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\DOI{10.5802/crchim.339}
\datereceived{2024-05-17}
\daterevised{2024-07-09}
\dateaccepted{2024-09-06}
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\dateposted{2025-04-16}
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\CDRsetmeta{articletype}{review}

\title{Inorganic chemical biology and metal complexes in cells: from the 
design of cellular models to evaluate antioxidant 
activity to the characterization of metal complexes in cells}

\alttitle{Ch\'{e}mobiologie inorganique et complexes m\'{e}talliques en
contexte cellulaire : de la conception de mod\`{e}les cellulaires pour
\'{e}valuer l'activit\'{e} antioxydante \`{a} la caract\'{e}risation de
complexes m\'{e}talliques dans les cellules}

\author{\firstname{Clotilde} \lastname{Policar}\CDRorcid{0000-0003-0255-1650}\IsCorresp}
\address{Laboratoire Chimie physique et chimie du vivant, CPCV,
UMR8228, D\'epartement de chimie de l'ENS-PSL, \'Ecole normale sup\'erieure,
PSL University, Sorbonne Universit\'e, CNRS, 75005 Paris, France}
\email[C. Policar]{clotilde.policar@ens.psl.eu}

\author{\firstname{Christine} \lastname{Rampon}\CDRorcid{0000-0002-1444-3166}}
\addressSameAs{1}{Laboratoire Chimie physique et chimie du vivant, CPCV,
UMR8228, D\'epartement de chimie de l'ENS-PSL, \'Ecole normale sup\'erieure,
PSL University, Sorbonne Universit\'e, CNRS, 75005 Paris, France}

\author{\firstname{Alice} \lastname{Balfourier}\CDRorcid{0000-0002-4801-1388}}
\addressSameAs{1}{Laboratoire Chimie physique et chimie du vivant, CPCV,
UMR8228, D\'epartement de chimie de l'ENS-PSL, \'Ecole normale sup\'erieure,
PSL University, Sorbonne Universit\'e, CNRS, 75005 Paris, France}

\author{\firstname{Michel} \lastname{Volovitch}\CDRorcid{0000-0002-7488-764X}}
\addressSameAs{1}{Laboratoire Chimie physique et chimie du vivant, CPCV,
UMR8228, D\'epartement de chimie de l'ENS-PSL, \'Ecole normale sup\'erieure,
PSL University, Sorbonne Universit\'e, CNRS, 75005 Paris, France}

\author{\firstname{Sophie} \lastname{Vriz}\CDRorcid{0000-0003-2029-5750}}
\addressSameAs{1}{Laboratoire Chimie physique et chimie du vivant, CPCV,
UMR8228, D\'epartement de chimie de l'ENS-PSL, \'Ecole normale sup\'erieure,
PSL University, Sorbonne Universit\'e, CNRS, 75005 Paris, France}

\author{\firstname{H\'{e}l\`{e}ne Charlotte} \lastname{Bertrand}\CDRorcid{0000-0002-3841-022X}}
\addressSameAs{1}{Laboratoire Chimie physique et chimie du vivant, CPCV,
UMR8228, D\'epartement de chimie de l'ENS-PSL, \'Ecole normale sup\'erieure,
PSL University, Sorbonne Universit\'e, CNRS, 75005 Paris, France}

\author{\firstname{Nicolas} \lastname{Delsuc}\CDRorcid{0000-0001-5570-8311}}
\addressSameAs{1}{Laboratoire Chimie physique et chimie du vivant, CPCV,
UMR8228, D\'epartement de chimie de l'ENS-PSL, \'Ecole normale sup\'erieure,
PSL University, Sorbonne Universit\'e, CNRS, 75005 Paris, France}

\thanks{ENS-PSL, PSL University, CNRS, MITI-CNRS, Sorbonne University
(SU), ANR-15-CE07-0027 MAGIC; ANR-16-CE18-0017-01 SATIN;
ANR-16-CE07-0025 Metallopepzyme; DEI20151234413 (Fondation pour la
recherche m\'{e}dicale 2016), BACTMAN and ANACOMDA (Mission pour les
initiatives transverses et interdisciplinaires MITI-CNRS), Association
Fran\c{c}ois Aupetit (AFA, research fellowship 2015), ANR 21-CE18-0053-02
MOBIDIC, ANR 20-CE07-0039-01-CATMAN, ANR 23-CE23 METALINFY; Idex PSL
Qlife project Main ANR Q-life ANR-17-CONV-0005; CEFIPRA project no.
6505-1; EMERGENCE SU EMRG-24 TOTEM; ANR-22-PEBI-0003 (PEPR)}

\thanks{\textbf{Note}. Invited review on the occasion of the award to
CP of the \textit{Prix de la F\'{e}d\'{e}ration Gay-Lussac 2022} of the
French \textit{Acad\'{e}mie des sciences}}

\keywords{\kwd{Inorganic chemical biology}
\kwd{Oxidative distress}
\kwd{Integrity of complexes in cells}
\kwd{Speciation}
\kwd{Superoxide dismutase mimetics or mimics}
\kwd{Catalase mimics or mimetics}}

\altkeywords{\kwd{Ch\'{e}mobiologie inorganique} \kwd{Stress
oxydant}\kwd{Int\'{e}grit\'{e} des complexes en environnement
cellulaire}\kwd{Sp\'{e}ciation}\kwd{Mimes de superoxyde
dismutase}\kwd{Mimes de catalase}} 

\begin{abstract}
Metal complexes are increasingly used as metallo-probes or
metallo-drugs. Their characterization in cellular or biological
environments poses specific challenges related to possible
decoordination, metal exchange, fixation of additional ligands, or
precipitation. These phenomena can hardly be foreseen and it is
important to integrate the design of these complexes with their study
in cells, from characterization in cells (quantification, speciation,
integrity of the complex in cells, location) to bioactivity. Redox
homeostasis and oxidative stress are strongly associated with
biochemistry of metal ions. Metalloenzymes protecting against oxidative
stress have evolved such as catalase or superoxide dismutase. Bioactive
antioxidant metal complexes can be designed as catalysts bioinspired
from these natural antioxidant enzymes protecting the cell against
oxidative stress. To characterize their activity, it is important to
develop dedicated cellular models to evaluate their ability to restore
normal cell life from a situation compromised by oxidative stress. This
article describes cellular models and assays that have been developed
in that context as well as approaches to interrogate the complexes'
nature in cells.\looseness=-1
\end{abstract}

\begin{altabstract}
Les complexes m\'{e}talliques sont de plus en plus utilis\'{e}s comme
m\'{e}tallo-sondes ou m\'{e}tallo-m\'{e}dicaments. Leur
caract\'{e}risation dans un environnement cellulaire ou biologique pose
des probl\`{e}mes sp\'{e}cifiques li\'{e}s aux possibles
d\'{e}coordination, \'{e}change de m\'{e}taux, fixation de ligands
suppl\'{e}mentaires ou pr\'{e}cipitation. Ces ph\'{e}nom\`{e}nes sont
difficilement pr\'{e}visibles et il est donc important d'int\'{e}grer
la conception de tels complexes \`{a} leur \'{e}tude dans les cellules,
\`{a} partir de la caract\'{e}risation dans les cellules
(quantification, sp\'{e}ciation, int\'{e}grit\'{e} du complexe dans les
cellules, localisation) \`{a} la bio-activit\'{e}. L'hom\'{e}ostasie
redox et le stress oxydatif sont fortement associ\'{e}s \`{a} la
biochimie des ions m\'{e}talliques. Des m\'{e}talloenzymes
prot\'{e}geant contre le stress oxydatif ont \'{e}t\'{e}
d\'{e}velopp\'{e}es, telles que la catalase ou la superoxyde dismutase.
Les complexes m\'{e}talliques antioxydants bioactifs peuvent \^{e}tre
con\c{c}us comme des catalyseurs bio-inspir\'{e}s de ces enzymes
antioxydantes naturelles prot\'{e}geant la cellule contre le stress
oxydatif. Pour caract\'{e}riser leur activit\'{e}, il est important de
d\'{e}velopper des mod\`{e}les cellulaires d\'{e}di\'{e}s afin
d'\'{e}valuer leur capacit\'{e} \`{a} restaurer une vie cellulaire
normale \`{a} partir d'une situation o\`{u} le stress oxydatif l'a
compromise. Cet article d\'{e}crit les mod\`{e}les cellulaires et les
essais qui ont \'{e}t\'{e} d\'{e}velopp\'{e}s dans ce contexte, ainsi
que les approches permettant d'interroger la nature de ces complexes
dans les cellules. Nous y discutons aussi comment jouer sur l'inertie
pour am\'{e}liorer la bioactivit\'{e}.
\end{altabstract}

\shortrunauthors

\maketitle
{\vspace*{3pc}}

\twocolumngrid

\end{noXML}

\section{Introduction} 

Metal complexes have long been used as therapeutic agents. In the
modern era, arsphenamine, toxic to syphilis-causing \textit{Treponema
pallidum}, or antimony compounds, toxic to leishmania, have been
successfully developed as metal-based drugs in the early
20{th} century. However, most modern medicines are
organic molecules. There has been a revival of inorganic medicinal
chemistry with the discovery that \textit{cis}-Pt can be toxic to cells
with high division rate, with application as anticancer agent. Most of
these compounds are meant to be used in living organisms and exert
their activity at a cellular level. Because cells are compartmented,
bioactivity can vary depending on the subcellular distribution and on
the organelle reached. Cells are a very particular environment, with
high viscosity, molecular overcrowding impacting diffusion~\cite{bib1},
and abounding with metal cations and Lewis bases, which are strong
competitors for metal cation coordination. This is why the use of metal
complexes raises new specific challenges associated with possible
decoordination, metal exchange, etc., that participate to their
metabolization, with the possibility of precipitation when the metal
cation is released. Predicting these exchanges is not easy due to the
complexity of biological media. Exploring the integrity of a complex in
cells is key to understanding its activity. Thus, it is important to
integrate the design of metal complexes with their study in a cellular
context, an approach for which the terms \textit{cellular inorganic
chemistry} or \textit{inorganic chemical biology} can be coined~\cite{bib2}.

Most of the metal complexes studied in the literature for their
bioactivity are used as anticancer, antibacterial or antifungal agents,
with requirements of selectivity---for cancerous cells,
bacteria or fungi---versus normal healthy cells of the host.
Hitherto, most bioanalyses have focused on evaluating the toxicity
towards cancer cells, microorganisms, parasites, or fungi, which should
be selective relative to vertebrate noncancerous cells. There is
another class of metal complexes meant to restore the normal cellular
life or rescue cells from a pathological or intoxication situation.
This is the case for instance of functional mimics of rhodanese, an
enzyme that detoxifies cyanide by catalyzing its conversion to
thiocyanate~\cite{bib3,bib4}. Other catalytic metal-based drugs~\cite{bib5} can be developed to protect the cell from oxidative
damages, as catalytic antioxidants mimicking protective enzymes such as
superoxide dismutase (SOD) or catalase (CAT), called SOD mimics and CAT
mimics~\cite{bib6,bib7,bib8,bib9}. Like the corresponding enzymes,
these complexes are meant to catalytically react with reactive oxygen
species (ROS), and do not act as inhibitors of ROS production. They
must be nontoxic redox catalysts and they are meant to restore the
basal activity in cells under oxidative distress. Their
characterization therefore requires the development of specific
non-routine strategies to evaluate their bioactivity, which is to
restore normal cell life from a situation where it has been 
compromised~\cite{bib2,bib10,bib11,bib12}. Note also that, as activation of
paramagnetic dioxygen ($\mathrm{S}=1$) can be performed by paramagnetic species,
there is a strong intertwining between biochemistry of metal ions and
metal complexes and that of dioxygen and ROS. This is why, in parallel
with the development of SOD and CAT mimics, we have
developed a particular interest in cellular models related to
pathologies of oxidative stress that will be described in the
following.

\section{Oxidative stress, distress and eustress}

Dioxygen (O$_{2}$) is necessary for aerobic life. Its ground
state is $\mathrm{S}=1$ with two unpaired electrons in the highest occupied
molecular orbital (HOMO). This diradical ground state nature (triplet
state $\mbox{}^{3}$O$_{2}$) explains its slow reaction
with organic molecules (mostly $\mathrm{S}=0$)~\cite{bib13}. O$_{2}$
sequential monoelectronic reductions lead to superoxide
(${\mathrm{O}_{2}}^{{\bullet}-}$), hydrogen peroxide (H$_{2}$O$_{2}$) or
hydroxyl radical (HO$^{{\bullet}}$), and then H$_{2}$O (see~Figure~\ref{fig1}). The partially reduced
species---${\mathrm{O}_{2}}^{{\bullet}-}$,  H$_{2}$O$_{2}$ and
HO$^{{\bullet}}$---are much more reactive than dioxygen itself, with no
spin-state kinetic barrier for the reaction with singlet molecules, and
hence they are called \textit{reactive oxygen species} (ROS). They are
continuously produced in biological systems by endogenous mechanisms,
and their concentration is maintained to low enough levels by
protective pathways such as superoxide dismutase or catalase, proteins
carved by evolution that catalyze respectively (7) and
(8)\footnote{There has been so far no protective system identified for
HO$^{\bullet}$: this radical reacts with a rate only limited by
diffusion, meaning that the first encounter with a molecule leads to an
efficient reaction (H-abstraction notably) and there is no possibility
for this radical to diffuse to a protective system.}.

\begin{figure*}
{\vspace*{2pt}}
\includegraphics{fig01}
{\vspace*{2pt}}
\caption{\label{fig1}Oxidation of water (1) and reduction of dioxygen
(2). Reduction cascade of O$_{2}$ leading to reactive oxygen species
ROS (3) to (6). Dismutation of ${\mathrm{O}_{2}}^{{\bullet}-}$ (7) and
H$_{2}$O$_{2}$ (8). Note that the superoxide radical anion can be noted
${\mathrm{O}_{2}}^{{\bullet}-}$ or $\mathrm{O}_{2}^{-}$, the mention of the
radical is not mandatory, as it is not in O$_{2}$ that could be noted
$\mathrm{O}_{2}^{\bullet\bullet}$ if we want to emphasize its diradical nature.}
{\vspace*{2pt}}
\end{figure*}



In the last two decades, it has become clear that cellular responses to
redox imbalance are more complex than previously assumed. On the one
hand, cells facing redox imbalance often react with an overshoot
effect, as first evidenced in the case of reoxygenation after 
hypoxia~\cite{bib15}. On the other hand, it turned out that moderate oxidative
imbalance (essentially due to spatially and temporally controlled
H$_{2}$O$_{2}$ production) is used at different scales by cells and
tissues as a signaling device to perform physiological tasks. Such
signaling by H$_{2}$O$_{2}$ was, for instance, observed to be necessary
for cytoskeleton rearrangement needed during migration~\cite{bib16,bib17}, leukocyte navigation towards a wound~\cite{bib18},
neural stem cell maintenance and neurogenesis~\cite{bib19,bib20}, or
tissue regeneration in fish or amphibian~\cite{bib21,bib22}. Among ROS,
H$_{2}$O$_{2}$ is clearly suited to this signaling function due to its
comparatively long half-life and thus diffusion properties, moderate
reactivity, and membrane crossing ability via a subfamily of
aquaporins. That is why H$_{2}$O$_{2}$ has been particularly studied as
signaling molecule. To study its spatial distribution in real time, new
genetically-encoded sensors have been developed (reviewed 
in~\cite{bib23}, see also part 2.3 for the HyPer probe)~\cite{bib24,bib25}. These improved sensors allow analyzing the dynamics
of redox balance in live organisms (reviewed in~\cite{bib26}). More
recently they were used to revisit H$_{2}$O$_{2}$ dynamics and roles in
neuronal differentiation~\cite{bib27}, brain patterning~\cite{bib28},
neuroinflammation~\cite{bib29}, Shh signaling in the developing floor
plate~\cite{bib30}, appendage regeneration~\cite{bib31}, and in
hypoxia/reoxygenation~\cite{bib32}. Linking these findings with the
observation that activation of several growth factors was accompanied
by H$_{2}$O$_{2}$ production, H. Sies proposed the new concept of
``oxidative eustress''~\cite{bib33,bib34,bib35}. According to this
concept, H$_{2}$O$_{2}$ is a central redox signaling molecule in
physiological contexts, transient and localized bursts of low levels of
H$_{2}$O$_{2}$ (in the 1--10~nM range) being required for
fundamental developmental and homeostatic processes from intracellular
decisions to intercellular communication and organism patterning. In
contrast, ``oxidative distress'' corresponding to supraphysiological
levels of H$_{2}$O$_{2}$ (above 100~nM) definitely leads to various
damages, with an intermediate range of concentration (from 10 to 100
nM) leading to adaptive stress responses. 

\section{A range of biological models to evaluate antioxidant activity}

Metal complexes are of paramount importance for redox reactions, and
they occupy a prominent position in the growing research field to fight
oxidative stress. Metal complexes can be designed as antioxidants
bioinspired by natural cellular antioxidants, such as SOD or CAT,
leading to SOD mimics or CAT mimics able to catalyze the dismutation of
${\mathrm{O}_{2}}^{{\bullet}-}$ and H$_{2}$O$_{2}$, respectively. Their
chemical design has been discussed in the literature by us and 
others~\cite{bib6,bib7,bib8,bib9,bib36,bib37,bib38,bib39} and we will not
focus on that aspect in the present article.

The evaluation of these mimics goes first through the characterization
of their reactivity with either ${\mathrm{O}_{2}}^{{\bullet}-}$ or
H$_{2}$O$_{2}$ out of any cellular context, which we call
\textit{intrinsic activities}; this term refers therein to the reaction
kinetics with ${\mathrm{O}_{2}}^{{\bullet}-}$ or H$_{2}$O$_{2}$, possibly
the catalytic constant in case of a true catalytic SOD/CAT mimic. But
what will be mainly discussed in this section is the evaluation of the
\textit{bioactivities in cells}.

Many biological assays have been developed to characterize antioxidant
activity of SOD and CAT mimics, ranging from fluorescent probes to
SOD-deficient organisms~\cite{bib12,bib40,bib41}. We have been
interested in the study of metal-based antioxidants in the context of
inflammatory bowel diseases (IBDs). In that context, we have been
developing three cellular models to evaluate the bioactivities of
metal-based catalytic antioxidants, SOD or CAT mimics:

(a) activated macrophages~\cite{bib11,bib42}.

(b) intestinal epithelial cells made highly sensitive to bacterial
lipopolysaccharide (LPS, HT29-MD2 cells); the strong inflammation
induced by LPS, presumably mediated by oxidative stress, is mitigated
in the presence of SOD or CAT mimics~\cite{bib2,bib10,bib11,bib43}.

(c) HyPer-HeLa cells genetically engineered to express a
H$_{2}$O$_{2}$-sensing protein~\cite{bib11}; in these models we have
characterized the bioactivity of a range of Mn-complexes described as
SOD mimics in the literature~\cite{bib11}.

\subsection{Evaluation of the effect of antioxidants on the superoxide
(${\mathrm{O}_{2}}^{{\bullet}-}$) flow generated by macrophages}

Macrophages are immune cells meant to produce ROS against invading
bacteria. High ROS flow can be induced upon stimulation with bacterial
lipopolysaccharide (LPS), interferon ${\upgamma}$ (IFN-${\upgamma}$)
and phorbol 12-myristate 13-acetate (PMA) (typically 24h, slow
activation of the inducible NO synthase (iNOS) by LPS and
IFN-${\upgamma}$ followed by PMA, for the fast activation of the NADPH
oxidase (NOX) (1h))~\cite{bib42,bib44,bib45}. Quantification of
${\mathrm{O}_{2}}^{{\bullet}-}$ concentration is made possible by using
ferricytochrome \textit{c} as a selective ${\mathrm{O}_{2}}^{{\bullet}-}$
UV--vis marker. As cytochrome \textit{c} is not able to penetrate
cells, only the ${\mathrm{O}_{2}}^{{\bullet}-}$ released extracellularly
is measured~\cite{bib42,bib46}. Murine macrophages RAW 264.7 are first
activated by LPS and IFN-${\upgamma}$ for 24 hours. Cells are then
incubated with PMA, ferricytochrome \textit{c} and a chosen
concentration of antioxidant that is nontoxic for the cells (typically
estimated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay) in an homemade noncolored medium~\cite{bib11}. The
absorbance of the supernatant at 550~nm, where there is a band for
ferrocytochrome \textit{c}, gives an estimate of the amount of
${\mathrm{O}_{2}}^{{\bullet}-}$ in the extracellular medium (typically
after 1h of incubation with ferricytochrome \textit{c}) (see Figure~\ref{fig2}). This assay was shown to provide a positive result with
antioxidants not able to enter cells~\cite{bib11,bib42}. This is the
case of the commercially available CuZnSOD, an efficient catalyst of
the dismutation of ${\mathrm{O}_{2}}^{{\bullet}-}$ into dioxygen and
H$_{2}$O$_{2}$ with a kinetics close to diffusion limit. The CuZnSOD
overall negative charge at physiological pH and its large size prevent
its cell penetration when incubated in the extracellular medium. For
that reason, we have labeled this experiment ``extracellular activity
assay''~\cite{bib11,bib42}.

\begin{figure}
{\vspace*{2pt}}
\includegraphics{fig02}
{\vspace*{2pt}}
\caption{\label{fig2}Visible spectrum (wavelength in nm) of ferri- and
ferrocytochrome \textit{c} showing the peak at 550~nm used for quantification.}
\end{figure}


The activity seen in this assay could be due either to (a) an
inhibition of the ${\mathrm{O}_{2}}^{{\bullet}-}$ production or (b) to
the quenching of the superoxide produced (through a SOD-like activity).
We have shown that the SOD mimic \textbf{Mn1} (see Figure~\ref{fig5})
was not an inhibitor of ${\mathrm{O}_{2}}^{{\bullet}-}$ production using
an electrochemical quantification of ROS (H$_{2}$O$_{2}$,
ONOO$^{-}$, NO, $\mathrm{NO}{_{2}}^{-}$) with a
possible back-calculation of the ${\mathrm{O}_{2}}^{{\bullet}-}$
produced, which was similar with and without the \textbf{Mn1}
incubation~\cite{bib42}. These results show that the reduction of the
${\mathrm{O}_{2}}^{{\bullet}-}$ flow is not due to an inhibition of
${\mathrm{O}_{2}}^{{\bullet}-}$ \mbox{production,} but to a direct reaction of
\textbf{Mn1} with ${\mathrm{O}_{2}}^{{\bullet}-}$. Note that the results
in electrochemistry and by ferricytochrome \textit{c} quantification
were complementary, as the electrochemistry provides information on the
overall ${\mathrm{O}_{2}}^{{\bullet}-}$ production, not modified by the
SOD mimic \textbf{Mn1}, and cytochrome \textit{c} assay gives the
extracellular flow of ${\mathrm{O}_{2}}^{{\bullet}-}$ over a one-hour
period, efficiently reduced by the presence of a SOD\break
mimic~\cite{bib42}.




\subsection{Epithelial intestinal cells as cellular models to estimate
intracellular antioxidant and anti-inflammatory effects}

Oxidative stress is known to play a key role in triggering
inflammation~\cite{bib47} and inflammatory bowel diseases~\cite{bib48}.
Intestinal epithelial cells (IECs) have adapted to the presence of
commensal bacteria with a limited response to bacterial
lipopolysaccharide (LPS) of its regular receptor (TLR4). Low or absent
expression of the myeloid differentiation 2 (MD2) receptor, LPS
co-receptor to TLR4, underlies LPS tolerance in IECs, and MD2 blood
circulating levels have been shown to be associated with LPS
responsiveness~\cite{bib49,bib50}. The HT29 intestinal epithelial cell
line has been stably transformed to overexpress the MD2 protein in the
extracellular environment. The HT29-MD2 cell line thus exhibits a high
LPS-responsiveness, increased more than 100-fold compared to the HT29
cell line~\cite{bib50,bib51}. The release of interleukin 8 (IL8), but
also the expression of cyclooxygenase 2 (COX2) and the related
secretion of prostaglandin E2 (PGE2), were \mbox{coordinately} stimulated by
LPS in HT29 cells expressing MD2. We have also shown that LPS
stimulation for six hours induces overexpression of an active
mitochondrial MnSOD, but not of the cytosolic CuZnSOD nor
CAT~\cite{bib2,bib10,bib11,bib52}. This is in line with LPS
challenge--induced ROS production~\cite{bib53} at the mitochondria and
clearly incriminates ${\mathrm{O}_{2}}^{{\bullet}-}$. The
overexpression of MnSOD has been interpreted as a result of cellular
feedback to mitochondrial ${\mathrm{O}_{2}}^{{\bullet}-}$ increase
induced by LPS. In this cellular model, metal-based antioxidants
displaying a SOD-like activity have shown anti-inflammatory activity,
as measured by IL8 secretion and COX2 expression after six-hour
incubation with LPS~\cite{bib2,bib10,bib11,bib52}. The expression of
the MnSOD was reduced when LPS was co-incubated with a SOD
mimic~\cite{bib2,bib10,bib11,bib43,bib52,bib54}, showing that competent
SOD mimics are able to complement
for~\mbox{MnSOD.}

A typical result with \textbf{Mn1} is shown in Figure~\ref{fig3}. The
anti-inflammatory activity of \textbf{Mn1} has been compared to the
5-aminosalicyclic acid (\textbf{5-ASA}, see Figure~\ref{fig5}), an
organic anti-inflammatory agent used as the first therapeutic line in
IBD~\cite{bib2,bib55}. As shown on {Figure~\ref{fig3}}, \textbf{5-ASA}
is less efficient against inflammation than \textbf{Mn1}. A comparison
of its antioxidant activity with other antioxidants, ranging from Mn
porphyrins to organic nitroxide is discussed below ({Table~\ref{tab1}},
\mbox{Section~{\ref{sec2.4}}} and Figure~\ref{fig7}), showing that
\textbf{Mn1} is among the best cell-efficient antioxidants.


\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}Anti-inflammatory and antioxidant activity of SOD mimic \textbf{Mn1}
and comparison with 5-ASA (see Figure~\ref{fig5} for the structure of
\textbf{Mn1} and
5-ASA).
{Left:} Anti-inflammatory activity in intestinal epithelial
cells activated with LPS: comparison with 5-ASA (see
Figure~\ref{fig5}).  IL8 secretion measured by ELISA in supernatants
of HT29-MD2 incubated 7 h under several conditions: C: control; LPS
(0.1~${\upmu}$g/mL) was added after 1 h of incubation with \textbf{Mn1}
(100~${{\upmu}}$M), \textbf{5 ASA}: 100~${{\upmu}}$M, 1 mM and 10 mM.
Data represent means ${\pm}$ SD for 2 replicates, $^{**} p&lt;0.01$ and
$^{*} p &lt; 0.05$ versus LPS.
{Right:} Antioxidant activity, measured indirectly by
overexpression of MnSOD and CuSOD in comparison with actin.
Representative experiment in Western blot (WB) after a 7~h incubation
under several conditions, LPS (0.1~${\upmu}$g${\cdot}$mL$^{-1}$) added after 1 h and incubated
for 6 h: C for control; LPS; LPS ${+}$ \textbf{Mn1} (100~${\upmu}$M); LPS ${+}$
MnCl$_{2}$ (100~${\upmu}$M). Adapted with permission from~\cite{bib2}
Copyright 2017 American Chemical Society.
These results are representative of typical experiments, performed many
times by different experimentalists in slightly different{\unskip\break}
conditions~\cite{bib2,bib10,bib11,bib52,bib54}.}
\end{figure*}

\subsection{{HeLa HyPer cells}}

The detection of ROS using fluorescence probes is one of the most
straightforward strategies to correlate the bioactivities of
antioxidants with their intrinsic catalytic activities. However, for
more reactive ROS such as the hydroxyl radical
($^{\bullet}$OH) and ${\mathrm{O}_{2}}^{{\bullet}-}$,
commercially available selective probes are scarce. Very interestingly,
for H$_{2}$O$_{2}$ detection, a genetically encoded ratiometric sensor
called HyPer has been developed, in HeLa cells among
others~\cite{bib56}. HyPer is a circularly permuted yellow fluorescent
protein~(cpYFP) emitting at 530~ nm, integrated into the regulatory
domain of
the bacterial H$_{2}$O$_{2}$-sensing protein OxyR (regulatory domain of
OxyR, OxyR-RD). The oxidation of HyPer thiols by H$_{2}$O$_{2}$ induces
the formation of disulfide bridges that leads to a modification of the
spectral properties of the YFP. More precisely, a ratiometric
modification of the excitation spectrum of HyPer, which possesses two
excitation maxima at 420 and 488~nm, is induced. Thus, by measuring the
normalized ratio of the intensity
$\mathrm{I}_{(405/530)}/\mathrm{I}_{(491/530)}$ by confocal
fluorescence microscopy, it is possible to monitor specifically
H$_{2}$O$_{2}$ concentration in cells (see Figure~\ref{fig4}).
H$_{2}$O$_{2}$, being not as reactive as
${\mathrm{O}_{2}}^{{\bullet}-}$, is able to diffuse across cytoplasmic
membrane, mainly through aquaporines~\cite{bib57,bib58}, and endogenous
H$_{2}$O$_{2}$ diffuses outside cells. Consequently, when these HeLa
HyPer cells are incubated with antioxidants mimicking CAT, the decrease
in fluorescence ratio observed may be composite: it may be due to
dismutation of endogenous H$_{2}$O$_{2}$ inside or outside the cells.
For instance, incubation of HeLa HyPer with the recombinant heme CAT
has been shown to reduce H$_{2}$O$_{2}$ concentration although it does
not cross cells membrane~\cite{bib59}. When antioxidants possessing a
CAT-like activity~\cite{bib11}, including peptidyl Cu complexes
(Figure~\ref{fig5})~\cite{bib25}, were incubated with HeLa HyPer cells,
a clear decrease in HyPer fluorescence has been observed, showing that
they were efficient in this biological context
\cite{bib11,bib25}.

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}H$_{2}$O$_{2}$ levels in HeLa HyPer cells
monitored by fluorescence for 60~min after treatment with H$_{2}$O
(control cells), CATm1 peptide (250~{${\upmu}$}M), CuSO$_{4}$ 
(500~{${\upmu}$}M), and complex CATm1-Cu$^{2+}$ 1:2 (250 {${\upmu}$}M). The
right panel shows the mean ratio for 2 or 3 independent experiments.
Adapted with permission from~\cite{bib25}. Copyright 2021 American
Chemical Society.}
\end{figure*}

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}Chemical structures of the molecules discussed in
this article. ({A}) \textbf{5-ASA}, \textbf{CATm1}, \textbf{Mangafodipir}
and \textbf{Calmangafodipir}. ({B}) Structure of the antioxidants
screened by the three-cell assay.}
\end{figure*}

\subsection{Conclusion on the three cellular models}\label{sec2.4}

We have recently used the above-mentioned extracellular assays to
screen a range of antioxidants metal complexes known as SOD or CAT
mimics (see Figure~\ref{fig5})~\cite{bib11}.


We have compared their intrinsic activities, defined as their catalytic
activities measured out of any cellular context, with their
bioactivities in these assays (see Table~\ref{tab1}).


\begin{figure*}
\includegraphics{fig06}
\bcaption{\label{fig6}Screening of antioxidants with the three-cellular
assay. Adapted with permission from~\cite{bib11}. Copyright 2021
Elsevier. {(A) Evaluation of antioxidants on murine macrophages RAW
264.7.} Relative values in comparison with activated cells arbitrarily
set at 100\%. Macrophages were incubated with IFN-${\upgamma}$
(1~U${\cdot}$mL$^{-1}$) and LPS (1~ng${\cdot}$mL$^{-1}$) for 24 h at
37~{\textdegree}C in DMEM followed by an incubation with
ferricytochrome \textit{c} (100~${\upmu}$M), PMA (800~nM) and
antioxidants (100~${\upmu}$M) at 37~{\textdegree}C for 1 h in a
homemade medium devoid of phenol red (see~\cite{bib11} exp. Part). Data
are mean ${\pm}$ SEM of three independent experiments with
($\mbox{}^{**}$) $p &lt; 0.01$ and ($\mbox{}^{*}$) $p&lt; 0.05$ versus
activated cells. (B) H$_{{2}}${O}$_{{2}}$ {levels measured in HyPer
HeLa cells.}}{5}{ Cells
were incubated for 1 h at 37~{\textdegree}C in HEPES (30~mM pH 7.5)
under 5\% CO$_{2}$ without (control) or with antioxidants at
100~${\upmu}$M. Fluorescence images were recorded after excitation at
405 nm and 491~nm and the ratio of the emission at 530~nm
I(491/530)/I(405/530) was measured for several cells. The ratio was
arbitrarily set to 1 for control cells, i.e., cells incubated with
HEPES only. Data are mean ${\pm}$ SEM of several cells (from 48 to 163
cells) with $^{****}p &lt; 0.0001$, $^{***}p &lt; 0.001$, $^{**}p &lt; 0.01$ and
$^{*}p &lt; 0.05$ versus control cells. ns means non-significant. (C)
Quantification of the inflammatory marker IL8 by ELISA after incubation
of HT29-MD2 cells with the antioxidants at 10 or 100 ${\upmu}$M.
Antioxidants were incubated for 6 h at 37~{\textdegree}C in
supplemented DMEM in presence of LPS (0.1~${\upmu}$g${\cdot}$mL$^{-1}$)
except for control where neither antioxidants nor LPS were added. The
absorbance measured for activated cells was arbitrary set to 100\%.
Data represent mean ${\pm}$ SEM of two independent experiments for
\textbf{MnTnBuOE-2-PyP}$^{\mathbf{5+}}$ and \textbf{M40403} and three
independent experiments for \textbf{Mito-TEMPOL}, \textbf{Mn1},
\textbf{EUK-134}, \textbf{MnTE-2-PyP}$^{\mathbf{5+}}$ and
\textbf{MnTBAP}$^{\mathbf{3-}}$ with $^{****}p&lt; 0.0001$, $^{***}p &lt;
0.001$, $^{**}p&lt; 0.01$ and $^{*}p &lt; 0.05$ versus activated cells.}
{\vspace*{4pt}}
\end{figure*}

\nocite{bib11,bib60,bib54,bib38,bib61,bib62,bib63,bib64,bib65,bib66,bib67,bib68,bib69,bib70,bib71,bib72,bib73,bib74,bib75,bib76,bib77,bib78}

\begin{table*}
\caption{\label{tab1}Intrinsic dismutation kinetic constants and
cellular activity of antioxidants}
%\fontsize{8.5}{10.2}\selectfont\tabcolsep-0.5pt
%\begin{tabular}{lccccccccc}
\begin{tabular}{c}
\tbody
\inlinefig{fx01}
%\thead
% &amp; \multicolumn{2}{c}{SOD activity} &amp; CAT activity &amp;  \morerows{1}{Ref.} &amp;
%\morerows{1}{\parbox[t]{1.8cm}{\centering Macrophages (extracell)}}
%&amp; \multicolumn{2}{c}{HT29-MD2} &amp;
%\morerows{1}{\parbox[t]{1.1cm}{\centering HeLa  HyPer}} &amp;
%\morerows{1}{\parbox[t]{.9cm}{\centering Ref. (cell assay)}}\\
%\cline{2-3}\cline{7-8}
%&amp;
%\parbox[t]{2cm}{\centering $k_{\mathrm{McCF}}$ (10$^{6}$~M$^{-1}{\cdot}\mathrm{s}^{-1}$)} &amp;
%\parbox[t]{2cm}{\centering $k_{\mathrm{cat}}$  (10$^{6}$~M$^{-1}{\cdot}\mathrm{s}^{-1}$)} &amp;
%\parbox[t]{1.6cm}{\centering $k_{\mathrm{cat}} \mathrm{H}_{2}$O$_{2}$ ($\mathrm{M}^{-1}{\cdot}\mathrm{s}^{-1}$)$^{f}$}
%&amp;  &amp;  &amp;
%{10~${{\upmu}}$M} &amp; 100~${{\upmu}}$M &amp;   &amp;  \vspace*{2pt}\\
%\endthead
%Mito-TEMPOL &amp; {0.0297$^{\mathrm{a}}$} &amp; {} &amp; none &amp; {} &amp; 0 &amp; ${-}$ &amp; ${-}$ &amp; ${+}$ &amp; \cite{bib11} \\
%\rowcolor{gray!35}\textbf{Mn1} &amp; {7.0$^{\mathrm{b}}$} &amp; {6.2$^{\mathrm{c}}$ SF} &amp; 2.3$^{\mathrm{g}}$
%&amp; {\cite{bib60}} &amp; ${+}$ &amp; ${-}$ &amp; ${{+}{+}}$ &amp; ${+}$ &amp; \cite{bib11} \\
%\rowcolor{gray!35}\textbf{Mn1C} &amp; {3.4$^{\mathrm{c}}$} &amp; {nd} &amp; nd &amp;
%{\cite{bib54}} &amp; nd &amp; ${+}$ &amp; ${{+}{+}}$ &amp; {nd} &amp; \cite{bib54} \\
%\rowcolor{gray!35}\textbf{M40403} &amp; {3.55$^{\mathrm{b}}$} &amp;
%{1.9$^{\mathrm{d}}$ SF} &amp; 8.2 &amp; {\cite{bib38,bib61,bib62}} &amp; ${+}$ &amp; ${+}$ &amp;
%${+}$ &amp; ${-}$ &amp; \cite{bib11} \\
%\textbf{EUK-134} &amp; {0.602$^{\mathrm{b}}$} &amp; {nd} &amp; 13.0$^{\mathrm{h}}$
%&amp; {\cite{bib38,bib63,bib64}} &amp; ${+}$ &amp; ${-}$ &amp; ${-}$ &amp; ${+}$ &amp; \cite{bib11}\\
%\textbf{MnTE-2-PyP}$^{\textbf{5+}}$ &amp; {57$^{\mathrm{b}}$} &amp; {54 PR} &amp;
%63.3 &amp; {\cite{bib38,bib65}} &amp; ${+}$ &amp; ${-}$ &amp; ${-}$ &amp; ${++}$ &amp; \cite{bib11} \\
%\rowcolor{gray!35}{\textbf{MnTnBuOE-2-PyP}$^{\textbf{5+}}$} &amp; 68$^{\mathrm{b}}$ &amp; {} &amp; {88.5} &amp;
%\cite{bib38,bib66} &amp; ${+}$ &amp; ${-}$ &amp; ${+}$ &amp; ${{+}{+}}$ &amp; {\cite{bib11}} \\
%\textbf{MnTBAP}$^{\mathbf{3-}}$ &amp;
%{0.00145$^{\mathrm{b}}$} &amp; {none$^{\mathrm{d}}$ SF} &amp; 5.8 &amp;
%{\cite{bib62,bib66,bib67}} &amp; ${-}$ &amp; ${-}$ &amp; ${-}$ &amp; ${-}$ &amp; \cite{bib11} \\
%MnCl$_{2}$ &amp; {1.3$^{\mathrm{b}}$} &amp; {1.9$^{\mathrm{b}}$ PR} &amp; none &amp;
%{\cite{bib62,bib68,bib69}} &amp; ${+}$ &amp; {nd} &amp; ${-}$ &amp; ${-}$ &amp; \cite{bib11} \\
%Mn(ClO$_{4}$)$_{2}$ &amp; {1.3$^{\mathrm{b}}$} &amp; {} &amp;  &amp;
%{\cite{bib70,bib71}} &amp; nd &amp; {nd} &amp; nd &amp; {nd} &amp;  \\
%CuZnSOD &amp; {692--1995$^{\mathrm{b}}$} &amp; {2000$^{\mathrm{e}}$ SF} &amp;  &amp;
%{\cite{bib62,bib72,bib73,bib74,bib75}} &amp; ${{+}{+}}$ &amp; {(100 U/mL)} &amp; ${-}$ &amp;
%{nd} &amp;  \\
%MnSOD &amp; {} &amp; {ca.\ 3000 PR} &amp;  &amp; {\cite{bib76}} &amp; nd &amp; {nd} &amp; nd &amp; {nd} &amp;  \\
%Catalase  &amp; {} &amp; {} &amp; 1.5 $\times$ 10$^{6}$ &amp; {\cite{bib38}} &amp; nd &amp; {nd} &amp; nd &amp; {nd} &amp;  \\
%\parbox[t]{2.5cm}{Self-dismutation (pH 7.5)}\vspace*{2pt}&amp; {} &amp; {0.3} &amp;  &amp; {\cite{bib77,bib78}} &amp; nd &amp; {nd} &amp; nd &amp; {nd} &amp;
%\botline
\end{tabular}
\tabnote{$k_{\mathrm{McCF}}$ refers to the rate
constant calculated from the McCord and Fridovich assay~\cite{bib37,bib71}.}
\tabnote{$^{\mathrm{a}}$Ferricytochrome \textit{c}, phosphate buffer
(50~mM, pH~8). $^{\mathrm{b}}$Ferricytochrome \textit{c}, phosphate
buffer (50~mM, pH~7.8). $^{\mathrm{c}}$HEPES (60~mM, pH~7.4).
$^{\mathrm{d}}$ Phosphate buffer (50~mM, pH~7.4). 
$^{\mathrm{e}}$Phosphate buffer (50 mM, pH 7.8). $^{\mathrm{f}}$Unless specified,
catalytic constants were measured by polarography using a Clark-type
electrode in Tris buffer (50~mM, pH~7.8). $^{\mathrm{g}}$HEPES (100
mM, pH 7.4). $^{\mathrm{h}}$Value found for \textbf{EUK-8} instead of
\textbf{EUK-134} which differs only by the two methoxy substituents. SF
means stopped-flow and PR pulse radiolysis. $^{\mathrm{i}}$Cellular
assays: $++/+/-$ refers to the level of activity in comparison with
activated condition (macrophages and HT29-MD2) or basal H$_{2}$O$_{2}$
(HeLa HyPer) and they correspond to the color code in
Figure~\ref{fig6}. In blue: the best compounds identified as active in
the three-cell assay.}
\end{table*}

Overall, three groups were identified for each of the assay, namely
very efficient (${{+}{+}}$), efficient (${+}$) and weakly active or inactive (${-}$)
antioxidants ({Figure~\ref{fig6}}). As anticipated, the less
intrinsically active antioxidants proved the most inactive in the cell
assays. For instance, \textbf{MitoTempol} and
\textbf{MnTBAP}$^{\mathbf{3-}}$, which show a
$k_{\mathrm{cat}}$ for superoxide activity lower than the
rate of self-dismutation of superoxide and that are therefore not true
SOD-like catalysts, were found inactive with the RAW model and HT29-MD2
assays. These two are probably able to react once with
${\mathrm{O}_{2}}^{{\bullet}-}$ but their molecular structure or redox
potential prevent cycling back and catalysis. In other words, they are
stoichiometric antioxidants. The observation of their weak cellular
activity is probably linked to their stoichiometric nature, as opposed
to the higher activity catalytic antioxidants. In the extracellular
assay (raw 264.7), the CuZnSOD was found to be very active as expected,
but it was not active in the HT29-MDA assay: we can deduce that this
assay (HT29-MD2) probes an intracellular antioxidant effect. For the
HT29-MD2 assay, only \textbf{M40403} and
\textbf{MnTnBuOE-2-PyP}$^{\mathbf{5+}}$ were found active
at both 10~${{\upmu}}$M and 100~${{\upmu}}$M, whereas
\textbf{Mn1} was only active at 100~${{\upmu}}$M. Interestingly,
more inert analogues of \textbf{Mn1}, \textbf{Mn1C} and \textbf{Mn1CP}
were found to remain active at 10~${{\upmu}}$M~\cite{bib54},
confirming the idea that the lack of \mbox{activity} of \textbf{Mn1} at 10
${{\upmu}}$M was due to its dissociation in cells (see below). A
further study showed that, when \textbf{Mn1} dissociates in cells, it
leads to the Zn-analogue, which was found inactive both in the HeLa
HyPer and HT29-MD2 assays~\cite{bib43}. Another interesting example is
the case of \textbf{MnTnBuOE-2-PyP}$^{\mathbf{5+}}$ and
\textbf{MnTE-2-PyP}$^{\mathbf{5+}}$, showing a similar
intrinsic SOD activity and cell penetration but different 
bioactivities~\cite{bib11}, probably due to different subcellular
localizations.

Overall, the best compounds identified as active in the three-cell
assay are those highlighted in blue in {Table~\ref{tab1}}.

As we have already discussed in other cases~\cite{bib2,bib79,bib80},
the overall efficacy of a metal-based compound relies on several
parameters. {{Figure~\ref{fig7}}} shows radar plots revealing
the link between intrinsic activity (bottom part) and cellular
activities (upper part) determined in the three-cellular assays. The
choice of axis was guided by the idea of 
\mbox{maximizing} the {differences} in
the series explored, with favorable effects leading to a larger value
on the axis (see caption of {Figure~\ref{fig7}} for details).

\begin{figure*}
\includegraphics{fig07}
\caption{\label{fig7}Performances of metal-based antioxidants compared
using a radar representation for \textbf{MitoTEMPOL}, \textbf{Mn1},
\textbf{M40403}, \textbf{EUK-134}, \textbf{MnTBAP}$^{\mathbf{3-}}$,
\textbf{MnTnBuOE-2-PyP}$^{\mathbf{5+}}$, and
\textbf{MnTE-2-PyP}$^{\mathbf{5+}}$ after incubation at 100
${{\upmu}}$M. Upper-left radar: parameters involved in this
representation. Axis c1: HeLa, 100/F (F ${=}$ normalized fluorescence
ratio, see Figure~\ref{fig6}B). Axis c2: Macrophages, 100/F-red (F-red:
\% of ferricytochome \textit{c} reduction, see Figure~\ref{fig6}A).
Axis c3: HT29-MD2, 100-I (I ${=}$ IL8: \% of activation by LPS,
see~Figure~\ref{fig6}C).}
{\vspace*{-1.6pt}}
\end{figure*}

The cellular assays have been chosen for their complementarity, with
the macrophage assay measuring the effect on the superoxide
extracellular flow, HeLa Hyper measuring the effect on H$_{2}$O$_{2}$,
and HT29-MD2 measuring an intracellular effect on inflammation induced
by oxidative stress.

As shown on the upper-left insert in {Figure~\ref{fig7}}, the
upper part of the pentagons deciphers the biological effects on cells,
whether extracellular (HeLa and macrophages, middle part of the
\mbox{pentagon}) or {intracellular} (HT29-MD2 cell assay, at the apex of the
pentagon), linked to oxidative stress in general.

The lower part reveals the intrinsic SOD and CAT activity. The left
side of these pentagons is associated to the catalase-type activities
(intrinsic and in the HeLa cell assay) and the right side with the
anti-superoxide activities (intrinsic and in the macrophage assay).

The larger surface in the lower area, the higher the intrinsic
activities. The larger the surface in the upper area, the better the
activity in the three cells model. The larger the overall surface, the
higher the antioxidants activities, intrinsic and in cells.

As shown by the analysis of {Table~\ref{tab1}} (see above), a
low intrinsic activity leads to a weak cellular activity---see
\textbf{Mito-TEMPOL} and \textbf{MnTBAP}$^{\mathbf{3-}}$
with the very small chart surface. For all compounds, the macrophage
and HeLa cell assays, which probe a bioactivity in an extracellular
medium, mirror well the intrinsic catalytic activities since cell
penetration is not required. However, a large intrinsic activity (lower
part of the pentagon) does not necessarily go with a high intracellular
activity (HT29-MD2, apex of the pentagon). A loss of activity when going to cells or
biological environment in general can be due to a low \mbox{bioavailability,}
because of a weak penetration, poor stability in cells (see above,
discussion in the case of \textbf{Mn1}), or formation of a less active
ternary complex with cellular ligands.

\textbf{MnTnBuOE-2-PyP}$^{\mathbf{5+}}$ is the most active antioxidant
followed by \textbf{MnTE-2-PyP}$^{\mathbf{5+}}$ and \textbf{Mn1}
and this reflects on the surfaces of their pentagons being larger. The
two porphyrins \textbf{MnTnBuOE-2-PyP}$^{\mathbf{5+}}$ and
\textbf{MnTE-2-PyP}$^{\mathbf{5+}}$ are associated with a high
intrinsic catalase and SOD activity. They show a similar good activity
in the HeLa and macrophage assays but a weaker one in the HT29-MD2
assay. Note that the difference in activity between
\textbf{MnTnBuOE-2-PyP}$^{\mathbf{5+}}$,
\textbf{MnTE-2-PyP}$^{\mathbf{5+}}$ and \textbf{MnTBAP}$^{\mathbf{3-}}$
cannot be ascribed to a difference in cell penetration. Indeed,
\textbf{MnTBAP}$^{\mathbf{3-}}${,} anticipated to penetrate weakly due
to its high negative charge, was paradoxically penetrating better in
HT29-MD2 than \textbf{MnTnBuOE-2-PyP}$^{\mathbf{5+}}$ and
\textbf{MnTE-2-PyP}$^{\mathbf{5+}}$ (see Figure~\ref{fig8}). \textbf{Mn1} was
the most penetrating molecule.

\begin{figure}
\includegraphics{fig08}
\caption{\label{fig8}Quantification by inductively coupled plasma mass
spectrometry (ICP-MS) (black columns) and UV spectrometry (gray
columns, only for porphyrins) of Mn content in cell lysates of HT29-MD2
stimulated cells, incubated with or without compounds at 100
${{\upmu}}$M in DMEM supplemented with 10\% heat-inactivated FBS and
LPS (0.1~${{\upmu}}$g/mL). Values are given for a single experiment.
For ICP-MS, values were corrected according to the Zn content of
control cells to compare the data. Reprinted with permission from~\cite{bib11}. Copyright 2021 Elsevier.}
\end{figure}

\textbf{Mn1} has a very weak catalase activity and good SOD activity:
this is shown by the surface on the chart representation being shifted
to the right side. In contrast, the activity of
\textbf{MnTnBuOE-2-PyP}$^{\mathbf{5+}}$,
\textbf{MnTE-2-PyP}$^{\mathbf{5+}}$ is more balanced
between CAT and SOD, with a left-right symmetry of the filled inner
surface.

These results on cells stress the importance of characterizing the
bioactivity within cellular models. Of course, a compound needs to show
a good intrinsic activity to be active in cells. But other parameters
are at play, such as cell penetration, localization, stability, and
inertia, in link with speciation in cells.

Other studies on Mn porphyrins have demonstrated that these compounds
upregulate MnSOD, CAT, glutaredoxins (GRXs), peroxiredoxins (PRDXs) and
other antioxidants. These proteins are under control of NRF2, a
transcription factor known to activate genes through the antioxidant
response element (ARE). NRF2 is continuously produced in cells and
binds to KEAP1 (Kelch-like ECH-associated protein, ECH ${=}$ enoyl-coenzyme
A (CoA) hydratase) with a particular conformation that promotes its
continuous degradation by the proteasome (see Figure~\ref{fig9}). The
modification of the Cys in KEAP1 (upon oxidative or chemical stress
induced by electrophiles for instance) leads to an inhibition of NRF2
degradation: the newly synthesized NRF2 \mbox{accumulates} in the cytosol and
translocates to the nucleus where it activates genes leading notably to
an overexpression of SOD, CAT, glutathione reductase, \mbox{heme-oxygenase}
(HO-1), and NAD(P)H dehydrogenase quinone (NQO1)~\cite{bib81}. Note
that a similar activation of the ARE has also been proposed for
nitroxides~\cite{bib82} which react stoichiometrically with
${\mathrm{O}_{2}}^{{\bullet}-}$.

\begin{figure*}
\includegraphics{fig09}
\caption{\label{fig9}Schematic representation of the two possible pathways on cellular
activity of SOD mimics. Pathway 1 corresponds to the direct effect of
SOD mimics, with their ability to catalytically dismutate superoxide. A
similar pathway could be ascribed to CAT mimics as well as they reduce
H$_{2}$O$_{2}$. Pathway 2 is associated to a pro-oxidant effect of SOD
mimics: as they produce H$_{2}$O$_{2}$, they can activate the
NRF2-signaling ARE pathway involving the biosynthesis of antioxidant
enzymes such as SOD. (Adapted with permission from~\cite{bib9}.)
Copyright 2022 Elsevier.}
\end{figure*}

SOD mimics can then act through two pathways (see Figure~\ref{fig9})~\cite{bib9}: (A) by reducing the ${\mathrm{O}_{2}}^{{\bullet}-}$
concentration and (B) by increasing H$_{2}$O$_{2}$ concentration
leading to an induction of the ARE. The balance between these two
pathways could depend on the speed of dismutation
($k_{\mathrm{{cat}}}$, intrinsic activity) in
comparison with the cellular capacity to detoxify H$_{2}$O$_{2}$ and
also on the H$_{2}$O$_{2}$ cell content. A fast dismutation delivers a
high shot of H$_{2}$O$_{2}$ concentration activating the antioxidant
cellular response (pathway B, pro-oxidant). Pathway A may occur as soon
as the SOD mimics enter the cells or the mitochondria but is also prone
to decrease in time due to the SOD mimic degradation. In contrast,
Pathway B requires time to activate protein synthesis (from 30 min to
several hours), but this has a long-lasting effect as it works by
boosting the cellular antioxidant feedback.
We have recently shown by a proteomic cysteine-redoxomic analyses in
HT29-MD2 that LPS challenge generates an early oxidative stress (15 min
time-scale) leading to inflammation (hour time-scale) with a
cellular-feedback activating the antioxidant response. These effects
are mitigated by \textbf{Mn1} with an early limitation of the cysteine
oxidations as early as 15 min~\cite{83}.

In a way, compounds commonly known as SOD mimics can be separated into
two classes~\cite{bib9}: (1)~compounds directly acting on
${\mathrm{O}_{2}}^{{\bullet}-}$, by catalyzing its dismutation, are truly
mimicking SOD activity and should show an early cellular activity and
\mbox{(2) compounds,} such as porphyrins, acting in fact as pro-oxidants,
activate the cellular antioxidant enzymatic arsenal through
transcription factors, such as NRF2, that promote gene expression of
antioxidant proteins (SOD, CAT{\ldots}). Their action takes longer to
activate, as it depends on cellular protein biosynthesis, but also
lasts longer.

{\vspace*{-2pt}}

\subsection{Of cells and mice}

{\vspace*{-2pt}}

\subsubsection{Mice models in link with inflammatory bowel diseases}

{\vspace*{-2pt}}

It has been shown that inflammation and oxidative stress are strongly
intertwined in inflammatory bowels diseases (IBDs)~\cite{bib83}. In
addition, IBDs are associated with a weakening of the \mbox{antioxidant}
defenses: the content and/or activity of SODs was shown significantly
reduced in plasma~\cite{bib84,bib85,bib86} or in tissues homogenates
from resection specimen of IBD patients as compared to healthy 
subjects~\cite{bib87,bib88}. In this context, managing oxidative stress using
SOD mimics is thus an interesting therapeutic alternative to existing
treatments~\cite{bib89,bib90}. Toward this goal, we have used an IBD
mouse model in which colitis is induced by administration of
2,4-dinitrobenzenesulfonic acid (DNBS). Starting the same day as DNBS
administration, \textbf{Mn1} was administered daily by gavage in a
basic buffer, to try to limit metal decoordination in the acidic
stomach \mbox{environment.} Interestingly, mice treated with \textbf{Mn1}
showed a significantly reduced weight loss in comparison with untreated
mice, supporting the idea that accelerating superoxide dismutation is a
valuable strategy against inflammatory pathologies~\cite{bib2}.
Recently, we have shown that the anti-inflammatory activity of \textbf{Mn1}
could be preserved in this mice colitis model, without using the
buffer, when \textbf{Mn1} was loaded in bacteria (called ``chemically
modified bacteria'')~\cite{90}.

\subsubsection{Side effects of anticancerous agents: mitigation of
neuropathy by SOD mimics}

As already mentioned, low levels of ROS play important roles in normal
cells and are involved in the regulation of cellular signaling and
proliferation~\cite{bib91}. Higher levels of ROS along with
deregulation of antioxidant pathways have been observed in cancer 
cells~\cite{bib91,bib92}. The differences in cellular redox states between
normal and cancer cells and the role of ROS and their regulation in
tumorigenesis have received much attention to explore and develop new
therapeutic and preventive cancer-targeted strategies~\cite{bib93}. In
the context of anticancer therapy, Mn-porphyrins SOD mimics have been
studied in clinical trials for radioprotection of normal tissue and
organ and for chemosensitization of cancer cells, their anticancer
activity has been reviewed elsewhere~\cite{bib94,bib95}.\looseness=1

Clinically used chemotherapeutics are characterized by dose-limiting
off-target toxicities. Treatment with Oxaliplatin (Oxa), a reference
drug in the treatment of digestive tract tumors, especially colorectal
cancer and gastric cancer, is associated with the development of
peripheral neuropathy (Oxa-induced peripheral neuropathy, OIPN) in a
majority of patients in its acute form, which may evolve into a chronic
form severely impacting the patients' quality of life. OIPN is a
frequent cause of treatment arrest and a major hurdle limiting Oxa's
clinical use with no clinically effective treatment or prevention
therapy~\cite{bib96,bib97}.

OIPN affects nerves and induces damages in peripheral nerves and dorsal
root ganglia, causing pain, impaired sensations, or involuntary
movements. The associated molecular mechanisms are complex and were
shown to involve neuroinflammation, oxidative stress, and mitochondrial
dysfunction. Antioxidants and modulators of redox balance have been
studied as adjuvants in Oxa treatments in in vivo studies and were
shown to have neuroprotective effects~\cite{bib98,bib99,bib100}.
\textbf{Mangafodipir} (see Figure~\ref{fig5}) is a Mn(II) complex
developed for its activity as a contrast agent in magnetic resonance
imaging (MRI), associated with the release of Mn(II) from the ligand,
but which also shows SOD-like activity~\cite{bib101,bib102}. In this
context, the first phase III neuroprotection trial in patients with
colorectal cancer involved \textbf{calmangafodipir} (CaM;
PledOx$^{\text{\textregistered}}$; PledPharma AB/Egetis Therapeutics AB, see
Figure~\ref{fig5}), although negative results were reported~\cite{bib103}.

We recently explored the relevance of SOD mimics derived from
\textbf{Mn1} in combination treatments with Oxa in reducing
OIPN~\cite{bib104,bib105}. In vitro, \mbox{antitumoral} activity was assessed
by the viability and ROS \mbox{production} of tumor cell lines (CT16, HCT116,
HT29) and murine fibroblasts (NIH3T3). In vivo, a murine model of
colorectal cancer with subcutaneous injection of CT26 cells in Balb/c
mice was used. The tumoral growth was monitored, and OIPN was primarily
assessed using a behavioral Von Frey test reflecting nociception and
mechanical sensitivity (tactile pressure on the paw). In vivo, the
association of \textbf{Mn1} derivatives to Oxa did not counteract Oxa's
antitumoral activity, significantly decreased OIPN, and improved global
clinical tolerance of Oxa. Additional effects were measured by cold
plate tests, specific functional neuromuscular assays, and electron
{microscopy} analyses of sciatic nerves. Further \mbox{studies} are on
the way to characterize Pt accumulation in organs, peripheral nervous
system, and tumors by inductively coupled plasma mass spectrometry
(ICP-MS), and neuropathy markers by immunohistology (hind paws, sciatic
nerves). Our studies showed that \textbf{Mn1} derivatives are promising
in preventing the appearance of sensitive axonal neuropathy and
neuromuscular disorders induced by Oxa~\cite{bib104,bib105}, as they
reduce Oxa-induced neurosensitive and neuromuscular alterations in vivo
and prevent Oxa-induced peripheral demyelination. We are now developing
covalent conjugates between MnSOD mimics and Oxa under the shape of
Pt(IV) prodrugs and evaluating them for {antitumoral} activity and
neuropathy prevention in mice models.

\section{Speciation in cells: complex integrity and physical form}

As exemplified by the results reported above, a key information when it
comes to the design of bioactive metal complexes and rationalization of
their bioactivity is their speciation. Speciation of an element is
defined by the International Union of Pure and Applied Chemistry
(IUPAC) as its distribution among defined chemical species, including
molecular structure or physical form~\cite{bib106,bib107}. Determining
the integrity of a metal complex within a biological system, or its
physical form (solute, precipitate), is part of this description and
several examples are given below.

Because it is not easy to anticipate which molecular structure is
active in the biological environment, it is important to explore
experimentally the nature of metal-based species present in cells. The
recent development of analytical and imaging techniques~\cite{bib108,bib109}, 
including X-ray fluorescence~\cite{bib9,bib110,bib111,bib112}, nano secondary ion mass spectrometry
(nanoSIMS)~\cite{bib108,bib109}, ICP-MS and its laser ablation version
for imaging (LA-ICP-MS)~\cite{bib108,bib113,bib114}, ion mobility
spectrometry \mbox{coupled} to mass spectrometry (IMS-MS)~\cite{bib43}, Energy
Dispersive X-ray (EDS or EDX)~\cite{bib115}, or laser-induced breakdown
spectroscopy (LIBS)~\cite{bib116}, have bolstered our capacity of
picturing the cellular content and distribution of metal based species
and metal ions. We here delineate two original ways of examining the
integrity of metal-complexes in cells, through X-fluorescence imaging
and mass-based approaches. Molecular and chemical design tools have
also been developed by chemists to improved stability and inertia of
metal compounds~\cite{bib117,bib118,bib119,bib120,bib121}. In most
cases, ligand exchange is being detrimental to the activity, but it can
be also favorable and purposely designed, with a carrier metal complex
delivering an active ligand, protected by metal coordination before
reaching its target and then released in the desired cellular
environment~\cite{bib122,bib123}.

\subsection{Exploring complex integrity and speciation by
``unconventional'' methods}

\subsubsection{X-ray fluorescence imaging}

Exploring speciation in cells using a single imaging technique in a
single experiment has the advantage of limiting sample manipulation
(lysis, extraction, dilution, etc.),
that could lead to metal decoordination, as much as possible. One
original possibility is to use X-ray fluorescence (XRF), which enables
the detection of heavy elements such as metal ions and imaging them
using raster scanning of cells or biological tissue sections. If the
ligand is appended with a heavy substituent, it is possible to map this
heavy element as well as the metal ion and thus determine whether they
colocalize in ${{\upmu}}$-XRF or not. This has been done for
instance with a Br-substituted ligand~\cite{bib124}.

We have functionalized a Mn-based SOD mimic with a rhenium-based moiety
(see Figure~\ref{fig10}). This type of Re-based complexes has been
developed by us~\cite{bib126} and others, as UV--vis fluorescent probes.
Interestingly, they also show IR and Raman characteristic absorptions
and rhenium (Re) can be detected using
X-fluorescence~\cite{bib112,bib125}. These probes are thus multimodal
probes active in UV--vis fluorescence, IR, Raman, and X-fluorescence.
The distribution of the Mn in cells incubated with \textbf{Mn1} is
homogeneous in the cell (not shown)~\cite{bib2} and is different from
that of \textbf{Mn1Re} with an accumulation into perinuclear organelles
(see Figure~\ref{fig10})~\cite{bib10}. The Re-probe is \mbox{positively}
charged and hydrophobic: it may drive \textbf{Mn1Re} into lipid-rich
organelles like mitochondria characterized by a negative
potential~\cite{bib126}. The bioactivity in the HT29-MD2 assay
(described above) of \textbf{Mn1} and \textbf{Mn1Re} was similar,
although \textbf{Mn1Re} was overall more cell-penetrant than
\textbf{Mn1}. Their anti-inflammatory activity is thought to be
associated to a reduced LPS-induced mitochondrial oxidative stress.
Interestingly, both \textbf{Mn1} and \textbf{Mn1Re} showed the same
quantity accumulated in mitochondria, in line with their similar
bioactivity and that bioactivity being associated with an effect in
mitochondria.


\begin{figure*}
\includegraphics{fig10}
\caption{\label{fig10}From ref~\cite{bib10}. (1) Elemental distribution
of P, K, Mn, Re, and Zn in an HT29-MD2 cell incubated with
\textbf{Mn1Re}. The phosphorus (P, K${\upalpha}$-line), and zinc (Zn,
K\ss{}z-line, and not K${\upalpha}$ to avoid overlapping with Re
L${\upalpha}$) maps are used to identify the nucleus area. The overlay
(top right) corresponds to the manganese (Mn) (K${\upalpha}$-line)
(magenta) and rhenium (Re) (L\ss{}-line that does not overlap with
other lines) (green) maps. The regions corresponding to an overlap of
both elements are displayed in white. Intestinal epithelial cells
HT29-MD2 were incubated for 2 h with \textbf{Mn1Re} (100 mM, 0.02\%
DMSO) before cryofixation and freeze-drying. Images were recorded on
the 2-ID-D beamline of the APS synchrotron (excitation at 12.0 keV;
integration time 2 s per pixel; pixel size 200~nm).  (2) Confocal
fluorescence images of HT29-MD2 of two different cells incubated with a
mitochondrial marker (Mitotracker{\texttrademark}, fluorescence) and
X-fluorescence maps of \textbf{Mn1Re} (Re, X-fluorescence). Intestinal
epithelial cells HT29-MD2 were incubated for 1.5 h with \textbf{Mn1Re}
(100~${{\upmu}}$M, 0.02\% DMSO) before the addition of
Mitotracker{\texttrademark} deep red (200~nM, 30~min), washing with
50~mM EDTA, cryofixation and freeze-drying. Confocal fluorescence
images of Mitotracker{\texttrademark} deep red (ex 633~nm, em
645--750~nm) were recorded with open pinhole. X-fluorescence (Re)
images were recorded on the Nanoscopium beamline of SOLEIL synchrotron
(excitation at 14.1~keV; integration time 2.4~s/pixel; pixel size
300~nm). Scale bar 3~${{\upmu}}$m. (3) C: control HT29-MD2 cells,
\textbf{Mn1}/\textbf{Mn1Re}: HT29-MD2 cells incubated with
\textbf{Mn1}/\textbf{Mn1Re} (10 and 100~${{\upmu}}$M, 6 h). IL8
secretion in HT29-MD2 cells. Intestinal epithelial cells HT29-MD2 were
incubated for 7 h under different conditions indicated in the figure.
LPS (0.1~${{\upmu}}$g{${\cdot}$}mL$^{-1}$) was added at the end of the
first hour. Data represent means $+/-$ SEM for 7--10 independent
experiments. ($^*$) $p &lt; 0.05$, ($^{**}$) $p &lt; 0.01$ vs.\ LPS condition.
(4) C-control cells, \textbf{Mn1}/\textbf{Mn1Re}-HT29-MD2 cells
incubated with \textbf{Mn1}/\textbf{Mn1Re} (100 ${{\upmu}}$M, 6 h).
Total Mn content was determined in acid-digested cell lysates by
titration using EPR. Data represent mean $+/-$ SEM for 4 independent
experiments. ($^{*}$) $p &lt; 0.05$, ($^{**}$) $p &lt; 0.01$ versus C. Total
Mn content in mitochondria-enriched fractions was determined by ICP-MS.
Mitochondria were isolated using a mitochondria isolation kit (Thermo
Fisher Scientific). Data represent mean $+/-$ SEM for 7 independent
experiments; see~\cite{bib11} for details. Schemes in blue and
pink: molecular structure of \textbf{Mn1} and \textbf{Mn1Re} mentioned
in this figure.}
\end{figure*}

\subsubsection{Metal speciation studied by mass spectrometry (MS) and
ion mobility spectrometry (IMS)}

The detection and quantification of exogenous metal complexes are
crucial to understand their activity in biological environments. In
this regard, Mn(II) complexes are a challenge because of their low
association constants, high lability, and rapid metal
exchange~\cite{bib2,bib127,bib128}. We have recently shown that liquid
chromatography coupled to mass spectrometry (LC-MS) in cell lysates can
be used to detect and quantify Mn-based SOD mimics as long as they are
not too labile, with the simple use of a calibration
curve~\cite{bib54}. For the more labile ones, metal ions leaking from
the LC-MS detection system were detrimental, with an Mn--Zn exchange,
especially at low concentrations of the Mn complexes. As direct
infusion showed no metal exchange, the leakage of metal ions from the
separation part (LC) was incriminated to be responsible for the metal
ion exchange (Mn--Zn) in the complex. Another separation technique,
namely ion mobility, was then explored to separate labile metal
complexes of the first transition series.

Ion mobility spectrometry (IMS) was first developed in the 1970s to
detect traces of organic molecules in gas phase~\cite{bib129}. This
technique uses separation according to the size of the molecule within
an electric field and within a gas flow that pushes the molecules
forward or backward depending on set-up. Their velocity is linked to
their collision cross section (CCS). In IMS-MS, the detection is
achieved by mass spectrometry. IMS-MS is a powerful technique used to
characterize differences in folding of proteins upon ligand binding~\cite{bib130} 
or of peptides upon metal ion binding~\cite{bib131}.

Recently, we have shown that IMS-MS is powerful enough to discriminate
between very similar low molecular weight (LMW) ligands folding around
metal ions across the first metal transition series~\cite{bib43}: ion
mobility correlates well with ion radius (Figure~\ref{fig11}). Then, IMS-MS
was efficiently used to detect these labile metal complexes in cell
lysates. They were quantified using a calibration curve with a standard
made of a heavy ligand (with six $^{13}$C) and a Co(II), metal ion
chosen to lead to the least metal exchanges. The use of this heavy
ligand-based Co complex, added just before IMS-MS analysis, allowed us
to probe the metal exchange that occurred in the analytical system
rather than in the cell lysates~\cite{bib43}.


\begin{figure*}
{\vspace*{-3pt}}
\includegraphics{fig11}
{\vspace*{-3pt}}
\caption{\label{fig11}Collision cross-section of complexes
\textbf{M(enPI}$_{\mathbf{2}}$\textbf{P)} (M(II) coordinated by ligand
enPI$_{2}$P) in \AA{}$^{2}$ plotted against the ionic radius of metal
cations in \AA{}. The $\mathrm{r}_{\mathrm{ion}}$ of any M(II)
corresponds to a high-spin pentacoordination, except for Fe(II)
(high-spin hexacoordination). The $\mathrm{r}_{\mathrm{ion}}$ for
Cu(II) is also shown (in yellow) for a high-spin tetracoordination. The
$\mathrm{r}_{\mathrm{ion}}$ values were taken from
\url{http://abulafia.mt.ic.ac.uk/shannon/ptable.php}. Adapted with
permission from~\cite{bib43}. Copyright 2022 Wiley.}
{\vspace*{-3pt}}
\end{figure*}


In the cellular context, there are a lot of Lewis bases that can
coordinate to endogenous metal ions. The Irvin--Williams 
series~\cite{bib132} describes the range of association constants for
divalent cations of the first transition row associated with
unconstrained ligands. They show a trend of increasing association
constant from left (Mn(II)) to right (Cu(II) and Zn(II)).
Interestingly, Giedroc and Lisher have proposed a very enlightening
idea, classifying cations as ``competitive'' (Zn(II), Cu(I), the redox
state in cells) or ``bioavailable'' (Mn(II) but also, to a weaker
extent, Fe(II))~\cite{bib128}. Cu(I) and Zn(II) are more tightly
controlled in cells than Mn(II). They show a higher association
constant for most biological ligands. The existence of a pool of
loosely bound Cu(I) and Zn(II) is very elusive,  if only because hard
to define. With a concentration of ca.\ 10$^{-19}$ M estimated for this
pool, as defined by the threshold of detection by endogenous metal
sensors~\cite{133,134,135}, this is not even one ion in a 2 pL cell, a
typical volume for one eukaryotic cell. In contrast, the loosely bound
pool is very abundant in Mn(II) (10$^{-5}$ M corresponding to more than
10 millions Mn(II) ions in 2 pL)~\cite{bib128}.

In a mixture where several metal ions M and several ligands L are
competing, the primarily formed LM complex(es) are those showing the
highest $\mathrm{K}_{\mathrm{LM}}$ association constants. The ligands and
metals ions remaining after this pairing are those less prone to
associate. Hence, paradoxically, the stability threshold in cells is
more stringent for Cu-complexes, which will find strong ligand
competitors in cells, than for Mn-complexes, which will in contrast
find Mn(II) highly bioavailable in cells. Furthermore, the loosely
bound abundant pool of Mn(II) will contribute to shift the equilibrium
M ${+}$ L ${=}$ LM toward the formation of the complex L--Mn(II). To that
extent, and paradoxically, complexes of Mn(II), such as \textbf{Mn1},
\textbf{Mn1P}, \textbf{Mn1C} and \textbf{Mn1CP}, are probably ideal to
be used in cells, as the ligand is weak enough not to compete with
endogenous ligands for Cu or Zn (proteins), and probably strong enough
to possibly remove Mn from the loosely bound endogenous Mn pool.


{\vspace*{-2pt}}

\subsection{Improving inertia against metal exchange}

As stated earlier, the use of metal complexes involving coordination
bonds raises specific difficulties in terms of stability in biological
contexts, associated with metal exchanges, ligand addition (into a
ternary complex for instance) or ligand exchanges, which is in addition
to questions raised by other metabolization pathways common with
organic molecules. This can clearly impact the pharmacokinetics
properties of metal complexes--based drugs. Indeed, the first
metabolization pathway of such drugs is metal decoordination, in
particular for complexes involving an open-chain ligand. Dissociation
may occur via different pathways, such as spontaneous decoordination
and acid-catalyzed or metal-assisted decomplexation involving 
exchange with a competitive metal ion~\cite{bib127}. Thermodynamic
stability and kinetic inertness are thus key parameters to take into
account when designing complexes for biological applications. In order
to improve the \mbox{biological} \mbox{properties} of Mn(II)-based SOD mimics,
inspired by the work of T\'{o}th et~al.~\cite{bib120}, we have
increased the inertness of the complexes by rigidifying the ligand
central structure. To do so, the central ethane diamine bridge has been
replaced by a trans cyclohexyl-1,2-diamine~\cite{bib54}. We expected
that a weaker elasticity of the ligand, as defined by Comba~\cite{bib133}, 
would reduce the kinetics of metal decoordination by
limiting the opening--closing breathing. Indeed, we have shown that the
rigidified ligand led to complexes with slower metal exchanges with
divalent competitive cations~\cite{bib128} such as
Cu$^{2+}$, Zn$^{2+}$, Ni$^{2+}$,
and Co$^{2+}$. Very interestingly, this improved inertness
was associated with similar to better anti-inflammatory and antioxidant
properties in the HT29-MD2 cell line. These activities were also
retained at lower concentration (10~${{\upmu}}$M, \textbf{Mn1C},
\textbf{Mn1CP}), than what is observed for the complex built with the
unconstrained ligand (100~${{\upmu}}$M, \textbf{Mn1}). Increasing
the inertness of complexes is consequently a valuable strategy to
improve their bioactivities~\cite{bib54}.

{\vspace*{-2pt}}
\subsection{Equilibrium between soluble and insoluble phases}
{\vspace*{-2pt}}

In addition to soluble species, it has also been observed that
accumulated metals can form dense phases in biological systems,
suggesting the \mbox{formation} of insoluble phases. This is particularly
striking in the case of metal-accumulation diseases, such as
hemochromatosis, i.e., iron (Fe) overload, or Wilson diseases, i.e.,
copper overload. In the \mbox{pathological} contexts of metal overload,
tissues that accumulate significant quantities of metals, such as the
liver, generally present dense deposits of a few to few hundred
nanometers. In the case of Fe, these phases can be identified as
ferritin, the iron storage protein, resulting in the formation of
crystalline objects around 8~nm, containing different crystalline
phases~\cite{bib134}. However, not all metal ions undergo finely tuned
biomineralization, and other transition metals could, when they
accumulate, result in less organized phases, with differences in size,
crystallinity, and composition. For example, in Wilson disease, while
dense areas can easily be observed by transmission electron microscopy
imaging, the diversity of the observed structures is important, and
does not allow their nature to be clearly identified~\cite{bib135}.

Interestingly, in a recent work published with Gazeau and Carn, we
elucidated that exogenous metal ions could be subjected to precipitation as
insoluble phases~\cite{bib136,bib137}. These observations stem from the
initial description of the biodegradation of gold (Au) nanoparticles
inside cell lysosomes. Not only was it found that Au nanoparticles
evolve and dissolve over time, but that the released metal ions
recrystallize under a unique ultrastructure, forming curved leaves
composed of self-assembled nanometer-sized Au clusters. This was
surprising as Au particles have been considered inert or
non-degradable. However, this result was consistent with some old
literature dedicated to soluble ionic Au compounds used for the therapy
of rheumatoid arthritis~\cite{bib138}. In patients' biopsies and in
animals, similar Au assemblies were observed due to Au accumulating in
the tissues during treatment. These findings suggest that repeated
exposure to an exogenous element in a soluble form can trigger its
precipitation inside tissues as new solid phases with a common
structure, regardless of the initial speciation of the metal (soluble,
nanoparticular Au), the cell type considered, and the host species.

These phases present brand new challenges for speciation, as they
present complex composition (mixed amorphous and crystalline phases
incorporating endogenous elements, metabolites, or proteins) with
complex reactivity, that can varies with the surrounding biological or
chemical environment. They might also be highly sensitive to minor
changes in their chemical environment, and particularly to change in
the redox environment. Indeed, in the case of gold nanoparticles'
biodegradation, oxidative stress was a key element to enable gold
nanoparticles dissolution, and inhibition of intralysosomal ROS
production was found to slow down degradation process~\cite{bib136}.
Thus, lysosomal change in redox potential or pH might be of major
importance in biodissolution processes, but also to the formation of
insoluble phases.

If soluble--insoluble phase equilibrium raises new challenges to
describe metals speciation, it also offers new opportunities for their
characterization, as their density allows their observation using
transmission or scanning electron microscopy, even if caution is called
for as these highly energetic beams can destabilize the samples while
observing \cite{bib137,bib139}.

This phenomenon of metal ions precipitation in biological matrices
provides food for thought when considering how cells can deal with
metal ions that are essential for their metabolism but become toxic at
high concentration, such as iron and copper. Interestingly, it also
raises questions about the metabolization of metal ions that are
naturally present in ultra--trace levels, but become elevated due to
exposure resulting from therapeutic, occupational, accidental or
pollution-related activities.
Indeed, precipitation could be a way for
the cells to mitigate metal ion reactivity, limiting the proportion of
chemically active atoms to the atoms accessible at the surface (which
can be different from all of the surface atoms because of protein
adsorption on the dense phase), thus also moderating their potential
adverse effects.

{\vspace*{-2pt}}

\section{Conclusion}

{\vspace*{-2pt}}

Because speciation matters, integrating the design of metal complexes
meant to be bioactive with their study in a cellular context is quite
important and fruitful toward the development of therapeutic
application for metal complexes. For this integrated approach, the
terms cellular inorganic chemistry or inorganic chemobiology can be
coined. It goes with a particular interest in subcellular metal
imaging. In addition, as paramagnetic species such as metal centres
perform activation of paramagnetic dioxygen, there is a strong intertwining
between biochemistry of metal ions and that of dioxygen. In addition,
metalloproteins and metal complexes are used to combat pathologies
related to oxidative stress.

As it encompasses chemical conception, characterization, and cell
studies, including chemical \mbox{analyses} and evaluation of bioactivities,
this approach in inorganic chemical biology requires skills in organic
(ligands) and inorganic (complexes) syntheses, physical and analytical
chemistries in intricate biological media, cell biology and bioimaging,
with special interests in monitoring metal distribution and cell state,
and more specifically redox state.

In this article we have described first the cellular models that we
have developed for evaluating the bioactivities of these complexes.
Those three cellular models can be used for the screening of
metal-based antioxidants, to have a better idea of their propensity to
act on superoxide or hydrogen peroxide concentration, and of their
ability to be an efficient antioxidant in a biological context. The
second part deals with the nature of the metal species in cells and of
their subcellular distribution. The question of the integrity of metal
complexes is intrinsically linked to the broader question of metal
speciation, which aims to determine the complete array of metal-based
species present, including insoluble phases formed by precipitation.
Interestingly for the design of Mn-complexes, it is possible to play
with the inertia to enhance bioactivity through a higher in-cell\break
stability.

{\vspace*{-4pt}}

\section*{Abbreviations}
%{\vspace*{-7pt}}
\mbox{}\noindent
\begin{inftab}
\begin{tabular}{@{}l@{\hskip6pt}l}
\tbody
ARE &amp; {Antioxidant response element}\\
CaM &amp; {Calmangafodipir}\\
CAT &amp; Catalase\\
COX2 &amp; Cyclooxygenase 2\\
Cys &amp; cysteine\\
DMEM &amp; Dulbecco's modified eagle medium\\
DNBS &amp;  2,4-dinitrobenzenesulfonic acid\\
EDS/EDX &amp;  \parbox[t]{5.5cm}{\raggedright Energy-dispersive X-ray spectroscopy}{\vspace*{2pt}}\\
\textbf{EUK-134} &amp;  See Figure~\ref{fig5}\\
FBS &amp;  fetal bovine serum\\
GRX &amp;  glutaredoxin\\
HT29(-MD2) &amp;  \parbox[t]{6cm}{\raggedright Human colon cancer cell line (stably transfected to over
express MD2, the co-receptor of TLR4 for LPS)}\vspace*{2pt} \\
HeLa &amp;  Cancerous cell line\\
HEPES buffer &amp;  \parbox[t]{5cm}{\raggedright 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,
$\mathrm{p}K_{\mathrm{a}}$ (25~{\textdegree}C) ${=}$ 7.48}{\vspace*{2pt}}\\
HO &amp;  Heme oxygenase\\
HyPer &amp;  Hydrogen peroxide sensor\\
IBD &amp;  Inflammatory bowel diseases\\
\inftabbreak
\parbox[t]{2cm}{\raggedright ICP-MS/LA-ICP-MS} &amp;  
\parbox[t]{5cm}{\raggedright Inductively coupled plasma mass spectrometry/laser
ablation inductively coupled plasma mass spectrometry}\vspace*{2pt}\\
IEC &amp;  Intestinal epithelial cells\\
IFN-${\upgamma}$ &amp;  Interferon ${\upgamma}$\\
IL8 &amp;  Interleukin 8\\
IUPAC &amp;  \parbox[t]{5cm}{\raggedright International union of pure and applied chemistry}\vspace*{2pt}\\
IMS/MS &amp;  \parbox[t]{5cm}{\raggedright Ion mobility spectrometry coupled
to mass spectrometry}{\vspace*{2pt}}\\
KEAP1 &amp;  \parbox[t]{5.5cm}{\raggedright Kelch-like ECH-associated protein}\\
LC &amp;  Liquid chromatography\\
LIBS &amp;  \parbox[t]{5cm}{\raggedright Laser-induced breakdown spectroscopy}\vspace*{2pt}\\
LPS &amp;  Lipopolysaccharide\\
LMW &amp;  Low molecular weight\\
\textbf{MitoTempol} &amp;  See Figure~\ref{fig5}\\
MD2 &amp;  \parbox[t]{5.5cm}{\raggedright Myeloid differentiation 2 (receptor)}\\
\parbox[t]{2cm}{\raggedright\textbf{Mn1}, \textbf{Mn1C}, \textbf{Mn1CP}, \textbf{MnTxxx},
\textbf{M40403}}\vspace*{2pt} &amp;  See Figure~\ref{fig5}\\
MS &amp;  Mass spectrometry\\
MTT &amp;  \parbox[t]{5.5cm}{\raggedright 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide}\vspace*{2pt}\\
NAD(P) &amp;  \parbox[t]{5.5cm}{\raggedright Nicotinamide adenine dinucleotide (phosphate)}\vspace*{2pt}\\
(i)NOS &amp;  (inductive) NO-synthase\\
NOX &amp;  NADPH oxidase\\
NQO-1 &amp;  \parbox[t]{5.5cm}{\raggedright NAD(P)H Quinone Dehydrogenase 1}{\vspace*{2pt}}\\
NRF2 &amp;  \parbox[t]{5.5cm}{\raggedright Nuclear factor (erythroid-derived 2)-like 2}\vspace*{2pt}\\
OIPN &amp;  \parbox[t]{5.5cm}{\raggedright Oxa-induced peripheral neuropathy}\vspace*{2pt}\\
Oxa &amp;  OxaliPt\\
OxyR(-RD) &amp;  \parbox[t]{5.5cm}{\raggedright Bacterial peroxide sensor (regulatory domain)}\vspace*{2pt}\\
PDRX &amp;  peroxiredoxin\\
PMA &amp;  Phorbol 12-myristate 13-acetate \\
PGE2 &amp;  Prostaglandin E2\\
RAW 264.7 &amp;  Murine macrophages\\
ROS &amp;  Reactive oxygen species\\
Shh &amp;  Sonic hedgehog\\
SIMS &amp;  Secondary ion mass spectrometry\\
\parbox[t]{2cm}{\raggedright SOD, CuZnSOD and MnSOD} &amp;  
\parbox[t]{5.5cm}{\raggedright Superoxide dismutase, Copper-Zinc superoxide
dismutase, manganese superoxide dismutase; SOD1: SOD cytosolic (Cu-Zn);
SOD2: MnSOD, mitochondrial}\vspace*{2pt}\\
\inftabbreak
TLR4 &amp;  Toll-Like-Receptor 4\\
Tris  buffer &amp; \parbox[t]{5.5cm}{\raggedright 2-amino-2-(hydroxymethyl) propane-1,3-diol, $\mathrm{p}K_{\mathrm{a}}$ 
(20~{\textdegree}C) ${=}$ 8.3}\vspace*{2pt}\\
XRF &amp; X-fluorescence\\
(cp)YFP &amp; \parbox[t]{5.5cm}{\raggedright (circularly permuted) yellow fluorescent protein}
\end{tabular}
\end{inftab}

\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}

The authors want to acknowledge ENS-PSL, PSL University, CNRS,
MITI-CNRS, Sorbonne University (SU) and the other bodies that funded
this work. ANR-15-CE07-0027 MAGIC; ANR-16-CE18-0017-01 SATIN;
ANR-16-CE07-0025 Metallopepzyme; DEI20151234413 (Fondation pour la
recherche m\'{e}dicale 2016), and BACTMAN and ANACOMDA (Mission pour
les initiatives transverses et interdisciplinaires MITI-CNRS), the
Association Fran\c{c}ois Aupetit (AFA, research fellowship 2015), ANR
21-CE18-0053-02 MOBIDIC, ANR 20-CE07-0039-01-CATMAN, ANR 23-CE23
METALINFY; Idex PSL Qlife project Main ANR Q-life ANR-17-CONV-0005;
CEFIPRA project no. 6505-1; EMERGENCE SU EMRG-24 TOTEM;
ANR-22-PEBI-0003 (PEPR).

\CDRGrant[ANR]{ANR-15-CE07-0027}
\CDRGrant[ANR]{ANR-16-CE18-0017-01}
\CDRGrant[ANR]{ANR-16-CE07-0025}
\CDRGrant[FRM]{DEI20151234413} 
\CDRGrant[ANR]{21-CE18-0053-02}
\CDRGrant[ANR]{ANR 20-CE07-0039-01-CATMAN}
\CDRGrant[ANR]{ANR 23-CE23}
\CDRGrant[ANR]{ANR-17-CONV-0005}
\CDRGrant[CEFIPRA]{6505-1}
\CDRGrant[SU]{EMRG-24 TOTEM}
\CDRGrant[ANR]{ANR-22-PEBI-0003}

\section*{Acknowledgements}

{Servier Medical Art}, licensed under CC BY 4.0, is acknowledged for
cell image used in TOC.

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