In situ modulation of cell behavior via smart dual-ligand surfaces

Article abstract of DOI:10.1021/la503521x

Due to the highly complex nature of the extracellular matrix (ECM), the design and implementation of dynamic, stimuli-responsive surfaces that present well-defined ligands and serve as model ECM substrates have been of tremendous interest to biomaterials,

Full text of DOI:10.1021/la503521x

Article
pubs.acs.org/Langmuir
In Situ Modulation of Cell Behavior via Smart Dual-Ligand Surfaces
†
†
†
†,‡
,†,‡
Abigail Pulsipher, Sungjin Park, Debjit Dutta, Wei Luo, and Muhammad N. Yousaf*
†Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States
‡Department of Chemistry and Biology, York University, Toronto, Ontario M3J 1P3, Canada
*
S Supporting Information
ABSTRACT: Due to the highly complex nature of the extracellular matrix
ECM), the design and implementation of dynamic, stimuli-responsive
(
surfaces that present well-defined ligands and serve as model ECM substrates
have been of tremendous interest to biomaterials, biosensor, and cell biology
communities. Such tools provide strategies for identifying specific ligand−
receptor interactions that induce vital biological consequences. Herein, we
report a novel dual-ligand-presenting surface methodology that modulates
dynamic ECM properties to investigate various cell behaviors. Peptides
PHSRN, cRGD, and KKKTTK, which mimic the cell- and heparan sulfate-
binding domains of fibronectin, and carbohydrates Gal and Man were
combined with cell adhesive RGD to survey possible synergistic or antagonist
ligand effects on cell adhesion, spreading, growth, and migration. Soluble
molecule and enzymatic inhibition assays were also performed, and the levels
of focal adhesion kinase in cells subjected to different ligand combinations
were quantified. A redox-responsive trigger was incorporated into this surface strategy to spontaneously release ligands in the
presence of adhered cells, and cell spreading, growth, and migration responses were measured and compared. The identity and
nature of the dual-ligand combination directly influenced cell behavior.
INTRODUCTION
for inducing synergistic effects on cell adhesion and migration.
Furthermore, conflicting reports of whether PHSRN alone is
capable of supporting cell adhesion have been a topic of debate
■
The extracellular matrix (ECM) is a highly dynamic, insoluble
aggregate of collagens, proteoglycans, structural glycoproteins,
and elastin that provides structural support for the adhesion,
growth, differentiation, migration, and survival of mammalian
15−18
over the past decade.
Although α5β1 and αVβ3 integrins serve as the primary cell
surface receptors that mediate adhesion, syndecan-4, a
transmembrane heparan sulfate proteoglycan (HSPG), is a
1
−3
cells.
Improper cell attachment and migration have been
implicated in cancer cell metastasis and other diseased states,
20,21
4
−7
coreceptor for FN.
A heparan sulfate (HS) binding domain
including fibrosis.
For a cell to undergo migration, it must
spans FNIII12‑14. Simultaneous interactions of syndecan-4 and
α5β1 integrin with FNIII12‑14 and FNIII8‑10, respectively,
induces downstream signaling events, leading to the activation
of focal adhesion kinase (FAK) and extracellular signal-
regulated kinase (ERK) with subsequent complete cell
attachment and enhanced spreading via focal adhesion complex
first adhere to another cell or the ECM through cell surface
8
receptor−ligand interactions. Integrins and syndecans, which
are transmembrane proteins, represent the most common cell
surface receptor families that facilitate cell adhesion to the ECM
9
−11
and transduce extra- and intracellular signals.
Fibronectin (FN) is a predominant ECM glycoprotein that
contains three homologous globular domainstypes I, II, and
IIIand possesses a number of interaction sites for both
1
2,20,21
(
FAC) formation.
A few HS binding domain mimics
have been tested; however, similar to the synergistic effect of
RGD and PHSRN on cell adhesion, these small molecules or
sequences are less efficient in promoting cell attachment alone.
Such mimics contain the sequence B-B-B-X-X-B, where B is a
basic amino acid (e.g., Arg or Lys) and X is a hydropathic
12
integrins and syndecans. As such, FN plays an important role
in cell adhesion, growth, migration, and differentiation and is
critical to cellular processes, including embryogenesis and tissue
1
3
repair. A number of cell types bind to FN regions that span
the 8th to 10th type III (FNIII8‑10) cell-binding domain. Arg-
Gly-Asp (RGD), found in FNIII , was identified as the
22−24
amino acid (e.g., Ser, Tyr, or Thr).
Conflicting hypotheses
regarding the role of HS binding sequences on virus attachment
have been reported. The Lys-Lys-Thr-Lys (KKTK) motif,
found in the human adenovirus (hAd) fiber shaft, serves a
minimal role in binding HSPGs but is significant to virus
1
0
minimal cell attachment sequence of α5β1 and αVβ3 integrin
1
4
recognition. A synergy site that presents Phe-His-Ser-Arg-Asn
PHSRN) was then identified in FNIII and shown to enhance
(
9
FN’s association with α5β1 integrins, mediating cell adhesion
1
5−18
and migration.
same plane of FN, connected by a flexible 30-40 Å linker.
Spatial orientation and positioning of these signals are crucial
RGD and PHSRN are presented on the
Received: September 3, 2014
Revised: October 13, 2014
Published: November 6, 2014
19
©
2014 American Chemical Society
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Article
25
infection and trafficking into the nucleus. Two separate works
demonstrated that different hAd types, which lacked the KKTK
2
6,27
motif, were able to attach and infect hepatic cells in vivo.
However, little is known concerning the role of HS binding
sequences on cell adhesion and migration and its possible
synergistic effects, if any, with RGD.
Due to the complex nature of the ECM, identifying all the
diverse small molecules and ligand−cell surface receptor
combinations that induce specific biochemical processes
28,29
remains challenging.
Rather than performing in vitro
studies with large native FN (∼440 kDa), which is purified
from blood plasma and tends to denature or adsorb in
unnatural orientations and conformations on surfaces,
researchers have sought to discover alternative approaches. As
such, tremendous effort has been extended to creating model
substrates that mimic the ECM using structurally well-defined,
1
8,30
decoupled biomolecules, including RGD and PHSRN.
Such surfaces enable the spatial and temporal presentation of
well-defined ligands for the interrogation of biospecific ligand−
cell surface receptor interactions, providing great tools for
applications in cell biology, biotechnology, and tissue engineer-
ing.
Over the past decade, self-assembled monolayers (SAMs) of
alkanethiolates on gold have proven to be smart, dynamic, and
stimuli-responsive model surfaces for a number of cell
3
1−34
biological investigations.
“Dynamic” refers to the in situ
Figure 1. Simplified schematics of (A) cell adhesion to ECM protein
FN via integrin interaction with FNIII9‑10 cell-binding domains,
cytoplasmic proteins, and the actin cytoskeleton to form a FAC and
(B) a dynamic dual-ligand ECM model substrate. SAMs of alkyne-
control of cell behavior in response to an applied external
stimulus. Liu et al. demonstrated that cells attach to the E
35
isomer of RGD-conjugated azobenzene SAMs. However,
when irradiated with UV light, azobenzene adopted the Z
conformation, masking RGD, and the cells detached. Using a
photodeprotection strategy, Lee and colleagues selectively
exposed and conjugated SAM regions with RGD to probe
cell adhesion, polarization, and migration behaviors. Morev-
over, Lamb and Yousaf reported a dynamic and switchable
strategy based on electrochemically controlled hydroquinone
terminated tetra(ethylene glycol) (alkyne-EG SH) and tetra(ethylene
4
glycol)-terminated (EG SH) alkanethiols are generated on gold
4
substrates and reacted with hydroquinone- and azide-functionalized
cell adhesive peptide RGD (HQ-RGD-N ). HQ is considered “off”
3
36
and can be turned “on” for oxyamine (OA) ligand conjugation by
electrochemical oxidation. Ligands that are functionalized with OA
groups (i.e., peptides, KKKTTK-OA, PHRSN-OA, RGD-OA, cRGD-
OA, and sugars, Man-OA, Gal-OA) react and conjugate to Q-
presenting surfaces under physiological conditions. Cells are cultured
and observed on surfaces displaying cell adhesive RGD and variable
biomolecule. In situ electrochemical reduction dynamically releases the
variable biomolecule, and cellular response to this environmental
change is monitored.
(
HQ) SAMs, in which the affinity of RGD ligands was
37
altered. Upon the application of a specific oxidative (Ox) or
reductive (Red) electrochemical potential, the HQ could be
turned “off” and “on” to reveal or hide RGD ligands for cell
attachment studies. Furthermore, in a mild, reducing environ-
ment, HQ has been shown to release its covalently bound
ligand in the presence of living cells for subsequent
immobilization and release cycles with negligible affects on
generate K(N )-C linker-G(HQ)GRGDSS, where the N
3
3
6
moiety is conjugated to alkyne-terminated SAMs via click
chemistry and the HQ serves as a conjugation site for a variety
of oxyamine-containing ligands. Synergy peptide PHSRN, high
affinity cyclic RGD (cRGD), putative HS-binding sequence
KKKTTK, and monosaccharides galactose (Gal) and mannose
(Man) were functionalized with oxyamine groups and surveyed
for potential synergistic or antagonistic effects with cell-
adhesive RGD. Fibroblasts (Fbs) were seeded to the different
ECM mimics substrates, with or without HQ-RGD, and the
number of attached cells, spreading area, morphologies, and
migration rates were tabulated. Inhibition and competitive
binding studies were also performed in which soluble FN,
cRGD, and HS were added. Chondroitinase ABC and
heparinase I and II were also delivered to Fbs in culture to
determine whether HS-binding KKKTTK exhibited a syner-
gistic effect on cell adhesion and spreading. FAK protein levels
were also detected and quantified. Furthermore, the ligands
were released in the presence of cells, providing the dynamic
component to our system, and cell adhesion, morphology, and
migration rates were again examined. To our knowledge, this is
3
8,39
cell behavior and viability.
In vivo, ECM proteins and cell
surfaces are constantly being remodeled and modified, where
ligands are subjected to different compositions and orientations
for biomolecular recognition. Therefore, model substrates that
can mimic and modulate the highly evolving ECM would serve
as great analytical tools, providing insight into the mechanisms
of cell adhesion and migration in real time.
Herein, we report a dynamic, redox-responsive strategy to
immobilize and release ligands in the presence of cells for cell
adhesion, spreading, morphology, and migration studies
(
Figure 1). Electrochemistry enables the complete quantitative
control over ligand density and provides a dynamic molecular
switch for the combinatorial discovery of ligand effects. Two
bioothogonal coupling methodologies, click and oxime
chemistry, were incorporated, and an HQ- and azide (N )-
functionalized RGD peptide (HQ-RGD) was synthesized to
modulate the ECM. Commercially available lysine-N and
derivatized glycine-HQ were incorporated into Ser-Ser-Asp-
Gly-Arg-Gly-C6 linker via solid-phase peptide synthesis to
3
3
1
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Article
the first report that uses a density-controlled, bioorthogonal,
and stimuli-responsive model ECM to probe ligand−cell
surface integrin and syndecan interactions in situ. The ability
to switch ligands for the combinatorial screening of synergistic
of antagonistic ligand effects provides a platform that would be
of tremendous significance to the biosensor and biomaterials
research communities.
Scheme 1. Synthetic Scheme of HQ-RGD: (A) Solution
a
Synthesis of Fmoc-glycine-HQ and (B) Solid-Phase Peptide
b
Synthesis of HQ-RGD Using MBHA Resin
MATERIALS AND METHODS
■
Chemicals and Reagents. All chemicals and reagents were of
analytical grade and used without further purification. Common
chemicals were obtained from Fischer Scientific (Pittsburgh, PA) or
Sigma-Aldrich (St. Louis, MO) unless specified. Rink amide 4-
methylbenzylhydrylamine (MBHA) resin, amino acids [Fmoc-Arg-
(
Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-
OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Lys(N )-
3
OH], Boc-aminooxyacetic acid, Fmoc-ε-Ahx-OH, and HBTU were
purchased from Anaspec (San Jose, CA). Antibodies antipaxillin and
antivinculin and Cy-2 goat anti-mouse IgG were purchased from BD
Biosciences (San Jose, CA) and Jackson ImmunoResearch Labo-
ratories, Inc. (West Grove, PA), respectively. Fluorescent dyes DAPI
and phalloidin and penicillin/streptomycin were obtained from
Invitrogen (Carlsbad, CA). Fluorescence mounting medium was
purchased from Dako (Carpinteria, CA). Swiss albino 3T3 mouse
fibroblasts were obtained from the UNC-CH Tissue Culture Facility
(Chapel Hill, NC). Heparan sulfate, heparinase I and II, and
chondroitinase ABC were obtained from Sigma-Aldrich (St. Louis,
MO).
Syntheses. Alkyne-terminated tetra(ethylene glycol) alkanethiol
4
0
(
(
alkyne-EG SH, 7) was synthesized as previously reported. Tetra-
4
ethylene glycol)-terminated alkanethiol (EG SH, 8) was prepared as
4
41
previously described. Rhodamine-oxyamine (Rhod-OA) was synthe-
sized as previously demonstrated. Galactose- and mannose-oxyamine
5 and 6, respectively) were also synthesized as previously reported.
42
a
4
3
Reagents and conditions: (i) DHP (4.5 equiv), HCl (cat.), THF, 16
(
h, 67.4%; (ii) tert-butyllithium (2 equiv), 1,6-dibromohexane (2
Synthetic routes and a list of molecules are respectively shown in
Schemes 1 and 2.
equiv), dry THF, 0−25 °C, 20 h, 50.6%; (iii) NaN (1.5 equiv), DMF,
3
2
h, 80 °C; and (iv) Fmoc-propargylglycine (0.5 equiv), CuSO ·5H O
4 2
1
,4-Bis((tetrahydro-2H-pyran-2-yl)oxy)benzene (A). To a solution
(1.5 equiv), NaAsc (1.5 equiv), DMF/H O/EtOH (2:1:1), 12 h,
2
b
of hydroquinone (6.0 g, 54.5 mmol) in tetrahydrofuran (THF, 40 mL)
were added 3,4-dihydropuran (20.8 mL, 245.3 mmol, 4.5 equiv) and
three drops of concentrated HCl (cat.). The mixture was stirred for 16
h at room temperature. The reaction contents were then diluted with
ethyl acetate (EtOAc, 20 mL) and extracted with 1 M sodium
bicarbonate (3 × 25 mL) and brine (1 × 25 mL) and concentrated to
6
1.1%. Reagents and conditions: (i) piperidine (20%); (ii) repetition
of DIEA (3 equiv), HBTU (3 equiv), Fmoc-amino acid (SSDGRG, 3
equiv), DMF, then piperidine (20%); (iii) DIEA (3 equiv), HBTU (3
equiv), Fmoc-Gly-HQ (3 equiv), DMF, then piperidine (20%); (iv)
DIEA (3 equiv), HBTU (3 equiv), Fmoc-C -linker (3 equiv), Fmoc-
6
Lys-N (3 equiv), piperidine (20%), acetic anhydride (10%), then
3
afford a white solid, A. The solid product was dried under vacuum for
1
TFA/H
2
O (9.75/0.25, 2 h), ether suspension, centrifugation (2 ×
an additional 3 h (10.23 g, 67.4%). H NMR (400 MHz, CDCl , δ):
3
2
000 rpm, 10 min), dissolve and freeze in H O (10 mL), lyophilize.
2
1
−
=
.58−1.67 (m, 6H, J = 36 Hz; −CH −), 1.85−1.88 (m, 4H, J = 12 Hz;
2
Overall yield of 54 mg, 65%.
CH −), 2.00−2.03 (m, 2H, J = 8 Hz; −CH −), 3.60−3.62 (m, 2H, J
2
2
8 Hz; −CH −), 3.96−3.98 (m, 2H, J = 8 Hz; −CH −), 5.32−5.34
2
2
(
t, 2H, J = 7 Hz; −CH−), 7.00 (s, 4H; Ar−H).
2
,2′-((2-(6-Azidohexyl)-1,4-phenylene)bis(oxy))bis(tetrahydro-2H-
2
,2′-((2-(6-Bromohexyl)-1,4-phenylene)bis(oxy))bis(tetrahydro-
pyran) (C). To a solution of B (1.90 g, 4.32 mmol) in DMF at 80 °C
was added sodium azide (0.56 g, 8.64 mmol, 2 equiv). The mixture
was stirred for 2 h at 80 °C and was then cooled, diluted with DCM
2
H-pyran) (B). To a stirring solution of A (3.0 g, 10.8 mmol) in dry
THF (100 mL) at 0 °C was added tert-butyllithium (1.5 M in pentane,
10.1 mL, 13.0 mmol, 1.2 equiv) dropwise. After the base addition, a
(
20 mL), extracted with H O (3 × 25 mL), dried over MgSO , and
concentrated to afford a pale yellow oil, C (1.72 g, 99.9%). No further
2
4
white precipitate formed. The mixture was stirred for 2 h at 0 °C and
then warmed to room temperature for 3 h. 1,6-Dibromohexane (3.3
mL, 21.6 mmol, 2 equiv) was then added, and the reaction was stirred
for 16 h to afford a pale yellow liquid. The mixture was then diluted
1
purification was required. H NMR (400 MHz, CDCl , δ): 1.38−1.39
3
(
1
m, 4H, J = 4 Hz; −CH −), 1.59−1.61 (m, 10H, J = 8 Hz; −CH −),
2
2
.84−1.85 (m, 4H, J = 4 Hz; −CH −), 1.95−1.97 (m, 2H, J = 8 Hz;
2
+
‑
with EtOAc (20 mL); extracted with NH Cl (2 × 25 mL), H O (1 ×
4
2
−CH −), 2.57−2.61 (t, 2H, J = 16 Hz; −CH −), 3.23−3.26 (m, 2H, J
2
2
5
0 mL), and brine (1 × 50 mL); dried over MgSO ; and concentrated
4
= 12 Hz; −CH −), 3.56−3.61 (m, 2H, J = 16 Hz; −CH −), 3.86−
2
2
to a pale yellow oil. Silica flash column chromatography (Hex/EtOAc/
DCM, 8:1:1) was employed to purify B (2.4 g, 50.6%). H NMR (400
3
−
.96 (m, 2H, J = 40 Hz; −CH −), 5.28−5.30 (m, 2H, J = 8 Hz;
2
1
CH −), 6.80−6.82 (m, 2H, J = 8 Hz; Ar−H), 6.99−7.01 (d, 1H, J =
2
MHz, CDCl , δ): 1.40−1.42, 1.46−1.47 (d × m, 4H, J = 8 Hz;
3
8 Hz; Ar−H).
−
−
−
CH −), 1.49 (m, 8H; −CH −), 1.86−1.88 (m, 6H, J = 8 Hz;
2
2
2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-(6-(2,5-bis-
((tetrahydro-2H-pyran-2-yl)oxy)phenyl)hexyl)-1H-1,2,3-triazol-4-yl)-
propanoic Acid (Fmoc-Gly(HQ), D). To a solution of C (1.64 g, 4.51
mmol, 2 equiv) and Fmoc-propargylglycine (0.76 g, 2.26 mmol) in
DMF (20 mL) and EtOH (5 mL) was added a solution of CuSO4·
CH −), 2.02 (m, 2H; −CH −), 2.60−2.62 (t, 2H, J = 8 Hz;
2
2
CH −), 3.41−3.44 (m, 2H, J = 12 Hz; −CH −), 3.61−3.62 (m, 2H,
2
2
J = 4 Hz; −CH −), 3.96−3.99 (m, 2H, J = 12 Hz; −CH −), 5.31 (s,
2
2
2H; −CH −), 6.85−6.87 (m, 2H, J = 8 Hz; Ar−H), 7.02−7.04 (d, 1H,
2
J = 8 Hz; Ar−H).
5H O (0.85 g, 3.38 mmol, 1.5 equiv) and sodium ascorbate (0.67 g,
2
1
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2
.5, Labconco Corp., Kansas City, MO) overnight to afford a white
Scheme 2. List of Molecules and Surface Groups Used in
This Study
a
2+
solid, 1 (54 mg, 65%). HRMS (m/z) HQ-RGD: [M] calcd fo+r
C H N O 615.32, found 616.47. HRMS (m/z) RGD-OA: [M]
52
84 19 16
calcd for C H N O 676.34, found 676.31. HRMS (m/z) PHSRN-
2
5
46 11 11
+
OA: [M] calcd for C H N O 923.52, found 923.56. HRMS (m/
z) KKTTK-OA: [M] calcd for C H N O 917.59, found 917.46.
38
67 16 11
+
4
0
79 13 11
Preparation of Gold-Coated Substrates and Monolayers.
2
Glass coverslips (75 × 25 mm ) were immersed into a piranha solution
(
1:3 (v:v) concentrated H SO :30% H O ; use with caution) for 4 h,
2 4 2 2
followed by rinsing with deionized H O and EtOH. Gold substrates
2
were prepared by electron-beam deposition (Model VE-100,
Thermionics Laboratory, Inc., Port Townsend, WA) of titanium (5
nm) and then gold (12 nm for cell work and 50 nm for
electrochemical measurements). The gold-coated slides were cut
2
into 1 × 2 cm pieces. To form SAMs on gold, the slides were
immersed in an ethanolic solution containing the alkanethiols (1 mM
of 5% alkyne-EG SH for cell studies and 100% alkyne-EG SH for
4
4
electrochemical characterization) for at least 16 h. Once removed from
solution, the surfaces were rinsed with EtOH and dried with an air
stream before use.
HQ-RGD Immobilization. After monolayer formation with 1 mM
of 1% alkyne-EG SH/EG SH, a solution containing 10 mmol HQ-
4
4
RGD, 15 mmol CuSO ·5H O, and 15 mmol NaASc (1:1:1) in H O
4
2
2
and EtOH (3:1) was added to the substrates and allowed to react for
0 min. Substrates were then rinsed with EtOH and dried with a
9
stream of air, and HQ-RGD immobilization was confirmed by cyclic
voltammetry (CV).
Electrochemical Activation. Electrochemical experiments were
performed using a BAS 100B/W electrochemical analyzer (Bio-
analytical Systems, Inc., West Lafayette, IN) in a 1 M HClO4
electrolyte solution with an Ag/AgCl electrode serving as the
reference, the gold monolayer as the working electrode, and a Pt
wire as the counter electrode. Surfaces were scanned at a rate of 100
mV/s from −100 to 850 mV to activate “on” (quinine form).
Ligand Immobilization and Release. Peptides and sugars were
a
The following molecules are depicted: (1) hydroquinone- and azide-
functionalized RGD, HQ-RGD; (2) cyclic RGD-functionalized
oxyamine, cRGD; (3) PHSRN-functionalized oxyamine, PHSRN;
added to RGD-Q-presenting surfaces (20 mM in H O) and allowed to
2
(
4) KKKTTK-functionalized oxyamine, KKKTTK; (5) galactose-
react for 90 min. Immobilized ligands were confirmed using the same
electrochemical parameters listed above. The ligands were released by
applying potential for 12 cyclic scans (−100 to 850 mV) in PBS buffer
(pH 7) and were characterized using similar conditions.
functionalized oxyamine, Gal; (6) mannose-functionalized oxyamine,
Man; (7) alkyne-terminated tetra(ethylene glycol) alkanethiol, alkyne-
EG SH; and (8) tetra(ethylene glycol)-terminated alkanethiol,
4
EG SH.
Cell Culture and Surface Seeding. Swiss albino 3T3 mouse
fibroblasts were cultured in Dulbecco’s modified Eagle medium
containing 10% calf bovine serum and 1% penicillin/streptomycin.
When passaging or seeding, a 1 mL solution of 0.05% trypsin in 0.53
mM EDTA was employed to remove cells from the tissue culture
4
3
.38 mmol, 1.5 equiv) in H O (10 mL) and EtOH (5 mL). The
2
reaction formed a cloudy orange precipitate and was stirred for 16 h at
room temperature. The mixture was diluted with DCM (30 mL),
5
plastic. For passaging, cells (10 cells/mL) were then added to a new
culture flask containing fresh media and placed in the incubator (37
extracted with H O (4 × 100 mL), stirred in a solution containing
EDTA (2 × 50 mL, 10 mM in H O) for 10 min, dried over MgSO ,
2
2
4
°
C, 5% CO ) to grow and divide. For surface seeding, cells were
2
and concentrated. The colorless oil was then purified via silica flash
column chromatography using a gradient of MeOH/DCM (0−5%
5
3
resuspended in serum-free medium (10 cells/mL) and diluted to 10
cells/mL with serum-free medium. The cells were then seeded to
surfaces for 2 h, and after 2 h, serum-containing media was added to
promote cell growth.
Cell Staining. After 2 h of cell growth, the cells were fixed with
formaldehyde (3.2% in PBS) and then permeated (PBS containing
MeOH) to afford a white solid, D (1.02 g, 61.1%). HRMS (m/z):
+
[
M] calcd for C H N O 738.36, found 738.38. HPLC: t = 15.2
4
2
50
4
8
r
min, 0.1% TFA and 5−50% H O/ACN over 25 min.
2
Solid-Phase Peptide Syntheses (SPPS). Rink amide MBHA
resin (0.127 g, 0.1 mmol) was delivered to an automated glass
chamber for peptide synthesis. The amino acids listed above were
measured (0.30 mmol) and diluted in 5 mL of DMF (10 mL for Ser
and Gly) and also delivered to peptide synthesizer chambers. The
following reagents were prepared in DMF: 0.1 M HBTU, 0.1 M DIEA,
and 20% piperidine. The peptide synthesizer (C S Bio Co., Peptide
Synthesizer Division, Menlo Park, CA) was programmed to generate
the following sequences, separately, over the course of 12 h: resin-S-S-
D-G-R-G-G(HQ)-C (linker)-K(N )-amide (1), resin-S-D-G-R-G-
0
.1% Triton X-100). Two fluorescent dye mixtures were made with
the following components: 10 mmol of phalloidin (1.6 μL), 10 mmol
of antipaxillin or antivinculin (1 μL), and 5% normal goat serum with
0
.1% Triton X -100 (397.4 μL), or 10 mmol of phalloidin (10 μL), 10
mmol of Cy-2 (1.25 μL), 1 mmol of DAPI (μL), and 5% normal goat
serum with 0.1% Triton X-100 (487.8 μL). Cells were immersed in
each staining solution for 1 h. The substrates were then secured with
their gold-coated side down in fluorescence mounting medium (Dako,
Carpinteria, CA), which enhances the visualization of cells when
viewed under a fluorescent microscope, on a glass coverslip.
6
3
C (linker)-ONH (2), resin-N-R-S-H-P-K-C (linker)-ONH (3), and
6
2
6
2
resin-K-T-T-K-K-K-C (linker)-ONH (4). Following elongation, the
6
2
resin was washed with DMF and DCM repeatedly and then cleaved
from the resin in an N -bubbling solution of H O/TFA (10 mL,
Fluorescence Microscopy. Fluorescent images were obtained
with a Nikon Eclipse TE2000-E inverted microscope (Nikon USA,
Inc., Melville, NY) and a Plan Fluor 40× oil immersion objective (1.30
NA, Nikon USA). Immersion oil was purchased from Carl Zeiss
MicroImaging, Inc. (Thornwood, NY) and the lens paper was
2
2
0
.25:9.75) for 1 h. The filtrate was drained into a tube containing cold
ether to form a white precipitate that was then centrifuged (2 × 2000
rpm, 10 min), dissolved in H O (10 mL), and lyophilized (FreeZone
2
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6
obtained from Fisher. Image analysis was performed using MetaMorph
software (Molecular Devices, Downingtown, PA).
Focal Adhesion Kinase (FAK) Assay. Fbs (10 cells/mL) were
incubated for 4 h on the following substrates at 2% ligand density:
HQ-RGD + PHSRN, KKKTTK, or cRGD. After incubation, the FAK
protein levels were detected and quantified using an ELISA kit
(Invitrogen, Camarillo, CA), following the manufacturer’s instructions.
Briefly, the cells were removed from each substrate with trypsin,
centrifuged (1000 rpm, 5 min), resuspended in ice-cold PBS (2 mL),
centrifuged (1000 RMP, 5 min), and lysed. Then, 50 μL of the FAK
detection antibody was added and incubated with 50 μL of each cell
sample for 3 h at room temperature, after which the samples were
aspirated and washed with PBS (4 × 2 mL). The samples were then
incubated with 100 μL of HRP anti-rabbit antibody for 30 min at room
temperature and then washed (4 × 2 mL PBS). One hundred
microliters of Stabilized Chromagen was then added for 30 min at
room temperature, after which 100 μL of stop solution was then added
to each sample. The optical densities were measured at 450 nm using a
Beckman Du-640 spectrophotometer (GMI, Ramsey, MN) and
compared to the standard FAK concentrations.
Cell Adhesion Assay. The number of adhered cells were
measured 2 h after seeding on the following substrates at 2% ligand
density: ±HQ-RGD and Gal, Man, PHSRN, KKKTTK, and cRGD.
3
Two hundred microliters of cells (∼10 cells/mL) was added to each
2
substrate (1 cm ), which was subsequently incubated at 37 °C and 5%
CO for 2 h. Two random regions of four different substrates for each
2
ligand combination were imaged at a 4× resolution using a Nikon
Eclipse TS100 (Nikon USA, Inc., Melville, NY). The attached cells
were then counted. The data are expressed as the mean ± SEM of
eight replicates.
2
Cell Area Determination Assay. Cell areas (μm ) were measured
h after seeding on the following substrates at 2% ligand density:
2
±
HQ-RGD and Gal, Man, PHSRN, KKKTTK, and cRGD. Two
3
hundred microliters of cells (∼10 cells/mL) was added to each
2
substrate (1 cm ), which was subsequently incubated at 37 °C and 5%
CO for 2 h. The cells were then fixed as previously reported, and two
2
random cell areas from four different substrates for each ligand
combination were imaged at a 20× resolution using a Nikon Eclipse
TS100 (Nikon USA, Inc., Melville, NY). Image analyses (Nikon
Eclipse TE2000-E inverted microscope; Nikon USA, Inc., Melville,
NY),was carried out with MetaMorph software (Molecular Devices,
Downingtown, PA). The data are expressed as the mean ± SEM of
eight replicates.
Cell Migration Rate Determination. Cells were cultured and
seeded at concentrations of approximately 103 cells/mL onto the
following substrates at 2% ligand density, as described: HQ-RGD and
HQ-RGD + Gal, Man, PHSRN, KKKTTK, or cRGD. Migration rates
were determined after recording the movement of cells for 18 h in
bright-field mode (Nikon Eclipse TE2000-E inverted microscope;
Nikon USA, Inc., Melville, NY). Eight randomly selected cells (four
cells per substrate condition, two substrates per condition) were
imaged each hour for 18 h, and the displacements from their original
adherent position were tracked and calculated using MetaMorph
software (Molecular Devices, Downingtown, PA). The data are
expressed as the mean ± SEM of eight replicates.
RESULTS AND DISCUSSION
■
Dynamic Surface Design and Ligand Selection Ration-
ale. The development and use of smart materials that have
switchable or stimuli-responsive properties have proven to be
important for a number of biological studies ranging from
fundamental basic cell biology research to biomedical implants
31−34
and tissue engineering scaffolds.
Therefore, we aimed to
modulate the dynamic ECM with redox-responsive surfaces to
survey ligand effects on cell behavior. FN is an abundant
glycoprotein that promotes cell adhesion to the ECM via
integrin (i.e., transmembrane, cell surface receptors) inter-
actions (Figure 1A). Two key peptide sequences, Arg-Gly-Asp
(1, RGD, Scheme 2) and Phe-His-Ser-Arg-Asn (3, PHRSN,
Scheme 2), located in FNIII10 and FNIII , respectively, were
identified as cell-binding ligands. Polymers, nanoparticles, and
other biomaterials are routinely functionalized with both linear
9
Electrochemical Characterization. HQ-RGD was immobilized
to alkyne-terminated SAMs (100%, 1 mM in EtOH, 16 h), as
previously described. The immobilization was confirmed using a BAS
(
K ∼ μM) and cyclic (2, cRGD; K ∼ nM, Scheme 2) forms of
RGD to promote integrin recognition and subsequent cell
d
d
1
00B/W electrochemical analyzer (Bioanalytical Systems, Inc., West
44,45
attachment.
PHSRN is described as a synergy ligand with
Lafayette, IN) in a 1 M HClO electrolyte solution with an Ag/AgCl
4
RGD and interacts simultaneously with α β integrins to
electrode serving as the reference, the gold monolayer as the working
electrode, and a Pt wire as the counter electrode. Samples were
scanned at 100 mV/s ranging from −100 to 850 mV. To immobilize
oxyamine-containing ligands, the substrates were activated and turned
on after performing a linear scan from −100 to 850 mV at 100 mV/s.
After ligand immobilization, the redox reversible oxime signal was
confirmed using CV with the parameters just mentioned. The ligands
were then release, as described, and confirmed by CV.
5 1
15−18
mediate cell adhesion and migration.
Furthermore, an HS
binding domain, located within FNIII12‑14, was identified and
promotes cell surface syndecan and integrin corecognition and
interaction. With the FN structure in mind, we hoped to
reproduce the combined ligand effects of coupling PHSRN and
cRGD with RGD on cell adhesion, spreading, morphology, and
migration. As a new ligand, we chose to survey the combined
effects of RGD and Lys-Lys-Lys-Thr-Thr-Lys (4, KKKTTK,
Scheme 2), which bares four positive charges, on cell behavior.
We assumed that KKKTTK would mediate electrostatic
interactions with negatively charged cell surface HSPGs,
mimicking the HS binding domain on FN and producing a
synergistic effect on cell adhesion, growth, and migration.
Detailed peptide structures are displayed in Scheme S2 in the
Supporting Information.
4
Enzymatic Adhesion Assay. Fbs (∼10 cells/mL) were
suspended in serum-free media and treated separately with heparinase
I and heparinase II (0.025 U/mg), chondroitinase ABC (0.025 U/mg),
or no enzyme (control) for 30 min at room temperature. Cells were
then seeded to substrates presenting 2% HQ-RGD and 2% HQ-RGD
+
KKKTTK for 2 h, after which the surfaces were fixed. Two random
regions of four different substrates for each ligand combination were
imaged at a 4× resolution using a Nikon Eclipse TS100 (Nikon USA,
Inc., Melville, NY). The attached cells were then counted. The data are
expressed as the mean ± SEM of eight replicates.
Cell Detachment Assay with Soluble Molecules. Fbs (104
cells/mL) were seeded in a suspension of serum-free media on the
following substrates at 2% ligand density for 4 h: HQ-RGD + PHSRN,
KKKTTK, or cRGD. After 4 h, several soluble molecules, GRGDS,
cGRDSF, HS, and FN (0.1 μM), were added for 1 h. The Fbs were
then fixed. Two random regions of three to four different substrates for
each ligand combination were imaged at a 4× resolution using a Nikon
Eclipse TS100 (Nikon USA, Inc., Melville, NY). The attached cells
were then counted. The data are expressed as the mean ± SEM of 5−8
replicates.
We also chose to investigate the dual-ligand effects of RGD
with galactose (5, Gal, Scheme 2) and mannose (6, Man,
Scheme 2) monosaccharides due to a few published works. Du
et al. reported the enhancement of hepatocyte adhesion using a
hybrid Gal/RGD monolayer via hepatic asialoglycoprotein
46
receptor interactions. Fbs that express the mannose receptor,
which contains a FNII domain, have been shown to exhibit
specificity in binding to type I, III, and IV collagens to facilitate
47
cell−ECM adhesion. Thus, we hypothesized to observe an
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increase in cell adhesion and spreading and a decrease in
migration when presented with RGD.
The general dynamic redox-responsive surface strategy to
present two ECM ligands is represented in Figure 1B,C, and a
more detailed schematic is provided in Scheme S1 (Supporting
Information (SI)). Mixed EG SH/alkyne-EG SH SAMs
4
4
(
9
Scheme 2) were formed on gold substrates in a ratio of
9:1 (1 mM total in EtOH, 16 h). Both alkanethiols are
resistant to nonspecific protein adsorption and cell adhesion,
which is extremely important when conducting biospecific
4
8−50
ligand−receptor interaction studies.
Here, EG SH (99%
4
density) serves as the inert background and alkyne-EG SH (1%
4
density) provides an alkyne terminal group for ligand
immobilization via click chemistry with an azide-containing
RGD ligand. The molecule density was maintained at 1% to
ensure that only the specific interactions between the ligands in
question with Fb cell surface receptors occur.
The redox-responsive trigger, in the form of a hydroquinone/
quinone (HQ/Q) couple, was built into an Fmoc-protected
glycine residue (E, Scheme 1A) and was compatible with
routine solid-phase peptide synthesis (Figure S1, SI). E was
incorporated into an RGD-containing peptide (1, HQ-RGD,
Schemes 1 and 2) that was capped with an azide-functionalized
Figure 2. Number of adhered cells after 2 h of culture on the following
substrates: single ligands (green) cRGD, Man, KKKTTK, Gal, or
PHSRN; dual ligands (blue) HQ-RGD + cRGD, Man, KKKTTK, Gal,
or PHSRN. Statistical analyses were performed with respect to HQ-
RGD: *p < 0.001 and **p < 0.01.
lysine residue for coupling to the 1% alkyne-EG SH SAMs via
4
click chemistry (20 mM in H O/EtOH (3:1), cat. CuSO ·
2
4
5
H O and NaAsc, 90 min). We have previously shown and
2
extensively characterized the immobilization and release of
oxyamine (OA)-functionalized ligands (i.e., peptides, small
molecules, and carbohydrates) to and from HQ/Q SAMs on
gold substrates for a number of biotechnological applications
3
seeded to surfaces (∼10 cells/mL, 2 h), fixed, imaged, and
counted after 2 h. As shown in Figure 2, all ligands were able to
support cell adhesion to varying degrees. The data are
represented as the average number of attached cells per 4×
frame (eight random regions). HQ-RGD and cRGD demon-
strated similar affinity for attracting Fbs when compared to the
surfaces presenting Man, Gal, KKKTTK, and PHSRN, which
exhibited almost a 2-fold reduction in the amount of attached
Fbs. Additionally, the Fbs were more loosely adhered and
adopted a rounder morphology on substrates presenting Man,
Gal, KKKTTK, and PHSRN. However, when these ligands
were combined with and immobilized to SAMs with 1% Q-
RGD (2% ligand total), the amount of cells increased
significantly, approximately 2-fold, resembling the results
observed with 2% HQ-RGD. When Man and Gal were coupled
to HQ-RGD, Fbs behaved similarly to when in the presence of
cell adhesive RGD; similar numbers of attached cells and mor
phologies were observed. When PHSRN was coupled to HQ-
RGD, a synergistic effect on the amount of cells adhered and
over cell spreading and morphology was observed, although, at
surface value, the actual number of attached cells did not
increase significantly from the substrates with 2% HQ-RGD.
Surprisingly, KKKTTK coupled to HQ-RGD showed a
dramatic increase in the amount of attached Fbs when
compared to HQ-RGD- and KKKTTK-presenting substrates,
indicating a profound synergistic effect between KKKTTK and
RGD on adhesion. A marked 0.5-fold increase from HQ-RGD
substrates was observed on KKKTTK + HQ-RGD.
37−39
and cell behavioral studies.
As shown in Figure 1B, after
HQ-RGD-N3 immobilization to alkyne-EG SH SAMs, the
4
substrates are oxidized [Ox] using linear sweep voltammetry to
the corresponding Q (1 M HClO , −100 to 850 mV, 100 mV/
4
s), which then reacts rapidly and chemoselectively at room
temperature and under physiological conditions (20 mM in
PBS, pH of 7, 2 h) to form an oxime conjugate. The oxime
bond is stable in all pH ranges until application of a reducing
potential at a pH of 7, at which the ligands are spontaneously
3
8,39
cleaved, regenerating the original HQ-RGD-tethered SAM.
Ligand immobilization and release to and from HQ-RGD-
presenting SAMs were characterized by electrochemistry and
fluorescence microscopy (Figures S2 and S3, respectively, SI).
A key feature of this system is that both HQ-RGD and the
corresponding oxime conjugate exhibit signature redox signals
that can be monitored and quantified by cyclic voltammetry
(
CV) in terms of ligand immobilization, release, and density,
characteristics that are significant when designing a platform for
biological investigation. Thus, this dual-ligand ECM and
combinatorial screening strategy possesses the following
features: a dynamic, molecular redox-responsive trigger for
immobilizing and releasing structurally well-defined ligands in
the presence of cells; density control over ligands; and rapid,
chemoselective, and bioorthogonal coupling reactions (i.e.,
Huisgen cycloaddition and oxime chemistry).
Cell Adhesion Assays. Before testing our dual-ligand ECM
system, we first verified whether Fbs could adhere and healthily
spread on substrates presenting HQ-RGD and each decoupled
ligand (Figure 2). As such, HQ-RGD and cRGD, Man, Gal,
PHSRN, and KKKTTK ligands were immobilized to alkyne-
EG SH (20 mM in H O/EtOH (3:1), cat CuSO ·5H O and
Cell Spreading, Morphology, and Migration Assays.
Upon adhering to the ECM or another cell, a cell undergoes
the following sequential events: spreading, actin cytoskeleton
organization, and focal adhesion (FA) formation (Figure 1A).
The cell then waits in anticipation of receiving and processing
extracellular signals and migrates from various epithelial layers
to target locations, where it differentiates to form a specialized
cell that comprises different tissues and organs.
4
2
4
2
NaAsc, 90 min) and HQ-terminated (20 mM in PBS, 2 h)
SAMs, respectively, at a 2% ligand density. Fbs cells were then
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Similar immobilization and fixing conditions were employed
to survey the possible synergistic or antagonistic effects of
coupling cell adhesive RGD with a number of peptides and
carbohydrates on cell spreading, morphology, and migration
rate. Cell areas were measured, and FAs and actin were
visualized by staining for vinculin (antivinculin and Cy-2,
green) and F-actin (phalloidin, red) after 2 h of culture on all
surfaces (Figure 3A,B). Cells were then observed to migrate on
substrates during an 18-h period via life-cell recordings, and
migration rates were calculated (Figure 3C). Notably, the focus
of this work was to observe the possible synergistic or
antagonist effects of an artificial, dual-ligand ECM. Our cell
attachment and spreading results in Figures 2 and 3A,
respectively, demonstrate significant increases in the size and
number of cells adhered to dual-ligand-presenting SAMs when
compared to substrates displaying single HQ-RGD, cRGD,
PHSRN, KKKTTK, Gal, and Man ligands. Thus, cell migration
behavior was only examined on dual-ligand surfaces.
As shown in Figure 3A,B, when compared to HQ-RGD, Fbs
were extremely well spread on both cRGD and HQ-RGD +
cRGD presenting surfaces. These results are not surprising,
because the RGD sequence in native FN is located on a β-turn,
and thus, the cyclic form of RGD has a nanomolar affinity for
integrin binding, compared to linear RGD, which has
5
1,52
micromolar cell-binding affinity.
Cells exhibited a 30%
2
2
increase (HQ-RGD, 630 μm ; cRGD, 850 μm ; HQ-RGD +
cRGD, 850 μm ) in area from HQ-RGD with a more
2
pronounced and intricate network of actin striations and FAs
on the periphery and along the actin extensions of the main cell
body (right panel, Figure 3B). These morphological character-
istics are indicative of well-adhered, healthy cells. Moreover,
when comparing Fb migration on HQ-RGD (7.2 μm/h), cells
exhibited a 2-fold decreased rate (3.1 μm/h) on HQ-RGD +
cRGD, corroborating the adhesion, spreading, and morphology
data (Figure 3C).
Synergistic effects of coupling HQ-RGD + PHSRN on Fb
spreading and morphology were observed, verifying the results
from the cell adhesion assay. An approximate 4-fold increase in
2
average cell area from PHSRN (180 μm ) to HQ-RGD +
2
PHSRN (700 μm ) was recorded, indicating that PHSRN,
which resembles FNIII , does not alone sustain adequate
9
attachment and growth. This result was expected due to the
18,30
lower binding affinity of integrins for PHSRN.
Moreover,
cells were larger on average than those on HQ-RGD alone (630
2
μm ). The cell morphologies were drastically different upon
observing Figure 3A,B. Cells on PHSRN alone remained small
with rounded ruffling features at the cell periphery and little to
no FA formations, as compared to cells on HQ-RGD. However,
when coupled to HQ-RGD, cells spread but appeared to be in a
migratory state; the actin cytoskeleton was not fully extended
and striated and FAs were not strongly pronounced. These
cellular characteristics were further verified with the migration
data represented in Figure 3C. Cells on HQ-RGD + PHSRN
(
11.9 μm/h), demonstrated a 66% increase in migration rate
Figure 3. Representative cell areas (A), morphologies (B), and
migration rates (C) after 2 h of culture on the following substrates
presenting single ligands (green) cRGD, Man, KKKTTK, Gal, or
PHSRN and dual ligands HQ-RGD (blue) + cRGD, Man, KKKTTK,
Gal, or PHSRN. Cells in part B were stained for actin (red, phalloidin),
nucleus (blue, DAPI), focal adhesions (FAs, green, antivinculin/Cy 2).
Migration rates were calculated from an 18-h period using live-cell
recording and imaging software. Each bar represents the mean ± SEM
(n = 8). Statistical analyses were performed with respect to HQ-RGD:
*p < 0.001 and **p < 0.01.
when compared to cells on HQ-RGD (7.2 μm/h). These
results demonstrate a relationship between increased migration
rate and cell ruffling dynamics, suggesting that our cell findings
on RGD/PHSRN substrates were induced by ligand effects.
These data corroborate previous reports showing that PHSRN
causes ECM invasion of Fbs and keratinocytes and stimulates
corneal epithelial cell migration, accelerating wound healing in
5
3,54
mice and rabbits, respectively.
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Although marked differences were observed in the Fb
ligand ECM substrates. In Figure 4A, the addition of soluble
FN had significant effects on the cells that were adhered to HQ-
spreading, areas, and morphologies on Gal and HQ-RGD + Gal
2
(
420−600 μm ) and Man and HQ-RGD + Man (380−610
2
μm ), no overall synergistic effects were observed when
2
compared to HQ-RGD alone (630 μm ) (Figure 3A). This
conclusion was also apparent from the images in Figure 3B.
Similar to PHSRN, Fbs on Gal and Man adopted small and
round shapes with no noticeable FA formations and organized
actin cytoskeleton. Ruffling characteristics are typical of cells
that are feeling and sensing their environment. The increase in
cell area after Man and Gal immobilization to Q-RGD is most
likely due to the presence of cell adhesive RGD. The cells
appear more spread with FA formations at the tips of spikey
actin extensions. However, the cell bodies remain rounded, and
the actin cytoskeleton is not well structured; it appears that
these monosaccharide ligands may be partially masking the
adhesive properties of RGD. When observing the migration
data, the rate differences were minimal when comparing cells
on HQ-RGD and HQ-RGD + Man or Gal substrates (7.2, 7.4,
and 7.5 μm/h, respectively). Interestingly, Fbs migrated at
approximately the same rate on both RGD and carbohydrate-
presenting SAMs (Figure 3C).
As previously mentioned, KKKTTK weakly supported the
attachment of Fbs. However, when compared to the average
2
area of cells on HQ-RGD (630 μm ), cells on KKKTTK alone
2
exhibited a comparable average area of 560 μm . Although no
defined FAs were formed on the cell peripheries (left panel,
Figure 3B), the actin cytoskeleton appeared to be partially
organized, with striated patterns and small extensions. This area
may be due to the electrostatic interactions, generated from the
positive lysine residues at physiological conditions and highly
negatively charged cell surface. When coupled to HQ-RGD, the
average cell area on KKKTTK + HQ-RGD increased by
approximately 50% that of cells on HQ-RGD alone, indicating a
synergistic effect of spreading and growth. When imaged, cells
maintained a rounded, spread, dense cell body, as seen with
cells on KKKTTK alone; however, the appearance of strongly
pronounced FAs at the cell periphery and increased actin
organization was observed (left panel, Figure 3B). When
considering the difference in cell migration rates, Fbs on HQ-
RGD + KKKTTK demonstrated a 20% decrease from that of
cells on HQ-RGD alone. The data was statistically significant (p
Figure 4. Cell adhesion inhibition assays. (A) Soluble competitive
inhibition studies with (orange) FN, (blue) cRGD, and (light purple)
HS and (B) enzymatic treatment of cells with (olive green)
chondroitinase ABC and (dark purple) heparinase I/II on the
following substrates: HQ-RGD and HQ-RGD + cRGD, KKKTTK,
or PHSRN. Each bar (mean ± SEM) represents an average of five to
4
eight trials (∼10 cells/mL).
RGD and HQ-RGD + KKKTTK; an approximate 75%
reduction of attached cells was observed. Thirty percent of
the Fbs on HQ-RGD + cRGD detached from the surface after
FN addition, and only 10% lifted from HQ-RGD + PHRSN, as
expected. Therefore, RGD alone and the combination of RGD
with KKKTTK or cRGD do not exhibit as strong of a
synergistic effect on promoting strong cell attachment as
originally anticipated. For all substrates, the addition of soluble
cRGD and HS had minimal effects on Fb detachment. The high
binding affinity small peptide and negatively charge oligosac-
charide were not strong enough to detach the cells from the
dual-ligand ECM mimics, most likely due to already established
cell surface integrin− and syndecan−ligand interactions. On the
basis of the other evidence in this section, RGD + KKKTTK
remains a good dual-ligand ECM platform, but is not as ideal as
natural ligand FN.
We also investigated the enzymatic effects of heparinase I
and II (Hep I/II) and chondroitinase ABC (chon ABC) on
cleaving the HS and CS chains of PGs that are found on Fb
membranes. These enzymes were incubated with Fbs at 0.025
units/mg in serum-free media for 1 h, after which the cells were
added to HQ-RGD and HQ-RGD + KKKTTK (2%) for 2 h.
The Fbs were then fixed, counted, and compared to the
<
0.0001), indicating that the presence of KKKTTK does
exhibit a combined ligand effect. These significant morpho-
logical distinctions and decreased migration rate further
support the synergistic effects of RGD (FNIII ) and KKKTTK
1
0
(
FNIII12‑14) in promoting cell surface integrin and syndecan
interactions.
Ligand Inhibition Studies on Cell Adhesion. Because
the ligand combinations HQ-RGD + cRGD, HQ-RGD +
KKKTTK, and HQ-RGD + PHSRN demonstrated an effect on
Fb adhesion, several soluble molecule and enzyme inhibition
studies were performed. We first surveyed the number of
attached cells on HQ-RGD + cRGD, HQ-RGD + KKKTTK,
and HQ-RGD + PHSRN substrates (2%) before and after the
addition of FN, cRGD, and HS (0.1 mmol in PBS). FN is a
natural adhesion protein in the ECM, and we choose cRGD
because integrins have a high binding (nM) affinity for the
ligand. HS is a repeating disaccharide that possesses a
carboxylate and varying sulfate groups. Thus, at physiological
conditions, the overall net charge of HS is highly negative. We
assumed that addition of soluble FN, cRGD, and HS would
interfere with the binding interactions of Fbs and our dual-
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controls (−Hep I/II or −Chon ABC). Figure 4B presents the
number of adhered cells after enzyme treatment. As shown,
HQ-RGD showed little change in attracting cells to the surface
with the following results: no treatment, 59; +Hep I/II, 57; and
cells on +PHSRN when compared to HQ-RGD. The combined
ligand effects of RGD with KKKTTK on enhancing cell
adhesion and FA formation, as determined by the increased
FAK concentration, were again verified. Thus, RGD +
KKKTTK is a good dual-ligand ECM mimic.
+
chon ABC, 55. This could be due to the fact that only a net
charge of +1 is present in the same ligand area (arginine in HQ-
RGD). Therefore, cleaving the HS and CS groups from cell
surfaces did not have an effect on integrin-mediated cell
attachment. However, when compared to HQ-RGD +
KKKTTK, Fbs experienced a 50% decrease in adhesion after
enzymatic treatment of Hep I/II and chon ABC with the
following results: no treatment, 83; +Hep I/II, 41; +chon ABC,
Modulation of Cell Behavior via a Dynamic ECM
Model Surface. The final study in this work concerned the
dynamic release of immobilized ligands in the presence of cells.
Fb spreading areas, migration rates, and morphologies were
investigated after releasing PHSRN, Gal, Man, KKKTTK, and
cRGD from separate HQ-RGD-presenting SAMs. The data are
presented in Figure 6A−C. Remarkably, after ligand release and
5
3. Furthermore, the results corroborated the previous
adhesion results; more cells adhered to surfaces due to the
synergistic effects of HQ-RGD and KKKTTK. The inhibition
in cell adhesion after Hep I/II and chon ABC treatment is most
likely due to the decreased electrostatic interactions of the
negatively charged cell surface with positively charged dual-
ligand ECM (+5 net charge in one concentrated area), as well
as less interaction with syndecan-4 surface receptors.
Focal Adhesion Kinase Assay. To confirm the results
from inhibition and morphological assays, we detected and
quantified the FAK levels in cells subject to HQ-RGD + cRGD,
HQ-RGD + KKKTTK, and HQ-RGD + PHSRN substrates.
FAK serves a major role in cell adhesion, spreading,
differentiation, migration, division, and apoptosis. Evidence of
enhanced FAC was observed in morphological data of the dual-
ligand ECM systems listed above. Thus, Fbs were incubated on
these substrates for 4 h, and using an ELISA kit and
spectrophotometry, the FAK levels were quantified. The lysates
of each cell population were diluted to measure the FAK level
range and generate a linear relationship between the optical
density and FAK concentration. The total FAK concentration
was then determined according to each substrate. As shown in
Figure 5, higher FAK concentrations were observed in Fbs after
Figure 6. Cell behavioral responses to the dynamic release of ECM
ligands. Cell (A) areas, (B) migration rates, and (C) morphologies
after releasing (blue) cRGD, Man, KKKTTK, Gal, or PHSRN from
HQ-RGD. Fbs were cultured on the dual ECM ligand-presenting
surfaces for 2 h, after which the ECM ligands were electrochemically
released (PBS, pH 7, 12 cyclic scans: −100 to 850 mV, 100 mV/s) and
incubated for an additional 2 h. Migration rates were calculated from
an 18-h period using live-cell recording and imaging software after
ligand release. Each bar (mean ± SEM) represents an average of eight
3
trials (10 cells/mL).
4
h adjusted time, the cells more or less adopted similar
phenotypes (Figure 6C), motility (Figure 6B), and spreading
areas (Figure 6A) to those of substrates that presented HQ-
RGD. The Fbs that were subject to Man, Gal, and PHSRN
release reorganized and extended their actin protrusions,
spreading out to adopt similar sizes and migration rates.
Similarly, the cells adhered to substrates in which KKKTTK
and cRGD were released reorganized their actin cytoskeleton,
focal adhesion assemblies, and stress fibers and contracted
slightly to adapt to their new ligand stimulus, HQ-RGD. Thus,
these results demonstrate the powerful nature of this surface
platform in modulating the dynamic ECM environment, where
ligands, proteins, and small molecules are constantly being
hidden and revealed to cells.
Figure 5. Total FAK (ng/mL) concentrations measured in cells on the
following substrates: (black) HQ-RGD and HQ-RGD + (blue) cRGD,
(pink) KKKTTK, or (green) PHSRN. Each bar (mean ± S.E.M.)
represents an average of three trials from the same batch dilution
6
(
∼10 cells/mL).
ligand immobilization to HQ-RGD SAMs when compared to
substrates only baring HQ-RGD (35 ng/mL, black), with
+
cRGD (59 ng/mL, red) and +KKKTTK (58 ng/mL, blue)
surfaces possessing similar FAK levels, followed by +PHSRN
50 ng/mL, green) functionalized substrates. Therefore, the
(
FAK assay confirmed the morphological data; more FAs were
formed in the cells on +KKKTTK and +cRGD and less in the
1
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Article
CONCLUSIONS
ACKNOWLEDGMENTS
■
■
In summary, we developed a model substrate for in situ cell
biological studies that dynamically modulates the ECM. A small
library of biomolecules, Gal, Man, PHSRN, cRGD, and
KKKTTK, was synthesized to bear an oxyamine group that
reacts rapidly and chemoselectively with carbonyl moieties.
This reaction is also bioorthogonal and can be performed in the
presence of cells without inducing any side reactions with
proteins and lipids. A cell adhesive HQ-containing RGD was
This work was supported by the Carolina Center for Cancer
Nanotechnology Excellence, the Burroughs Wellcome Founda-
tion (Interface Career Award), the National Science
Foundation (Career Award), National Science and Engineering
Research Council of Canada (NSERC), and the Canadian
Foundation for Innovation (CFI). The authors thank J. Park for
HPLC help, Brian Matthew for HPLC and MS assistance, and
Nathan Westcott for valued advice.
also synthesized and immobilized to bioinert alkyne-EG SH
4
SAMs to provide the ketone (in the form of electrochemically
oxidized quinone) for dual-ligand display, and cell adhesion,
spreading and growth, migration, and adhesion inhibition were
measured. An added benefit of this platform included the
redox-responsive trigger that turns the system on and off. Such
a feature allows modulation of the dynamic ECM environment,
where ligands, proteins, and small molecules are constantly
being hidden and revealed to cells. In surveying the synergistic
or antagonistic effects of the immobilized and released ligands
to and from HQ-RGD, many key results were observed. When
comparing the ligands, the number of attached cells increased
for all ligands when immobilized to HQ-RGD. However, HQ-
RGD + KKKTTK showed a dramatic increase of 50%,
indicating a possible synergistic effect of simultaneous cell
surface integrin and syndecan-4 interactions with the cell- and
HS-binding domain mimics of FN. Furthermore, the formation
of more FA in the morphological data was observed in the
images and confirmed by the increased FAK levels from control
substrates (HQ-RGD). The decreased migration rates and
soluble and enzymatic inhibition assays also demonstrated that
KKKTTK serves as a synergistic ECM ligand with RGD in
promoting cell attachment, spreading, and division. When
cRGD and PHSRN were investigated, results similar to those
reported previously in the literature were observed, and Man
and Gal had no effect on cell adhesion, spreading, and
migration. Thus, not only does this dynamic dual-ligand ECM
enable the immobilization and release of ligands in the presence
of cells, but it also provides a platform for the combinatorial
screening of ligands to further probe the synergistic or
antagonistic effects of cell adhesive RGD with other molecules.
This surface strategy can be applied to nanoparticles for the
redox-state-dependent delivery and release of therapeutics and
ABBREVIATIONS USED
■
Alkyne-EG SH, alkyne-terminated tetra(ethylene glycol) alka-
4
nethiol; ECM, extracellular matrix; EG SH, tetra(ethylene
4
glycol) alkanethiol; FA, focal adhesion; Fbs, fibroblasts; FN,
fibronectin; cRGD, cyclic Arg-Gly-Asp; Gal, galactose; HQ,
hydroquinone; HQ-RGD, hydroquinone Arg-Gly-Asp; Kd,
dissociation constant; KKKTTK, Lys-Lys-Lys-Thr-Thr-Lys;
Man, mannose; OA, oxyamine; Q, quinone; PHSRN, Phe-
His-Ser-Arg-Asn; SAMs, self-assembled monolayers.
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Corresponding Author
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