Selective imaging of damaged bone structure (microcracks) using a targeting supramolecular Eu(III) complex as a lanthanide luminescent contrast agent

Article abstract of DOI:10.1021/ja908006r

(Chemical Equation Presented) The synthesis and photophysical evaluation of a new supramolecular lanthanide complex is described which was developed as a luminescent contrast agent for bone structure analysis. We show that the Eu(III) emission of this complex is not pH dependent within the physiological pH range and its steady state emission is not significantly modulated by a series of group I and II as well as D-metal ions and that this agent can be successfully employed to image mechanically formed cracks (scratches) in bone samples after 4 or 24 h, using confocal laser-scanning microscopy.

Full text of DOI:10.1021/ja908006r

Published on Web 11/13/2009
Selective Imaging of Damaged Bone Structure (Microcracks) Using a
Targeting Supramolecular Eu(III) Complex As a Lanthanide Luminescent
Contrast Agent
Brian McMahon,† Peter Mauer,‡ Colin P. McCoy,§ T. Clive Lee,‡ and Thorfinnur Gunnlaugsson*,†
School of Chemistry, Center for Synthesis and Chemical Biology, Trinity College Dublin, Dublin 2, Ireland,
Department of Anatomy, Royal College of Surgeons in Ireland, St. Stephen’s Green, Dublin 2, Ireland, and School
of Pharmacy, Queen’s UniVersity of Belfast, 97 Lisburn Road, Belfast, BT9 7BL U.K.
Received September 21, 2009; E-mail: gunnlaut@tcd.ie
Scheme 1. Synthesis of 1 (Free Ligand) and Corresponding Eu(III)
Complexes 1.Eu and 1.Eu.Na
The sensing and imaging of biological matter using lanthanide
luminescent supramolecular sensors,1,2 or imaging agents,3,4 has
attracted significant attention in recent years. The photophysical
properties of ions such as Eu(III) and Tb(III), which possess long-
lived excited states and line-like emission bands, occurring at long
wavelengths, makes these ions excellent candidates for use in such
applications, as they overcome short-lived background emission
and light scattering from biological matter.5,6 To date, lanthanide
complexes have been designed for both luminescent cellular and
tissue imaging.7 However, the development of such luminescent
agents for larger biological structures, such as bones,8 has, to the
best of our knowledge, not been achieved to date.
Bone is a rigid structure consisting of canals, Haversian systems,
canaliculi, and resorption cavities. Being a dynamic organ it undergoes
continuous repair, shaping, and molding.9 The formation of microc-
racks in bones (25-300 µm in length in human bones) is usually
caused by repetitive loading and stress and can have extreme biological
effects and hence medical conditions, such as osteoporosis.10 Due to
the presence of organic matrix and crystalline hydroxyapatite in bones,
it can be difficult to distinguish such damage from healthy bone. Hence,
the development of novel contrast agents which have the ability to
penetrate the bone matrix can enable a greater understanding of this
complex morphology.10,11 Herein we present an example of a
luminescent lanthanide bone-targeting imaging agent,12 1.Eu.Na,
designed for the selective Eu(III) imaging of damaged bone at
physiological pH. We foresaw that 1.Eu.Na, which possess three
iminodiacetate moieties,12 would be able to bind selectively to exposed
Ca(II) sites within the hydroxyapaptite lattice of the bone, enabling
the luminescent imaging of this exposed damage.
Figure 1. Changes in the Eu(III) luminescence of 1.Eu.Na upon excitation
of the naphthalene antenna as a function of pH (I ) 0.1 M NEt4HClO4
(TEAP)). Insert: The changes at 616 nm vs pH.
aqueous buffered pH 7.4 (20 mM HEPES) solution upon excitation
at the antenna, as well as upon direct excitation of the metal center of
both 1.Eu and 1.Eu.Na. The excited state lifetimes in H2O and D2O,
upon excitation at 282 nm, gave τH O ) 0.576 ms and τD O ) 1.744
2
2
The synthesis of 1.Eu.Na, Scheme 1, was achieved in few steps
from 2,13 which was reacted with 3 equiv of diethyl 2,2-(2-
chloroacetyl-amino)diacetate, 3, in refluxing MeCN solution, in the
presence of Et3N for 7 days. Purification using alumina column
chromatography (gradient elution 100f80:20 CH2Cl2/CH3OH) af-
forded 1 in 54% yield, which was reacted with 1 equiv of Eu(CF3SO3)3
in freshly distilled MeCN for 15 h (Figure S1b), giving 1.Eu in 69%
yield (see H NMR in Figure S1). Alkaline hydrolysis of 1.Eu in
MeOH/H2O gave 1.Eu.Na in 90% yield after precipitation from dry
diethyl ether.
The design of 1.Eu.Na envisaged indirect excitation of the Eu(III)
ion via the covalently attached naphthalene antenna, enabling the
sensitization of the Eu(III) 5D0 excited state, which upon relaxation to
the 7FJ (J ) 0, 1, 2, 3, 4) ground state gives rise to characteristic line-
like Eu(III) emission.1,5 Indeed, metal centered emissions occurring
at 580, 595, 616, 655, and 701 nm (Figure S2) were observed in
ms from which the hydration state q ≈ 1 (Figure S3) was determined,
indicating that 1.Eu.Na possessed one metal bound water molecule,
the Eu(III) ion being overall nine coordinated. The effect of pH on
both the ground and the singlet excited states of the antenna and the
Eu(III) excited states were analyzed so as to quantify the pH
dependence of 1.Eu.Na within the physiological pH range. While the
pH dependence changes in the absorption spectra of 1.Eu.Na (Figure
S4a) were minor, the fluorescence emission (Figure S4b) and the
Eu(III) emission, Figure 1, were significantly modulated as a function
of pH (λEX ) 282 nm). The analysis of the Eu(III) emission for the
∆J ) 2 transition (see inset in Figure 1) clearly demonstrates that the
Eu(III) emission was ‘switched on’ within the physiological pH range,
being only ‘switched off’ below pH 5 or above pH 8.14 To further
investigate the effectiveness of 1.Eu.Na as a potential contrast agent,
its luminescence response toward group I and II as well as several
biologically relevant d-metal ions was determined (as their MCl2 salts)
within the concentration range 0-0.1 mM in buffered pH ) 7.4 (20
mM HEPES) solution. In general, only minor changes were observed
in the Eu(III) emission of 1.Eu.Na (Figures S5-S10), in the presence
1
† Trinity College Dublin.
‡ Royal College of Surgeons in Ireland.
§ Queen’s University of Belfast.
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17542 J. AM. CHEM. SOC. 2009, 131, 17542–17543
10.1021/ja908006r  2009 American Chemical Society

C O M M U N I C A T I O N S
exposure, Figure 2d, the emission from the healthy bone surface is
almost negligible (in comparison to the scratched surface), with an IE
≈ 28, Figure 2e, making the distinction between healthy and damaged
bone clear.
The images shown in Figure 2b-d clearly demonstrate the ability
of 1.Eu.Na to selectively bind and hence image the damaged bone
sites, likely occurring through chelation to exposed Ca(II) sites within
these cracks. To confirm this, the ability of 1.Eu to bind to such
scratched bone regions was also investigated. The confocal fluorescence
laser-scanning microscopy images (Figure S13) indeed showed that
1.Eu did bind to the scratched areas. However, the contrasting ability
is much diminished relative to 1.Eu.Na, with an EI of only 2 being
determined after 24 h of exposure (Figure 2f), an order of magnitude
smaller IE than that for 1.Eu.Na. Furthermore, unlike the case for
1.Eu.Na, no significant change was seen in the Eu(III) emission
intensity between samples exposed to 1.Eu for 4 and 24 h, respectively
(Figure S14). This clearly highlights the important role that the
iminodiacetate moieties in 1.Eu.Na play to selectively bind the agent
to the damaged hydroxyapaptite lattice, resulting in effective imaging
of such bone damage.
Figure 2. Confocal laser-scanning microscopy images of bone sample
immersed in a 1 × 10-3 M solution of 1.Eu.Na (pH 7.4, 20 mM HEPES, 135
mM KCl): (a) Reflected light image: 0 h, (b) Control, (c) 4 h, (d) 24 h. Bar )
150 µm. (e) Contrast between Eu(III) emission from 1.Eu.Na inside and outside
the scratch after 24 h. (f) The contrast observed using 1.Eu (1 × 10-3 M) after
24 h.
of these ions, with the exception of Co(II) and Ni(II) (Figures S9-10)
which resulted in quenching of the Eu(III) emission.
In summary, we have developed the first example of a cyclen based
Eu(III) complex, incorporating the iminodiacetate functionalities (as
selective Ca(II) binding motifs), as a lanthanide luminescent contrast
agent for bone structure analysis. We are in the process of further
studying the properties of 1.Eu.Na and other related Eu(III) and Gd(III)
structures as bone imaging agents.
We next evaluated the ability of 1.Eu.Na to act as a luminescent
imaging agent for bone cracks. To demonstrate this, several bovine
tibia specimens were sectioned and polished to give mechanically
smooth surfaces (see Supporting Information), which should have low
or no affinity for 1.Eu.Na and, hence, would not result in positive
luminescent imaging of such areas. The same samples were then
scratched using established protocols,11,12 exposing fresh Ca(II) sites.
These specimens were then treated with a 1 × 10-3 M aqueous solution
of 1.Eu.Na (in 135 mM of KCl at pH 7.4) for a period of 0 to 24 h.
For comparison, polished and scratched bone samples were also treated
with 1.Eu. Using steady-state fluorescence, the Eu(III) emissions from
the samples containing ‘smooth’ and scratched bone surfaces were
first recorded using specimens that were stained for 4 and 24 h. The
results clearly demonstrated that the Eu(III) emission from the scratched
areas was more intense than that of the undamaged surface, with all
of the D0 f FJ transitions being clearly visible, even after 4 h of
staining. Similarly, using 1.Eu, some Eu(III) emission was also visible.
However, in comparison to 1.Eu.Na, the emission was significantly
less visible (Figures S11-12). Furthermore, by analyzing the intensity
ratios of the various Eu(III) ∆J transitions, it was clear that the ratio
between ∆J ) 1 and 2 was significantly greater for 1.Eu than for
1.Eu.Na, for which the ratio was almost 1:1. This would suggest that
1.Eu bound to the scratched surface in a different manner to 1.Eu.Na,
which is to be expected, as in 1.Eu the Ca(II) chelating moieties are
blocked.
The samples were next imaged using confocal fluorescence laser-
scanning microscopy. The overall results are shown in Figure 2 (Figure
2a shows the bone scratch as a reflected light image), where in Figure
2c and 2d the emission (recorded at 616 nm) arising from the bone
surface of the same sample after 4 and 24 h staining/exposure to
1.Eu.Na is clearly visible, being most pronounced within the scratched
areas. In contrast, Figure 2b shows no such emission arising from a
bone surface that was exposed to 1.Eu.Na for 24 h, after which it was
scratched and imaged. Hence, the presence of exposed Ca(II) sites is
a prerequisite for the successful binding of 1.Eu.Na to the bone surface
and, hence, imaging of any damaged bone surface. It is also clear from
Figure 2b-d that the emission contrast between the healthy bone
surface and the scratched areas enhances significantly with increasing
1.Eu.Na exposure time. While Figure 2c shows that the Eu(III)
emission from the crack is clearly distinguishable from the surrounding
undamaged bone after only 4 h of exposure, where the intensity
enhancement (IE) (at 616 nm) within the scratched area is only twice
that measured for the unscratched area. In contrast, after 24 h of
Acknowledgment. We thank IRCSET (Postgraduate award to
B.M.), TCD, QUB, and RCSI for financial support and The
University of Canterbury, New Zealand, for a 2009 Erskine
Fellowship to T.G.
Supporting Information Available: Synthesis and characterization
of all novel compounds, Figures S1-13. This material is available free
of charge via the Internet at http://pubs.acs.org.
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