Angewandte
Chemie
DOI: 10.1002/anie.201300170
Xenon Biosensor
129
Molecular Imaging of Cancer Cells Using a Bacteriophage-Based Xe
NMR Biosensor**
Krishnan K. Palaniappan, R. Matthew Ramirez, Vikram S. Bajaj, David E. Wemmer,
Alexander Pines, and Matthew B. Francis*
The accurate detection and localization of clinically relevant
biomarkers in vivo is a great challenge for molecular imaging,
Molecular imaging agents that leverage these character-
[11]
istics, generally called Xe biosensors, have been developed.
They consist of a Xe-binding host molecule, commonly
[1]
requiring high sensitivity and molecular specificity. This is
particularly true for screening applications, where the ability
to image disease progression non-invasively could improve
patient outcome. Magnetic resonance imaging (MRI) is
a ubiquitous, non-invasive imaging technique with sub-milli-
[
12]
cryptophane-A (CryA), attached to targeting groups for
[
13]
localization.
a distinct chemical shift apart from that of aqueous Xe
Xenon bound by CryA (Xe@CryA) has
[
11]
(Xe@water). Its rapid and reversible encapsulation is the
basis of an indirect detection scheme in which the small
Xe@CryA spin pool is saturated by frequency-selective
radiofrequency (RF) pulses and transferred by exchange to
the larger Xe@water spin pool, an example of amplification
[
2]
meter spatial resolution, but its use in molecular imaging has
been limited by its poor sensitivity when imaging molecules
[
1,3]
other than water.
This has led to the development of
contrast agents and MRI methods that improve sensitivity by
modulating the local magnetic environment of protons in
by CEST. This results in a decrease in the signal relative to
[
4]
[11b]
water, including gadolinium chelators, iron-oxide parti-
a control experiment (Figure 1a).
Combined with the
[
5]
[6]
cles, and chemical exchange saturation transfer (CEST).
signal enhancement of hyperpolarization, Xe biosensors can
achieve the detection thresholds necessary for molecular
1
3
3
More recently, approaches that use hyperpolarized C, He,
and Xe nuclei have been developed and used in clinical
studies.
1
29
[14]
imaging. To improve the sensitivity further we used multi-
[7]
valent systems in which many CryA hosts are assembled onto
a single carrier molecule, a concept initially applied with
In the experiments below, we used xenon (Xe) as a sensor
medium. Xe is an attractive option for MRI-based molecular
[
15]
paramagnetic relaxation and CEST agents.
We have
[
8]
imaging because it is chemically inert, has low toxicity, is
soluble in water and tissue, and can be hyperpolarized (hp) to
demonstrated this strategy for Xe biosensors with branched
[
16]
[17]
dendrimers and viral capsids, producing constructs that
were detectable by hyperCEST at sub-picomolar concentra-
tions.
[9]
increase its signal more than 10000-fold. Thus, even low
concentrations of dissolved Xe give an NMR signal compa-
rable to that of water, and there is no Xe background in vivo.
These favorable properties of Xe MRI have already been
In previous studies of Xe biosensors biological binding
events were measured in solution. This was first achieved with
biotin-functionalized biosensors binding streptavidin
1
29
[10]
demonstrated in humans after inhalation of hp Xe gas.
[
11,14a,18]
beads,
and subsequently with the detection of DNA
[
19]
hybridization, enzymatic cleavage by matrix metallopro-
teinase-7, ligand binding to human carbonic anhydrase
and an a b integrin, and peptide complex formation with
a major histocompatibility complex protein.
[
20]
[21]
[
+]
[+]
[
22]
[
*] Dr. K. K. Palaniappan, R. M. Ramirez, Dr. V. S. Bajaj,
2
b 3
Prof. Dr. D. E. Wemmer, Prof. Dr. A. Pines, Prof. Dr. M. B. Francis
Department of Chemistry, University of California
Berkeley, CA 94720-1460 (USA)
[23]
Once the
[22,24]
cellular compatibility of Xe biosensors was established,
1
29
Xe NMR spectroscopy was performed with cells after
targeting them with micromolar concentrations of a trans-
E-mail: mbfrancis@berkeley.edu
[
+]
R. M. Ramirez, Dr. V. S. Bajaj, Prof. Dr. A. Pines,
Prof. Dr. M. B. Francis
[
25]
ferrin-functionalized biosensor. While that study detected
biosensor binding by measuring the Xe@CryA chemical shift,
non-specific binding was also observed.
Materials Sciences Division
Lawrence Berkeley National Laboratory
Berkeley, CA 94720-1460 (USA)
Here, we report a multivalent Xe biosensor that uses
single-chain antibodies to target cell surface biomarkers. We
further specifically demonstrate its ability to specifically
recognize these biomarkers in living cells and at concentra-
tions required for molecular imaging. To accomplish this, we
used fd filamentous bacteriophage that display single-chain
antibody variable fragments (scFvs) on their minor coat
Prof. Dr. D. E. Wemmer
Physical Biosciences Division
Lawrence Berkeley National Laboratory
Berkeley, CA 94720-1460 (USA)
+
[
] These authors contributed equally to this work.
[
**] This work was supported by grants from the U.S. Department of
Defense Cancer Research Program (grant number BC016995,
M.B.F.) and by the U.S. Department of Energy, Office of Basic
Energy Sciences, Division of Materials Science and Engineering
under contract number DE-AC02-05CH112 (A.P.).
[
26]
proteins (p3, Figure 1b). The rod-like body of the fd phage,
which has 4200 identical copies of the major coat protein (p8),
can be modified with proteins or synthetic molecules to create
[
27]
new materials.
Additionally, through the use of phage
display techniques, filamentous phage that display proteins as
Angew. Chem. Int. Ed. 2013, 52, 4849 –4853
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4849