Ultrasmall particle of iron oxide-RGD peptidomimetic conjugate: synthesis and characterisation

Article abstract of DOI:10.1016/j.bmcl.2010.01.150

Ultrasmall particles of iron oxide (USPIOs) coated with 3,3′-bis(phosphonate)propionic acid were covalently coupled to a home-made Arg-Gly-Asp (RGD) peptidomimetic molecule via a short oligoethyleneglycol (OEG) spacer. The conjugation rate was measured by X-ray photoelectron spectroscopy (XPS). The particle size and magnetic characteristics were kept. Our novel conjugate targeted efficiently Jurkat cells (increase of 229% vs the control).

Full text of DOI:10.1016/j.bmcl.2010.01.150

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1861–1865
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Ultrasmall particle of iron oxide—RGD peptidomimetic conjugate: synthesis
and characterisation
Vincent Rerat a, Sophie Laurent b, Carmen Burtéa b, Benoît Driesschaert a, Vincent Pourcelle a,
Luce Vander Elst b, Robert N. Muller b, Jacqueline Marchand-Brynaert a,
*
a Unité de Chimie Organique et Médicinale, Université Catholique de Louvain, Bâtiment Lavoisier, Place Louis Pasteur 1, B-1348 Louvain-la-Neuve, Belgium
b Department of General, Organic and Biomedical Chemistry, NMR and Molecular Imaging Laboratory, University of Mons, Belgium
a r t i c l e i n f o
a b s t r a c t
Ultrasmall particles of iron oxide (USPIOs) coated with 3,30-bis(phosphonate)propionic acid were cova-
lently coupled to a home-made Arg-Gly-Asp (RGD) peptidomimetic molecule via a short oligoethylene-
glycol (OEG) spacer. The conjugation rate was measured by X-ray photoelectron spectroscopy (XPS). The
particle size and magnetic characteristics were kept. Our novel conjugate targeted efficiently Jurkat cells
(increase of 229% vs the control).
Article history:
Received 16 December 2009
Revised 27 January 2010
Accepted 29 January 2010
Available online 4 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Iron particle
RGD peptidomimetic
Magnetic labeling
Jurkat cell
XPS
Non invasive diagnosis methods and cellular labelling for bio-
medical researches represent important application domains of
medical imaging (MI). Recent developments in this field rely upon
the discovery of functional radiotracers and contrast agents tar-
geted at particular cell types, such as tumor cells and migrating
respectively. For magnetic resonance imaging (MRI), cyclic RGD
peptides have been coupled to Gd3+ complexes11 as positive con-
trast agents (effect on T1, longitudinal relaxation time) and to iron
oxide nanoparticles12 as negative contrast agents (effect on T2,
transverse relaxation time).
vascular endothelial cells.1 These cells express
a
vb3 integrin trans-
membrane receptors2 that are involved in their migration, inva-
sion, proliferation and survival. The vb3 integrin recognizes
The growing interest in non peptide RGD mimics (peptidomi-
metics), as orally bioavailable
avb3 antagonists for therapeutical
a
purposes,13 stimulates the interest to use these molecules in MI
applications. RGD peptidomimetics conjugated to 111In-14 and
89Yb-DOTA complexes,15 and to Gd-DTPA (Gd-diethylenetriamine
pentaacetate) complexe,16 have been developed to target the
matrix proteins containing the cell-adhesion tripeptide motif
Arg-Gly-Asp (RGD)3 of which the biologically active conformation
is known thanks to the cyclic peptide named Cilengitide (cyclo-
[RGDfN(Me)V]-)4 featuring high selectivity and binding affinity in
the nanomolar range.
avb3 receptor (see discussion in Supplementary data).
To our knowledge, iron oxide nanoparticles have not been con-
Accordingly, RGD cyclic peptides have been conjugated to vari-
ous carriers for selective gene5 and drug delivery6 in cancer che-
motherapy, but also linked to radioactive labels and contrast
agents for selective cell imaging.7 For instance, conjugation to
DOTA (1,4,7,10-tetraazadodecane-N,N0,N00,N000-tertraacetic acid)
and radiolabelling with 111In,8 conjugation to 5-carboxylate-2,20-
bipyridine (BPy)9 or 6-(2-(2-sulfonatobenzaldehyde)hydrazino)-
nicotinyl (HYNIC) and radiolabelling with 99mTc,10 have been
described to image tumors by positron emission tomography
(PET) and single photon emission computed tomography (SPECT),
jugated to RGD peptidomimetics till now. However, small particles
of iron oxide possessing superparamagnetic properties are the
most commonly used systems for the magnetic labelling of cul-
tured cells and their detection by MRI.17 In this letter, we describe
the grafting of a home-made RGD peptidomimetic on ultrasmall
particles of iron oxide (USPIO) coated with a thin layer of 3,30-
bis(phosphonate)propionic acid,18 and the determination of the
grafting rate by X-ray photoelectron spectroscopy (XPS). The RGD
peptidomimetic—USPIO conjugate has been characterized by dy-
namic light scattering (DLS), magnetometric and relaxometric pro-
files, and its capacity for targeting leukemic cells in vitro.
The structure of our functional magnetic contrast agent that tar-
gets
avb3-displaying cells is shown in Scheme 1: the carboxylated
* Corresponding author.
E-mail address: Jacqueline.marchand@uclouvain.be (J. Marchand-Brynaert).
iron particle is connected to the RGD peptidomimetic via an OEG
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.150

1862
V. Rerat et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1861–1865
O2N
R1O
1, R1 = H
CO2tBu
HN
HN
O
SO2
1
2
, R =
O
N
N
3, R1 =
CF3
4, R2 = H
R2HN
O
CO2R3
SO2
O
2
5
NHBoc
NH2
H
, R =
O
O
5
5
N
N
O
O
O
O
6, R2 =
7, R2 =
3
a
b
, R = tBu;
CF3
3
, R = H
O
Me
3
O
-O
O
H
O
P
-O
N
FenOm
O
O
O
-O
-O
CO2H
SO2
N
O
O
N
P
O-
O
O
H
O
P
O
HN
O-
O-
O
HO
P
O
O-
H
N
CF3
Particle
OEG-spacer
RGD-mimic
Scheme 1. Synthesis of USPIO–petidomimetic conjugate.
(oligoethylene glycol) spacer; amide linkages are used for the
assembly of the three building blocks.
The peptidomimetics 4b, 6b, and 7b have been previously eval-
uated for selectivity and binding affinity towards isolated human
The peptidomimetic design stems from previous works of our
a
vb3 and
a
IIbb3 receptors by using standard competitive assays.20c
team19 dedicated to surface modification of polymer substrates
Results showed that all the tested compounds are
a
vb3 selective
for promoting cellular attachment. The (
L
)-tyrosine scaffold 1 was
and active in the (sub)nanomolar range with IC50 values of 0.1
(4b), 0.7 (6b), and 0.3 nM (7b). The presence of an OEG spacer does
not disturb the biological activity of the peptidomimetic core
(compare 4b with 6b), and the amine end-function of the spacer
is not involved in the recognition process (compare 7b with 6b).
Hence, the graftable RGD peptidomimetic 6b is a good candidate
for conjugation to USPIO.
The iron particles are surface modified with bis-(phospho-
nate)propionic acid molecules (about 1.6% of organic material vs
FenOm). The phosphonate grips strongly complex the metal core
while the carboxyl functions remain available for coupling reac-
tions (see Fig. 1). The USPIO reactivity has been assayed by using
a water soluble carbodiimide (WSC) as activating reagent (namely
N-(3-dimethylamino)propyl N0-ethylcarbordiimide hydrochloride)
and lysine as molecular probe, in a one-pot grafting protocol.23
The excess of reagents and self-coupling products were removed
by dialysis of the particles. After freeze-drying, the modified parti-
cles were analyzed by XPS for determining their surface atomic
composition.24 The native USPIO showed Fe, O, P and C atoms,
used to introduce the basic motif (Arg mimic) and the OEG spacer
respectively at the para- and meta-positions of the aromatic ring, in
agreement with a recent modeling study.20 In this work, we used
the RGD peptidomimetic 6b, ready for grafting on materials; it fea-
tures the tetrathydro-naphthyridinyl moiety21 as the Arg basic mi-
mic and a spacer-arm of six OEG units. The complete synthetic
protocols toward 6b are given as Supplementary data. Briefly, the
known precursor 119b (t-butyl-(S)-3-(40-hydroxy-30-nitro-phenyl)-
2-(30-trifluoromethyl-1-benzenesulfonylamino)-propionate) was
reacted with 3-acetyl-1-propanol under Mitsunobu conditions
(DIAD, Ph3P, THF, 20 °C, 17 h) to give the corresponding phenol
ether 2 (57%). Then, a Friedländer condensation reaction (2-ami-
no-3-pyridin-carboxaldehyde, (L)-proline, EtOH, reflux, 48 h) led
to the naphthyridin derivative 3 (55%). Catalytic hydrogenation
(Pd/C, H2 1 atm., EtOH, 20 °C, 17 h) reduced simultaneously the ni-
tro group and the naphthyridin ring to furnish compound 4a
(R3 = tBu, 95%). t-Butyl ester deprotection (TFA, DCM, 20 °C, 2 h),
afforded the RGD peptidomimetic 4b (R2 = R3 = H, 95%) devoid of
spacer-arm, for biological evaluation.
The spacer-arm was independently prepared according to stan-
dard protocols; the use of OEG spacers is a well established strat-
egy for the construction of bifunctional integrin-binding
devices.22 The peptide-like coupling of peptidomimetic 4a and
2-(2-(2-(2-(2-(2-t-butoxycarbonylaminoethoxy)-ethoxy)-ethoxy)-
ethoxy)-ethoxy)-ethoxy)-acetic acid made use of Py-Bop
(Benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium hexa-
fluorophosphate) as activating reagent (iPr2EtN, DMF, 20 °C,
15 h). Compound 5a (R3 = tBu) was isolated in 33% yield after col-
umn chromatography, and then fully deprotected (TFA, DCM,
20 °C, 4 h) to quantitatively furnish the target-molecule 6b
(R3 = H). The reference peptidomimetic, 7b, was similarly prepared.
Figure 1. Magnetometric profiles.

V. Rerat et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1861–1865
1863
Table 1
Surface grafting on USPIO—XPS analysis
Entry
Ligand
Conditions
(WSC; dialysis)
N/C ꢁ 100
Derivatization
from XPSa
b
%
Nbrc
1
2
3
4
5
6
7
8
Lysine
Lysine
Lysine
Lysine
Lysine
Lysine
Mimic 6b
GRGDS
20 °C, 1 h; 48 h
20 °C, 2 h; 48 h
20 °C, 4 h; 48 h
20 °C, 6 h; 48 h
40 °C, 1 h; 48 h
40 °C, 2 h; 48 h
20 °C, 2 h; 24 h
20 °C, 2 h; 24 h
6.35 (0.43)
5.84 (0.36)
5.22 (0.98)
6.23 (0.73)
5.77 (0.42)
6.68 (1.08)
5.00 (nd)
11.8
10.6
9.3
11.5
10.5
12.5
5.0
6.6
5.9
5.2
6.4
5.9
7.0
2.8
1.9
7.19 (0.90)
3.2
a
Corrected values obtained by subtraction of the blank. Mean of three inde-
pendent samples with standard deviation into parentheses.
b
Percentage calculated as described in Ref. 25.
Number of molecules grafted per particle; see Ref. 26.
c
while the lysine-coupled USPIO revealed the presence of supple-
mentary N atoms, in larger amounts as compared to the blank sam-
ples. The grafting rates calculated from the experimental N/C
atomic ratios25 are collected in Table 1 (entries 1–6). The percent-
ages of derivatization remained in the range of 9–12%, correspond-
ing to 5–7 molecules of lysine per particle,26 whatever the coupling
conditions had been (20 °C for 1 h, 2 h, 4 h, 6 h and 40 °C for 1 h,
2 h). This means that the coupling occurs quite rapidly and is well
reproducible. The conjugation protocol validated with lysine
(20 °C, 2 h) was further applied for the coupling of the peptidomi-
metic molecule 6b (Fig. 1) and the commercial pentapeptide Gly-
Arg-Gly-Asp-Ser (GRGDS) considered as the reference ligand of
Figure 2. Relaxometric profiles.
and r2 of 72.3 mMꢀ1 sꢀ1 at 20 MHz and r1 of 19.0 mMꢀ1 sꢀ1 and
r2 of 71.8 mMꢀ1 sꢀ1 at 60 MHz (37 °C). The grafting of GRGDS pep-
tide or of peptidomimetic 6b on the nanoparticle surface does not
change significantly the relaxometric properties.
The magnetic labelling of cells expressing the
performed with Jurkat T lymphocytes. For preliminary validation,
avb3 integrin was
a
vb3 receptor.27 The derivatization rates recorded by XPS analysis
were quite lower, about 3–5% corresponding to 2–3 molecules of
vb3 ligand per particle (Table 1, entries 7 and 8).23,25,26 Interest-
Jurkat cells are usually considered as good cell model of avb3 inte-
grin expression,30 but not as a model of certain pathology although
such cells are cancerous cells (acute T cell leukemia). In previous
studies, this cell model was routinely employed to validate various
a
ingly, by using the experimental F/C atomic ratio we calculated a
derivatization rate for USPIO-g-Mimic6b of 4.9% corresponding to
an average of 2.7 molecules of RGD peptidomimetic per particle,
in good agreement with the result of Table 1 (entry 7).
a
vb3 integrin-targeted imaging probes.16,31 Jurkat cells are known
to express the
avb3 receptors under stimulation with phorbol 12-
myristate 13-acetate (PMA, 50 ng/mL, 3 h, 37 °C, 5% CO2). Cells
(1.5 ꢁ 106 cells/mL), stimulated or not (negative control), were
incubated in USPIO-conjugate solutions (0.5 mM) during 2 h at
25 °C. After washing out the excess of particles, cells were seeded
in a gelatin matrix for measuring T2 (CPMG pulse sequence, Bruker
Minispec Mq-60, 60 MHz, 37 °C). The efficiency of USPIO capture
by cells is determined by the Rn2orm values (where R2 = 1/T2, while
Rn2orm is the normalized R2 which is calculated by subtracting the
R2 of cells free of USPIO from R2 of the cells incubated with iron
oxide nanoparticles), the highest values corresponding to the best
cell targeting. Results of Figure 3 showed a clear difference be-
tween activated cells (PMA) and non activated ones (CONtrol).
The native particles gave some unspecific cell adhesion, indepen-
dently of their activation state, which is justified by the absence
of any stealth coating. After conjugation to the RGD peptidomimet-
ic 6b, the particles were more efficiently trapped by the activated
cells (increase of 229% as compared to CON cells; p <0.05). This tar-
geting effect was also visible in the case of particles conjugated to
the reference peptide GRGDS, but the peptide appeared less active
than the peptidomimetic probably because the reaction of conju-
gation to USPIO could have altered the affinity of the reference
peptide for the target. With its nanomolar binding affinity, the
new RGD peptidomimetic 6b represents an interesting candidate
ligand for contrast agent vectorization. For sensitive MRI applica-
tions (i.e., detection of low biomolecule concentration) iron oxide
superparamagnetic agents28 are very promising after rendering
them enough furtive to circumvent the inevitable capture by the
phagocytic cells of the reticuloendothelial system. The novel US-
PIO-RGD peptidomimetic 6b could thus assist the robust tumor cell
Physico-chemical properties of the novel USPIO conjugates,
namely USPIO-g-Mimic6b and USPIO-g-GRGDS, were measured
to verify the keeping of particle size and magnetic characteristics28
(Table 2): hydrodynamic mean diameter was determined by
dynamic light scattering (DLS, see Supplementary data); from mag-
netometric (Fig. 1) and relaxometric profiles (Fig. 2), the magneti-
zation at saturation (Ms) and Fe microcrystal radius were obtained
(see Supplementary data). As already reported,29 the magnetomet-
ric mean diameter is lower than the relaxometric one (Table 2) be-
cause of the distribution of the crystal sizes which influences the
mean size obtained by various methods. This size dispersion is also
responsible for the lower relaxometric specific magnetization as
compared to the magnetometric one.
The physicochemical properties of the USPIO-g-Mimic 6b in-
cluded a r1 of 35.0 mMꢀ1 sꢀ1 and a r2 of 72.1 mMꢀ1 sꢀ1 at
20 MHz and a r1 of 17.5 mMꢀ1 sꢀ1 and a r2 of 73.4 mMꢀ1 sꢀ1 at
60 MHz (37 °C). For the USPIO-g-GRGDS, r1 was of 37.2 mMꢀ1 sꢀ1
Table 2
Characteristics of USPIO conjugates
Entry
Sample
Da (nm)
Rb (nm)
Method A/B
Ms (A m2/kg)c
Method A/B
1
2
3
Native USPIO
USPIO-g-Mimic 6b
USPIO-g-GRGDS
24
37
21
4.71/5.20
4.78/5.51
4.67/5.16
66.12/58.4
62.51/54.0
66.7/56.9
a
Apparent hydrodynamic diameter measured by DLS.
Microcrystal radius determined from magnetometric profile (method A) or from
b
detection by MRI,32 possibly taking advantage of the
mediated endocytosis phenomenon for particle uptake.33 Optimiz-
avb3 receptor-
NMRD profile (method B).
c
Magnetization at saturation.

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V. Rerat et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1861–1865
Figure 3. Targeting Jurkat cells with USPIO conjugates. PMA for activated cells and CON for control cells.
19. (a) Biltresse, S.; Attolini, M.; Marchand-Brynaert, J. Biomaterials 2005, 26, 4576;
(b) Biltresse, S.; Attolini, M.; Dive, G.; Cordi, A.; Tucker, G. C.; Marchand-
Brynaert, J. Bioorg. Med. Chem. 2004, 12, 5379.
ing the conjugation yield on USPIO coated with an adequate stealth
layer (i.e., pegylation) is currently under investigation.
20. (a) Rerat, V. Ph.D. Thesis, Université catholique de Louvain (Louvain-la-Neuve),
2008 (http://hdl.handle.net/2078.1/20874).; (b) Marchand-Brynaert, J.
Cardiovascular Pathol. 2008, 17, 276; (c) Rerat, V.; Dive, G.; Tucker, G. C.;
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Chem. 2009, 53, 7029.
21. Kinney, W. E.; Teleha, C. A.; Thompson, A. S.; Newport, M.; Hansen, R.;
Ballentine, S.; Ghosh, S.; Mahan, A.; Grasa, G.; Zanotti-Gerosa, A.; Dingenen, J.;
Schubert, C.; Zhou, Y.; Leo, G. C.; McComsey, D. F.; Santulli, R. J.; Maryanoff, B. E.
J. Org. Chem. 2008, 73, 2302.
22. (a) Owen, R. M.; Carlson, C. B.; Xu, J.; Mowery, P.; Fasella, E.; Kiessling, L. L.
ChemBioChem 2007, 8, 68; (b) Pilkington-Miksa, M. A.; Sarkar, S.; Writer, M. J.;
Barker, S. E.; Shamlou, P. A.; Hart, S. L.; Hailes, H. C.; Tabor, A. B. Eur. J. Org.
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Acknowledgements
This work was supported by the F.R.S.-FNRS and the ARC Pro-
gram 05/10-335 of the French Community of Belgium. The support
and sponsorship accorded by COST Action D38, EMIL NoE of the
FP6 of the EC, and Encite (Contract No. 201842) are kindly
acknowledged. We thank Ir. Michel Genêt for XPS facilities (UCL-
CIFA) and advices. V.R. was F.R.I.A. (Belgium) fellow.
23. Coupling protocol: USPIO material was received from Guerbet laboratories
(Aulnay-sous-Bois, France). The particles feature carboxylated groups on their
surface according to Ref. 18. They are suspended in water in order to obtain the
[Fe] concentration of 0.407 M. USPIO (1 mL, 0.407 mmol) was introduced in a
flask previously washed with 37% HCl, milliQ water, acetone, and ether, and
gently stirred with a magnetic bar. Lysine (8.13 ꢁ 10ꢀ2 mL of a solution of
4.5 mg LysꢂHCl in 25 mL milliQ water; 0.0814 mmol) and water soluble
carbodiimide (WSC, 54 mg, 0.28 mmol) were successively added, very slowly.
The mixture was left under stirring at 20 °C or 40 °C, for 1 h to 6 h (see Table 1).
After dialysis against milliQ water (membrane of 12–14,000) during 48 h, the
suspension was freeze-dried, and the resulting powder analyzed by XPS (see
Supplementary data for the table of results). The so-called blank samples were
similarly prepared, but with omitting the carbodiimide. Control samples were
similarly prepared but with omitting the lysine. These samples were also
analyzed by XPS (see Supplementary data). For the coupling of peptidomimetic
6b, we used USPIO (0.5 mL, 0.203 mmol), a 10ꢀ3 M solution of 6b in water–
DMSO, 95:5 (4.06 ꢁ 10ꢀ2 mL) and WSC (27 mg, 0.14 mmol); the mixture was
reacted for 2 h at 20 °C and dialyzed during 24 h. For the coupling of GRGDS
peptide, we applied the above protocol with a 10ꢀ3 M solution of peptide in
water.
Supplementary data
Materials and methods, general synthesis of RGD peptidomi-
metics (2, 3, 4a, 5a, 6b), NMR spectra, USPIO derivatization rates
and comments on avb3 receptor are provided. Supplementary data
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2010.01.150.
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[(90 ꢁ 3) + (10 ꢁ 9)] = 20/(270 + 90) = 0.0555.
The
percentages
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