A molecular toolkit for the functionalization of titanium-based biomaterials that selectively control integrin-mediated cell adhesion

Article abstract of DOI:10.1002/chem.201301478

We present a click chemistry-based molecular toolkit for the biofunctionalization of materials to selectively control integrin-mediated cell adhesion. To this end, α5β1-selective RGD peptidomimetics were covalently immobilized on Ti-based materials, and the capacity to promote the selective binding of α5β1 was evaluated using a solid-phase integrin binding assay. This functionalization strategy yielded surfaces with a nine-fold increased affinity for α5β1, in comparison to control samples, and total selectivity against the binding of the closely related integrin αvβ3. Moreover, our methodology allowed the screening of several phosphonic acid containing anchoring units to find the best spacer-anchor moiety required for establishing an efficient binding to titanium and to promote selective integrin binding. The integrin subtype specificity of these biofunctionalized surfaces was further examined in vitro by inducing selective adhesion of genetically modified fibroblasts, which express exclusively the α5β1 integrin. The versatility of our molecular toolkit was proven by shifting the cellular specificity of the materials from α5β1- to αvβ3-expressing fibroblasts by using an αvβ3-selective peptidomimetic as coating molecule. The results shown here represent the first functionalization of Ti-based materials with α5β1- or αvβ3-selective peptidomimetics that allow an unprecedented control to discriminate between α5β1- and αvβ3-mediated adhesions. The role of these two integrins in different biological events is still a matter of debate and is frequently discussed in literature. Thus, such bioactive titanium surfaces will be of great relevance for the study of integrin-mediated cell adhesion and the development of new biomaterials targeting specific cell types. Copyright

Full text of DOI:10.1002/chem.201301478

FULL PAPER
DOI: 10.1002/chem.201301478
A Molecular Toolkit for the Functionalization of Titanium-Based
Biomaterials That Selectively Control Integrin-Mediated Cell Adhesion
Florian Rechenmacher,[a] Stefanie Neubauer,[a] Carlos Mas-Moruno,[a, g]
Petra M. Dorfner,[b] Julien Polleux,[c] Judith Guasch,[d] Bert Conings,[e]
Hans-Gerd Boyen,[e] Alexander Bochen,[a] Tariq R. Sobahi,[f] Rainer Burgkart,[b]
Joachim P. Spatz,[d] Reinhard Fꢀssler,[c] and Horst Kessler*[a, f]
Abstract: We present a click chemistry-
based molecular toolkit for the bio-
functionalization of materials to selec-
tively control integrin-mediated cell ad-
hesion. To this end, a5b1-selective
RGD peptidomimetics were covalently
immobilized on Ti-based materials, and
the capacity to promote the selective
binding of a5b1 was evaluated using a
solid-phase integrin binding assay. This
functionalization strategy yielded surfa-
ces with a nine-fold increased affinity
for a5b1, in comparison to control
samples, and total selectivity against
the binding of the closely related integ-
rin avb3. Moreover, our methodology
allowed the screening of several phos-
phonic acid containing anchoring units
to find the best spacer–anchor moiety
required for establishing an efficient
binding to titanium and to promote se-
lective integrin binding. The integrin
subtype specificity of these biofunc-
tionalized surfaces was further exam-
ined in vitro by inducing selective ad-
hesion of genetically modified fibro-
blasts, which express exclusively the
a5b1 integrin. The versatility of our
molecular toolkit was proven by shift-
ing the cellular specificity of the mate-
rials from a5b1- to avb3-expressing fi-
broblasts by using an avb3-selective
peptidomimetic as coating molecule.
The results shown here represent the
first functionalization of Ti-based mate-
rials with a5b1- or avb3-selective pep-
tidomimetics that allow an unprece-
dented control to discriminate between
a5b1- and avb3-mediated adhesions.
The role of these two integrins in dif-
ferent biological events is still a matter
of debate and is frequently discussed in
literature. Thus, such bioactive titanium
surfaces will be of great relevance for
the study of integrin-mediated cell ad-
hesion and the development of new bi-
omaterials targeting specific cell types.
Keywords: biomaterials · cell adhe-
sion · click chemistry · integrins ·
peptidomimetics · surface coating ·
titanium
Introduction
ensuing cell adhesion process.[1] By recognizing certain ECM
proteins through defined binding motifs, integrins enable
cell migration, proliferation and survival.[2] As a conse-
quence, they play key functions in cellular processes that re-
The interaction of integrins with extracellular matrix (ECM)
proteins is crucial in the formation of focal contacts and the
[a] Dr. F. Rechenmacher,+ Dr. S. Neubauer,+ Dr. C. Mas-Moruno,
Dr. A. Bochen, Prof. Dr. H. Kessler
[e] Dr. B. Conings, Prof. Dr. H.-G. Boyen
Institute for Materials Research, Wetenschapspark 1
3590 Diepenbeek (Belgium)
Institute for Advanced Study (IAS) and
Center for Integrated Protein Science (CIPSM)
Department Chemie, Technische Universitꢀt Mꢁnchen
Lichtenbergstrasse 4, 85747 Garching (Germany)
E-mail: Kessler@tum.de
[f] Prof. Dr. T. R. Sobahi, Prof. Dr. H. Kessler
Chemistry Department, Faculty of Science
King Abdulaziz University
P.O. Box 80203, Jeddah 21589 (Saudi Arabia)
[b] Dr. P. M. Dorfner, Priv.-Doz. Dr. R. Burgkart
Orthopedic and Trauma Surgery, Klinikum rechts der Isar
Technische Universitꢀt Mꢁnchen, Ismaninger Strasse 22
81675 Mꢁnchen (Germany)
[g] Dr. C. Mas-Moruno
Current address:
Biomaterials, Biomechanics and Tissue Engineering Group
Department of Materials Science and Metallurgical Engineering
Technical University of Catalonia (UPC), Av. Diagonal 647
08028 Barcelona (Spain)
[c] Dr. J. Polleux, Prof. Dr. R. Fꢀssler
Max Planck Institut fꢁr Biochemie
[+] These authors contributed equally to this work.
Department of Molecular Medicine
Am Klopferspitz 18, 82152 Martinsried (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201301478: Detailed synthesis of
all compounds, analytical data for compound characterization, meth-
ods for the competitive solid phase integrin binding assay, the cell ad-
hesion assay and QCM-D experiments.
[d] Dr. J. Guasch, Prof. Dr. J. P. Spatz
Department of New Materials and Biosystems
Max Planck Institute for Intelligent Systems
Heisenbergstrasse 3, 70569 Stuttgart (Germany)
and
Department of Biophysical Chemistry, University of Heidelberg
INF 253, 69120 Heidelberg (Germany)
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quire cell attachment or growth of tissue.[3] The integrin sub-
types avb3 and a5b1 have been described to mediate and
influence cell attachment, adhesion, proliferation and differ-
entiation.[4] Thus, functionalization of surfaces with ligands
that display affinity for these integrin subtypes has been ex-
tensively pursued in order to promote cell adhesion and to
improve the biointegration of implant materials.[4,5] The
most prominent minimal binding motif is the RGD-tripep-
tide,[6] recognized by almost a dozen of integrin heterodim-
ers, including a5b1 and avb3. Since these integrins are
widely expressed in many cell types, surface coating with
RGD epitopes is commonly used to provide strong mechani-
cal contacts between the surface and the adherent cells and
to enhance the biointegration of coated materials.[5,7] In this
regard, the use of cyclic RGD peptides with high affinity for
avb3 has shown to enhance osteoblast adhesion in vitro[8–10]
and improve bone recovery in vivo.[11] Alternatively, engi-
neered fibronectin fragments containing the RGD motif and
the synergy domain PHSRN have been used to promote
a5b1-mediated osteoblast adhesion, differentiation and in
vivo osseointegration.[12] Interestingly, the biological effects
observed in these studies were correlated with the capacity
of the coated-molecules to interact with avb3 or a5b1. Al-
though it is now known that these two integrins have dis-
tinct roles in promoting cell adhesion, proliferation and dif-
ferentiation,[13] their specific function in these biological
processes remains to be elucidated.[3c] Contradictory results
in the literature further illustrate this fact. For instance, it
has been reported that blocking avb3 enhances the differen-
tiation of mesenchymal stem cells into osteoblasts,[14] where-
as, in another study, avb3-active cyclic RGD peptides were
shown to promote osteogenesis in cultured mesenchymal
stem cells.[15] Thus, further research needs to be pursued in
this area. However, most of current investigations in the
field of biomaterials rely on the use of biomolecules (pepti-
des and proteins) that show activity for both avb3 and a5b1
integrins, thus precluding a clear discrimination between
their biological functions.
It became evident to us that an optimal strategy to func-
tionalize a large number of materials should allow for the
modification of the coating molecule without the necessity
of extensive chemical treatments. Thus, we envisaged the
use of a molecular toolkit based on click chemistry[18] as a
chemoselective, straight-forward strategy to immobilize pep-
tidomimetics on materials of different surface chemistry. In
this study we selected phosphonic acids as anchor molecules
and Ti6Al4V as surface material. Our strategy focused on
the functionalization of the a5b1-selective RGD peptidomi-
metic 1[19] with an azide group at the para position of the ar-
omatic ring (compound 2, Scheme 1). This position was
chosen for modification based on previous docking studies,
which demonstrated that the isopropoxy group is not in-
volved in the interaction with the a5b1 integrin.[20] Indeed,
the resulting azide-derived compound 2 retained the original
biological activity for a5b1 as well as the selectivity against
avb3 (Table 1), confirming our functionalization strategy
was not detrimental for biological activity. This a5b1 ligand
2 constitutes the main “tool” of our molecular kit, and it
will readily and selectively react with any anchor bearing an
alkyne group via a 1,3-dipolar cycloaddition[21] (compounds
3–11, Scheme 1). As anchor units, nine lysine-based phos-
phonic acids were used in our toolkit for the investigation of
the optimal spacer length and the number of phosphonates
required for efficient binding to Ti6Al4V and to exert the
desired biological responses. Compound 2 can be further
modified via click chemistry with arbitrary alkynes for dif-
ferent applications.
The synthesis of the different, benzyl-protected phosphon-
ic anchors was performed on the solid phase[22] via standard
Fmoc procedure (detailed synthesis see Supporting Informa-
tion).[23] After cleavage from the resin, the free carboxyl
group was coupled with propargylamine, followed by click
chemistry in solution and deprotection of the phosphonates
with trifluoroacetic acid (Scheme 1).
Table 1. Synthesized antagonists and their IC50 values determined in a
solid phase integrin binding ELISA. All compounds were referenced to
Cilengitide.[24]
Results and Discussion
Entry
IC50 (a5b1) [nm]
IC50 (avb3) [nm]
1
2
8
12
2.8Æ0.57
4.4 Æ0.47
1.0Æ0.11
169Æ38
79Æ5.5
>1000
>1000
>1000
0.76Æ0.08
1.52Æ0.12
0.55Æ0.05
To circumvent this limitation, we have recently described
for the first time the functionalization of gold nanoarrays
with thiol derivatives of RGD-based peptidomimetics with
high activity and selectivity towards avb3 and a5b1, as
promising agents for functionalizing biomaterials.[16] Howev-
er, thiols may not be suitable for coating other materials,
where different chemical anchors are preferable.[5] This is
the case of Ti and its alloys, common materials used in med-
icine, for which the use of phosphonic acids and phospho-
nates has shown to efficiently immobilize biomolecules to
the titanium oxide layer of the material.[17] Indeed, the use
of phosphonic acids as anchors of RGD peptides on
Ti6Al4V showed better biological performances compared
to the thiol-functionalized molecules.[10]
13
Cilengitide
15.5Æ1.8
Next, phosphonates 3–11 were coated on Ti6Al4V discs
and the binding of isolated integrin a5b1 was determined by
means of a solid-phase binding assay based on a modified
ELISA (Figure 1A).[25] Compounds 3–5 did not show any in-
crease in integrin a5b1-binding activity in comparison to
control samples such as uncoated discs incubated in PBS
(Figure 1). The peptidomimetic 6 slightly enhanced the at-
tachment of integrin a5b1, though insufficiently for applica-
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Titanium-Based Biomaterials
FULL PAPER
tion in surface coating. The
poor activity of this first series
of compounds may rely on the
fact that the spacer unit be-
tween the phosphonic group
and the molecule is too short, a
critical feature required for the
correct accessibility and presen-
tation of coating molecules to
integrins.[9,10,26] For this reason,
we selected longer spacer sys-
tems in the molecules 7–11. Re-
markably, compounds 8–10
showed a significant nine-fold
increase in promoting a5b1 in-
tegrin binding compared with
control discs. This result indi-
cates that a high a5b1 integrin
binding activity is achieved re-
gardless of the number of phos-
phonic acids (2, 3, or 4) as long
as a minimum spacer length is
provided. The inactivity of com-
pound 7 (only one phosphonic
acid) could be attributed to a
weak interaction of the mole-
cule with the titanium surface,
or to steric hindrance of the
phosphonic group and ineffi-
cient binding. To investigate
this issue we synthesized com-
pound 11 which is similar to
compound 7, but lacks a part of
the lysine tree and thus pro-
vides better accessibility to the
anchor moiety. In our ELISA-
like assay, 11 showed an a5b1
integrin binding on titanium
similar as 8–10. As a conse-
quence, we hypothesized that
the extra lysine branching unit
present in compound 7 may
exert steric interactions with
the phosphonic acid thereby
precluding an efficient attach-
ment to the surface. Although
we have shown that only one
phosphonic acid is required for
efficient binding to titanium, it
should be mentioned that tetra-
phosphonates may provide
a
more stable binding of the bio-
molecules to the surface during
sterilization treatments necessa-
ry for clinical applications.[9]
However, this was not subject
of our studies.
Scheme 1. Conjugation of azide-functionalized a5b1-selective ligand 2 and avb3-selective ligand 13 with differ-
ent phosphonate anchors via click chemistry. i) Alkyne (1 equiv), CuSO4 (1.2 equiv), Cu (40 equiv), H2O/
tBuOH 1:1; ii) 95% TFA, 2.5% triisopropylsilane, 2.5% H2O.
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H. Kessler et al.
immobilized
(Figure 3).
peptidomimetic
Interestingly,
Figure 3 shows that a5b1-ex-
pressing cells started adhering
and spreading on TiO2 nanoar-
rays coated with compound 8
after few minutes upon seeding.
On the contrary, avb3-express-
ing fibroblasts failed to adhere
on the surfaces, even after sev-
eral hours of incubation, giving
a striking proof of activity and
selectivity.
To prove the robustness of
our molecular toolkit, we exam-
ined whether our strategy could
be applied to an avb3-selective
integrin ligand. Thus, we select-
Figure 1. Integrin binding ELISA on Ti6Al4V. A) a5b1 Integrin binding on surfaces coated with compounds
3–11. B) Selectivity proof with compound 8 for a5b1 (blue) and avb3 (red). PBS = phosphate buffered saline,
negative control.
To check whether the selectivity profile of these peptido-
mimetics was retained upon immobilization on Ti6Al4V
discs, compound 8, one of the most potent a5b1-binding li-
gands within the series (Figure 1A), was selected and its ca-
pacity to promote the binding of isolated avb3 was deter-
mined. As shown in Figure 1B, this compound displayed no
significant increase in the binding of integrin avb3 in com-
parison to control samples. The data obtained from the
solid-phase binding assays on Ti6Al4V correlate with the
IC50 values determined for these RGD-peptidomimetics and
confirm that our functionalization strategy retains the bio-
logical activity and selectivity of the original compounds.
Furthermore, we were able to prove the binding of com-
pound 8 to a TiO2 surface with X-ray photoelectron spectro-
scopy (XPS) and quartz crystal microbalance with dissipa-
tion monitoring (QCM-D, see Supporting Information).
Figure 2 shows the XPS spectrum of a substrate incubated
for 4 h, which was demonstrated to be sufficient to get a
maximum coverage. The spectrum exhibits a peak at 133 eV,
characteristic for P 2p, thereby confirming the presence of 8
on the TiO2 layer. Additionally, we proved that longer incu-
bation times (>15 h) do not influence the already function-
alized TiO2 substrates, which is in agreement with the re-
ported high stability of the Ti-O-P bond.
ed the peptidomimetic 12, which shows a subnanomolar IC50
in avb3 integrin binding, while keeping an affinity for a5b1
by two orders of magnitude lower (Table 1). This compound
was modified with an azide to yield compound 13 (see
Scheme 1) and “clicked” to a series of alkyne-based phos-
phonic acids. However, due to the increased hydrophobicity
of compound 12 compared to 1, the resulting analogues
showed a poor solubility in aqueous media. To overcome
this issue, we designed the peptidomimetic 14, introducing
four phosphonate groups and a small polyethylene glycol
linker in the anchor moiety to improve its water solubility.
The biological activity and selectivity of compound 14 was
evaluated using the same cellular experiment as previously
described. In contrast to the effects showed by the integrin
a5b1 antagonist 8, avb3-rescued fibroblasts adhered imme-
diately on the titanium nanoparticles coated with the integ-
rin avb3 antagonist 14, while a5b1-rescued cells were
unable to bind to them (Figure 3).
As previously introduced, there is an extensive amount of
work focusing on the functionalization of titanium-based
materials. However, to the best of our knowledge, none of
Having demonstrated the feasibility of our approach by
means of an assay using isolated integrins, we aimed at
studying the biological potency of these ligands in a cellular
model. To this end, we performed adhesion assays on func-
tionalized titanium oxide nanoarrays with genetically modi-
fied fibroblast cell lines, either expressing a5b1 or avb3 in-
tegrins. TiO2 nanoparticles with a lateral spacing about
50 nm and a diameter of about 10 nm served as anchoring
points for the terminal phosphonate groups of compound
8.[27] After passivating the glass substrate with polyethylene
glycol (PEG) and functionalizing the nanoparticles with 8,
avb3 or a5b1 integrin-expressing fibroblasts were seeded in
low serum-containing (0.5% FBS) medium in order to
reduce non-specific protein adsorption onto the substrate,
and, thus allowing the cells to exclusively interact with the
Figure 2. XPS spectrum of a TiO2 film after functionalization with a5b1-
selective compound 8 (25mm in water) for 4 h at room temperature and
rinsing with water.
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Titanium-Based Biomaterials
FULL PAPER
Science of Complex Interfaces) of the Elite Network of Bavaria for fund-
ing, IAS (Institute for Advanced Study) of Technische Universitꢀt Mꢁn-
chen, CIPSM (Center for Integrated Protein Science Munich), Deutsche
Forschungsgemeinschaft (DFG, Project PO 1727/1-1) and Max Planck
Society for financial support. We gratefully thank for the technical and fi-
nancial support from King Abdulaziz University KAU (grant No. HiCi/
25-3-1432). The work was part of the SFB/TRR 79. Part of the research
leading to these results has received funding from the European Union
Seventh Framework Program (FP7/2007–2013) under grant agreement
No. NMP4-LA-2009-229289 NanoII and No. NMP3-SL-2009-229294
NanoCARD. J.P.S. is the Weston Visiting Professor at the Weizmann In-
stitute of Science. J.P.S. is part of the excellence cluster CellNetworks at
the University of Heidelberg.
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Acknowledgements
We thank Bund der Freunde der TU Mꢁnchen e.V., IGSSE (Internation-
al Graduate School of Science and Engineering), CompInt (Materials
Chem. Eur. J. 2013, 00, 0 – 0
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Received: April 18, 2013
Published online: && &&, 0000
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www.chemeurj.org
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Titanium-Based Biomaterials
FULL PAPER
Biomaterials
F. Rechenmacher, S. Neubauer,
C. Mas-Moruno, P. M. Dorfner,
J. Polleux, J. Guasch, B. Conings,
H.-G. Boyen, A. Bochen, T. R. Sobahi,
R. Burgkart, J. P. Spatz, R. Fꢀssler,
H. Kessler* .................... &&&& — &&&&
A Molecular Toolkit for the Function-
alization of Titanium-Based Biomate-
rials That Selectively Control Integrin-
Mediated Cell Adhesion
Selective click: Based on a click chem-
istry derived molecular toolkit, an
a5b1 integrin selective ligand was con-
jugated to a variety of alkyne-modified
spacer/anchor systems to investigate
integrin binding on coated titanium
(see scheme). The strategy was addi-
tionally extended to develop a phos-
phonate-functionalized avb3-selective
ligand and selectivity in mediating
integrin subtype specific cell adhesion
was shown for both subtype selective
ligands on TiO2 nanoarrays.
Chem. Eur. J. 2013, 00, 0 – 0
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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ÞÞ
These are not the final page numbers!