Dynamic Interfaces between Cells and Surfaces: Electroactive Substrates that Sequentially Release and Attach Cells

Article abstract of DOI:10.1021/ja038265b

An electroactive substrate that combines dual dynamic properties is demonstrated. A monolayer is patterned to first release an immobilized ligand, and therefore adherent cells, on application of an electrical potential. Subsequently, electrical oxidation

Full text of DOI:10.1021/ja038265b

Published on Web 11/14/2003
Dynamic Interfaces between Cells and Surfaces: Electroactive Substrates
that Sequentially Release and Attach Cells
Woon-Seok Yeo, Muhammad N. Yousaf, and Milan Mrksich*
The UniVersity of Chicago, Department of Chemistry, Institute for Biophysical Dynamics,
5735 South Ellis AVenue, Chicago, Illinois 60637
Received September 1, 2003; E-mail: mmrksich@uchicago.edu
An important theme in surface chemistry is the design of
interfaces between cells and materials. This work has had an
important impact in basic studies to understand the adhesion of
cells and in many applied areas, including biomaterials and tissue
engineering.1 An exciting recent focus has been the development
of substrates that are dynamic and offer real-time control over the
presentation of ligands to attached cells. Early examples of dynamic
substrates have relied on the use of thermally responsive2 and
photoactive3 gels to manipulate the adsorption of proteins and
therefore the adhesion of cells. We have previously described
electroactive substrates that directly switch ligand activities on or
off in response to electrical potentials.4 In this communication, we
describe an important advance by demonstrating a substrate that
combines two dynamic properties: the release of one ligand
followed by the immobilization of a second ligand.
We demonstrate our approach using a self-assembled monolayer
(SAM) that incorporates an O-silyl hydroquinone moiety to present
Figure 1. (A) Structures for the functionalized alkanethiol used to prepare
a peptide ligand (E*-RGD, Figure 1). The Arg-Gly-Asp peptide is
a ligand for cell surface integrin receptors and serves to mediate
the adhesion of cells while the surrounding tri(ethylene glycol)
groups serve to prevent the nonspecific attachment of cells to the
monolayer.5 The O-silyl hydroquinone ether is electroactive and
provides for selective release of the peptide from the substrate. The
release is triggered by applying an electrical potential to the
substrate, effecting the oxidation of this group to yield the
corresponding benzoquinone with hydrolysis of the silyl ether. The
resulting benzoquinone group undergoes a selective immobilization
reaction with a diene-tagged peptide (RGD-Cp, Figure 1A) by way
of a Diels-Alder reaction and, therefore, provides the basis for a
second dynamic activity. The benzoquinone is redox active and
can be reduced to the hydroquinone, which prevents immobilization
of the diene-tagged ligand. In this work, we demonstrate these two
dynamic properties by observing the effects of ligand modulation
on the adhesion of cells.
We first characterized the electrochemical modulation of the
monolayer. We prepared a self-assembled monolayer comprising
a diethylisopropylsilyl hydroquinone-terminated alkanethiolate and
a tri(ethylene glycol)-terminated alkanethiolate in a ratio of 1:3
(Figure 2A). MALDI-TOF mass spectrometry of this monolayer
showed two major peaks at m/z 694 and m/z 1014 (Figure 2B, left).6
The first peak corresponds to the symmetric disulfide derived from
a tri(ethylene glycol)-terminated alkanethiolate, and the second peak
corresponds to the mixed disulfide derived from a diethylisopro-
pylsilyl hydroquinone-terminated alkanethiolate and a tri(ethylene
glycol)-terminated alkanethiolate. An electrical potential of 550 mV
was applied to an identical monolayer for 5 min to generate the
benzoquinone followed by a potential of -550 mV for 30 s to
reduce the benzoquinone to the hydroquinone.7 MALDI-TOF
revealed a new peak at m/z 886 corresponding to the mixed disulfide
derived from a hydroquinone-terminated alkanethiolate and a tri-
dynamic substrates (E*-RGD) and the cyclopentadiene moiety (RGD-Cp)
used to selectively immobilize ligand. (B) A monolayer presenting the
O-silyl hydroquinone undergoes electrochemical oxidation to give a
benzoquinone, with hydrolysis of the silyl ether and selective release of
the RGD ligand. The resulting benzoquinone reacts with RGD-Cp by way
of a Diels-Alder reaction, which selectively immobilizes the second ligand.
The RGD peptide mediates the adhesion of cells.
(ethylene glycol)-terminated alkanethiolate (Figure 2B, right). The
electrochemical generation of benzoquinone was verified with cyclic
voltammetry, which showed the voltammetric waves for the
benzoquinone redox pair and the loss of the benzoquinone on
addition of cyclopentadiene (see Supporting Information).8 This
result confirms that benzoquinone groups were generated by the
electrical potential and consumed through Diels-Alder reaction
with cyclopentadiene. As a final control experiment, we synthesized
an analogue of E*-RGD that does not contain the alkanethiol moiety
and performed a bulk electrolysis in phosphate-buffered saline
(PBS) at pH 7.4. Extraction of the aqueous solution with dichloro-
methane gave the corresponding benzoquinone in ∼90% yield
(Figure 2C).
To demonstrate the application of this strategy to manipulating
the adhesion of cells, we patterned a monolayer into circular regions
(220 µm in diameter) with hexadecanethiol and modified the
remaining regions with E*-RGD at a density of 0.02% mixed with
a tri(ethylene glycol)-terminated alkanethiolate (Figure 3).9 This
patterned substrate was treated with a solution of fibronectinsa
protein that contains the RGD peptide and mediates cell adhesions
which resulted in the adsorption of protein only to the circular
regions. Swiss 3T3 fibroblast cells were incubated on the substrate
for 2 h.10 An optical micrograph showed that cells adhered to the
monolayer and were evenly distributed across the regions presenting
fibronectin and RGD peptide (Figure 3A). To selectively release
RGD peptides outside the circular features, we applied an electrical
potential of 550 mV to the monolayer for 5 min.11 Cells on these
9
14994
J. AM. CHEM. SOC. 2003, 125, 14994-14995
10.1021/ja038265b CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
by the electrochemical treatment as described in Figure 1B. We
next replaced the cell medium with the serum-free medium
containing RGD-Cp at a concentration of 1 mM for 1 h and then
replaced the medium with serum-containing medium. Following
the Diels-Alder mediated immobilization of the peptide, cells began
to migrate from fibronectin-coated circular regions onto surrounding
regions of the substrate (Figure 3C). After 14 h of culture, the
original circular pattern was no longer evident, and after 24 h, cells
were distributed evenly over the entire surface. When the quinone
was electrically reduced to give the hydroquinone before addition
of RGD-Cp, cells did not migrate and remained localized to the
circular pattern. Further, cells did not respond to the immobilization
of an inactive RGE peptide (see Supporting Information). This
example demonstrates that the electrochemical strategy for both
releasing and attaching ligands from/to the monolayer is effective
in complex cell culture media.
The demonstration of a dynamic substrate that combines two
functions makes a significant advance on previous reports of
substrates that can modulate ligand activities. The ability to effect
multiple changes in the bioactivity of a substrate will be especially
useful in studies of heterotypic cell-cell interactions where the fate
of a given cell depends on the identities and periods of exposure
to neighboring cells. The dynamic substrates will also find use in
microfluidic lab-on-a-chip systems by allowing active use of
channels to process analytes in a sample. More broadly, this
example illustrates a flexible strategy for using synthetic and
physical organic chemistry to design and prepare surfaces that
display complex functions.
Figure 2. Characterization of the electrochemical oxidation of O-silyl
hydroquinone groups on the monolayer. (A) A monolayer presenting
diethylisopropylsilyl hydroquinone groups was treated with an electrical
potential of 550 mV for 5 min followed by a potential of -550 mV for 30
s to give the hydroquinone. (B) A MALDI-TOF mass spectrum for the
initial monolayer displayed a peak at m/z 1014 corresponding to the mixed
disulfide derived from a diethylisopropylsilyl hydroquinone-terminated
alkanethiolate and a tri(ethylene glycol)-terminated alkanethiolate (left).
After electrochemical treatment, the original peak was absent and gave rise
to a new peak at m/z 886 corresponding to the hydroquinone-terminated
disulfide (right). (C) Bulk electrolysis of a model compound gave the
corresponding benzoquinone.
Acknowledgment. This work was supported by National
Science Foundation and the National Institutes of Health.
Supporting Information Available: Synthesis and experimental
details for E*-RGD, cyclic voltamograms showing electroactive cleav-
age, and a control experiment with RGE-Cp (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) (a) Mrksich, M. Chem. Soc. ReV. 2000, 29, 267. (b) Hubbell, J. A. Curr.
Opin. Biotechnol. 1999, 10, 123.
(2) (a) Okano, T.; Yamata, N.; Sakai, J.; Sakurai, Y. Biomaterials 1995, 16,
297. (b) Collier, T. O.; Anderson, J. M.; Kikuchi, A.; Okano, T. J. Biomed.
Mater. Res. 2002, 59, 136. (c) Shimizu, T.; Yamato, M.; Kikuchi, A.;
Okano. T. Biomaterials 2003, 24, 2309.
(3) (a) Hern, D. L.; Hubbell, J. A. J. Biomed. Mater. Res. 1998, 39, 266. (b)
Elbert, D. L.; Hubbell, J. A. Biomacromolecules 2001, 2, 430.
(4) (a) Hodneland, C. D.; Mrksich, M. J. Am. Chem. Soc. 2000, 122, 4235.
(b) Yeo, W.-S.; Hodneland, C. D.; Mrksich, M. ChemBioChem 2001, 2,
590. (c) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 5992. (d) Yousaf, M. N.; Houseman, B. T.; Mrksich,
M. Angew. Chem., Int. Ed. 2001, 40, 1093.
(5) Chemistry and Biological Applications of Polyethylene Glycol; Mrksich,
M., Whitesides, G. M., Eds.; ACS Symposium Series 680; American
Chemical Society: Washington, DC, 1997; pp 361.
(6) The monolayers were analyzed on a Voyager-DE Biospectroscopy mass
spectrometer using 2,5-dihydroxybenzoic acid (1 µL of 10 mg/mL solution
in acetonitrile) as a matrix. Sodium adducts of disulfides are the major
species observed in MALDI spectra of SAMs: see (a) Su, J.; Mrksich,
M. Angew. Chem., Int. Ed. 2002, 41, 4715. (b) Su, J.; Mrksich, M.
Langmuir 2003, 19, 4867-4870. (c) Trevor, J. L.; Lykke, K. R.; Pellin,
M. J.; Hanley, L. Langmuir 1998, 14, 1664.
(7) Electrochemistry was performed with a Bioanalytical Systems CV-50W
potentiostat using PBS (pH 7.4) as the electrolyte. All experiments used
a custom-designed electrochemical cell with the monolayer as the working
electrode, a Pt wire as the counter electrode, and an Ag/AgCl reference
electrode.
Figure 3. Demonstration of a dynamic substrate that combines two dynamic
properties: (i) the release of RGD ligands and, thus, the release of cells,
(ii) the immobilization of RGD ligands and, hence, migration and growth
of cells. A monolayer was patterned into circular regions that present
fibronectin and surrounded by RGD ligands tethered by way of an
electroactive linker (E*-RGD). (A) Swiss 3T3 fibroblast cells adhered and
spread evenly over entire substrate. (B) An electrical potential of 550 mV
was applied to the substrate for 5 min, and the substrate was incubated for
4 h. Cells were efficiently released only from the E*-RGD regions. (C)
Treatment of the monolayer with RGD-Cp resulted in ligand immobilization
and initiated cell migration from fibronectin regions onto remaining regions.
After 24 h, cells were distributed evenly over the substrate.
(8) (a) Yousaf, M. N.; Mrksich, M. J. Am. Chem. Soc. 1999, 121, 4286. (b)
Kwon, Y.; Mrksich, M. J. Am. Chem. Soc. 2002, 124, 806.
regions immediately began to adopt a round shape and detach from
the monolayer. An optical micrograph showed that most cells on
E*-RGD regions were released, while cells on the fibronectin-coated
circular regions were not affected by the electrical potential and
remained on the monolayer (Figure 3B). This result indicates that
the RGD ligand was released from the O-silyl ether tethered SAM
(9) The preparation of patterned substrates is described in ref 4d.
(10) Swiss Albino 3T3 cells (ATCC, Rockville, MD) were grown in Dulbecco’s
Modified Eagle Medium (DMEM) containing 10% fetal bovine serum
and 1% penicillin/streptomycin. All cultures were maintained at 37 °C in
a humidified 5% CO2 atmosphere.
(11) DMEM media containing serum, pH 7.4, used as solvent and electrolyte.
JA038265B
9
J. AM. CHEM. SOC. VOL. 125, NO. 49, 2003 14995