Carborane-β-cyclodextrin complexes as a supramolecular connector for bioactive surfaces

Article abstract of DOI:10.1039/c4tb01489h

Supramolecular chemistry provides an attractive entry to generate dynamic and well-controlled bioactive surfaces. Novel host-guest systems are urgently needed to provide a broader affinity and applicability portfolio. A synthetic strategy to carborane-peptide bioconjugates was therefore developed to provide an entry to monovalent supramolecular functionalization of β-cyclodextrin coated surfaces. The β-cyclodextrin·carborane-cRGD surfaces are formed efficiently and with high affinity as demonstrated by IR-RAS, WCA, and QCM-D, compare favourable to existing bio-active host-guest surface assemblies, and display an efficient bioactivity, as illustrated by a strong functional effect of the supramolecular system on the cell adhesion and spreading properties. Cells seeded on the supramolecular surface displaying bioactive peptide epitopes exhibited a more elongated morphology, focal adhesions, and stronger cell adhesion compared to control surfaces. This highlights the macroscopic functionality of the novel supramolecular immobilization strategy. This journal is

Full text of DOI:10.1039/c4tb01489h

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Schimer, P. Jonkheijm, L. G. Milroy, P. Cigler and L. Brunsveld, J. Mater. Chem. B, 2014, DOI:
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ARTICLE
Carborane-β-cyclodextrin complexes as
supramolecular connector for bioactive surfaces
P. Neirynck,a J. Schimer,b P. Jonkheijm,c L.-G. Milroy,a P. Cigler,b* and L.
Brunsvelda*
Supramolecular chemistry provides an attractive entry to generate dynamic and well-
controlled bioactive surfaces. Novel host-guest systems are urgently needed to provide a
broader affinity and applicability portfolio. A synthetic strategy to carborane-peptide
bioconjugates was therefore developed to provide an entry to monovalent supramolecular
functionalization of β-cyclodextrin coated surfaces. The β-cyclodextrin•carborane-cRGD
surfaces are formed efficiently and with high affinity as demonstrated by IR-RAS, WCA,
and QCM-D, compare favourable to existing bio-active host-guest surface assemblies, and
display an efficient bioactivity, as illustrated by a strong functional effect of the
supramolecular system on the cell adhesion and spreading properties. Cells seeded on the
supramolecular surface displaying bioactive peptide epitopes exhibited a more elongated
morphology, focal adhesions, and stronger cell adhesion compared to control surfaces. This
highlights the macroscopic functionality of novel supramolecular immobilization strategy.
βCD and carborane is used for chromatographic separations21,22
and for the solubilization of carborane complexes containing
platinum(II)-based DNA intercalators.23,24
Introduction
Supramolecular host-guest chemistry has recently emerged as a
versatile entry for the reversible immobilization of
biomolecules on surfaces with retention of activity. For
example, functional proteins and peptide epitopes modified
with
a
ferrocene moiety have been immobilized on
cucurbit[7]uril (CB7) surfaces with applications to protein
arrays.1–3 Similarly, beta-cyclodextrin (βCD) monolayers have
been widely studied for the immobilization of ferrocene-labeled
proteins or peptides via the ferrocene-βCD host-guest
binding.4,5 However, the relatively weak binding of βCD to
ferrocene necessitates for multivalent interactions to enable
efficient surface immobilization on βCD monolayers.6,7 Rapid
and efficient supramolecular protein and cell adhesion thus
requires new guest molecules with alternative chemotypes and
strong binding affinities to βCD-functionalized surfaces.
Carboranes are icosahedral cluster compounds consisting of
boron, carbon and hydrogen atoms. Their exceptional chemical
stability, caused by pseudo-aromatic delocalization of electrons,
as well as their high resistance to biological degradation
predisposes carboranes to various biomedical applications.
Their high boron content renders carboranes useful for boron
neutron capture therapy,8 while their well-defined structure and
Figure 1. Bioactive surfaces via the supramolecular assembly of carborane-β-
cyclodextrin complexes on gold or glass (not shown). A β-cyclodextrin monolayer
is supramolecularly coated with a bioactive peptide sequence using the strong
monovalent recognition of a carborane conjugated to the cyclic RGD motif. The
functionality of the supramolecular platform is evidenced at the macroscopic
level via the subsequent, substrate selective, recruitment, adhesion, and
distinctive hydrophobic properties makes them useful spreading of cells.
molecular scaffolds for drug development,9,10 including as
pharmacophores with tunable geometry and peripheral
substitution for the construction of various tight-binding
enzyme inhibitors such as carbonic anhydrase11 and HIV
protease.12 Within the supramolecular field carboranes13–17 and
metallacarboranes18–20 are highly appreciated due to their
ability to form strong non-covalent complexes with
cyclodextrins. For example, the host-guest interaction between
Here we use 1,2-closo-carborane (Cb) as a monovalent
supramolecular guest molecule for the efficient non-covalent
immobilization of biologically active peptides on βCD surfaces
(Figure 1). We demonstrate the potential utility of the approach
for the generation of biomaterials and cell adhesion applications
by immobilizing integrin-binding peptides as a means to
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selectively enhance adhesion and cell spreading of C2C12 cells TFA/H2O/triisopropylsilane (95/2.5/2.5, %v/v). Purification by
to the supramolecular functionalized βCD monolayer.
preparative scale HPLC (gradient 15-50 % MeCN in 40
minutes; Rt = 17 mins) afforded 65 mg of as white powder
upon lyophilization (42 % yield, purity > 95%). Note that the
addition of acetone to leads to stable thiazolid-2-one.
3
Materials and methods
3
Analytical HPLC Rt = 18.5 min. HRMS (ESI+): calculated for
TBTU (O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
tetrafluoroborate) was supplied by Iris Biotech. The βCD
derivatives were a kind gift from Dr. Alejandro Mendez Ardoy
(University of Twente, The Netherlands). Amino acids were
supplied by Novabiochem. Other chemicals and solvents were
1
C6H21ON2SB10 [MH]+ 279.22997. Found 279.23010. H NMR
(400 MHz, CD3CN) δ 8.11 (bs, 1H), 4.33 (bs, 1H), 4.22 (t, J =
5.4 Hz, 2H), 3.98 (qd, J = 15.3, 6.7 Hz, 2H), 3.06 (ddd, J =
20.7, 14.9, 5.3 Hz, 2H), 2.85–1.35 (m, 12H). 13C NMR
(101 MHz, CD3CN) δ 168.46 (s), 75.86 (s), 62.02 (s), 55.52 (s),
45.03 (s), 26.07 (s). 11B NMR (128 MHz, CD3CN, decoupled) δ
-2.99 (s), -5.88 (s), -9.99 (s), -11.86 (s), -13.20 (s).
cRGD-maleimide 4 and cRAD-maleimide 5. cRGD and
cRAD were synthesized according to previous literature.2 20
mg of peptide was reacted with NHS-activated maleimide
(synthesized according to previous literature, see SI)26 (1.4 eq.)
in dry DMF (1 mL) for 1 h at rt in the presence of DIPEA (4
eq.). The solvents were then removed in vacuo and the peptide-
maleimide conjugates were purified by preparative-RP HPLC
(gradient 10 - 25% MeCN, 0.1 % HCO2H in 20 min) to afford
purchased from Sigma-Aldrich. Carboranes
1 to 3 were purified
using column chromatography on silica (Sigma, pore size 60 Å,
70-230 mesh, 63-200 μm). The peptide conjugates were
purified using preparative scale RP-HPLC Waters Delta 600
(flow rate 7 mL/min, gradient shown for each compound -
including Rt) with column Waters SunFire C18 OBD Prep
Column, 5 µm, 19 x 150 mm. Compound purity was
determined by analytical Jasco PU-1580 HPLC (flow rate 1
mL/min, invariable gradient 2-100 % MeCN in 30 minutes, Rt
shown beside each compound) with column Watrex C18
Analytical Column, 5 µm, 250 x 5 mm. Compounds were
characterized using HRMS at LTQ Orbitrap XL (Thermo
Fisher Scientific) and NMR (Bruker Avance I™ 400 MHz).
cRGD-maleimide
and 28%, respectively, both as white powders.
HPLC Rt = 2.55 min. MS (ESI+): calculated for C34H46N10O10
4
and cRAD-maleimide
5
in yields of 25%
4
: Analytical
Products
4 to 7 were purified using RP-HPLC on a Shimadzu
[MH]+ 755.79 Found 755.67.
5: Analytical HPLC Rt = 2.55
HPLC equipped with surveyor PDA (C18 preparative column
from Phenomenex (21.20 x 150 mm), flow rate 15 mL/min).
Analysis was performed using LCQ Fleet from Thermo
Scientific on a C18 column equipped with surveyor AS and
PDA. Eluent conditions (CH3CN/H2O/0.1% HCO2H) for 15
min run: 0-1 min, isocratic, 5 % CH3CN; 1-10 min, linear
gradient, 5 – 100 %; 10-11 min, isocratic, 100 %; 11-12 min,
linear gradient, 100 - 5 %; 12-15 min, isocratic, 5 % CH3CN,
flow rate 0.1 mL/min.
min. MS (ESI+): calculated for C35H49N10O10 [MH]+ 769.79
Found 769.67.
Cb-cRGD 6 and Cb-cRAD 7. 5.8 mg of cRGD-maleimide
(resp. 3 mg of cRAD-maleimide) was dissolved in PBS (30
mM sodium phosphate, 50 mM NaCl, pH 7.4) and added to
1
(1 eq.) dissolved in 1 mL DMF. The reaction was stirred at
room temperature for 1 h and the solvents were removed in
vacuo. Purification was performed using preparative RP-HPLC
(gradient 20-50% MeCN, 0.1 % HCO2H in 30 min). Yields for
Cb-cRGD
Analytical HPLC Rt = 4.48 min. MS (ESI+): calculated for
C40H67B10N12O11S [MH]+ 1033.21. Found 1032.75.
6 and Cb-cRAD 7 were 63 and 5 %, respectively. 6:
Synthesis of carborane-cRGD and carborane-cRAD conjugates
Aminoethyl-o-carborane hydrochloride (1). 1.79 g (1.0 eq,
7
:
14.7 mmol) of decaborane (KatChem) was dissolved in 50 mL
of dry toluene along with 2.75 g (1.0 eq, 14.7 mmol) of 2-
(prop-2-yn-1-yl)isoindoline-1,3-dione. 1.286 g (0.5 eq, 7.36
mmol) of 1-butyl-3-methylimidazolium chloride was added and
the reaction mixture refluxed overnight. The toluene was then
evaporated and the organic slurry extracted 3x with Et2O (50
mL). Organic phases were combined and evaporated to dryness.
The product was further recrystallized from hot DCM to obtain
pure product at 41 % yield (1.821 g, 8.72 mmol). The next two
steps in synthesis were conducted as described previously and
the data collected was identical to previously reported data.25
Carborane-cysteine (3). 530 mg (1.2 eq, 1.14 mmol) of Boc-
Cys(Trt)-OH was weighed out in a round-bottom flask and
dissolved in 3 mL of DMF. TBTU (367 mg, 1.2 eq, 1.14 mmol)
and DIPEA (367 µL, 3.5 eq, 3.34 mmol) were then added and
the reaction mixture left stirring for 15 min after which
Analytical HPLC Rt = 4.49 min. MS (ESI+): calculated for
C41H70B10N12O11S [MH]+ 1047.25. Found 1046.75.
Surface chemistry
βCD immobilization on glass coverslips. Glass coverslips
were sonicated for 10 min in Hellmanex, then twice for 5 min
in H2O, dried under N2 flow and exposed to O2 plasma for 30 s.
Surfaces were thoroughly washed with H2O, then with EtOH
and dried under N2 flow. Surfaces were placed in a vacuum
dessicator
overnight
with
(trimethoxysilyl)
propyl-
ethylenediamine (TPEDA). The next day, the surfaces were
washed with EtOH, dipped in dry toluene and then dried.
Surfaces were incubated in a 1 mM toluene solution of 1,4-
phenylene diisothiocyanate at 50 oC for 2 h under N2
atmosphere, washed with toluene, EtOH and water, and
o
subsequently incubated for 2 h at 50 C with a 1 mM solution
(aminoethyl)-o-carborane hydrochloride
1 (200 mg, 1.0 eq,
of per-6-amino-b-cyclodextrin (βCD-7NH2,
8, Scheme 1) in
0.95 mmol) was added in one portion. All volatiles were
evaporated after 12 h and the organic slurry dissolved in 20 mL
of EtOAc. This solution was then washed 2x with a 10%
solution of KHSO4 (20 mL), 2x with a saturated solution of
NaHCO3 (20 mL) and once with brine. The organic layer was
then dried and evaporated. The crude product was purified by
column chromatography (hexane:EtOAc 5:1, Rf = 0.35; UV
H2O.27 Finally, the surfaces were washed sequentially with
H2O, EtOH and then thoroughly dried under N2 flow.
Where applicable, substrates were then incubated for 3 h with
75 µL of a 100 µM aqueous solution of the carborane-peptide
conjugate, rinsed with H2O and dried under N2 flow.
βCD immobilization on gold substrates. Prior to use in QCM-
D experiments, resonators were activated for 15 s using a
piranha solution (H2SO4/H2O2, 3:1, %v/v). Surfaces were then
extensively washed with H2O, EtOH, and then incubated in a 1
detection). 350 mg (0.57 mmol) of the protected product
2 was
obtained in a 65% yield. The trityl- and boc- protecting groups
were then cleaved off by treating
2 for 1 h with 1 mL of
mM
solution
of
heptakis{6-deoxy-6-[12-
2 | J. Name., 2012, 00, 1-3
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Journal Name
Journal of Materials Chemistry B
ARTICLE
(thiododecyl)undecanamido]}--cyclodextrin (βCD-7S,
9,
Results and discussion
Scheme 1) for efficient immobilization28,29 in CHCl3/EtOH 2/1,
heated at 60 oC for 1 h, then left at room temperature overnight,
under a N2 atmosphere. They were then rinsed with EtOH and
dried under N2 flow. The same protocol was followed for the
preparation of substrates for IR-RAS analysis. Where
applicable, the substrates were then incubated for 3 h with 75
µL of a 100 µM aqueous solution of the carborane-peptide
conjugate, rinsed with H2O and dried under N2 flow.
Synthesis
Our aim was to develop conditions to couple the carborane to
biomolecules, which might be compatible with a broad range of
molecules including peptides and proteins. Therefore thiol-
functionalized carboranes31,32 33,34 were explored to react with
,
maleimide modified peptides under mild conditions. Direct
connection of the thiol to the Cb cage,31,32 is expected to lead to
steric hindrance regarding βCD binding and peptide
conjugation. A water-soluble Cb bearing a thiol group attached
Characterization of βCD – carborane-peptide surfaces.
Fourier
Transform
Infrared
Reflection
Absorption
Spectroscopy (FT-IR-RAS) measurements utilized 200 nm gold
Si wafers, 2x2 cm. Polarized FT-IR-RAS spectra of 1000 scans via a short linker,33,34 would constitute a more beneficial
with a resolution of 2 cm-1 were obtained using a Thermo starting point. Therefore, the 1-aminomethyl-1,2-closo-
Scientific TOM optical module.
carborane precursor was first prepared in three steps starting
from decaborane B10H14 (see Scheme 1). We modified the
previously published synthesis25 by implementing a recently
described acetylene insertion methodology, which is performed
Water contact angle measurements were performed on a Krüss
G10 contact angle measuring instrument, equipped with a CCD
camera. Images were analyzed using the Drop Shape Analysis
software version 1.90.0.2 and ImageJ Contact Angle plug-in.
QCM-D studies. QCM-D data were measured using a Q-Sense
E1 with a peristaltic pump, Ismatec Reglo Digital M2-2/12.
Gold-coated QCM-D resonators QSX 301 with a resonance
frequency of 4.95 MHz +/- 0.05 MHz were purchased from
LOT-QuantumDesign. All solutions of Cb-cRGD were
prepared using PBS buffer. Measurements were performed at
20 oC, with a flow of 50 µL/min. Prior to the binding of the Cb-
in ionic liquid.35 The aminomethyl-carborane
1
was then
coupled to the protected cysteine Boc-Cys(Trt)-OH via TBTU
activation and the desired product, , obtained upon treatment
3
with TFA/H2O/triisopropylsilane (95/2.5/2.5, %v/v) without
evidence of thiol capping. Peptide activation was performed at
pH 7 to favor selective coupling of the NHS-activated
maleimide to the lysine, providing
4 and 5. Reactions between
the Cys-functionalized Cb and the maleimide-cRGD and
maleimide-cRAD were performed in a 1:1 (v/v) mixture of
RGD 6, surfaces were equilibrated by flowing over PBS buffer
until a stable baseline was obtained.
DMF/PBS at pH 7-7.5 to afford the target compounds
6 and 7.
Cell culture and adhesion studies
C2C12 cells, from a mouse myoblast cell line, were used at
passage between 15 and 20 for the cell experiments. 80%
confluent T25 or T75 flasks of C2C12 were trypsinized,
centrifuged and redispersed in DMEM medium supplemented
with penicillin/strep, NEAA, as well as 10% FBS for culturing
and 0% FBS for surface incubation experiments.
Glass substrates coated with βCD and carborane-peptide were
dipped in and out into 70% EtOH and rinsed twice with PBS.
Cells in suspension in 0% FBS supplemented DMEM media
were seeded on the substrates (20 000 cells/mL, 3 mL/well) and
o
left to adhere for 1 h at 37 C and 5% CO2. Surfaces were then
gently washed twice with PBS, cells were fixed for 10 min with
10% formalin and then rinsed three times with PBS.
Cells were incubated with blocking solution (0.1% Triton, 0.5%
w/w BSA in PBS pH 7.4) for 1 h at room temperature or
o
overnight at 4 C. Surfaces were then incubated with Paxillin
1:500 in blocking buffer for 1 h, washed 3 times for 10 min
with blocking buffer, and incubated for 1 h with secondary
antibody-Alexa 488 (1:500) and phalloidin-Alexa 546 (1:500)
in blocking buffer. Finally the surfaces were washed once for
10 min with blocking buffer and twice with PBS, incubated for
10 min with DAPI in PBS (1:1000), rinsed with PBS three
times, and then stored at 4 oC.
Imaging was performed using an Olympus IX71 fluorescence
microscope, at 40x magnification. Five pictures per substrate
were taken for each of the three repetitions and analysis of the
cell adhesion was performed using CellProfiler.30 Cells that
could not be recognized by the software or that did not fall
completely in the field of view were discarded from the
analysis. On average, between 30 and 40 cells per substrate per
set remained for analysis, corresponding to approximately 100
cells per condition. Results were normalized towards the
average value obtained for each experiment set for the βCD
control surface. All experiments were performed in triplicate.
Scheme 1. Structures of βCD-7NH2 827 and βCD-7S 928,29 and synthesis of
carborane-thiol and peptide-carborane conjugates, (cRGD) and (cRAD). a)
2-(prop-2-yn-1-yl)isoindoline-1,3-dione, [BMIM]Cl, toluene, 110 °C; b) NaBH4, i-
3
6
7
PrOH/H2O; c) AcOH/H2O, HCl, 75 °C; d) Boc-Cys(Trt)-OH, TBTU, DIPEA, DMF; e)
TFA/H2O/TIS; f) DMF/PBS 1/1 %(v/v),
methylimidazolium, TBTU = O-(Benzotriazol-1-yl)-N,N,N
tetrafluoroborate, DIPEA N,N-diisopropylethylamine, DMF
dimethylformamide, TIS = triisopropylsilane.
1
h, rt. BMIM
,N -tetramethyluronium
N,N-
=
1-Butyl-3-
′
′
=
=
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stretch of amides present in βCD-7S structure as well as in the
cRGD peptide conjugates, while the intense broad peak at 3345
cm-1 is characteristic of the presence of secondary OH groups.
A peak at 2582 cm-1 (B-H) (Figure 3 top, arrow) is observed in
the case of βCD + Cb-cRGD, which is indicative of
complexation between βCD and Cb and has also been observed
for a similar system in solution.37,38 Both surface analyses
provide convincing evidence for the immobilization of
carborane-peptide conjugates to the βCD-7S gold monolayers
via the βCD•carborane complexation.
Surface characterization
Water contact angle (WCA) measurements were performed to
provide information on changes in the hydrophilicity of the
surface upon successive monolayer formation (Figure 2). A
large decrease in contact angle was observed – from 95 to 49
degrees – after functionalization of gold surface with βCD-7S,
which display several OH groups and thus increase the
hydrophilicity of the surface. Subsequent incubation with Cb-
cRGD, 6, resulted in an a small increase in WCA of the polar
surface from 49 to 56 degrees, in agreement with previously
reported values for RGD functionalized surfaces.2
Host-guest surface complexation
Gold
βCD
βCD + Cb-cRGD 6
Quartz Crystal Microbalance with Dissipation monitoring
(QCM-D) measurements were performed for a more detailed
96.8o +/- 1.4
48.6o +/- 0.2
55.1o +/- 0.9
and quantitative analysis of the affinity of Cb-cRGD (6) for
βCD monolayers. In general, a change at the surface of a quartz
crystal sensor, for example via binding of a compound, results
in a measurable change in the vibration frequency of the sensor.
Various concentrations of Cb-cRGD (6) in PBS, ranging from
Figure 2. Water contact angle values for gold, βCD monolayer and βCD
complexed with Cb-cRGD (n=4), with a representative picture. A high WCA
angle value indicates a hydrophobic surface.
10 to 500 µM, were flown over gold crystals pre-functionalized
with βCD-7S (Figure 4a). Dissipation remained within 10% of
the change in frequency value, indicating the formation of a
rigid film at the resonator surface and allows the Sauerbrey
model to be applied.39 The change in frequency of the 5th
6
resonance was plotted versus the concentration of
and the resulting graph could be fitted with a Langmuir model,
providing a Kd value of 178 µM +/- 39 µM for the interaction
6 (Figure 4b)
1.16
1.14
1.12
1.10
1.08
1.06
1.04
1.02
1.00
0.98
CD
CD + Cb-cRGD
of
6 for the βCD monolayer, via the Cb mediated host-guest
interaction.
a
2
0
3600
3400
3200
3000
2800
2600
)
2400
2200
2000
-2
Wavelength (cm-1
-4
1.10
CD
-6
10uM
1.09
1.08
1.07
1.06
1.05
1.04
1.03
1.02
1.01
1.00
0.99
CD + Cb-cRGD
50uM
100uM
250uM
500uM
-8
-10
0
500
1000
1500
2000
2500
Time (s)
b
10
2000
1800
1600
1400
1200
1000
Wavelength (cm-1
)
Figure 3. FT-IR-RAS of βCD (red) and βCD + Cb-cRGD,
6 (black) on gold, in two
5
different regions (top: 3600 – 2000 cm-1, bottom: 2000 - 1000 cm-1). The arrow
shows a characteristic peak of a βCD•carborane complex.
To get more insight into the formation of the βCD•Cb complex
0
on gold, substrates functionalized with bCD-7S28,29 and further
0
100
200
300
400
500
[Cb-cRGD] (M)
incubated with Cb-cRGD
6 were studied by Infrared Reflection
Absorption Spectroscopy (IR-RAS). The IR spectrum of βCD36
in solution exhibits characteristic absorption peaks at 1053,
1088, 1157, 1204, 1241 and 1267 cm-1 – corresponding to
different stretching (CO and CC), and bending modes (COH,
OCH and CCH), which are also observed on the gold surface
(Figure 3). Sharp peaks at 1654 cm-1 (βCD) and 1661 cm-1
(βCD + Cb-cRGD) were observed corresponding to the C=O
Figure 4. QCM-D data forbinding of Cb-cRGD (6) to βCD-7S coated quartz
crystals. a) Fifth resonance frequency overtone (Δf5) for various concentrations
of Cb-cRGD (10, 50, 100, 250, 500 uM). b) Change in frequency of the fifth
overtone versus Cb-cRGD concentration. Fit was performed using Origin,
Langmuir fit, resulting in a Kd value of 178 µM +/- 39 µM.
4 | J. Name., 2012, 00, 1-3
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The affinity of carborane
6 for the βCD monolayer compares A more in-depth analysis of the cell adhesion was performed
using CellProfiler30 to obtain a quantitative difference in
cellular morphological properties under the different surface
immobilization conditions (Figure 6). Similar studies have been
performed to correlate qualitative and quantitative aspects of
cell pictures.48,49 A workflow chart providing information about
e.g. cell area, perimeter or eccentricity was run and data were
analyzed using the software GraphPad Prism. A repeated-
measures one-way analysis of variance (one-way ANOVA) test
favorably with other known guests of βCD, such as ferrocene
and adamantane. Carborane binds to βCD with 5-fold greater
affinity than aminomethylferrocene derivatives and is therefore
better suited for monovalent surface immobilization.4
Lithocholic acid binds to βCD with high binding affinity in
solution (Kd = 1.2*10-6 M), but has limited potential for βCD
surface interactions due to the guest protruding the βCD at the
smaller ring, resulting in lowered affinities.40 The affinities of
carborane and adamantane for
a
βCD monolayer are was applied on the normalized averages for each repetition and
comparable.41 However, carborane introduces to the system a
unique quality: high content of boron, which in principle can be
further utilized for quantification of conjugation yields using
sensitive spectral method such as inductively atomic emission
spectrometry with inductively coupled plasma (ICP AES), as
has been shown for boron-containing BODIPY dyes.42
each condition. As already indicated by the simple visual aspect
and observation of the focal adhesions, statistically noticeable
differences in eccentricity, perimeter, form factor, compactness
and ratio between the major and minor axis lengths was only
observed for the βCD monolayer complexed with Cb-cRGD
compared to βCD; there is a significant difference (p < 0.05)
between the control surface βCD and the active surface (βCD
with Cb-cRGD).
Cellular evaluation of the surfaces
Strong and directional supramolecular surface immobilization
strategies provide substantial opportunities for biomedical
applications. To explore the potential of the βCD-Cb complex
in this respect, the ability of surface-immobilized Cb-cRGD
conjugates to induce specific cell adhesion was studied using
the C2C12 mouse myoblast cell line. C2C12 cells express
various integrin receptors, including αvβ3, which is known to
bind to cRGDfK, as used in 6 43,44
and show clear phenotypic
,
changes to the environment.45,46 For these experiments, cells
were passaged at 80% confluence to avoid differentiation.
While the surface characterization was performed on gold
surfaces coated with βCD (vide supra), glass surfaces are more
suitable for fluorescence microscopy studies and were thus
favored for the cell experiments. Surfaces featuring a βCD
monolayer and a βCD monolayer complexed with bio-inactive
conjugate Cb-cRAD
7 were used as reference surfaces.
Cyclodextrins are composed of oligomerized glucose and
therefore do not specifically discourage cell adhesion, but lack
a specific molecular entity to enhance cell spreading, such as
the bioactive epitope cRGD. Cells seeded on either the control
βCD or βCD + Cb-cRAD substrates remained round and did
not form proper focal adhesions (Figure 5a,b). However, cells
seeded on the βCD + Cb-cRGD surfaces became strongly
anchored to the surface, evident already within 1 h of seeding,
with pronounced stretching of actin filaments as a consequence
of cell and focal adhesion (Figure 5c). These results show that
the cells specifically recognize the RGD sequence through
binding to integrins, and that the differences in cell morphology
observed between the βCD + Cb-cRGD and βCD + Cb-cRAD
surfaces are a specific consequence of the difference in integrin
binding affinities between the supramolecular immobilized
cRGD and cRAD.47
Figure 6. Statistical analysis of the cell experiments with CellProfiler30 and
GraphPad Prism. Data were normalized and averaged for each repetition
towards the control βCD.
Some of the morphological characteristic changes strongly
correlate with one another. For example, the eccentricity
describes the elliptical character of the cell morphology (Figure
6a): an increase in eccentricity describes a shape that transitions
from a circle, through an ellipse, to a line. In line with this, the
ratio of the major axis length divided by the minor axis length
will be higher in the case of an elongated cell compared to a
cell displaying a more rounded morphology (Figure 6b). These
observations can specifically be made for the supramolecular
adhered cells; the eccentricity increases from 1 for βCD to 1.12
for the Cb-cRGD surface. The ratio of the axis lengths also
increases, from 1 to 1.14. The form factor is defined as
4π*area/perimeter2: this value will be equal to 1 for a circle and
will decrease as the perimeter of the cell increases (Figure 6c).
An increase in the perimeter (from 1 to 1.24) (Figure 6d) can be
observed which correlates with a decrease in the form factor (1
to 0.76). These results confirm the qualitative observation from
the pictures and thus the functional effect of the supramolecular
system on the cell adhesion and spreading properties: cells are
Figure 5. Scale bar: 50 m. C2C12 seeded on glass surfaces coated with a) βCD,
b) βCD and Cb-cRAD, c) βCD and Cb-cRGD, fixed after 1 h and stained for nucleus
(DAPI) and actin (Phalloidin). Focal adhesions are exemplary indicated by the
white arrows.
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Journal of Materials Chemistry B
Page 6 of 9
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Conclusions
Supramolecular systems offer great opportunities for the
development of dynamic and well-controlled biocompatible
surfaces and coatings. Existing host-guest elements require
optimization regarding affinity and applicability. Here, we
reported the synthesis of a carborane derivative mono-
functionalized with cysteine for conjugation to biologically
relevant molecules, such as peptides, under mild conditions.
The utility of the approach was demonstrated by conjugating
the cysteine-carborane derivative to cRGD analogs via Michael
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Acknowledgements
This research forms part of the Project P4.02 Superdices of the
research program of the BioMedical Materials institute, co-
funded by the Dutch Ministry of Economic Affairs, Agriculture
and Innovation. Co-funded by Netherlands Organisation for
Scientific Research via Gravity program 024.001.035 and ERC
grant 259183 – Sumoman (PJ). The work of PC and JS was
supported by MSMT CR grant No. LH11027. Dr. Alejandro
Mendez Ardoy is thanked for the synthesis of the bCD
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Laboratory of Chemical Biology and Institute of Complex Molecular
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The supramolecular carborane-β-cyclodextrin system allows for effective monovalent immobilization of
biologically active peptides resulting in efficient cell adhesion and spreading.

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