Robert Granet

Gautier René Faure

Mark-Arthur Ndong Ntoutoume

Jean Pierre Mbakidi

Yann David

Bertrand Leger

Vincent Liagre

Sol

Mark-Arthur Ndong Gauthier

Jean-Pierre Ntoutoume

David Y Mbakidi

Enhanced cytotoxicity of gold porphyrin complexes after inclusion in cyclodextrin scaffolds adsorbed on polyethyleneimine-coated gold nanoparticles

Keywords:

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Bioorganic & Medicinal Chemistry Letters

Colorectal cancer is the third most common cancer worldwide and is responsible for almost 30% of cancer deaths. 1 Combination chemotherapy is usually given when surgery has failed. First line chemotherapy for colorectal cancer is generally based on 5fluorouracil or irinotecan in combination with oxaliplatin. 2 However, oxaliplatin, as well as cisplatin, which are among the most active drugs against colon cancer, may generate severe side effects along with induction of drug resistance; these drawbacks hamper a long term clinical use of these chemicals. 3 For decades-long, gold derivatives have been used against various diseases. 4-7 Au III and Pt II lead to tetracoordinated complexes with the same square planar geometry. One could expect for Au III complexes an activity comparable to that of cisplatin. In fact, a number of Au III coordinates, tested against a broad spectrum of tumor cells, actually displayed inhibitory effects, although less prominent than those reported with platinum complexes. 7 On the other hand, Au III coordinates have been shown to act upon more cellular or molecular targets as compared to cisplatin, e.g. inhibition of thioredoxin reductase, proteasome inhibition and modulation of specific kinases. 8,9 Moreover, they have displayed anti-cancer activity against both cisplatin-and multidrug-resistant xenografts. 10 Among these compounds, gold porphyrins complexes are particularly stable and have been the subject of some studies. [11][12][13][14][15] For example, Chen et al showed that five gold(III)-tetraphenylporphyrin derivatives are more cytotoxic that cisplatin against seven selected human cancer cell lines. In particular, gold(III) tetraphenylporphyrin complexe was the more efficient. 12

A R T I C L E I N F O A B S T R A C T

Article history:

Recently, our team showed that gold (III) 15 Among the many gold (III) complexes of tetraarylporphyrin, tetraphenylporphyrin is one of the more lipophilic macrocycle and one of the most active of these compounds. 13 However this large activity enhancement is counterbalanced by the poor solubility of these complexes in aqueous media. A way to circumvent this difficulty could consist in a noncovalent association of the porphyrin complex with a carrier with a hydrophilic surface. 16,17 Moreover, in vivo, this carrier could selectively vectorize the active substance to the tumor, thanks to the enhanced permeation and retention (E.P.R.)

effect. 18 Nanoparticles with a size comprised between 20 and 100 nm are selectively retained by the leaky structure of the tumor neovascularization and, conversely, are not absorbed by healthy tissues. Moreover, positively charged nanoparticles are prone to be actively endocytosed by cancerous cells. 19,20 Consequently, we resolved to design gold nanoparticles coated with a layer of polyethyleneimine (PEI). A one-pot synthesis of gold nanoparticles protected by polyethyleneimine was envisaged,

since it has been recently shown that PEI can be used as both a catalyst and a capping agent for the synthesis of gold nanoparticles. 21,22 Unfortunately these gold nanoparticles suffer from poor stability unless high molecular weight PEIs are being used, but high molecular weight PEIs are somewhat toxic to normal cells. 23 So we envisaged to graft a thiol function on a low molecular weight PEI in order to obtain a stable binding of PEI to the gold surface, thus enhancing the stability of the nanoparticles.

The layer-by-layer technique allowed us to introduce a capping with anionic β-cyclodextrin which would be able to encapsulate hydrophobic tetraarylporphyrins. Cyclodextrins (CDs) are cyclic oligosaccharides consisting of six (α-), seven (β-) or eight (γ-) Dglucopyranose units linked by α-(1,4) glycosidic bonds. They are well known to form stable inclusion complexes with a variety of guest molecules 24 . β-CD is the most commonly used, due to its relatively easy synthesis, its low price and also to the large number of polar molecules which can fit into its internal cavity.

Metallation of porphyrins by gold salts is usually realized in acetic acid at reflux for several hours. [11][12][13] However, in a recent work on the metallation of porphyrins by lanthanides, we showed that this reaction was greatly facilitated by microwave (MW) activation. 25 In addition, we showed that the reaction yield was largely improved by lithium ions. So, we thought to apply this methodology, in the presence of lithium ions, for the metallation of porphyrins by gold salts. 25,26 The synthesis of the different gold porphyrins and their encapsulation in cyclodextrin-capped gold nanoparticle is presented in scheme 1.

Scheme 1. General pathway of AuNP@PEI@CD@Porphyrin synthesis

Tetraphenylporphyrin (H 2 TPP, 1) was synthesized according to the Adler and Longo method starting from benzaldehyde and pyrrole. 27 The unsymmetrical 5-(4-acetoxyphenyl)-10, 15, 20triphenyl porphyrin (H 2 TPPOAc, 2) was obtained by the mixed aldehydes method of Little. 28 Para-acetoxybenzaldehyde was synthesized by reaction of para-hydroxybenzaldehyde and acetic anhydride with DMAP (4-DimethylaminoPyridine) as catalyst. 15 Porphyrins were characterized by UV-Visible, NMR and mass spectrometry. In previous works, we have reported a catalytic effect of lithium ions on the metallation of porphyrins with copper, 26 erbium and gadolinium. 25 Moreover it has been shown that a considerable enhancement of the reaction yield was obtained by microwave irradiation. 25 The goal of this work was to study these parameters on the metallation of tetraphenyl The percentage of metallation increases linearly with the amount of added lithium. This result is in accordance with the effect previously reported for the metallation of tetratolylporphyrin with erbium or gadolinium. 25 Moreover, microwave activation considerably reduces reaction time: only 3 minutes are required for a complete reaction instead of two hours at reflux as reported in the literature. [11][12][13] Gold metallation resulted in the formation of AuTPPCl (3) and AuTPPOAcCl (4).

One-pot synthesis of gold nanoparticles with high molecular weight polyethyleneimine (25 kDa PEI) has been recently reported; these particles benefit from a satisfying stability. 21,22 Unfortunately, this kind of PEI can hardly be considered for in vivo treatment, due to their significant toxicity. 23 Our previous works showed that the use of low molecular weight PEI (from 0.6 to 2 kDa) which displays low toxicity, led in the formation of unstable nanoparticles. 16 So, we decided to provide PEI with a thiol function, able to anchor the PEI chains onto the gold particle surface. In order to minimize the toxicity of polyamines against normal cells we chose to work with a very low molecular weight PEI (1.2 kDa). PEI 1200 was reacted with 3acetamidotetrahydro-2-thiophenone in dichloromethane at RT (Room Temperature) for 24 hours. After evaporation of dichloromethane and dissolution in water, a dialysis with a membrane with a 500 Da cutoff was realized. A degree of substitution of 1.3 was determined by elemental analysis (see Supplementary Data). Nanoparticles were synthesized by adding various amounts of PEI-thiol to a solution of KAuCl 4 at 90 °C for 30 minutes. The average diameters of the resulting AuNP@PEI, determined by UV-Visible spectroscopy 28 and by transmission electron microscopy (Fig. 2) are reported in Table 1. Particles diameter slightly drops while PEI-thiol concentration increases. of the different nanoparticles (AuNP@PEI, AuNP@PEI@CD, AuNP@PEI@CD@AuTPPCl, and AuNP@PEI@CD@AuTPPOAcCl) determined by Dynamic Light Scatting (Fig. S1, S2 and S3), are reported in table S1.

AuNP@PEI size observed by DLS corresponds to a diameter of 30 nm (Table S1). Addition of β-cyclodextrin resulted in an increased size (up to 45 nm) and a second increase was observed (AuTPPCl, AuTPPOAcCl and AuNP@PEI@CD@AuTPPCl, AuNP@PEI@CD@AuTPPOAcCl) is reported in Fig. 4 for each cell line.

% cell viability

Concentration (µM)

0 AuNP@PEI@CD@AuTPPOAcCl AuNP@PEI@CD@AuTPPOAcCl AuNP@PEI@CD@AuTPPCl AuNP@PEI@CD@AuTPPCl Controls conducted in presence of gold nanoparticles coated with PEI and cyclodextrin (AuNP@PEI@CD) showed that these particles did not exhibit any cytotoxicity (up to a concentration corresponding to a value of 5 µM in porphyrin) against either of the two cell lines. IC 50 deduced from these curves are shown in Table 2.

Table 2: IC50 of free AuTPPCl (3), AuTPPOAcCl (4) and AuNP@PEI@CD@AuTPPCl and AuNP@PEI@CD@AuTPPOAcCl complex after 48h Incubation in HT-29 and HCT-116 cells. a Concentration corresponding to a value of 5µM of PS.

IC 50 (µM of PS)

Cell lines

AuTPPCl AuNP@PEI@CD@AuTPPCl AuTPPOAcCl AuNP@PEI@CD@AuTPPOAcCl AuNP@PEI@CD a HT-29 30 The introduction of the acetoxy group does not modify significantly the cytotoxicity of this molecule. Encapsulation of either of the two gold porphyrin resulted in strong increases in cytotoxicity (3 and 5 fold) against the HT-29 cell line. Smaller effects (1.5 and 2 fold) were observed against HCT-116; however this cell line is more sensitive to the free compounds (0.69 -0.74 µM). These results are comparable to previous findings where encapsulation of curcumin by cyclodextrin-cellulose-nanocrystals complexes resulted in a 3 fold increase in cytotoxicity against HT-29 cell line. 16 We describe in the present work and for the first time, a new, easy and efficient method for the synthesis of gold porphyrins which uses microwave activation and the catalytic effect of lithium ions. These gold porphyrin complexes where successfully encapsulated in β-cyclodextrin-capped gold-PEI nanoparticles.

This encapsulation resulted in an enhancement of gold porphyrin cytotoxicities against two colon cancer lines by increasing the drug penetration into the cells. This new platform (AuNP@PEI@CD) could be a very efficient new anti-cancer hydrophobic drug delivery system, for in vivo application.

  j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m Enhanced cytotoxicity of gold porphyrin complexes after inclusion in cyclodextrin scaffolds adsorbed on polyethyleneimine-coated gold nanoparticles Robert Granet a , René Faure a , Gautier Mark-Arthur Ndong Ntoutoume a , Jean Pierre Mbakidi a , David Yann Leger a , Bertrand Liagre a , Vincent Sol a* a Université de Limoges, Laboratoire PEIRENE, EA 7500, F-87000 Limoges-France

  porphyrins by gold salts. All the experiments were conducted in acetic acid at 100 °C with a reaction time of 3 min and with 2 equivalents of KAuCl 4 . The lithium amount added varied from 5 to 100 equivalents. The amount of metallated porphyrin was determined by UV-Visible spectroscopy by measuring absorbance at 526 nm (metallated) versus non metallated (acidic form) at 660 nm. The percentage of metallation of H 2 TPP as a function of Li + equivalents is shown in Fig. 1.

Figure 1 :

 1 Figure 1: Percent metallation as function of Li + eq at 100 °C

Figure 2 :

 2 Figure 2: TEM images of gold nanoparticles (AuNP@PEI) with ratio ( mPEI/mAu = 0.46)Finally, commercial sulfated β-cyclodextrin was added to a solution of AuNP@PEI in water and the mixture was left 1 hour at RT, letting the negatively charged cyclodextrin to bind the positively charged AuNP@PEI. The resulting nanoparticles (AuNP@PEI@CD) were purified by centrifugation and resuspended in DI (Dionized) water. AuNP@PEI@CD were characterized by UV-Visible (Fig.3), dynamic light scattering (DLS) and zeta potential measurements. The metallated porphyrin was then encapsulated into cyclodextrin. The amount of entrapped porphyrin was calculated, after centrifugation, by subtracting the amount of porphyrin contained in the supernatant (determined by UV-Visible) from the total amount of porphyrin introduced.

Figure 3 :

 3 Figure 3: UV-Visible spectra of gold nanoparticles AuNP@PEI@CD alone and with encapsulated gold porphyrin AuTPPOAcCl. Encapsulated AuTPPCl and AuTPPOAcCl represented 6 and 9.5% (w/w) of the total mass of the respective nanoparticles. The acetate moiety contributes to an enhanced encapsulation yield of the porphyrin into the cyclodextrin ring. The measured diameters

(

  up to 70 nm) after inclusion of gold porphyrins. AuNPs bearing polyethyleneimine exhibited a large positive surface charge (ζ potential) of 43.2 mV. After addition of the negatively charged βcyclodextrin this value dropped down to 16 mV but remained relatively unchanged after addition of gold porphyrins. Gold porphyrins (compounds 3 and 4) and nanoparticles bearing gold porphyrins were evaluated for their cytotoxicity against two colorectal cancer cell lines, HT 29 and HCT-116. Cell viability was assessed by the 3-(4,5-dimethyl-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) staining method after a 48 hour incubation. Cell viability versus gold porphyrin concentration

Figure 4 :

 4 Figure 4: Cell viability of HT-29 (a) and HCT-116 (b) for 48h in the presence of increasing amounts of gold porphyrins (3 and 4) and the same porphyrins entrapped in gold nanoparticles. Each value represents the mean±SD of three separate experiments.The concentration of AuNP@PEI@CD@AuTPPCl and AuNP@PEI@CD@AuTPPOAcCl were expressed as equivalent of gold porphyrin (3 or 4) concentration.

Table 1 : Diameter of gold nanoparticles (AuNP@PEI) determined by UV-vis spectroscopy and TEM

 1

	Ratio (mPEI / mAu)	0.36	0.4	0.46
	UV-Visible (d.nm ; λ.nm)	46 ; 530	40 ; 527	22 ; 523
	TEM (d.nm)	35	40	15

Acknowledgements

We thank the 'Conseil Regional de Nouvelle Aquitaine' for financial support. The authors are indebted to Dr. Michel Guilloton for help in editing the manuscript, Pierre Carles (CARMALIM-IRCER-Limoges) for MET analysis and Yves Champavier from BISCEm core facility for NMR analysis.


Clarisse Mariet
email: clarisse.mariet@gmail.com

Axel Vansteene

Marion Losno

Julien Pellé

Jean-Philippe Jasmin

Anthony Bruchet

Gwendolyne Hellé

Microfluidics devices applied to radionuclides separation in acidic media for the nuclear fuel cycle

Keywords: solvent extraction, extraction chromatography, solid phase extraction, monolith, functionalization

Miniaturization of analytical devices provides new perspectives for safety improvement, intensification, and diversification of processes (reduction of matter shipment, reduction of stored materials, energy savings, reaction conditions that are inaccessible with macroscopic reactors…). This reduced scale satisfies most of the criteria of green analytical chemistry as well as the principle of minimization of hazards associated to radioactive samples, thanks to a major reduction of sample quantities, reactants, wastes, and analysis time, and to automation. An increasing number of specific microsystems or "labs-on-a-chip" have been developed with this ambition, particularly for radionuclides purification and separation steps, and for their analyses in complex and hard-to-handle samples. This article illustrates the development of microsystems for elemental separations in acidic media prior to isotopic or elemental measurements by mass spectrometry or a radiometric analysis method for nuclear-related activities, and underlines the main challenges to take up.

Introduction

The current nuclear reactor fuel consists of a mixture of oxides of uranium and plutonium or uranium oxide. After its stay in reactor, the fuel still contains a significant amount of valuable material which are interesting to recover. Moreover, the nuclear reactions that produced energy have also created other elements, fission products and minor actinides, which make the spent fuel highly radioactive. This is the reason for which the fuel is treated in order to separate material to recycle (uranium, plutonium) from other radioactive elements that constitute waste and condition waste in a safe and inert form. The hydrometallurgical recycling and the storage of the waste need conducting chemical 2 analyses of the solutions after nuclear fuel dissolution [START_REF] Burger | Gamma Radiolysis. Tributyl phosphate and its diluent systems[END_REF][START_REF] Burr | The radiolysis of tributyl phosphate[END_REF][START_REF] Schulz | Science and technology of tributyl phosphate[END_REF]. The precise and accurate chemical analysis of nuclear spent fuel (determination of the concentration levels of Uranium, Plutonium and some Fission Products) represents a critical part of materials control and accountancy and plays an essential role in designing future nuclear fuels cycle, reprocessing cycle, after dissolution, and throughout the process for environmental regulatory reasons or non-proliferation monitoring.

To obtain low uncertainties using Thermo-Ionization Mass Spectrometry (TIMS) or Multi-Collection Inductively Coupled Plasma Mass Spectrometry (MC-ICPMS), mono-elemental samples are mandatory. Obtaining pure fractions of each radionuclides remains difficult because nuclear spent fuel samples exhibit an extreme chemical diversity stemming from several schemes of neutron capture, fissions or activation reactions that occur in reactors. This leads to the formation of the so-called transuranium radionuclides (Np, Pu, Am, Cm) as well as a wide variety of fission products constituted of Lanthanides (Nd, Sm, Eu, Gd), alkalines (Cs) and alkaline earth metals (Sr). Then, analysis of radionuclides is done according to operating protocols including the sequence of several separation/purification operations before performing the measurement by liquid scintillation, gamma spectrometry, X spectrometry or inductively coupled plasma mass spectrometry (ICP -MS) in order to remove interferences. Solid phase extraction (SPE) and liquid-liquid extraction (LLE) [START_REF] Durham | The absorption of plutonium by anion resins, Chalk River, Ontario[END_REF][START_REF] Navratil | Ion exchange technology in spent fuel reprocessing[END_REF], performed in concentrated nitric or hydrochloric acid to form anionic and neutral complexes and stabilize oxidation degrees, pose a significant number of constraints specific to the sample (very irradiating which involves work in glove box, very corrosive), and the required precision. In addition, these separations are difficult to automate, involve the prolonged presence of an experimenter and generate large quantities of waste [START_REF] Bruchet | Centrifugal microfluidic platform for radiochemistry: potentialities for the chemical analysis of nuclear spent fuels[END_REF]. Methods dedicated to fast elementary or isotopic analyses, automated, consuming very little solvent, and targeting the desired compounds must be developed to acquire more numerous reliable data at a lower cost. The development of radiochemical separations in acidic media at the micrometric scale is part of the ALARA (As Low As Reasonably Achievable) approach for the reduction of waste and the handling of dangerous chemicals. The major interest of microsystems lies in their ability to integrate a series of operations on a small plate. Operations are faster and the same device brings together several identical processes in parallel. In addition, such a system takes in charge several steps without external intervention between the different stages. Thanks to micro-devices, the volumes of solvents and samples are reduced and operations are linked without human intervention, which limits the losses and contamination of samples.

If numerous microsystems have been developed and are already traded for medical diagnosis [START_REF] Arshak | A review of low-power wireless sensor microsystems for biomedical capsule diagnosis[END_REF][START_REF] Mouradian | Lab-on-a-chip: applications in proteomics[END_REF][START_REF] Ríos | Miniaturization through lab-on-a-chip: Utopia or reality for routine laboratories? A review[END_REF] or chemical synthesis [START_REF] Watts | The application of micro reactors for organic synthesis[END_REF][START_REF] Mason | Greener approaches to organic synthesis using microreactor technology[END_REF] since 2000, few studies deal with very acidic samples like nuclear spent fuels or leach solutions issued from metallurgical processes. Unlike typical microfluidic devices that perform under about neutral pH conditions for biological and environmental applications, devices for radiochemical separations must be chemically compatible with strong mineral acids, and must also contribute negligibly to the trace element analytical blank. Reviews on the use of microfluidic devices have been published in several fields [START_REF] Mark | Microfluidic lab-on-a-chip platforms: requirements, characteristics and applications[END_REF][START_REF] Squires | Microfluidics: fluid physics at the nanoliter scale[END_REF][START_REF] Atencia | Controlled microfluidic interfaces[END_REF][START_REF] Gravesen | Microfluidics -a review[END_REF][START_REF] Abou-Hassan | Microfluidics in inorganic chemistry[END_REF][START_REF] Reyes | Micro total analysis systems. 1. Introduction, theory and technology[END_REF][START_REF] Ciceri | The use of microfluidic devices in solvent extraction[END_REF]. However, none have highlighted the use of microfluidic devices in very corrosive and complex media. Here, we have reviewed the state-of-the-art of microfluidic tools with a specific focus on the micro-devices used or designed for radionuclides separation in very acidic media.

Liquid-liquid extraction of radionuclides in acidic media

Very selective, liquid-liquid extraction is the preferred separation method of nuclear chemistry.

Nuclear and pharmaceutical industries allowed its first industrial developments in the 1940s, then the oil and petrochemical industry in the 1955s-1965s. The real boom in the area of the hydrometallurgical allowing recovery of metals in various aqueous solutions (copper, uranium, gallium, rare earth, ...) happened in 1960 [START_REF] Cote | Extraction liquide-liquide -Présentation générale[END_REF]. The solvent is made of thinner containing or not an extractant (organic molecule having an affinity for the element to be transferred). If the organic synthesis often uses the solvent extraction by spreading a species from one phase to the other by simple transfer [START_REF] Kralj | Integrated continuous microfluidic liquid-liquid extraction[END_REF][START_REF] Zhao | Liquid-liquid two-phase mass transfer in the T-junction microchannels[END_REF], in radiochemistry radioelement extraction is still implementing an extractant. In addition to the diffusion of the element there is a chemical reaction [START_REF] Cote | Extraction liquide-liquide -Bases physico-chimiques des procédés[END_REF]. In this article, only liquid-liquid extraction has been considered.

Since its origins, to save natural resources and given the specific constraints related to radioactive and ionizing radiation, the nuclear industry prioritized raw material economy, recycling and minimization of the final waste, environmental footprint reduction and control of the risks of exposure for workers. This has led to the establishment of the recycling of the fuel, which allows to recover 96 % materials and a minimization of the production of final waste. Now, continuous improvement of these processes is intended to improve their efficiency to decrease the cost, quantities of waste and improving safety around several axes: i) the development of new extractants for selective extraction of uranium ores in order to reduce the environmental impact of mining; ii) the development of new solvents for recycling of fuel which will be more robust, more compact and more efficient; iii) the miniaturization of systems, on the one hand, for the screening of new extractants, obtaining thermodynamic and kinetic data, the optimization of the parameters (temperature, pH, concentrations) of extraction processes and, on the other hand for the analysis of effluents, waste, environmental samples... Two types of miniaturized systems are identified in the literature regarding the liquid-liquid extraction of radionuclides. The so-called 'chaotic' reactors promote mixing of the phases [START_REF] Xie | Two-stage feedback miniextractor with high-throughput via selective dimension scale-out[END_REF][START_REF] Xu | High-throughput production of droplets using mini hydrodynamic focusing devices with recirculation[END_REF] and allow operation at counter-current of two immiscible phases [START_REF] Xu | Review of microfluidic liquid-liquid extractors[END_REF]. By definition, the specific interfacial area of these systems is not known. Their dimensions are rather millimeter and flow rates are tens or even hundreds of mL.h -1 and they found application in the design of process engineering.

Regarding lab on chips, they promote laminarity allowing accurate control of the specific interfacial area. Their dimensions are micrometer and flow rates are less than 10 mL.h -1 , which explains their applications are analysis and R & D. Liquid-liquid extraction microsystems provide many advantages such as large interfacial areas [START_REF] Hibara | Interfacial phenomena and fluid control in micro/nanofluidics[END_REF], short diffusion distance [START_REF] Hibara | Interfacial phenomena and fluid control in micro/nanofluidics[END_REF][START_REF] Shui | Multiphase flow in microfluidic systems -Control and applications of droplets and interfaces[END_REF][START_REF] Zhao | Two-phase microfluidic flows[END_REF], high surface/volume ratio [START_REF] Minagawa | Integration of a wet analysis system on a glass chip: determination of Co(ii) as 2-nitroso-1-naphthol chelates by solvent extraction and thermal lens microscopy[END_REF],

process intensification [START_REF] Kolar | Priest Microfluidic solvent extraction of rare earth elements from a mixed oxide concentrate leach solution using Cyanex® 572[END_REF] and possible integration of many operations stages on a single compact chip [START_REF] Hellé | Liquid-liquid extraction of uranium(VI) with Aliquat(R) 336 from HCl media in microfluidic devices: Combination of micro-unit operations and online ICP-MS determination[END_REF][START_REF] Tokeshi | Continuous-flow chemical processing on a microchip by combining microunit operations and a multiphase flow network[END_REF][START_REF] Maruyama | Liquid membrane operations in a microfluidic device for selective separation of metal ions[END_REF]. Solvent extraction in microchannels is a contact of two immiscible phases in a microchannel to promote the extraction of an element from one of the two phases towards the other.

Only the case of the microsystems is treated in this article.

Two types of flows are implemented in microsystems: parallel flows and segmented flow (a phase is dispersed in the other) (Fig. 1). They also used a commercial glass made microsystem with Y-junction but with an unbalanced channel profile to ensure parallel phases flow. The equilibrium was achieved within 10 seconds of contact between the 2 phases. Recently, extraction of Pu(IV) was studied by Yamamoto et al. [START_REF] Yamamoto | Evaluation of plutonium(IV) extraction rate between nitric acid and tri-n-butylphosphate solution using a glass chip microchannel[END_REF] between nitric acid and a 30 % TBP in n-dodecane organic phase in stratified flows. These studies highlight that stratified flows solvent extractions in microsystems present the advantage to allow a precise control of the contact times of the two phases, especially short contact times, to lead to high speed and breakthroughs allow to work with automated microsystems which can be used in parallel processing to increase the throughput or in multiplexed processing of separation/purification steps coupled to a detection system [START_REF] Tokeshi | Continuous flow chemical processing on a microchip using operations and a multiphase flow network[END_REF][START_REF] Thorsen | Microfluidic large-scale integration[END_REF]. To take advantage of a combination of micro-unit operations and multiphase flow network, liquid-liquid extraction of the uranium(VI) in HCl media by Aliquat® 336 coupling with stripping in the same microsystem was performed [START_REF] Hellé | Liquid-liquid extraction of uranium(VI) with Aliquat(R) 336 from HCl media in microfluidic devices: Combination of micro-unit operations and online ICP-MS determination[END_REF].

In the majority of the aforementioned studies, the detection was performed at the outlet of the microchannel by mass spectrometry on each phase recovered pure, so the interface was positioned at the center of the microchannel (Fig. 2-b). To set the interface position, the values of flow rates have to be adjusted considering viscosities values of the two immiscible phases [START_REF] Malengier | Optimizing performance of liquid-liquid extraction in stratified flow in micro-channels[END_REF]. During solvent extraction, the two phases viscosities are held constant because the extracted element is much less concentrated than the nitric acid or the extractant in the aqueous phase or in the organic phase, respectively. Then, when the radionuclide, is initially present at several tens of g.L -1 in the aqueous phase, viscosities values are modified during the extraction process [START_REF] Bruus | Theoretical microfluidics[END_REF] and a change of the position of the interface between the two phases can be expected. This could involve a decrease of the quality of the separation at the outlet of the microsystem [START_REF] Cubaud | Separation of highly viscous fluid threads in branching microchannels[END_REF]. Hence, in 2017, Jasmin et al. [START_REF] Tokeshi | Continuous-flow chemical processing on a microchip by combining microunit operations and a multiphase flow network[END_REF] studied the extraction of U(VI) present at several tens g.L -1 from the nitric acid to the organic one (30% v/v TBP)

and showed the influence of the modification of the phases' viscosities on the flow regime. For nuclear or hydrometallurgical applications, this problem has to be considered.

Segmented flows

In addition to stratified flows, phases can be put in contact by establishing segmented flow in T or flow-focusing junctions (Fig. 3). With segmented flow, specific interfacial area close to 11000 m -1 [START_REF] Bruus | Theoretical microfluidics[END_REF][START_REF] Dessimoz | Liquid-liquid two-phase flow patterns and mass transfer characteristics in rectangular glass microreactors[END_REF] The acquisition of kinetic data is of great interest in the screening of new extractants preformed to improve the future fuel reprocessing [START_REF] Danesi | The kinetics of metal solvent extraction[END_REF][START_REF] Yagodin | Interfacial phenomena in solvent extraction[END_REF]. Most often, the determination of the chemical reaction coefficients is performed with macroscopic tools which can be classified into two categories: i) tools characterized by a high dispersion of phases, but for which the interfacial area is unknown; ii) tools with controlled interfacial area for which the turbulence and dispersion are weak. Among them, a distinction can be made between tools in which the two immiscible phases are continuous (Lewis cell [START_REF] Lewis | The Mechanism of Mass Transfer of Solutes across Liquid-liquid Interfaces -Part I: The Determination of Individual Transfer Coefficients for Binary Systems[END_REF], Nitsch cell [START_REF] Nitsch | Zur Kinetic der Zinkionenextraktion aus Wasser in dithizonbeladene solventien[END_REF], rotating diffusion [START_REF] Albery | Interfacial transfer studied with a rotating diffusion cell[END_REF] and those in which one phase is dispersed and the other continuous (single drop technique) [START_REF] Nitsch | Die Behandlung der stoffübertragung zwischen flüssigen Phasen als problem der grenzflächenreaktionen[END_REF]). These tools have disadvantages. First, they necessitate a significant consumption of reagents that can be costly, the interfacial area is sometimes poorly controlled, and most often cannot be adjusted. The development of a fast and accurate new kinetic data acquisition methodology is an important issue. In order to be effective, the device used must have three key properties: (1) constant and well known specific interfacial area; (2) fast mixing of the two immiscible liquid phases, in order to operate in the kinetic regime, as opposed to the diffusion regime;

(3) rapid and high-purity phase settling and separation [START_REF] Nichols | Toward mechanistic understanding of nuclear reprocessing chemistries by quantifying lanthanide solvent extraction kinetics via microfluidics with constant interfacial area and rapid mixing[END_REF].

In this context segmented flows in microsystems are new tools for kinetics studies [START_REF] Vansteene | Segmented micro-flows as a tool for optimization of mass transfer in liquid-liquid extraction: application at the extraction of europium (III) by a malonamide[END_REF]. Specific interfacial area can be modulated by changing flow conditions (droplet size can be adjusted [START_REF] Xu | Correlations of droplet formation in T-junction microfluidic devices: from squeezing to dripping[END_REF][START_REF] Cubaud | Droplet breakup and viscosity-stratified flows in microchannels[END_REF][START_REF] Vansteene | Towards chip prototyping: A model for droplet formation at both T and X-junctions in dripping regime[END_REF])

and mass transfer is controlled mainly by the movements of internal forced convection to gout in movement. This impacts the mass transfer. Indeed, in microchannels, the velocity field inside the moving drop is not uniform and the renewal of the reagents at the interface is improved [START_REF] Dessimoz | Liquid-liquid two-phase flow patterns and mass transfer characteristics in rectangular glass microreactors[END_REF][START_REF] Burns | The intensification of rapid reactions in multiphase systems using slug flow in capillaries[END_REF][START_REF] Ufer | Liquid-liquid slug flow capillary microreactor[END_REF][START_REF] Wegener | Fluid dynamics and mass transfer at single droplets in liquid/liquid systems[END_REF][START_REF] Tsaoulidis | Effect of channel size on mass transfer during liquid-liquid plug flow in small scale extractors[END_REF][START_REF] Song | A microfluidic system for controlling reaction networks in time[END_REF][START_REF] Tsaoulidis | Extraction of dioxouranium(VI) in small channels using ionic liquids[END_REF][START_REF] Tsaoulidis | Dioxouranium(VI) extraction in microchannels using ionic liquids[END_REF].

Therefore, mass transfer is favored. This is the background for the recent studies of radionuclides extraction in segmented flows. For instance, segmented flows were implemented in Teflon capillaries for the extraction of uranyl from nitric media by Darekar et al. [START_REF] Darekar | Solvent extraction in microbore tubes with UNPS-TBP in dodecane system[END_REF][START_REF] Nirvik | Solvent extraction and stripping studies in microchannels with TBP nitric acid system Solv[END_REF] and Tsaoulidis and al. [START_REF] Tsaoulidis | Extraction of dioxouranium(VI) in small channels using ionic liquids[END_REF][START_REF] Tsaoulidis | Dioxouranium(VI) extraction in microchannels using ionic liquids[END_REF].

Extraction of U(VI) by TBP in segmented flow nitric media was studied by Tsaoulidis et al. [START_REF] Tsaoulidis | Extraction of dioxouranium(VI) in small channels using ionic liquids[END_REF][START_REF] Tsaoulidis | Dioxouranium(VI) extraction in microchannels using ionic liquids[END_REF] who diluted TBP to 30% (v/v) in ionic liquids, the organic phase being the continuous one. The influence of the residence time on the mass transfer for fixed flow rates was highlighted. Extraction of U(VI) increases with the time of residence. For an aqueous phase such as [HNO3] = 3 mol.L -1 , extraction reaches its equilibrium value of 91% in just 14 seconds (compared with 20 minutes in batch). Thereafter, Darekar, et al. [START_REF] Darekar | Solvent extraction in microbore tubes with UNPS-TBP in dodecane system[END_REF][START_REF] Nirvik | Solvent extraction and stripping studies in microchannels with TBP nitric acid system Solv[END_REF] studied the influence of the residence time of the drops on the transfer of material.

Nichols et al. [START_REF] Nichols | Toward mechanistic understanding of nuclear reprocessing chemistries by quantifying lanthanide solvent extraction kinetics via microfluidics with constant interfacial area and rapid mixing[END_REF] determined mass flow rates of 13 lanthanides and yttrium using a Teflon capillary to generate a segmented flow between an aqueous phase (diethylenetriaminepenta acetic acid and ammonium citrate) and an organic phase containing the extractant acid di-(2-ethylhexyl)phosphoric (HDEHP). A system that mimics the TRUEX (TRansUranium EXtraction) [START_REF] Horwitz | The TRUEX process -a process for the extraction of the transuranic elements from nitric acid wastes utilizing modified PUREX solvent[END_REF] liquid-liquid extraction process was studied in a flow-focusing junction by Nemer et al. [START_REF] Nemer | Drop mass transfer in a microfluidic chip compared to a centrifugal contactor[END_REF]. The extraction of neodymium (Nd 3+ ) from aqueous droplets to dodecane using the complexant thenoyltrifluoroacetone (HTTA) and ligand TBP was chosen as a mass-transfer system model. Parameters including diffusion and distribution coefficients were obtained to better understand the contactor behavior.

Nirvik et al. [START_REF] Nirvik | Solvent extraction and stripping studies in microchannels with TBP nitric acid system Solv[END_REF] performed extraction and stripping in a combined microsystem. µ-LLE experiments were carried out in a T-junction serpentine microchannel and a split-and-recombine microchannel with TBP-nitric acid system. Stripping experiments were carried out in the T-junction serpentine microchannel. The effects of residence time and the organic/aqueous ratio on overall volumetric masstransfer coefficients were discussed. Flow patterns in microchannels were observed to have more insight into experimental trends. For the same average velocity, the split-and-recombine microchannel was found to give higher overall volumetric mass-transfer coefficients. Overall volumetric masstransfer coefficients observed for the two microchannels were compared with overall volumetric masstransfer coefficients reported for conventional extractors.

The extraction of Eu(III) from nitric acid solutions was studied using anionic liquid solution

([CMPO] = 0.2 M-[TBP] = 1.2 M) /1-butyl-3-methylimidazoliumbis[(trifluoromethyl)sulfonyl] amide; ([C4min][NTf2]
) as the extraction phase [START_REF] Li | Intensified Eu(III) extraction using ionic liquids in small channels[END_REF]. The investigations were carried out with the aqueous solution as the dispersed phase. The interfacial area in plug flow and the velocity profiles within the plugs, distribution and mass transfer coefficients were studied.

Conclusion on solvent extraction in microsystems

As shown with the study of the bibliography (Table 1), in most cases µ-LLE developments have been carried out in glass microsystems with Y junctions implementing parallel flows because such flow configuration allows deported analysis by collection of the two phases separately [START_REF] Hibara | Interfacial phenomena and fluid control in micro/nanofluidics[END_REF][START_REF] Kashid | Microstructured reactors for multiphase reactions: State of the art Ind[END_REF][START_REF] Fries | Liquid extraction of vanillin in rectangular microreactors[END_REF].

However micro-droplets exhibit several properties that justify the use of these systems in research over classical macroscale reactors or miniaturized continuous flow microfluidic systems. Indeed, with segmented flow, recirculation of the compounds within the plugs of the dispersed phase and slugs of the continuous phase is responsible for a huge improvement in the global mass transfer [START_REF] Burns | The intensification of rapid reactions in multiphase systems using slug flow in capillaries[END_REF]. The transport of the compounds within their respective phases is operated both by diffusion and advection.

So, segmented flows may be used to retrieve or acquire kinetic data for various extracting molecules.

U(VI) in hydrochloric acid extracted by

Aliquat 336 [START_REF] Hellé | Towards numerical prototyping of labson-chip: modeling for liquid -liquid microfluidic devices for radionuclides extraction[END_REF] Am(III) in nitric acid extracted by CMPO [START_REF] Ban | Extraction of Am(III) at the interface of organicaqueous two-layer flow in a microchannel[END_REF] Pu(IV) in nitric acid extracted by TBP [START_REF] Yamamoto | Evaluation of plutonium(IV) extraction rate between nitric acid and tri-n-butylphosphate solution using a glass chip microchannel[END_REF] Nd [START_REF] Dessimoz | Liquid-liquid two-phase flow patterns and mass transfer characteristics in rectangular glass microreactors[END_REF][START_REF] Zhang | Ju Solvent extraction of Nd(III) in a Y type microchannel with 2-ethylhexyl phosphoric acid-2-ethylhexyl ester[END_REF] [70]

Tm, Gd, Dy, Y par le PC-88A [START_REF] Maruyama | Liquid membrane operations in a microfluidic device for selective separation of metal ions[END_REF][START_REF] Nishihama | Separation of lanthanides using micro solvent extraction system[END_REF][START_REF] Ooe | Development of on-line solvent extraction system with microchips for heavy element chemistry[END_REF] Eu in nitric acid extracted by DMDBTDMA [START_REF] Hellé | Liquid-liquid extraction of uranium(VI) with Aliquat(R) 336 from HCl media in microfluidic devices: Combination of micro-unit operations and online ICP-MS determination[END_REF][START_REF] Hellé | Liquid-liquid microflow patterns and mass transfer of radionuclides in the systems Eu(III)/HNO3/DMDBTDMA and U(VI)/HCl/Aliquat® 336[END_REF][START_REF] Hellé | Towards numerical prototyping of labson-chip: modeling for liquid -liquid microfluidic devices for radionuclides extraction[END_REF] Eu in nitric acid extracted by CMPO [START_REF] Li | Intensified Eu(III) extraction using ionic liquids in small channels[END_REF] Lanthanides and Am stripping [START_REF] Nichols | Toward mechanistic understanding of nuclear reprocessing chemistries by quantifying lanthanide solvent extraction kinetics via microfluidics with constant interfacial area and rapid mixing[END_REF][START_REF] Launiere | High precision droplet-based microfluidic determination of americium(III) and lanthanide(III) kinetics solvent extraction separation[END_REF]

Solid phase extraction (SPE)

Liquid chromatography for the separation of elements is currently used in radiochemistry for elemental and isotopic analyses protocols. The protocols on extraction chromatographic resins are widely implemented on radioactive samples for purified actinides or others elements like lanthanides [START_REF] Horwitz | Separation and preconcentration of uranium from acidic media by extraction chromatography[END_REF][START_REF] Fitzpatrick | The removal of uranium from acidic media using ion exchange and/or extraction chromatography[END_REF][START_REF] Horwitz | Separation and preconcentration of actinides by extraction chromatography using a supported liquid anion exchanger: application to the characterization of high-level nuclear waste solutions[END_REF][START_REF] Chiarizia | Radiolytic stability of some recently developed ion exchange and extraction chromatographic resins containing diphosphonic acid groups[END_REF][START_REF] Horwitz | Novel extraction of chromatographic resins based on tetraalkyldiglycolamides: characterization and potential applications[END_REF][START_REF] Marinov | Sorption of rare-earth elements and Ac on UTEVA resin in different acid solutions[END_REF]. Common methods for actinide matrix removal use chromatographic resin beds that are several milliliters in volume. The difficulty of the miniaturization of solid phase extraction in microsystem lies in the integration of the stationary phase in microchannel. Indeed, the stationary phase should allow the retention of the solutes, presenting a maximum exchange capacity and, often, be resistant to concentrated acids used in radiochemistry. Moreover, a sufficient resolution to obtained purified fraction of each radionuclide is needed. For radiochemical application in acidic media, two routes were considered: filling of the microchannels by particles [START_REF] Wegener | Fluid dynamics and mass transfer at single droplets in liquid/liquid systems[END_REF] or a stationary phase synthesized in -situ [START_REF] Bruchet | Centrifugal microfluidic platform for radiochemistry: potentialities for the chemical analysis of nuclear spent fuels[END_REF][START_REF] Losno | Photochemical synthesis and versatile functionalization method of a robust porous poly(ethylene glycol methacrylate-co-allyl methacrylate) monolith dedicated to radiochemical separation in a centrifugal microfluidic platform[END_REF][START_REF] Losno | Separation and preconcentration of actinides from concentrated nitric acid by extraction chromatography in microsystems[END_REF].

Particulate stationary phase in microchannel

Two companies have addressed the manufacture of chromatographic microsystems as a simple scaledown technology transfer, from the usual chromatographic columns (inner diameter of 4.6 mm) to microchannels of hundreds of microns or less, integrating particulate stationary phases. Agilent

Technologies has developed a "top-down" approach by integrating the injection loop and the separation column into a microsystem in polyimide [START_REF] Yin | Microfluidic chip for peptide analysis with an integrated HPLC column, sample enrichment column, and nanoelectrospray tip[END_REF]. This device helped to significantly improve the robustness of the instrumentation, its ease of use and the reduction of the risk of leaks. In terms of performance, the integration of part of the injector in the microsystem minimizes external dispersion phenomena and improves separation [START_REF] Yin | Microfluidic chip for peptide analysis with an integrated HPLC column, sample enrichment column, and nanoelectrospray tip[END_REF][START_REF] Yin | The fundamental aspects and applications of Agilent HPLC-Chip[END_REF]. Eksigent society has adopted the same approach for its chip [START_REF] Hebert | Using 200 μm ID cHiPLC columns for increased sample throughput in peptide quantitation[END_REF]. However, these two commercial chips found application in the field of biology.

To the best of our knowledge, to date, no commercial microsystem dedicated to the separation of metals or radionuclides has been developed and only one study implementing particulate stationary phase in microchannel was reported. Gao et al. [START_REF] Gao | Solid-phase extraction microfluidic devices for matrix removal in trace element assay of actinide materials[END_REF] designed 100-μL and 20-μL solid-phase separation columns in microfluidic devices built in thermoplastic polyvinyl chloride (PVC, Fig. 4).

The microcolumns, packed by centrifugation with specific resins, provide efficient actinide matrix removal from trace element impurities. Commercially-available anion exchange and chromatographic resins were chosen to separate traces impurities from concentrated thorium or uranium matrices in strong nitric acid solutions, while trace impurities passed through quantitatively for analysis by inductively coupled plasma -optical emission spectroscopy (ICP-OES). Actinides were subsequently recovered using dilute hydrochloric acid solution. The filling of the micro-colonnes was performed with the open system and phases particulate were held in place thanks to the presence of sintered at both ends of microcolonnes. Flow rates ranged from 20 to 100 µL.min -1 . UTEVA resin (column 2). Scale is illustrated using a US dime coin (17.9 mm diameter) [START_REF] Gao | Solid-phase extraction microfluidic devices for matrix removal in trace element assay of actinide materials[END_REF].

Monolithic stationary phase synthesized in-situ in microchannel

Monolithic stationary phases in chromatography and electrochromatography emerged in the early 1990s [START_REF] Guiochon | Monolithic columns in high-performance liquid chromatography[END_REF][START_REF] Svec | Monolithic materials: promises, challenges, achievements[END_REF]. These materials gained popularity because they are formed from a single piece of porous material that includes large flow through pores enabling separations at high flow rates achieved at low back pressures. Organic monoliths can be easily prepared within the confines of a microchannel (mold) by photo-initiated polymerization of a mixture of monomers (functional and crosslinker), porogenic solvents, and a free radical initiator. Organic monoliths combine a good mechanical resistance by covalent anchored to the walls of the microchannels, good control of porosity and permeability, and wide possibilities of chemical functions. Reviews [START_REF] Svec | Monolithic materials: promises, challenges, achievements[END_REF][START_REF] Svec | Less common applications of monoliths: preconcentration and solid phase extraction[END_REF][START_REF] Potter | Porous polymer monoliths for extraction: diverse applications and platforms[END_REF][START_REF] Namera | Monolith as a new sample preparation material: recent devices and applications[END_REF] demonstrated that organic polymer-based monoliths for off-line SPE can be prepared in formats including the conventional syringe cartridges, pipette tips, stir bars, and spin columns. For on-line SPE, however, the in-tube or microchip formats are mandatory, and include nuts and ferrules for fitting the column to the analyzers and flow-through detectors. Then, monolithic columns for extraction of metal ions from environmental water and biological matrices (human hair, urine, and serum) were prepared by functionalizing the stationary phase with complexing functionalities [START_REF] Masini | Porous monoliths for on-line sample preparation: A review[END_REF].

The strong acidity of the mobile phases used in radiochemistry leads to the use of organic monoliths for their chemical resistance.

Inspired from the study of Lafleur et al. [START_REF] Lafleur | Pre-concentration of trace metals on centrifugal microfluidic discs with direct determination by laser ablation inductively coupled plasma mass spectrometry[END_REF] who have elaborated a monolithic microcolumn inserted into a centrifuge microsystem for the separation and analysis of V, Pb, Cu and Co in water to drink, Bruchet et al. [START_REF] Bruchet | Centrifugal microfluidic platform for radiochemistry: potentialities for the chemical analysis of nuclear spent fuels[END_REF] developed an original design of microfluidic platform integrating four rectangular microchips made of thermoplastic (cyclo olefin copolymer COC). The microchips, dedicated to anionexchange chromatographic separations, integrated a localized monolithic stationary phase as well as injection and collection reservoirs (Fig. 5). For a simplified simulated nuclear spent fuel sample composed of non-radioactive isotopes of Eu and U in usual proportion for Uranium Oxide nuclear spent fuel, analytical results were consistent with the conventional procedure (extraction yield for Europium of about 97 %). The use of the centrifugal microfluidic platform allowed to reduce the volume of liquid needed by a factor 250. Thanks to their unique "easy-to-use" features, centrifugal microfluidic platforms are potential successful candidates for the downscaling of chromatographic separation of radioactive samples (automation, multiplexing, easy integration in glove-boxes environment and low cost of maintenance). A quaternary ammonium was covalently bound to the monolith thanks a thermal reaction. However, the functionalization was not robust in nitric acid media and the functionalization procedure was suitable only for the grafting of tertiary or secondary amines. The separation of Eu, Th and U was performed in the simple microsystem functionalized with TBP with only 30 µL of reagents compared to mL used in classical SPE with flow rates set at 2 μL min -1 .

Compared to the protocol carried out for classic SPE [START_REF] Yamaura | Sequential separation of actinides and lanthanides by extraction chromatography using a CMPO-TBP/XAD7 column[END_REF], there is a significant reduction in volumes handled (Table 2). The procedures of Gao et al. [START_REF] Gao | Solid-phase extraction microfluidic devices for matrix removal in trace element assay of actinide materials[END_REF] is faster (about 30 minutes) than the one made on TBP impregnated monolith, but possible parallelization of separation in centrifugal microsystem allows to compensate for this difference. Compared to the classic SPE, a microsystem of extraction chromatographic incorporating a monolithic stationary phase dimensioned based on the detection limit of the measurement technique allows to make separations with volumes samples less than 100 µL and at flow rates lower than 100 µL.min -1 .

Thanks to this tool, much less effluent will be generated by analysis that demand continually believes and the cost of waste management in nuclear laboratories will be reduced.

Conclusion

In 2011, George M. Whitesides noted microfluidics, and the concept of the lab-on-a-chip, have had a spectacularly successful 15-year run of science and technology [START_REF] Whitesides | What comes next?[END_REF]. He asked "what comes next?"

Henceforth, this review is indicative of the rise of the development of lab on chips dedicated to radiochemistry. In the nuclear field, chemical and physico-chemical analysis and screening of new extractant now consider environmental issues in the development of a technique or a method of analysis. At every stage of an analytical process, the questions focus on:

-the reduction of volumes of reagents, solvents and samples;

-the reduction of waste production;

-the reduction of the consumption of energy, water, gas...

-the increase of speed and accuracy of the analytical response.

To meet current and future needs, the preferred development areas relate to: i) the implementation of parallelizable and automatized sample preparation methods. Microfluidic tools already significantly reduce analytical effluent generated by the methods currently used in laboratories. However, their parallelism and their automation must still be subject to developments requiring the collaboration of chemists, control engineers, programmers.

(iii) the development of MicroTAS (miniaturized total analytical system). In radiochemistry, developed microsystems are coupled to spectrometers of laboratories for elemental or isotopic analyses. Now, the effort should focus on integration of the measure directly in the lab on a chip. It will lead to a drastic decrease of the volumes of samples and reagents.

Fig. 1 .Fig. 2 .

 12 Fig. 1. (a) Parallel flows (b) segmented flow.

Fig. 3 .

 3 Fig. 3. Schematic of (a) a T-junction and (b) a flow-focusing junction used to establish segmented flows. In white the continuous phase and in black the segmented phase.

Fig. 4 .

 4 Fig. 4. Assembled 20-μL solid-phase extraction microfluidic device, filled with AG MP-1 resin (column 1) and

Fig. 5 .

 5 Fig. 5. Design of the microchips: C1 ion-exchange column, C2 restriction channel, C3 vent channel, R1 injection reservoir, R2 collection reservoir and chromatographic connection, R3 vent and chromatographic connection, E1entry port and E2 vent port of the injection reservoir. The centrifugal platform is presented in[START_REF] Bruchet | Centrifugal microfluidic platform for radiochemistry: potentialities for the chemical analysis of nuclear spent fuels[END_REF].

Table 1 :

 1 Synthesis of the literature review of extraction of nuclear systems studied in microsystems

			Segmented flow
	Extracted analyte	Parallel flows	Organic continuous	Aqueous
			phase	continuous phase

Table 2 :

 2 Performance comparison of different SPE systems of radionuclides in acidic media

	Separation	Separative	Column	Sample	Time	Media	Reference


Hugo Salmon

Rabah Gahoual

Pascal Houzé

Tayssir Ibrahim

Michel Bessodes

Daniel Scherman

Johanne Seguin

Nathalie Mignet
email: nathalie.mignet@parisdescartes.fr

Dr J S Author

Europium labeled lactosylated albumin as a model workflow for the development of biotherapeutics

Keywords: targeted imaging, nanomedicines, bioconjugates development, lanthanide chelates, time resolved fluorescence

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

2

of the fluorescence decay of the lanthanide, we performed measurements directly on the cell media without any further treatment. Finally, biodistribution in mice was confirmed by ex vivo measurements.

Background

The use of proteins for therapeutic purpose has proven to be an unprecedented success over the last two decades and has spurred the growth of the biopharmaceutical industry. The number of proteins approved and technologies involved continue to increase. [START_REF] Dimitrov | Therapeutic Proteins[END_REF] One of the most advanced technologies is the chemical modification of proteins to incorporate additional functionalities. In particular, the use of chemical modification has successfully produced antibody-drug conjugates that combine the epitope specificity of immunoglobulin G (IgG) with a potent cytotoxic drug covalently bonded to the peptide backbone. [START_REF] Beck | Strategies and challenges for the next generation of antibody-drug conjugates[END_REF] Currently, no rigorous method exists to relate the chemical identity of a protein to its biological one, especially by evaluating its in vivo outcome. [START_REF] Coty | Characterization of nanomedicines: A reflection on a field under construction needed for clinical translation success[END_REF] Nanotechnology Characterization Laboratories (NCL) have been created both in the US and Europe for the development of standardized analytical methods. [START_REF]Nanotechnology Characterization Laboratory : Assay cascade protocols[END_REF][START_REF]EUNCL Nanomedicine Characterisation Laboratory : Assay Cascade[END_REF] As a result of the inherent complexity of proteins, partial modifications to the peptide backbone require detailed characterization after the modification process. Indeed, the addition of a chemical compound, covalently bonded to the peptide backbone, could potentially destabilize the tertiary and/or quaternary structure of the native protein. Therefore, it could potentially result in a drastic modification of the physico-chemical properties and functionality of the protein. For instance, protein aggregation has been found to be primarily due to partial unfolding. [START_REF] Roberts | Therapeutic Protein Aggregation: Mechanisms, Design, and Control[END_REF] Consequently, intensive research was recently conducted to establish the analytical methodologies required to achieve a comprehensive characterization of the different levels defining the structure of therapeutic proteins. [START_REF] Fekete | Chromatographic, Electrophoretic, and Mass Spectrometric Methods for the Analytical Characterization of Protein Biopharmaceuticals[END_REF][START_REF] Gahoual | Cutting-edge capillary electrophoresis characterization of monoclonal antibodies and related products[END_REF] These properties should provide a global understanding of the structure of the developed therapeutic protein to assess the formation of endogenous species such as aggregates and/or other types of degradation products. [START_REF] Berkowitz | Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars[END_REF] Thus, from a development perspective, the implementation of a dedicated analytical workflow should also provide robust information regarding potential protein conjugates to optimize the modification process. Such a methodology could be useful to nanomaterial development ranging from ongoing fundamental research to preclinical evaluations.

Prior to the clinical phase, the biodistribution of therapeutic proteins should be assessed, especially when the protein strategy involves specific targeting. In general, biodistribution is performed by positron emission tomography (PET) or single-photon emission tomography (SPET) imaging due to their high sensitivity, or even single-photon emission computed tomography (SPECT) imaging where both resolution and sensitivity is obtained. However, labelling conditions are a main concern since they can alter protein structure or induce aggregation. [START_REF] Richard | Amphiphilic perfluoroalkyl carbohydrates as new tools for liver imaging[END_REF] Moreover, the use of radioisotopes requires additional safety and experimental conditions that are not available in all laboratories. In this context, we investigated the shift to lanthanide labelling as a means of anticipating labelling conditions and protein characterization. As such, Europium (Eu) appears to be a good candidate for substitution. Eu (III) is readily available, does not require a restricted lifetime compared to conventional fluorophores, and has also demonstrated strong photoluminescent capacity. [START_REF] Binnemans | Interpretation of europium(III) spectra[END_REF] Eu (III) forms complexes with metal cation chelating agents like diethylenetriamine-pentaacetic acid (DTPA) or tetraazacyclododecane-tetraacetic acid (DOTA), so it could be used to replace 99m Tc or 68 Ga to obtain preliminary in vitro and in vivo data.

For liver disease patients, the assessment of hepatic function plays a decisive role in developing the appropriate therapeutic strategy, which might include irreversible measures like partial resection and liver transplantation. [START_REF] Clavien | Strategies for Safer Liver Surgery and Partial Liver Transplantation[END_REF] Patients with primary and secondary liver diseases demonstrate a reduced number of asialoglycoprotein (ASGP) receptors, expressed mainly on the hepatocyte sinusoidal surface. [START_REF] Marshall | Measurement of circulating desialylated glycoproteins and correlation with hepatocellular damage[END_REF] ASGP receptors allow for the internalization of ASGP through receptor mediated endocytosis, so they are a relevant indicator of functional hepatocytes. The quantification of liver uptake using radiolabeled ASGP, in the format of 99m Tc-DTPA-galactosyl serum albumin ( 99m Tc-GSA), proved to be a reliable and noninvasive approach for the evaluation of liver function. [START_REF] De Graaf | 99mTc-GSA Scintigraphy with SPECT for Assessment of Hepatic Function and Functional Volume During Liver Regeneration in a Rat Model of Partial Hepatectomy[END_REF] We have previously described a novel one-pot preparation protocol of 99m Tc-DTPA-lactosyl serum albumin (LACTAL). [START_REF] Scherman | Nouveaux conjugues, utilisables a des fins therapeutiques, et/ou a titre d'agent de diagnostic et/ou d'imagerie et leur procede de preparation[END_REF] For LACTAL preparation, two different chemical compounds were conjugated to the protein.

DTPA was incorporated onto the HSA peptide backbone to form complexes with the 99m Tc.

Targeting of the ASGP receptor was provided by the grafting of lactose moieties. The labelling process maintained the stability of human serum albumin (HSA) to provide consistent liver targeting. Radiolabeled HSA showed interesting liver targeting capabilities. In addition, the detected signal in scintigraphy provided semi-quantitative information regarding the uptake kinetics. [START_REF] Chaumet-Riffaud | Synthesis and application of lactosylated, 99mTc chelating albumin for measurement of liver function[END_REF] In this work, we first implemented Eu (III) for the substitution of 99m Tc to the LACTAL protein conjugate, providing a daily labor practice tool. We performed detailed characterization using orthogonal analytical techniques. We estimated the mean number of each type of chemical compound (propyl-DTPA and propyl-lactose) effectively added to HSA.

The formation of endogenous aggregates due to the modification of the protein was evaluated.

LACTAL was successfully used for liver scintigraphy imaging, so it demonstrates the relevance of Eu (III) use as a fluorescent label. A panel of in vitro experiments were conducted to demonstrate that Eu (III) can successfully form complexes with the chelating agent grafted to the protein and do not provide cytotoxicity. Concomitantly, in vivo biodistribution was tracked to demonstrate the applicability of Eu (III) fluorescence in biological media. These experiments also allowed for the determination of whether specific hepatocyte targeting could be maintained when Eu (III) replaced the 99m Tc moiety in the probe. We finally introduced an original workflow spanning the synthesis and development of LACTAL.Eu to in-vivo validation, and offer it as a guideline to develop biomolecular conjugates for imaging applications, specifically using lanthanide cryptates. Each step included go/no-go tests and standard methods to illustrate the workflow. We emphasized analytical validation to quantify grafting and colloidal stability.

Methods

Chemicals reagents were obtained from Sigma-Aldrich ((Breda, The Netherlands), solvents were purchased from Carlo Erba (Val-de-Reuil, France). Maleimido-propyl (1-(β-D-LActosyl)-3-maleimidopropionamide) lactose was synthetized as described earlier.

Asymmetrical flow field flow fractionation coupled to multi-angle light scattering

analysis, UV and RI (AF4-MALS-UV-RI) characterization. The AF4 controlling system was an Eclipse Dual Tec separation module from Wyatt Technology Europe, Germany, suitable for aqueous systems. The mobile phase was delivered using a Thermo Fischer scientific Dionex Ultimate 3000 HPLC (Manchester, UK) natively equipped with a vacuum degasser, an auto-sampler and a UV detector. Between the auto-sampler and HPLC pump, mobile phase was filtered in-line by a 0.1 µm durapore PVDF membrane in a PEEK in-line filter. This channel was an SC series (15 cm long) with a wide type spacer of 350 µm. The membranes used for separation were made of regenerated cellulose, Wyatt Technology. Detection was realized using UV at a wavelength of 280 nm. Concomitantly, refractive index quantifications were performed by means of a T-Rex differential refractometer detector using a 660 nm laser source (Wyatt Technology). Mobile phase was NaNO3 10 mM NaN3 0.02 % in milli-Q water degassed by vacuum pump and filtered using stericup filter units.

Bioconjugates were eluted with a two-step separation. First step -focalization -was an isoforce (constant flow rate) of 3 mL/min during 5 min and second was an exponential decrease of the separative force from 3 mL/min to 0 mL/min in 9 min. The preliminary 5 min focusing step at 3 mL/min limits diffusion effect to maximize the separation resolution. The aggregation comparative study used standard clinical HSA as a control and all solutions were injected at 2 mg/mL in 50 µL. MALS detection was achieved with a DAWN module from Wyatt Technology Europe. It was equipped with a Qels module for DLS supplementary analysis at angle 9 of the MALS detector. Data analysis was achieved on Astra 6.1, Wyatt Technology.

Mass Spectrometry (MS) characterization. MS experiments were performed using a

Voyager DE pro mass spectrometer from Applied Biosystems (Darmstadt, Germany). This instrument implements a matrix assisted laser desorption (MALDI) ionization source and a time-of-flight (TOF) mass analyzer. The matrix used was composed of α-Cyano-4hydroxycinnamic acid (HCCA) purchased from Sigma-Aldrich (Breda, The Netherlands).

Based on bicinchoninic acid assay results, the samples were diluted to a final concentration of 1 µg/µL in milli-Q water. A sample volume of 1 µL was mixed with 3 µL of HCCA (10 mg/mL in H2O/ACN), then 1 µL of this mixture was deposited on a stainless-steel plate.

The droplet was left to dry at room temperature. MS measurements were performed in positive ionization and linear mode. Ion transfer parameters were optimized using the clinical grade human serum albumin. MS spectra were acquired in the m/z range 8400 -101000 Th.

Each MS spectrum was obtained through the summation of 500 laser shots. MS data treatment was performed using Voyager Data explorer software (Applied Biosystems, Foster city, CA).

The average mass, for the detected species was determined by considering the different charge states detected, usually from 1 to 3.

Ricin agglutinin test:

Different concentration of Lactal.Eu or HSA-DTPA.Eu and ricin agglutinin (1 g/l) were prepared by mixing equal volumes. The turbidity was then measured at 450 nm with a Victor2 spectrophotometer plate reader from Perkin-Elmer. HEPG2 internalization. HEPG2 cells were grown into DMEM supplemented with lglutamin (29.2 mg/mL), penicillin (50 U/mL), streptomycin (50 U/mL), and 10 % fetal bovine serum. The day before the experiment, the cells were seeded into 24 well culture plates at 3 different concentrations (10 5 , 2x10 5 , 4x10 [START_REF]EUNCL Nanomedicine Characterisation Laboratory : Assay Cascade[END_REF] ) overnight at 37 °C under 5 % CO2. Twenty-four hours later HSA-DTPA.Eu and LACTAL.Eu (6.2 µM DTPA) were added for four hours at Six-week-old female (≈20 g) BALB/c J mice (Janvier) were inserted in a rodent restrainer and HSA-DTPA.Eu or LACTAL.EU were injected into the mouse tail vein (200 µl, 2.7 mg/ml for LACTAL.Eu and 2 mg/mL for HSA-DTPA.Eu). One mouse was dedicated as a control, 5 were injected per product. Twenty minutes after injection we proceeded to euthanasia, perfused PBS into the heart left ventricle and gathered the first drops of concentrated blood by cutting the right atrium. PBS injection was achieved using a peristaltic pump at 5 mL/min. After 25-30 mL perfused in mice to remove blood, the organs appeared bleached. We then collected and weighed the liver, spleen, lungs and kidney.

Probe extraction. After collecting the tissues, we immersed organs in Luciferase Cell Culture Lysis 5x Reagent (125 mM Tris (pH 7.8) with H3PO4, 10 mM CDTA, 10 mM DTT, 50 % glycerol and 5 % Triton X-100) mixed with a Red Blood Cell Lysis Solution 1X Miltenyi Biotec. We took into consideration each organ's measured relative density (liver 0.99, spleen 1.01, kidney 0.97, lung 0.60). We performed tissue homogenizing using a Precellys Evolution manufactured by Bertin Instruments and supplied by Ozyme (Montigny-Le-Bretonneux, France). Lysis kits were 2 and 7 mL tubes, containing 1.4 mm ceramic beads (CK14). 4 cycles of 30 s were achieved at 6500 rpm with samples being maintained 1 min on ice between each cycle to avoid bubble formation and heat degradation.

Results

Synthesis and analytical assessment of LACTAL.Eu. LACTAL was verified based on amine thiolation using Traut's reagent followed by thiol maleimide reaction (Figure 1) and was improved compared to previous art [START_REF] Chaumet-Riffaud | Synthesis and application of lactosylated, 99mTc chelating albumin for measurement of liver function[END_REF] in favor of lactose binding over DTPA. First by optimizing the thiolation of lysine hot spots (Figure 1A), then by introducing the lactose with a 15 min delay before DTPA, -Figure 1B. Remaining free maleimide-DTPA may chelate free Eu (III), therefore it had to be removed from the sample before the addition of Eu (III) using ultrafiltration. To evaluate the eventual presence of free DTPA, Eu (III) was added to the filtrates and the solution was measured by time-resolved fluorimetry (TRF) to ensure that all free DTPA had been washed out (Figure S1). 4 filtration cycles led to a more than 99.9 % drop in the fluorescent signal. The final medium of the chelates was saline buffer to avoid competitive complexation with ions such as phosphate during conservation which could lead to Eu (III) transfer. [START_REF] Byegård | The stability of some metal EDTA, DTPA and DOTA complexes: Application as tracers in groundwater studies[END_REF] We then evaluated the global synthesis yield and aggregation distribution. The average efficiency of synthesis could be first evaluated by a bicinchonic acid assay (BCA) -Figure S2. Below R < 80 %, the reaction efficiency was considered unsatisfying and led to the optimization of reactive quantities, ultracentrifugation time and speed and membrane material used, mainly to avoid precipitates. LACTAL synthesis with optimized conditions reached upto 90 %.

Figure 2B

summarizes the distribution of sizes up-to trimer sizes and aggregates (usually above 10 nm aggregated objects) and confirms that 80 % of LACTAL as well as HSA-DTPA remained under its monomeric form. This hyphenated analysis could also be a substitute to protein quantification by UV or RI detection by achieving preliminary calibrations. This technique also revealed that DTPA grafting seemed to generate more aggregates as LACTAL and HSA-DTPA observe a 10-14% fraction whereas lactosylated and non-modified albumin displayed better stability. This is most likely due to DTPA being highly sensitive to pH variations. An optimization of buffer pH and salinity could improve performances.

The ligand quantification average value could be achieved using commercial assay kits as described previously: thiol can be evaluated using the Ellman's reagent and maleimide using a reverse glutathione assay. [START_REF] Chaumet-Riffaud | Synthesis and application of lactosylated, 99mTc chelating albumin for measurement of liver function[END_REF] The accuracy and specificity of these assays vary greatly depending on reagents. While an average ligand concentration is provided, there is no information about the monomer polydispersity induced by grafting. We hereby proposed to base our study on physical analysis methods and separation techniques.

We used hyphenated method AF4 (or SEC)-MALS-UV-RI performed using light scattering to obtain aforementioned aggregate quantification and molar weight increase of each synthesis product in a single run. From the Zimm scattering plot, we extracted the molar mass increase and compared it to MS values -see Figure 2C. We plotted in Figure 2A the MW function of elution volume and observed a slight variation on the monomer peak due to inhomogeneity generated by synthesis mechanism. This homogeneity was even more evident when we studied aggregates, whose MW varied between 200 kDa and 1 MDa. Gyration radii of samples could also be monitored from Zimm plot above 10nm in Figure 2D. This allowed for the control of aggregate sizes and confirmation that they remained between 10 and 100 nm.

Averaged MW values for each HSA monomer (HSA, HSA-Lactose, LACTAL and HSA-DTPA) were summed up in Table 1 (LS column).

The average number of each compound, effectively coupled to HSA, was also determined using MALDI-TOF mass spectrometry which allowed for the estimation of induced polydispersity from a few µg of sample as well as the MW increase to assess the grafted mass. Thus, as illustrated by Figure 3A, the MS spectrum corresponding to the clinical HSA gave an average mass of 66447 Da which agrees with the theoretical mass of HSA considering the performances of the instrument. The characterization of the HSA-DTPA conjugate allowed measurement of a mean mass of 76210 Da which represents an additional mass of 9763 Da compared to the native HSA (Figure 3B). Using this mass difference, it was possible to estimate the attribution of a mean grafting value of 12 DTPA per protein. Also, the results indicated that 15 additional lysines underwent thiolation but did not react with the maleimide-DTPA. The experiment performed on the LACTAL.Eu conjugate allowed for measurement of an average mass of 76610 Da as emphasized in Figure 3C. The mass difference compared to native HSA was attributed to the incorporation of 7 DTPA and 7 lactoses onto the peptide backbone of the protein. Also, like the previous conjugates, 12 additional lysines were found to be modified through thiolation but did not ultimately undergo incorporation of either one of the ligands. The results achieved using MALDI-TOF mass spectrometry demonstrated that it is possible to obtain relevant and consistent information in a straightforward manner regarding the conjugation process, which enables critical evaluation of the performance of the conjugation reaction. MALDI-TOF MS characterization could be therefore used to optimize the reactions conditions. Indeed, pH conditions were adjusted, and nitrogen degassing was implemented to the preparation protocol to improve the number of ligands effectively coupled with the protein.

In particular, MS analysis was performed on HSA after the thiolation reaction. Using optimal conditions, MS analysis provided a mass of 69360 Da for thiolated HSA which corresponds to an average number of 27 thiols effectively added to the protein, and potentially available for reaction with a maleimide (Figure S3).

We determined HSA and DTPA dn/dc to be 0.1731 and 0.1574 mL/g respectively. The literature value of lactose is 0.150 mL/g. [START_REF] Theisen | Refractive Increment Data-Book: For Polymer and Biomolecular Scientists[END_REF] Exploiting both UV and RI quantification, we could quantify proteins, ligands (modifiers) and total bioconjugate MW in a straightforward manner -see Figure S4.

We achieved titration of DTPA on LACTAL and HSA-DTPA products with Eu(III) itself with the Eu / DTPA complexation pK being around 22.5 and having quick kinetics. [START_REF] Byegård | The stability of some metal EDTA, DTPA and DOTA complexes: Application as tracers in groundwater studies[END_REF] When not complexed and free in solution, Eu 3+ ions are quenched by OH -. Progressively increasing their quantity leads to a drastic slope break in fluorescence response when these ions stop being chelated by DTPA, as observed in Figure S5. This provides the equivalent amount of grafted DTPA, concluding the analytical steps with chelating moieties quantification. A quantity of 2 -4 DTPA is enough for a sensitive measurement with Eu 3+ . Considering that Eu has a low quantum efficiency fluorescence and is proposed in this study as a substitute for Tc or Gd chelates to evaluate their biodistribution, it should require fewer DTPA moieties. In case of insufficient TRF signal, the pH and eventually the synthesis kinetics and quantities can be adjusted.

In vitro evaluation. We introduced the lactose moiety to the protein to provide a targeting ligand that would retain the protein in tissues expressing the asialoglycoprotein receptor.

Since this receptor is mainly located at the apical sites of hepatocytes, [START_REF] Mizuno | Cellular Distribution of the Asialoglycoprotein Receptor in Rat Liver Implications for Hepatic Accumulation of Desialylated Lymphocytes[END_REF] we aimed to study the interaction between LACTAL.Eu and hepatocytes; however for this , we had to find a way of measuring this interaction.. As the protein bore a DTPA moiety for scintigraphy, we also used europium complexed DTPA to measure interaction between LACTAL.Eu and cells.

Prior to the cell experiment, it is necessary to qualitatively confirm that the targeted receptor interacts with the grafted ligand, which in this case was lactose. As shown in Figure 4A, we used the ricin agglutinin test [START_REF] Zentz | Binding of galactose and lactose to ricin. Equilibrium studies[END_REF] and showed that the LACTAL.Eu did induce an absorbance increase (dark circle) , meaning an agglutination of the ricin only when the lactose was present on the protein and this was reversed in the presence of free lactose in the suspension (dark square).

In vitro models are often criticized compared to vivo models for failing to replicate the precise cellular conditions of an organism. In particular, receptors expression varies greatly depending on cell culture conditions and cell lines. [START_REF] Nakaya | Regulation of asialoglycoprotein receptor synthesis by inflammation-related cytokines in HepG2 cells[END_REF] Conventional western blot and immunofluorescence provided a qualitative measure of receptor expression (Figure S6).

Moreover, to acquire a fine quantification of ASGPR expression, we used flow cytometry and compared three hepatocyte cell lines (BWTG3, HEPG2 and TIB75) and 1 fibroblast cell line (3T3) -see Figure S7. The optimal percentage of ASGPR expression was obtained with HEPG2 lines was 29.4 % expression. However, this parameter can be optimized by the culture conditions, particularly to amplify the internalization response demonstrated in the next step.

We loaded LACTAL.Eu and HSA-DTPA.Eu respectively onto hepatocyte (HEPG2) cells expressing the ASGPR receptor as verified. The advantage of europium in this case is that signals could be measured in the cell lysates to give quantitative data based on a preliminary calibration in the media. The specific interaction between the LACTAL.Eu and HEPG2 cell line was tested after an incubation of 4h at 37 °C as shown on Figure 4B. As expected, a significant difference was obtained with the lactose conjugated with albumin for a cells quantity of 2x10 5 and 4x10 5 per well. Finally, we assessed the toxicity of LACTAL.Eu and HSA-DTPA compound by measuring mitochondrial activity -see Figure S8. No major toxicity were observed with BNL and INH-3T3 cell lines after 24h.

In vivo evaluation.

Optimizing the extraction of the biomolecule pushes the probe's limits of detection. In the case of TRF, we increased the number of parameters, including delay time, gate time and fluorescence measurement. Since TRF measurements of chelated Eu (III) are linear, the limit of detection of the spectrophotometer is directly related to noise level, background and sensitivity, i.e. slope. Though plate-readers usually have factory parameters, the literature reveals a great diversity of detection protocols when dealing with time resolved fluorescence. [START_REF] Mignot | A top-down synthesis route to ultrasmall multifunctional Gd-based silica nanoparticles for theranostic applications[END_REF] Figure S9 demonstrated this attempt to empirically optimize the signal. A standardized homogenization procedure optimizes the reproducibility and performances of protein extraction from tissue and avoids probe fouling due to heat. We optimized various commercial lysis buffers (Figure S10) and combined the advantages of a standard lysis buffer with a red blood cells lysis buffer, as erythrocytes absorb at 620 nm. Papain protease was added to maximize extracted probes from proteins while avoiding eventual quenching which confirmed the relevance of a delayed fluorescence approach.

The main criterion for ex-vivo quantification is sufficient hepatic cellular uptake to confirm targeting. The use of Eu (III) time resolved assays and large Stokes shift overcame tissues' auto fluorescence, contributing to the effort of improving the limit of biodistribution quantification. We evaluated LACTAL.Eu uptake versus HSA-DTPA.Eu in a group of mice using ex-vivo TRF. As shown in Figure 5, a significant difference of 9.6 % of injected dose (ID) was observed between the accumulation of the HSA-DTPA.Eu and the LACTAL.Eu probes in the liver (p < 0.01, ***). Other organs and blood did not display any specific response between the two products. The grafting of lactose had a significant effect on LACTAL.Eu distribution and corroborated its targeting capacities.

We finally designed a rationalized development workflow using our experience with LACTAL.Eu development combined with conjugated proteins [START_REF] Scherman | Nouveaux conjugues, utilisables a des fins therapeutiques, et/ou a titre d'agent de diagnostic et/ou d'imagerie et leur procede de preparation[END_REF] and our knowledge of therapeutic protein characterization. [START_REF] Coty | Characterization of nanomedicines: A reflection on a field under construction needed for clinical translation success[END_REF] This workflow highlights key criteria for the rigorous evaluation of the developed conjugate and elimination of unsatisfactory candidates. We used a conditional decision methodology (Figure 6) to optimize the procedure for in vivo characterization and ultimately facilitate clinical translation.

Discussions

Among the factors influencing biodistribution and sensitivity of the probe in the litterature, the first is the substrate type (protein, inorganic, nanoparticle…) and its chemical characteristics, such as size, shape, and charge. [START_REF] Blanco | Principles of nanoparticle design for overcoming biological barriers to drug delivery[END_REF] Second, the conjugation pathway used for ligand binding should be considered. Third, the colloidal stability and medium used for the synthesis, conservation, or in vivo injection of the biomolecular probe could impact the size distribution and number of grafted ligands with consequences for targeting function, biodistribution [START_REF] Blanco | Principles of nanoparticle design for overcoming biological barriers to drug delivery[END_REF] and detection. [START_REF] Kijanka | Influence of aggregation and route of injection on the biodistribution of mouse serum albumin[END_REF] Finally, the specificity of the ligands and the properties of the imaging agent conjugated on the particle surface may influence the recognition and binding to the specific receptors, [START_REF] Thomas | Ultrasmall AGulX theranostic nanoparticles for vascular-targeted interstitial photodynamic therapy of glioblastoma[END_REF] increase the circulation half-life [START_REF] Gref | Stealth" corona-core nanoparticles surface modified by polyethylene glycol (PEG): Influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption[END_REF] and define the imaging sensitivity and modalities (epifluorescence, scintigraphy, TRF or FRET…).

The signal exhibited on the mass spectra from Figure 3B demonstrated a reasonable peak width increase compared to the signal of native HSA which strongly suggests that the entity produced was not completely homogenous. Finally, the intensity of the signal was significantly lowered compared to native HSA. This observation was attributed to an increased capacity of the conjugate to absorb the energy delivered by the MALDI laser.

Though AF4-MALS-UV-RI offers a wider range of information, this technique has a nonnegligible difference with MS values, particularly for lactosylated molecules with 4kDa for HSA-lactose. MALS data relies on the analyte optical constants i.e. refractive index increment dn/dc and extinction coefficient ε. For small mass increases, below 10 % of initial mass, the optical constant of the native protein alone predominated but in our case we also needed to specify the ligand's coefficients. We compared in Table 1 (Protein Conjugate (PC) column) this alternative quantification of ligands with other ligand quantification in better agreement with MS spectrum for total mass. It seemed the modifier values were less accurate for LACTAL products which is due to the presence of two different grafting mechanisms whereas the model supposed only one. Though LS coupled to separation methods provided a low trueness value compared to mass spectrometry, the technique can be improved by knowing the modifiers' optical constants. Containing more information on conjugates homogeneity, polydispersity and sizes, this detection method constitutes a straightforward tool particularly useful for drug screening that can be used intermittently. Coupled to separative methods, it offers a straightforward manner to run several synthesis conditions and to obtain most of the product properties to achieve analytical validation. Depending on the nature of the probe, alternative quantifications method could be considered such as fluorescence or radioluminescence.

Lanthanide chelation allows for TRF detection, increasing measurement sensitivity and overcoming background noise issues inherent to biological tissues. This approach can motivate the development of other biomolecular agents and provide better understanding of connections between probe synthesis and biological identities. The specificity of the LACTAL.Eu for the ASGPR was demonstrated in vitro by interaction with the receptor using a ricin agglutinin test and in vitro against HEPG2 cell line. We took advantage of the TRF to perform preliminary biodistribution studies on healthy mice directly in the cell media. If we consider the previous result obtained with the LACTAL probe, [START_REF] Chaumet-Riffaud | Synthesis and application of lactosylated, 99mTc chelating albumin for measurement of liver function[END_REF] the percent of injected dose found in the liver was close to the 29.0 % found in this study. This method offers the possibility to use Eu in daily laboratory practice to pre-evaluate compounds intended for MRI, scintigraphy and biotherapies. The biodistribution results also agree with our recently published complementary approach exploiting advantages of Cy5 fluorescent dye, offering other imaging applications albeit with less sensitive detection methods and efficiency issues in biological media [START_REF] Lemdani | Assessment of the targeting specificity of fluorescent albumin conceived as a preclinical agent of the liver function[END_REF] . The analytical workflow implemented here was designed based on physical detections and separative techniques to determine the optimal reaction conditions and characterize the influence of the additional chemical compounds on the protein. This workflow offers a rigorous methodology for the efficient development of conjugated proteins for targeted imaging, taking advantage of absolute physical analytical characterization techniques. We also contribute to the nanomedicine community important need to understand the relation between chemical and biological identity of imaging agents 3 . Finally, our strategy was envisioned for LACTAL.Eu, i.e. albumin lanthanide cryptates, but could be generalized for other biomolecules with different conjugation mechanisms, targeting and imaging probes. [START_REF] Hermanson | Bioconjugate Techniques[END_REF] To our knowledge, this workflow is the first published systematic protocol to optimize bioconjugates for targeted imaging.

17

 17

Maleimide-DTPA ( 2 ,

 2 2′-(1-carboxy-2-(carboxymethyl)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-10-oxo-2,5,8,11-tetraazatridecane-5,8-diyl) diacetic acid) was purchased from Chematech (France). Human serum albumin compliant with European safety regulation was purchased as Ydralbum from LFB (France) as a 200mg/mL solution in normal saline. Disposable 0.22 µm Stericup filter units were purchased from Merck-Millipore (Molsheim, France). Ultra-centrifugal concentrators Vivaspin 5, 30 kDa MWCO and Vivaspin 500, 30 kDa MWCO were from Sartorius. Dilutions in water were achieved using milliQ water, 18.2 MΩ.cm-1.

  37 °C. Europium signals were measured in the cells lysate with a spectrofluorometer (Victor, Perkin Elmer). The amount of DTPA in LACTAL.Eu or DTPA.Eu in the cell lysate was calculated to the respective value obtained from the cells taken as 100%with calibration curves realized in the same condition. In vivo study. Experiments were conducted following the Guidelines for Care and Use of Laboratory Animals of European directive No. 2010-63 and French decree No. 2013-118 and in agreement with a regional ethics committee for animal experimentation (Animal Ethics Committee of Paris Descartes No. 34 , CEEA 34) under the project number APAFiS # 13636.

Figure 1 :

 1 Figure 1: Schematic representation of the preparation of lactosylated DTPA serum albumin

Figure 2 :

 2 Figure 2: AF4-MALS-UV-RI analysis of HSA-DTPA, HSA-Lactose and LACTAL in red,

Figure 3 : 21 Figure 4 :

 3214 Figure 3: MALDI-TOF MS spectra corresponding to (A) clinical grade HSA, (B) HSA-

Figure 5 : 9 Liver 22 Figure 6 :

 59226 Figure 5: Biodistribution of HSA-DTPA.Eu (grey) and LACTAL.Eu (black) in Balbc/J mice

Table 1 .

 1 Molar weight increase after moieties grafting. We compare Mass Spectrometry, Light Scattering and Protein conjugate deduced from Light Scattering method.

	Product	MS [kDa]	LS [kDa] a	PC [kDa] a
				Total	Modifier
	HSA	66.5	68.1±0.5	X	X
	HSA-lactose	75.5	79.5±1.2	X	X
	LACTAL	76.6	76.85±0.5	76.7±0.5%	13.2±2.5%
	HSA-DTPA	75.7	74.5±0.56	75.5±1.7%	9+-12%
	a n=3

Acknowledgements

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors would like to thank the LIOPA optical animal imaging platform, school of Pharmacy (UMS 3612 CNRS) Life Imaging Facility of Paris Descartes University (Plateforme Imageries du Vivant), supported by France Life Imaging (grant ANR-11-INBS-0006), Infrastructures Biologie-Santé and the GDR-AIM. The authors would also like to thank INP Purpan FFF European Platform and Stéphanie Terme from Wyatt Technology France for fruitful discussions on FFF separation. For their support and fruitful discussions on the manuscript, the authors gratefully acknowledge Ruth Yoo and Nicholas DiStasio. Finally, the authors would like to acknowledge the Mass Spectrometry facility from Institut de Biologie Paris Seine for the constructive discussions on MS analysis.

Appendix A. Supplementary data

The following are the supplementary data related to this article: