P Gros

A Bergel

M Comtat

Paul Sabatier

ELECTROCHEMICALLY ASSISTED CATALYST FOR ENZYMATIC GLUCOSE OXIDATION

A catalyst was made by entrapping glucose oxidase in a polymeric film deposited on an electrode surface. Its efficiency was studied by measuring the transformation of glucose by oxygen into gluconic acid. A numerical model was elaborated and validated with respect to experimental data. The electrochemical step affected the enzymatic catalysis in two ways: the reactant oxygen was regenerated, and the product, hydrogen peroxide which is a glucose oxidase inhibitor, was consumed. By monitoring these two concentrations, the electrochemical assistance significantly enhanced catalyst efficiency.

INTRODUCTION

The modification of electrode surfaces represents an important domain of applied electrochemistry, whose techniques and application fields are continuing to widen. Until now, electrode surface modifications have essentially been used to increase the selectivity and the detection performances of electrochemical sensors (Ivaska, 1991;Imisides et al., 1991), but they could also offer promising opportunities for synthesis processes. As a general rule, the method consists in immobilizing proven homogeneous catalysts on the electrode in order to use their intrinsic properties in association with the new possibilities given by the electrochemical intervention: fine concentration control of some species, continuous electrochemical regeneration of some reactants, or easy extraction of the catalyst from the solution at the end of the process [START_REF] Linford | Electrochemical Science and Technology of Polymers[END_REF]. Compared to a classic catalysis, this kind of electrochemical assistance allows the micro-environment of the catalyst to be monitored finely, by intervening directly in the core of the catalytic body.

A modified electrode was designed here, in order to assist electrochemically an enzymatic transformation. The enzymatic catalyst glucose oxidase was immobilized by entrapment inside a polymeric film formed at the electrode surface. Polymerization was performed by electrochemical oxidation of the pyrrole monomer, which is an increasingly common method used for electrode modification (Foulds and Lowe, 1986;Bartlett and Whitaker, 1987;Fortier et al., 1990). Among other advantages, a particular interest of the method is that it allows the thickness of the polymeric film to be controlled precisely (Holdcroft and Funt, 1988).

The entrapped glucose oxidase enzyme catalysed the oxidation of glucose by dissolved oxygen into gluconic acid:

GOD Glucose + O 2 + H 2 O -------> gluconic acid + H 2 O 2 (1)
When no potential was applied, the electrode only acted as an inert support, as would be the case with classic immobilized enzyme. However, when a suitable potential was applied, the hydrogen peroxide, which was produced by the enzymatic reaction, underwent oxidation at the electrode:

H 2 O 2 -------> O 2 + 2H + + 2e (2)
In this case, the electrochemical reaction (2) should enhance the enzymatic reaction rate (1) in two ways:

-by regenerating the reactant oxygen inside the catalytic film, -by consuming the hydrogen peroxide, which would have drastically inhibited and denatured the glucose oxidase.

The aim of this work is to quantify the enhancement effect of electrochemical assistance on a catalytic reaction.

Several groups have already discussed the interest of associating an electrochemical step with an enzymatic reaction.

Most of the studies consisted of experimental approaches aimed at demonstrating the feasibility of such processes [START_REF] Lortie | [END_REF]Cantet et al., 1995). A few specific electrochemical reactors have also been investigated (Fassouane et al., 1990;Grimes and Drueckhammer, 1993;Devaux et al., 1995) and a few theoretical works have dealt with modelling (Bauer et al., 1981;Bergel and Comtat, 1986;Labrune and Bergel, 1992). Nevertheless, most of the systems considered concerned homogeneous reactions or, when immobilized enzymes were used, this was only a means of retaining the enzyme near the electrode, without any regard to optimization (Mellor et al., 1992). To our knowledge, this is one of the first theoretical investigations dealing with the immobilisation of an enzyme in a polymer film in a view to the elaboration of a bioelectrochemical reactor.

The first part of the study is devoted to the elaboration of a theoretical model. The experimental data obtained with several modified electrodes with different film thicknesses were used to validate the model. The confrontation of the experimental and theoretical approaches achieved in this first part also led to a better description of the enzyme distribution inside the polymeric film. The model was used in the second part to quantify the improvement of the transformation reaction induced by the electrochemical regeneration of oxygen. This is discussed on the basis of the curves plotting the effectiveness factor as a function of the Thiele modulus. Furthermore, as predicted by the theoretical concentration profiles of hydrogen peroxide inside the catalytic film, the experiments conducted over longer periods of time in a batch reactor confirmed that the stability of glucose oxidase was increased when the hydrogen peroxide concentration in the film decreased.

EXPERIMENTAL SECTION

General

Glucose oxidase 220 U mg -1 (E.C. 1.1.3.4) type VII from Aspergillus niger, -D-glucose, potassium perchlorate and pyrrole were purchased from Sigma. All electrolytic solutions were prepared in aqueous phosphate buffer 0.1 mol l -1 pH 7.0. The electrode modifications were carried out in a one-compartiment cell at room temperature using a 0.03 cm 2 area platinum rotating disk working electrode. Enzymatic glucose oxidation was achieved in a laboratory scale batch reactor with a 4 cm 2 area platinum working electrode. In all cases an EGG model 362 potentiostatgalvanostat was used and connected to a Sefram x-y recorder. All potentials were measured versus a saturated calomel electrode (SCE). A large surface area platinum grid was used as counter electrode and placed opposite the working electrode to obtain homogeneous polymeric films. The glucose oxidase activity was measured spectrophotometrically according to the Sigma protocol. The unit is defined as the amount of enzyme necessary to catalyse the oxidation of one micromole of -D-glucose in one minute at 35°C with oxygen as electron acceptor.

Preparation of the modified electrode

The modified electrode was prepared by anodic electropolymerization of pyrrole 0.1 mol l -1 in phosphate buffer also containing perchlorate ions 0.1 mol l -1 as counter anions and glucose oxidase 1.5 mg ml -1 . The solution was first deaerated and a nitrogen flux was maintained during experiments to avoid the oxidation of the monomer by dissolved oxygen. The working electrode was immersed in the solution one minute before starting and one minute after the end of electropolymerization to ensure the adsorption of the glucose oxidase at the platinum and polymer surfaces. Polypyrrole films were grown galvanostatically with a current density value of 600 A cm -2 to keep a constant polymerization rate during electrolysis. The thickness of the polymeric films  p was varied between 10 nm and 1 m by changing the electrolysis time and was controlled during electropolymerization by means of the charge passed (Holdcroft and Funt, 1988). The polypyrrole films were electrochemically overoxidised after being synthesised to obtain an inert and non-conducting matrix. This was performed by cycling the potential of the working electrode twice between -0.3V/SCE and 1.2V/SCE at the sweep rate of 5 mV s -1 in the deaerated phosphate buffer.

Glucose oxidation

The rotating disk electrode modified by a polypyrrole film containing the glucose oxidase was immersed in a three electrode cell containing 5 ml of aerated phosphate buffer. The rotation speed of the working electrode was 500 rpm and its potential was held at 0.55V/SCE to ensure fast oxidation of the hydrogen peroxide produced by the enzymatic reaction. When a stable background current was reached (< 100 nA), 125 l of a 200 mmol l -1 glucose solution was injected to finally obtain a 5 mmol l -1 glucose solution. The current stabilized at a steady value in about thirty seconds.

In the case where the enzymatic reaction was achieved in the batch reactor, 2.5 ml of a 20 mmol l -1 glucose solution was introduced into the reactor. The solution was stirred with a magnetic bar. As soon as the modified electrode was immersed in the glucose solution, its potential was held at 0.4V/SCE. Under this condition the glucose solution was not found to be directly oxidised at the platinum electrode during at least 10 hours. The variation of the glucose concentration was measured enzymatically by using the Glucose [HK] reagent purchased from Sigma.

THEORY

Figure 1 represents the modified electrode structure highlighting the different locations of the enzyme entrapped in the polymeric matrix. Glucose oxidase was assumed to be uniformly distributed inside the polypyrrole film.

Previous experimental studies showed that the enzyme could also be adsorbed at the electrode and at the polymer surfaces (Bartlett and Caruana, 1992;Hämmerle et al., 1992;Gros and Bergel, 1995). Glucose and oxygen diffused from the bulk of the solution through the polymeric film and were simultaneously involved in the enzymatic reaction. Depending on wheter an electrode potential was applied or not, the hydrogen peroxide produced by the reaction underwent oxidation at the electrode or not.

Modelling was based on the steady state mass balance equations for the three species in the polymer. These differential equations involved both the mass transport of the species in the matrix and the enzymatic reaction kinetics. Assuming an enzymatic catalysis of the "ping-pong" type, the steady state mass balance equations for glucose, oxygen and hydrogen peroxide are respectively: In the case of electrochemically assisted enzymatic catalysis, experiments showed that applying an electrode potential of at least 0.4 V/SCE allowed the electrochemical oxidation of hydrogen peroxide to be mass transport controlled. In this case, the hydrogen peroxide concentration at the electrode surface is zero:

D g p d 2 C g d z 2 - r max 1 + K g /C g + K o /C o = 0 (3) D o p d 2 C o d z 2 - r max 1 + K g /C g + K o /C o = 0 (4) D h p d 2 C h d z 2 + r max 1 + K g /C g + K o /C o = 0 (
C h | z=0 = 0 (9)
Furthermore the mass flux densities of oxygen and hydrogen peroxide at the electrode surface are equal and opposite, according to equation 2:

D o p dC o d z | z=0 = -D h p dC h dz | z=0 (10)
The expression of the current induced by the electrochemical step took account of the hydrogen peroxide flux arising from the polymer, and of the hydrogen peroxide produced at the platinum surface by the adsorbed enzyme:

I = 2 F A ( D h p dC h d z | z=0 + r max ad 1 + K g /C g | z=0 + K o /C o | z=0 ) ( 11
)
The adsorption of the enzyme at the polypyrrole surface was considered in the corresponding boundary conditions.

Taking account of a linear concentration profile in the hydrodynamic diffusion layer, the equations are:

D g p dC g d z | z=dp + r max ad,p 1 + K g /C g | z=dp +K o /C o | z=dp = D g b  d ( C g b -C g s ) (12) D o p dC o d z | z=dp + r max ad,p 1 + K g /C g | z=dp +K o /C o | z=dp = D o b  d ( C o b -C o s ) (13) D h p dC h d z | z=dp - r max ad,p 1 + K g /C g | z=dp +K o /C o | z=dp = D h b  d ( C h b -C h s ) (14)
The glucose concentration in the bulk of the solution C g b was freely chosen. The oxygen C o b and hydrogen peroxide C h b concentrations were assumed to be always equal to 0.24 mmol l -1 and 0 mmol l -1 respectively.

Using dimensionless variables (Leypoldt and Gough, 1984) three principal dimensionless numbers were introduced (Coulson et al., 1979;[START_REF] Villermaux | Génie de la réaction chimique. Conception et fonctionnement des réacteurs[END_REF]: the Thiele modulus relating to glucose species which is the ratio of the potential enzymatic reaction rate without internal diffusion limitation to the maximum diffusion rate in the polymer:

 g 2 = r(C g s , C o s ) .  p 2  g . D g p . C g s (15)
the mass transfer Biot number for all three species, which represents the ratio of the external diffusion (in the solution) to the internal diffusion (in the polypyrrole film):

Bi j = D j b .  p  j .  d . D j p ( 16
)
and the effectiveness factor defined as the ratio of the effective reaction rate in the polymer to the enzymatic reaction rate without internal diffusion:

 = D g b  d ( C g b -C g s ) r(C g s , C o s ) .  p (17)
The numerical process was carried out by a classic finite difference method. An iterative calculation was necessary because of the non-linearity of the enzymatic kinetics.

RESULTS AND DISCUSSION

Model validation

Figure 2 shows the amperometric responses (discrete points) recorded for the oxidation of a 5 mmol l -1 glucose solution at the rotating disk electrode modified by polypyrrole films of different thicknesses. Glucose oxidase was entrapped in the polymeric matrix and adsorbed at the platinum and at the polymer surfaces as described above. The current increased rapidly in the first part of the curve and then decreased when the thickness of the film went beyond A similar experiment performed with the same modified electrode with the glucose oxidase only adsorbed at the polymer surface led to the determination of the value of the parameter r max ad,p . The other parameters adjusted were the maximum activity of the enzyme in the polymeric matrix r max and the diffusion coefficient of glucose in the polymer D g p . All other physico-chemical parameters were taken from the literature or determined by independent experiments, as described previously (Gros and Bergel, 1995), and always had the same values for all experiments. Figure 2 shows that the theoretical model succeeded well in fitting the experimental results.

Efficiency of the catalytic processs

The model was further used to predict the evolution of catalyst efficiency as a function of the Thiele modulus and thus to estimate the effect of the electrochemical assistance. The effectiveness factor variation is plotted in Figure 3.a. in the case of a 20 mmol l -1 glucose solution and where no potential was applied to the modified electrodes.

The continuous line shows the theoretical results obtained in the case where only the enzyme entrapped inside the polymeric matrix is involved in the calculation. This curve clearly highlights the two different types of behaviour as a function of the film thickness. For Thiele modulus values less than 0.25 the effectiveness factor is constant and its value is near 1. The reaction rate is limited by the enzymatic kinetics. When the Thiele modulus is more than 0.6 however, the effectiveness factor decreases strongly when the thickness of the polymer increases. The diffusion of glucose inside the film becomes therefore the rate determining factor. The dotted line corresponds to the theoretical calculations taking account of the glucose oxidase both entrapped inside the film and adsorbed at the platinum and at the polymer surfaces. The comparison of the two curves shows that the efficiency of the electrode increases in the presence of the adsorbed glucose oxidase for low Thiele modulus values only. In this case, kinetics is rate determining and the enzyme adsorbed either at the electrode or at the polymer surfaces significantly affected catalyst efficiency. The effectiveness factor reached a value higher than 1 because the maximum enzymatic reaction rate, which was estimated without internal diffusion in the calculation of the effectiveness factor, did not take account of the adsorbed enzyme layers. For Thiele modulus values greater than 0.6 this effect remained secondary. In the case where the potential is applied, experimental results have shown for instance that the adsorbed enzyme layers contributed for less than 10% of the amperometric response due to the oxidation of the glucose at a 250 nm thick polymer modified electrode (Gros and Bergel, 1995). the concentration profiles of the oxygen and of the hydrogen peroxide respectively in a 250 nm thick polypyrrole film with and without electrochemical assistance. Figure 4.a. shows that the average concentration of oxygen, that is the co-substrate of the enzymatic reaction, is greater when the potential is applied whereas the consumption of hydrogen peroxide at the electrode leads to a lower average concentration. These concentration profiles also illustrate the fact that the simplification assumption generally used in previous modellings, e.g. no external diffusion, negligible oxygen and hydrogen peroxide concentration gradient... are not valid for the general case.

Influence of the electrochemical assistance

Distance from electrode (nm) The enzymatic rate expression used in the model only took account of the concentrations of the reactants, oxygen and glucose. As a consequence, the influence of the electrochemical assistance was only estimated by the modifications which affected the oxygen concentration in the polymeric film. It was not possible to predict theoretically the influence of the hydrogen peroxide consumption without using a more complex kinetic expression, which should take account of the enzyme inhibition and/or denaturation by hydrogen peroxide. For this reason, an experimental investigation was carried out with a thin modified electrode. The thickness of the polymer, about 10 nm, corresponded to the Thiele modulus range where no influence of the electrochemical step was expected by the model, because the enzymatic kinetics is the rate determining factor. Nevertheless, the transformation ratios obtained experimentally after a 6 hour electrolysis of a 20 mmol l -1 glucose solution varied from 25% without the electrochemical step to 50% when the potential was applied. This difference can only be explained by a change in the enzymatic rate. The hydrogen peroxide consumption significantly decreased the enzyme inhibition and/or denaturation, which resulted in greater catalyst efficiency. The kinetics of the enzyme denaturation will have to be determined precisely so that this effect can be quantified by the model.

CONCLUSION

On the basis of an electrode modification technique, which is commonly used in the field of analytical chemistry, a method for designing catalysts is proposed here. The electrochemical step, which was thus added to the catalytic pathway, allowed the partial monitoring of the concentrations of the electroactive species in the catalyst body. In the example considered, a reactant was generated, while an inhibitory product was consumed. The two actions were demonstrated both theoretically and experimentally. The electrode modification domain, which is growing continuously, should afford more and more new possibilities for catalyst design. The conducting polymers could for instance help to improve the fine control of the concentrations inside the catalyst. In this case, the electrochemical step could occur throughout the polymeric structure, rather than being limited to the electrode surface.

Notation

A = electrode surface area.

Bi j = mass transfer Biot number for the species j.

C j = concentration of the species j.

D j = diffusivity of the species j.

Figure 1 .

 1 Figure 1. General kinetic scheme for the polypyrrole glucose oxidase (GOD) modified electrode.

  Figure 2. Variation of the amperometric response of the modified electrode as a function of the film thickness. ____ theoretical curve; experimental results.

Figure 3 .

 3 Figure 3. Influence of the Thiele modulus on the effectiveness factor.

Fig. 3

 3 Fig. 3.a. Influence of the location of the glucose oxidase. ___ enzyme only entrapped in the polymeric matrix; _ _ _ enzyme entrapped in the polymer and adsorbed at the electrode and the polymer surfaces.

Fig. 3

 3 Fig. 3.b. Influence of the electrochemical step. ___ without electrochemical assistance; _ _ _ with electrode potential.

Figure 3

 3 Figure 3.b. shows the influence of the electrochemical assistance on the efficiency of enzymatic catalysis. The continuous line is the same as that in figure 3.a. whereas the dotted line was obtained in the case where the potential was applied at the electrode. Figure 3.b. clearly shows that the electrochemical step has no influence on the effectiveness factor for low Thiele modulus because the enzymatic kinetics is the rate determining process. For high Thiele modulus, application of the potential, leading to the regeneration of the oxygen, significantly improves the efficiency of the catalysis. In this case the overall rate is limited by the internal diffusion and therfore depends strongly on the concentration of the species in the polymer. This result is confirmed by Figure 4.a. and 4.b. showing

Figure 4 .

 4 Figure 4. Concentration profile of oxygen (Fig. 4.a) and of hydrogen peroxide (Fig. 4.b) in a 250 nm thick polypyrrole film. ____ without electrochemical assistance; _ _ _ with electrode potential.

F

  Menten constant relative to the species j. r max = activity of the glucose oxidase in the polymer. r max ad = activity of the glucose oxidase adsorbed on the electrode surface.r max ad,p = activity of the glucose oxidase adsorbed on the polymer surface.

Acknowledgments

We thank Elf Aquitaine (Research Group of Lacq) and the C.N.R.S. (program PIRTEE-ECOTECH-ADEME) for financial support.


Dr Karine Porte

Dr Maxime Riberaud

Rémi Châtre

Dr Davide Audisio

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

Frédéric Taran
email: frederic.taran@cea.fr

Pr S Papot

Bioorthogonal Reactions in Animals

Keywords: In vivo chemistry, chemical biology, bioorthogonal chemistry, click chemistry, pretargeting

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In 2003, Bertozzi and co-workers introduced for the first time the concept of bioorthogonal chemistry. [1] Since then, the interest in this kind of chemistry has continued to grow exponentially and has become an essential tool in chemical biology. [2] Initially developed to understand and modulate biological fundamental mechanisms, bioorthogonal chemistry has recently found potential applications in the fields of diagnosis and targeted therapies. A rapid survey of the literature clearly indicates a dramatic increase in the number of publications over the last 10 years dealing with biorthogonal reactions on and inside living cells but chemistry within living animals, that is more complex to carry out, remains by far less explored. Several excellent perspective reviews have already treated some aspects of the emerging applications of in vivo chemistry. [3] Herein, we present an update of the recent examples of biorthogonal chemistry accomplished in animals to understand biological processes as well as to develop innovative diagnosis or therapeutic tools.

By definition, bioorthogonal reactions are compatible with biological media, display incredible selectivity and ideally involve stable, inert, non-natural chemical functions. However, these parameters alone are not sufficient for successful in vivo chemistry. Biostability, non-toxicity, favourable pharmacokinetics of bioorthogonal reactants and high kinetics of the reaction are also mandatory prerequisites. However, despite the importance of these parameters, only few studies have been performed to demonstrate the real in vivo bioorthogonality of the reactions and the biostability of the reactants. [4] Bioorthogonal reactions can be classified in three main categories: ligation, [5] cleavage [6] and clickand-release reactions. [7] Among the ligation reactions that have found a wide scope of in vivo applications (Figure 1a), the Tetrazine (Tz)/Trans-cyclooctene (TCO) ligation is the most described in the literature as the consequence of its favourable kinetics providing the most significant in vivo results. Furthermore, the Strain-promoted Azide-Alkyne (SPAAC) and Sydnone-Alkyne (SPSAC) cycloaddition reactions have also been successfully investigated for in vivo applications. Click and release reactions offer the possibility not only to link two molecules together but also to release a compound of interest, providing many new perspectives. Nowadays, only three click-and-release reactions have been successfully used in animals. The most widely used reaction is the one involving Tz and TCO derivatized by a carbamate adjacent to the olefin. Described for the first time by M. Robillard and co-workers in 2013 (Figure 1b), [8] this reaction has been recently improved in order to combine high kinetics and yields of released molecules. [9] The second reaction, which has been described in 2017 by our team, in vivo chemistry needs:

correspond to [3+2] cycloaddition followed by a retro Diels-Alder between Iminosydnones and Cyclooctynes (Strain-Promoted Iminosydnone-Cycloalkyne Cycloaddition, SPICC). [10] Very recently, Franzini et al. showed the applicability of the reaction between tetrazines (Tz) and isonitriles (IsoN) for the decaging of drugs. The reaction yields 4-iminopyrazoles, which upon hydrolysis and subsequent -elimination successfully release fluorophores or active drugs in zebrafish embryos. [11] 2. In vivo chemistry to study fundamental biological mechanisms

Glycome imaging in zebrafish and mice

After their pioneer work on Staudinger ligation (SL) [12], [13] and copper-catalyzed click chemistry [14], [15] to detect azide-labeled biomolecules on cells, Bertozzi and co-workers made the first step toward the use of bioorthogonal reactions in living systems. They brilliantly demonstrated that this chemistry can be a powerful tool to understand fundamental living mechanisms. By using an unnatural sugar containing an azide moiety, they developed an imaging method of glycans in zebrafish embryos. [16] After metabolic incorporation of peracetylated N-azidoacetylgalactosamine (Ac4GaINAz), addition of DIFO probes allowed the imaging of glycans into zebrafish embryos via SPAAC (Figure 2).

Using this methodology, the same group was also able to study the dynamic behaviour of glycans during zebrafish development by using up to three different dyes over time. This robust approach of metabolic labeling with Ac4GaINAz followed by detection via SPAAC, revealed differences in the cell-surface expression, but also in tissue distribution of glycans during zebrafish embryogenesis. Few years later, Wu and co-workers, reported the use of CuAAC ligation reaction for imaging of fucosylated glycans during zebrafish early embryogenesis. [START_REF] Soriano Del Amo | [END_REF] However, while this metal- catalysed strategy permitted efficient glycans labelling in zebrafish, [18], [19] its transfer in more complex living systems such as mice remains problematic. Indeed, in the case of this three components-based reaction, the azide, alkyne and copper catalyst have to be targeted in the same tissue to reach efficient concentrations, which is highly challenging. Furthermore, the potential toxicity of the copper is still an issue for the exploitation of this bioorthogonal reaction in mammals. In 2010, Bertozzi and co-workers demonstrated that the bioorthogonal ligation reaction between cyclooctynes and azides can occur under the physiological conditions prevailing in living mice. [START_REF] Chang | Proc. Natl. Acad. Sci[END_REF] First, the mice were injected with Ac4ManNAz once a day for one week to metabolically incorporate azides to cell-surface sialoglycoconjugates (Figure 3). After metabolic labelling, the corresponding azido sialic acids (SiaNAz) reacted in vivo through SPAAC reaction with a panel of cyclooctynes functionalized with a water-soluble FLAG peptide epitope tag (DYKDDDDK) to facilitate the detection of the ligation products. Finally, mice were euthanized and the splenocytes isolated to study the presence of cycloadducts by flow cytometry analysis. These experiments demonstrated that the biorthogonal reaction occurred in vivo at the surface of cells with an efficacy that was dependent of the structure of the cyclooctyne conjugate. The obtained outcomes showed that in addition to the intrinsic reactivity of bioorthogonal reagents, their pharmacokinetics played also a crucial role for cell labelling in vivo. Indeed, DIFO-FLAG which possesses the best reactivity with azides in vitro was less efficient than PHOS-FLAG, a reagent with a lower inherent reactivity with azides (Staudinger ligation), for the bioorthogonal labelling of splenocytes in vivo.

In vivo click to image the microbiota

In 2015, Kasper and co-workers, employed for the first time bioorthogonal reaction for in vivo imaging and tracking of hostmicrobiota interactions via metabolic labelling of gut anaerobic bacteria. [START_REF] Geva-Zatorsky | [END_REF] In this study, the authors used DIBO derivatives to track living bacteria. In a first instance, they incorporated azide functional groups into polysaccharides of B. fragilis by supplementing bacterial growth medium with azide-modified tetraacetylated-N-azidoacetylgalactosamine (GalNAz, Figure 4a). The subsequent incubation of cyclooctyne derivatives into the culture medium allowed the fluorescent labelling of B. fragilis by copper-free click reaction. After this first proof of concept, this strategy was adapted to visualize in real-time bacteria dissemination along the intestine in live mice (Figure 4b). Thus, by combining metabolic oligosaccharide engineering and bioorthogonal click chemistry, the authors reported an efficient method to detect and track live B. fragilis in the host.

In vivo enzyme profiling

Recently, bioorthogonal reactions emerged as efficient tools for profiling the activity of enzymes in vivo and understanding the role of these proteins in physiological and pathological processes. Dubikovskaya and co-workers described in vivo ligation between D-cysteine (D-Cys) and 2-cyanobenzothiazoles (CBT) to generate D-luciferin substrates for bioluminescent imaging of protease activity in living mice. [22] The authors tested this approach for a real time imaging of apoptosis associated with caspase 3/7 activities in living mice. The activity of caspase 3/7 was induced in the mice by injection of lipopolysaccharide (LPS) and D-galactosamine (d-GalN). Then, a combination of CBT and protected peptide substrate Asp-Glu-Val-Asp-D-Cys (DEVD-(D-Cys)) were injected to the mice (Figure 5). The caspase 3/7 activity in mice triggered the release of free D-Cys which upon bioorthogonal reaction with CBT allowed the formation of luciferin substrates with subsequent light emission from luciferase. The light was detected using a charge-coupled device (CCD) camera. In these experiments, the controlled caspase 3/7-catalysed luciferin formation produced a better light signal than that obtained using commercially available DEVD-aminoluciferin substrate, therefore showing the potential of this approach for both the quantification and imaging of protease activities in living animals. 3 In vivo imaging for diagnostic purposes

Pretargeting strategies

During the past 30 years, numerous pretargeting methods have been developed, with several preclinical results for both radiotherapy and radioimaging purposes. Pretargeting is a two steps strategy, which consist in (1) administering modified antibodies (mAbs) that can bind a specific biological target, and (2) injecting a radiolabelled probe that can react with the mAbstarget complex (Figure 6). The first pretargeting approach was described at the end of the 80's by Hnatowich et al. [23] . They used the biotin-streptavidin interaction to target agarose-streptavidin beads with 111 In-Biotin in living mice. However, this strategy was abandoned in phase II due to the streptavidin toxicity induced by the non-specific binding to endogenous biotin.

In 2011, van Dongen and co-workers [24] were the first to attempt a bioorthogonal approach for tumour pretargeting in living mice using the Staudinger ligation (SL) reaction. However, results did not live up to expectations probably due to the slow kinetics of the ligation reaction.

A few years later, the groups of Kim and Lee proposed to employ the SPAAC reaction instead of SL in order to improve the kinetics of the ligation. [25] For this purpose, they prepared glycol chitosan nanoparticles containing azides which they radiolabelled in vivo with 64 Cu-DOTA-Lys-PEG4-DBCO for PET imaging. With this approach, they were able to localize tumours in mice bearing SCC7 xenografts.

In 2013, Robillard and co-workers synthetized anti-CD20 mAbs functionalized with azides and a series of 177 Lu-labelled DOTA linked to cyclooctyne derivatives with the aim to evaluate their capability to react in mice. [26] Unfortunately, control experiments carried out in mice without tumours highlighted the low efficiency of the SPAAC reaction. Shortly afterward, Kim and co-workers also used the SPAAC reaction to develop a pretargeting approach based on the permeability and retention effect (EPR) with mesoporous silica nanoparticles as the targeting unit instead of mAbs. [27] These nanoparticles were previously functionalized by dibenzocyclooctyne (DBCO) and administered into tumour-bearing mice. Once non-targeted nanoparticles eliminated from the blood (24 h after their i.v. administration), a 18 F-labelled azide was injected and the accumulation of radioactivity was followed by PET-CT imaging. Under these conditions, an accumulation of the radioactivity at the tumour site was observed while much less radioactivity was detected in the control group that did not received the pretargeting agent. These results indicated that SPAAC can be successfully used for tumour pretargeting but preferentially using nanoparticles with high density of biorthogonal functions at their surfaces.

Undoubtedly, the most efficient reaction for pretargeting is the inverse-electron-demand Diels-Alder reaction (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine derivatives (Tz). The first use of TCO/Tz reaction for pretargeting application in living mice was described in 2010 by Rossin et al. (Figure 7, entry 1). [28] They injected a non-internalizing CC49 mAbs functionalized with PEG12-TCO moieties to mice-bearing colorectal tumors (LS174T). After a clearance time of 24 hours, a radiolabeled-111 In-DOTA-PEG12-Tz was then administered. Radioactivity monitoring by SPECT-CT proved the efficacy of IEDDA reaction for pretargeting purposes. Furthermore, pioneer work from Weissleder in 2012 demonstrated the importance of the clearance kinetics of the radiolabeled secondary agent. [29] Their work indicated indeed that long circulating derivatives such as polymers might strongly increase the in vivo labelling efficiency.

Several other experiments were conducted by many teams using different tumour models, mAbs-TCO, Tz-based radioligands and different radioisotopes ( 111 In, 18 F, 99m TC, 64 Cu, 212 Pb 177 Lu, 225 Ac, 68 Ga). In Figure 7 we selected some of the successive improvements in the pretargeting strategies developed for imaging purposes. [30] An issue that arises with pretargeting strategies is that a significant portion of the injected antibody remains in circulation, causing a reduction in target-to-background ratios. In 2013 Robillard et al. used for the first time a masking TCO agent to neutralize circulating antibodies in order to improve the pretargeting approach (Figure 7, entry 2). [31] The idea was to inject a clearing Tz-based reagent, which can specifically react with the free circulating TCO-mAbs in the blood stream (and not at the tumor site), in order to mask these mAbs before the injection of the Tz-based radioligand. They demonstrated the value of this concept with a Tz-based-galactose clearing agent (CA) which allowed an improvement of 125-fold of the tumour/blood ratio. Following these results, several other teams tried to improve CAs by using different clearing moiety such as albumin, polystyrenebased particles, or dextran polymers. [32], [32], [33], [34], [35], [36] IEDDA-based pretargeting approach was implemented by Lin's team with the use of TCO-functionalised nanoparticles. [37] They first administrated the nanoparticles into mice with tumours and, after a delay of 24h, injected a 64 Cu-radiolabelled Tz-DOTA complex and followed the reaction by PET imaging. They found a strong PET signal at the tumour site, but also in the liver due to trapping of the nanoparticles by the mononuclear phagocyte system or trans-chelation of 64 Cu from DOTA in vivo.

In addition to the radiodetection or NIR detection, Valliant and co-workers extended the TCO/Tz pretargeting approach to ultrasound imaging (Figure 7, entry 7). [38] They first injected TCOconjugated mAbs VEGFR2-positive (modified EGFR antigen) murine xenografts and after 24 hours of clearance delay, they administered intravenously Tz-functionalized microbubbles and monitored the reaction by ultrasound imaging. They demonstrate high retention of the microbubbles at the tumour site with significant contrast enhancement compared to mice without TCO-mAb pre-treatment. Despite all these encouraging results, pretargeting approaches based on the TCO/Tz reaction has not reached clinical trials yet. The main problem remains the TCO isomerization into its inactive form (cis-cyclooctene) that cannot react with the tetrazine moiety. Nevertheless, some teams tried to minimize these side reactions by modulating some parameters of the mAbs such as the PEG linker length, the number of TCO per antibody and their localisation on the immunoconjugate. The Tz structure was also tuned to improve pharmacokinetics. For more details on the recent advancements up to the year 2019 on the TCO/Tz pretargeting approach, we direct the readers to consult the recent review published by Rondon and Degoul. [39] Figure 8. in vivo pretargeting studies involving SPSAC approach Besides the above-described reactions, our team recently evaluated the SPSAC reaction for in vivo PET imaging (Figure 8). [40] We demonstrated that this strain promoted reaction between 4-chloro-sydnones and [ 18 F]F-PEG2-DBCO can occur in vivo with a good kinetic (k = 300 L mol -1 s -1 ). PET imaging of mice bearing subcutaneous A431 xenografts showed a specific tumour uptake but hepatobiliary elimination of the [ 18 F]F-PEG2-DBCO was very low, leading to a low imaging contrast. With further optimization of the labelled cyclooctyne pharmacokinetics, we do hope that this strategy becomes a practical tool for pretargeting imaging.

Cancer imaging using metabolic glycoengineering

Among the chemical biology approaches to study cancers, metabolic glycoengineering (MGE) is one of the most used alternatives to the classic pretargeting. Specific changes in glycan profiles, such as overexpression of sialic acid in tumour tissues suggest that glycans could be employed in clinical diagnostics. [41] MGE involves the introduction of non-natural chemical groups into the surface of cells by metabolization of specific monosaccharides bearing a bioorthogonal functional group. Among them, the azide moiety is the most utilized because its incorporation into the tumour glycan is well established through metabolization of the monosaccharide Ac4ManNAz. Cancer cells decorated by multiple azide functions can then be functionalized and detected through either the SPAAC or SL reactions.

Brindle and co-workers brilliantly demonstrated for the first time an in vivo bioorthogonal metabolic labelling strategy allowing the detection of sialylated cell surface glycans by radio-and fluorescence imaging (Figure 9, entry 1). [42] They intraperitoneally injected Ac4ManNAz, which was metabolized in azidosialic acid and incorporated at the surface of cancer cells. After 24h, they injected a biotinylated phosphine (bPhp) which can react by SL with the azidosialic acids present at the surface of tumour cells. Finally, the biotin moiety was detected by subsequent intravenous injection of a fluorescent or radiolabeled avidin derivative. Because SL and SPAAC reactions are rather slow, targeting strategies to increase the local concentrations of either the phosphine or cyclooctyne partners might improve the efficiency of the attachment to the azides incorporated into tumor glycans. Koo and co-workers [43] were the first to follow this strategy by introducing azide groups into tumor-A549-bearing mice through intra-tumoral injection of Ac4ManNAz followed by i.v administration of DBCO encapsulated in liposomes (Figure 9, entry 2). The reaction was monitored by fluorescence and showed a significantly higher accumulation of DBCO-liposome in mice pretreated by Ac4NanNAz, thereby proving the efficacy of the in vivo SPAAC reaction. The selective introduction of azide groups into cancer cells while sparing healthy cells is also a very challenging task. Several Ac4ManNAz analogues have been developed (Figure 9, Entries 3-8) to favour intravenous rather than intratumoral injection. [44] For instance, Shim and co-workers developed a cancer cell-specific azide labelling strategy by designing an Ac3ManNAz derivative (RR-S-Ac3ManNAz) that can be cleaved by cathepsin B overexpressed in tumours to release Ac3ManNAz. [45] They intravenously injected RR-S-Ac3ManNAz in HT-29-bearing mice, followed by administration of DBCO-Cy5.5. They successfully monitored the fluorescence signal in the tumour with a good signal compared to non-pre-treated mice. Based on the same approach Wang and co-workers exploited two cancer-overexpressed proteases: histone deacetylase and cathepsin L which can selectively deprotected 1-O-protected-Ac3ManNAz (DCL-AAM) to incorporate azides to cancer cells (Figure 9, entry 8). [46] Lee's team synthetized chitosan nanoparticles (CNPs) coated by Ac4ManNAz to improve the accumulation of Ac4ManNAz moiety at the tumour site via the EPR effect (Figure 9, entry 3). [47] They intravenously injected Ac4ManNAz-CNPs and found a 2-fold higher azide expression on the tumour site than with direct injection of Ac4ManNAz. Cheng's group also adapted MGE to ultrasound-labelling by using Ac4ManNAz-loaded microbubbles (Figure 9, entry 4). [48] They injected these microbubbles in 4T1-bearing-mice and by applying high-amplitude ultrasound released Ac4ManNAz from them selectively in the tumour. After the injection of DBCO-Cy5, fluorescence monitoring confirmed the efficiency of the process but, unfortunately, this strategy can be used only if the tumour location is well-known.

Based on a different approach, Goun and co-workers reported the development of a bioluminescent glucose-uptake probe for glucose absorption imaging both in vitro and in vivo (Figure 10). [49] Figure 10. Imaging and quantification of D-glucose uptake in 4T1-Luc-bearing Swiss nu/nu mice using the GAz and CLP. GAz was injected 24 h after CLP.

They designed a triarylphosphine luciferin cleavable ester (CLP) which can accumulate in cells and "decaged" luciferin in presence of a modified-azido-glucose (GAz) via SL reaction. The luciferin can further react with Luciferase to produce light, which can be monitored by a specific camera imaging system. They demonstrated the application of their probe by imaging cancer in mice bearing xenograft tumour of 4T1-luc and 4T1-luc GLUT1 knockouts. They observed lower light signal in GLUT1 knockout mice in comparison to 4T1-luc controls. In addition, they proved that this method can be useful to find new GLUT inhibitors in vivo. They also brilliantly demonstrated that this approach is comparable to PET imaging in term of sensitivity.

In vivo biorthogonal chemistry for therapeutic applications 4.1 Biorthogonal systems for selective drug delivery in targeted tissues

The selective activation of non-toxic prodrugs within diseased tissues represents an attractive alternative to standard treatments with the aim to enhance their efficacy, while limiting their adverse effects. In this context, the use of bioorthogonal cleavage or clickand-release reactions for drug decaging at desired locations offers stimulating perspectives for developing more selective therapeutic strategies, since this triggering method is independent of endogenous chemical and biochemical markers (e.g. pH, enzymes…). Along the past decade, such approaches have been investigated in vivo through various protocols involving either the preimplantation or the targeting of at least one of the bioorthogonal partners in the tissues of interest. Similar strategies have been also developed for the selective activation of masked probes at specific locations. These studies were mainly conducted to demonstrate that the selected bioorthogonal reactions could occur in animals, thus allowing to envision therapeutic perspectives. The major advances in this field are summarized in the following paragraphs. By exploring palladium-catalysed depropargylation reactions in biological medium, Bradley and Uniciti-Broceta were the first who highlighted the potential of bioorthogonal cleavage processes in a living organism for biomedical applications (Figure 11, entry 1). [50] In their study, the authors showed that biocompatible Pd 0 -resins implanted in the yolk sac of zebrafish embryos displayed catalytic activity enabling the local turn on of a fluorescent probe. This pioneer work was extended few years later by Weissleder and co-workers who reported the activation of an allyloxycarbonyl-protected doxorubicin prodrug within sarcoma HT1080 xenografts in mice (Figure 11, entry 2). [51] This team developed palladium nanoparticles that efficiently accumulated in tumours following their intravenously administration. The subsequent injection of the prodrug induced inhibition of tumour growth with minimal side effects compared to standard therapy, therefore testifying the selective drug decaging in malignant tissues.

With the purpose to design a novel protocol for cancer treatment, Mejia-Oneto and Royzen studied the activation of a TCO-based doxorubicin prodrug by an alginate hydrogel functionalized with Tz moieties (Figure 11, entry 3). [52] It is worth mentioning that polymeric implants bearing bioorthogonal functions had already been studied in mice for in vivo imaging. [53], [54] The biomaterial was first injected next to HT-1080 fibrosarcoma xenografts implanted in nude mice. The prodrug was then administered daily for ten days and its therapeutic efficacy was compared to that of the free doxorubicin used at the maximum tolerated dose. In these experiments, the prodrug was found significantly more efficient than the parent drug, inducing total and lasting remission of tumours in of mice. Furthermore, none overt sign of toxicity was observed in the animals treated the prodrug while mice under therapy doxorubicin a body This study provided evidence that the TCO/Tz click-and-release reaction has proceeded selectively in the vicinity of the tumor mass, thereby resulting in the efficient release of the chemotherapeutic agent. This concept was also assessed for the therapy of infectious diseases by the same groups (Figure 11, entry 4). [55] In this case, the Tz-modified alginate gel was implanted along with staphylococcus aureus bacteria into the thighs of neutropenic mice. The infected animals then received a systemic dose of a TCO-based prodrug of vancomycin, a FDA-approved antibiotic. Quite remarkably, with such a treatment the bacterial infection completely disappeared within 24 hours.

Recently, the Franzini's team described 3-isocyanopropyl substituents as new bioorthogonal masking groups. These latter can be indeed removed by reacting with Tz, thereby leading to the activation of fluorescent probes or the release of drugs (Figure11, entries 5 and 6). [11] Preliminary in vitro evaluations showed that this click-and-release reaction was suitable for the decaging of amine-, phenol-and thiol-containing drugs under physiological conditions. In vivo assays were then carried out in zebrafish embryos implanted with a Tz-modified polystyrene bead. First, the fishes were exposed to a 3-isocyanopropyl-derivatized resorufin pro-fluorescent probe. In these experiments, the detection of resorufin fluorescence within zebrafishes confirmed the in vivo Tz-mediated decaging of the probe. A prodrug of mexiletine, a sodium channel blocker that provokes cardiac arrhythmia and decreases heart rate, was then administered in the animals. This treatment resulted in a slowdown of heart rate in fishes, comparable to that observed with the free drug, hence demonstrating the potential of this approach of drug decaging in living organisms.

As an extension of this work, the same team revealed that tetrazylmethyl (TzMe) protecting groups can be cleaved in the presence of isonitriles to deprotect amine or phenol moieties (Figure 11,entry 7). [56] By combining TzMe derivatives with Tzresponsive 3-isocyanopropyls described above, they demonstrated that two fluorophores can be unmasked by a single bioorthogonal reaction. This double release process was evidenced in zebrafish embryos through the concomitant turn on of two different fluorescent probes. While this study represents a remarkable achievement, the location where the bioorthogonal reaction occurred in the fishes was however not controlled.

Antibody-drug conjugates (ADCs) represent a new class of oncology therapeutics with seven ADCs already approved by the American Food and Drug Administration (FDA) and more than eighty under clinical evaluation. [57] Following their pioneer work on Tz/TCO click-and-release reaction, [8] Robillard and co-workers proposed to use this bioorthogonal process for the selective cleavage of ADCs. [58], [59] In contrast to the vast majority of ADCs that are internalized inside cancer cells prior to exert their cytotoxic activity, Robillard developed non-internalizing ADCs designed to selectively accumulate on the surface of targeted cancer cells. With such a system, the bioorthogonal reaction leads to the extracellular release of the drug that can subsequently penetrate passively within surrounding cancer cells, even the ones that do not express the targeted surface marker. This concept was first explored in mice bearing TAG72-expressing colon xenografts, with a CC49 antibody linked to doxorubicin via TCO (Figure 12, entry 1). [58] Figure 12. Antibody-drug conjugates and nanoparticles designed for the selective delivery of drugs following bioorthogonal activation After the selective accumulation of ADCs in tumours, the administration of a Tz activator led to the release of doxorubicin in malignant tissues. However, in these experiments, the observed anticancer activity was relatively modest. Indeed, the therapeutic efficacy was impeded by the large size of ADCs that limited their penetration within tumours. Furthermore, a supplementary step devoted to the elimination of non-targeted ADCs was necessary to avoid unselective Tz-mediated release of doxorubicin, therefore increasing the complexity of the therapeutic protocol. To circumvent these drawbacks, Robillard and co-workers developed a diabody-based ADC that exhibits a high tumour uptake and very low retention in healthy tissues (Figure 12, entry 2). [59] In this approach, they also chosen to target the monomethylauristatin E (MMAE) that is far more potent than doxorubicin. As a result, when it was used in combination with a Tz activator, this novel ADC induced a strong anticancer activity in colon and ovarian tumour models in mice. In contrast, the same diabody loaded with the protease-responsive linker employed in the FDA approved Adcetris was not effective for the treatment of such tumours. Thus, this study suggested for the first time that non-internalizing ADCs, bearing bioorthogonal click-and-release systems, could represent a valuable alternative to ADCs already used in clinic for the therapy of some malignancies.

Very recently, Shao and Liu designed an ADC including a fluoride-sensitive cleavable linker that allowed the release of MMAE in the presence of a cancer-imaging probe phenylalanine trifluoroborate ([ 18 F]Phe-BF3). [60] The originality of this strategy relies on the targeting of each bioorthogonal partners at the tumour site. Indeed, experiences conducted in mice grafted with HER2-positive cancer cells showed the accumulation and retention of both the probe and the trastuzumab-MMAE conjugate, thereby conducting to the selective release of the anticancer drug (Figure 12, entry 3). This result in hands, the authors pursued their investigations using the same linker system to develop bioorthogonal-responsive nanoparticles transporting gasdermin A3 (Figure 12, entry 4). This latter is a pore-forming protein that triggers pyroptosis-induced inflammation, thereby causing antitumor immunity. Proof of principle has been demonstrated in immunocompetent mice with 4T1 mammary xenografts. In these assays, administration of the biorthogonal system led to a total disappearance of the tumour as the consequence of the antitumor immune response.

Our teams also proposed a double tumour targeting strategy using micelles that can be cleaved by a biorthogonal click-andrelease SPICC reaction. [61] The micelles were constructed with an iminosydnone amphiphile moiety designed to fragment in the presence of cyclooctynes (Figure 13). In this fashion, the clickand-release process triggers the disassembly of the micelle, hence allowing the controlled delivery of their content. In addition, to avoid the decomposition of untargeted micelles outside the tumour site, Papot and Taran conceived a -glucuronidaseresponsive pro-activator. Due to its high hydrophilicity, this latter cannot penetrate inside the micelles to react with iminosydnone amphiphiles. However, tumour-associated -glucuronidase can activate the pro-activator to release a hydrophobic cyclooctyne suitable to enter readily micelles and launch their decomposition. This concept was evaluated in mice bearing subcutaneous KB mouth epidermal carcinoma xenografts with micelles loaded with a fluorophore. Selective accumulation of the micelles in tumours was observed 24h following their i.v. administration. The subsequent injection of the pro-activator triggered micelles disassembly in malignant tissues demonstrating that the expected sequential enzymatic and bioorthogonal activation process occurred in living mammal.

Bioorthogonal chemistry for site-specific targeting of nanoparticles

Nanoparticles have emerged as promising tools for various biomedical applications since they can provide improved pharmacokinetics and multifunctionality compared to the majority of small molecules. Within this framework, the efficient targeting of nanoparticles to the disease site is an important issue that remains to be solved for enhancing their potential as diagnostic and therapeutic agents.

As early as 2012, Kim and co-workers proposed to increase the selectivity of nanoparticles for malignant tissues using bioorthogonal chemistry in combination with MGE. [62] For this purpose, they first carried out intra-tumoral injection of Ac4ManNAz in mice with A549 lung xenografts. Mice received then fluorescent DBCO-conjugated liposomes that accumulated in tumours as the result of their cooper-free click reaction with the azide groups beforehand generated on the surface of cancer cells. Interestingly, once bound on cell surface, nanoparticles were internalized providing evidence that such a targeting approach could be useful for intracellular delivery.

Two years later, the same group reported a two-step tumour targeting strategy for nanoparticles also based on MGE and click chemistry (Figure 14, entry 1). [47] However, in contrast to their previous study, Kim and co-workers avoided the intra-tumoral administration of Ac4ManNAz by pre-targeting the azide-modified sugar using glycol chitosan nanoparticles. Intravenous injection of these latter in mice bearing tumours resulted in the generation of azides on the surface of cancer cells due to the selective delivery of Ac4ManNAz through the EPR effect followed by MGE. In a second time, nanoparticles decorated with BCN and a photosensitizer were administered to the same animals. In this case, the formation of covalent bonds between the nanoparticles and the azides-modified cancer cells allowed to enhance their accumulation in tumours, compared to the targeting based only on the EPR effect. Furthermore, laser irradiation of mice treated with the two-step tumour targeting strategy led to a significant antitumor response correlated excessive local generation of cytotoxic singlet oxygen. This outcome highlighted the potential of this tumour targeting approach for photodynamic therapy.

More recently, the Yao's team investigated a similar targeting protocol for the chemo-photothermal therapy of breast tumours (Figure 14, entry 2). [63] They developed DBCO-functionalized nanocomposites designed for the simultaneous delivery of doxorubicin and a photosensitizer (zinc phthalocyanine). When administered in mice with breast xenografts previously treated by MGE, these nanoparticles showed tumour-specific accumulation, thereby inducing a high tumour-inhibition rate. Moreover, laser irradiation increased the tumour surface temperature to 57°C. On the other hand, such a temperature elevation was not observed in mice that were not pretreated by MGE. Thus, these results clearly demonstrated the advantages brought by the in vivo click reaction on the efficiency of photothermal therapy.

In an elegant approach, Zheng, Li and Cai constructed nanoparticles coated with azide-labelled T cell membrane designed to target natural antigen present at the surface of cancer cells by both immune recognition and biorthogonal chemistry (Figure 14, entry 3). [64] Since the sole recognition of T cell receptors is limited by tumours heterogeneity, they proposed this dual targeting strategy as a promising alternative to enhance the accumulation of nanoparticles in malignant tissues. This concept was evaluated in mice bearing Raji tumours pre-treated with Ac4ManN-BCN. Accumulation in tumours of N3-labelled nanoparticles loaded with a photosensitizer was 1.5-fold higher than that of the unlabelled ones, 48 h post-injection. Such a result demonstrated unambiguously that the click reaction between the nanoparticles and cancer cells enhanced the efficiency of the targeting. Once again, the increased concentration of nanoparticles within tumours significantly improved the efficacy of photothermal therapy, when mice were submitted to NIR irradiation.

Pretargeting approach for Radiotherapy

Encouraged by the results observed for imagery purposes using mAb, most pretargeting radioimmunotherapy (PRIT) strategies are based on the same techniques. The bispecific antibody (bsAb) strategy was extensively investigated in PRIT and some of them reached clinical studies. For instance, Kraeber-Bodere et al. developed a method involving 131 I-hapten with bsAb (directed against CEA antigen) which enabled to detect and treat 70 % of the CEA-expressed tumoral lesions. [65] The biotin streptavidin interaction was also explored in clinical studies using 90 Y-biotin as the therapeutic agent [66] but, as previously mentioned, these two techniques have several disadvantages.

In a recent study, SPAAC reaction was evaluated in PRIT using the antibody rituximab (directed against CD20 antigen) in lymphoma cells (Figure 15, entry 1). [67] Au and co-workers functionalized this antibody with DBCO moiety and they synthetized in parallel an 90 Y-dendrimer (PAMAM: Poly(amidoamine)) functionalized with azide functions. In this study, they observed a tumour regression in all mice, with 67% of mice becoming tumour free. This success was correlated with the high number of azide functions in the labelled dendrimer which could potentially increase the efficiency of the SPAAC reaction.

Naturally, the Tz/TCO reaction was investigated for PRIT with several radionuclides, such as 177 Lu, 225 Ac and 212 Pb for example, and showed promising results. The first preclinical proof-ofconcept of PRIT strategy involving Tz/TCO reaction was successfully performed by Zeglis and co-workers (Figure 15, entry 2). [68] The authors designed a two-steps strategy with a first i.v. injection of 5B1-TCO mAb (directed against CA 19-9) in mice bearing subcutaneous pancreatic tumours followed, 72 h later, by i.v. injection of the tetrazine derivative 177 Lu-DOTA-PEG7-Tz. They demonstrated a slow-down of tumour growth in treated mice compared to the control group. They also demonstrated a regression of tumour volume for the highest dose (32 MBq). One year later, the same group demonstrated a survival rate of 100 % in subcutaneous colorectal model with their 177 Lu-DOTA-PEG7-Tz derivative in pre-treated huA33-TCO mice (Figure 15, entry 3). [69] In 2019, Rondon and co-workers applied a PRIT method using lutetium-177 in a disseminated model of peritoneal carcinomatosis, for either SPECT imaging or radionuclidetargeted therapy (Figure 15, entry 4). [70] They demonstrated a significant reduction of tumour growth in treated mice compared to the control group that received only saline or 40 MBq of Tz-PEG7-DOTA-177 Lu alone.

Compared to beta-emitters, alpha-emitters can deliver a much more toxic radiation dose in a shorter distance. This characteristic can be evaluated with the linear energy transfer (LET, ratio between the of energy emitted and the distance travelled). A higher LET value is advantageous for micro metastases treatment. The first reported Tz-alpha-emitter was labelled with 212 Pb (t1/2 = 10.6 h, Figure 15, entry 5). [33] The tumour growth rate was reduced using Tz-PEG10-DOTA-212 Pb in pretreated CC49-TCO mice, compared to mice either treated with a labelled antibody or pretreated with mismatching mAb-TCO. Control experiments showed no reduction of the tumour growth rate when only saline buffer or CC49-TCO were administered. Unfortunately, this study was not further investigated for clinical application since a high kidney uptake was observed (2.5% ID/g 3h p.i.).

Poty and co-worker also demonstrated the ability of 225 Ac in PRIT strategy to selectively deliver the radioactivity to the tumour while reducing the haematotoxicity associated with the classical RIT methods (Figure 15 15, entry 6). [71]

Bioorthogonal chemistry for the clearance of bioactive molecules

In vivo bioorthogonal chemistry has also been studied to modify 'on demand' the biological activity of drugs. Indeed, long acting and metabolically stable bioactive molecules can produce adverse effects. Consequently, the design of strategies offering the possibility to interrupt the action of some drugs at the desired time is of great interest.

In this context, Krezel and Wagner elaborated a novel approach for neutralizing the activity of an anticoagulant drug in mice (Figure 16, entry 1). [72] For this purpose, they first synthesized an azide-containing analogue of Warfarin, a wellknown anti-vitamin K anticoagulant compound, and a hydrophilic clearing agent including a BCN moiety. Then, they demonstrated that the Warfarin analogue exhibited anticoagulant activity in mice. However, administration of the clearing agent led to inactivation of the anticoagulant derivative and induced its plasma elimination via renal clearance. This effect was the consequence of the in vivo formation of the clicked product that does not possess any anticoagulant activity.

The development of biorthogonal clearing agents was also envisioned for therapeutic applications employing targeted antibodies. This field was pioneered by Robillard et al. within the framework of their efforts at designing pretargeting strategies for radiotherapy of solid tumours (Figure 16, entry 2). [31] 16. Bioorthogonal startegies devoted to the 'on demand' clearance of bioactive molecules.

They investigated a CC49 antibody functionalized by TCO and programmed to react with a radiolabelled Tz selectively in tumour tissues. However, evaluation of this approach in mice failed due to a low tumour-to-blood ratio resulting from the click reaction between the Tz derivatives and untargeted circulating antibodies. To prevent this issue, the authors designed a clearing agent derivatized from albumin including several galactose and Tz moieties. The use of this latter led to a doubling of the Tz tumour uptake and a 125-fold improvement of the tumour-toblood ratio. Indeed, once in the blood steam, the clearing agent reacted with TCO attached to the CC49 antibody to direct it to the liver while reducing its plasmatic concentration. Along the same line, Simberg and co-workers conceived a set of nanocarriers decorated with bioorthogonal functional groups such as TCO and Tz, with the aim to accelerate the elimination of IgG therapeutic antibodies (Figure 16, entry 3). [73] However, while the click reaction between the two biorthogonal partners occurred in vivo, antibodies were not completely eliminated from the blood, even after multiple injection of the clearing agents. These results pointed out the need of further investigations to optimize antibodies clearance using bioorthogonal chemistry.

Summary and Outlook

In less than two decades, bioorthogonal chemistry led to a new paradigm in chemical sciences, showing that living organisms can be used as reaction vessels in which it is possible to control chemical bonds forming and breaking, in spite of the complex conditions prevailing within biological media. While still in its infancy, in vivo chemistry offers already great promises to better understanding biological mechanisms in real time as well as for the development of novel biomedical applications. The recent advances in this field highlighted some of the potential issues that have to be taken into account for designing efficient bioorthogonal systems, programmed to operate in live animals. Whereas kinetics of the reactions remains a key factor due to in vivo high dilution conditions, biodistribution and circulation time of the reactants play also major roles for the success of chemistry in living organisms. Hence, it is not surprising that many bioorthogonal reactions have been used in combination with delivery systems or targeted strategies. Furthermore, one can expect such combined approaches should develop even more in a near future. These progresses will be of particular interest for therapeutic strategies in which a stringent control of bioorthogonal reactivity will be needed in specific tissues. The discovery of new bioorthogonal reactions will be also necessary to widen the scope of applications of in vivo chemistry. To date, it is clear that the number of reactions available remains limited, representing a substantial hurdle for further breakthroughs in this area. Increasing the diversity of biorthogonal tools will probably enable to interact with biological processes in an even more precise manner. Along this line, one could then envision to design multistep synthetic strategies occurring entirely within living organisms.

In summary, the development of bioorthogonal chemistry in animals has already conducted to remarkable achievements letting imagine numerous applications in humans, particularly for diagnosis and therapeutic purposes. However, since this field of research has emerged only few years ago, its potential could be broader than previously anticipated with unexpected applications.

Table of Contents

MINIREVIEW

Performing and controlling nonnatural chemical reaction inside living organisms is one of the most difficult challenge chemists may have to face. Here we review the recent update in the development of biorthogonal tools useful not only for basic research in chemical biology but also for diagnosis and therapeutic applications.

Bioorthogonal reactions in animals

Figure 1 .

 1 Figure 1. Main bioorthogonal reactions used for in vivo applications.

Figure 2 .

 2 Figure 2. Schematic representation of cell-surface glycans imaging during zebrafish development.

Karine

  Porte joined the group of Dr. Taran for her PhD degree in chemistry at the Atomic Energy and alternative Energy Commission (CEA) in 2016. Her PhD research was focus on the use of bioorthogonal chemistry for drug delivery. After obtaining her PhD in September 2019, she moved to a postdoctoral position in the group of Prof. Frank Glorius at the University of Münster. Davide Audisio holds a PhD from the University Paris-Sud. After a postdoctoral experience at the Max-Planck-Institut für Kohlenforschung (Germany), in 2012 he joined the chemistry department of Eli Lilly & Co (UK). Next, he joined the CEA and since 2016, he is in charge of the Laboratory of Carbon-14 Labeling. In 2019, he was recipient of an ERC Consolidator Grant devoted to the development of late-stage radiocarbon labeling. Maxime Ribéraud received his master degree from Poitiers University in 2019 and he is currently working toward his PhD degree in organic chemistry at the Atomic Energy and alternative Energy Commission (CEA). His current research focuses on developing new methodes for the detection and the quantification of tumor receptors by bioorthogonal chemistry. 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). His research interests include the design of smart drug delivery systems for cancer chemotherapy, in vivo chemistry, functional interlocked systems and prebiotic chemistry. Rémi Châtre works toward obtaining his PhD degree at the Institut de Chimie des Milieux et Matériaux de Poitiers (IC2MP). His PhD research focused in the development of bioorthogonal approaches for modifying serum albumin in vivo to improve therapeutic efficacy of albumin drug conjugates.. Dr. Taran secured a PhD in chemistry at the Paris XI University under the supervision of Dr. Charles Mioskowski and then moved to a postdoctoral position with Prof. Sir Derek Barton at Texas A&M University. Since 1998, he works for the French Alternative Energies and Atomic Energy Commission (CEA) located at Saclay, close to Paris and is now in charge of the department of bioorganic chemistry.

Figure 3 .

 3 Figure 3. Schematic representation of the chemistry in mice. Mice were injected with Ac4ManNAz to allow metabolic labeling of glycans with SiaNAz. The injection of a cyclooctyne-FLAG conjugate induce the in vivo covalent labeling of azido glycans. Structure of DIFO-FLAG for SPAAC reaction and PHOS-FLAG for Staudinger ligation.

Figure 4 .

 4 Figure 4. a) Schematic representation of bacteria labeling by copper-free click chemistry. b) In vivo visualization of B. fragilis in the instestine. Whole body imaging by IVIS of Cy7-labeled B. fragilis in conventional specific-pathogen-free (SPF) and germ free (GF) mice.

Figure 5 .

 5 Figure 5. Formation of luciferin substrates in vivo by selective reactions of cyanobenzothiazoles (CBT) with D-Cysteine. Imaging of enzymatic activity in mice by D-aminoluciferin formation in vivo with (a) or without (b) caspase 3/7 activation.

Figure 6 .

 6 Figure 6. Schematic representation of the mostly use pretargeting strategy for radioimagery or radiotherapy using bioorthogonal functions.

Figure 7 .

 7 Figure 7. Highlighted in vivo pretargeting studies involving TCO/Tz approach.

Figure 9 .

 9 Figure 9. Imaging tumor cell using SPAAC or reactions Staudinger Ligation (SL) via a MGE approach.

Figure 11 .

 11 Figure 11. Therapeutic strategies designed for the controlled release of drug via bioorthogonal cleavage or click-and-release reactions.

Figure 13 .

 13 Figure 13. Strategy for double-targeted delivery in tumours. The approach takes advantage of the biorthogonal click-and-release SPICC reaction to deliver the content of nanoparticles.

Figure 14 .

 14 Figure 14. Strategy combining MGE and click chemistry for site-specific targeting of nanoparticles.

Figure 15 .

 15 Figure 15. Overview of in vivo PRIT in mice

  Karine Porte, Maxime Riberaud, Rémi Châtre, Davide Audisio, Sébastien Papot and Frédéric Taran Page No. -Page No.

Acknowledgements

This work was funded in part by the French Research National Agency (ANR-19-CE06-0006-01), the Fondation pour la Recherche Médicale (DCM20181039570), the Ligue Nationale contre le Cancer (Comités Vienne and Deux-Sèvres) and the Région Nouvelle Aquitaine (2017-1R30234-00013189).