Magneto-optical nanomaterials: A SPIO-phthalocyanine scaffold built step-by-step towards bimodal imaging

Article abstract of DOI:10.1039/c3cc41898g

A SPIO-phthalocyanine nanohybrid is developed as a bimodal contrast agent for Optical and Magnetic Resonance Imaging. The organic coating was covalently attached onto SPIO in a step-by-step approach. Each coated-SPIO was thoroughly characterized. The hydrodynamic size of the SPIO-Pc is ca. 60 nm with a coverage of ca. 690 Pc/SPIO.

Full text of DOI:10.1039/c3cc41898g

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Magneto-optical nanomaterials: a SPIO–phthalocyanine
scaffold built step-by-step towards bimodal imaging†
Cite this: Chem. Commun., 2013,
49, 7394
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Julien Boudon, Jeremy Paris, Yann Bernhard, Elena Popova,
Received 13th March 2013,
Accepted 21st June 2013
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Richard A. Decreau* and Nadine Millot*
DOI: 10.1039/c3cc41898g
www.rsc.org/chemcomm
A SPIO–phthalocyanine nanohybrid is developed as a bimodal contrast (i.e. the so-called imaging window);2 (ii) a fluorescence quantum
agent for Optical and Magnetic Resonance Imaging. The organic coating yield (FF (ZnPc) = 0.3) in the range of known fluorophores,9 and a
was covalently attached onto SPIO in a step-by-step approach. Each high molar extinction coefficient (e = 105 MÀ1 cmÀ1), resulting in
coated-SPIO was thoroughly characterized. The hydrodynamic size of high brightness (e  FF), making ZnPc very competitive compared
the SPIO–Pc is ca. 60 nm with a coverage of ca. 690 Pc/SPIO.
to other near-IR emitting fluorophores.9 (b) Moreover, ZnPc is one
of the most photostable fluorophores reported to date (degrada-
Bimodal Medical Imaging relying on Magnetic Resonance Imaging tion quantum yield of 10À6, i.e. about 30 times more robust than
(MRI),1 coupled to Optical Imaging (OI),2 may improve the accu- rhodamine or Cy5).6,10 † (c) Our experience in SPIO6,11 and in the
racy of a diagnosis3 by combining the sensitivity of OI4 and the step-by-step immobilization of functional porphyrin/porphyrinoid
resolution of MRI.5 Reported hybrid MRI–OI probes bear MRI and models on gold12 prompted us to investigate a similar strategy for
OI imaging probes, but their design does not usually address the immobilization of ZnPc onto SPIO (Fig. 1).‡ Each coated-SPIO
either of the following issues: (a) the difference in sensitivity between was thoroughly characterized by an array of analytical techniques,
the two methods (mM–mM for MRI compared with nM–pM for OI) which allowed us to keep track of the precise composition of the
is not addressed when a fluorophore is bound to one MRI-active coated-SPIO at a given step. We stuck to the highest standards of
metal complex (1 : 1 ratio),4 but is more relevant when the probe characterization of hybrid materials.
(Cy5.5, fluorescein, rhodamine) is immobilized on nanoparticles
(NPs) (CLIO, SPIO, Gd-doped NPs), because it contains multiple
paramagnetic centers;4–6 (b) the photostability of fluorophores; (c) a
step-by-step construction of reported magneto-optical nanohybrids
was not carried out, hence the precise composition of the coating
on the nanoparticle may not be accurate enough. Conjugate 1 may
overcome these drawbacks: (a) combining Super Paramagnetic
Iron Oxide (SPIO; T2-weighted MRI contrast agents)6,7 and the zinc
phthalocyanine fluorophore (ZnPc)8 together in a single bimodal
contrast agent may better address the difference in sensitivity
SPIO nanoparticles were prepared according to the classical
between MRI and OI (a favorable ratio of one optical probe Massart method.6 † The NPs obtained were crystallized in the
per a large number of magnetic probes). ZnPc has appealing spinel phase a = (8.381 Æ 0.002) Å. The NPs crystallite size was
optical properties:8 (i) an absorption band in the 650–800 nm obtained using X-Ray Diffraction dXRD = (9.1 Æ 0.2) nm and the
region where light penetration in tissues is the greatest mean diameter using Transmission Electron Microscopy dTEM
=
(9 Æ 3) nm (on 100 spinel crystallites at least). These NPs are
superparamagnetic at room temperature: a blocking temperature
of 170 K is measured for the applied field of 100 Oe (Field Cooling,
FC, and Zero Field Cooling, ZFC, Fig. 2A).† The agglomerate size
dTEM = (25 Æ 2) nm correlates with the hydrodynamic diameter
determined using Dynamic Light Scattering (DLS) dDLS = (44 Æ 1) nm.
a Laboratoire Interdisciplinaire Carnot de Bourgogne (ICB),
´
UMR 6303 CNRS-Universite de Bourgogne, BP 47870, F-21078, Dijon Cedex,
France. E-mail: Nadine.Millot@u-bourgogne.fr
b
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Institut de Chimie Moleculaire de l’Universite de Bourgogne (ICMUB),
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UMR 6302 CNRS-Universite de Bourgogne, BP 47870, F-21078, Dijon Cedex,
The BET specific surface area of the powder is S = (103 Æ 1) m2 gÀ1
.
France. E-mail: Richard.Decreau@u-bourgogne.fr
´
`
´
Groupe d’etude de la matiere condensee (GEMaC), UMR 8635 CNRS-UVSQ,
z-Potential measurements (Fig. 2B–A) indicated an Isoelectric Point
F-78035 Versailles, France
(IEP) at pH 7.4 and maximum potentials reached 30 mV. FTIR data†
† Electronic supplementary information (ESI) available: Experimental procedures,
spectroscopic details, additional figures and references. See DOI: 10.1039/c3cc41898g are consistent with those of previous studies.13
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7394 Chem. Commun., 2013, 49, 7394--7396
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consistent with the modification of the NP surface by introdu-
cing azide groups (IEP variation) at the end of short ethylene
oxide chains (slight shielding effect). The XPS measurements
are in correlation with other characterizations: the N 1s peak
corresponds to the N–C contribution; the C 1s peak decom-
À
poses into three different contributions, C–N and C–C/C–H
À
À
À
À
components are in equal proportions meaning that the C–N
contribution increased because of the presence of the azide
groups on the particle surface.
Phthalocyanine 2 was synthesized according to the synthetic
pathway described in Fig. 1 and ESI.† Briefly, the mixed con-
densation of compound 3 with phthalonitrile in the presence of
a zinc salt led to a statistical mixture containing the alkyne-
bearing A3B-ZnPc 2 and the click-unreactive A4-ZnPc counter-
part. This mixture was purified by a series of washings, leading
to the removal of the non-phthalocyanine material.‡
As for SPIO–NH2 and SPIO–N3, the working hypothesis was to
ensure a successful coupling and to supply a proof of concept,
i.e. to achieve a significant coverage in Pc to reliably characterize
SPIO–Pc 1. Cu-catalyzed Azide–Alkyne Coupling (CuAAC)14 between
Fig. 1 Step-by-step synthesis. Top and right: SPIO–NH2, SPIO–N3; bottom left: Pc 2,
and SPIO–Pc conjugate 1.
Free hydroxyl groups (TGA gave 7 OHÀ nmÀ2, 12 mmol mÀ2) on the alkyne-containing species 2 and the azide-containing SPIO–N3
the surface of bare SPIO NPs were reacted with 3-aminopropyl- was achieved in the presence of a Cu salt and sodium ascorbate to
triethoxysilane (APTES) to afford amine-functionalized SPIO NPs. afford conjugate 1.‡ To ensure that no material was adsorbed a
Both the average size and size distribution of agglomerates are series of thorough washing–filtration was performed by resuspend-
increased when compared to bare SPIO NPs (dTEM = (35 Æ 8) nm ing the SPIO–Pc material in THF, magnetically precipitating down
and dDLS = (91 Æ 2) nm). The BET specific surface area of the the adsorbents, removing the supernatant and ultrafiltering the
SPIO–NH2 powder increased to S = (148 Æ 1) m2 gÀ1. z-Potential mixture. The observed agglomerate mean size is about dTEM
=
measurements (Fig. 2B–B) indicated an IEP at pH 9.8, which (56 Æ 5) nm (Fig. 2D).‡ The agglomerate size of conjugate 1 is in
shifted higher because of the presence of amine functions at agreement with the DLS agglomerate size which is known to be less
the NP surface and preserving maximum potential values. The than 100 nm according to the biomedical application for which it
elemental analysis suggests about 5 NH2 per nm2 (8.3 mmol mÀ
)
was designed.15 Commercial contrast agents are indeed a compro-
2
on SPIO–NH2. XPS measurements showed that the C 1s peak mise between rapid elimination in kidneys (monodisperse state) or
decomposes into C–C/C–H, C–N and O–CQO contributions and no cell incorporation (very large agglomerates). z-Potential measure-
À
À
À
À
that the N 1s peak only corresponds to the N–C contribution.† The ments indicated (a) an IEP around pH 8.1 close to that of the
À
latter are correlated by the FTIR measurements† that exhibit the previous functionalization stage (SPIO–N3), and (b) a distinct
characteristic band of secondary amines and protonated amine shielding effect due to the high proportion of grafted ZnPc. This
bands. SPIO–NH2 nanohybrids were subsequently coupled with is consistent with the Inductively Coupled Plasma Atomic Emission
azide/carboxyl terminated-PEG chains by EDC coupling, to afford Spectroscopy (ICP-AES) analysis that indicates a significant Zn : Fe
azido-functionalized SPIO (SPIO–N3). The observed size and ratio of about 1 : 24. However, all azide groups were not reacted
dispersion state are given by TEM observations: the agglomerate with alkynylphthalocyanine 2. Given the characterization data,
sizes are dTEM = (73 Æ 25) nm.†
our calculation indicates about 2 ZnPc per nm2 (3.3 mmol mÀ2);
The SPIO–N3 NPs were characterized using FTIR spectro- i.e. ca. 690 ZnPc per NP (ca. 15 700 Fe atoms).† This corresponds to
scopy, and the spectra exhibited a characteristic azide stretch at about 40% coverage when compared to SPIO–NH2. The ZnPc steric
2100 cmÀ1 (Fig. 2C–C). z-Potential measurements (Fig. 2B–C) bulk could be at stake here leading to non-quantitative coverage of
indicated an IEP shifted down to pH 8.4, along with a 10 mV the SPIO–NH2 NP surface. XPS analysis showed that the C 1s peak
decrease in the maximum potential. These observations are decomposes into three different contributions† and the N 1s peak
Fig. 2 Characterization of SPIO–Pc and precursors: (A) FC and ZFC measurements between 5 and 320 K; (B) z-potential and (C) FTIR (both labeled A: SPIO–OH,
B: SPIO–NH2; C: SPIO–N3; D: SPIO–Pc); (D) SPIO–Pc TEM; (E) SPIO–Pc XPS contributions of nitrogen 1s; (F) SPIO–Pc fluorescence.
c
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is shared into two contributions (Fig. 2E). The appearance of a new actual photostable fluorophores;† and (c) by proceeding in a
N 1s contribution at 397 eV is thus attributed to the presence step-by-step manner using classical reactions of organic synthesis
of ZnPc (NZnPc–C contribution). The C–C/C–H component also (silanization, acylation, cycloaddition) with the following func-
À
À
À
increased because of the presence of the ZnPc aromatics. FTIR tional groups or molecules found at the SPIO surface: hydroxyl
(Fig. 2C–D) spectra showed the disappearance of the azide stretch groups (bare SPIOs), amine groups (first stage coating), azide
(nas N3) at 2100 cmÀ1, which indicates that the reaction with the groups (second stage coating), and phthalocyanines (third stage
alkyne occurred, leading to the formation of a triazole linker. coating), respectively. These reactions proceeded smoothly and
UV-Visible spectroscopy in ethanol† showed the characteristic ZnPc each SPIO end-product was well identified and thoroughly char-
absorption bands: Q bands (667, 640 and 603 nm) and the Soret acterized by an extensive number of spectroscopic techniques.
band (344 nm). At high surface coverage in 2, the SPIO–Pc spectrum Based on preliminary data (fluorescence and relaxivity measure-
in ethanol is still comparable to that of the monomeric ZnPc with ments), we are confident on the relevant nature of 1.
no significant shift indicating that no p-stacking occurred; hence,
JB and RAD thank the Burgundy Regional Council, CRB
the optical properties of immobilized (1) and free (2) A3B-ZnPc are (FABER Programs), RAD thanks CNRS for Chaire d’Excellence;
comparable to each other and to that of the parent A4-ZnPc species.‡ JB and YB thank CRB (JCE, FABER grants respectively). EP acknowl-
The relevant indicator of the step-by-step nanoparticle-coating edges financial support from ‘‘C’Nano IdF’’ network (grant no.
achievement is the surface coverage rates that are expressed as a IF-08-1453/R). This work was supported by the 3MIM agreement
function of the available surface groups in the previous step. At the (CNRS, uB and CRB), and labelled by the PharmImages consortium.
different stages of conjugate 1 elaboration, coverage rates were Dr Olivier Heinz is acknowledged for XPS measurements.
determined to be: (a) 7 OHÀ per nm2 (12 mmol mÀ2) on initial bare
Notes and references
SPIO; (b) 5 NH2 per nm2 (8.3 mmol mÀ2) on SPIO–NH2 (71% yield);
(c) 5 N3 per nm2 (8.3 mmol mÀ2) on SPIO–N3 (quantitative yield
assuming that all amines were acylated);† and (d) 2 ZnPc per nm2
(3.3 mmol mÀ2) on SPIO–Pc (40% yield). Therefore, the overall yield
is ca. 30% grafting on bare SPIO NPs. This was achieved because of
the low steric hindrance of neighboring ZnPc and the positioning of
the short PEG chains (9 ethylene oxide units) that probably do not
imbed the azide function inside the organic layer.
Subsequent spectroscopic studies were performed to address
the imaging capabilities of each probe in the well characterized
SPIO–Pc conjugate 1: fluorescence (ZnPc probe for OI) and relaxi-
vity (SPIO probe for MRI) measurements. Upon excitation of 1 at
600 nm in ethanol solution, a fluorescence emission (Fig. 2F) at
673 nm was observed, which is comparable to the fluorescence
emission of the parent free ZnPc 2. The results seem to indicate
that there is no Aggregation Caused Quenching (ACQ) due to
‡ Pc groups have been immobilized on other NPs for other applications
in a direct (as opposed to step-by-step) approach.23,24 The remaining
A4-ZnPc coproduct that does not bear the alkyne moiety could not be
clicked, hence it was easily washed away after the coupling reaction
with SPIO NPs. A mixture of ZnPc and SPIO NPs treated without the
Cu catalyst did not lead to the SPIO–Pc conjugate. Hence, this indicates
that these spectroscopic observations such as UV-Vis do not reflect an
artifact, i.e. adsorption of ZnPc onto NP, but a covalent linkage instead.
1 C. Ru¨menapp, B. Gleich and A. Haase, Pharm. Res., 2012, 29, 1165–1179.
2 V. J. Pansare, S. Hejazi, W. J. Faenza and R. K. Prud’homme, Chem.
Mater., 2012, 24, 812–827.
3 C. I. Olariu, H. H. P. Yiu, L. Bouffier and T. Nedjadi, et al., J. Mater.
Chem., 2011, 21, 12650–12659.
4 J. Cheon and J.-H. Lee, Acc. Chem. Res., 2008, 41, 1630–1640.
5 O. Veiseh, J. W. Gunn and M. Zhang, Adv. Drug Delivery Rev., 2010,
62, 284–304.
6 L. Maurizi, H. Bisht, F. Bouyer and N. Millot, Langmuir, 2009, 25,
8857–8859.
7 S. Mornet, S. Vasseur, F. Grasset and E. Duguet, J. Mater. Chem.,
2004, 14, 2161–2175.
8 N. Sekkat, H. v. d. Bergh, T. Nyokong and N. Lange, Molecules, 2011,
17, 98–144.
¨
p-stacking and Forster Resonance Energy Transfer (FRET) quench-
ing in the organic solvent.ÀT1he relaxivity measÀu1rements led to R2
and R1 values of 73 mMFe sÀ1 and 3 mMFe
s
À1, respectively
9 L. D. Lavis and R. T. Raines, ACS Chem. Biol., 2008, 3, 142–155.
(R2/R1 = 24).† These values are reminiscent of currently available T2 10 T. Nyokong, Coord. Chem. Rev., 2007, 251, 1707–1722.
contrast agents.16 The thickness of the organic coating on the SPIO
core is known to affect the R2 relaxivity, and the outer ‘‘hydro-
11 L. Maurizi, F. Bouyer, J. Paris and F. Demoisson, et al., Chem.
Commun., 2011, 47, 11706–11708.
´
12 J. P. Collman, N. K. Devaraj, R. A. Decreau and Y. Yang, et al.,
phobic phthalocyanine belt’’ may prevent, to a certain extent, the
access of water molecules to the inner superparamagnetic iron
oxide core of 1.17 In addition magnetic measurements showed that
SPIO–Pc remain superparamagnetic and their blocking tempera-
ture decreases to 105 K indicating the decrease in dipolar magnetic
interactions due to organic shells (Fig. 2A). Hence the hydrophilic
Science, 2007, 315, 1565–1568.
13 S. Mornet, J. Portier and E. Duguet, J. Magn. Magn. Mater., 2005, 293,
127–134.
14 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed.,
2001, 40, 2004–2021.
15 F. M. Kievit and M. Zhang, Acc. Chem. Res., 2011, 44, 853–862.
16 S. Srivastava, R. Awasthi, D. Tripathi and M. K. Rai, et al., Small,
2012, 8, 1099–1109.
nature of 1 may be increased upon tuning down the Pc coverage 17 L. E. W. LaConte, N. Nitin, O. Zurkiya and D. Caruntu, et al., J. Magn.
Reson. Imaging, 2007, 26, 1634–1641.
18 D. Yoo, J.-H. Lee, T.-H. Shin and J. Cheon, Acc. Chem. Res., 2011, 44, 863–874.
rate (or reacting the remaining azides with alkyne-PEG). Finally,
conjugate 1 may be considered as a putative theranostic agent
´
19 R. A. Decreau, J. P. Collman, Y. Yang and Y. Yan, et al., J. Org. Chem.,
(i.e. a species that can be used to perform imaging and therapy
simultaneously)12,18,19 † because it could be used in hyperthermia
(SPIO)20,21 and photodynamic therapy (ZnPc).8,22
2007, 72, 2794–2802.
20 S. S. Kelkar and T. M. Reineke, Bioconjugate Chem., 2011, 22, 1879–1903.
21 M. De, S. S. Chou, H. M. Joshi and V. P. Dravid, Adv. Drug Delivery
Rev., 2011, 63, 1282–1299.
´
In summary, SPIO NPs were successfully functionalized 22 R. A. Decreau, M. J. Richard, P. Verrando and M. Chanon, et al.,
J. Photochem. Photobiol., B, 1999, 48, 48–56.
23 R. Lin, L. Zhou, Y. Lin and A. Wang, et al., Spectroscopy, 2011, 26, 179–185.
24 T. Stuchinskaya, M. Moreno, M. J. Cook and D. R. Edwards, et al.,
three consecutive times (a) by setting a higher proportion of
magnetic probes with respect to the optical ones to address the
difference in sensitivity; (b) by using zinc phthalocyanines as
Photochem. Photobiol. Sci., 2011, 10, 822–831.
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7396 Chem. Commun., 2013, 49, 7394--7396
This journal is The Royal Society of Chemistry 2013