Sergey Korchak1, Wolfgang Kilian1, Leif Schröder2, and Lorenz Mitschang1
1Physikalisch-Technische Bundesanstalt (PTB), Braunschweig and Berlin, Germany, 2Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany
Synopsis
The reversible binding of xenon to host
structures is fundamental to the development of novel contrast agents employing
hyperpolarized xenon and chemical exchange saturation transfer (HyperCEST) for
molecular imaging. The rates for entering and leaving the host depend on atomic
details and affect the obtainable contrast rendering them pivotal for the
selection of hosts and optimization of imaging methods. However, different
exchange processes may apply whose contributions are difficult to assign. Exchange
spectroscopy experiments are proposed which enable straightforward
disentanglement of the exchange kinetics and quantification of individual
contributions. The approaches are exemplified for the cryptophane-xenon
host-guest system.Purpose
Determination and quantification of the kinetics
for the reversible binding of xenon to host molecules to enable development of
effective contrast agents and optimization of contrast.
Method and Results
The remarkable MR properties of xenon and its
ability to reversible binding to suitable host structures like container
molecules or nanostructures is currently heavily explored for novel approaches
to molecular imaging. Upon functionalization of the host biomarkers or tissue
can be targeted specifically while the NMR readout can report about the state
of the system under investigation with sensitivity enhanced by orders of
magnitude because of hyperpolarization of xenon and application of HyperCEST$$$^{1,2}$$$. The binding kinetics underlying the method and its application comprises
contributions from different exchange mechanisms: a simple dissociative
process, where xenon enters a free host or leaves one behind, and a degenerate
process where bound and unbound xenon interchange their binding status directly
without detour of an intermediate free host$$$^{3,4}$$$. Their
differentiation by hand of a single NMR spectrum is not possible. However, by
employing exchange spectroscopy in combination with variation of xenon
concentration the overall kinetics can be disentangled and individual
contributions quantified. For demonstration the binding kinetics of xenon to
the host molecule cryptophane-A (CrA) in pure water at room temperature was
analyzed. In a gas mixture with He and N$$$_2$$$ hyperpolarized xenon was
bubbled through the aqueous CrA solution ($$$6.4\,\mu$$$M) until xenon
saturation to an extent given by Henry´s Law was achieved, i.e. by xenon
partial pressure the amount of unbound xenon in the solution, [Xe], could be
controlled. Subsequently, either of the pulse sequences in Fig.$$$\,1$$$ was applied
to achieve saturation or magnetization transfer in between the pools of unbound
and CrA-bound xenon, $$$M_{\text{Xe}}$$$ and $$$M_{\text{CXe}}$$$, respectively. In all
cases, perturbation of the one pool (saturation, or magnetization inversion and
subsequent free exchange) leads to depletion of the other pool because of
exchange and the accompanied signal decay can be determined by a series of
experiments with variable perturbation period. In dynamic equilibrium and under
the mild condition $$$M_{\text{Xe}}\,\gg\,M_{\text{CXe}}$$$ in case of
experimental schemes II and III which can be easily achieved by a sufficiently
high xenon partial pressure, linear dependencies of the depletion rate (I and
II) or a related quantity (III) in the unbound xenon concentration are
obtained. In Fig.$$$\,2$$$ data from experiments I to III are displayed together with
linear fittings according to the linear relations in Fig.$$$\,1$$$. While saturation
as well as magnetization transfer from the unbound to CrA-bound xenon pools
(schemes I and II) allow for direct quantification of the rates of degenerate
exchange, $$$k$$$, and complex dissociation, $$$c$$$, by determination of slope and
intersection of the fit, in case of HyperCEST (scheme III) prior knowledge of
the relaxation rate, R$$$_{1\text{Xe}}$$$, and CrA concentration, [C$$$^{tot}$$$],
are required for that purpose. The rates obtained in this manor are displayed
in Tab.$$$\,1$$$, the values and error estimates indicate high accuracy for every of
the proposed experiments and their respective precision. By the weighted
average of the results of experiments I to III the kinetic rate coefficients
for complex dissociation and for degenerate exchange are for aqueous solution
at 298 K determined as $$$c\,=\,(\,20.0\,\pm\,0.3\,)\,$$$s$$$^{-1}$$$ and $$$k\,=\,(\,5800\,\pm\,280\,)\,$$$M$$$^{-1}\,$$$s$$$^{-1}$$$, respectively. The identification of
exchange mechanisms and the quantification of their individual contributions to
the overall kinetics as proposed here may, because of simplicity in
interpretation and data evaluation and by the numerical quality of the results,
prove helpful in efforts towards a complete characterization and optimization of
parameters governing molecular imaging approaches using hyperpolarized xenon$$$^5$$$.
Acknowledgements
This work was funded by the Bundesministerium
für Bildung und Forschung (01EZ1010A) and the European Metrology Research
Programme participating countries within EURAMET and the European Union (EMRP
grant HLT-10).The authors thank C. Witte at FMP for generous support in the
experimentation.References
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