Ivanna Amarsy
email: ivanna.amarsy@chimieparistech.psl.eu

Sébastien Papot
email: sebastien.papot@univ-poitiers.fr.

Gilles Gasser
email: gilles.gasser@chimieparistech.psl.eu

Stimuli-Responsive Metal Complexes for Biomedical Applications

Keywords: Diagnostic tools, Drug Release, Medicinal Inorganic Chemistry, Metals in Medicine, Selective Delivery 4-aminobenzyl alcohol, ADEPT: antibody direct enzyme prodrug therapy, β-gal: βgalactosidase, β-lac: β-lactamase, β-gluc: β-glucuronidase, BR: Biotin receptor, CA prodrug therapy, DO3A: 1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane, Food and drug administration, GSH: glutathion, 7-HMC: 7-hydroxy-3-methylcoumarin, methyl trans-2-hydroxycinnamate, MRI: magnetic resonance imaging, NO: nitric oxide, PARACEST: paramagnetic chemical exchange saturation transfer, PET: positron emission tomography, QM: quinone methide, ROS: Reactive oxygen species, SPECT: Single photon emission computed tomography

Metal-based compounds have been widely used for biomedical applications. Their unique characteristics make them attractive for both therapeutic and diagnostic purposes. However, numerous issues including toxicity, poor aqueous solubility, and unfavorable biodistribution hamper their widespread use. To overcome these drawbacks, the concept of metal-based prodrugs emerged. This field is particularly developed for applications in oncology. More precisely, tumor-associated stimuli (e.g., pH variation, redox activity, enzyme overexpression, etc.) have been exploited to trigger the selective delivery of active metal-based drugs to the tumor site. The main advances in this area are discussed in this review.

Biographies

Introduction

Metals are present in all living organisms and play a fundamental role in a wide range of biological processes such as cofactors, enzymes (e.g., superoxide dismutase) or metal salts (iron, zinc, calcium, etc.). [START_REF] Carver | Essential Metals in Medicine: Therapeutic Use and Toxicity of Metal Ions in the Clinic[END_REF]2] This is likely one of the reasons why the use of metals for biomedical applications has attracted great interest for many centuries. For instance, the use of gold for therapeutic purposes has been reported in both the Egyptian and Chinese cultures about 4500 years ago. [3] However, it was only after the discovery of salvarsan, an arsenic-based antimicrobial agent effective against syphilis, by Paul Ehrlich in 1912 that the study of metals as potential drugs took off. [4] One of the major breakthroughs that marked the interest in metallodrugs was the discovery of cisplatin in 1965 through serendipity by Barnett Rosenberg and Loretta VanCamp. [START_REF] Jones | Medicinal Applications of Coordination Chemistry[END_REF] Cisplatin and its derivatives are currently among the most widely used drugs for cancer therapy. [4,6,7] Nowadays, a large number of metal complexes are employed for various medical applications (Figure 1). The two pnictogens, antimony and bismuth, are frequently prescribed as antimicrobial and antiparasitic agents, and also in the treatment of gastrointestinal disorders (i.e., the bismuth subsalicylate) (Figure 2). [4] Silver is commonly incorporated in creams and aqueous solutions to fight infections resulting from burns. [4] Gold is employed as an antiarthritic (auranofin) [START_REF] Jones | Medicinal Applications of Coordination Chemistry[END_REF]9] (Figure 2) and even explored as a potential agent against infectious diseases and cancer. [START_REF] Carver | Essential Metals in Medicine: Therapeutic Use and Toxicity of Metal Ions in the Clinic[END_REF] Aside from their use in drugs, metal complexes are also utilized as diagnostic tools.

The dominant isotope, also called the "Workhorse of Diagnostic Nuclear Medicine" in SPECT and PET imaging, is technetium-99m, with twenty-eight 99m Tc imaging agents approved by the FDA so far. As for MRI scans, the most used metal is gadolinium, with few examples also approved by the FDA. [4] To demonstrate the importance of this metal ion, it is worth mentioning that 500,000 doses are given every day around the globe. One important issue that must be overcome in order to use this metal is the high toxicity of the free Gd(III) ion. In this context, the design of biocompatible complexes should prevent the release of the free metal ion in vivo, hence reducing its toxicity. [10][11][12]

A

Pt

Cl Cl Compared to purely organic compounds, metal-containing derivatives present unique characteristics that confers several advantages for the design of bioactive molecules. First, metal centers exhibit various geometry (e.g., pyramidal, trigonal bipyramidal, octahedral, etc.) providing a greater stereochemical diversity than a tetrahedral carbon atom. [START_REF] Jones | Medicinal Applications of Coordination Chemistry[END_REF]13,14] This diversity increases de facto the number of biological targets, [15] increases the possible interaction modes, and allows a better selectivity (e.g., targeting one specific kinase on the 518 coded by the human genome). [15,16] Second, the redox potential of metals allows them to manipulate biological processes (e.g., production of ROS). [3,4,14] It can also confer spectroscopic (e.g., Mossbauer spectroscopy), magnetic, or radioactive properties that find applications in therapy or diagnosis. [4] However, as it is the case with their organic counterparts, the use of metal complexes in the clinic is still fraught with numerous issues including toxicity, poor aqueous solubility, and unfavorable biodistribution. [START_REF] Carver | Essential Metals in Medicine: Therapeutic Use and Toxicity of Metal Ions in the Clinic[END_REF], [START_REF] Jones | Medicinal Applications of Coordination Chemistry[END_REF] For example, the widely used cisplatin lacks of selectivity leading to a severe kidney toxicity that limit its therapeutic efficacy. [START_REF] Carver | Essential Metals in Medicine: Therapeutic Use and Toxicity of Metal Ions in the Clinic[END_REF][START_REF] Jones | Medicinal Applications of Coordination Chemistry[END_REF]17,18] With the aim to overcome these limitations, the concept of metal-based prodrugs was proposed in the 2000s. [19,20] As introduced earlier by Albert, [21] a prodrug is usually defined as a medication or compound that is metabolized into a pharmacologically active drug once administered in a living organism. This approach was specifically explored in cancer chemotherapy with the design of prodrugs that can be activated exclusively in malignant tissues. Within this framework, most of the anticancer prodrugs developed so far include a specific trigger that induces the release of the active drug in response to a tumor-associated stimulus (e.g., pH, redox, enzyme, etc.). [20,22,23] (Figure 3) The best illustrations of this strategy are the antibodydrug conjugates (ADCs) bearing a stimuli-responsive linker, with several of them already approved for various applications in oncology. [24][25][26] The prodrug concept has been considered in the case of metallodrugs and some of them have already shown promising results with an increased selectivity and reduced side effects in clinical trials. [27], [28] For example, it was postulated that the Ruthenium complex NAMI-A, which has reached phase II clinical trial, was activated upon reduction in cells, -we note that this suggestion is under debate. [29,30] Several Pt(IV) complexes, like Straplatin, which are activated upon reduction, entered clinical trial. In addition, the approved Pt(II) complexes (e.g, cisplatin, carboplatin, oxaliplatin, etc.) and the liposome nanoparticle formulation of cisplatin, Lipoplatin, which successfully finished Phase III as treatment for non-small cell lung cancer adenocarcinomas, are other examples of prodrugs per se -they undergo aquation before binding to DNA. [31] In this review, we present the different stimuli-responsive metal complex delivery systems investigated to date for both therapeutic and diagnostic purposes. While photosensitizers (PSs) are sometimes considered as prodrugs, photodynamic therapy (PDT) will not be discussed here since many recent reviews on this topic have already appeared. [32,33] We note that for certain topic, only a few selected examples are described since this review aims more to highlight principles. Figure 3. General scheme of selective delivery of metal complexes to the tumor site.

Enzyme-responsive metal complexes

The design of stimuli-responsive systems that can be activated by an enzyme selectively present in targeted tissues represents a very attractive strategy for the development innovative diagnostic and therapeutic agents. In this approach the active species (e.g., imaging probe, drug, etc.) can be turned on in a catalytic manner, thereby offering a substantial advantage compared to non-catalytic activation processes. Thus, enzyme-responsive metal complexes have been investigated for both imaging and therapeutic purposes. [23] In this context, a particular attention has been paid to the targeting of glycosidases, such as β-galactosidase and β-glucuronidase that are overexpressed in some diseased tissues (e.g. tumors), [34] and β-lactamase, a bacterial enzyme that can be pre-targeted using DEPT (Directed Enzyme Prodrug Therapy) strategies. [35] 2.1. β-Galactosidase a. Imaging Magnetic resonance imaging (MRI) is an alternative to optical microscopy and offers the possibility to study biological structures in 2D or 3D in a non-invasive manner. These images are obtained using the magnetic resonance signal of the nucleus of water's protons. The signal intensity obtained is a function of the water concentration and both the longitudinal relaxation time T1 and the transverse relaxation time T2. The interaction of water molecules with the metal ion leads to a decrease in the T1 of the complex, resulting in a higher contrast image of the observed object. In 1997, Meade et al. exploited this imaging property by designing the first enzyme-responsive contrast agent. [36] They studied the two Gd 3+ complexes 1a (EGad) and 1b (EGadMe) that included a galactoside on their 9 th coordination site (Scheme 1). In the presence of β-galactosidase, the glycosidic bond was cleaved, allowing the interaction of water molecules with the paramagnetic ion. This variation in the coordination sphere of the lanthanide decreased the T1 relaxation time of the complexes resulting in an increased intensity of the observed signal (image enhancement). In the course of preliminary experiments conducted in vitro, the complex 1a showed only an increase of 20% in relaxivity, which was not enough to pursue the investigations in vivo. In contrast, upon β-galactosidase activation, the relaxivity with the complex 1b increased by a factor of 3 (Rcleaved : 2.72, Runcleaved : 0.903). [37] Indeed, the presence of the methyl group conferred to this complex a higher rigidity by preventing the rotation of the galactoside and consequently limiting the access of water to the Gd 3+ ion. This enzyme-responsive metal complex was tested in vivo on living xenopus laevis embryos. A 57% enhancement in signal intensity was observed where β-gal was expressed, showing the potential of this contrast agent for the monitoring of enzymatic activity.

In parallel to better understand the pharmacology of 1b contrast agent, the authors replaced the Gd(III) ion with 111 In as the radiolabeled agent. [38] Since galactosides have the ability to target the asialoglycoprotein receptor, they studied the selective uptake of this imaging agent in receptor-rich cells both in vitro and in vivo. These experiments demonstrated that the galactoside moiety acts as a targeting group directing the complex in asialoglycoprotein receptor-enriched tissues. However, 1b also appeared as a poor substrate for the activating enzyme, hence limiting its potential for the detection of β-galactosidase activity. To overcome this drawback, the authors developed the MRI contrast agent 2 including a selfimmolative linker that exhibited fast enzymatic kinetics (Scheme 2). The insertion of a pendent carboxylic group on the linker allowed the saturation of the metal coordination sphere, making it inactive. [39] Once hydrolyzed by β-gal, the self-immolative linker underwent an electron cascade, releasing the Gd(III) chelate agent and enabling one water molecule to coordinate. The activity of 2 was demonstrated in transgenic mice expressing β-gal.

N N N N O O O O O O H N O O O HN O O 5 Gd 3+ N N N N O O O O O O H 2 N Gd 3+ O H H β-gal 2 O OH HO HO OH CO 2
Scheme 2. Mechanism of the MRI contrast agent 2.

In 2007, Wang et al. reported the complex 3 beared a novel β-galactosidase-sensitive selfimmolative linker. [40] Once activated, the latter reacted with nucleophilic moieties of either human serum albumin (HSA) or β-galactosidase to generate the corresponding bioconjugates In 2008, Chauvin and their co-workers developed enzyme-responsive MRI and PARACEST (Paramagnetic Chemical Exchange Transfer) probes derived from the DOTA ligand. [41,42] These off/on probes can overcome the lack of sensitivity of these imaging techniques and target molecules present in low (nanomolar) concentrations. The probes 5 consist of a DOTA ligand encapsulating either Gd 3+ or Yb 3+ and is linked to a β-galactosidase-responsive self-immolative benzylcarbamate moiety (Scheme 4). In the presence of β-galactosidase, the cleavage of the glycosidic bond triggered the self-decomposition of the linker, leading to the release of the corresponding complex. The structural modification of the lanthanide derivative resulted in the modification of the MRI and PARACEST properties of the complexes, therefore allowing the detection of enzymatic activity. One of the drawbacks of in vivo MRI images is the presence of background signals intrinsic from tissues. To overcome this limitation, Engelmann's team developed the bimodal 1 H and 19 F MRI probe 6 (Scheme 5). [43] To quench the fluorine signal in the absence of β-gal, the authors exploited the phenomenon of intramolecular shortening of 19 F relaxation times by paramagnetic lanthanide metal ions: the so-called Paramagnetic Relaxation Enhancement (PRE) effect.

Enzymatic cleavage of the probe released the fluorine moiety, thereby increasing its relaxation time and consequently the 19 F MRI signal (Scheme 5). In vitro experiments were carried out with 6 (0.96 mM) on melanoma cells expressing βgalactosidase (LacZ/B16) and B16 cells without the targeted enzyme. An acquisition of the 19 F MRI images showed that no 19 F MRI signal could be detected in the samples after incubating B16 cells with probe 6 (either 15 min or 2 h). In contrast, a signal of increasing intensity was observed after incubation of LacZ/B16 cells in the presence of 6. These results indicated that 6 was efficiently and specifically converted within β-galactosidase-expressing LacZ/B16 cells.

Based on the same principle, Kikuchi et al. developed the new 19 F MRI probe 7 (Scheme 6). [44] With this design, the transverse relaxation time T2 of the trifluoromethyl group was reduced by the PRE effect. As observed by 19 F NMR, enzymatic cleavage of the galactoside increased the T2 of the trifluoromethyl moiety, therefore enhancing the MRI signal. In vitro studies were conducted on HEK293T cells expressing and not expressing β-gal. An increase of the 19 F NMR signal was recorded only in cells expressing β-gal. These results demonstrated the selectivity of the probe 6 for the enzyme and its potential for the detection of the β-galactosidase activity.

However, due to hydrophilicity of 6, the cell membrane needed to be permeabilized for observing the probe activation, which represents a major obstacle for the use of this imaging technology in vivo. The same authors exploited the properties of gold nanoparticles (AuNPs) to develop new imaging techniques for enzyme detection. First, they designed gold nanoparticles bearing lipoic acid chains with and without glycosidase-responsive ends (Scheme 7). [45] In the presence of the corresponding enzyme (β-gal), hydrolysis of the glycosidic bond followed by selfdecomposition of the linker led to the release of a positively charged ammonium. The free ammonium can then interact with anionic lipoate on the neighboring nanoparticle, resulting in electrostatic aggregation. This aggregation induces a colorimetric change from wine red to blue that can be observed with the naked eye. This imaging technique that allows the detection of glycosidase activity in real time presents the advantage of being simple and highly sensitive.

N N N N O O O O O O Gd 3+ H N O O CF 3 O PRE effect β-gal N N N N O O O O O O Gd 3+ H N O O CF 3 O N N N N O O O O O O Gd 3+
Kikuchi et al. also designed a β-gal metal-enhanced near infrared (NIR) fluorescence sensor based on functionalized gold nanoparticles (AuNPs). [46] The aim of this study was also to improve the detection of enzymes in vivo for diagnosis purposes.

Metal-enhanced fluorescence (MEF) is due to the localized surface plasmon resonance (LSPR) of noble metals that can increase the fluorescence of a fluorophore when in close proximity (>5nm). It has been shown that β-gal can shift the LSPR absorption spectrum of the AuNPs from visible to the NIR region. [45] Based on these two properties, the authors synthesized nanoparticles decorated with fluorophore ligands (Lip-Cy5.5m), PEG400, and either galactoside or aldehyde ends (Scheme 8). In the presence of β-gal, the hydrolysis of the galactoside moiety followed by the decomposition of the linker led to the release of an amine that then reacted with the aldehyde of a neighboring particle. Through this fashion, the inter-nanoparticle distance decreased, and the LSPR shifted into the NIR regions. This fluctuation enhanced the electromagnetic resonance coupling and thus the fluorescence.

The NIR fluorescence was enhanced by a factor 7.8 showing the potential of such an activatable probe for enzyme detection. This concept can be useful for on-demand imaging techniques such as in vivo enzyme detection and early diagnostic imaging.

The use of nanomaterials as drug delivery systems is an approach that has been widely explored.

This allows for the masking of the drug activity during its transport until it reaches the diseased tissues, therefore minimizing side effects. However, the unselective drug leakage, a process observed with the vast majority of nano-carriers, represents an important drawback for this targeting strategy both for therapeutic and diagnosis purposes. First, the authors compared the difference in relaxivity properties of 9 and 10 between a competitive extracellular anion mimic solution and human serum. An increase of 240% in relaxivity in the human serum compared to high concentrated anion buffer was observed.

β-Glucuronidase

Furthermore, the complex 9 was 27% brighter than 10 in the serum, which should be sufficient for in vivo imaging. The complex composition of human serum did not allow to ascribe these results to any particular component of the serum. Kinetic studies using bovine liver βglucuronidase monitored by magnetic resonance correlated the change in T1 as function of enzyme incubation time. These experiments demonstrated the efficiency of this contrast agent in a complex biological environment.

b. Therapy

Platinum-based drugs are chemotherapeutic agents currently used for cancer therapy. However, their poor water solubility as well as their lack of selectivity for tumor cells strongly limit their therapeutic efficacy. In order to overcome these drawbacks, Reedjik et al. developed the hydrophilic glucuronide prodrug 11 designed for the selective delivery of platinum derivatives within malignant tissues (Scheme 10). [48] β-Glucuronidase is indeed overexpressed in the microenvironment of almost all solid tumors. [49] Thus, selective hydrolysis of the glucuronide 11 should lead to the release of the platinum-based drug 12 in the tumor extracellular space.

The latter will then penetrate passively inside surrounding cancer cells to exert its cytotoxic activity. The enzymatic cleavage of prodrug 11 was monitored by 1 H-NMR. The authors demonstrated the relatively fast hydrolysis of the glucuronide to generate the platinum drug. This proof of principle showed for the first time that platinum complexes can be efficiently released upon βglucuronidase activation, hence highlighting the potential of this approach to improve the selectivity of cancer chemotherapy. Unfortunately, no biological experiment was undertaken to confirm the validity of this targeting strategy. ). [50] The probe was composed of a 19 F group, a cephalosporin moiety, and the Gd 3+ complex. With this design, the large magnetic moment of Gd 3+ reduced the T2 of the probe and removed the 19 F MRI signal.

In the presence of β-lac, the cephalosporin β-lactam ring was hydrolyzed, leading to the release of both the Gd 3+ complex and the 19 F moiety that turn on the 19 F MRI signal. Using this probe, β-lactamase activity on cell surface was successfully visualized. However, intermolecular PRE was still observed after the enzymatic cleavage, hence lowering the MRI signal intensity.

b. Therapy β-Lactamase-responsive prodrugs can also be used to release a cytotoxic agent for therapeutic purposes. Thus, with the aim to deliver an anticancer agent selectively at the tumor site,

Hanessian and Wang developed an ADEPT system employing β-lactamase as the triggering enzyme for releasing a platinium complex selectively in malignant tissues (Scheme 12). [51] For this purpose, they designed prodrugs 14 bearing different platinium complexes. With this construct, it was anticipated that the enzymatic reaction should lead to the release of the Ptbased drug attached at the 3'-position of cephalosporin residue. Scheme 12. A). General mechanism of the ADEPT system using β -lactamase and Pt-drug B). Structure of the two platinum prodrugs.

In this study, two potent antitumor agents have been investigated, the 4'-carboxyphthalato (1,2cyclohexanediamine) platinum (DACCP) and the 1,2-diamino cyclohexane (DACH) platinum complex.

The β-lactamase hydrolysis of the DACCP derivative was studied by [START_REF] Carver | Essential Metals in Medicine: Therapeutic Use and Toxicity of Metal Ions in the Clinic[END_REF] H NMR in deuterated buffer solutions. These experiments showed the efficient release of the carboplatinium subunit 15 and 16.

While significant advances have been made in the field of enzyme-responsive metal complexes in past years, some progresses are still needed in order to transfer this approach towards the clinic. Too few experiments have been undertaken in vivo so far for estimating the potential of this approach. The next generations of enzyme-responsive metal complexes could be inspired by their organic counterparts that are currently used in humans. For instance, ADCs including an enzyme-sensitive linker enable both the selective accumulation and release of the drug in malignant cells. Thus, enzyme-responsive systems devoted to the delivery of metal complexes could be combined to a targeting warhead like an antibody to enhance their potential as therapeutic or diagnosis agents.

Other Triggers

Based on tumor chemical specificities, non-enzymatic stimuli-responsive triggers were also developed. These triggers can respond to exogenous stimuli such as light or endogenous stimuli like ROS or pH variation.

Chemical Reduction /Redox Activation

a. Imaging

Since glutathione (GSH) is present in high concentration in cells of certain disease tissues such as cancer, it has been investigated as an endogenous stimulus for probe activation. GSH can act as a reductive agent or a nucleophile. [52,53] Within this framework, Zhao et al. reported the development of the fluorescent probe 17 designed to target mitochondrial GSH in living cells (Scheme 13). [54] The probe included a para-dinitrophenoxy benzyl pyridinium moiety at the meso position on the BODIPY fluorophore. The pyridinium linker was used as a mitochondrial targeting moiety as well as an electronic sink to turn off the fluorescence of the fluorophore. In the first instance, the authors used the 2,4-dinitrophenoxy group for H2S detection. However, it turned out that the pyridinium group allowed the selective recognition of GSH instead of H2S.

A plausible explanation would be the formation of electrostatic interactions between the biothiols and the cationic pyridinium. Therefore, only GSH would reach the reactive site due to the longer chain. Several tests were then carried out to prove the efficiency of this probe. The authors demonstrated that fluorescence increased with the concentration of GSH (up to 32-fold increase with 100 µM of glutathione). In the presence of various biothiol species, GSH demonstrated the fastest response in fluorescence. The detection limit was described at 109 nM of glutathione, hence allowing intracellular imaging. The probe was found to be non-toxic for HeLa cells (cervical cancer cells), and confocal laser scanning microscopic fluorescent imaging showed the localization of the probe in mitochondria.

Hlava's group also developed GSH cleavable conjugates of amino-BODIPY dye to monitor drug release via an OFF-ON fluorescent system. [55] They conjugated an amino-BODIPY dye to several 2-phenyl-3-hydroxy-4(1H)-quinolinone (known for their anticancer activity) through the symmetrical 19 or asymmetrical 18 self-immolative linker (Scheme 14). The measured fluorescence spectra showed a lower intensity for the conjugate probe than for the released BODIPY. The cleavage of each conjugate in the presence of GSH was confirmed by HPLC and LC/MS analysis, demonstrating the release of both the BODIPY moiety and the drug. These experiments indicated that measuring the fluorescence of BODIPY reflected the release of the drug. The authors also demonstrated that this probe system can be useful for the delivery of other non-fluorescent drugs (different from quinolinone), highlighting the broad application of this sensing strategy. The efficiency of probe activation was also demonstrated in HeLa cells. However, it was shown that not only glutathione present in biological medium can induce the cleavage but also cysteine. Therefore, this system lacks selectivity and its application for in vivo imaging is rather limited. Pt(IV) complexes have been widely studied as prodrug system delivering active Pt(II) complexes after cellular reduction. [56][57][58][59] However, in this this review, we only discussed the examples when an active metal complex is released. Recently, Zhu et al. designed the Pt(IV)-based sensor 20 for investigating the reduction of the platinum (IV) prodrug to platinum (II) in real-time (Scheme 15). [60] They demonstrated that the enhanced fluorescence had a linear relationship with the percentage of reduced-Pt and correlated to the cytotoxicity on several cancer cells. However, the sensor concentration and its nearby environment can influence the measurement of the fluorescence intensity. Moreover, no in vivo experiment has been performed to investigate the penetration and precision of this sensor on living organism.

b. Therapy

Straddling the line between imaging and therapy, Gao et al. developed the novel prodrug system 21 containing a targeting agent (i.e., biotin), an MRI complex (i.e., Gd-DOTA), an anticancer drug (i.e., camptothecin -CPT) along with a disulfide self-immolative linker (Scheme 16). [61] Because of the overexpression of the sodium-dependent multivitamin transporters (SMVT) in some cancer cells, biotin is used as targeting unit to boost the drug efficiency; SMVT is known to transport pantothenic acid (vitamin B5) and biotin. [62] Gd-DOTA is a commonly used MRI contrast agent, enabling accurate diagnosis and real-time monitoring. CPT is a powerful antitumor drug, that is very efficient, but it can also bring harmful side effects due to its lack of selectivity for cancer cells. The disulfide linker provides selectivity for the release as the cleavage will take place in the reductive environment present inside tumor cells (i.e., high concentration of reductive glutathione -GSH). Moreover, the paramagnetic complex would quench the fluorescence of CPT and will turn on after the cleavage. It allows for a complementary approach to monitor drug release. CPT was revealed by comparing the tumor sizes. Overall, this prodrug system showed great results for both the accurate diagnosis and the precise treatment of tumors. Furthermore, it could be adapted to the targeting of others anticancer drugs.

In 2016, Mokhir and coworkers reported an aminoferrocenne prodrug activated by reductive glutathione. [63] The system was based on a previous model activated by H2O2 instead of GSH. [64] Their first compound 22a was composed of an aminoferrocene moiety linked to an aryl boronic ester (Scheme 17). The cleavage was induced by H2O2 highly present in cancer cells. To improve the efficiency of prodrug 22a, the boronic-ester trigger was replaced by an azide residue, which is activated in a reductive environment. The reduction of 22b produces an amine that can then undergo 1,6-elimination followed by a spontaneous decarboxylation. The two resulting products are the aminoferrocene/Fe salts, which can catalyst the generation of reactive oxygen species (ROS), and the aza-para-quinone methide, which alkylates glutathione and hence inhibits the antioxidative system of cells. These two products work in synergy to increase the oxidative stress and cause cell death. The cytotoxicity of compound 22b was evaluated on the human promyetotic leukemia cell line HL-60. The complex was found to be more toxic than the previous drug 22a bearing the boronic ester (IC50: 27 ± 4 µM versus >50 µM). While this prodrug system works in vitro, no in vivo experiments were conducted, so this system does not provide a full assessment. Furthermore, the compound was not significantly more efficient in hypoxic conditions (IC50: 23 ± 2 µM versus 27 ± 4 µM in normoxic conditions) meaning that the prodrug 22b is not useful for the treatment of hypoxic tumors.

In 2020, Huang et al. developed a novel strategy relying on a biorthogonal Pt(II) prodrug and a dual theranostic effect. [65] Unlike Pt Cytoplasmic reductants in cancer cells (e.g., ascorbic acid) reduce the prodrug to form, on one hand, a Pt(II) complex and, on the other hand, a propargyl compound. The Pt(II) complex can then act as theranostic drug and catalyzes the depropargylation to liberate NO in situ. This system provides synergistic, antiproliferative activity against cancer cells. The prodrug exhibited a better toxicity (IC50: 0.23 ± 0.01 µM) against human ovarian cancer A2780 cells compared to free cisplatin (IC50: 1.08 ± 0.06 µM) and the Pt(IV) complex alone (IC50: 1.69 ± 0.09 µM). The compound 23 was also found to be less toxic (IC50: 119.1 ± 7.98 µM) against normal epithelial IOSE80 cells than the two free complexes (IC50: 8.420 ± 0.57 µM for cisplatin and 18.47 ± 1.36 µM for the Pt(IV) complex). Finally, the researchers showed cancer cell growth inhibition induced by the prodrug on a zebrafish embryo model, demonstrating its in vivo activity. Overall, this new biorthogonal cleavage reaction-based prodrug could be expanded to other active molecules of interest. Moreover, the selectivity against cancer cells, due to the use of a Pt(IV) complex, allows it to overcome the side effects generated by Pt(II)based drugs.

It is well-known that Pt(II) drugs, especially cisplatin, displays harmful side effects and chemoresistance. Pt(IV) prodrugs are a useful strategy to overcome this limitation. Recently, Ang and their coworkers developed the codrug delivery system 24 using a Pt(IV)-prodrug (Scheme 19). [66] For connecting the complex to the bioactive drug, the authors used a 4aminobenzyl alcohol (4ABA) self-immolative linker, enabling a controlled and traceless codrug release. The intracellular reduction of the platinum complex leads to a decarboxylation and an Cytotoxicity of the prodrug systems containing A or B were evaluated on HeLa cervical carcinoma cells. Incubation of the prodrugs showed a better toxicity (IC50 (A): 2.4 ± 0.1 µM and IC50 (B): 5.5 ± 1.0 µM) than each drug alone or when co-injected. For the prodrug system C, the antiproliferative efficacy was measured on Pt-sensitive A2780 and Pt-resistant A2780cis ovarian carcinoma cells. Unfortunately, the prodrug was not more cytotoxic than cisplatin against A2780 cells, likely due to its slow 1,6-elimination kinetics. However, the prodrug showed great efficiency against the Pt-resistant A2780 cell line (IC50 (C): 5.2 ± 1.5 µM versus IC50 (cisplatin): 15.8 ± 2.9 µM). This strategy still needs to be improved and evaluated in vivo, but it has already showed promising preliminary results. In contrast, the cytotoxicity of the Ru complex alone was found to be lower on the resistant cell lines A2780 cis and A2780 ADR (IC50, 480nm = 3.41 -4.67 μM, IC50, 595 nm = 4.47 -7.10 μM),

clearly showing the beneficial effect of the conjugate 25 to overcome drug resistance. We note that the tremendous work of Hambley and co-workers on the release of active moieties from a Co(III) complex that is reduced to a Co(II) is not discussed in this review since the active species is not the metal-based compound. [67][68][69]

Acidic pH activation

The decrease of extracellular pH in tumors is one of the characteristics allowing for the targeting of malignant cells using a prodrug strategy. [19,23,[START_REF] Swietach | The chemistry, physiology and pathology of pH in cancer[END_REF] In 2006, Meade and their co-workers designed the first complex 26 combining an MRI contrast agent and an anticancer drug (doxorubicin) via an acid-labile hydrazone linker (Scheme 21). [71] Doxorubicin is a well-known anticancer agent exhibiting high efficiency, but it also has toxic side-effects. Few doxorubicin prodrugs using acid-labile linkers have been reported to date. [START_REF] Beretta | Anthracycline Chemistry and Biology II[END_REF] The linker in 26 is cleaved by the lysosomal acidic pH, allowing intracellular drug release. In previous systems, this release was measured indirectly by high-performance liquid chromatography and cytotoxicity studies.

To overcome this limitation, Meade and their co-workers designed the first prodrug-procontrast agent 26 (i.e., a doxorubicin-Gd(III) conjugate shown in Scheme 21). [73] The contrast agent is called procontrast agent because it is inactive before the drug release. When exposed to low pH, the prodrug releases the doxorubicin, and the MRI contrast agent undergoes a change in relaxivity, allowing for imaging.

O HO

H 2 N OH O OH O N O O OH OH N H O N N N N O O O O Gd 3+ H 3 O + NH 2 N H O N N N N O O O O Gd 3+
O HO The hydrolytic stability of the compound at pH 7.4 and 4.5 was assessed by relaxometry at 37°C. A decrease in relaxivity was observed at pH 4.5. 90% of doxorubicin was released within 16h, as observed by reversed-phase high-performance liquid chromatography. This lead compound opens up the door for a new class of prodrugs. [74] Improvements are still needed for allowing detection at lower concentrations. For this purpose, relaxivities must be improved. In vivo experiments have not been reported yet.

Recently, HSA-based drug delivery systems tethered to a metallodrug have emerged. [75][76][77][78][79][80][81] The large hydrophobic cavity in the HSA IIA subdomain is constituted of two N-donor residues, namely Lys199 and His242, which can be linked to a metal complex. In the acidic tumor microenvironment, the N-donor residues become protonated, which decreases their ability to coordinate with the metal. Through this fashion, the metallodrug is released (Scheme 22) . [82] Lys199 -NH 2 This concept was then extensively exploited by Yang, Liang and their co-workers using several

Cu-based drugs. The authors played on different parameters (e.g., using two copper centers, [83] replacing the ligand, [80,81] modifying the leaving groups, [79] adding a folate moiety for targeting, [84] creating nanoparticles, etc. [85] ) to improve the efficiency, selectivity and solubility of the drug.

The best Cu-based prodrug developed so far is the HSA complex 28 (Scheme 23). [81] Its cytotoxicity was evaluated in vitro on A549 cells (adenocarcinoma human alveolar basal A plausible explanation for these promising results is the better cellular uptake and selectivity of release in cancer cells of the prodrug 28 compared to the complex 27 due to the presence of the HSA moiety. This concept was shown to be effective with copper complexes and more recently with iron complexes. [78] Other metal centers remain to be evaluated. Despite these promising results, to the best of our knowledge, no pH-responsive prodrugs entered into clinical trial for now. [86]

Activation by the high production of ROS

Most cancer cells exhibit enhanced reactive oxygen species (ROS) production, including under aqueous conditions, undergoes spontaneous 1,6-elimination to release the ferrocene derivative and a p-quinone methide (QM). Boronic acid is also formed, which is non-toxic. QM is a glutathione scavenger, thus inhibiting antioxidative system of cells. The ferrocene moiety acts as a catalyst for ROS generation. This prodrug system leads to a double antiproliferative action in cancer cells.

Mokhir and their coworkers explored this system with different substituents R' and R'' on the prodrug (Scheme 24) to modulate the cell-membrane permeability and thus cell cytotoxicity on human promyelocytic leukemia (HL-60). [87] The best compound was 29H with an IC50 : 9± 2 µM compare to 28D-G with an IC50 between 24 and 55 µM. In contrast, the ferrocene alone was not toxic (IC50 >200 µM). Importantly, 29H exhibited no toxicity (up to 100 µM) towards nonmalignant fibroblasts. After this first discovery, this research group attempted to improve their prodrug system. To do so, in 2013, they found out that 30 (Scheme 24) had an IC50 of 1.4 ± 2.1 µM on primary chronic lymphocytic leukemia (CLL) cells, which is similar to 29H (IC50 of 1.5 1 µM) . [64] In 2015, they tested 29H, which was the best compound found at this time, against two prostate cancer cell lines, namely LnCaP and DU-145. Interesting IC50 values were found for 29H (IC50 (LnCaP) = 11-17 µM ; IC50 (DU-145) = 18-27 µM) compared to the N-unsubstituted prodrug 29D, which was not toxic towards both cell lines (IC50 >50 µM). These values correlated with an increase of intracellular ROS amount by 11-18 fold compared to that observed with the ferrocene alone. [88] Preliminary in vivo studies also showed a strong concentration-dependent growth inhibition of the tumor after treatment with 29H.

B O O R' O O N R'' Fe H 2 O 2 OH R' O O N R'' Fe O R' O O N R'' Fe CO 2 NH R'' Fe O R' Fe 2+ /Fe 3+
However, at low micromolar concentration of the prodrugs, the activation by ROS is not efficient enough. Since lysosomes contain a higher concentration of ROS than the cytoplasm, Mokhir et al. designed prodrug analogues of 29H containing an alkylated piperidine fragment to target the lysosomes (Scheme 25). [89] The acidic environment of the lysosome protonates the piperidine, leading to the trapping of the prodrug in this organelle. The authors studied the toxicity of 31 (Scheme 25) against human blood cancer cell lines (i.e., BL-2 and Jurkat-cells). ROS generation is also more important in the mitochondria than in the cytoplasm. That is the reason why Mokhir's team explored a hybrid of 29H that accumulates in mitochondria after ROS activation in cells. [90] Since the first model envisioned was not very efficient, the authors designed new prodrugs 32 and 33 (Figure 4), which first accumulate in mitochondria before being activated. [91] Unfortunately, ROS produced in mitochondria were shown to be insufficient for activation of the ROS-responsive prodrugs. In parallel, Mokhir and their co-workers also developed Pt(IV) prodrugs based on this ROSactivated prodrug system. Pt(IV) prodrugs are known to overcome the usual side effects of Pt(II) drugs and to show activity against cisplatin-resistant cancer cells. Unfortunately, extracellular reduction of Pt(IV) prodrugs decreased their anticancer activity. To overcome this limitation, the group designed a Pt(IV)-based prodrug 34 containing an aminoferrocene (AmFc) moiety, which can be activated by ROS thanks to the boronic pinacol ester fragment (Scheme 26). The first model described in 2017 used a cisplatin-Pt(IV) complex and showed nice preliminary results (IC50(34) = 2.5 ± 0.5 µM on human carcinoma A2780 cells and IC50(34) = 6 ± 1 µM towards cisplatin-resistant cell line A2780cis). [92] Yet, the activity was not better than cisplatin alone (IC50(cisplatin) 2.1 µM). To improve the model, the authors used an-oxaliplatin based Pt(IV) prodrug instead of a cisplatin one (compound 35 in Scheme 26) and shortened the linker by one methylene group between the AmFc moiety and the Pt(IV) to improve the intramolecular e --transfer (Scheme 26). [93] The prodrug 35 showed a better cytotoxicity than 34 on A2780 cells (IC50(35) = 0.4 ± 0.1 µM) and against A2780cis cells (IC50(35) = 0.7 ± 0.2 µM)

and was found to be not cytotoxic for normal cells. In vivo experiments still need to be conducted to assess the full potential of this compound.

H 3 N Pt H 3 N Cl Cl O O O N N N N O O B O O H 2 O 2 2x QM

Photo-deprotection

The use of a light-triggered prodrug strategy is an effective approach to provide temporal and spatial control of the drug release. Unlike photodynamic therapy (PDT), this light-mediated strategy does not rely on the presence of oxygen in the tumor, allowing hypoxic solid tumors to be targeted. This strategy is based on a photoremovable moiety, which releases the active drug upon light irradiation. This technique was widely used to release organic compounds, sometimes using metal-based photo-cages like Ru(II) polypyridyl complexes -we note that we do not discuss in this article the use of metal complexes in photo-activated chemotherapy (PACT) since this topic has been recently reviewed and since we focus on the release of active metal complexes. [94,95] To the best of our knowledge, it was only in 2013 that the first metal complex, namely an inert Ru(II) complex, was photo-released upon light irradiation. [96] Indeed, Gasser et al. first developed a Ru(II) polypyridyl complex [Ru(dppz) 2 (CppH)] 2+ , which was found to be extremely cytotoxic on different cell lines. [97] Knowing that the carboxylate function on the pyrimidine ring was essential for the cytotoxic activity, the authors hide it with a photocleavable moiety (i.e., DMNPB: 3-(4,5-dimethoxy-2-nitrophenyl)-2-butyl) to form 35

(Scheme 27). The cytotoxic evaluation was conducted on cervical cancer (Hela), bone cancer (U2OS), and non-cancerous lung fibroblast (MRC-5) in the dark and under 350 nm light irradiation (2.58 J . cm -2 ). In the dark, the prodrug 36 was found, as anticipated, to be not toxic, The same group then investigated this prodrug strategy on ruthenium and rhenium complexes conjugated to targeting peptides, such as bombesin, to boost the cellular uptake by malignant tissues and add cellular specificity. [98,99] The Re compound presented better activity than the Ru complex, but optimization are still required, especially for the choice of the wavelength to allow deeper tissue penetration for in vivo experiments. In 2016, the same team developed the first light-activable organometallic histone deacetylase (HDACs) inhibitor. [100] HDAC is a key enzyme in cancer as well as metabolic syndromes and neurodegenerative diseases, making HDAC inhibitors very attractive. SAHA (suberoylanilidehydroxamic acid) was approved to treat cutaneous T-cell lymphoma (CTCL), although it presents severe side effects and low efficiency on solid tumors. This is the reason why the authors designed a photo-labile HDAC inhibitor 38 combined with a ferrocene, on which the hydroxamic acid function was photocaged with a PLPG (i.e., 1-(bromomethyl)-4,5-dimethoxy-2-nitro-benzene) moiety (Scheme 28).

HDACs inhibition was evaluated on HDAC1, HDAC2, and HDAC6 enzymes in the dark and upon UV-A irradiation (350 nm; 2.79 J.cm -2 ). IC50 values determined upon light irradiation were similar to SAHA and Fc-SAHA. This first evaluation opened the field to other photoactivable HDACs inhibitors. Braber et al.

developed a photoactivable Pt(IV) complex 39 conjugated to an HDAC inhibitor (SubH:

suberoyl-bis-hydroxamic acid) (Scheme 29). [101] The prodrug 39 was then evaluated on human ovarian cell lines A2780 and resistant cell lines A2780cisR. It showed high toxicity values after irradiation at 365 nm (IC50(39) = 3.3 ± 0.3 µM versus IC50(cisplatin) = 18 ± 1 µM on A2780 cells and IC50(39) = 3.9 ± 0.5 µM versus IC50(cisplatin) = 45 ± 2 µM against A2780cisR cells).

These promising results need to be further explored, notably with in vivo experiments. Recently, Zhu and their co-workers developed a photoactivable carboplatin prodrug 41 linked to a BODIPY as a photoabsorber (Scheme 30). [102,103] Upon light activation, a transfer of energy occurs allowing the Pt(IV) center to be reduced. [104] The cytotoxicity of the prodrug 41 was higher on human breast carcinoma (MCF-7) cells and showed better activity under green light To minimize the cytotoxicity of metal complexes in the dark, Lo and their co-workers designed photoactivable Iridium(III) complexes 42a-b connected to a poly(ethyleneglycol) (PEG) chain (Scheme 31). [105] The authors used a nitroveratryl group as a photolabile protecting group between the complex and the PEG chain. PEGylation was shown to significantly decrease the cytotoxicity activity of luminescent transition metal complexes. [106,107] In this study, the cytotoxicity was evaluated on HeLa cells with the irradiation wavelength at 365 nm. In the dark, compounds 42a and 42b were found to be less toxic than the complex alone [IC50(42a) = 36.2 We note that a system combining both pH-and light-activation using a ruthenium complex was published (Scheme 32). [106] The acidic pH surrounding cancer cells activates the prodrug by weakening the bond between the phenanthroline spectator ligand and the metal center.

Photodissociation then occurs under irradiation at 450 nm (blue light) releasing the active complex. This system shows promising results to minimize off targets effects (IC50: 4 μM with 450 nm light compared to cisplatin IC50: 2 μM on MCF-7 breast cancer).

Summary and Outlook

In summary, stimuli-responsive metal-based prodrugs are promising tools to overcome the inherent drawbacks of metal-based drugs such as poor aqueous solubility, toxicity, and unfavorable biodistribution. The use of tumor-associated stimuli to selectively deliver metal complexes within malignant tissues showed promising results for both therapeutic and diagnostic applications. In this review, we reported different activation modes (endogenous or exogenous), allowing for site-specific delivery of the complexes. To date, to the best of our knowledge, none of the prodrugs discussed in this review has entered clinical trial, possibly due to a lack of activity in vivo or some biodistributions issues. The future directions of these prodrugs system will be to overcome these challenges by improving their accumulation in targeted tissues and controlling the release more precisely. Moreover, the exact mechanism of action or the behavior in biological environment are still not fully understood. Their understanding would certainly help to develop more efficient prodrugs. However, we are confident that in the near future, stimuli-responsive metal-based prodrugs could become an efficient complementary method for the diagnosis and treatment of cancer.

Ivanna

  Amarsy received her B.Sc. in Biology and Chemistry from Paris-Saclay University (Paris, France) in 2018 and her M.Sc. in organic chemistry from the same institute in 2020. She is currently a Ph.D. student in the research group of Prof. Gilles Gasser at Chimie Paris-Tech, PSL University (Paris, France) in co-supervision with Prof. Sébastien Papot from the University of Poitiers (France) studying the design and synthesis of metal-based anticancer complexes delivery system. Sébastien Papot is Professor of organic chemistry at the University of Poitiers (France) where he is heading the Programmed Molecular Systems Team within the IC2MP (Institut de Chimie des Milieux et des Matériaux de Poitiers). Sébastien was the recipient of the Pierre Fabre Award for Therapeutic Innovation (2014). He was the President of the French Medicinal Chemistry Society (SCT, 2019-2020). Sébastien is also the co-founder and CSO of Seekyo, a biotechnology company developing new generation cancer treatments. His research interests include the design of smart drug delivery systems, in vivo chemistry, functional interlocked systems and prebiotic chemistry. Gilles Gasser started his independent scientific career at the University of Zurich (Switzerland) in 2010 before moving to Chimie ParisTech, PSL University (Paris, France) in 2016 to take a PSL Chair of Excellence. Gilles was the recipient of several fellowships and awards including the Alfred Werner Award from the Swiss Chemical Society, an ERC Consolidator Grant, the European BioInorganic Chemistry (EuroBIC) medal and the Pierre Fabre Award for therapeutic innovation from the French Société de Chimie Thérapeutique. He is a Fellow of the Royal Society of Chemistry since 2021. Gilles' research interests lay in the use of metal complexes in different areas of medicinal and biological chemistry.

Figure 2 .

 2 Figure 2. Examples of metallodrugs (A) bismuth subsalicylate (B) cisplatin (C) auranofin.

Figure 1 .

 1 Figure 1. Representative diagram of the different applications of a metallodrug.

Scheme 1 .

 1 Scheme 1. General mechanism of the galactosidase-responsive contrast agent: EGad and EGadMe.

4 ,

 4 namely Gd[(DOTA-FP)(H 2 O)]-HSA and Gd[(DOTA-FP)(H 2 O)]-β-gal respectively, via the mechanism depicted on Scheme 3. A higher relaxivity and T1 relaxation time was observed with the HSA-bound chelate agent compared to Egad 1a. This new generation of MRI contrast agents showed an enhanced signal intensity in the presence of bio-macromolecules such as βgal and HSA allowing for the monitoring of enzymatic activity in tumor expressing β-gal.

Scheme 3 .

 3 Scheme 3. New generation of Gd-contrast agent: Gd[(DOTA-FP)(H 2 O)]-HSA or -β-gal.

2 Scheme 4 .

 24 Scheme 4. Lanthanide MRI and PARACEST probes developed by Chauvin et al.

Scheme 5 .

 5 Scheme 5. Visualization of the PRE effect and activation of 6.

Scheme 6 .

 6 Scheme 6. Mechanism of the Gd-DFP-Gal probe.

2 Scheme 7 .

 27 Scheme 7. General mechanism of gold nanoparticles enzyme-responsive probes allowing colorimetric assay for glycosidase activity detection.

Scheme 8 .

 8 Scheme 8. Metal-enhanced fluorescence mechanism of functionalized gold nanoparticles.

  Scheme 9. β-Glucuronidase-catalysed release of Gd(III)DO3A.

Scheme 10 .

 10 Scheme 10. Hydrolysis of a glucuronide-based Platinum prodrug.

2. 3

 3 . β-Lactamase a. Imaging Kikuchi et al. designed the 19 F MRI probe 13 based on the PRE effect enabling the monitoring of β-lactamase activity expressed on the surface of living cells (Scheme 11

Scheme 11 .

 11 Scheme 11. Structure of the Gd-FC-lac probe and its enzymatic hydrolysis.

17 Scheme 13 .

 1713 Scheme 13. Mechanism of the BODIPY-GSH probe from Zhao group.

Scheme 14 .

 14 Scheme 14. Fluorescence monitoring system of drug release.

20 Scheme 15 .

 2015 Scheme 15. BODIPY probe for the real-time detection of Pt(II) release.

Scheme 16 . 5 .

 165 Scheme 16. Mechanism of the release of camptothecin from the biotin-based Gd-contrast agent 5.

Sscheme 17 .

 17 Aminoferrocenne prodrug 22b activated by GSH.

Scheme 18 .

 18 Scheme 18. Mechanism of the dual theranostic effect of prodrug 23.

1, 6 - 2 Scheme 19 .

 6219 Scheme 19. Illustration of release of the Platinum and the bioactive drug.

  In a similar manner, Gasser, Gibson and their co-workers worked on a Ru(II)-Pt(IV) conjugate 25 (Scheme 20). Reduction of the platinum center releases both a Ru(II) polypyridine complex and cisplatin. The Ru(II) complex can act as a photodynamic therapy photosensitizer to generate singlet oxygen upon irradiation either at 480 or even 595 nm. The cytotoxicity of the conjugate was evaluated towards human ovarian carcinoma (A2780), human cisplatin resistant ovarian carcinoma (A2780 cis), and human doxorubicin resistant ovarian carcinoma (A2780 ADR) cell lines in the dark or upon light irradiation. The results were compared with both the Ru(II) complex alone and with cisplatin. The cytotoxicity in the dark of the conjugate 25 (IC50 = 0.98 -01.38 μM) was higher compared to the Ru complex (IC50 = 15.35 -18.60 μM) and cisplatin (IC50= 4.54 -19.53). The irradiation drastically improved the cytotoxic profile of 25 providing IC50 values in the nanomolar range (IC50, 480nm= 0.08 -0.10 μM, IC50, 595nm= 0.16 -0.19 μM).

25 cisplatin 20 .

 2520 Scheme 20. Release mechanism of the bioconjugate 25.

  Scheme 21. Acidic-mediated release of the Gd-contrast agent and doxorubicin.

Scheme 22 .

 22 Scheme 22. General release mechanism of HSA-based metal drugs.

Scheme 23 :

 23 Scheme 23: Formation of the HSA-Cu(Bp44 mT) prodrug followed by the release in acidic environment of the metal-based drug.

30 Scheme 24 .

 3024 Scheme 24. Structures and mechanisms of ROS-sensitive ferrocene prodrugs.

31 2 Scheme 25 .

 225 Scheme 25. Illustration of the lysosome-targeting ROS-responsive prodrug system.

33 Figure 4 .

 334 Figure 4. Structures of prodrugs 32 and 33.

Scheme 26 .

 26 Scheme 26. Structures of compounds A) 34 and B) 35. C) Release mechanism in the presence of ROS of 35.

while 37 was [IC50( 36 )Scheme 27 .

 3627 Scheme 27. Structure of 36 and its photodeprotection to form the cytotoxic [Ru(dppz)2(CppH)] 2+ .

38 Scheme 28 .

 3828 Scheme 28. light-activatable histone deacetylase inhibitor p-Fc-SAHA.

Scheme 29 .

 29 Scheme 29. Structure of 39 and its photodeprotection to form 40.

  irradiation than carboplatin (IC50(41) = 15.7 ± 1 µM versus IC50(carboplatin) = 642.6± 51.4 µM). The cytotoxicity of 41 under light irradiation was 39 times higher than carboplatin alone under irradiation. The phototoxicity index (PI) of 11 expresses the better efficiency of the prodrug upon light irradiation than in the dark (IC50(41) = 173.4 ± 8.8 µM in the dark).

41 Scheme 30 .

 4130 Scheme 30. Photoactivatable BODIPY-Platinum prodrug 41.

± 3 . 2 Scheme 31 :

 3231 Scheme 31: Photo-release of the prodrugs 42a-b

Scheme 32 :

 32 Scheme 32: Ruthenium complex combining both pH-and light-activation.

Ackowledgments

This work was financially supported by an ERC Consolidator Grant PhotoMedMet to G.G. (GA 681679), has received support under the program "Investissements d'Avenir" launched by the French Government, and was implemented by the ANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.). S.P. thanks La Ligue contre le Cancer (Ligue nationale, comités Vienne and Deux-Sèvres) and La Région Nouvelle Aquitaine for their financial support.

Graphical Abstract

Let's make metal-based drugs more selective! The main advances in the use of metal-based prodrugs are discussed in this review.


Su-Young Lee
email: su-young.lee@sophia.inria.fr

Frédéric Mallet
email: frederic.mallet@sophia.inria.fr

Robert De Simone
email: robert.de_simone@sophia.inria.fr

Dealing with AADL End-to-end Flow Latency with UML MARTE

AADL and MARTE are both modeling formalisms supporting the analysis of real-time embedded systems. We investigate how MARTE, with its Time Model facilities, can be made to represent faithfully AADL periodic/aperiodic tasks communicating through event or data ports, in an approach to end-to-end flow latency analysis.

Introduction

Modern embedded systems must support the deployment of heterogeneous applications onto heterogeneous architectures. At design time the targeted execution platform may still be speculative, or the same applications may tentatively be deployed onto various flexible architectures. Therefore, early performance estimation of the pairing, under imposed real-time constraints, is highly desirable. Such analysis requires a model of both the application and the architecture, and effective means to define the mapping of applicative functions onto architecture resources and services.

AADL and MARTE are two such modeling frameworks. AADL (Architecture Analysis & Design Language) [START_REF]SAE: Architecture Analysis and Design Language (AADL)[END_REF] was developed as a standard of the Society of Automotive Engineers (SAE), whereas MARTE (Modeling and Analysis of Real Time and Embedded systems) [START_REF]OMG: UML profile for Modeling and Analysis of Real-Time and Embedded systems (MARTE)[END_REF] is a recent OMG UML profile. Despite their many similar features (and MARTE being more detailed on the modeling aspects), AADL provides specific communication schemes between tasks, that need to be represented in MARTE: AADL tasks may be periodic or aperiodic, and in the former case of harmonic or independent periods; communication between tasks may use event-data or pure-data ports (with events triggering the recipient task behavior, while pure data are only sampled and used as such whenever the consuming tasks is otherwise activated).

Representing all these kinds of communications (periodic vs. aperiodic, event-triggered vs. sampled data) in MARTE is not only a challenge, but also an opportunity to provide timed semantics inside the modeling framework (and not aside, as separately provided semantic interpretation to time attributes). We build this semantic construction using MARTE Time Model [START_REF] André | Modeling Time(s)[END_REF], which is intended exactly for this: specifying in a formal way new timed domains of computation and communication.

We exemplify this approach by dealing with the same example as used in AADL [START_REF] Feiler | Flow Latency Analysis with the Architecture Analysis and Design Language (AADL)[END_REF] to explain the computation of end-to-end flow latencies (and various other related features) in a case of three threads with various rates (periodic or not) and connected through even-data or pure-data links in several locations. We show how these formulas are derived from time relations that are integral parts of the MARTE model.

A brief AADL overview

AADL supports the modeling of application software components (thread, subprogram, process), execution platform components (bus, memory, processor, device) and the binding of software onto execution platform. Each model element (software or execution platform) must be defined by a type and comes with at least one implementation. Type and implementation declarations also provide a set of properties to characterize model elements. For threads, the AADL standard properties include the dispatch protocol (periodic, aperiodic, sporadic, background), the period (if the dispatch protocol is periodic or sporadic), the deadline, the minimum and maximum execution times, along with many others.

AADL end-to-end flows explicitly identify a datastream from sensors to the external environment (actuators). Figure 3 represents such a flow. Note that this model is really an excerpt of the complete model and implies that the threads are declared within the context of one or many processes and bound to an execution host, i.e., a processor. All this contextual information is absolutely required to make the latency analysis since the execution platform and the topology determines the actual parallelism available. When executing on a single processor platform, the threads have to be serialized whereas a dual processor platform offers more parallelism. There are three kinds of ports: data, event and event-data. Data ports are for data transmissions without queuing. Connections between data ports are either immediate or delayed. Event ports are for communications of events that may be queued. The size of the queue may induce transfer delays that must be taken into account when performing latency analysis. Event data ports are for message transmission with queuing, here again the queue size may induce transfer delays. On Figure 3, all components have event-data ports represented as a solid triangle (as for data ports) with its shadow (as for event ports).

MARTE for AADL

The emerging UML2 Profile for MARTE is expected to be the basis for UML representation of AADL models [START_REF] Faugère | MARTE: Also an UML Profile for Modeling AADL Applications[END_REF]. The adopted MARTE OMG specification provides guidelines in this direction. The main goal of this paper is to further investigate how specific AADL concepts required for end-to-end flow latency analysis can be represented in MARTE. As such, this work is not (yet?) included in the official standard annex.

The idea here is to define, once and for all, a model library for AADL with MARTE. The end-user is not expected to enter into each and every detail about this library and most of the time he should be able to use it as a black box. The following section illustrates the use of this library on two selected examples. For brevity, we only present here the model elements required for dealing with the latency analysis example.

AADL application software components with MARTE.

The first step is to create classifiers to represent AADL threads. We use the stereotype SwSchedulableResource from the Software Resource Modeling sub-profile (see Figure 4). Only classifiers for periodic and aperiodic threads are shown here. The properties deadlineElements and periodElements are explicitly identified so as to help model transformation tools to extract the right property. This makes it easier for the transformation tools to be language and domain independent. Hence, the exact spelling of the property names does not matter as long as they are referenced by the stereotype properties. Note that contrary to AADL only periodic threads have a property called period. The MARTE equivalent to the AADL type Time is NFP_Duration, defined in the

AADL hardware components with MARTE.

The Hardware Resource Modeling subprofile is used to model AADL hardware components (bus, memory, processor, and device). Possible equivalents using MARTE are given in Figure 6. Actually specific classifiers should be customized depending on the physical characteristics of the hardware components (throughput or latency of memories, clock speed of processors, etc.). The latency analysis performed here does not use any specific property, apart from the device latency, so these classifiers need not being specialized further.

AADL ports with MARTE.

UML component diagrams provide ports and connectors to connect components. The queuing policy should rather be represented on the algorithm itself, i.e., on a UML activity diagram. Activities are composed of actions. Ordering in which the actions are executed are given by a control flow. Data communications between the actions are represented with object flows. By default, an object flow has a queue, the size of which can be parameterized with its property upperBound. So object flows can be used to represent AADL communications using either event or event-data ports. UML allows the specification of a customized selection policy to select which one of the tokens stored in the object node is selected. Unfortunately, the selection behavior must select only one token making it impossible to represent the AADL dequeue protocol AllItems. This protocol dequeues all items from the port every time the port is read. Thus, only the dequeue protocol OneItem is supported.

To model data ports, UML provides DataStore nodes. On these nodes, the tokens are never consumed thus allowing for multiple readings of the same token. Using a data store node with an upper bound equal to one is a good way to represent communications through data ports.

On The difference between immediate and delayed communications is addressed in the next sub section, since it is not really a structural matter but rather a temporal aspect.

AADL MoCC with MARTE.

Aside the model elements, the time semantics of these elements must be defined. On one hand, the model of computation, i.e., when the processing starts, finishes or is aborted. On the other hand, the model of communications, i.e., what kind of communication is used. The MARTE Time subprofile, inspired from the theory of tags systems [START_REF] Sangiovanni-Vincentelli | A framework for comparing models of computation[END_REF], provides a set of general mechanisms to define MoCC. These modeling aspects should be hidden to end-users and we show here how to use MARTE, as a model architect, to build a partial The language to be used on these clock constraints is called Clock Constraint Specification Language (CCSL) and is defined as an annex of the MARTE specification.

Overall two kinds of communications are possible in AADL. Data-driven communications, where the execution of a given task is triggered by the availability of the data produced by the preceding (according to the ordering defined by the control flow) task(s). Sampled communications, where pure data are only sampled and used as such whenever the task is otherwise activated. Note that the nature (event, event-data, or data) of the ports involved in the communication is not enough to determine its kind.

For instance, data-driven communications exist in chains of aperiodic tasks (devices or threads) connected by event or event-data ports. They also exist with synchronous periodic tasks connected by dataports through an immediate connection. In that latter case, the consuming task becomes aperiodic and its execution is triggered by the completion of the producing task. The CCSL clock relation alternatesWith models data-driven communications.

step1.finish alternatesWith step2.start

Eq. 1 illustrates a data-driven communication from step1 to step2. Note that this constraint is not symmetrical since the completion of step1 may cause the execution of step2, but not the converse.

Sampled communications occurs in various cases. For instance, when two asynchronous tasks (whether periodic or not) communicate, but also when two synchronous periodic tasks are connected by data-ports through a delayed connection.

step2.start = step1.finish sampledTo ^step2 (2)

Eq. 2 illustrates a sampled communication from step1 to step2. ^step2 represents the activation condition of step2. If step2 is a periodic thread, its activation condition can be defined using the CCSL relation isPeriodicOn (see Eq. 3-4). c 100  idealClk discretizedBy 0.01 [START_REF] André | Modeling Time(s)[END_REF] ^step2 isPeriodicOn c 100 period=10 [START_REF] Feiler | Flow Latency Analysis with the Architecture Analysis and Design Language (AADL)[END_REF] idealClk is defined in the MARTE Time library and stands for a dense chronometric (related to physical time) perfect (with no jitter or any other flaw) clock. Eq. 3 defines c 100 by discretizing idealClk. The default unit of idealClk is the second (s), so c 100 is a 100-hz discrete chronometric clock. ^step2 is periodic on c 100 with period 10 (Eq. 4), that makes ^step2 a 10hz discrete chronometric clock.

Had step2 been an aperiodic thread, its activation condition would have been an unbound logical clock.

An example

The AADL description

In this section we combine all these elements into a complete model that derives from [START_REF] Feiler | Flow Latency Analysis with the Architecture Analysis and Design Language (AADL)[END_REF]. It is displayed using the OSATE Eclipse plug-in environment for AADL [7] in Figure 8.

Figure 8. The System in OSATE

This flow starts from a sensor (an aperiodic device instance) and sinks in an actuator (also aperiodic) through three process instances. Each process executes a single thread. The two devices are part of the execution platform and communicate via a bus (db1) with two processors (cpu1 and cpu2), which host the three processes with several possible bindings. All processes are executed by either the same processor, or any other combination. The actual binding is not represented on this figure as we have ignored the effects of communications, just as in the original example [START_REF] Feiler | Flow Latency Analysis with the Architecture Analysis and Design Language (AADL)[END_REF]. The component declarations and implementations are not presented here. The full AADL code is available in [START_REF] Feiler | Flow Latency Analysis with the Architecture Analysis and Design Language (AADL)[END_REF].

The MARTE representation (synchronous sampling flow case)

We start by describing the model algorithm with an UML activity diagram (see Figure 9, upper-most part). All communications are through event-data ports with infinite queues. Two actions (acquire and release) have been added as the behavior of devices, compare with Since all threads are periodic and synchronous, the first three communications (from acquire to step1, from step1 to step2, and from step2 to step3) are sampled communications. The last communication (from step3 to release) is data-driven, since the device is aperiodic.

clk  idealClk discretizedBy P (5)

Eq. 5 declares the common clock to all periodic threads. Figure 10 represents the CCSL constraints and an equivalent graphical representation. Green arrows represent the communications. Dashed arrows represent the instant relations. Plain arrows denote strict precedence whereas empty arrows denote nonstrict precedence. The first two lines can then be interpreted as follows. The instant at which the action acquire (Ds) finishes (Ds.finish) is located between two ticks of the clock, let clk i be this instant (Eq. 6).

(i  *) (Ds.finish  ]clk i , clk i+1 ]) [START_REF] Sangiovanni-Vincentelli | A framework for comparing models of computation[END_REF] This implies that the action step1 must follow the tick clk i+1 (Eq. 7).

step1.start 

[clk i+1 , clk i+2 [) (7)
That is the definition of an asynchronous sampling. The data emitted by the asynchronous device Ds is sampled by the synchronous thread t1, according to its clock clk.

The AADL binding mechanism finds its equivalent in the MARTE allocation package. First, actions and object nodes are allocated (dashed arrows on Figure 9) to software components. Second, software components are allocated to execution platform model elements.

All these annotations (stereotypes) can be extracted using model-driven engineering techniques and fed into time analysis tools, including AADL latency analysis tool. Then, we go a bit further than AADL, by bringing back the latency analysis results into UML and MARTE in the form of timing diagrams (Figure 11). The timing diagram represents a family of possible schedules for a given execution flow and a given pair application/execution platform. timing diagrams to have these "oblique" lines, but it is a convenient notation to represent intermediate communication states between two steady processing states (e.g., between Ds and t1). Assuming, as in [START_REF] Feiler | Flow Latency Analysis with the Architecture Analysis and Design Language (AADL)[END_REF], that the sampling delays are always maximal, we get the same formulas (reproduced below) as the AADL latency analysis tool.

The MARTE representation (Mixed Event-data flow case)

We study here a second possible configuration extracted from [START_REF] Feiler | Flow Latency Analysis with the Architecture Analysis and Design Language (AADL)[END_REF] that only differs by making aperiodic the thread t2 (Figure 12). Few other cases involving data ports are studied in [START_REF] André | Modeling of Immediate vs. Delayed Data Communications: from AADL to UML MARTE[END_REF]. In that configuration, the communication from step1 to step2 becomes data-driven. The CCSL constraint is adapted accordingly (Figure 13). We also get a different timing diagram (Figure 14) and different flow latency formulas.

Conclusion

We have shown how MARTE could be used to model mixed systems with both periodic and aperiodic tasks, which is a big issue while modeling embedded systems. We have compared MARTE and AADL in this matter, highlighting MARTE capabilities to make the computation formulas explicit. Two different configurations are illustrated.

More generally, MARTE and its time model could be used to model various timed models of computation and communication.

Figure 1 .

 1 Figure 1. Declaration and implementation of both a process and a thread.

Figure 1

 1 Figure1presents the declarations of one process type and one thread type. Each of these declarations

Figure 2

 2 illustrates the case where a process executes two threads (t1 and t2) sequentially.

Figure 2 .

 2 Figure 2. Two threads executed sequentially.

Figure 8 ,

 8 detailed later, gives a more faithful view of the complete model.

Figure 3 .

 3 Figure 3. Flow extracted from an AADL model.

Figure 4 .

 4 Figure 4. AADL thread declarations.

  MARTE::BasicNFP_Types (Non UML&AADL'08. Belfast, Northern Ireland. April 2008, pp. 228-233 The original publication is available from http://dx.doi.org/10.1109/ICECCS.2008.14 © 2008 IEEE Functional Property Types) model library. An NFP_Duration value is defined as a tuple containing a real value and a time unit, among others.

  Figure 5 shows examples of thread instances, the left one being periodic and the right one being aperiodic.

Figure 5 .

 5 Figure 5. Two thread instances. NFP_Duration only supports the description of duration values associated with an ideal chronometric clock, which is exactly what AADL supports. Had we wanted to support logical clocks we would have used the templateable TimedValueType defined in the MARTE::Time model library.

Figure 6 .

 6 Figure 6. AADL hardware components.

Figure 7 ,

 7 the upper part shows an event-based communication with a queue size of 4. The lower part illustrates a data-based communication.

Figure 7 .

 7 Figure 7. Event or data communications.

  Figure 3.

  UML&AADL'08. Belfast, Northern Ireland. April 2008, pp. 228-233 The original publication is available from http://dx.doi.org/10.1109/ICECCS.2008.14 © 2008 IEEE

Figure 9 .

 9 Figure 9. MARTE model, all periodic case.AADL software (Figure9, middle part) and execution platform (Figure9, lower part) components are modeled with composite structure diagrams using the classifiers defined in Section 3. The information extracted from the MARTE stereotypes helps defining adequate clock constraints (Figure10) following the methodology defined in Section 3.4.

Figure 10 .

 10 Figure 10. CCSL constraints, all-periodic case.

Figure 11 .

 11 Figure 11. Timing diagrams, all periodic case.Computation execution times (bold horizontal lines) are equal to the latency for devices and range between the MinimumExecutionTime and the Deadline for threads. Oblique lines linking two computation lines represent the communications and the sampling delays. For sampled communications, this amounts to wait for the next tick of the receiver clock. The maximal sampling delay is when the communication waits for the full sampling period because the previous tick has just been missed. It is not normative in UML

Figure 12 .

 12 Figure 12. MARTE model, mixed case.

Figure 13 .

 13 Figure 13. CCSL constraints, mixed case.

Figure 14 .

 14 Figure 14. Timing diagram, mixed case.

  UML&AADL'08. Belfast, Northern Ireland. April 2008, pp. 228-233 The original publication is available from http://dx.doi.org/10.1109/ICECCS.2008.14 © 2008 IEEE MoCC suitable for AADL. To specify the time constraints with MARTE we use the stereotype

	ClockConstraint	that	extends	the	metaclass
	UML::Constraint.


Lorris Saoudi

Hugues Girard

Jean-Charles Arnault

Colloidal stability of synthetic hydrogenated nanodiamonds in water

Colloidal stability of synthetic hydrogenated nanodiamonds in water

L. Saoudi 1 , H. A. Girard 1 , J. C. Arnault 1 1 Université Paris-Saclay, CEA, CNRS, NIMBE, CEDEX, 91 191 Gif sur Yvette, France Two main processes are used to synthesize nanodiamonds (NDs): (i) by detonation (DND) giving rise to 5 nm mono-dispersed but highly defective nanoparticles, or (ii) by milling of bulk diamond (natural or synthetic) into polydispersed nanoparticles (MND). The latter preserves the crystallinity of bulk diamond and similar semi-conductor properties suitable for energy related applications. Able to host NV or SiV color centers, these MND are also currently under study for nanomedecine or quantum applications [START_REF] Nunn | Brilliant blue, green, yellow, and red fluorescent diamond particles: synthesis, characterization, and multiplex imaging demonstrations[END_REF]. These applications imply a fine control of colloidal properties, especially for a hydrogenated surface chemistry. Hydrogenated DND colloidal stability is well understood but its hydrogenated MND counterpart still needs further comprehension for a stability over time.

State of the art research has demonstrated that sp 2 carbon content, generated by a prior vacuum annealing, is essential to confer the colloidal stability to MND hydrogenated under pure H2 in mild conditions [START_REF] Ginés | Positive zeta potential of nanodiamonds[END_REF]. The present study reports on another route towards colloidal hydrogenated MND, which relies on the direct treatment of untreated MND at high annealing temperature (750°C) and under hydrogen pressures ranging from 10 mbar to atmospheric pressure. Our results show the possibility to obtain stable suspensions of hydrogenated MND over several months. Colloidal properties were investigated through DLS, zetametry and SAXS measurements. A thorough analysis of surface chemistry and morphology (FTIR, XPS, Raman, SEM, XRD) enables us to correlate stability of H-MND to their surface chemistry. Finally, the origin of the colloidal stability of hydrogenated DND and MND will be discussed.


est destinée au dépôt

Radiolysis on electrolytes of batteries: an efficient and quick screening for a fast selection of electrolytes

Yanis Levieux-Souid, a Jean-Frédéric Martin, b Recently we demonstrated that the highly reactive species generated by radiolysis (the chemical reactivity induced by the interaction between matter and ionizing radiation) of an electrolyte are the same as those generated during the cycling of lithium-ion batteries (LIBs) using similar solvents. [START_REF] Ortiz | [END_REF][2][3][START_REF] Varenne | Sustainable Energy & Fuels[END_REF] Furthermore, radiolysis generated these species in measurable amounts at time scales significantly shorter than that of electrochemical cycling in a battery cell (minutes vs. days) and enabled proposing reaction mechanisms and measuring rate constants. [START_REF] Ortiz | [END_REF][2][3] In order to provide safe and reliable lithium-ion batteries, the knowledge of aging phenomena is crucial. Among the different parts of the system prone to aging, the behavior of the electrolyte is a key parameter. The screening of different electrolytes is then of major interest. However, screening studies on electrolytes are time-consuming because they require numerous cycles in batteries; they are thus barely found. We demonstrate here that radiation chemistry is a valuable tool to screen very quickly, within a few hours, various electrolytes. Comparison with literature data providing classification of batteries by their performance efficiency showed that the H2 production under electrolyte irradiation allowed obtaining the same stability order. It allowed a direct comparison with experiments performed on pouch-cells. Therefore, we evidenced that irradiation experiments on electrolytes alone, lasting a few hours without any manufacturing operations such as the fabrication of electrochemical cells, makes it possible to reproduce trends observed during battery cycling. This work attests to the interest of radiolysis studies for the effective, reliable and fast screening of electrolytes.

  Philippe Moreau, c Nathalie Herlin-Boime a and Sophie

	Le Caër a*
	a NIMBE, UMR 3685 CEA, CNRS, Université Paris-Saclay, CEA Saclay F-91191 Gif-sur-Yvette
	Cedex, France.
	b CEA, LITEN, DEHT, STB, LM F-38054 Grenoble Cedex 9, France.
	c Institut des Matériaux Jean Rouxel, IMN, Université de Nantes, CNRS, 2 rue de la Houssinière, BP
	32229 F-44322 Nantes Cedex 3, France
	*email: sophie.le-caer@cea.fr

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