Supramolecular control of cell adhesion via ferrocene-cucurbit[7]uril host-guest binding on gold surfaces

Article abstract of DOI:10.1039/c3cc37592g

Supramolecular control of adhesion of cells is demonstrated using synthetic integrin binding RGD peptide-ferrocene conjugates that were immobilized via host-guest chemistry onto cucurbit[7]uril coated gold surfaces. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc37592g

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Supramolecular control of cell adhesion via
ferrocene–cucurbit[7]uril host–guest binding
on gold surfaces†
Cite this: Chem. Commun., 2013,
49, 3679
Received 18th October 2012,
Accepted 12th March 2013
a
b
Pauline Neirynck,z Jenny Brinkmann,z Qi An,b Daisy W. J. van der Schaft,c
Lech-Gustav Milroy,a Pascal Jonkheijm*b and Luc Brunsveld*a
DOI: 10.1039/c3cc37592g
www.rsc.org/chemcomm
Supramolecular control of adhesion of cells is demonstrated using
synthetic integrin binding RGD peptide–ferrocene conjugates that
were immobilized via host–guest chemistry onto cucurbit[7]uril
coated gold surfaces.
Supramolecular construction processes provide innovative plat-
forms for the study of fundamental aspects of cell biology, as a
basis for the engineering of biomaterials with self-adaptive cell
interface properties.1 On surfaces, structured interfaces can be
formed with molecular precision – on a scale most relevant to
cellular function – through the spatial or temporal localization
Fig. 1 Coating of gold surfaces with a CB[7] monolayer, pre-incubation with
Fc-cRGD and reference compound Fc-cRAD, and subsequent endothelial cell
of ligands.2,3 With the appropriate combination of ligands, the adhesion to the supramolecularly functionalized surfaces.
surface functionality can be tuned to specifically manipulate
and monitor cell adhesion and migration. This offers unique
opportunities for the development of biomaterials for cell- correct supramolecular host–guest combination, displaying the
growth engineering.4 The use of semi-synthetic proteins, RGD motif, enable enhanced molecular induced endothelial
growth factors or peptides in various patterns can direct adhe- cell adhesion and wound recovery in a controlled manner.
sion and migration of e.g. endothelial cells. Supramolecular
To investigate supramolecular control of endothelial cell
chemistry would allow for potentially reversible or stimuli- adhesion, the ferrocenylamine-CB[7] based host–guest system
responsive surfaces with interesting properties,3,5 which are was selected (Fig. 1).9–11 The (ferrocenylmethyl)-trimethyl-
currently being integrated into biological systems6,7 and bio- ammonium cation binds to CB[7] in solution with a Kd in the
materials with intended regenerative medicine applications.8 range of 1011 Mꢀ1 12
Here, important progress towards such supramolecular-based surfaces and forms
.
CB[7] spontaneously adsorbs onto gold
stable self-assembled monolayer
a
bio-interfaces is reported, based on the non-covalent immobi- (SAM).13,14 Such supramolecular CB[7]-SAMs have been used
lization of ferrocene-labelled cyclic RGD (Fc-cRGD) peptides for the site-selective immobilization of fluorescent proteins that
onto gold surfaces coated with a CB[7] (cucurbit[7]uril) mono- were monovalently labelled with ferrocenylamine. Although the
layer using host–guest interactions. Only surfaces featuring the binding affinities to CB[7] SAMs might differ from solution,
strong and reversible binding to CB[7]-coated gold surfaces was
a Laboratory of Chemical Biology, Department of Biomedical Engineering,
Eindhoven University of Technology, Den Dolech 2, 5612 AZ, Eindhoven,
The Netherlands. E-mail: l.brunsveld@tue.nl; Fax: +31 40-247-8367
b Molecular Nanofabrication Group, MESA+ Institute for Nanotechnology,
Department of Science and Technology, University of Twente, P. O. Box 217,
7500 AE, Enschede, The Netherlands. E-mail: p.jonkheijm@utwente.nl
c Soft Tissue Biomechanics & Tissue Engineering, Department of Biomedical
Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands
† Electronic supplementary information (ESI) available: Synthesis of peptide
conjugates, preparation of cucurbituril coated surfaces, supramolecular host–
guest surface immobilization studies, IR-RAS, WCA and XPS analysis, hUVEC
culture and microscopy. See DOI: 10.1039/c3cc37592g
observed.15 This surface immobilization system bodes well for
exploration of cellular applications such as cell adhesion studies,
and is potentially attractive due to the ease of CB[7] SAM
formation and the low toxicity of gold.
Crucial to the high affinity of this system is the amino
functionality present in the ferrocene guest molecule. While
the ferrocene ring system fills the lipophilic CB[7] cavity, the
protonated amino functionality protrudes from the cavity,
where it makes stabilizing electrostatic interactions with the
polar carbonyl groups located at the CB[7] rim.12 To enable
studies of endothelial cell adhesion on such CB[7]-coated surfaces,
‡ These authors contributed equally.
c
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RGD peptides16–19 were attached to the key ferrocenylamine infrared reflection absorption spectroscopy (IR-RAS), water
moiety via an oligoethylene glycol (OEG)-based linker (1). The contact angle measurements (WCA) and X-ray photoelectron
use of OEG was suitable for the envisioned purposes due to its spectroscopy (XPS). The morphological properties of the CB[7]-
biocompatibility and inertness to gold surfaces.20 The choice of coated gold surface monolayer have been previously described.14
the linker length was based on previous investigations regarding Cyclic voltammetry (CV) measurements and dynamic force
the immobilization of ferrocenylamine-labelled fluorescent pro- spectroscopy (DFS) suggested that CB[7] covers ca. 40–50% of
teins onto CB[7]-SAMs.15
the gold surface,13,14 and atomic force microscopy (AFM)
It was anticipated that a suitable strategy for attaching the showed that the surface adsorbed CB[7] molecules are acces-
ferrocenylamine guest molecule (1) to RGD peptides21 would sible for the immobilization of ferrocene-labelled proteins via a
require special consideration of the specific design of the specific monovalent CB[7]–ferrocene interaction.15 Here, the
guest molecule. Firstly, the ferrocenylamine group was initially IR-RAS spectrum of the CB[7]-coated gold surface after incuba-
developed for peptide synthesis as an amine protective group, tion with Fc-cRGD 5 and subsequent blocking with (oligo-
and is typically cleaved under acidic conditions (TFA–b-thio- ethylene glycol)6-thiol (OEG6–SH) revealed additional peaks at
naphthol–CH2Cl2, 2–4 h).22 Furthermore, compatibility with 1670–1700 cmꢀ1 (amide CQO bonds) and 1116 cmꢀ1 (C–O–C),
standard amide coupling chemistry21 would in all likelihood which are characteristic of the presence of the peptide–
require further modification (at least one additional step) ferrocene conjugate and ethylene glycol, respectively (ESI†).
or laborious protection–deprotection steps of the ferrocenyl The WCA of the CB[7]-SAMs increased from 321 to 581 upon
secondary amine functionality. Thus, an approach that intro- incubation with Fc-cRGD 5, which is indicative of the immobi-
duced the ferrocenylamine guest into a fully unprotected pep- lization of the ferrocene–peptide conjugate and is in agreement
tide sequence was preferred to enable a divergent late stage with previously reported values for similar surfaces.15 XPS
attachment of the ferrocenylamine moiety.23 Therefore a strategy shows the expected C/N/O ratio of 3/2/1 for CB[7]-SAMs, and
based on copper-catalyzed azide–alkyne click chemistry24 was the appearance of an Fe signal upon incubation with Fc-cRGD 5
chosen. The novel ferrocenylamine-PEG-azide 1 (Fig. 2) was (ESI†). The surface characterization data thus demonstrate
readily prepared in one pot, via in situ imine formation followed the successful immobilization of the ferrocene–peptide conju-
by reduction using NaBH4. Cyclic RGDfK, and RADfK25 as gates onto the CB[7]-SAMs via a ferrocene–CB[7] host–guest
reference, were synthesized using standard solid-phase peptide interaction.
synthesis methods26 and then coupled to pentynoic acid to
Human umbilical vein endothelial cells (hUVECs) were used
form the alkyne derivatives 2 and 3. Ligation of 1 to either 2 or 3 as a model system to study the cellular response to supra-
was achieved using CuSO4 and sodium ascorbate in the molecularly immobilized peptides 4 and 5. Cells were seeded
presence of copper. This afforded the guest molecules Fc-cRAD on CB[7]-SAMs using different chemical surface functionaliza-
(4) and Fc-cRGD (5), respectively, in useful yields after purifica- tions, including blocking against unspecific interactions, and
tion using reversed-phase-HPLC (ESI†).
the cell response was monitored at different time points over a
CB[7] coated gold surfaces were prepared according to period of 48 h (Fig. 3a, 24 h, and Fig. S6–S9, ESI†). The blocking
previous methods.11,13–15 Immobilization of the ferrocene– step using OEG6–SH was evaluated at different steps in the
peptide conjugates onto the CB[7] coated surfaces was per- surface preparation protocol. Addition of the short ethylene
formed by immersion in aqueous solutions (50 mM) of 4 or 5 for glycol chain directly after the incubation of the surface with the
3 h. After washing, the surfaces were characterized using ferrocenylamine-peptides, via immersion in a 0.1 mM solution
of OEG6–SH for 2 min, was most effective (see ESI† for optimi-
zation of blocking conditions).
Supramolecular coating with Fc-cRGD 5 resulted in efficient
adhesion and growth of hUVECs on the supramolecular
surfaces as evidenced by the increase in the surface coverage
and cell density after 24 h (Fig. 3a, (ii)). In contrast, surfaces
pre-incubated with Fc-cRAD 4 showed less spreading of cells
and a significantly reduced number (3-fold, ESI†) of adhered
cells (Fig. 3a, (i)). Similarly, reference surfaces using bare gold
(Fig. 3a, (iii)), CB[7]-only (Fig. 3a, (iv)), CB[7] with a ferrocenyl-
PEG3 linker or only 5 (ESI†) showed only minor cell adhesion
and a more round cell morphology. After 48 h, a closed
monolayer of cells was formed on the surfaces with CB[7] and
Fc-cRGD 5, whereas this state could not be reached in any of the
control experiments including surfaces with CB[7] and Fc-cRAD 4.
A live-dead assay confirmed that the hUVECs remain viable on
CB[7]-SAMs incubated with Fc-cRGD 5 compared to control
tissue culture plates (TCP) and fibronectin coated TCP (ESI†).
Fig. 2 Synthesis of ferrocene–peptide conjugates 4 and 5 via copper-catalyzed
azide–alkyne cycloaddition of azide 1 to alkynes 2 or 3. Reagents and conditions:
A wound assay on surfaces with CB[7] and Fc-cRGD 5 showed full
recovery of the cell monolayer within 8 h, indicating effective
(i) 1 (1.0 eq.), CuSO4 (30 mol%) sodium ascorbate (50 mol%) copper ribbon,
40 1C, 24 h.
c
3680 Chem. Commun., 2013, 49, 3679--3681
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co-funded by the Dutch Ministry of Economic Affairs, Agriculture
and Innovation. Co-funded by ERC grant 204554 – SupraChemBio
(LB) and ERC grant 259183 – Sumoman (PJ). We thank Dr
S. Sahebali for help with statistical analysis, L. Olijve for help
with NMR measurement, J. van Dongen for high resolution
mass spectrometry measurements, S. Krabbenborg, MSc, for
the electrode surfaces and G. Kip for XPS measurements.
Notes and references
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Fig. 3 Scale bar: 200 mm. (a) hUVECs seeded on gold surfaces pre-incubated
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`
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c
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Chem. Commun., 2013, 49, 3679--3681 3681