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\DOI{10.5802/crchim.335}
\datereceived{2024-05-17}
\daterevised{2024-08-26}
\dateaccepted{2024-09-04}
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%\copyeditor{Reeja}

\dateposted{2024-11-27}
\begin{document}

\begin{noXML}

%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{French/Nordic Special Issue on Materials and Coordination Chemistry}
%\def\cdr@specialtitle@french{Num\'ero sp\'ecial franco-nordique sur les mat\'eriaux et la chimie de coordination}
%\makeatother

\CDRsetmeta{articletype}{research-article}

\title{Cherenkov radiation energy transfer from
[\textsuperscript{18}F]-fluorodeoxyglucose to subphthalocyanine fluorophores}

\alttitle{Transfert de l'\'{e}nergie de la radiation Cherenkov 
du [\textsuperscript{18}F]-fluorodeoxyglucose aux fluorophores de type
subphthalocyanine}

\author{\firstname{Vivian} \lastname{Lioret}}
\address{Universit\'{e} de Bourgogne, Institut de Chimie Mol\'eculaire ICMUB,
Sciences Mirande, 21000 Dijon, France}
%\email{vivianlioret.vl@gmail.com}

\author{\firstname{Richard A.} \lastname{Decr\'eau}\CDRorcid{0000-0003-4205-0092}\IsCorresp}
\addressSameAs{1}{Universit\'{e} de Bourgogne, Institut de Chimie Mol\'eculaire ICMUB,
Sciences Mirande, 21000 Dijon, France}
\email[R. A. Decr\'eau]{Richard.Decreau@u-bourgogne.fr}

\keywords{\kwd{Cherenkov radiation energy transfer
(CRET)}\kwd{Subphthalocyanine}\kwd{Cherenkov luminescence imaging
(CLI)}}

\altkeywords{\kwd{Transfert de l'\'{E}nergie de la radiation Cherenkov
(CRET)}\kwd{Subphtalocyanine}\kwd{Imagerie par luminescence Cherenkov
(CLI)}}

\begin{abstract}
Cherenkov radiation (CR) is an optical light that is emitted during the
decay of beta-particle-emitting radionuclides, such as \textsuperscript{18}F, which is
present in [\textsuperscript{18}F]-fluorodeoxyglucose, a well-known tracer used in
positron emission tomography. The CR emission occurs from relaxation of
molecules of the dielectric medium surrounding the emitted
beta-particle. The Cherenkov radiation energy transfer (CRET) to
a CR acceptor, such as subphthalocyanine, was analyzed. 
Subphthalocyanine is a unique concave-shaped 14\tralicstex{\textit{π}}{$\rmpi$}-electron
fluorophore with optical properties. The relevance of such a CR
emitter/acceptor couple to performing CRET is discussed.
{\vspace*{-3pt}}
\end{abstract}

\begin{altabstract}
La Radiation Cherenkov (CR) est une lumi\`{e}re optique qui est
\'{e}mise au cours de la d\'{e}croissance radioactive de
radio\'{e}l\'{e}ments \'{e}metteurs beta, tels que le \textsuperscript{18}F que
l'on retrouve dans un traceur bien connu pour la Tomographie d'emission
de positron (PET) en clinique. Le transfert de l'\'{e}nergie de la
radiation Cherenkov (CRET) a \'{e}t\'{e} \'{e}tudi\'{e} avec un
accepteur de CR acceptor, telle que les subphthalocyanines, un
fluorophore particulier \`{a} 14 electrons \tralicstex{\textit{π}}{$\rmpi$} de forme concave et
avec de bonnes propri\'{e}t\'{e}s optiques. La pertinence d'un tel
couple emetteur/acceptor de CR pour faire du CRET est discut\'{e}.
\end{altabstract}

\thanks{CNRS ``Chaire d'Excellence'', Burgundy/Burgundy
Franche-Comt\'e (BFC) Regional Councils (FABER, 3MIM, PARI, 
respectively), French Ministry of Higher Education, Research and 
Innovation for a scholarship,  ANR-EQPX-IMAPPI, PO-FEDER-FSE Bourgogne,
Cancerop\^ole Est (CLI Emergence Project), SATT-SAYENS (CLI-PDT)}

\thanks{\textbf{Note.} This proof-of-concept study was performed within
the framework of a PhD thesis~\cite{8}.}

\maketitle
\vspace*{-1pt}

\twocolumngrid

\end{noXML}

\section{Introduction} \label{sec1}

{

Cherenkov radiation (CR) is emitted during radionuclide decay when a
beta particle is released at a speed that causes the surrounding
molecules from the dielectric medium (along the track of the particle)
to attain an excited/polarization state. Subsequent relaxation of the
surrounding molecules proceeds with light emission, which is called
Cherenkov radiation. Overall, such a CR emission occurs when the energy
of the beta particle is so high that it can travel even faster than
light in a given dielectric
medium~\cite{1,1b,1c,1d,1da,1db,1e,5a,1f,1g,1h,1i,1j} (which is a medium that
could be polarized by an electric field). Hence an analogy can be
drawn: the noise emitted when airplanes break the sound barrier may
somehow compare with CR, which is \mbox{emitted} when beta particles
break the light barrier in a dielectric medium.

\begin{figure*}
{\vspace*{3pt}}
\includegraphics{fig01}
{\vspace*{3pt}}
\caption{\label{fig1}(A)~CR emitter:
[${}^{18}$F]-fluorodeoxyglucose ([${}^{18}$F]-FDG). Inset (left): CR as
seen in pools that cool nuclear rods. (B)~CR
acceptor: subphthalocyanine (\textbf{SUB}).}
{\vspace*{2pt}}
\end{figure*}

The history of CR~\cite{1,1b,1c,1d,1da,1db,1e,5a,1f,1g,1h,1i,1j} 
began from its prediction by Oliver~Heaviside in 1888,
then Arnold Sommerfeld in 1904, and followed by its first observation by Marie
Curie who detected its emission from radium samples in the 1910s.
Later in the 1920s, Lucien Mallet demonstrated that it was emitted
from transparent bodies and could record its spectrum. Then Pavel
Cherenkov, a graduate student of Sergei Vavilov at the Lebedev
Institute (FIAN), demonstrated its origin and
properties and that it consists of a continuous spectrum.
Ilya Frank and Igor Tamm subsequently performed the theoretical
interpretation of this emission, and they were awarded the Nobel Prize
in 1958 together with Pavel Cherenkov. About 100 years after the first
observation of CR, the development of a sensitive camera allowed the
use of CR in a variant of optical imaging named Cherenkov luminescence
imaging (CLI), first in 2009 in preclinical settings~\cite{2,2b,2c,2d}
and later in 2013 in clinical settings~\cite{3,3b}.\looseness=-1

Cherenkov radiation is an alternative light source to perform
fluorophore photoactivation, which originates from atomic decay
(radionuclides) and surrounding molecules relaxation. Cherenkov
radiation energy transfer (CRET) to fluorophores is expected to be
efficient as long as there is a good match/overlap between the CR
emission spectrum and the fluorophore absorption
spectrum~\cite{4,4b,4c,4d,4e,4f,4g}, which roughly falls in the
300--600~nm window of the electromagnetic spectrum. It is required that
the optical properties of both the CR emitter and the CR acceptor are
acceptable. This means a high CR quantum yield for the former and high
brightness B for the latter (with high values of either one or both of
its components: fluorescence quantum yield $\rmPhi_{\mathrm{F}}$ and
molar coefficient absorption~$\rmvarepsilon$). These matters related
to CRET will be addressed on choosing two partners of
interest for future biomedical applications, such as
${}^{\mathbf{18}}\mathbf{F}$ as the CR emitter and subphthalocyanine
(\textbf{SUB}) as the CR \mbox{acceptor}\break (Figure~\ref{fig1}).

}

\section{Results and discussion} \label{sec2}
\subsection{Synthesis and radiosynthesis of CR emitter and CR
acceptor} \label{sec2.1}
\subsubsection{CR emitter: fluorine-18 (${}^{18}$F) in
[${}^{18}$F]-fluorodeoxyglucose [${}^{18}$F]-FDG}
\label{sec2.1.1}

Fluorine-18 (${}^{18}$F) is the most widely used radionuclide for
positron emission tomography (PET) imaging in clinical
settings~\cite{5,5b,5c,5d}.\ Fluorine-18 emits a positron ($\rmbeta^+$)
that annihilates on reaction with an electron to produce two gamma rays
that are detected and used for PET imaging purposes. The actual study
focuses on the ability of the positron to trigger the emission of CR
with the surrounding dielectric medium. [${}^{18}$F]-fluorodeoxyglucose
([${}^{18}$F]-FDG) is synthesized by electrophilic \textit{or}
nucleophilic fluorination~\cite{5,5b,5c,5d} (Figure~\ref{fig2}).
In electrophilic
fluorination, tri-O-acetyl-D-glucal reacted with [${}^{18}$F]-F$_2$ or
[${}^{18}$F]-CH$_3$CO$_2$F to produce a mixture of fluorinated
molecules. From among these molecules, the difluoroglucose derivative was
isolated and subsequently hydrolyzed to form [${}^{18}$F]-FDG (in 8\%
yield in 2~h). In nucleophilic fluorination, the [${}^{18}$F]-F$^-$ ion
is in the presence of Kryptofix 222{\texttrademark}, a cyclic crown
ether that binds the potassium ion, which prevents the formation of
poorly reactive [${}^{18}$F]-KF and makes [${}^{18}$F]-F$^-$ more
reactive in the nucleophilic substitution. The
radioactive anion will displace the triflate leaving group in
tetra-O-acetyl-triflate mannose to form [${}^{18}$F]-FDG.\looseness=-1

\begin{figure*}
{\vspace*{4pt}}
\includegraphics{fig02}
{\vspace*{4pt}}
\caption{\label{fig2}[${}^{18}$F]-fluorodeoxyglucose ([${}^{18}$F]-FDG)
synthesis, following the electrophilic~(A) and nucleophilic~(B)
fluorination reactions~\cite{5,5b,5c,5d}.}
{\vspace*{4pt}}
\end{figure*}

\subsubsection{CR acceptor: subphthalocyanine} \label{sec2.1.2}

Subphthalocyanines (\textbf{SUB}, also reported with the acronym SubPc in the
literature) are 14-${\rmpi}$ electron
macrocycles that are reported as fluorophores of interest for
fluorescence imaging~\cite{6,6b}. Their shape is unique; they have a
domed concave structure, which is an appealing feature in biomedical
\mbox{applications} because it does not favor aggregation, a
deactivation phenomenon in fluorescence imaging. 

The first step in \textbf{SUB} platform synthesis 
(Figure~\ref{fig3}) is the dicyanobenzene
cyclotrimerization reaction that is typically achieved in
1,2-dichlorobenzene as a solvent under inert atmosphere in the presence
of boron trichloride to form golden sparkling chlorosubphthalocyanine
dye (SUB-Cl, \textbf{1}) of approximately 58\% yield.\ Subsequent
reaction of \textbf{1} with phenol/hydroxyaryl-containing reagents
leads to the replacement of the axial chlorine atom with the
corresponding aryloxy substituent of interest, also of \mbox{60--64\%}
yield (which corresponds to 35--37\% overall yield in \textbf{2} and
\textbf{3}).\ The first rationale of such an addition
was to further address the three-dimensional feature of the dye, that
is, subsequently preventing aggregation. The second rationale was to
conveniently afford a chemical functionality for further derivatization
of \textbf{SUB} platforms as with nitrophenoxy substituent in SUB-PH
\textbf{2} (which could be reduced into an amine for further
conjugation). The third rationale was to append a UV/blue \mbox{absorbing}
fluorophore to produce a SUB-COUM \textbf{3} dyad, which could undergo
more CR absorption and contribute to its transfer toward a less
energetic wavelength potentially less absorbed by biological\break tissues. 

\begin{figure*}
{\vspace*{4pt}}
\includegraphics{fig03}
{\vspace*{4pt}}
\caption{\label{fig3}Syntheses of subphthalocyanine (\textbf{SUB}) targets
bearing three distinct axial features: a chlorine atom
(\textbf{SUB-Cl}, \textbf{1}), a nitrophenoxy group (\textbf{SUB-PH},
\textbf{2}), and a coumarin (\textbf{SUB-COUM}, \textbf{3})~\cite{6,6b}.}
{\vspace*{4pt}}
\end{figure*}

\subsection{Luminescence studies: CR emitter,\hfill\break CR
acceptor, and their combination to\hfill\break achieve CRET} \label{sec2.2}

\subsubsection{CR emitter: ${}^{18}\mathrm{F}$} \label{sec2.2.1}

\subsubsection*{Theory~\cite{5d}}

\begin{figure}
\includegraphics{fig04}
{\vspace*{-2pt}}
\caption{\label{fig4a}Emission of the Cherenkov Radiation (CR) from
radionuclides, and as it is seen in pools that cool nuclear rods.}
{\vspace*{-2pt}}
\end{figure}

\begin{itemize}
\item Fluorine-18 (${}_{\xx09}^{18}$F) is a ``neutron-deficient'' or
``proton-rich'' radionuclide that decays by emitting a positron
($\rmbeta^+$) particle, a neutrino ($\rmnu$), and its daughter radionuclide
${}_{\xx0\mathbf{8}}^{18}$O (the atomic number of which is one unit less
than that of its starting ${}_{\xx09}^{18}$F parent):
{\[
{}_{\xx09}^{18}\mathrm{F}\rightarrow {}_{\xx0\mathbf{8}}^{18}\mathrm{O}
+\rmbeta^++\rmnu
\]}\unskip
\item ${}^{18}$F decay follows the \textit{radioactivity decay law}
(Equation~(\ref{eq1})):
{\begin{equation} \label{eq1}
A_t=A_{0}\mathrm{e}^{-\lambda t}
\end{equation}}\unskip
{\textit{Parameters of the equation:}}
$A_{t}$, activity at time $t$;
$A_{0}$, activity at $t = 0$;
$\lambda$, decay constant---it is the probability of disintegration per
unit time and it is related to the \mbox{half-life} (0.693/$t_{1/2}$);
$t_{1/2}$, \mbox{half-life} of the radionuclide, the time the starting
activity drops to one half---it is related to activity~$A$
and the number of radioactive atoms~$N$;
activity of a radionuclide, or radioactivity, is the disintegration
rate: $A=\lambda N=-{\mathrm{d}N}/{\mathrm{d}t}$.\vspace*{.25pc}\Tg</p>
\Tg<p>{\hfill\break}Equation~(\ref{eq1}): radioactivity decay law.
\vspace*{.5pc}

\item To achieve CR emission (Figure~\ref{fig4a}), the energy of the beta particle emitted by a
radionuclide during the decay process has to be beyond the Cherenkov
threshold. Cherenkov emission occurs above this threshold, which is a function of
the nature of the medium. When the medium is water, the Cherenkov
threshold is 264~keV. The energy of the beta particle emitted by
a radionuclide such as fluorine-18 (${}^{18}$F) is 634~keV, which makes it a
good candidate for CLI because it is about 2.5-fold
beyond the Cherenkov threshold~\cite{1,1b,1c,1d,1da,1db,1e,5a,1f,1g,1h,1i,1j}.

\item The number of Cherenkov photons emitted follows the equation
reported by Ilya Frank and Igor Tamm (Equation~(\ref{eq2}))
\cite{1,1b,1c,1d,1da,1db,1e,5a,1f,1g,1h,1i,1j}:
{\begin{equation} \label{eq2}
\frac{\mathrm{d}N}{\mathrm{d}x} = 2\rmpi\rmalpha \left(\frac{1}
{\lambda_{1}}-\frac{1}{\lambda_{2}}\right)\left(1-\frac{1}{\beta^{2}
\eta^{2}}\right)
\end{equation}}\unskip
\raisebox{1pc}{}\noindent
{\textit{Parameters of the equation:}}
${\mathrm{d}N}/{\mathrm{d}x}$, number of photons per unit of path
length whose wavelength is between the $\lambda_1$ and
$\lambda_2$ intervals; $\rmalpha$,~fine structure constant (1/137);
$\beta$,~velocity of the particle (in the medium) divided by that in
vacuum (c); $\eta$,~refractive index (RI).\vspace*{.25pc}\Tg</p>
\Tg<p>{\hfill\break}Equation~(\ref{eq2}): determination of the number
of Cherenkov photons generated during radionuclide decay.
\vspace*{.4pc}

\item The number of optical photons (400--800~nm) per nuclear decay is 
reported to be 0.8--1.4 photons per decay of ${}^{18}$F 
atom on a 50~$\upmu$m step path~\cite{2,2b,2c,2d}. A Monte Carlo
simulation showed that CR emitted from ${}^{18}$F is confined within
0.3~mm~\cite{2,2b,2c,2d}.
\vspace*{.4pc}

\item \textbf{CR yield is a function of three parameters:}\Tg</p>
\Tg<p>{\hfill\break}{$^*$\ A---energy of the beta particle}\Tg</p>
\Tg<p>{\hfill\break}{$^*$\ B---activity loaded}\Tg</p>
\Tg<p>{\hfill\break}{$^*$\ C---RI of the medium}
\vspace*{-.2pc}
\end{itemize}

\subsubsection*{Experiment}

Three parameters governing radiance/CR yield were examined throughout
the studies. The emission of CR was recorded both on a
spectrofluorometer and on an optical imager.

\paragraph*{${}^*$ A---energy of the beta particle}

Experiments were conducted on the spectrofluorometer. The fluorescence
cell was filled with a solution of a [${}^{18}$F]-FDG (34.5~MBq) and
recording was achieved on a bioluminescence mode (no laser irradiation)
with maximum open slits and maximum scans. Under the experimental
conditions, a spectrum develops, which is UV/blue weighted
(Figure~\ref{fig4}).
The CR profile is the same from one radionuclide to another. When the
energy of the beta particle is raised, such as ${}^{90}$Y ($E_{\rmbeta}
= 2.2$~MeV), the luminescence intensity is raised compared to
${}^{18}$F ($E_{\rmbeta} = 0.63$~MeV). Hence, ${}^{90}$Y appears as a
more appealing CR emitter than ${}^{18}$F.\ However, the latter was
chosen because of its ready availability and for cost reasons.

\begin{figure*}
\includegraphics{fig05}
{\vspace*{4pt}}
\caption{\label{fig4}Cherenkov radiation emission spectra of
[${}^{18}$F]-FDG (34.5~MBq) and [${}^{90}$Y]-YCl$_3$ (13.2~MBq)~\cite{4d}.}
{\vspace*{.4pc}}
\end{figure*}

\paragraph*{${}^*$ B---activity loaded}

96-well plates were filled with 5--25~MBq (0.135--0.676~mCi)
[${}^{18}$F]-FDG solution and the remainder of the medium with
water.\ CLI \mbox{luminescence} studies were performed on the
\textit{optical imager}, with images recorded just a few minutes after
sample deposition, using an open filter mode. Photon emission is a
function of activity: a rise in activity (with no change in medium
composition) subsequently leads to a corresponding rise in radiance.
The radiance of several activities has been measured by detecting all
emitted photons (no filter used) for 1~s (Figure~\ref{fig5}). 

\begin{figure}
\includegraphics{fig06}
\caption{\label{fig5}Measured radiance as a function of activity in
[${}^{18}$F]-FDG~\cite{8}.}
\end{figure}

\paragraph*{${}^*$ C---RI of the medium}

{

The value of the measured radiance varied depending on the nature of
the \mbox{co-solvent.} The co-solvent with the highest RI
led to the highest radiance as expected from Equation~(\ref{eq1})
(Figure~\ref{fig6}).
Hence, when DMSO with RI ${=}$ 1.477 was used as the co-solvent, a
radiance was measured that was 1.2--1.5 times as much as that of other
co-solvents, the RIs of which were lower, that is, in the 1.3 range.
Conversely a striking observation could be made: although the methanol RI
value was slightly lower (1.328) than that of serum (1.34), its
radiance evolved the other way around. This may possibly be explained
because the RI value is the average of values recorded on the whole
UV--vis window. Although the RI value of methanol measured at
400~nm is noticeably higher (1.374) (Figure S1) than that measured at 700~nm (1.320), the
reported value (1.331)~\cite{7} corresponds to the \mbox{average} value of all
other measured RIs at various wavelengths. Hence, at 400~nm, which corresponds
to a region of the spectrum where CR emission is more intense, the RI
value for methanol (1.374) is higher than that of water (1.34).

}

\begin{figure*}
{\vspace*{-2pt}}
\includegraphics{fig07}
{\vspace*{-.65pc}}
\caption{\label{fig6}Measured radiance of solutions containing
[${}^{18}$F]-FDG emitter (35~$\upmu$L solution) and a co-solvent
(65~$\upmu$L) such as serum~\textbf{(1)}, DMSO~\textbf{(2)}, and
CH$_3$OH~\textbf{(3)}~\cite{8}.  Radiance values are also reported in a
diagram depicted in Figure~S2.}
{\vspace*{-2pt}}
\end{figure*}

\subsubsection{CR acceptor: $\mathbf{SUB}$ (fluorescence studies)}\label{sec2.2.2}

The optical properties of subphthalocyanine measured in toluene
indicate a maximal absorption at 562~nm and emission at 572~nm (i.e.\ 
10~nm Stokes shift).\ It was noted that the fluorescence quantum yield
$\rmPhi_{\mathrm{F}}$ measured in toluene of the subphthalocyanine
dropped when the axial chlorine atom was substituted (0.32 for
\textbf{1}, 0.17 and 0.19 for \textbf{2} and \textbf{3},
\mbox{respectively}).

\subsubsection{CRET studies} \label{sec2.2.3}
\paragraph*{Rationale}

Hence, studies reported in Sections~\ref{sec2.2.1} (Figure~\ref{fig4})
and~\ref{sec2.2.2} (Figure~\ref{fig7}) indicate that the spectral
overlap between the CR acceptor and the CR emitter is optimum
(Figure~\ref{fig8}A). Hence, subsequent CRET studies on the optical
imager could be performed, the concept of which is depicted in
\mbox{Figure}~\ref{fig8}B,C, and CLI images displayed in 
Figure~\ref{fig9}.

\begin{figure*}
\includegraphics{fig08}
{\vspace*{-.55pc}}
\caption{\label{fig7}Top: Overlay of selected emission filters on the
optical imager with subphthalocyanine \textbf{SUB-PH~2} absorption
spectrum (black) and emission spectrum (red) measured in DMSO/H$_2$O
60/40~vol. Bottom: Subphthalocyanine \textbf{SUB-COUM 3} spectra:
absorption (blue), emission (orange), and excitation (gray) spectra
recorded in pure DMSO~\cite{8,6,6b}.}
{\vspace*{-.2pc}}
\end{figure*}

\begin{figure*}
{\vspace*{-1pt}}
\includegraphics{fig09}
{\vspace*{-1pt}}
\caption{\label{fig8}\fontsize{9.6}{12}\selectfont(A)~Overlay of the Cherenkov radiation emission
spectrum from [$^{90}$Y]-YCl$_3$ (13.2~MBq) (see Figure~\ref{fig4}) and
the absorption spectrum of subphthalocyanine \textbf{SUB-COUM 3}
(measured in DMSO) (see Figure~\ref{fig7}). (B,C)~Depiction of CRET
from CR emitter to CR acceptor(s) (fluorophore(s)), subsequently
followed by fluorescence emission (from SUB fluorophore only), which is
enhanced when COUM antenna is present and undergoes additional CRET
transfer and subsequent intramolecular FRET/TBET transfers at the SUB
platform (with no expected residual fluorescence emission from COUM,
previous studies suggested~\cite{8,6b}).}
{\vspace*{-1pt}}
\end{figure*}

\begin{figure*}
\includegraphics{fig10}
\caption{\label{fig9}Cherenkov luminescence imaging studies:
measured radiances of solutions containing [${}^{18}$F]-FDG (10~MBq) CR
emitter only (column~1), or CR emitter mixed with subphthalocyanines
\textbf{SUB-PH 2} (column~2) and \textbf{SUB-COUM 3} (column~3).
Radiances were measured with filters centered at 520, 570,
and 620~nm, respectively.}
\end{figure*}

\paragraph*{Experiment}

{\advance\baselineskip by .4pt

It was performed in 96-well plates and consisted in mixing a solution
of the CR emitter to a solution of the CR acceptor. Hence, a few
microliters of a [${}^{18}$F]-FDG solution that corresponds to
approximately 10~MBq (0.270~mCi) was added to a solution of 1~mM of
subphthalocyanine \textbf{SUB-PH 2} or \textbf{SUB-COUM 3}. As the
radionuclide half-life has to be taken into account at all times
(${}^{18}$F half-life is 109.8~min) and because the [${}^{18}$F]-FDG
provider did not furnish the same volumetric activity (depending on
multiple parameters such as the time of delivery and the starting time
of the experiment), extreme care had to be taken with the volume added
to a well. Hence, adding 10~MBq could vary from 15~$\upmu$L up to 
45~$\upmu$L depending on the volumetric activity of the  stock solution
of the purchased radionuclide.  To ensure the medium maintains the same
composition (and hence the same RI), all controls were diluted the same
way.

The first well was filled with [${}^{18}$F]-FDG CR emitter (column~1),
the second and third with both CR emitter and subphthalocyanine CR
acceptor \textbf{SUB-PH 2} (column~2) or \textbf{SUB-COUM 3}
(column~3), respectively. [${}^{18}$F]-FDG was provided in solution in
physiological serum, in which subphthalocyanines \textbf{2} and
\textbf{3} are not soluble. As a result, the latter were dissolved in
an organic solvent that could remain miscible with water, such as DMSO.
Such a co-solvent was also added to the FDG-containing well to ensure
that conditions remain comparable. 

\paragraph*{Results}

A first series of radiance measurements were achieved using the
\textbf{520} ${\pm}$ \textbf{20 nm} filter. Radiance measured in wells
from column 1 that contain \textit{only} CR emitters without \textbf{SUB}
probes was 1.5-fold higher than that measured in wells from columns 2
and 3 that contain \textit{both} CR emitters and \textbf{SUB} probes.
This filter overlays a window where the CR spectral emission is
significant, which explains why the radiance measured in wells from 
CR-emitter-only column~1 is high. Conversely, the \textbf{SUB} platform
absorbs CR in this window as a result of a good spectral overlap of
both while \textbf{SUB} is not yet detected in such a window. Overall, this 
explains such a drop in radiance from CR-only wells compared to wells
containing CR-absorbing molecules.

}

A second and third series of radiance measurements were achieved using
\textbf{570} and \textbf{620} ${\pm}$ \textbf{20 nm} filters,
respectively. Such windows shift away from the main CR emission
peak. As a result, the radiance corresponding to CR emission only
(column~1) keeps on dropping from 570 to 620~nm. Conversely, these
filters are overlaid with \textbf{SUB} platform emission spectra, which
explains why the radiance is raised in columns~2 and 3, compared to that
in column 1 (control). Hence, the most spectacular result shows that
the radiance corresponding to subphthalocyanine \textbf{SUB-COUM 3}
emission is raised by 2.1-fold compared to the radiance to CR-only emitting
well. This is because the \textbf{SUB} platform is equipped with a
coumarin antenna. Subphthalocyanines not equipped with a \textbf{COUM}
antenna do not display such a rise in radiance. Earlier results with a
non-radioactive light source showed that \mbox{setting} the irradiation at the
\textbf{COUM} antenna (360~nm) led to an emission corresponding to that
of the \textbf{SUB} platform and not from that corresponding to
\textbf{COUM} emission. This suggests a high energy transfer efficiency
yield in the form of intramolecular F\"{o}rster resonance
energy transfer (FRET) or through bond energy transfer
(TBET)~\cite{4f,6b}. It is first crucial to point out, as mentioned in
the previous section, that the fluorescence quantum yields measured for
all substituted subphthalocyanines remained comparable.\ This means that
such differences in radiance \mbox{measured} from \textbf{SUB-PH 2} to
\textbf{SUB-COUM 3} is the result of CRET and not from the
subphthalocyanine~$\rmPhi_{\mathrm{F}}$.\looseness=1

\section{Conclusion} \label{sec3}

This proof-of-concept study showed that CR is an alternative light
source to photoactivate fluorophore platforms upon CRET, and indicated
that the chosen CR emitter/acceptor pair in the actual study
(${}^{\mathbf{18}}$\textbf{F/SUB}) was relevant. Regarding the CR
emitter (${}^{\mathbf{18}}$\textbf{F} in [${}^{18}$F]-FDG), despite its
potency to emit CR (0.8--1.4~optical photons 
(400--800~nm)/decay per 50~$\upmu$m step size~\cite{2,2b,2c,2d},
also indicated by
 the energy of its emitted beta particle
[${E}_{\rmbeta} = 634$~keV]) that appears to be not as high as that
of other radionuclides (up to threefold as less as ${}^{90}$Y), it was
\mbox{straightforward} to perform its CLI imaging on an optical imager at
10~MBq and to record its emission spectrum on a spectrofluorometer.
Moreover, ${}^{\mathbf{18}}$\textbf{F} remains easily accessible and
cheap because of its wide availability in the clinical field.
Regarding the CR acceptor (\textbf{SUB}), despite its brightness (i.e.,
referring to $\rmPhi_{\mathrm{F}}$ and $\rmvarepsilon$) that appears to be
not as high as that of other fluorophores (12,500 versus 73,000--88,000 with
fluorescein), its absorption spectrum overlaps well with the CR
emission spectrum while its unique conical shape makes \textbf{SUB}
appealing with respect to preventing aggregation and to envisioning
future molecular assemblies.\ Hence, such a
\textbf{SUB}/${}^{\mathbf{18}}$\textbf{F} pair enabled achieving
CRET and subsequent \mbox{fluorescence} emission from \textbf{SUB}.
Furthermore, such a proof-of-concept study showed that appending an
additional fluorophore onto \textbf{SUB} with a good spectral overlap
with both \textbf{SUB} and CR allowed achieving additional CRET
transfers at both \textbf{COUM} and \textbf{SUB} fluorophores within
the dyad together with intramolecular ET transfers (FRET/TBET). This
subsequently allowed further rise in radiance toward the targeted
near-infrared (NIR) window. Such an approach is of significant
relevance to in vivo imaging purposes for two reasons: (a)~CR may be
considered an alternative to exogenous lasers classically used in
fluorophore photoactivation in standard optical \mbox{imaging,} where the
\mbox{radionuclide} becomes an embarked (on board) light source, which has advantages
with respect to signal-to-background ratios~\cite{2,2b,2c,2d}; (b)~the
rationale of the study could be extended to other dyads with much
larger {pseudo-Stokes-shifts} (than that of SUB-COUM~3) in order to achieve
fluorescence emission farther in the NIR window where tissues are more
transparent. Hence, fluorophores with absorption bands falling within
the 300--600~nm window are a good match to CR emission spectrum (albeit
residual CR emission is still detected way beyond
600~nm)~\cite{4,4b,4c,4d,4e,4f,4g}. Moreover, such a study suggests that
CRET to other photoactivatable platforms other than fluorophores may
potentially be envisioned~\cite{4,4b,4c,4d,4e,4f,4g}. 

\section{Experimental section} \label{sec4}
\subsection{Materials and methods} \label{sec4.1}

All spectroscopic measurements conducted to thoroughly characterize
\textbf{SUB} molecules were performed on the PACSMUB (P\^{o}le Chimie
Mol\'{e}culaire) platform at the University of Burgundy.

\subsubsection{Nuclear magnetic resonance spectroscopy{\hfill\break} (NMR)}

Measurements were performed on
a Bruker at 300~MHz or 500~MHz (${}^1$H), 75~MHz or 125~MHz
(${}^{13}$C), and 96~MHz (${}^{11}$B) in CDCl$_3$ with the chemical
shifts reported as $\rmdelta$ in ppm relative to TMS (residual chloroform
from deuterated \mbox{chloroform} chemical shift was set at 7.26~ppm,
and coupling constants expressed in Hz). The following abbreviations
were used to describe spin multiplicity: s ${=}$ singlet, d ${=}$ doublet,
t ${=}$ triplet, and m ${=}$ multiplet.

\subsubsection{UV--visible spectroscopy}

UV--vis measurements were performed on a Shimadzu UV-2550
spectrophotometer in glass cuvettes of 1 $\times$ 1 $\times$ 3~cm (1~cm
path) and on an Agilent Cary 50 or Cary 60.

\subsubsection{Mass spectrometry}

(a)~Matrix-assisted laser desorption/ionization time of flight mass
spectrometry (MALDI-TOF MS): Measurements were performed on Ultraflex
II LRF 2000 (Bruker), using dithranol or DHB as a matrix or on a
microflex LRF (Bruker). Solutions were \mbox{prepared} by dissolving 1~mg of
compound in 1~mL of the appropriate solvent. (b)~Electrospray
ionization mass spectrometry (ESI MS). Measurements were performed on
LTQ Orbitrap XL (Thermo Scientific) coupled to HPLC UltiMate 3000
(Dionex). Solutions were prepared by dissolving 1~mg compound into 1~mL
of appropriate solvent and then diluted 100 times with methanol.

\subsubsection{High-pressure liquid chromatography separation and
analysis (HPLC)}

\textit{System~A}: HPLC-MS (Hypersil C18 column, 2.6~$\upmu$m, 2.1
$\times$ 50~mm) with H$_2$O 0.1\% FA as eluent A and CH$_3$CN 0.1\% FA
as eluent B (linear gradient from 5 to 100\% of B [5~min] and 100\% of
B [1.5~min]) at a flow rate of 0.5~mL/min. UV detection was achieved at
201, 290, 550, 690, and 750~nm. \textit{System~B}: HPLC (Hypersil C18
column, 5~$\upmu$m, 10 $\times$ 250~mm) with H$_2$O 0.1\% FA as eluent
A and CH$_3$CN 0.1\% FA as eluent B (linear gradient from 20 to 60\% of
B in 40~min) at a flow rate of 3.5~mL/min. UV detection was achieved at
700 and 780~nm.

\subsubsection{Fluorescence measurement and quantum yield}

Fluorescence measurements were performed on Horiba Jobin Yvon Fluorolog
spectrofluorometer (software FluorEssence).

\textbf{Cherenkov luminescence imaging studies: CR detection and CRET
studies} were performed at the CGFL Preclinical Imaging and
Radiotherapy Platform (PIRP). All protocols strictly comply with
standards of radioprotection and regulations of the \textit{French
Nuclear Safety Authority} (ASN). CLI studies were performed using
[${}^{18}$F]-FDG as a source of fluorine-18 radionuclide.
[${}^{18}$F]-FDG was provided by Curium (Cyclopharma), and the sample
activity was measured upon reception on site. Studies were performed as
follows: (a)~Agilent Cary Eclipse spectrofluorometer (sensitivity:
signal-to-noise measurements of Raman band of water 1/700) in quartz
cuvette 1 $\times$ 1 $\times$ 3~cm (1~cm path). (b)~IVIS Lumina III
optical imager using 96-well plate (200~$\upmu$L).

\subsection{Synthesis and radiosynthesis} \label{sec4.2}

[${}^{18}$F]-fluorodeoxyglucose (${}^{18}$F-FDG) was purchased from
Cyclopharma, which produces it on site in Dijon. Its radiosynthesis was
performed on site following a reported method~\cite{5,5b,5c,5d}.
Organic syntheses of subphthalocyanines SUB-Cl, SUB-PH, and SUB-COUM were
previously reported~\cite{6,6b} and optimized as follows.

\subsubsection{Synthesis of
B-chloro{\ubreak}[subphthalocyaninato]boron(III):
$\mathrm{SUB}$-$\mathrm{Cl~1}$} \label{sec4.2.1}

A 1~M solution of BCl$_3$ in hexane (20~mL, 20~mmol) was slowly added
under nitrogen atmosphere to a solution of phthalonitrile (1.06~g,
8.27~mmol) in dry dichlorobenzene (45~mL). Next, hexane was removed on
heating the mixture at 70~{\textdegree}C for 30~min. A condenser was
subsequently appended to the flask, and heating of the reaction mixture
was raised to 180~{\textdegree}C and carried on for 1.5~h. The color of
the mixture that was initially light milky yellow turned to dark purple
on heating. On cooling, a precipitate formed, which was isolated by
filtration. It was subsequently washed with methanol and pentane and then
dried under reduced pressure to obtain \textbf{SUB-Cl 1} (700~mg,
58\%). ${}^1$H NMR (500~MHz, CDCl$_3$, 300~K, 
Figure~S3): $\rmdelta$ (ppm) ${=}$ 7.95 (m,
6H), 8.90 (m, 6H). HR-MS ESI: m/z 431.0966 [M$+$H]$^+$ (calcd for
C$_{24}$H$_{13}$BClN$_6^+$: 431.0978). HP-LC analysis (system~A):
retention time\break 5.83~min.

\inlinefig{fx01}

\subsubsection{Synthesis of
B-(4-nitrophenoxy){\ubreak}[subphthalocyaninato]boron(III):
$\mathrm{SUB}$-$\mathrm{PH~2}$} \label{sec4.2.2}

To a solution of \textbf{SUB-Cl 1} (50~mg, 0.12~mmol) in toluene (3~mL)
was added 4-nitrophenol (81~mg, 0.58~mmol). The reaction mixture was
heated under reflux conditions for 3 days and monitored by LCMS. The
solvent was removed under reduced pressure. Then the crude product was
subjected to a short alumina gel column chromatography (eluent:
dichloromethane) to remove the excess of unreacted phenol, yielding
37~mg (60\%) of pure \textbf{SUB-PH 2}. ${}^{1}$H NMR (300~MHz,
CDCl$_3$, 300~K, Figure S4): $\rmdelta$ (ppm) ${=}$ 5.37 (d, ${}^3J = 9.2$~Hz, 2H),
7.64 (d, ${}^3J = 9.2$~Hz, 2H), 7.89 (m, 6H), 8.84 (m, 6H).
${}^{13}$C NMR (75~MHz, CDCl$_3$, 300~K): $\rmdelta$ (ppm) ${=}$ 118.3,
122.1, 125.0, 129.9, 130.7, 141.3, 151.2, 158.5. ${}^{11}$B NMR
(96~MHz, CDCl$_3$, 300~K): $\rmdelta$ (ppm) ${=}$\ ${-}$14.93 (s, 1B).
UV--vis (THF), $\lambda_{\mathrm{max}}$ (nm) 
\mbox{($\varepsilon \times
10^3~\text{L}{\cdot}\text{mol}^{-1}{\cdot}\text{cm}^{-1}$)} ${=}$ 304
(56.7), 562 (86.0). HR-MS ESI: $\text{m/z} = 533.1414$ [M-e]$^+$ (calcd
for C$_{30}$H$_{16}$BN$_{7}$O$_3^+$: 533.1402). $\rmPhi_{\mathrm{F}}$
(THF) ${=}$ 0.17 (reference: rhodamine 6G in solution in ethanol).

\inlinefig{fx02}

\subsubsection{Synthesis of
B-(coumarinoxy){\ubreak}[subphthalocyaninato]boron(III):{\hfill\break}
$\mathrm{SUB}$-$\mathrm{COUM~3}$} \label{sec4.2.3}

To a solution of \textbf{SUB-Cl 1} (30~mg, 0.07~mmol) in toluene
(4~mL) was added 7-hydroxycoumarine (57~mg, 0.35~mmol). The reaction
mixture was heated under reflux conditions for 2 days and monitored
by LCMS. Thereafter, the solvent was removed under reduced pressure and
the crude product was subjected to alumina gel column chromatography
using dichloromethane as the eluent to yield 25~mg (64\%) of pure
\textbf{SUB-COUM 3} as a purple solid. ${}^{1}$H NMR (500~MHz,
CDCl$_3$, 300~K, Figure S5):
$\rmdelta$ (ppm)  ${=}$  5.27 (d, $J = 2.2$~Hz, 1H), 5.33 (dd,
$J = 8.5$, 2.3~Hz, 1H), 6.08 (d, $J = 9.4$~Hz, 1H), 6.83 (d, $J =
8.4$~Hz, 1H), 7.36 (d, $J = 9.4$~Hz, 1H), 7.93 (m, 6H), 8.87 (m, 6H).
${}^{13}$C NMR (125~MHz, CDCl$_3$, 300~K): $\rmdelta$ (ppm)  ${=}$  161.25,
156.62, 155.03, 151.61, 143.30, 131.07, 130.22, 128.29, 122.48, 116.31,
113.35, 113.11, 106.41, 77.41, 77.16, 76.91. HR-MS ESI: m/z 557.1527
[M$+$H]$^+$ (calcd for 
$\mathrm{C}_{33}\mathrm{H}_{18}\mathrm{BN}_6\mathrm{O}_3^+$: 557.1528). HP-LC
analysis (system~A): retention time 5.68~min, 100\% MeCN, 0.1\% TFA.
$\rmPhi_{\mathrm{F}}$ (THF) ${=}$ 0.19 (reference: rhodamine 6G in solution
in ethanol).

\inlinefig{fx03}

\subsection{Luminescence studies} \label{sec4.3}
\subsubsection{Fluorescence} \label{sec4.3.1}
\paragraph*{Fluorescence measurement and quantum yield}

Fluorescence measurements were performed on the Horiba Jobin Yvon Fluorolog
spectrofluorometer (software FluorEssence) at 25~{\textdegree}C or
37~{\textdegree}C (using a temperature control system combined with
water circulation), with standard fluorometer cells (Labbox, LB~Q,
light path: 10~mm, width: 10~mm, chamber volume: 3.5~mL). Fluorescence
quantum yields were calculated using rhodamine 6G in ethanol
($\rmPhi_{\mathrm{F}} = 0.96$), Nile blue in ethanol ($\rmPhi_{\mathrm{F}}
= 0.27$), and indocyanine green in DMSO ($\rmPhi_{\mathrm{F}} = 0.11$) as
reference. Excitation was performed at 488~nm (rhodamine 6G), 610~nm
(Nile blue), or 725~nm (indocyanine green) for both the sample and the 
reference. 
Emission spectra were recorded upon excitation (at a given wavelength) 
at absorbance intensity between 0.02 and 0.09 (a.u.). Fluorescence
quantum yields ($\rmPhi_{\mathrm{F}}$) were determined by the comparison
method, using the following equation:
{\[
\rmPhi_{\mathrm{F}} = \rmPhi_{\mathrm{F}}(\text{Std})\times
\left(\frac{\eta}{\eta(\text{Std})}\right)^{2}\times
\left(\frac{1-10^{-\mathrm{Abs}}}{1-10^{-\mathrm{Abs(Std)}}}\right)
\times \left(\frac{A(\text{Std})}{A}\right)
\]}\unskip
Here, Std corresponds to standard; $\rmPhi_{\mathrm{F}}$ and
$\rmPhi_{\mathrm{F}}$(Std), fluorescence quantum yields; $\eta$ and
$\eta$(Std), RIs of solvent; $A$ and $A$(Std), areas
under the fluorescence curves; Abs and Abs(Std), absorbances at
excitation wavelength (488~nm).

\subsubsection{Cherenkov luminescence imaging studies:{\hfill\break} CR
detection and CRET studies} \label{sec4.3.2}

(a)~On the Agilent Cary Eclipse spectrofluorometer (measurement
parameters: bioluminescence mode, gate time of 10~s, 20~nm
emission slit, 3~nm of data interval, Savitzky smoothing factor~5):
(i)~CR detection: a solution of radioactive species ([${}^{18}$F]-FDG
or [${}^{90}$Y]-YCl$_3$) was mixed with saline solution (0.9\% NaCl) to
attain a total volume of 1~mL. (ii)~CRET studies (radiofluorescence
measurements): 400~$\upmu$L of fluorophore ($10^{-3}$~M in a water
miscible solvent) was mixed with a solution of radioactive species
([${}^{18}$F]-FDG or [${}^{90}$Y]-YCl$_3$), the volume of which was
varied because it depends on the desired level of radioactivity to
introduce in the cuvette. Subsequent addition of saline solution (0.9\%
NaCl) was made to attain an overall volume of 1~mL. (b)~On the IVIS
Lumina II optical imager (measurement parameters: bioluminescence mode,
on the open filter mode or with filters, with an acquisition time from
1~s to 1~min). CR detection and CRET studies: The experiment was performed in
triplicate on addition of the following solution in each
subphthalocyanine-containing well: 10~$\upmu$L of the subphthalocyanine
stock solution (1~mM in DMSO), 32~$\upmu$L [${}^{18}$F]-FDG
corresponding to 10~MBq (in solution in physiological serum, the volume
of which was varied because it depends on the desired level of
radioactivity to introduce in wells), and 58~$\upmu$L of DMSO to
complete the final volume up to 100~$\upmu$L. To control wells were added
10~MBq [${}^{18}$F]-FDG and 68~$\upmu$L DMSO.

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Funding}

CNRS ``Chaire d'Excellence'' (RD, 2010--2015) funds allowed to initiate
the Cherenkov Luminescence Imaging (CLI) program. The following are
also acknowledged: Burgundy/Burgundy Franche-Comt\'e (BFC) Regional
Councils (FABER grant (spectrofluorimeter), \textit{3MIM} grant
(optical imager), PARI Dual Regional/Universit\'e de Bourgogne
Innovation Grant (through ``Pharmacoimagerie et Agents
Th\'eranostiques'' project), French Ministry of Higher Education,
Research and Innovation for a scholarship (VL)), both ANR-EQPX-IMAPPI
and European Union (PO-FEDER-FSE Bourgogne) for their contribution in
purchasing radionuclides, Cancerop\^ole Est and SATT-SAYENS for
subsequent funding in subsequent development of the Cherenkov
Luminescence Imaging (CLI) and Cherenkov Photodynamic Therapy (CLI-PDT)
research programs.

{\vspace*{-2pt}}

\section*{Acknowledgments}

{\vspace*{-2pt}}

The following are acknowledged: PACSMUB platform for access to NMR and
Mass spectrometers, Preclinical (PIRP) Platform at CGFL for leaving
access to their labs.

{\vspace*{-2pt}}

\section*{Supplementary data}

{\vspace*{-2pt}}

Supporting information for this article is available on the journal's
website under \printDOI\ or from the author.   

\CDRsupplementaryTwotypes{supplementary-material}{\cdrattach{crchim-335-suppl.pdf}}

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