cells cultured in 3D hydrogels
To investigate the effect of bioactive sequences on the 3D culture of
7

J.Y. Chia et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116345
a
c
b
150
100
50
f
d
e
0
6
2
S
)
N
)
i
1
D
R
K
i
C
-
S
G
F
2
H
R
)
F
-
(
P
-
K
2
i
)
2
F
K
F
K
i
(
F
F
F
(
F
(
Hydrogels
Fig. 7. Confocal imaging of sfGFP-MCF-7 cells at Day 5 in 3D hydrogels. Confocal images of sfGFP-MCF-7 cells embedded within hydrogels on day 5. Dead cells were
stained with propidium iodide (PI). (a) (FFiK)2 (b) (FFiK)2-RGDS (c) (FFiK)2-PHSRN (d) (FFiK)2-AG73 (e) (FFiK)2-C16. (f) The diameter of 3D spheroids formed in
(FFiK)2, (FFiK)2-RGDS, (FFiK)2-PHSRN and (FFiK)2-C16 hydrogels. Scale bar is 100 µm.
MCF-7 cells, we focused on E-cadherin, a cell adherent protein, because
MCF-7 cells cultured in the (FFiK)2 hydrogel formed spheroids, but MCF-
7 cells cultured in the (FFiK)2-AG73 hydrogel did not form spheroids. E-
cadherin and actin filaments were visualized by immunostaining after
10 days of cultivation in each hydrogel (Fig. 8). In the case of the (FFiK)2
hydrogel, E-cadherin localized on the cell membrane of MCF-7 cells and
formed tight adhesion between cells (Fig. 8a). E-cadherin is a mediator
of cell-cell adhesion,39 and actin filaments mediate cell shape, spreading
and migration.40,41 F-actin stained with phalloidin-iFluoro633 co-
localized well with E-cadherin. These results correspond to a previous
report on spheroid formation by cancer cells.42 In cases of the (FFiK)2-
RGDS and (FFiK)2-PHSRN hydrogels, the expression and localization of
E-cadherin and F-actin were similar to the case of the (FFiK)2 hydrogel
(Fig. 8b, c). There was no significant difference in the expression level of
E-cadherin among the (FFiK)2, (FFiK)2-RGDS and (FFiK)2-PHSRN
hydrogels (Fig. S9). These results indicate that the short sequences RGDS
and PHSRN do not have a significant effect on the spheroid formation of
MCF-7 cells in 3D hydrogels. In the case of the (FFiK)2-C16 hydrogel,
although E-cadherin localized on the cell membrane, including cell-cell
interfaces, F-actin was not observed at cell-cell interfaces (Fig. 8e). In
addition, filopodium-like structures were observed on MCF-7 cells
(Fig. S10b). Since C16 promotes cancer cell migration,38 actin filaments
might not be observed at cell-cell interfaces.
filopodium formation.34 Filopodium-like structures were also observed
on MCF-7 cells (Fig. S10a), therefore, AG73 could affect cell adhesion
and invasion through filopodia in the tumor environment and subse-
quently disrupt the cell assembly.
4. Conclusion
We successfully functionalized the self-assembling peptide (FFiK)2
with bioactive sequences derived from fibronectin and laminin to confer
biological functions on (FFiK)2-based hydrogels. Not only short se-
quences (RGDS and PHSRN) but also long sequences (AG73:
RKRLQVQLSIRT and C16: KAFDITYVRLKF) could be conjugated with
(FFiK)2 on the solid support. FTIR spectral study revealed that all (FFiK)2
derivatives could assemble into β-sheet structures. TEM observations
revealed that (FFiK)2-RGDS could self-assemble into uniform nanofibers.
Although other (FFiK)2 derivatives could not form nanofibers alone, co-
assembly with the parent (FFiK)2 allowed them to form nanofibrous
structures and hydrogels displaying bioactive sequences. In cell exper-
iments, functionalized hydrogels showed superior adhesion of MCF-7
cells to the (FFiK)2 hydrogel. MCF-7 cells were successfully encapsu-
lated in all hydrogels and cultured over 7 days without significant
cytotoxicity. In addition, all hydrogel scaffolds were stable under the cell
culture conditions over 7 days. During cell proliferation, MCF-7 cells
formed spheroids in the (FFiK)2, (FFiK)2-RGDS and (FFiK)2-PHSRN
hydrogels. Spheroid formation was also observed in the (FFiK)2-C16. In
contrast, MCF-7 cells did not form spheroids and migrate in the (FFiK)2-
AG73 hydrogel. Immunostaining experiments revealed that cell-cell
interaction was disrupted by AG73, causing cell migration in the
hydrogel. Based on these results, we could identify the proper bioactive
sequences RGDS and PHSRN to control spheroid formation and AG73 to
induce metastatic activity of MCF-7 cells in 3D hydrogels. Recently, it
In contrast, E-cadherin did not localize to the cell membrane in MCF-
7 cells cultured in the (FFiK)2-AG73 hydrogel (Fig. 8d). In addition, tight
adhesion between cells was not formed, and F-actin was not observed in
many cells. The expression level of E-cadherin in the (FFiK)2-AG73
hydrogel decreased compared to that in the (FFiK)2 hydrogel. These
results indicate that the AG73 sequence disrupts cell-cell interaction by
down regulating E-cadherin and F-actin proteins. The AG73 sequence
binds to syndecan receptors on breast cancer cells and increases the
8

J.Y. Chia et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116345
Data availability
The data required to reproduce these findings are available upon
request.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116345.
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