Chemical functionalization of bioceramics to enhance endothelial cells adhesion for tissue engineering

Article abstract of DOI:10.1021/jm301092r

To control the selective adhesion of human endothelial cells and human serum proteins to bioceramics of different compositions, a multifunctional ligand containing a cyclic arginine-glycine-aspartate (RGD) peptide, a tetraethylene glycol spacer, and a gallate moiety was designed, synthesized, and characterized. The binding of this ligand to alumina-based, hydroxyapatite-based, and calcium phosphate-based bioceramics was demonstrated. The conjugation of this ligand to the bioceramics induced a decrease in the nonselective and integrin-selective binding of human serum proteins, whereas the binding and adhesion of human endothelial cells was enhanced, dependent on the particular bioceramics.

Full text of DOI:10.1021/jm301092r

Article
pubs.acs.org/jmc
Chemical Functionalization of Bioceramics To Enhance Endothelial
Cells Adhesion for Tissue Engineering
Franco̧
ise Borcard,§ Davide Staedler,§ Horacio Comas,§ Franziska Krauss Juillerat,†
Philip N. Sturzenegger,† Roman Heuberger,# Urs T. Gonzenbach,† Lucienne Juillerat-Jeanneret,*,⊥
and Sandrine Gerber-Lemaire*,§
§Institute of Chemical Sciences and Engineering, EPFL, CH-1015, Lausanne, Switzerland
†Nonmetallic Inorganic Materials, ETHZ, CH-8093 Zurich, Switzerland
̈
#RMS Foundation, CH-2544 Bettlach, Switzerland
⊥University Institute of Pathology, CHUV-UNIL, CH-1011, Lausanne, Switzerland
S
* Supporting Information
ABSTRACT: To control the selective adhesion of human endothelial cells
and human serum proteins to bioceramics of different compositions, a
multifunctional ligand containing a cyclic arginine-glycine-aspartate (RGD)
peptide, a tetraethylene glycol spacer, and a gallate moiety was designed,
synthesized, and characterized. The binding of this ligand to alumina-based,
hydroxyapatite-based, and calcium phosphate-based bioceramics was
demonstrated. The conjugation of this ligand to the bioceramics induced a
decrease in the nonselective and integrin-selective binding of human serum
proteins, whereas the binding and adhesion of human endothelial cells was
enhanced, dependent on the particular bioceramics.
Lys] (cRGDfK) unit linked to a gallate functionality through an
ethylene glycol spacer. The chemical functionalization of the
bioceramics was monitored by X-ray photoelectron spectros-
copy (XPS). Three different bioceramics, i.e., aluminum oxide
(alumina), tricalcium phosphate (TCP), and hydroxyapatite
(HA), were investigated. HA was used as a reference material as
it is a highly studied bioscaffold and human umbilical vein
endothelial cells (HUVEC) as an endothelial cell model to
evaluate the adhesive properties and biocompatibility of the
bioceramics functionalized with the cRGDfK-gallate ligand.
INTRODUCTION
■
The development of cellularized bone implants is a promising
approach for the treatment of large bone defects. A limitation
associated with large bone substitutes is the ability to develop a
functional vascular system. The vascularization of implants is
required to ensure the formation of stable and functional
structures and to provide nutrients to the cells of the implants.1
Different strategies can be applied to allow the vascularization
of implants, which include the incorporation of pro-angiogenic
growth factors into the biomaterials,2−7 the generation of pro-
angiogenic materials mimicking the extracelullar matrix,8−10
and the preseeding of endothelial cells in the implants prior to
implantation.11−13 The arginine-glycine-aspartate (RGD)-based
peptide sequences are widely used for the recognition of
integrins to selectively target endothelial cells. In particular,
cyclic RGD peptides are specific ligands for αvβ3 integrins
which are highly expressed by human endothelial cells and are
involved in the adhesion of human endothelial cells to
bioceramics.14,15
Our approach explores methodologies to enhance the
adhesion of human endothelial cells to biocompatible ceramics
for further applications in tissue engineering.16 We recently
demonstrated the ability of pyrogallol and catechol moieties to
ensure stable anchoring of organic molecules on alumina
bioscaffolds through complexation of the aluminum present in
the inorganic matrix.17 We disclose herein the surface
modification of bioceramics of different chemical composition
with organic ligands containing the cyclo[Arg-Gly-Asp-D-Phe-
RESULTS
■
Synthetic Pathways toward Organic Ligands for the
Functionalization of Bioceramics. Synthesis of Compound
2. A model compound containing the cRGDfK peptide
conjugated to biotin was first prepared, in order to ascertain
that human endothelial cells are able to recognize the cRGDfK
peptide modified on the lysine residue. The synthesis started by
a coupling reaction between a protected c(t-Bu)RG(Pbf)DfK
peptide 1 (Supporting Information) obtained by solid-phase
peptide synthesis using Fmoc protected amino acids18 and a
OSu-activated biotin, in DMF at room temperature (rt) in the
presence of triethylamine. Cleavage of all protecting moieties
under acidic conditions and sequential washings with water,
Received: April 30, 2012
Published: August 16, 2012
© 2012 American Chemical Society
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Scheme 1. Synthesis of Compound 2
Scheme 2. Synthesis of the Multifunctional Ligand 11
methanol, and dichloromethane provided the desired com-
moiety to allow conjugation to the bioceramics was
synthesized. PEG derivative 3 was coupled with a t-Bu-
semiprotected glutamic acid 4 in the presence of PyBOP as a
coupling reagent as previously described16 to afford amide 5,
pound 2 (Scheme 1).
Synthesis of the Multifunctional Ligand 11. A chemical
ligand containing the cRGDfK recognition entity and a gallate
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which was subsequently treated with piperidine to give amine 6
in 73% yield (two steps). Coupling with triacetoxy gallic acid 7
in the presence of PyBOP delivered intermediate 8 in moderate
yield. The tert-butyl ester was cleaved under acidic conditions
to liberate the carboxylic acid 9 which was reacted with
protected peptide 1 in the presence of PyBOP and (i-Pr)2NEt.
Sequential cleavage of the peptide protecting moieties with
TFA and deprotection of the three hydroxyl groups of the
gallate entity with hydrazine provided the desired ligand 11,
which was purified by HPLC (Scheme 2).
Preparation and Characterization of the Ligand-
Functionalized Bioceramics. Dense bioceramic disks from
alumina, HA and TCP were prepared under pressure and
sintering procedures. Then the bioceramics were functionalized
with model ligands 12a−12c bearing either pyrogallol or
catechol moieties (Scheme 3). Using the methodology
absence or the presence of compound 2 (Figure 2C,D), since
the interaction of the integrin with its ligand will modify the
cytoskeletal network.19 The colocalization of actin fibers and
vinculin demonstrated the specific interaction between
compound 2 and cellular integrins. Then the HUVEC were
exposed to either free biotin as a negative control or to
increasing concentrations (2.5, 5, 10 μM) of compound 2 for
assessing the recognition of this modified cRGDfK peptide by
the integrins expressed by the treated cells. The HUVEC were
first exposed to compound 2 for 30 min and then treated with
Alexafluor-labeled streptavidin to visualize the presence of the
biotin residue from compound 2 by fluorescence microscopy
(Figure 2E,F). This experiment confirmed the recognition of
compound 2 by the HUVEC, thus proving that the
modification of the lysine residue of cRGDfK is not detrimental
to the affinity toward integrins. Then the HUVEC were
exposed to the ligand 11 under similar experimental conditions
(Figure 2G,H), demonstrating that ligand 11 was efficiently
recognized by the HUVEC integrins (Figure 2H). This ligand
was thus further investigated for the functionalization of the
bioceramics.
Scheme 3. Chemical Structure of the Model Ligands 12a−
a
12c
Chemical Functionalization of the Bioceramics and
Adhesion of HUVEC. The HA, TCP, and alumina bioceramics
were functionalized with ligand 11 under the same
experimental conditions as for the model ligands 12a−12c.
Then the HUVEC (106 cells per bioceramic) were added to
either unfunctionalized or bioceramics functionalized with
ligand 11, for 2 h at 37 °C. After the washing and fixation
steps, the cells were stained with crystal violet. HUVEC
adhesion to the bioceramics was imaged with a stereo-
microscope and the surface area of the adhered cells was
quantified by the Image J software. First, the number of
adhered HUVEC was compared among the different
unfunctionalized bioceramics. Quantification of the number of
adhered cells showed that alumina bioceramics had better
adhesive properties for the HUVEC than HA or TCP
bioceramics (results not shown). Then, quantification of the
number of adhered HUVEC on the functionalized bioceramics
was performed. After functionalization with ligand 11, the
number of adhered HUVEC on HA bioceramic significantly
decreased, even at low concentrations of the ligand, compared
to unfunctionalized HA bioceramic (Figure 3A). However,
functionalization with ligand 11 at 2.5 μM concentration
resulted in the largest cell surface area, compared with
unfunctionalized HA bioceramics and functionalized HA
bioceramics with higher ligand concentrations (Figure 3B).
Functionalization of TCP bioceramics with ligand 11 resulted
in a significant increase of HUVEC adhesion to the bioceramic,
both in terms of the number of adhered cells and of cell surface
area (Figure 3C,D). The optimal concentration of ligand 11
was 5 μM for the functionalization of the TCP bioceramics. At
higher concentration (10 μM), a decrease in cell adhesion was
observed. For alumina bioceramics, the optimal ligand
concentration for functionalization was 2.5 μM. At higher
concentrations of ligand 11, HUVEC adhesion decreased
(Figure 3E,F). Thus, the functionalization of alumina and TCP
bioceramics with low concentration of ligand 11 induced an
increase of both the number of adhered HUVEC and the cell
size. For HA bioceramics, only the cell surface area was
increased upon functionalization with ligand 11 at 2.5 μM.
Adsorption of Human Serum Proteins on the Bioceramics.
The adsorption of human blood proteins onto the three
unfunctionalized or functionalized bioceramics was also
a
Synthesized as previously described in ref 17.
previously disclosed for the functionalization of alumina
bioceramics with organic ligands,17 surface modification of
HA and TCP bioceramics with compounds 12a−12c was
achieved through complexation of the calcium present in the
inorganic matrix by two adjacent hydroxyl groups of the
aromatic ring of catechol (12b) or pyrrogallol (12c).
The functionalization of HA and TCP bioceramics with the
ligands 12a−12c was monitored by XPS, following incubation
of the bioceramics with 12a−12c, in water for 24 h. Surface
analysis of the unfunctionalized (incubation with pure water)
and of functionalized HA and TCP bioceramics was performed
by XPS to determine the atomic composition of the samples.
As previously shown for alumina bioceramics,17 ligand 12a did
not show significant binding to HA and TCP bioceramics. The
peaks corresponding to fluorine and nitrogen atoms present in
the ligands are only visible for the bioceramics which were
treated with ligands 12b or 12c (Figure 1). Moreover the
binding energy of fluorine (688 eV) is consistent with a C−F
bond.
Thus, as previously observed with alumina-based bioceram-
ics,17 the functionalization of HA and TCP bioceramics only
occurred with ligands bearing catechol or pyrrogallol groups
(compounds 12b and 12c). Only the peaks corresponding to
the initial composition of the bioceramics were observed for the
bioceramics incubated with water or with 12a (Table 1).
Cell Assays. Recognition of Ligands 2 and 11 by HUVEC.
First, the expression of αvβ3 integrin by HUVEC was
demonstrated by immunofluorescence, using human lung
A549 cells as αvβ3 integrin-negative control cells (Figure
2A,B). The selective interaction between compound 2 and
integrins was proved by immunostaining of vinculin and
cytoskeletal actin after incubation of the HUVEC in the
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Figure 1. XPS spectra of unfunctionalized and functionalized bioceramics. XPS spectra for HA and TCP bioceramics incubated with water (A1-B1)
or compound 12a (A2-B2), 12b (A3-B3), and 12c (A4-B4).
determined. After incubation of the different bioceramics with
human serum for 1 h at 37 °C, the bioceramics were extracted
in boiling SDS buffer, and the proteins were resolved by
electrophoresis and analyzed by Western blotting using specific
antibodies. Functionalization of the bioceramics with the
optimal concentrations of ligand 11 (previously determined
through the evaluation of HUVEC adhesion) resulted in a
decrease of the binding of vitronectin, fibronectin, and
fibrinogen, which are involved in cell adhesion (Figure 4).
The adhesion of IGg and other serum proteins (Supporting
Information, Figure S1) was also decreased on the function-
alized bioceramics. Alumina and TCP bioceramics adsorbed
larger amounts of proteins compared to HA bioceramic.
Stability of the Chemical Coating on Bioceramics in
Cell Culture Medium. Samples of HA, TCP, and alumina
bioceramics, functionalized with compound 12c, were
incubated in Dulbecco’s Modified Eagle medium (DMEM)
supplemented with 10% fetal calf serum (FCS) for periods of 1
day and 14 days. The same experiment was performed on
unfunctionalized samples of HA, TCP, and alumina bio-
ceramics. Stability of the chemical functionalization over the
time was monitored by XPS analysis of the surface of the
bioceramic samples (Supporting Information, Table S1). After
14 days of incubation, partial release of the chemical coating
was observed for HA bioceramics (monitored by the
percentage of fluorine and nitrogen atoms). The coating of
TCP and alumina bioceramics was highly stable and was not
affected by incubation for 14 days in cell culture medium.
DISCUSSION
■
Implant vascularization is a major challenge in tissue engineer-
ing, tissue repair, and regenerative medicine. Failure to develop
a functional vasculature in large implants results in poor survival
of the cells which should colonize the implant. Different
approaches can be used to improve vascularization processes,
including the delivery over time of soluble pro-angiogenic
factors or the preintegration within the implant of vascular cells.
In this study, a general method for the coating of bioceramics
with ligands containing a targeting peptide for αvβ3 and αvβ5
integrins (cRGDfK) and an anchoring unit was developed. This
surface functionalization enhanced both the number of human
endothelial cells adhering to the materials and their spreading
on the surface. The use of pyrogallol or catechol moieties
allowed the efficient anchoring of the chemical ligands to the
surface of HA, TCP, and alumina bioceramics, as demonstrated
by the binding of compounds 12b and 12c to these materials.
After simple incubation of the bioceramics with aqueous
solutions of the ligands 12b and 12c, XPS analysis of the
resulting surfaces demonstrated the stable coating of HA and
TCP bioceramics. Complexation of the calcium present in the
inorganic matrix is most likely involved in the coating process.
Confirming previous data obtained with alumina bioceramics,17
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Table 1. Atomic Composition of the Bioceramics
a
Functionalized by Compounds 12a, 12b, or 12c
atomic concentrations [%]
water
12a
12b
12c
HA Bioceramic
F 1s
0.0
55.3
0.0
0.0
1.8
48.8
2.8
3.1
39.7
4.6
O 1s
N 1s
Ca 2p
C 1s
50.4
0.0
16.1
28.7
12.7
12.2
34.5
8.0
34.9
44.5
TCP Bioceramic
F 1s
0.0
58.9
0.0
0.0
2.9
52.6
2.1
6.1
41.1
4.8
O 1s
N 1s
Ca 2p
C 1s
55.8
0.0
16.3
24.8
16.9
14.1
28.4
10.2
37.8
27.3
Alumina Bioceramic
F 1s
0.0
51.7
0.0
0.0
51.6
0.0
6.4
35.2
5.2
7.4
24.2
7.5
O 1s
N 1s
Ca 2p
C 1s
Al 2p
3.8
4.3
1.9
1.0
17.4
27.1
21.0
23.1
36.3
15.0
52.5
7.4
a
The percentages of atomic concentrations were determined by XPS
for HA, TCP and alumina bioceramics functionalized with the
compound 12a, 12b, or 12c. The values previously obtained for
alumina bioceramic17 have been included here for comparison.
the pyrogallol group was superior to catechol moiety for the
functionalization of HA and TCP bioceramics and was thus
selected for the design of the functionalizing ligand.
Cell adhesion is mediated, in part, by the interaction of
integrins with RGD peptides.20 In particular, cRGDfK binds
specifically to αvβ3 and αvβ5 integrins which are highly
expressed by angiogenic endothelial cells.21 The synthesis of
a multifunctional ligand displaying a pyrogallol functionality for
conjugation to the bioceramics and cRGDfK for targeting
integrins at the surface of endothelial cells was thus envisaged.
A convergent pathway, using glutamic acid as template for the
sequential introduction of the binding units for both the
bioceramics and human endothelial cells and of an alkynyl
group allowing click-reaction with a fluorescent azido-
containing label, provided efficient access to the multifunctional
linker 11. Chemical modification of cRGDfK on the lysine ε
amino group did not affect its specific affinity for the integrins
expressed by the HUVEC, as demonstrated by the binding of
ligands 2 and 11 to the cells after only 30 min incubation time.
The HA, TCP, and alumina bioceramics were then function-
alized with ligand 11 via simple incubation in aqueous medium.
The adhesion and spreading of the HUVEC on the
functionalized bioceramics were compared to unfunctionalized
bioceramics. In the case of the HA bioceramic, the presence of
ligand 11 decreased the adhesion of the HUVEC to the
bioceramic in agreement with previously published informa-
tion.14,15 However, at low concentration of ligand 11, the cell
surface area was increased. It was previously hypothesized that
caspase activation occurred with the engagement of the
integrins, decreasing human endothelial cell survival.14,15 For
TCP and alumina bioceramics, an increase in the number of
adhered HUVEC and of the cell surface area was obtained
upon functionalization of the bioceramics with ligand 11, with
optimal ligand concentrations of 5 and 2.5 μM, respectively. At
Figure 2. Binding of compounds 2 and 11 to HUVEC expression of
the αvβ3 integrin by (A) A549 cells as negative control cells and (B)
HUVEC. Expression of vinculin and actin by the HUVEC nonexposed
(C) or exposed (D) for 45 min to compound 2 (5 μM). Nuclei were
labeled in blue with DAPI. The HUVEC were exposed for 30 min to
(E) free biotin (5 μM) as a control for nonspecific binding, or (F) to
compound 2 (5 μM), then to fluorescent Alexafluor-labeled
streptavidin to label biotin and to DAPI to label nuclei. The
HUVEC were exposed for 3 h to (G) HBSS, or (H) to ligand 11 (5
μM) in HBSS, then to DAPI and Alexafluor-azide (10 μM) in the
presence of CuSO4 (2 mM) and sodium ascorbate (1 mM).
Alexafluor-azide reacts with the alkyne moiety of ligand 11 through
a click-reaction promoted by the combination of CuSO4 and sodium
ascorbate.
higher concentrations of ligand 11, both the number of adhered
cells and the cell surface area decreased and became
comparable to the values observed for unfunctionalized
bioceramics. The different optimal concentrations determined
for the three bioceramics suggest that the adhesion of
endothelial cells is not only dependent on the nature of the
chemical functionalization at the surface of the biomaterials, but
also on the composition and topography of the materials.22
Consequently, comparable concentrations of the functionaliz-
ing ligand 11 do not have the same effect on the adhesion of
the HUVEC on the three bioceramics evaluated.
The adsorption of human serum proteins onto unfunction-
alized and functionalized bioceramics with ligand 11 was also
determined. The functionalization of the bioceramics with
ligand 11 decreased the adsorption of blood proteins able to
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Figure 3. Adhesion of HUVEC to unfunctionalized and functionalized bioceramics. Disks of HA (A, B), TCP (C, D), and alumina (E, F)
bioceramics functionalized with increasing concentrations (0−10 μM) of ligand 11 were incubated with HUVEC for 2 h in HBSS. The quantification
of cell adhesion was performed by analysis of stereomicroscopy pictures (A, C, E). Results are the mean SD of triplicates of three independent
experiments. The number of cells adhering to the bioceramics disks was compared using a Student’s t-test: *: p < 0.05; **: p < 0.01; ***: p < 0.001.
The quantification of the surface areas of the adhered cells was performed using Image J software followed by a Boxplot representation (B, D, F).
Cell surface areas on the bioceramics disks were compared using a Wilcoxon-signed rank test: *: p < 0.05; **: < 0.01; ***: p < 0.001. (A, B) HA
bioceramics; (C, D) TCP bioceramics; (E, F) alumina bioceramics.
bind integrins on the cell surface, such as vitronectin,
fibronectin, and fibrinogen, which suggests good osteoconduc-
tion potential for the functionalized bioceramics,14 but also of
immunoglobulins and serum albumin. TCP and alumina
bioceramics adsorbed human proteins at higher levels than
the widely used HA bioceramic, which represents interesting
properties for some specific developments of implants for tissue
engineering. In addition, chemical coating of TCP and alumina
bioceramic is highly stable over the time (at least 14 days),
which is promising for the long-term adhesion and proliferation
of endothelial cells on the biomaterial.
formation of a functional vascular system. A general method for
the functionalization of HA, TCP, and alumina bioceramics
with organic ligands containing cRGDfK as integrin-recognition
motif and a pyrogallol unit as anchoring moiety to bioceramics
was developed. Surface modification of the bioceramics was
ascertained by XPS analysis and the recognition of ligand 11 by
human endothelial cells was demonstrated. The combination,
in the same functionalizing ligand, of a targeting entity for the
recognition of integrins with a pyrogallol anchoring unit for
surface coating of the bioceramics and a reactive alkyne group
for visualization, represents a promising tool to improve the
adhesive properties of TCP and alumina bioceramics. The use
of a pyrrogallol moiety to ensure stable functionalization of the
bioceramics provides an interesting strategy as surface
modification is obtained by simple incubation in aqueous
medium. The potential of TCP and alumina bioceramics to
integrate human endothelial cells was significantly improved by
functionalization with ligand 11, as both the number and size of
CONCLUSION
■
The development of a functional vascular system in
biomaterials for large permanent bone implants is a prerequisite
to develop valid reconstruction materials allowing efficient
tissue repair. The adhesion of endothelial cells on the
biomaterials represents a challenge and is mandatory for the
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Figure 4. Adsorption of human serum proteins on the three bioceramics. Disks of HA, TCP, and alumina bioceramics either unfunctionalized or
functionalized with ligand 11 were incubated with normal human serum for 1 h at 37 °C. The proteins adsorbed on the disks were extracted in SDS,
resolved by electrophoresis and the presence of vitronectin, fibronectin, fibrinogen, and IgG was evaluated by Western blotting.
1s peak was calibrated at 285 eV and used as an internal standard to
compensate for any charging effects. Both curve fitting of the spectra
and quantification were performed with the CasaXPS software, using
relative sensitivity factors given by Kratos.
adhered endothelial cells were significantly higher than on
unfunctionalized bioceramics. Moreover, the high stability of
the chemical coating on TCP bioceramic over the time
represents and additional asset for this biomaterial to be further
developed for tissue engineering. The adhesion properties of
HA bioceramic could not be improved by this method. A
decrease of the adsorption of human serum proteins involved in
cell adhesion was observed on the three functionalized
bioceramics. However, functionalized alumina and TCP
bioceramics demonstrated higher adsorption of human proteins
than functionalized HA bioceramic, suggesting better bio-
compatibility of alumina and TCP bioceramics.
Chemical Syntheses. Synthesis of cyclo(Arg-Gly-Asp-D-Phe-Lys)-
Biotin (2). cyclo[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys] 1 (40 mg, 0.04
mmol, 1 equiv) was dissolved in dry DMF in the presence of
triethylamine (9 μL, 0.07 mmol, 1.5 equiv) and Biotin-OSu (17 mg,
0.05 mmol, 1.1 equiv). The reaction mixture was stirred overnight at
rt. The product was concentrated under reduced pressure to remove
the DMF. The residual product was dissolved in a solution of TFA/
water (1 mL/0.06 mL) and triisopropylsilane (0.03 mL) was added.
The reaction mixture was stirred at rt until total deprotection of the
peptide. After concentration and sequential washings with water,
methanol, and dichloromethane the desired product 2 was obtained as
a white powder (mass: 4 mg, yield: 12% for 2 steps). IR (film): 3435,
1660, 1315, 1015, 950, 705, 645, 590, 520, 500 cm−1. HRMS (ESI):
(m/z): calcd for C37H56N11O7S + H: 830.3983, found: 830.3965.
EXPERIMENTAL SECTION
■
General Procedures. Commercial reagents (Fluka, Aldrich, VWR,
Switzerland) were used without further purification. Anhydrous
solvents were obtained by filtration (PureSolv MD Series, Innovative
Technology). TLCs for reaction monitoring were performed on
Merck silica gel 60 F254 plates, and spots were revealed with UV light
and reaction with KMnO4 or ninhydrine. IR spectra were recorded on
a Perkin-Elmer-1420 spectrometer. 1H NMR spectra were recorded on
a Bruker ARX-400 spectrometer (400 MHz) using CDCl3 as solvent
and calibrated using the solvent’s residual signal at 7.27 ppm as an
internal reference. 13C NMR spectra were recorded on a Bruker ARX-
400 spectrometer (100.6 MHz) using CDCl3 as solvent and calibrated
using the solvent’s residual signal at 77.0 ppm as an internal reference.
Chemical shifts are expressed in parts per million (ppm) and coupling
constants (J) are in hertz. Mass spectra were obtained on a Nermag R-
10-10C spectrometer with chemical ionization (NH3) and mode m/z
(amu) [% relative base peak (100%)]. Semipreparative HPLC was
performed on a Waters Autopurification ZQ System equipped with a
2767 Sample Manager, a 2525 Binary Gradient Module and a 2996
Photodiode Array detector, coupled to Waters Micromass ZQ
analyzer. The HPLC purifications were performed on XTerra Prep
RP C18 (19 × 150 mm) columns, using reverse-phase conditions (2 to
100% acetonitrile with 0.1% TFA over 20 min). Purity of the
synthesized compounds was determined by HPLC and ESI-HRMS. All
compounds presented chemical purity > 95%. X-ray photoelectron
spectroscopy (XPS) data were collected by Axis Ultra and Axis Nova
instruments (Kratos analytical, Manchester, UK) under ultrahigh
vacuum conditions (<10−8 Torr) and using a monochromatic Al Kα
X-ray source (1486.6 eV). The source power was maintained at 225 or
150 W and the emitted photoelectrons were sampled from a 750 μm ×
300 μm area. The analyzer pass energy was 160 or 80 eV for survey
spectra and 40 eV for high-resolution spectra. The adventitious carbon
25
[α]D = −38 (c = 0.085, CH2Cl2).
Synthesis of tert-Butyl N-3,6,9,12-tetraoxapentadec-14-yn-1-yl-
N2-{[3,4,5-tris(acetyloxy)phenyl] carbonyl}-L-α-glutaminate (8). tert-
Butyl N2-[9A fluoren-9-ylmethoxy)carbonyl]-N-3,6,9,12-tetraoxa-pen-
tadec-14-yn-1-yl-L-α-glutaminate 5 (865 mg, 1.4 mmol, 1 equiv) was
dissolved in a solution of 20% piperidine in DMF at rt. The reaction
mixture was stirred for 1 h at rt and concentrated under reduced
pressure, and the intermediate product 6 was purified by flash column
chromatography (DCM/MeOH (98:2)) (mass: 409 mg, yield: 74%).
Acetoxygallic acid 7 (142 mg, 0.5 mmol, 1 equiv) and PyBOP (299
mg, 0.6 mmol, 1.2 equiv) were dissolved in dry DCM (2 mL). After 15
min, (i-Pr)2NEt (0.12 mL, 0.7 mmol, 1.5 equiv) followed by a solution
of tert-butyl-N-3,6,9,12-tetraoxa-pentadec-14-yn-1-yl-L-α-glutaminate 6
(200 mg, 0.5 mmol, 1 equiv) in DCM (2 mL) were added to the
reaction mixture. The reaction was stirred overnight at rt. The solvent
was removed under reduced pressure and the product 8 was purified
by flash chromatography (AcOEt/petroleum ether (7:1) to (9:1))
(mass: 134 mg, yield: 40%). IR (film): 3658, 3420, 3286, 2922, 1777,
1717, 1660, 1593, 1538, 1488, 1457, 1423, 1393, 1370, 1323, 1251,
1184, 1157, 1125, 1085, 1051, 949, 897, 833, 739, 615, 587, 555 cm−1.
1H NMR (400 MHz, CDCl3): δ= 7.66 (s, 2H, CH) 4.53 (m,1H, CH),
2
4.23 (d, 2H, J = 1.6 Hz, CH2), 3.71−3.38 (m, 16H, (CH2)6), 2.78−
2.35 (m, 3H, CH, CH2), 2.33 (s, 9H, (CH3)3), 2.30−2.09 (m, 2H,
CH2), 1.44 (s, 9H, (CH3)3). 13C NMR (100 MHz, CDCl3): 173.88
(Cq), 173.03 (Cq), 167.71 (Cq), 166.57 (Cq), 165.44 (Cq), 143.54
(Cq), 137.74 (Cq), 131.37 (Cq), 120.15 (C (aromatic)), 81.47 (Cq),
79.37 (d, CH), 75.08 (CH2), 71.12 (CH2), 69.44 (CH2), 69.40 (CH2),
69.29 (CH2), 69.10 (CH2), 67.82 (CH2), 58.36 (CH2), 54.25 (CH2),
38.98 (CH2), 31.72 (CH2), 28.01 (CH3), 20.49 (CH3). MS (ESI):
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HRMS (ESI): (m/z): calcd for C35H46N2O9+H: 695.3027, found:
695.3052. [α]D = −40 (c = 0.0025, CH2Cl2).
in Dulbecco’s Modified Eagle medium (DMEM) supplemented with
10% fetal calf serum (FCS) for periods of 1 day and 14 days. The
samples were drained on a piece of absorbent paper before being
soaked in fresh water (1.5 mL) for 5 min to remove the released
compound. This procedure was repeated three times and the samples
were then dried under a vacuum for 24 h and subsequently analyzed
by XPS. XPS data were collected by an Axis Nova instrument (Kratos
analytical, Manchester, UK) under ultrahigh vacuum conditions
(<10−8 Torr) and using a monochromatic Al Kα X-ray source
(1486.6 eV).
Cellular Assays. Cells and Cell Culture Reagents. HUVEC were
obtained from PromoCell (PromoCell, Heidelberg, Germany) and
grown in Endothelial Cell Growth medium 2 (PromoCell) containing
10% heat-inactivated fetal calf serum (FCS), penicillin/streptomycin
(Invitrogen, Basel, Switzerland) and supplemented with 2.5 mL of
SupplementMix (PromoCell). The human lung A549 adenocarcinoma
cell line is available from ATCC (American Tissue Culture Collection,
Manassas, VA, USA). The cells were grown in Dulbecco’s Modified
Eagle Medium (DMEM) containing 4.5 g/L glucose, 10% FCS, and
penicillin/streptomycin.
Immunofluorescence Staining for the αVβ3 Integrin. Cells were
grown for 24 h on a glass slide (Gerhard Menzel, Braunschweig,
Germany), fixed with a 4% PBS-buffered paraformaldehyde solution
containing 1% sucrose, 1 mM CaCl2, 1 mM MgCl2, and 0.1% NaN3 at
4 °C for 45 min. The cells were washed twice in PBS and
permeabilized with methanol at −20 °C for 10 min at 4 °C. Two
additional washings with PBS were performed followed by incubation
of the cells in 5% BSA (Sigma-Aldrich, Buchs, Switzerland) in PBS
containing 1 mM CaCl2, 1 mM MgCl2, and 0.1% NaN3, at rt for 1 h.
After two washings with PBS, the cells were incubated overnight at 4
°C with a murine antihuman αvβ3 integrin monoclonal antibody
(Millipore, Billerica, USA; dilution 1:150) in HBSS (Invitrogen)
containing 3% BSA, 0.1% Tween 20, and 0.1% NaN3 (complete
HBSS). The cells were washed three times with PBS for 5 min and
incubated at rt under slight agitation with the secondary fluorescent
antibody (antimouse Alexafluor 488 conjugate antibody, Invitrogen;
dilution 1:250) in complete HBSS. The cells were washed three times
with PBS for 5 min and the cell nuclei were labeled with a solution of
4′,6-diamidino-2-phenylindole (DAPI, Roche Diagnostics, Rotkreuz,
Switzerland, 1 μg/mL in PBS) at rt for 5 min. A final washing with
PBS was performed, and the evaluation of the αVβ3 integrin expression
was evaluated by fluorescence microscopy (Zeiss Axioplan 2, filters:
λex/λem 450−490/515−565 for Alexafluor 488; 365/420, for DAPI).
Evaluation of Vinculin and Cytoskeletal Actin after Incubation of
the HUVEC with Compound 2. Cells were grown for 24 h in BD
Falcon CultureSlides (BD Biosciences, Erembodegem, Belgium), then
exposed to increasing concentrations (0 to 10 μM) of compound 2 for
45 min at 37 °C in HBSS containing 0.1% BSA. The medium was
removed and the cells were washed twice with PBS, fixed in 4%
buffered paraformaldehyde for 10 min at rt. After two washings with
PBS, the cells were incubated for 5 min in 0.1% PBS-Triton. Two
additional washings with PBS were performed followed by incubation
of the cells in PBS containing 5% BSA at rt for 1 h. Then, the cells
were incubated overnight at 4 °C with a murine antivinculin
monoclonal antibody (Invitrogen, dilution 1:300) in a solution for
primary antibody (Calbiochem, Merck Biosciences, Darmstadt,
Germany). The cells were washed three times with PBS for 5 min
and incubated at rt for 1 h under slight agitation with a secondary
fluorescent antimouse Rhodamine C-conjugated antibody (Invitrogen;
dilution 1:250) in a solution for secondary antibody (Calbiochem).
The cells were washed three times with PBS for 5 min and the cell
nuclei and cytoskeletal actin were labeled with a solution of DAPI (1
μg/mL in PBS) and 2.5% Oregon Green 488 Phalloidin (Invitrogen)
at rt for 30 min. The cells were washed twice in PBS, and then the
chamber was removed from the glass slide with the chamber removal
device and the expression of vinculin and actin was evaluated by
fluorescence microscopy (Zeiss Axioplan 2, filters: λex/λem 450−490/
515−565 for Oregon Green 488; 365/420, for DAPI; 510−560/590
for Rhodamine C).
25
Synthesis of cyclo{N2-(L-α-aspartyl-D-phenylalanyl)-N6-{N-
(3, 6, 9, 12-tetraoxapentadec-14-yn-1-yl)-N2 -[(3, 4, 5-
trihydroxyphenyl)carbonyl]-L-α-glutaminyl}-L-lysyl-N5-[amino
(iminio)methyl]-L-ornithylglycyl} (11). tert-Butyl N-3,6,9,12-tetraox-
apentadec-14-yn-1-yl-N2-{[3,4,5-tris(acetyloxy)phenyl] carbonyl}-L-α-
glutaminate 8 (100 mg, 0.14 mmol, 1 equiv) was treated with a
solution of 4 M HCl in dioxane (4 mL) at 0 °C. After 15 min the
reaction mixture was allowed to reach rt and was stirred for 4 h. The
solvent was removed under reduced pressure and HCl was
coevaporated three times with Et2O to afford the intermediate
carboxylic acid 9 (mass: 87 mg, yield: 95%). N-3,6,9,12-tetraox-
apentadec-14-yn-1-yl-N2-{[3,4,5-tris(acetyloxy)phenyl]carbonyl}-L-α-
glutamine 9 (35 mg, 0.05 mmol, 1 equiv) and PyBOP (34 mg, 0.07
mmol, 1.2 equiv) were dissolved in dry DCM (0.4 mL) at 0 °C. After
15 min, a solution of cyclo[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys)] 1
(50 mg, 0.05 mmol, 1 equiv) in DCM (0.5 mL) containing (i-Pr)2NEt
(20 μL, 0.11 mmol, 2 equiv) was added to the reaction mixture at 0
°C. The reaction mixture was stirred for 2 h at rt. The product was
concentrated under reduced pressure and purified by flash column
chromatography (DCM/MeOH (9:1)). The resulting intermediate
was dissolved in a solution of TFA/water (1.2 mL/0.08 mL) at rt.
Triisopropylsilane (37 μL) was added. The reaction mixture was
stirred until total deprotection of cRGDfK peptide. The solution was
concentrated under reduced pressure and the residue was dissolved in
EtOH (0.1 mL). The reaction mixture was cooled to 0 °C and
hydrazine (1.22 μL, 0.025 mmol, 3 equiv) in EtOH (0.1 mL) was
added. The reaction mixture was stirred for 30 min at 0 °C,
concentrated under reduced pressure, and purified by HPLC (XTerra
Prep RP C18, 19 × 150 mm, Waters) to give the desired product (11)
as a dark oil (mass: 10 mg, yield: 18% for 3 steps). IR (film): 3255,
2570, 1650, 1615, 1525, 1450, 1470, 1395, 1380, 1355, 1285, 1220,
1155, 1135, 1105, 910, 840, 780, 755, 740, 655, 590, 565, 535, 520
cm−1. HRMS (ESI): (m/z): calcd for C50H71N11O17 + H: 1098.5108,
25
found: 1098.5106. [α]D = −38 (c = 0.004, CH2Cl2).
Materials. Preparation of the Bioceramics. Alumina was obtained
from Ceralox (HPA-0.5, Sasol North America Inc., Tucson, AZ, USA).
Hydroxyapatite (HA) (CAS 1306-06-5, purum p. a.) and β-tricalcium
phosphate (TCP) (CAS 7758-87-4, purum p. a.) were purchased from
Fluka (Switzerland). Dense bioceramic disks were prepared. For each
disk, 4.5 g of HA or of TCP, or 6.2 g of alumina powders were filled
into a cylindrical metallic matrix with a 30 mm inner diameter. TCP
powder was gently sprayed with water before filling the matrix. The
powders were unidirectionally compressed for 1 min at 15 kN in the
case of alumina and HA and at 20 kN in the case of TCP. Then, the
precompressed disks were isostatically pressed at 800 kN for 4 min to
reach maximum green density. Sintering of the disks was carried out
according to the following sintering schedules. In the case of HA and
TCP: 1 °C/min from rt to 1200 °C, 6 h dwell time at 1200 °C, 3 °C/
min from 1200 °C to rt. In the case of alumina: 1 °C/min from rt to
1575 °C, 2 h dwell time at 1575 °C, 3 °C/min from 1575 °C to rt.
Functionalization of the Bioceramics for XPS Measurements. A
small fragment of each material (ca. 0.1 cm3) was incubated in 1 mL of
a 1 mM aqueous solution of the corresponding linker (12a, b, or c), at
rt for 16 h, in the dark. The materials were drained on a piece of
absorbent paper before being soaked in fresh water (1.5 mL) for 5 min
to remove the nonattached linker. This procedure was repeated three
times and the samples were then dried under a vacuum for 24 h and
subsequently analyzed by XPS. XPS data were collected by an Axis
Ultra instrument (Kratos analytical, Manchester, UK) under ultrahigh
vacuum conditions (<10−8 Torr) and using a monochromatic Al Kα
X-ray source (1486.6 eV).
Stability of the Chemical Coating on Bioceramics. A small
fragment of each material (ca. 0.1 cm3) was incubated in 1 mL of a 1
mM aqueous solution of compound 12c, at rt for 16 h, in the dark.
The materials were drained on a piece of absorbent paper before being
soaked in fresh water (1.5 mL) for 5 min to remove the nonattached
linker. This procedure was repeated three times and the samples were
then dried under a vacuum for 24 h. The samples were then incubated
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HUVEC Labeling with Compound 2. Cells were grown for 24 h on
BD Falcon CultureSlides (BD Biosciences) and then exposed to
increasing concentrations (0−10 μM) of either compound 2 or free
biotin (Sigma-Aldrich), for 30 min at 37 °C, in HBSS containing 0.1%
BSA. The medium was removed and the cells were washed twice with
PBS, fixed in 4% buffered formaldehyde for 10 min at rt. After two
washings with PBS, the cells were labeled by incubation with
streptavidin-Alexafluor 488 (Invitrogen, dilution 1:2000, 2 mg/mL)
in PBS containing 1% BSA at rt for 30 min, in the dark. After two
additional washings with PBS, the cell nuclei were labeled with DAPI
(1 μg/mL in PBS) at rt for 5 min, in the dark. The cells were washed
twice in PBS, and then the chamber was removed from the glass slide
and the evaluation of cell recognition was performed by fluorescence
microscopy (Zeiss Axioplan 2, filters: λex/λem 450−490/515−565 for
Alexafluor 488; 365/420, for DAPI).
HUVEC Labeling with Ligand 11. Cells were grown for 24 h on BD
Falcon CultureSlides (BD Biosciences) and then exposed to increasing
concentrations of ligand 11 in HBSS containing 0.1% BSA, for 3 h at
37 °C. The medium was removed and the cells were washed twice
with PBS, fixed in 4% buffered formaldehyde for 10 min at rt. After
two washings with PBS, the cells were labeled by click reaction using
Alexafluor 488-azide (Invitrogen, 10 μM), CuSO4 (Sigma-Aldrich, 2
mM), and sodium ascorbate (Sigma-Aldrich, 1 mM) in PBS containing
1% of BSA, at rt for 30 min, in the dark. After two washings with PBS,
the cell nuclei were labeled with DAPI (1 μg/mL in PBS) at rt for 5
min, in the dark. The cells were washed twice in PBS, and then the
chamber was removed from the glass slide and the evaluation of the
cell recognition was performed by fluorescence microscopy (Zeiss
Axioplan 2, filters: λex/λem 450−490/515−565 for Alexafluor 488;
365/420, for DAPI).
Adhesion of HUVEC on the Bioceramics. Disks (d = 2.5 cm, h =
0.5 cm) of each of the three bioceramics were incubated in a solution
of ligand 11 at increasing concentrations (0, 2.5, 5, and 10 μM) for 24
at rt, in the dark under slight stirring. The disks were washed three
times with water and dried under a vacuum for 24 h. HUVEC were
trypsinized, counted, and centrifuged for 5 min after dilution in HBSS.
Three mL of the cell suspension (100 000 cells/mL) were added on
the disks of unfunctionalized or functionalized bioceramics in a six-well
plate (Costar, Corning, NY, USA) and incubated for 2 h at 37 °C. The
media were removed. The disks were washed once with PBS and
inserted in a new well, fixed with 4% buffered formaldehyde for 10 min
at rt, and then stained in a solution of crystal violet (Sigma-Aldrich,
1.5% glacial acetic acid, 0.05% crystal violet in water), for 5 min at rt.
Adhered cells were imaged by stereomicroscopy (MZ16 FA Leica/
DFC 480 Leica, 0.63X and 1.6X, zoom 72X) and the cell number and
surface areas were evaluated using the Image J software. The cell
surface areas were then represented by a Boxplot.
Statistical Analysis. Results were subjected to computer-assisted
statistical analysis using the R and RStudio softwares. The distribution
of data was tested by the Shapiro-Wilk test. The data that were from a
normally distributed population were compared using the Student’s t-
test, whereas data not normally distributed were compared using the
Wilcoxon signed-rank test. Differences of p < 0.05 were considered
significant.
ASSOCIATED CONTENT
* Supporting Information
■
S
Evaluation of microbial contamination of the bioceramics.
Evaluation of the total adsorption of human serum proteins.
Evaluation of the stability of the chemical coating on
bioceramics in cell culture medium. Detailed chemical
syntheses: Synthesis of cyclo[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-
Lys(Z)] (1); synthesis of cyclo(Arg-Gly-Asp-D-Phe-Lys)-Biotin
(2); synthesis of tert-butyl N-3,6,9,12-tetraoxapentadec-14-yn-
1-yl-N2-{[3,4,5-tris(acetyloxy)phenyl]carbonyl}-L-α-glutami-
nate (8); synthesis of cyclo{N2-(L-α-aspartyl-D-phenylalanyl)-
N6-{N-(3,6,9,12-tetraoxapentadec-14-yn-1-yl)-N2-[(3,4,5-
trihydroxyphenyl)carbonyl]-L-α-glutaminyl}-L-lysyl-N5-[amino-
(iminio) methyl]-L-ornithylglycyl} (11). This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*(L.J.-J.) Address: University Institute of Pathology, CHUV-
UNIL rue du Bugnon 25; CH-1011 Lausanne, Switzerland.
Phone: +41 21 314 7173; fax: +41 21 314 7115; e-mail:
lucienne.juillerat@chuv.ch (S.G.-L.) Address: Laboratory of
́ ́
Synthesis and Natural Products; Ecole Polytechnique Federale
de Lausanne (EPFL), ISIC, Batochime, CH-1015 Lausanne,
Switzerland. Phone: +41 21 693 93 72; fax: +41 21 693 93 55;
e-mail: Sandrine.Gerber@epfl.ch.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the Swiss National Science Foundation (Grant Nos.
CR23I3-124753 and CR22I2-140866) for financial support, Dr.
■
́
L. Menin, J. Artacho, M. Rey, Dr. P. Mieville, F. Sepulveda, and
C. Chapuis Bernasconi for excellent technical support and M.
Federico Tettamanti for the statistical advices.
Western Blotting Experiments of Bioceramic-Desorbed Human
Plasma Proteins. Fresh human blood in serum separation-containing
gel tubes (BD Falcon, Erembodegem, Belgium) was obtained from
leftovers of analytical blood with normal values. Tubes were
centrifuged for 10 min at 4000 rpm and serum was collected from
the upper phase. Disks of unfunctionalized and functionalized
bioceramics with ligand 11 were incubated with human serum at 37
°C, for 1 h under slight agitation, then washed three times with PBS
and proteins adsorbed on the materials were solubilized in boiling SDS
buffer (50 mM Tris buffer, 2% SDS, 5% β-mercaptoethanol), for 30
min under agitation. Desorbed proteins were resolved by SDS−PAGE
(6% and 8% polyacrylamide gels) and transferred onto a nitrocellulose
membrane (Whatman, Dassel, Germany) and then blotted with rabbit
polyclonal antihuman fibronectin (Millipore, diluted 1:5000), antihu-
man vitronectin (Santa Cruz Biotechnology, Heidelberg, Germany,
diluted 1:5000), antihuman IgG (Dako, Glostrup, Denmark, diluted
1:2000), or mouse monoclonal antihuman fibrinogen (Abcam,
Cambridge, UK, diluted 1:1000) antibodies. The membrane was
exposed for 60 min to horseradish peroxidase-conjugated antirabbit
(Sigma-Aldrich) or antimouse (Sigma-Aldrich) antibodies (diluted
1:5000) and visualized by enhanced chemiluminescence (ECL, GE
Healthcare, Amersham, UK).
ABBREVIATIONS USED
■
DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco’s
modified Eagle medium; FCS, fetal calf serum; HA,
hydroxyapatite; HBSS, Hank’s buffered salt solution; HEPE,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HUVEC,
human umbilical vein endothelial cells; OSu, oxy succinimide;
Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl;
PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexa-
fluorophosphate; RGD, arginine-glycine-aspartate; TCP, trical-
ciumphosphate; XPS, X-ray photoelectron spectroscopy
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