Phototriggering of cell adhesion by caged cyclic RGD peptides

Article abstract of DOI:10.1002/anie.200704857

Restrained potential: A caged cyclic peptide attached to a surface is able to trigger cell attachment to the surface with spatiotemporal definition upon exposure to light (λ = 351 nm). The peptide shows no integrin-binding activity in its caged form, but mediates cell adhesion effectively after irradiation (see optical microscopy image of cells on a surface irradiated through a mask in bands 100 μm in width). (Figure Presented).

Full text of DOI:10.1002/anie.200704857

Communications
DOI: 10.1002/anie.200704857
Cell–Surface Interactions
Phototriggering of Cell Adhesion by Caged Cyclic RGD Peptides**
Svea Petersen, JosØ María Alonso, Alexandre Specht, Portia Duodu, Maurice Goeldner, and
Aranzazu del Campo*
The controlled and selective adhesion of cells to surfaces is an
important issue in cell biology and tissue engineering. Differ-
ent strategies have been reported in which thermally,[1]
photochemically,[2,3] and electrochemically[4] responsive sur-
faces and materials are used to manipulate cell adhesion. A
more generic approach that would be suitable for any system,
independent of its chemical constitution, would be advanta-
geous. Such a strategy could not rely on material properties;
instead, the molecular interactions involved in cell attach-
ment must be controlled directly.
The design of a strategy to trigger the attachment event
needs to consider the sensitivity of cells to most triggering
sources (electric fields, chemical stimuli, pressure, and
temperature jumps). Light of wavelength above 320 nm
appears to be a convenient trigger, as its interaction with
biomolecular species is negligible. Light-controlled cellular
attachment requires the development of photosensitive
molecules able to mediate cellular adhesion and whose
activity changes upon irradiation. For this study, we selected
the RGD cell-adhesive peptide, well known to promote
integrin-mediated cell adhesion,[5,6] and modified it by
Scheme 1. Chemical structure of cyclo[RGD(DMNPB)fK] (DMNPB in
red) attached to the surface through the TEGlinker (green). The
caging group is released upon irradiation at 351 nm.
introducing a photolabile caging group on the carboxylic
acid side chain of the aspartic acid residue (Scheme 1). The
presence of the caging group may cause steric hindrance,
conformational constraint, or changes in the charge distribu-
tion of the peptide and thus prevent recognition of the
peptide by the integrins. Light irradiation releases the cage
from the peptide structure and restores the activity of the
peptide to enable in situ site and temporal control of cell
attachment. Cell-repellent surfaces modified with the caged
peptide (“off” state) can become cell-adhesive (“on” state)
upon irradiation with light of the appropriate wavelength and
intensity.
The selection of the caging position requires previous
knowledge of the structural characteristics of the RGD–
integrin binding site. In the particular case of the pentapep-
tide cyclo(-Arg-Gly-Asp-d-Phe-Val-) (cyclo(RGDfK)),
a
very active and selective ligand of integrin aVb3,[7] it has
been shown that the binding site involves two divalent cations,
and that the aspartate unit acts as a ligand for one of them.[8,9]
Therefore, we decided to introduce the caging group at this
position. It is also known that the amino acid in the fifth
position (Lys) does not have significant influence on the
activity of the peptide.[7] The free amine group of the Lys
residue has been used as anchoring position through which
the peptide can be coupled to surfaces.[10]
[*] S. Petersen,[+] Dr. J. M. Alonso,[+] Dr. A. del Campo
Max-Planck-Institut für Metallforschung
Heisenbergstrasse 3, 70569 Stuttgart (Germany)
Fax: (+49)711-689-3412
E-mail: delcampo@mf.mpg.de
3-(4,5-Dimethoxy-2-nitrophenyl)-2-butyl ester (DMNPB)
was selected as the photolabile caging group (lmax = 346 nm,
emax = 4100mÀ1 cmÀ1).[11] The caged Asp derivative DMNPB-
Asp-Fmoc (Fmoc = 9-fluorenylmethoxycarbonyl) and the
caged peptide cyclo[RGD(DMNPB)fK] were obtained and
S. Petersen,[+] A. Specht, P. Duodu, M. Goeldner
Laboratoire de Chimie Bioorganique UMR7175 CNRS
FacultØ de Pharmacie, UniversitØ Louis Pasteur Strasbourg
BP 24, 67401 Illkirch (France)
[+] These authors contributed equally to this research.
characterized
as
described
in
the
Supporting
[**] We thank Neo MPS Laboratories (Strasbourg, France) for synthe-
sizing the peptide, and Alexandra Goldyn, Henriette Ries, and Ralf
Kemkemer (research group of Prof. J. Spatz, MPI für Metal-
lforschung, Stuttgart, Germany) for help with the cell experiments.
The RGD peptide sequence is Arg-Gly-Asp.
Information.[11,12] Their UV spectra are shown in Figure 1.
The photolytic properties of the caged peptide in solution
were then determined quantitatively. Upon exposure for 2 h
to light of wavelength 364nm in neutral buffered solution, up
to 70% of cyclo[RGD(DMNPB)fK] disappeared, and up to
93% of the photolytic reaction product obtained was
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
3192
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 3192 –3195

Angewandte
Chemie
contact angle of the surface increased by 38 upon peptide
coupling to the linker. This value agrees with literature values
for RGD-modified silicon wafers.[16] Ellipsometry measure-
ments showed a small increase in the layer thickness, almost
within the error range.
We examined the kinetics of the photolysis of DMNPB at
the silica surface on the basis of the decay of the UV/Vis
absorption of the peptide-modified substrates at 351 nm after
different irradiation times (Figure 1b; see also the
Supporting Information). Irradiation cleaves the DMNPB
group from the surface layer. Subsequent washing removes
the chromophore from the surface. According to the absorb-
ance values, irradiation for 10 min at 351 nm cleaved 64% of
the chromophores. Longer irradiation times did not cause a
further significant decrease in absorption. We attribute the
remaining absorption to DMNBP entrapped as a photo-
product within the surface layer and unable to be removed by
washing.
The ability of the caged- and uncaged-RGD-modified
surfaces to attach cells selectively through RGD–integrin
recognition was tested in a cell-adhesion assay. Fibroblasts
3T3 were plated onto substrates modified with cyclo[RGD-
(DMNPB)fK] before and after irradiation, and onto sub-
strates modified only with the linker molecule as a negative
control. We then analyzed the adhesion of fibroblasts by
optical microscopy after different incubation times (3, 6, and
21 h) and counted the number of attached cells per unit area
(Figure 2). A very small number of attached cells (ca.
2000 cellscmÀ2) were observed for substrates modified only
with the linker molecule after incubation for 6 h; this number
increased to 5000 cellscmÀ2 after incubation for 21 h. This
result confirms that the TEG surface prevents nonspecific cell
adhesion. It is remarkable that the cell-repelling properties of
the surface remain almost unchanged after incubation for 21 h
in spite of the short length of the TEG spacer.
Figure 1. a) UV spectra of DMNPB-Asp-Fmoc, cyclo(RGDfK), linear
RGDfK, and cyclo[RGD(DMNPB)fK] in solution. b) UV absorption
measured at 351 nm for substrates modified with cyclo[RGD-
(DMNPB)fK] after irradiation at 351 nm for different periods of time
and washing. The decay in the absorption values corresponds to the
cleavage of the chromophore from the surface during exposure to
light.
identified as uncaged cyclo(RGDfK). A quantum yield of
0.34was determined (see the Supporting Information). [13]
To test specific RGD-mediated integrin binding to
surfaces, nonspecific interactions between the integrins or
any other molecule at the cell membrane and the surface
should be prevented. At the same time, the retention of
protein activity and cell function in the surface-immobilized
state is necessary. These requirements are fulfilled typically
by using surface-modification agents that contain oligo(ethyl-
ene glycol) (OEG) spacers.[14] Therefore, we synthesized a
bifunctional tetra(ethylene glycol) (TEG) linker containing a
triethoxysilane moiety at one end for anchoring to the silica
surface and an N-hydroxysuccinimide-activated carboxylic
acid functionality at the other end for coupling to the free
amine group of the Lys residue of the caged peptide.[15] The
structure of the linker is shown in Scheme 1, and its synthesis
is described in the Supporting Information.
Silica surfaces were modified with the linker by solution-
phase silanization. Silane layers of up to 1.75 Æ 0.05 nm in
thickness were obtained, and a static contact angle of 408 was
observed. The coupling of the peptide to the surface was
monitored by UV/Vis spectroscopy on modified quartz
substrates. The profile of the absorbance bands corresponding
to the DMNPB chromophore and the peptide bonds
appeared to be identical to the spectrum in solution. The
Figure 2. Cell density on surfaces modified with cyclo[RGD-
(DMNPB)fK] before and after exposure to light of wavelength 351 nm
for 10 min. Data are given for nonpatterned and patterned substrates.
For comparison, data for surfaces modified only with the linker
molecule are also shown.
Angew. Chem. Int. Ed. 2008, 47, 3192 –3195ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3193

Communications
The number of attached cells on the surfaces containing
caged cyclo[RGD(DMNPB)fK] is similar to the number of
cells attached to the substrate without the adhesive peptide, a
result that indicates the lack of bioactivity of the caged RGD
peptide. The number of cells attached to surfaces containing
active cyclo(RGDfK) was approximately 11 times higher
after incubation for 3 h and 13 times higher after incubation
for 6 h. This result serves as evidence that irradiation removes
the DMNPB cage and restores bioactivity. The maximum
selectivity (difference in the number of cells attached to
substrates modified with active RGD and substrates without
active RGD) was observed after incubation for 6 h. Longer
incubation times led to an increase in the number of cells
attached to the substrate, but decreased selectivity. This
decrease in selectivity may be a consequence of the adsorp-
tion of proteins from the medium on the surface. To improve
the selectivity, more-repellent surface layers (e.g. the use of
longer OEG chains) would be necessary.
These results show that cell attachment to surfaces can be
inhibited efficiently by blocking the carboxylic acid side chain
of the aspartic acid residue in the RGD motif. This conclusion
is supported by the fact that cells attached to RGD-containing
surfaces already show a high degree of spreading after 6 h,
whereas cells attached to caged-RGD-containing surfaces
need 21 h to start spreading. The use of the photosensitive
DMNBP cage as a trigger for light-modulated cell adhesion
provides much better results in terms of efficiency and
selectivity than other reported photoactivation strategies
based on the azobenzene unit.[17–19] However, the activation
process is not reversible with the caged compounds.
We then tested the possibility of using cyclo[RGD-
(DMNPB)fK] for in situ site-selective control of cell attach-
ment. Cells were first plated on substrates modified with
cyclo[RGD(DMNPB)fK], and the substrate was subse-
quently masked irradiated from the back side. Irradiation
generated stripes of activated RGD on the surface. Figure 3
shows microscopic images of plated cells after different
incubation times. The cells adhered preferentially to the
irradiated regions, which contain active RGD, and made a cell
pattern on the substrate. Cells on these samples were always
oriented statistically; a negligible effect of the pattern on cell
behavior was observed. Consequently, the results obtained
can be attributed solely to the presence or absence of RGD.
A new caged peptide able to phototrigger cell attachment
at surfaces has been developed. In its caged form, cyclo-
[RGD(DMNPB)fK], the peptide did not show any integrin-
binding activity. Upon activation with light, the peptide
effectively mediated cell adhesion to surfaces with spatio-
temporal definition. We envision numerous applications of
such systems: Further studies are in progress towards the
development of in situ patternable substrates for cell arrays,
triggerable cell-adhesive coatings for artificial scaffolds in
tissue engineering, and latent solution agents capable of
inhibiting, or even reverting, cell attachment to surfaces after
light activation.
Figure 3. In situ triggering of cell attachment to substrates with
cyclo[RGD(DMNPB)fK]. Cells were first plated onto substrates modi-
fied with the caged peptide and subsequently irradiated through a
mask. The optical microscopy images show cells adhering preferen-
tially to the irradiated regions (stripes of 100 mm in width), in which
the peptide is in its uncaged form. The images show fibroblasts after
incubation for 3 (a), 6 (b), and 21 h (c).
Keywords: cage compounds · cell adhesion · peptides ·
.
photolysis · responsive surfaces
[1] T. Okano, N. Yamada, M. Okuhara, H. Sakai, Y. Sakurai,
Biomaterials 1995, 16, 297.
[2] W. S. Dillmore, M. N. Yousaf, M. Mrksich, Langmuir 2004, 20,
7223.
[3] J. Nakanishi, Y. Kikichi, S. Inoue, K. Yamaguchi, T. Takarada, M.
Maeda, J. Am. Chem. Soc. 2007, 129, 6694.
[4] W. S. Yeo, C. D. Hodneland, M. Mrksich, ChemBioChem 2001,
2, 590.
Received: October 19, 2007
Revised: December 12, 2007
Published online: March 17, 2008
3194
www.angewandte.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 3192 –3195

Angewandte
Chemie
[5] J. D. Humphries, A. Byron, M. J. Humphries, J. Cell Sci. 2006,
119, 3901.
[13] J. W. Walker, G. P. Reid, J. A. McCray, D. R. Trentham, J. Am.
Chem. Soc. 1988, 110, 7170.
[6] U. Hersel, C. Dahmen, H. Kessler, Biomaterials 2003, 24, 4385.
[7] R. Haubner, R. Gratias, B. Diefenbach, S. L. Goodman, A.
Jonczyk, H. Kessler, J. Am. Chem. Soc. 1996, 118, 7461.
[8] D. Kirchhofer, J. Gailit, E. Ruoslahti, J. Grzesiak, M. D.
Pierschbacher, J. Biol. Chem. 1990, 265, 18525.
[14] J. Blummel, N. Perschmann, D. Aydin, J. Drinjakovic, T. Surrey,
M. Lopez-Garcia, H. Kessler, J. P. Spatz, Biomaterials 2007, 28,
4739.
[15] J. M. Alonso, A. Reichel, J. Piehler, A. del Campo, Langmuir,
2008, 24, 448.
[9] E. Ruoslahti, M. D. Pierschbacher, Science 1987, 238, 491.
[10] M. Kantlehner, P. Schaffner, D. Finsinger, J. Meyer, A. Jonczyk,
B. Diefenbach, B. Nies, G. Hꢀlzemann, S. L. Goodman, H.
Kessler, ChemBioChem 2000, 1, 107.
[11] A. Specht, J. S. Thomann, K. Alarcon, W. Wittayanan, D. Ogden,
T. Furuta, T. Kurakawa, M. Goeldner, ChemBioChem 2006, 7,
1690.
[16] A. R. El-Ghannam, P. Ducheyne, M. Risbud, C. S. Adams, I. M.
Shapiro, D. Castner, S. Golledge, R. J. Composto, Wiley Period-
icals Inc. 2004, 615.
[17] J. Auernheimer, C. Dahmen, U. Hersel, A. Bausch, H. Kessler, J.
Am. Chem. Soc. 2005, 127, 16107.
[18] M. Schutt, S. S. Krupka, A. G. Milbradt, S. Deindl, E. K. Sinner,
D. Oesterhelt, C. Renner, L. Moroder, Chem. Biol. 2003, 10, 487.
[19] A. G. Milbradt, M. Loweneck, S. S. Krupka, M. Reif, E. K.
Sinner, L. Moroder, C. Renner, Biopolymers 2005, 77, 304.
[12] J. S. Wood, M. Koszelak, J. Liu, D. S. Lawrence, J. Am. Chem.
Soc. 1998, 120, 7145.
Angew. Chem. Int. Ed. 2008, 47, 3192 –3195ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3195