Cherenkov Radiation-Mediated In Situ Excitation of Discrete Luminescent Lanthanide Complexes

Article abstract of DOI:10.1002/anie.201809783

Lanthanide luminescence, while ideal for in vivo applications owing to sharp emission bands within the optical window, requires high-intensity, short-wavelength excitation of small organic “antenna” chromophores in the vicinity of the lanthanide complex to access excited f-orbital states through intersystem crossing. Herein, we explored Cherenkov radiation of the radioisotopes ¹⁸F and ⁸⁹Zr as an in situ source of antenna excitation. The effective inter- and intramolecular excitation of the terbium(III) complexes of a macrocylic polyaminocarboxylate ligand (hydration number (q)=0, quantum yield (φ)=47 %) as well as its analogue functionalized to append an intramolecular Cherenkov excitation source (q=0.07, φ=63 %) was achieved. Using conventional small-animal fluorescence imaging equipment, we have determined a detection limit of 2.5 nmol of Tb(III) complex in presence of 10 μCi of ¹⁸F or ⁸⁹Zr. Our system is the first demonstration of the optical imaging of discrete luminescent lanthanide complexes without external short-wave excitation.

Full text of DOI:10.1002/anie.201809783

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Accepted Article
Title: Cherenkov Radiation Mediated In Situ Excitation of Discrete
Luminescent Lanthanide Complexes
Authors: Alexia G Cosby, Shin Hye Ahn, and Eszter Boros
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201809783
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Angewandte Chemie International Edition
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COMMUNICATION
Cherenkov Radiation Mediated In Situ Excitation of Discrete
Luminescent Lanthanide Complexes
[
a]
Alexia G. Cosby, Shin Hye Ahn, Eszter Boros*
Abstract: Lanthanide luminescence, while ideal for in vivo
applications owing to sharp emission bands within the optical
window, requires high intensity, short wavelength excitation of small
organic “antenna” chromophores in vicinity of the lanthanide
complex to access excited f-orbital states via intersystem crossing.
radioisotopes decay and emit charged particles, the
electromagnetic waves generated by the propagating particle
traveling in dielectric media result in phase interference,
[
7]
observed as CR. CR in aqueous solutions is continuous and
wavelength dependent, with maximum intensity below 400 nm.
This identifies discrete luminescent lanthanide complexes with
antennae as highly suitable acceptors for Cherenkov radiation
1
8
Here, we explored Cherenkov radiation of the radioisotopes F and
8
9
Zr as an in situ source of antenna excitation. The effective inter-
[
8]
and intramolecular excitation of the terbium(III) complexes of a
macrocylic polyaminocarboxylate ligand (q = 0, φ = 47%) as well as
its analogue functionalized to append an intramolecular Cherenkov
energy transfer (CRET).
The concept of CRET has been successfully explored to
excite Cu, Er, and CdTeSe based quantum dots and
fluorescein. However, organic fluorophores with favorable
Y
[
8-13]
excitation source (q
=
0.07,
φ
=
63%) was achieved. Using
conventional small animal fluorescence imaging equipment, we have
determined a detection limit of 2.5 nmol Tb(III) complex in presence
emission properties for biological applications absorb at longer
wavelengths (> 400 nm), which coincides with low intensity of
CR. We hypothesized that an application of CRET to discrete
luminescent lanthanide complexes would be feasible and ideal;
excitation of the lanthanide antenna occurs at short wavelengths
where CR exhibits maximum intensity, which produces efficient
intersystem crossing to yield the desired lanthanide-based
luminescence emission (Scheme 1).
1
8
89
of 10 µCi of F or Zr. Our system is the first demonstration of the
optical imaging of discrete luminescent lanthanide complexes
without external short-wave excitation.
Lanthanide-based luminescence represents an attractive
alternative to commonly employed, conventional organic
[
1-3]
chromophores.
trivalent lanthanides gives rise to sharp emission bands ranging
from green for Tb(III) (λem= 490, 545 nm), to red for Eu(III) (λem
13, 690 nm) and near-IR for Nd(III) (λem= 1080 nm). Due to the
The unique electronic configuration of
=
6
Laporte-forbidden nature of the f-f transitions, aromatic
chromophores (‘antennae’) are required for efficient excitation of
[
4-5]
metal-based luminescence after intersystem crossing.
The
antenna must possess triplet state energy comparable to
lanthanide acceptor states and allows efficient energy transfer
with excitation wavelengths of 250-350 nm. Because of the large
difference in antenna excitation and lanthanide emission
wavelength, the effective Stokes-shift is conveniently large and
minimizes inner filter effects; f-orbital centered deexcitation is
long lived and allows for time-resolved detection. The
applicability of luminescent lanthanides for bioassays requires
short wavelength excitation or time resolution of emitted
luminescence; the lack of tissue depth penetration of light below
Scheme 1. Schematic description of CR mediated excitation of a discrete,
luminescent lanthanide complex.
In order to furnish a lanthanide complex with significant φ
and comparable to organic fluorophores suitable for in vivo
applications, several design criteria were considered as
essential: 1. Incorporation of an antenna in vicinity of the metal
center to allow for efficient excitation and intersystem crossing to
the f-orbital centered excited state 2. Full coordinative saturation
5
00 nm wavelength negates in vivo optical imaging
[
6]
applications. A potential solution to this problem is the use of
an internal excitation source that obviates the need for high-
energy, external excitation.
2
to limit access of H O to the first coordination sphere, preventing
vibrational, radiationless deexcitation, 3. Hydrophilicity and high
kinetic inertness of the over-all complex to ascertain
biocompatibility, 4. Structural handles for functionalization for
covalent introduction of the CR source.
In this context, we propose the application of Cherenkov
radiation (CR) of radionuclides for the in situ excitation of the
antenna of discrete Eu(III) and Tb(III) complexes. As
Here, we tested Tb/Eu/La(III) complexes with and without
presence of an antenna to probe efficiency and detection limit
[a]
A. G. Cosby, Dr. S. H. Ahn, Prof. E. Boros
Department of Chemistry
Stony Brook University
18
89
for intermolecular excitation using 10-30 µCi F or Zr, which
corresponds to typical quantities of activity employed for in vivo
[
14-15]
PET imaging studies.
synthesized Tb(III) complex that allows for covalent
Subsequently, we designed and
100 Nicolls Road, Stony Brook, NY 11790
E-mail: eszter.boros@stonybrook.edu
a
8
9
attachment of the CR emitting isotope Zr, generating a Tb(III)
complex with an intramolecular CR source (Scheme 2).
Supporting information for this article is given via a link at the end of
the document.
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Angewandte Chemie International Edition
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COMMUNICATION
a commercial small animal fluorescence optical imaging scanner.
Radiance emission was collected between 515 – 840 nm and
quantified using ROI analysis. We observed increasing signal
intensity proportional to increasing concentration of lanthanide
for terbium(III) and europium(III) complexes; furthermore, the
-
-
emission intensity of complexes [Eu(L1)] and [Tb(L1)] is
enhanced by orders of magnitude when compared to the
-
-
corresponding [Eu(DOTA)] and [Tb(DOTA)] complexes (Figure
1
A, Figure S13, Supporting Information).
A
-
-
Scheme 2. Structure of lanthanide complexes [Ln(DOTA)] , [Ln(L1)] and
8
9
-
Zr[Ln(L2)] investigated in this work.
Table 1. Summary of hydration number, quantum yields and extinction
coefficient for complexes evaluated.
Complex
Hydration
number q
Quantum yield φ
Brightness B (ε
[
a]
–1
–1
× φ) [M cm
]
-
[
[
[
[
[
[
[
Tb(DOTA)]
0.96
1.00
1.00
0.00
0.02
0.00
0.07
0.1%
0.1%
-
20.0
1.96
-
B
-
Eu(DOTA)]
1
.5×106
Tb(L1)] , 10 µCi 89Zr
-
[
-
La(DOTA)]
[
Tb(L1)] , 10 µCi 18F
-
-
Tb(L1)] , 30 µCi 89Zr
-
[
Tb(L1)]
47%
1.5%
-
25345
633
-
[
Tb(L1)] , 30 µCi 18F
-
1
.0×106
-
Eu(L1)]
-
La(L1)]
-
5.0×105
Tb(L2)]
63%
44151
[
a] Quantum yield calculations were carried out as reported previously
as a reference standard in H O. The estimated relative
relative to Ru(bipy)
error is 10−15%.
3
2
2
0
0.0
0
100
200
300
400
500
Quantity of complex (nmol)
[
16]
89
Lanthanide complexes of DO3Apic (here referred to as L1)
Figure 1. (A) Images of phantoms to quantify radiance emerging from Zr (10
µCi) doped samples containing increasing quantities of lanthanide complex.
exhibit high thermodynamic stability and efficiently exclude water
from the inner hydration sphere. The picolinate arm provides
convenient bidentate donation and serves as an efficient
-
(
B) Quantified radiance for complex [Tb(L1)] in presence of 10 or 30 µCi of
Zr or F.
8
9
18
-
antenna to produce a quantum yield (φ) of 47% for [Tb(L1)]
-
(
Table 1). The corresponding [Eu(L1)] complex produced φ =
This indicates that excitation of the antenna by CR is the
-
1
.5% while [La(L1)] is not emissive. The Tb(III), Eu(III) and
dominant excitation pathway. As expected, complexes of
lanthanum show no enhanced emission in presence of a CR
emitting source (Figure 1A). The lanthanide-based emission is
La(III) complexes of 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetic acid (DOTA) were also synthesized and employed as
controls without antenna.
further confirmed by detection of the characteristic emission
-
bands
of
[Tb(L1)]
using
conventional
fluorescence
spectrophotometry (Figure S12, Supporting Information). The
picolinate antenna of L1 is not suited to produce a strongly
emissive Eu complex, which is reflected in the comparatively low
Solutions of 0.025 – 600 nmol complex in 500 µL of
phosphate buffered saline (PBS) were prepared; these
quantities are comparable to a typical dose of fluorophore
administered for targeted in vivo fluorescence imaging
-
radiance enhancement obtained for [Eu(L1)] (Figure 1A) and
[
17-19]
the low quantum yield value. We note a non-linear increase of
radiance especially at high concentrations of lanthanide,
indicating that a finite amount of photons is released by a given
studies.
To assess feasibility and efficiency of CR mediated
-
-
excitation, aqueous solutions of [Ln(DOTA)] and [Ln(L1)]
1
8
89
complexes were doped with 10 µCi F or Zr and imaged using
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Angewandte Chemie International Edition
10.1002/anie.201809783
COMMUNICATION
amount of activity and thus a limit of excitation is reached.
[
8]
-
To compare [Tb(L2)] with an intramolecular CR source with
1
8
89
-
-
Increase of activity to 30 µCi F or Zr in samples leads to
proportionally enhanced emission intensity, followed by
saturation of the maximum intensity (Figure 1B, Table S2,
Supporting Information).
[Tb(L1)] , we first carried out the direct radiolabeling of [Tb(L2)]
8
9
using Zr-oxalate. Radiolabeling proceeds quantitatively within
10 minutes at room temperature, producing the radiolabeled,
8
9
bimetallic complex
Zr[Tb(L2)]
(Figure 2). Subsequently,
-
CR mediated excitation of the antenna to indirectly produce
lanthanide luminescence emission opens possibilities for
multimodality and sensing applications in vitro and in vivo.
However, the absorption of short wavelength CR by proteins can
strongly attenuate efficient intermolecular excitation (Figure S16,
S17). To address this, we designed a Tb(III) complex with an
solutions with varying amounts of [Tb(L2)] (0.1 – 10 nmol) were
8
9
incubated with 10 µCi of Zr-oxalate, followed by HPLC analysis
8
9
-
to ascertain that Zr was fully complexed by [Tb(L2)] . Radiance
8
9
of the bimetallic Zr[Tb(L2)] construct in comparison with
-
[Tb(L1)] was collected and quantified. Phantom imaging
-
revealed analogous behavior of [Tb(L1)] in presence of 10 µCi
8
9
89
89
appended desferrioxamine to incorporate
Zr as an
of Zr-oxalate and Zr[Tb(L2)], with a detection limit as low as
8
9
intramolecular CR source. The synthesis of the corresponding
2.5 nmol for Zr[Tb(L2)] (Figure 3). This confirms that CR
mediated excitation of discrete Tb(III) complexes can be
achieved in both inter- and intramolecular fashion. We note that
L2 ligand was achieved through step-wise functionalization of
t
DO2A Bu with 1, followed by alkylation with 3. Debenzylation
8
9
-
and subsequent amidation with desferrioxamine mesylate yields
under these conditions, the ratio of Zr[Tb(L2)] to [Tb(L2)] is
approximately 1:10 - 1:1000 in solution, which represents a
typical ligand to complex ratio for radiometallated peptides and
5
, which is followed by deprotection of the tBu protective groups
-
and complexation with Tb(III) to afford [Tb(L2)] (Scheme 3).
-
[21]
[
Tb(L2)] exhibits a φ of 63% and a q = 0.07, thus representing a
antibodies utilized for targeted in vivo imaging. This raises the
-
suitable functionalized analogue of [Tb(L1)] (Table 1) and
question of which portion of emitted luminescence arises from
confirming complexation exclusively by the polyaza-macrocycle.
additional, intermolecular excitation processes. A plasma
incubation experiment of both complexes (Figure S21, Table S3)
-
confirms enhanced attenuation for [Tb(L1)] (-37% for 100 nmol)
whereas protein-mediated attenuation of emission was
8
9
minimized for Zr[Tb(L2)] (-8% for 100 nmol).
A
B 1
.6×105
Tb(L1)]-
89Zr[Tb(L2)]-
[
1
1
1
8
.4×105
-
Scheme 3. Synthesis of [Tb(L2)] : (i) K
2
CO
3 2 3
, MeCN, 70 °C, 18 h, (ii) 3, K CO ,
MeCN, 70 °C, 18 h (iii) 10% w/w Pd/C , H
2
, MeOH, rt, 3 h (iv) DFO-mesylate,
.2×105
HBTU, DIPEA, DMF, 40 °C, 18 h (v) TFA/DCM 1:1, rt, 20 h (vi) Tb(OTf)
pH 7.
3 2
, H O,
.0×105
.0×104
1
00
300
[
Tb(L2)]-
89Zr[Tb(L2)]
0
.0
2.5
5.0
7.5
10.0
12.5
7
5
2
5
0
5
0
2
00
Quantity of complex (nmol)
100
0
8
9
Figure 3. (A) Images of phantoms to quantify of radiance emerging from Zr
-
-
doped samples containing lanthanide complexes [Tb(L1)] and [Tb(L2)] at
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
Tb(III) concentrations ranging from 0.1-10 nmol. (B) Quantified radiance for
Time (min)
-
89
89
complexes [Tb(L1)] and Zr[Tb(L2)] with 10 µCi of Zr.
8
9
-
Figure 2. Overlay of HPLC traces of Zr[Tb(L2)] and [Tb(L2)] using analytical
method C.
Studies on distance and concentration effects between CR and
luminescent lanthanide are required as indicated by the detected
-
89
difference in emission intensity for [Tb(L1)] and Zr[Tb(L2)]
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Angewandte Chemie International Edition
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COMMUNICATION
observed at 10 nmol, specifically for intramolecular systems of
[
[
[
17]
18]
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[
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2
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In summary, we have successfully employed CR-emissive
radioisotopes to excite the antenna of discrete luminescent
lanthanide complexes in an inter- and intramolecular fashion.
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[20]
8
9
-
89
Solutions of ≥ 2.5 nmol Zr –doped [Tb(L1)] and Zr[Tb(L2)]
complex produce luminescence detectable by a conventional
small animal fluorescence imager. The in situ excitation of
discrete luminescent lanthanide complexes opens possibilities
for in vivo applications of lanthanide luminescence in tandem
with PET imaging or radiotherapy.
[
21]
B. M. Zeglis, J. S. Lewis, JoVE 2015, 96, e52521.
Experimental Section
Experimental details on chemical synthesis, sample preparation,
analytical methods and raw data of photophysical
measurements are provided in the Supporting Information.
Acknowledgements
E.B. acknowledges funding sources, specifically the NIH for a
Pathway to Independence Award (NHLBI R00HL125728-03), a
REACH award (U01HL127522 – 18150031) and Stony Brook
University for startup funds.
Prof. Peter Smith-Jones and Prof. Nashaat Turkman are kindly
1
8
acknowledged for generous donation of F. We thank Jaclyn
Ashcraft, Gregory Quevedo and Justin Devaraj for chemical
synthesis of precursors.
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COMMUNICATION
COMMUNICATION
Exciting isotopes: Cherenkov
radiation, emitted by clinically
utilized radioisotopes, was employed
as an in situ, inter- and
Alexia G. Cosby, Shin Hye Ahn, Eszter
Boros*
Page No. – Page No.
intramolecular excitation source for
biocompatible, discrete terbium(III)
complexes. The successful detection
of the emitted lanthanide
Cherenkov Radiation Mediated In
Situ Excitation of Discrete
Luminescent Lanthanide
Complexes
luminescence with conventional
bioimaging equipment is
demonstrated.
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