Sytze Buwalda
email: sijtze.buwalda@umontpellier.fr

Benjamin Nottelet
email: benjamin.nottelet@umontpellier.fr

Audrey Bethry
email: audrey.bethry@univ-montp1.fr

Robbert Jan Kok

Niels Sijbrandi
email: sijbrandi@linxispharmaceuticals.com

Jean Coudane
email: jean.coudane@umontpellier.fr

Reversibly core-crosslinked PEG-P(HPMA) micelles: Platinum coordination chemistry for competitive-ligand-regulated drug delivery

Keywords: PEG-P(HPMA), core-crosslinked micelle, coordination chemistry, controlled drug delivery, stimulus-responsive

Hypothesis. The presence of pendant thioether groups on poly(ethylene glycol)-poly(N(2hydroxypropyl) methacrylamide) (PEG-P(HPMA)) block copolymers allows for platinum-mediated coordinative micellar core-crosslinking, resulting in enhanced micellar stability and stimulus-responsive drug delivery.

Experiments. A new PEG-P(HPMA) based block copolymer with pendant 4-(methylthio)benzoyl (MTB) groups along the P(HPMA) block was synthesized by free radical polymerization of a novel HPMA-MTB monomer using a PEG based macro-initiator. As crosslinker the metal-organic linker [ethylenediamineplatinum(II)] 2+ was used, herein called Lx, which is a coordinative linker molecule that has been used for the conjugation of drug molecules to a number of synthetic or natural carrier systems such as hyperbranched polymers and antibodies.

Findings. The introduction of Lx in the micellar core results in a smaller size, a lower critical micelle concentration and a better retention of the hydrophobic drug curcumin thanks to coordination bonds between the central platinum atom of Lx and thioether groups on different polymer chains. The drug release from Lx crosslinked micelles is significantly accelerated under conditions mimicking the intracellular environment due to competitive coordination and subsequent micellar de-crosslinking.

Because of their straightforward preparation and favorable drug release characteristics, core-crosslinked Lx PEG-P(HPMA) micelles hold promise as a versatile nanomedicine platform.

Introduction

Polymeric micelles, prepared from amphiphilic block copolymers, are an important and extensively studied class amongst controlled drug delivery systems because of several attractive properties. Their hydrophilic PEG corona provides them with a stealth surface, whereas their hydrophobic core acts as a depot capable of solubilizing clinically relevant doses of hydrophobic drugs. [START_REF] Kwon | Block copolymer micelles as long-circulating drug vehicles[END_REF] However, due to their dynamic nature, classical non-crosslinked micelles may disintegrate prematurely in systemic circulation resulting from dilution in the bloodstream below the critical micelle concentration. [START_REF] Talelli | Core-crosslinked polymeric micelles: Principles, preparation, biomedical applications and clinical translation[END_REF] Furthermore blood components may adsorb onto the surface of nanoparticles, despite the presence of stealth PEG surface. [START_REF] Mahon | Designing the nanoparticlebiomolecule interface for "targeting and therapeutic delivery[END_REF] Such opsonisation of plasma proteins can trigger the activation of the complement immune system [START_REF] Hamad | Poly(ethylene glycol)s generate complement activation products in human serum through increased alternative pathway turnover and a MASP-2dependent process[END_REF] or may lead to enhanced clearance of the micelles. [START_REF] Alexis | Factors affecting the clearance and biodistribution of polymeric nanoparticles[END_REF] Recent developments in organic and polymer chemistry have facilitated the synthesis of block copolymers which contain functionalities allowing for alternative interactions in order to further stabilize the micelle, in addition to hydrophobic interactions. [START_REF] Shi | Physico-chemical strategies to enhance stability and drug retention of polymeric micelles for tumor-targeted drug delivery[END_REF] Among the different interactions, metal-ligand coordination has received increasing interest because of its high specificity and reversibility via competitive displacement. Xin et al. prepared self-assembling polymer conjugates containing a PEG chain as the hydrophilic segment, a triphenylmethyl group as the hydrophobic moiety and a catechol-bearing dopamine end group. [START_REF] Xin | Bioinspired coordination micelles integrating high stability, triggered cargo release, and magnetic resonance imaging[END_REF] The addition of Fe 3+ ions resulted in smaller micelles and a higher micellar stability due to coordinative core-crosslinking between Fe 3+ and catechol groups. Under acidic conditions, the release of doxorubicin from such Fe 3+ -catechol stabilized micelles was significantly faster compared to the release at pH 7.4 as a consequence of coordination collapse and particle de-crosslinking. The core-crosslinked micelles enabled increased doxorubicin accumulation in tumors and enhanced suppression of tumor growth in a mouse model. [START_REF] Xin | Bioinspired coordination micelles integrating high stability, triggered cargo release, and magnetic resonance imaging[END_REF] In addition, the presence of Fe 3+ endowed the micelles with visibility in magnetic resonance imaging (MRI).

Poly(hydroxyethyl methacrylate)-poly(2-((8-hydroxyquinolin-5-yl)methoxy)ethyl methacrylate)

(PHEMA-PHQ) diblock copolymers were prepared by Ren et al. via RAFT polymerization. [START_REF] Ren | synthesis, characterization and magnetic studies of the metalquinolate PHEMA-b-HQ polymer micelles[END_REF] These polymers could self-assemble in ethanol into micelles having a PHEMA shell and a PHQ core. The micelles were subsequently core-crosslinked by addition of Ni 2+ , Co 2+ or Nd 3+ ions resulting in coordination between the metal ions and the 8-hydroxyquinoline groups in the PHQ block. A study of the magnetic properties revealed that Ni 2+ -crosslinked PHEMA-PHQ micelles exhibit the features of a soft ferromagnet. The group of Zhuo synthesized poly(ethylene glycol)-poly(ethylenimine)-poly(εcaprolactone) (PEG-PEI-PCL) triblock copolymers through a condensation reaction between PEG-PEI diblock copolymer and monocarboxy-capped PCL. [START_REF] Dai | Polymeric micelles stabilized by polyethylenimine-copper (C2H5N-Cu) coordination for sustained drug release[END_REF] After self-assembly of the polymers in water, the micelles were crosslinked by addition of CuSO4 resulting in coordination between Cu 2+ ions and the amino groups in the PEI block. The stabilized micelles had a higher doxorubicin loading capacity and a more sustained drug release than non-stabilized micelles. Cao et al. reported on micelle-forming poly(ethylene glycol)-poly(urethane)-poly(ethylene glycol) triblock copolymers containing a selenium atom in the backbone of the hydrophobic poly(urethane) block. [START_REF] Cao | Coordination-responsive seleniumcontaining polymer micelles for controlled drug release[END_REF] Micellar core-crosslinking via coordination of Pt 2+ ions to the selenium-containing polymer resulted in a high stability at elevated electrolyte concentrations, as the Pt-crosslinked micelles kept their size of approximately 60 nm in 2 M NaCl while the size of non-crosslinked micelles increased above 1000 nm. When dithiothreitol was added, the release of doxorubicin occurred significantly faster due to competitive coordination of the platinum cations with dithiothreitol, resulting in micellar de-crosslinking. An analogous polymer, poly(ethylene glycol)-poly(urethane)-poly(ethylene glycol) containing a tellurium atom in the poly(urethane) block, was prepared by the same research group to enable the loading of platinum based drugs such as cisplatin. [START_REF] Cao | Tellurium-containing polymer micelles: Competitiveligand-regulated coordination responsive systems[END_REF] Micelles prepared from tellurium-containing polymers exhibited a significantly higher drug loading compared to control micelles prepared from polymers without tellurium. Via competitive coordination of biomolecules such as glutathione, the drugs could be released in a controlled manner. Furthermore, the release kinetics could be tuned by the competitive ligands involved due to their different coordination ability.

Amongst amphiphilic micelle-forming polymers, PEG-P(HPMA) based block copolymers have attracted much interest because of their biocompatibility, non-immunogenicity and the possibility for functionalization of the P(HPMA) block. [START_REF] Talelli | Micelles based on HPMA copolymers[END_REF] Because P(HPMA) itself is highly water-soluble, it can only be used as the core in polymeric micelles when it is chemically modified with hydrophobic moieties.

For example, micelle-forming PEG-P(HPMA) block copolymers have been described in which the P(HPMA) block was functionalized with oligolactate, [START_REF] Soga | Thermosensitive and biodegradable polymeric micelles for paclitaxel delivery[END_REF] benzoyl, [START_REF] Shi | Π-Π stacking increases the stability and loading capacity of thermosensitive polymeric micelles for chemotherapeutic drugs[END_REF] anthracene, [START_REF] Shi | Anthracene functionalized thermosensitive and UV-crosslinkable polymeric micelles[END_REF] ethylcarbonate [START_REF] Kasmi | Transiently responsive block copolymer micelles based on N-(2-hydroxypropyl) methacrylamide engineered with hydrolyzable ethylcarbonate side chains[END_REF] and lipoic acid groups. [START_REF] Wei | Reduction-responsive disassemblable core-cross-linked micelles based on poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide)-lipoic acid conjugates for triggered intracellular anticancer drug release[END_REF] PEG-P(HPMA-benzoyl) micelles developed in the group of Hennink showed excellent stability as well as a high paclitaxel loading due to polymer-polymer and polymer-drug π-π stacking effects. [START_REF] Shi | Π-Π stacking increases the stability and loading capacity of thermosensitive polymeric micelles for chemotherapeutic drugs[END_REF] These micelles induced complete regression of A431 epidermoid and MDA-MB-468 breast carcinoma xenografts in mice. [START_REF] Shi | Complete regression of xenograft tumors upon targeted delivery of paclitaxel via Π-Π stacking stabilized polymeric micelles[END_REF] The examples described above clearly demonstrate the advantages of micellar stabilization by coordinative crosslinking as well as the beneficial properties of PEG-P(HPMA) based block copolymers for the formation of micelles. This led us to combine in this work these 2 promising concepts by designing PEG-P(HPMA) block copolymers with hydrophobic 4-(methylthio)benzoyl (MTB) side groups that can stabilize the micellar core via coordinative crosslinking. As crosslinker we used the metal-organic linker [ethylenediamineplatinum(II)] 2+ , herein called Lx, which is a coordinative linker molecule that has been used for the conjugation of drug molecules to a number of synthetic or natural carrier systems such as hyperbranched polymers and antibodies. [START_REF] Sijbrandi | A novel bifunctional ADC linker benchmarked using 89Zr-desferal and auristatin Fconjugated trastuzumab[END_REF][START_REF] Waalboer | Platinum(II) as bifunctional linker in antibody-drug conjugate formation: Coupling of a 4-nitrobenzo-2-oxa-1,3-diazole fluorophore to trastuzumab as a model[END_REF][START_REF] Dolman | Dendrimer-based macromolecular conjugate for the kidney-directed delivery of a multitargeted sunitinib analogue[END_REF][START_REF] Dolman | Renal targeting of kinase inhibitors[END_REF] The properties of core-crosslinked micelles are presented and compared with those of uncrosslinked micelles. As a proof-of-principle for drug delivery applications, we demonstrate the capacity of the Lx-micelles to encapsulate and release curcumin, a poorly water-soluble drug exhibiting anti-cancer and anti-inflammatory properties. Lastly, the cytotoxicity of curcumin-loaded micelles is evaluated using the breast cancer cell line MCF-7. In summary, we show that core-crosslinking of PEG-P(HPMA-MTB) micelles with the Lx linker results in a smaller size, a lower CMC and a better retention of the hydrophobic drug. Importantly, the corecrosslinked micelles can be destabilized via competitive displacement under conditions mimicking the intracellular environment, resulting in significantly faster drug release. This study therefore confirms that the rationally designed, core-crosslinked PEG-P(HPMA-MTB) micelles are appealing as stimulusresponsive controlled drug delivery systems.

Experimental section

Materials

Dichloro(ethylenediamine)platinum(II) 99 %, silver nitrate ≥ 99.8 %, DL- [START_REF] Ulbrich | Polymeric drugs based on conjugates of synthetic and natural macromolecules: I. Synthesis and physico-chemical characterisation[END_REF] (HPMA) and 4-(dimethylamino)pyridinium-4-toluenesulfonate [START_REF] Wu | Synthesis of colloidal uranium-dioxide nanocrystals[END_REF] (DPTS) were synthesized as reported previously. Dichloromethane (DCM) and TEA were dried over calcium hydride and potassium hydroxide, respectively, and distilled prior to use.

Synthesis

Activated Lx complex [platinum(II)(chlorido)(N,N-dimethylformamido)(ethylenediamine)](NO3)2

The activated Lx complex was prepared according to a previously reported procedure. [START_REF] Waalboer | Platinum(II) as bifunctional linker in antibody-drug conjugate formation: Coupling of a 4-nitrobenzo-2-oxa-1,3-diazole fluorophore to trastuzumab as a model[END_REF] The reaction was allowed to proceed for 96 h in the dark at room temperature in an argon atmosphere.

The reaction mixture was filtered, washed with brine and dried with magnesium sulfate. The resulting solution was then stirred for 24 h in the presence of (aminomethyl)polystyrene. The resin was removed by filtration and HPMA-MTB was obtained as a brown solid after evaporation of DCM in vacuo. Yield:

1.50 g (42 %). The number-average and weight-average molar masses (Mn and Mw, respectively) and polydispersity (PDI, Mw/Mn) of the polymers were determined by size exclusion chromatography (SEC) using a Viscotek GPCmax VE2100 liquid chromatograph equipped with a Viscotek VE3580 refractive index detector operating at 35 °C. Tetrahydrofuran was used as the eluent and the flow rate was set up at 1.0 ml/min. Two LT5000L 300 x 7.8 mm columns operating at 29 °C were used. Molecular weights were determined using the conventional narrow standard calibration technique with polystyrene standards (600-1000000 g/mol).

FTIR spectra were recorded using a Perkin-Elmer Spectrum 100 FTIR spectrometer.

Preparation and characterization of micelles

For the preparation of 1 ml of a 0.5 w/v % solution of PEG-P(HPMA-MTB) micelles core-crosslinked via the Lx linker (Lx PEG-P(HPMA-MTB) micelles), typically 5 mg PEG-P(HPMA-MTB) was dissolved in 175 μl of activated Lx linker DMF solution (0.5 equivalents of Lx relative to MTB groups).

The conjugation reaction was allowed to proceed at 37 °C for 48 h, followed by evaporation of DMF under reduced pressure. The polymer-linker conjugates were subsequently dissolved in 2 ml of a mixture of acetone and water (1/1 v/v) to allow formation of core-crosslinked micelles via coordination chemistry, followed by evaporation of the acetone under atmospheric pressure for 48 h. Non-Lx PEG-P(HPMA-MTB) micelles (0.5 w/v %) were typically prepared by adding 1 ml of a polymer solution in THF (5 mg/ml) dropwise into 1 ml of water under magnetic stirring, followed by evaporation of THF under atmospheric pressure for 48 h.

The critical micelle concentration (CMC) of the PEG-P(HPMA-MTB) block copolymer was determined in the presence and absence of the Lx linker using 8,1-ANS as a fluorescent probe. Polymer solutions were prepared at different concentrations as described above. Polymer solution (200 μl) was pipetted into the wells of a black 96-well plate (Greiner), followed by 2 μl of a 5 mM 8,1-ANS solution in water. The plate was incubated for 18 h in the dark, after which the fluorescence was measured using a BMG Labtech Clariostar plate reader. The excitation and emission wavelengths were 355 and 520 nm, respectively. The fluorescence intensity was plotted against the polymer concentration and the intercept of the extrapolated straight lines was taken as the CMC.

Dynamic light scattering (DLS) of non-Lx and Lx PEG-P(HPMA-MTB) micellar solutions (0.5 w/v % in water) was performed to determine aggregate sizes. Experiments were carried out at 25 °C using a Malvern Zetasizer Nano ZS, a laser wavelength of 633 nm and a scattering angle of 173º. The sample positioning, attenuation selection and the measurement duration were run in automatic mode. Each measurement was the result of 10-15 test runs.

The morphology of non-Lx and Lx PEG-P(HPMA-MTB) micelles was evaluated by transmission electron microscopy (TEM). One drop of a micellar solution in water was applied to a carbon-coated copper grid and the water was evaporated overnight in air. Images were obtained with a JEOL 1200 EXII-120 kV transmission electron microscope equipped with an EMSIS Quemesa camera.

Preparation of curcumin-loaded micelles

For the preparation of a typical batch of curcumin-loaded Lx PEG-P(HPMA-MTB) micelles (polymer concentration 0.5 w/v %), 25 mg PEG-P(HPMA-MTB)-Lx conjugate (containing 20 mg polymer) was dissolved in 7 ml of a mixture of acetone and water (3/4 v/v) followed by addition of 1 ml of a curcumin solution in acetone (3 mg/ml) under magnetic stirring at 1000 rpm. The stirring was continued for 10 min, followed by evaporation of the acetone under atmospheric pressure for 48 h. The resulting curcumin-loaded micellar solution was filtered through 0.45 μm syringe filters to remove precipitated, unincorporated curcumin, yielding 4 ml of a clear, orange micellar solution.

Curcumin-loaded non-Lx PEG-P(HPMA-MTB) micelles (polymer concentration 0.5 w/v %) were typically prepared by adding 1 ml of a polymer THF solution (20 mg/ml) dropwise into 4 ml of water under magnetic stirring at 1000 rpm, followed by addition of 1 ml of a curcumin solution in acetone (3 mg/ml). The stirring was continued for 10 min, followed by evaporation of the acetone and THF under atmospheric pressure for 48 h. The resulting curcumin-loaded micellar solution was filtered through 0.45 μm syringe filters to remove precipitated, unincorporated curcumin, yielding 4 ml of a turbid, yellow micellar solution. The initial curcumin feed was 15 wt % relative to the polymer mass for both types of micelles.

Determination of drug loading and encapsulation efficiency

Drug loading (DL) and encapsulation efficiency (EE) were determined by adding 0.25 equivalent of acetone (v/v) to freshly prepared micellar solutions to break down the micelles and dissolve the initially loaded curcumin. Quantification was subsequently performed using a BMG Labtech Clariostar plate reader at 425 nm with reference to a calibration curve of curcumin in a mixture of acetone and water

(1/4 v/v). The DL and EE were calculated based on the following formulae:

DL (%) = 100 x (MC) / (MP + MC) EE (%) = 100 x (MC) / (MCi)
with MC = mass of curcumin in micelles, MP = mass of copolymer and MCi = mass of curcumin initially added during the preparation of micelles.

In vitro drug release assay

The release of curcumin from micelles was measured in water containing 0.1 w/v % Tween 80 and 0.0015 w/v % ascorbic acid at 37 °C under constant orbital shaking at 150 rpm. Tween is regularly used to provide water-solubility to curcumin in such assays. [START_REF] Gao | Improving the anticolon cancer activity of curcumin with biodegradable nano-micelles[END_REF] Ascorbic acid is a naturally occurring antioxidant, which is used to avoid the degradation of the released curcumin. [START_REF] Wang | Stability of curcumin in buffer solutions and characterization of its degradation products[END_REF] In a typical release study, 0.75 ml of curcumin-loaded micellar solution was injected in a dialysis device (Spectra-Por Float-A-Lyzer G2, MWCO 3.5 kDa) that was subsequently immersed in 15 ml of release medium at 37 °C in the dark. At specific time points, the entire release medium was removed and replaced with 15 ml fresh medium. Collected samples were diluted with 0.25 equivalent (v/v) of acetone and analyzed at 425 nm with reference to a calibration curve of curcumin in a mixture of acetone and water (1/4 v/v). The amount of released curcumin (RC) was calculated based on the following formula:

RC (%) = 100 x (MRC) / (MC)
with MRC = mass of released curcumin and MC = mass of curcumin in micelles.

To mimick the intracellular environment, 10 mM of dithioerythritol (DTE) was added to the release medium for selected experiments.

In vitro cell experiments

Cell culture

Human breast cancer cells (MCF-7) were purchased from ECACC. The cells were maintained in DMEM-F12 supplemented with fetal bovine serum (5 v/v %) and 1 % penicillin/streptomycin at 37 °C in a humidified incubator containing 5 % CO2. MCF-7 cells were tested to be free of mycoplasma.

Cytotoxicity of unloaded micelles

The cytotoxicity of unloaded micelles was assessed in MCF-7 cells using the lactate dehydrogenase activity assay (LDH assay, Pierce). For this assay, cells were seeded in a white 96-well plate in amounts of 1•10 4 cells per well and allowed to attach overnight. They were then treated with unloaded non-Lx PEG-P(HPMA-MTB) micelles or unloaded Lx PEG-P(HPMA-MTB) micelles (polymer concentrations 0.03 and 0.14 w/v % in cell medium, respectively). Cell viability was assessed at 1 h, 2 h, 4 h, 6 h and 24 h using the LDH assay according to the manufacturer's instructions. Briefly, equal amounts of cell supernatant and LDH reaction mixture were mixed and incubated for 30 minutes at room temperature.

Subsequently, the absorbance was read at 490 nm using a BMG Labtech Clariostar plate reader. Lysis buffer and cell culture medium were used as positive and negative control, respectively. The micellar formulations were prepared in distilled water and subsequently diluted using cell medium under laminar flowhood conditions to achieve the indicated concentrations and guarantee the absence of bacterial contaminations.

Bioactivity of curcumin-loaded micelles

The cytotoxicity of the micelles was assessed in MCF-7 cells using the CellTiter Glo assay (Promega). For this assay, cells were seeded in a white 96-well plate in amounts of 1•10 4 cells per well and allowed to attach overnight. They were then treated with free curcumin (0.03 μg/ml in cell culture medium), free curcumin which was solubilized with DMSO as a co-solvent (8 μg/ml dissolved in cell culture medium containing 0.1 v/v % DMSO), curcumin-loaded non-Lx PEG-P(HPMA-MTB) micelles (curcumin concentration 50 μg/ml, polymer concentration 0.03 w/v % in cell medium) or curcuminloaded Lx PEG-P(HPMA-MTB) micelles (curcumin concentration 8 and 50 μg/ml, polymer concentration 0.01 and 0.14 w/v % in cell medium respectively). Cell culture medium containing 0.1 v/v % DMSO or medium only was used as a control. Cell viability was assessed at 1 h, 2 h, 4 h, 6 h, 24 h, 48 h and 72 h using the CellTiter Glo luminescent cell viability assay according to the manufacturer's instructions. Briefly, CellTiter-Glo reagent was added in each well with a volume equal to the volume initially present in each cell culture well. After 10 minutes incubation at room temperature, luminescence was recorded on a BMG Labtech Clariostar. The luminescent signal is proportional to the amount of ATP present in cell medium after cell lysis and hence to the number of cells in the well. The micellar formulations as well as the aqueous solution of free curcumin were prepared in distilled water and subsequently diluted using cell medium under laminar flowhood conditions to achieve the indicated concentrations and guarantee the absence of bacterial contaminations.

Cellular uptake

The cellular uptake of curcumin-loaded micelles was assessed in MCF-7 cells. Briefly, 3•10 4 cells were seeded into each well of a Lab-Tek II chamber slide 8-well and incubated at 37 °C in 5 % CO2 for 3 days. Cells were then treated for 90 min with curcumin-loaded non-Lx PEG-P(HPMA-MTB) micelles or curcumin-loaded Lx PEG-P(HPMA-MTB) micelles. The curcumin concentration was 50 μg/ml for both formulations. To evaluate the cellular uptake, cell medium was replaced and observations were done directly employing an inverted fluorescence microscope (Leica DM IRB) using a magnification of 40x (excitation 488 nm, emission 525 nm).

Results and Discussion

Synthesis and characterization of PEG-P(HPMA-MTB) block copolymers with pendant 4- methacrylamide (Figure 1). This monomer contains a thioether group, which has been shown to form

robust, yet reversible coordinative bonds with the platinum atom in the Lx linker. [START_REF] Waalboer | Platinum(II) as bifunctional linker in antibody-drug conjugate formation: Coupling of a 4-nitrobenzo-2-oxa-1,3-diazole fluorophore to trastuzumab as a model[END_REF] After purification, the product was obtained in 42 % yield as a light brown solid. Its structure was confirmed by 1 H and [START_REF] Soga | Thermosensitive and biodegradable polymeric micelles for paclitaxel delivery[END_REF] C NMR spectroscopy (Figures S1 andS2, respectively). For the synthesis of amphiphilic AB block copolymers composed of PEG5K and a P(HPMA-MTB) block, we employed free radical polymerization using a monomethoxy-PEG substituted macro-initiator (Figure 1). (PEG)2-ABCPA was prepared following a previously reported procedure and its structure confirmed by [START_REF] Kwon | Block copolymer micelles as long-circulating drug vehicles[END_REF] H NMR (Figure S3). [START_REF] Neradovic | Thermoresponsive polymeric micelles with controlled instability based on hydrolytically sensitive N-isopropylacrylamide copolymers[END_REF] The SEC trace of (PEG)2-ABCPA (Figure S4) shows the presence of a small molecular weight shoulder that has the same retention time as PEG5K, which indicates that a small amount of PEG5K homopolymer was present in (PEG)2-ABCPA. The PEG5K probably originates from the macroinitiator synthesis, i.e. PEG5K that was either not coupled to ABCPA or PEG5K that was coupled to only one of the two available COOH groups of ABCPA, as shown previously. [START_REF] Shi | Π-Π stacking increases the stability and loading capacity of thermosensitive polymeric micelles for chemotherapeutic drugs[END_REF] Separation of the PEG5K homopolymer and (PEG)2-ABCPA (10 kg/mol) via commonly used separation techniques, which are based on differences in molecular weight, is very challenging. For example, methods such as preparative chromatography and dialysis have been reported to be impractical, expensive and not efficient. [START_REF] Fliervoet | Heterofunctional poly(ethylene glycol) (PEG) macroinitiator enabling controlled synthesis of ABC triblock copolymers[END_REF] For the synthesis of PEG-P(HPMA-MTB), the ratio between HPMA-MTP and (PEG)2-ABCPA in the feed composition was set at 22/1 (mol/mol). By comparing the C=C stretch absorption peak at 1665 cm - 1 (relative to the aromatic C-H bending absorption peak at 760 cm -1 ) in FTIR spectra of HPMA-MTB and the crude reaction mixture after polymerization, the conversion of HPMA-MTB was estimated to be 90 %, which is in good accordance with previous research concerning free radical polymerization of functionalized HPMA monomers. [START_REF] Shi | Π-Π stacking increases the stability and loading capacity of thermosensitive polymeric micelles for chemotherapeutic drugs[END_REF][START_REF] Shi | Fluorophore labeling of core-crosslinked polymeric micelles for multimodal in vivo and ex vivo optical imaging[END_REF] Because thermal decomposition of every (PEG)2-ABCPA leads to the formation of two PEG macroradicals, this corresponds to a theoretical degree of polymerization (DP) of approximately 10 for the P(HPMA-MTB) block. The polymer was obtained in high yield after purification (80 %). By comparing the 1 H NMR integrals of the peaks corresponding to the aromatic protons in the HPMA-MTB units and the main chain protons of PEG (Figure 2), an average DP of 9 was calculated for the P(HPMA-MTB) block (copolymer Mn 7500 g/mol). Similar to (PEG)2-ABCPA, the SEC trace of PEG-P(HPMA-MTB) (Figure S4) shows the presence of a small molecular weight population with the same retention time as PEG5K. Part of this population originates from PEG5K that was not coupled to ABCPA. In addition, primary radical recombination and chain transfer, which decrease the initiator efficiency, may also give rise to PEG5K homopolymers. This chain termination during polymerization can be suppressed by applying controlled radical polymerization techniques, including reversible addition-fragmentation chain transfer (RAFT) polymerization [START_REF] Stenzel | RAFT polymerization: an avenue to functional polymeric micelles for drug delivery[END_REF] and atom transfer radical polymerization (ATRP), [START_REF] Matyjaszewski | Atom transfer radical polymerization (ATRP): Current status and future perspectives[END_REF] which are the subject of current investigations. SEC analysis indicated a PDI of 1.35 for PEG-P(HPMA-MTB), which is relatively low for a polymer obtained by free radical polymerization. Similar values have been reported previously for PEG-P(HPMA) type diblock copolymers which were prepared via the (PEG)2-ABCPA macroinitiator route. [START_REF] Soga | Physicochemical characterization of degradable thermosensitive polymeric micelles[END_REF] The low PDI values may result from the relatively low DP of the polymers. PDIs of PHPMA based block copolymers obtained by controlled radical polymerizations may be even lower. [START_REF] Du | Poly(D,L-lactic acid)-blockpoly(N-(2-hydroxypropyl)methacrylamide) nanoparticles for overcoming accelerated blood clearance and achieving efficient anti-tumor therapy[END_REF] Because of the presence of PEG5K homopolymer, it is likely that calculation of the Mn of PEG-P(HPMA-MTB) via 1 H NMR integration (7.5 kg/mol) results in underestimation of the Mn. An additional indication is found in the Mn calculated via SEC, which gives a value of 11.3 kg/mol.

Although the molecular weight of PEG-P(HPMA-MTB) was determined using the conventional narrow standard calibration technique with polystyrene standards, which may result in overestimation of the Mn, [START_REF] Chaudhary | Biocatalytic solvent-free polymerization to produce high molecular weight polyesters[END_REF] it seems therefore plausible that the actual DP of the P(HPMA-MTB) block is larger than the theoretical DP of 10. Overall, the mixture of different polymers including diblock copolymers and homopolymers can form mixed micelles in aqueous solution, as shown by DLS analysis of the micelles (vide supra) indicating that there is only one size population with a relatively low PDI. Although the synthesis of PEG-P(HPMA) type block copolymers via the (PEG)2-ABCPA macroinitiator route may result in multimodal SEC distributions, these polymers have successfully been used for various biomedical applications including drug delivery [START_REF] Naksuriya | HPMAbased polymeric micelles for curcumin solubilization and inhibition of cancer cell growth[END_REF] and imaging. [START_REF] Shi | Fluorophore labeling of core-crosslinked polymeric micelles for multimodal in vivo and ex vivo optical imaging[END_REF] Moreover, this route is straightforward and cost-effective as it avoids the use of chain transfer agents, which may be costly and may require extensive purification due to toxicity and coloration issues (e.g. certain RAFT agents).

Preparation of micelles

Previously PEG-P(HPMA-Bz) block copolymers bearing pendant benzoyl groups along the P(HPMA) block have been shown to self-assemble in water to form micelles with a hydrophilic PEG corona and a P(HPMA) core which is stabilized by π-π stacking between the aromatic groups. [START_REF] Shi | Π-Π stacking increases the stability and loading capacity of thermosensitive polymeric micelles for chemotherapeutic drugs[END_REF] Following a similar self-assembly approach, PEG-P(HPMA-MTB) micelles were initially prepared in the absence of the Lx coordinative linker by adding a THF solution of polymer into water followed by evaporation of THF (Figure 3, top panel). In view of previous literature and the structural similarity between PEG-P(HPMA-Bz) and PEG-P(HPMA-MTB) it is our belief that π-π stacking plays an important role in PEG-P(HPMA-MTB) micelles. To further investigate and confirm the π-π stacking additional experiments are needed and underway. The critical micelle concentration (CMC) of the PEG-P(HPMA-MTB) block copolymer in aqueous solution was found to be 0.03 w/v % using a dye solubilization method employing the hydrophobic fluorescent probe 8,1-ANS (Figure S5). This value is higher than the previously reported CMC of PEG-P(HPMA-Bz) (0.0001 w/v %), [START_REF] Shi | Π-Π stacking increases the stability and loading capacity of thermosensitive polymeric micelles for chemotherapeutic drugs[END_REF] likely because of the substantially shorter P(HPMA) block length in case of PEG-P(HPMA-MTB) (3 vs. 17 kg/mol), resulting in less hydrophobic interactions and possibly less π-π stacking effects. Using dynamic light scattering (DLS) DH in 0.5 w/v % PEG-P(HPMA-MTB) aqueous solutions was found to be 160 nm with a PDI of 0.18 (Figure 4). With the aim of providing additional stability to the micellar core, next to hydrophobic interactions and possibly π-π stacking, [START_REF] Shi | Π-Π stacking increases the stability and loading capacity of thermosensitive polymeric micelles for chemotherapeutic drugs[END_REF] we subsequently introduced the coordinative Lx linker in the core of PEG-P(HPMA-MTB) micelles. Although covalent core-crosslinking can also provide stable micelles, it may be practically challenging because it requires additional reaction and purification steps. For this reason we selected coordinative core-crosslinking as it can be applied via a straightforward method under mild conditions, while still offering robust crosslinks in the micellar core. Importantly, coordinative corecrosslinks can be broken via competitive coordination by e.g. glutathione, which offers the possibility of stimulus-responsive drug release. Previously the Lx linker has been used for the attachment of various drug molecules to macromolecular carrier systems including antibodies [START_REF] Sijbrandi | A novel bifunctional ADC linker benchmarked using 89Zr-desferal and auristatin Fconjugated trastuzumab[END_REF][START_REF] Waalboer | Platinum(II) as bifunctional linker in antibody-drug conjugate formation: Coupling of a 4-nitrobenzo-2-oxa-1,3-diazole fluorophore to trastuzumab as a model[END_REF] and polymers. [START_REF] Dolman | Dendrimer-based macromolecular conjugate for the kidney-directed delivery of a multitargeted sunitinib analogue[END_REF] In these cases, the central platinum atom binds the functional moiety to the carrier molecule via coordination sites such as nitrogen and sulfur atoms with free electron pairs, while the other two coordination sites of platinum are made up permanently by an ethylenediamine bridge, which is a strong bidentate ligand that cannot be readily replaced by the mentioned monodentate ligands. On the other hand, the nitrate and chloride ligands that are initially present on Lx are weak ligands for platinum(II) and are readily replaced. [START_REF] Sijbrandi | A novel bifunctional ADC linker benchmarked using 89Zr-desferal and auristatin Fconjugated trastuzumab[END_REF][START_REF] Waalboer | Platinum(II) as bifunctional linker in antibody-drug conjugate formation: Coupling of a 4-nitrobenzo-2-oxa-1,3-diazole fluorophore to trastuzumab as a model[END_REF] In the present work we employ the Lx linker for the first time to act as a linker between two polymer molecules.

Lx crosslinked micelles were prepared by first coordinating the pendant thioether groups in the P(HPMA-MTB) block with Lx linker in a 2:1 ratio in DMF, resulting in substitution of the nitrate ligand on Lx by one thioether group (Figure 3, bottom panel). During this process, no gel formation in DMF was observed, demonstrating that no premature crosslinking took place. After DMF evaporation, the polymer-linker conjugates were dissolved in a mixture of acetone and water (1/1 v/v) as they were not soluble in water or any volatile organic solvent alone. The evaporation of the acetone induced the micellization of the polymer-linker conjugates in water, facilitating substitution of the second ligand of Lx, the chloride ligand, by a second thioether group on a different P(HPMA-MTB) chain in the hydrophobic core, resulting in coordinative crosslinks between the polymer molecules constituting the micelle. Using this straightforward preparation method, the CMC of Lx crosslinked micelles (termed Lx PEG-P(HPMA-MTB) micelles from here on) was found to be 0.009 w/v % (Figure S5), which is lower than the CMC of non-Lx PEG-P(HPMA-MTB) micelles (0.03 w/v %). The tendency of the polymers to form micelles at lower concentrations in the presence of Lx linker is a strong indication for Lx mediated coordinative core-crosslinking. Moreover, DLS measurements showed a shift of the micellar size to smaller dimensions upon the introduction of Lx from 160 nm to 60 nm (Figure 4, PDI of 0.26), reflecting a more condensed core in the micelles due to the coordinative crosslinking. TEM images (Figure S6) demonstrated the presence of spherical micelles with a diameter of approximately 120 nm (non-Lx PEG-P(HPMA-MTB) micelles) and 60 nm (Lx PEG-P(HPMA-MTB) micelles), which confirms the clear size difference found in the DLS experiments (Figure 4). To further investigate the coordinative crosslinking in the micellar core, a Lx PEG-P(HPMA-MTB) micellar solution prepared in D2O was analyzed by 195 Pt NMR spectroscopy. One main peak is observed at -3160 ppm (Figure S7). It is known from literature that the 195 Pt NMR chemical shift of Pt(II) compounds with a PtN2S2 coordination sphere is located between -3150 and -3250 ppm. [START_REF] Oehlsen | Reaction of polynuclear platinum antitumor compounds with reduced glutathione studied by multinuclear (1H, 1H-15N gradient heteronuclear single-quantum coherence, and 195Pt) NMR spectroscopy[END_REF][START_REF] Kasherman | Trans labilization of am(m)ine ligands from platinum (II) complexes by cancer cell extracts. JBIC[END_REF] Therefore, the peak at -3160 ppm likely relates to Pt which is coordinated by the ethylenediamine bridge and 2 thioether groups. Although the existence of coordination between thioether groups on the same polymer cannot be excluded, these 195 Pt NMR measurements are additional evidence for the existence of coordinative crosslinks between the polymer molecules constituting the micelle (Figure 3, bottom panel).

Curcumin incorporation and release

Curcumin is a naturally occurring low molecular weight compound which is extracted from the Curcuma Longa plant. Curcumin has various pharmacological activities, such as anti-inflammatory, anti-oxidant, anti-bacterial, anti-virus and anti-cancer effects. However, significant disadvantages of curcumin are its low water solubility (11 ng/ml [START_REF] Song | Curcumin-loaded PLGA-PEG-PLGA triblock copolymeric micelles: Preparation, pharmacokinetics and distribution in vivo[END_REF] ) and its poor stability under physiological conditions.

Furthermore, the absorption of curcumin in the gastro-intestinal tract is minimal and it is toxic at high concentrations. With the aim of improving its solubility and stability and making the drug applicable via intravenous injection, curcumin was loaded in non-Lx PEG-P(HPMA-MTB) and Lx PEG-P(HPMA-MTB) micelles via the self-assembly method. Figure 5 clearly shows the solubilizing effect of the micelles. In the absence of copolymer, curcumin is almost insoluble as shown in Figure 5A where precipitated curcumin is visible at the bottom of the vial and only slight coloration of the solution appears. In contrast, encapsulation of curcumin in the hydrophobic core of non-Lx PEG-P(HPMA-MTB) and Lx PEG-P(HPMA-MTB) micelles (Figure 5B and 5C) yields homogenous solutions. The incorporation of curcumin in the micelles did not have an effect on the micellar size, as the DH of non-Lx and Lx PEG-P(HPMA-MTB) micelles remained 160 and 60 nm, respectively (Table 1). This size difference is clearly visible in Figure 5, where Lx PEG-P(HPMA-MTB) micelles yield a clear solution whereas the non-Lx PEG-P(HPMA-MTB) micellar solution is turbid. Incorporation of curcumin in Lx PEG-P(HPMA-MTB) micelles results in a slight color change of curcumin, likely due to interaction of its conjugated electron system with the platinum. However, after release from the micelles the curcumin got back its original color (data not shown). Non-Lx PEG-P(HPMA-MTB) micelles exhibit a very high EE of 97 %, which may be explained by the presence of hydrophobic interactions and possibly π-π stacking 14 between the pendant MTB groups of the polymer and the aromatic groups of curcumin. Similar high EE and DL were reported for the incorporation of curcumin in PEG-P(HPMA-Bz) block copolymers bearing pendant benzoyl groups along the P(HPMA) block. [START_REF] Naksuriya | HPMAbased polymeric micelles for curcumin solubilization and inhibition of cancer cell growth[END_REF] Since the curcumin loading for the Lx PEG-P(HPMA-MTB) micelles was performed after the core-crosslinking, the latter may have acted as a physical barrier for the drug to reach the micellar core. The resulting slow kinetics of the curcumin diffusion into the core may explain the lower EE of 35 % for the Lx PEG-P(HPMA-MTB) micelles. Release studies were performed using the dialysis bag method in water at 37 °C. A low amount of Tween 80 (0.1 w/v %) was added to the release medium to facilitate solubilization of the released curcumin in water, as described in previous literature. [START_REF] Gao | Improving the anticolon cancer activity of curcumin with biodegradable nano-micelles[END_REF] Also a very low quantity of the physiologically relevant ascorbic acid (0.0015 w/v %) was dissolved in the release medium to avoid degradation of the drug. [START_REF] Wang | Stability of curcumin in buffer solutions and characterization of its degradation products[END_REF] Figure 6 shows that 55 % of the initially incorporated curcumin is released in 80 days from the non-Lx PEG-P(HPMA-MTB) micelles. For Lx PEG-P(HPMA-MTB) micelles, a biphasic release behavior was observed in which 20 % curcumin was released in a similar profile as observed for non-Lx PEG-P(HPMA-MTB) micelles during the first 20 days, after which curcumin release leveled off and increased only up to 30 % at day 80. This observed profile suggests that the major fraction of curcumin is retained better in the Lx PEG-P(HPMA-MTB) micelles as compared to non-Lx PEG-P(HPMA-MTB) micelles, which can be explained by the Lx mediated coordinative core-crosslinking as it prevents destabilization of the micelle and hence premature drug release. The release from Lx PEG-P(HPMA-MTB) micelles is comparable to the curcumin release from previously reported PEG-P(HPMA-Bz) micelles containing a long P(HPMA-Bz) block (16 kg/mol), in which a similar high degree of corecrosslinking was facilitated via hydrophobic interactions and π-π stacking effects. [START_REF] Naksuriya | HPMAbased polymeric micelles for curcumin solubilization and inhibition of cancer cell growth[END_REF] To corroborate that Lx-based crosslinking was involved in the drug retention, we modulated the release from Lx PEG-P(HPMA-MTB) micelles by adding a coordination-bond disrupting agent to the release medium. The tripeptide glutathione (GSH) is the most abundant reducing agent in mammalian cells having an intracellular concentration in the range of 2 to 10 mM, approximately 100 to 1000 fold higher than its concentration in the extracellular environment (2-20 μM). Tumor cells have at least a 4fold higher concentration of GSH as compared to normal cells, making GSH-sensitive micelles very attractive for tumor specific intracellular delivery. Apart from its reducing activity, GSH is also a good platinum coordinating ligand by virtue of its free thiol group. [START_REF] Gonzalo | Selective targeting of pentoxifylline to hepatic stellate cells using a novel platinum-based linker technology[END_REF] Because of the limited stability and activity of GSH under aerobic conditions, we selected the reducing agent dithioerythritol (DTE), the cis isomer of 2,3-dihydroxy-1,4-dithiolbutane (Cleland's reagent) as Lx-MTB disrupting agent. In the presence of 10 mM DTE, the release from Lx PEG-P(HPMA-MTB) micelles was significantly increased as compared to release from Lx PEG-P(HPMA-MTB) micelles in the absence of DTE and faster than the release from non-Lx PEG-P(HPMA-MTB) micelles (Figure 6). Since the thiol group on DTE is a better ligand for platinum than the thioether group on PEG-P(HPMA-MTB) and since DTE is present in excess, DTE can break existing coordinative bonds, destabilize the micelle and induce drug release. In vivo it is expected that the excellent drug retention capability of the Lx PEG-P(HPMA-MTB) micelles will lead to little premature drug release in circulation, whereas their small size may result in efficient accumulation in tumor tissue via the EPR effect. [START_REF] Cabral | Accumulation of sub-100 nm polymeric micelles in poorly permeable tumours depends on size[END_REF] In view of the demonstrated possibility of competitive coordination by DTE, it is hypothesized that the micelles are destabilized in the cytoplasm after competitive glutathione displacement, resulting in drug release and tumor cell death. After release of the payload, it is anticipated that the polymers can be excreted from the body via the renal pathway.

Due to hydrolysis of the ester groups connecting the side groups and the main chain, PEG-P(HPMA)

will likely be formed, as demonstrated for micelle-forming PEG-P(HPMA) block copolymers in which the P(HPMA) block was functionalized with oligolactates. [START_REF] Soga | Physicochemical characterization of degradable thermosensitive polymeric micelles[END_REF] Since PEG and P(HPMA) are also connected via a hydrolysable ester bond, this may result in the formation of P(HPMA) and PEG.

Although these polymers as such are non-degradable, elimination via the kidneys is possible in vivo because their molecular weights (5000 and 1300 g/mol, respectively) are below the renal thresholds. [START_REF] Yamaoka | Distribution and tissue uptake of poly(ethylene glycol) with different molecular weights after intravenous administration to mice[END_REF][START_REF] Seymour | Effect of molecular weight (mw) of N-(2hydroxypropyl)methacrylamide copolymers on body distribution and rate of excretion after subcutaneous, intraperitoneal, and intravenous administration to rats[END_REF] In vitro cell experiments

The cytotoxicity of empty and curcumin-loaded micelles was tested against the breast cancer cell line MCF-7. All tests were performed with associated controls to confirm the non-toxicity of various culture media used in the cytotoxicity experiments. In particular, the effect of 0.1% of DMSO as a co-solvent in the culture medium to ensure the solubility of free curcumin was assessed in accordance with recent work of our group. [START_REF] Samad | CL-PEG graft copolymers with tunable amphiphilicity as efficient drug delivery systems[END_REF] Also the effect of a slight dilution of the cell culture medium with water was tested as the micellar formulations were prepared in distilled water prior to the cell experiments. All media showed no toxicity (data not shown). It follows from Figure S8 that unloaded Lx PEG-P(HPMA-MTB) micelles show almost no cytotoxicity towards MCF-7 cells after 24 hours. The very low level of cytotoxicity may be explained by the presence of the platinum based linker, as unloaded non-Lx PEG-P(HPMA-MTB) micelles showed no cytotoxicity after 24 h. A similar slight cytotoxic effect of metal ions was found for PEG-PEI-PCL micelles which were core-crosslinked via coordination between Cu 2+ ions and the amino groups in the PEI block. [START_REF] Dai | Polymeric micelles stabilized by polyethylenimine-copper (C2H5N-Cu) coordination for sustained drug release[END_REF] Our experiments with unloaded micelles were performed using similar polymer concentrations as those employed in the experiments involving curcumin-loaded non-Lx and Lx PEG-P(HPMA-MTB) micelles (vide supra), namely 0.03 w/v % for non-Lx PEG-P(HPMA-MTB) micelles (i.e. the critical micelle concentration) and 0.14 w/v % for Lx PEG-P(HPMA-MTB) micelles. The higher polymer concentration may also be responsible for the slightly higher cytotoxicity of the unloaded Lx PEG-P(HPMA-MTB) micelles. In a subsequent experiment, we compared the cytotoxicity of non-Lx and Lx PEG-P(HPMA-MTB) micelles at the CMC of non-Lx PEG-P(HPMA-MTB) micelles w/v %). A higher curcumin concentration was used in these experiments due to the higher CMC of non-Lx PEG-P(HPMA-MTB) micelles compared to Lx PEG-P(HPMA-MTB) micelles. At a concentration of 50 μg/ml, non-Lx PEG-P(HPMA-MTB) micelles and Lx PEG-P(HPMA-MTB) micelles are able to induce almost full cell death within 6 hours (Figure 8). No significant differences between the two micellar formulations are observed in terms of cytotoxicity. In view of the low curcumin release at early timepoints (Figure 6) and the intracellular presence of curcumin 90 min after treatment (Figure S9) it is likely that both types of micelles are internalized and subsequently destabilized. Although at long timescales a clear difference in curcumin release behavior is observed in vitro between non-Lx and Lx PEG-P(HPMA-MTB) micelles (Figure 6), the intracellular micellar destabilization presumably occurs at the same rate for both formulations, resulting in similar cytotoxic effects. This result, in view of the better curcumin retention capacity of Lx PEG-P(HPMA-MTB) micelles (Figure 6), confirms that intracellular competitive coordination occurs for Lx PEG-P(HPMA-MTB) micelles, which allows similar release for both systems. as previously reported for drug-Lx-antibody conjugates [START_REF] Sijbrandi | A novel bifunctional ADC linker benchmarked using 89Zr-desferal and auristatin Fconjugated trastuzumab[END_REF][START_REF] Waalboer | Platinum(II) as bifunctional linker in antibody-drug conjugate formation: Coupling of a 4-nitrobenzo-2-oxa-1,3-diazole fluorophore to trastuzumab as a model[END_REF] and drug-Lx-polymer conjugates. [START_REF] Dolman | Dendrimer-based macromolecular conjugate for the kidney-directed delivery of a multitargeted sunitinib analogue[END_REF] The conjugates can potentially be loaded in high amounts in PEG-P(HPMA-MTB) micelles because of the possibility for coordinative bond formation between drug-Lx and a pendant thioether group on PEG-P(HPMA-MTB). The expected synergistic effects of i) a high drug loading, ii) the small size of Lx PEG-P(HPMA-MTB) micelles and iii) the demonstrated possibility of breaking coordinative bonds intracellularly via competitive displacement may result in a highly cytotoxic effect in vitro and in vivo.

Conclusions

PEG-PHPMA block copolymers constitute an appealing basis for micelles because of their biocompatibility, non-immunogenicity and the possibility for functionalization of the P(HPMA) block with groups that facilitate core-crosslinking to stabilize the micelle. In this regard, metal-ligand coordination is particularly attractive because of its specificity and reversibility via competitive displacement. In our present contribution, we combined for the first time these 2 promising concepts by synthesizing PEG-P(HPMA) block copolymers with 4-(methylthio)benzoyl (MTB) side groups that stabilize the micellar core via coordinative crosslinking. As crosslinker we used the platinum-organic Lx

(

  methylthio)benzoyl groups Amphiphilic block copolymers based on PEG and hydrophobized P(HPMA) represent an excellent basis for long-acting micelles because of their hydrolytic stability and biocompatibility. Two main strategies have been reported for the preparation of the hydrophobically modified P(HPMA) block: polymerization of functionalized HPMA or post-polymerization functionalization of pre-formed P(HPMA). The post-polymerization strategy allows for preparation and characterization of the polymer backbone prior to functionalization, but at the possible cost of a limited functional group density and a change in the chemical and physical state of the polymer depending on the post-polymerization reaction conditions. The functionalized monomer strategy, on the other hand, may yield polymers with a high number of functional groups along the P(HPMA) chain. For the present micellar system, we selected the functionalized monomer strategy in order to achieve a high number of HPMA side groups capable of forming coordination bonds with the Lx linker, which potentially leads to a high level of corecrosslinking. A new monomer, N-(2-(4-methylthio)benzoyloxypropyl) methacrylamide (HPMA-MTB), was synthesized by reaction of 4-(methylthio)benzoyl chloride with N-(2-hydroxypropyl)

Figure 1 .

 1 Figure 1. Synthesis scheme for the preparation of HPMA-MTB monomer (top panel), (PEG)2-ABCPA

Figure 2 . 1 H

 21 Figure 2. 1 H NMR spectrum of PEG-P(HPMA-MTB). Solvent: DMSO-d6. Signals corresponding to the

Figure 3 .

 3 Figure 3. Schematic representation of the formation of non-Lx PEG-P(HPMA-MTB) micelles (top

Figure 4 .

 4 Figure 4. Aggregate size distributions of non-Lx and Lx PEG-P(HPMA-MTB) micellar solutions (0.5

Figure 5 .

 5 Figure 5. Dispersion of curcumin in water (A). Curcumin-loaded non-Lx PEG-P(HPMA-MTB)

Figure 6 .

 6 Figure 6. Curcumin release from non-Lx PEG-P(HPMA-MTB) micelles (green open squares), Lx PEG-

Figure 7 .

 7 Figure 7. Viability of MCF-7 cells after treatment with an aqueous solution of curcumin (0.03 μg/ml in

Figure 8 .

 8 Figure 8. Viability of MCF-7 cells after treatment with curcumin-loaded non-Lx PEG-P(HPMA-MTB)

  Figure S2. 13 C NMR spectrum of N-(2-(4-methylthio)benzoyloxypropyl) methacrylamide (HPMA-

Figure S5 .

 S5 Figure S5. Determination of the critical micelle concentration (CMC) for non-Lx PEG-P(HPMA-MTB)

Figure S6 .

 S6 Figure S6. Representative TEM images of a non-Lx PEG-P(HPMA-MTB) micelle (left) and a Lx PEG-

Table 1 .

 1 Size, encapsulation efficiency (EE) and drug loading (DL) for curcumin-loaded non-Lx and Lx

	PEG-P(HPMA-MTB) micelles.
		DLS
				DL	EE
	Micelle type	DH	PDI	(%)	(%)
		(nm)
	Non-Lx PEG-P(HPMA-MTB)	160	0.13	13	97
	Lx PEG-P(HPMA-MTB)	60	0.17	5	35

Acknowledgements

This work was supported by a Marie Skłodowska-Curie Individual Fellowship to S.J.B. in the framework of the 'Horizon 2020' European Union Research and Innovation programme (grant #660953 'Lx micelles'). Stéphane Dejean (Department of Artificial Biopolymers, IBMM, Montpellier) and Franck Godiard (Platform for Electron and Analytical Microscopy, Montpellier University) are kindly acknowledged for help with the 1 H NMR and TEM experiments, respectively.

Following these results, we subsequently tested the cytotoxicity of curcumin-loaded micelles against MCF-7 cells. In a first set of experiments, we studied the solubilizing capacity of Lx PEG-P(HPMA-MTB) micelles and its effects on cell viability at the lowest possible polymer concentration, i.e. at the CMC (0.009 w/v %). Figure 7 shows that a 0.03 μg/ml solution of curcumin in cell medium (which was prepared by diluting a saturated aqueous curcumin solution) does not induce a significant reduction in MCF-7 cell viability, reflecting the poor solubility of curcumin in water. In contrast, the hydrophobic core of the Lx PEG-P(HPMA-MTB) micelles allows for a higher concentration of curcumin (set at 8 μg/ml in these experiments), resulting in a significant decrease in cell viability (4 % cells remaining after 72 h). The curcumin concentration of 8 μg/ml was selected based on our previous research, [START_REF] Samad | CL-PEG graft copolymers with tunable amphiphilicity as efficient drug delivery systems[END_REF] in which free curcumin (co-solubilized in cell medium by DMSO) showed an intermediate toxicity (ca. 50 %) at this concentration. Figure S9 shows that curcumin is present intracellularly 90 min after treatment of MCF-7 cells with curcumin-loaded Lx PEG-P(HPMA-MTB) micelles. Since the curcumin release is still negligible at these early time points (Figure 6), the results suggest that the Lx PEG-P(HPMA-MTB) micelles are internalized and intracellularly destabilized, resulting in curcumin release and cell death.

The curcumin-loaded Lx PEG-P(HPMA-MTB) micellar formulation is more cytotoxic than free curcumin (8 μg/ml) which was solubilized with the help of 0.1% DMSO as a co-solvent (20 % cell viability remaining after 72 h, Figure 7). One should also note that besides being less cytotoxic, free curcumin delivery using DMSO as a co-solvent, although useful as in vitro control, is not a realistic formulation for in vivo delivery, which further demonstrates the advantage of the Lx PEG-P(HPMA-MTB) micellar formulation. curcumin from Lx PEG-P(HPMA-MTB) micelles was significantly accelerated due to competitive coordination of the platinum atom in Lx with DTE, resulting in micellar destabilization. As opposed to previously reported P(HPMA) based micelles, [START_REF] Sponchioni | HPMA-PEG surfmers and their use in stabilizing fully biodegradable polymer nanoparticles[END_REF][START_REF] Krimmer | Synthesis and characterization of poly(ecaprolactone)-block-poly[N-(2-hydroxypropyl)methacrylamide] micelles for drug delivery[END_REF][START_REF] Du | Poly(D,L-lactic acid)block-poly(N-(2-hydroxypropyl)methacrylamide) nanoparticles for overcoming accelerated blood clearance and achieving efficient anti-tumor therapy[END_REF] Lx PEG-P(HPMA-MTB) micelles therefore offer the possibility for a stimulus-responsive drug release. These results confirm our hypothesis that platinummediated coordinative micellar core-crosslinking of PEG-P(HPMA-MTB) leads to enhanced micellar stability and stimulus-responsive drug delivery. The combination of a small size, a high stability under normal conditions and fast drug release under conditions mimicking the intracellular environment make the Lx PEG-P(HPMA-MTB) micelles promising materials in the field of nanomedicine. Future work should include optimization of the encapsulation efficiency of the Lx PEG-P(HPMA-MTB) micelles, investigation of the cellular uptake route as well as evaluation of the therapeutic efficacy of drug-loaded Lx PEG-P(HPMA-MTB) micelles in vivo.

Conflicts of interest

Declarations of interest: none. E-mail addresses: sijtze.buwalda@umontpellier.fr; benjamin.nottelet@umontpellier.fr; audrey.bethry@univ-montp1.fr; r.j.kok@uu.nl; sijbrandi@linxispharmaceuticals.com; jean.coudane@umontpellier.fr The curcumin concentration was 50 μg/ml for both formulations. Images were taken 90 min after treatment using a Leica DM IRB inverted microscope at 40 x magnification.

Supporting information for


A. Toutant Martin David
email: martin.david@promes.cnrs.fr

Adrien Toutant

Françoise Bataille

Françoise Bataille Assessment

ASSESSMENT AND COMPARISON OF LARGE EDDY SIMULATIONS IN ASYMMETRICALLY HEATED AND HIGHLY TURBULENT CHANNEL FLOWS

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

Introduction

Concentrated Solar Power (CSP) systems are capable of storing energy by use of thermal energy storage technologies and are then very attractive for largescale power generation. Solar power tower functioning with gas-pressurized solar receivers allow to reach very high fluid temperature and produce a high-quality energy. Moreover, to produce electricity, the solar receivers are included in a Brayton cycle that permits reaching are heat-to-power conversion efficiency. In these devices, the heat transfer to the fluid is critical since it directly impacts the efficiency of the cycle. As the heat transfer is strongly influenced by the flow parameters [START_REF] David | Numerical development of heat transfer correlation in asymmetrically heated turbulent channel flow[END_REF], accurate and accessible tools are required to study the solar receiver.

Large Eddy Simulations (LES) model the small scales in order to reproduce their effect of the larger ones, which are resolved. This Computational Fluid Dynamic (CFD) approach provides more accurate and detailed results than the ones of Reynolds Averaged Navier-Stokes (RANS) simulations. Their computational cost is also much more reasonable than the one of Direct Numerical Simulation (DNS). Hence, LES offers an interesting compromise. With the increase in computer performance, LES should become widely used in the industry. For that reason, the investigation of LES models in various conditions is valuable. A posteriori tests of Large Eddy Simulation are commonly performed in the literature [START_REF] Stolz | An approximate deconvolution model for large-eddy simulation with application to incompressible wall-bounded flows[END_REF][START_REF] Horiuti | The role of the Bardina model in large eddy simulation of turbulent channel flow[END_REF], Dupuy et al. 2019). LES at high Reynolds numbers are more recently studied [START_REF] Kremer | Large-eddy simulation of turbulent channel flow using relaxation filtering: Resolution requirement and Reynolds number effects[END_REF]Bogey 2015, Streher et al. 2021). The investigations of anisothermal flows are quite unusual despite their importance in the industry. Among those studies, very few take into account the coupling between the temperature and the dynamic [START_REF] Serra | Thermal Large Eddy Simulation in a Very Simplified Geometry of a Solar Receiver[END_REF], Dupuy et al. 2019[START_REF] David | Numerical development of heat transfer correlation in asymmetrically heated turbulent channel flow[END_REF].

To the author's knowledge, there is no study of LES in anisothermal channel flow heating from both sides in the literature. Nevertheless, this configuration is widely encountered in many engineering systems, such as heat exchangers or the gas-pressurized solar receivers. The present paper aims to fulfill this gap. Its outline is the following. In Section 1, the study configuration is detailed. Then, the results are given and discussed in Section 2. Lastly, a final section concludes.

Study configuration

Flow modeling

The Navier-Stokes equations are solved under the low Mach number proposed by Paolucci [START_REF] Paolucci | Filtering of sound from the Navier-Stokes equations[END_REF]. The ideal gas law is used to close the system. The thermal dilatation and the fluid property variations are taken into account. The Sutherland's law [START_REF] Sutherland | The viscosity of gases and molecular force[END_REF]) is used to computed the dynamic viscosity. To study various temperature profiles, i.e. various axial location in the solar receiver, a heat source, H s , is introduced in the energy equation. The two most significant subgrid terms highlighted by Dupuy et al. (Dupuy et al. 2019) are considered: a subgrid term related to the velocity-velocity correlation and another modeling the density-velocity correlation. Functional and structural models are studied. The Anisotropic-Minimum-Dissipation (AMD) model proposed by [START_REF] Rozema | Minimum-dissipation models for large-eddy simulation[END_REF] and the H (4) tensorial model version of the AMD model proposed by Dupuy (Dupuy et al. 2019) are investigated. As for the structural approach, the scalesimilarity model proposed by [START_REF] Bardina | Improved subgrid-scale models for large-eddy simulation[END_REF] is assessed. The same model is used to compute both the subgrid-scale stress tensor and the subgrid-scale heat flux. The tensorial AMD model is associated with the scalar AMD [START_REF] Abkar | Minimumdissipation scalar transport model for large-eddy simulation of turbulent flows[END_REF] model for the modeling of the subgrid-scale heat flux.

Numerical settings

The new generation of solar power towers functioning with gas-pressurized solar receivers allows to reach very high fluid temperature and, thus, to enhance the efficiency of the heat-to-power conversion. These absorbers are characterized by very complex flows. Indeed, they involve highly turbulent flows in order to enhance the wall heat flux and facilitate fluid heating. The solar receivers are a built-in cavity to lower the heat losses. For that reason, only one wall of the solar receiver is irradiated. This induces asymmetric heating of the fluid. To reproduce these conditions, large-eddy simulations are carried out in a fully developed three-dimensional turbulent channel flow asymmetrically heated, see Figure 1. The streamwise (x) for the LES. The streamwise and spanwise directions are discretized with constant cell size. In the wallnormal direction, the mesh follows a hyperbolic tangent law. The temperatures of the walls are 1300 K for the hot wall and 900 K for the cold wall. A streamwise volume force is added to the channel to maintain a

Investigated cases

Two different types of heating, representing the middle and the end of the solar receiver, are studied. They are performed at the same wall friction. In the first case, the mean fluid temperature is below the cold wall temperature, meaning that it is heated from the both walls. In the second case, the mean fluid temperature is between the two wall temperatures. These two configurations induce opposite directions of the velocity in the wall-normal direction near the cold wall. For practical reasons, the associated simulations are respectively denoted as low fluid temperature (LT) and high fluid temperature (HT). They respectively correspond to heat sources of H s = -54635 kW/m 3 and H s = 0. In the first case, the negative heat source removes heat from the flow which leads to the lowering of the fluid temperature. The Reynolds numbers, based on the hydraulic diameter and the bulk velocity, are 82 000 for the LT simulations and 60 000 for the HT simulations. The obtained friction Reynolds numbers are listed in Table 1. The bulk temperatures are respectively 1093 K and 883 K. The main characteristics of the meshes are given in Table 2.

Results and discussion

Functional models are traceless meaning that only the deviatoric Reynolds stresses can be reconstructed and compared to DNS data without filtering process:

u i u j dev = u i u j - 1 3 u k u k δ ij , (1)
where δ ij is the Kronecker symbol. To facilitate the comparison between LES and DNS, the modeled terms are systematically added to the associated quan-tity.

u i u j DN S,dev = u i u j LES,dev + τ SGS ij (U, ∆) dev , (2) with u i u j = U i U j -U i U j and u i u j LES = U i U j -U i U j .
τ ij is the subgrid-scale tensor associated with the Reynolds stresses. The coordinates x 1 , x 2 , x 3 and x, y, z as well as U 1 , U 2 , U 3 and U , V , W are used interchangeably for simplicity. The same procedure is applied to the correlations of velocity and temperature:

u i θ DN S = u i θ LES + π SGS i (U, T, ∆) , (3) with u i θ DN S = U i T -U i T and u i θ LES = U i T -U i T . π i is the density-velocity subgrid term.
In this section, the LES are assessed on the DNS performed in both heating conditions. The results are normalized thanks to the classical scaling "+". It involves the friction velocity and the friction temperature, respectively defined as U τ = ν∂U/∂y, and θ τ = φ w / (ρC p U τ ), where ν is the kinematic viscosity, C p is the heat capacity at constant pressure, and φ w is the conductive heat flux at the wall. The LES and DNS results are temporally averaged and spatially averaged along with the homogeneous directions. This combination of averages is denoted by • . The wallnormal profiles are displayed in the next. The firstorder statistics are firstly exposed then the secondorder statistics are investigated. In addition to the LES performed with turbulence models, a LES without a model is investigated and denoted "ILES".

First-order statistics

First-order statistics give the global behavior of the flow. Figure 2 exposes the wall-normal profiles of streamwise, wall-normal, and temperature. The results show that the streamwise velocity is mitigated in the simulation with low fluid temperature. The wallnormal velocity profile is substantially affected by the temperature distribution. Indeed, while it is negative and with the same magnitude in the case without source term, the wall-normal velocity is asymmetric in the low-temperature case. On the hot side, the magnitude is almost three time higher in the simulation with heat source. On the cold side, the wall-normal velocity is opposed between the two studied heating conditions. The dimensionless temperature profiles on the cold side traduce the difference between the two investigated cases. Regarding the LES results, the tendency of each profile is respected. The AMD model tends to overestimate the streamwise velocity profile while the other LES are relatively accurate. The wall-normal velocity is better predicted by the LES with functional models. The temperature profiles are quite well approximated by the tensorial AMD model. Overall, the quality of the estimation provided by each model is weakly dependent on the heating conditions.

Second-order statistics

The second-order statistics permit investigating the flow in detail. The velocity correlations give information on the turbulent scales. They are exposed in Figure 3. The correlations of streamwise, spanwise, and wall-normal velocities are quite similar: they follow a bell shape and reach a maximum of magnitude around y + = 13. The DNS carried out with a source term, "DNS [LT]" exhibits lower velocity fluctuations than the simulation "DNS [HT]" and the peak is reached slightly closer to the walls. The extrema of the u v + correlation are also mitigated when compared to the DNS without source term. The LES tend to overestimate the streamwise, spanwise, and wall-normal velocity correlations. The scale-similarity model seems to be not enough dissipative which explains its significant overestimation of the peaks. The tensorial AMD model produces the best results on these values. The tensorial formulation permits improving the results of the AMD model and is able to capture the anisotropy of the flow. The cross-velocity correlation is well approximated by all the LES models.

The correlations involving the temperature are given in Figure 4. The DNS results show that the magnitude of the correlation of streamwise velocity and temperature is mitigated in the case of the DNS with source term. The peaks are slightly shifted toward the walls. On the cold side, a secondary peak is observed close to the center of the channel. It is due to the temperature distribution. The correlation of wall-normal velocity and temperature is significantly influenced by the heat source. In the simulation with a lower bulk temperature than the cold wall temperature, the profile becomes positive on the cold side after y + = 450. On the hot side, it reaches a minimum at y + = 50. The temperature fluctuations are given on the bottom graph. They exhibit a peak around y + = 13, as for the velocity correlations. Once again, in the "DNS [LT]", the peak is reached slightly closer to the wall. In the simulation "DNS [HT]" a third peak is observed in the center of the channel while in the simulation with a heat source, it does not exist due to the highly asymmetric temperature fluctuations. The minimum is obtained for y + = 300. The AMD model and the tensorial AMD model produce satisfying results on the correlation of streamwise velocity and temperature. All the models produce quite good estimations of the correlation of wall-normal and temperature, the tensorial AMD model provides the most accurate estimation on both sides. The temperature fluctuations are poorly approximated by the LES. The AMD model is the closest to the DNS results.

The comparison of the LES performance on the second-order statistics shows that, as for the first-order statistics, the quality of the approximation provided by each model is weakly dependent on the heating conditions.

Conclusions

Direct Numerical simulations and Large Eddy simulations have been performed in asymmetrically heated turbulent channel flows in the operating conditions of solar receivers. Two different types of heating, representing the middle and the end of the solar receiver, have been studied thanks to the introduction of a heat source in the energy equation. In the first case, the mean fluid temperature is below the cold wall temperature, meaning that it is heated from the both walls. In the second case, the mean fluid temperature is between the two wall temperatures. Functional and structural models of LES have been studied in the two different heating conditions.

The results show that the physic of the flow is significantly impacted by the temperature profiles. The wall-normal velocity has opposite directions near the cold wall depending on the heating case. The fluctuations tend to be mitigated in the case of a mean fluid temperature below the cold wall temperature.

Regarding the performance of the LES, it seems that the scale-similarity model is not enough dissipative. The AMD and the tensorial version of the AMD model produce satisfying results. The tensorial AMD model is particularly valuable to estimate the velocity correlations due to its ability to capture the anisotropy of the flow. A0082A05099 made by GENCI. The authors acknowledge PRACE for awarding them access to Joliot-Curie at GENCI@CEA, France (PRACE Project : 2020225398). The authors gratefully acknowledge the CEA for the development of the TRUST platform. The authors gratefully acknowledge the Occitania region for their funding of the thesis grant.

Figure 1 :

 1 Figure 1: Channel flow geometry.

Figure 2 :

 2 Figure 2: Wall-normal profiles of mean quantities. The hot, respectively cold, profiles are plotted on the left, respectively right.

Figure 3 :Figure 4 :

 34 Figure 3: Wall-normal profiles of velocity correlations. The hot, respectively cold, profiles are plotted on the left, respectively right.

Table 1 :

 1 Friction Reynolds numbers.

	Name	Re c τ	Re h τ	Re mean τ
	LT simulations 1000 640	820
	HT simulations 970	630	800

Table 2 :

 2 Grid spacing of the DNS and LES meshes. The dimensionless cell sizes are computed at the cold wall (highest friction Reynolds number).

		Dimensionless cell size
	Name	∆ + x ; ∆ + y (0) ; ∆ + y (δ) ; ∆ + z
	DNS (LT)	10.9 ; 0.42 ; 5.4 ; 5.4
	DNS (HT)	10.7 ; 0.41 ; 5.3 ; 5.3
	LES (LT)	78 ; 1.1 ; 32 ; 43
	LES (HT)	77 ; 1.0 ; 32 ; 43

constant mass flow rate. TrioCFD software

[START_REF] Calvin | An objectoriented approach to the design of fluid mechanics software[END_REF]

) is used to perform simulations. Calculations are carried out operating a finite difference method in a staggered grid system.

Acknowledgments

This work was granted access to the HPC resources of CINES under the allocation 2020-


Organocatalyzed Asymmetric Allylic Alkylation Enables Synthesis of Chiral g-Lactones Bearing Vicinal Tertiary and Quaternary Stereocenters

Morgane Mando, Mathieu Fares, Christelle Kowandy, Fabienne Grellepois and Emmanuel Riguet* Université de Reims Champagne-Ardenne, CNRS, Institut de Chimie Moléculaire de Reims, UMR 7312, 51097 Reims, France. Supporting Information Placeholder ABSTRACT: The synthesis of enantioenriched 𝛼-aryl-𝛼'-allyl-𝛾-butyrolactones bearing vicinal tertiary and quaternary stereocenters through organocatalyzed asymmetric allylic alkylation is reported. The process demonstrated that weakly stabilized enolates derived from 𝛼-aryl-𝛾-butyrolactones can undergo regio-, diastereo-and enantioselective allylation using the proper activation of Morita-Baylis-Hillman (MBH) carbonates.

Investigation of catalytic asymmetric construction of stereocenters requires both the discovery of activation modes and the demonstration of their applicability to compounds exhibiting great structural diversity. It is well recognized that the second achievement can be highly challenging when the construction of quaternary stereocenters (i.e. carbon atoms bearing four different carbon substituents) is envisaged. 1 Asymmetric allylic alkylations (AAAs) are among the most popular processes for the construction of quaternary stereocenters. [START_REF] Behenna | Enantioselective Decarboxylative Alkylation Reactions: Catalyst Development, Substrate Scope, and Mechanistic Studies[END_REF] Continuous progress in AAA was made by the design of catalysts and ligands with the fittest stereoelectronic properties to improve stereoinduction. Nevertheless, this process often entails the emergence of over-represented classes of precursors of stabilized enolates (e.g. dicarbonyl compounds, 𝛼-aryloxoindole or even benzofuran-2(3H)-one). Exploration of the reactivity of uncommon classes of compounds is consequently highly desirable. 𝛼-Aryl-𝛾-butyrolactones embedded synthetic useful features in both the lactone function and the structural diversity available to the aryl moiety. Despite their easy availability, their usefulness in AAA was addressed only recently. Metal-mediated catalysis was first described. Palladium catalyzed decarboxylative asymmetric allylic alkylation (DAAA), whichs avoid the use of strong stoichiometric base, proved to be efficient for the construction of enantioenriched 𝛼-aryl-𝛼'-allyl-𝛾-butyrolactones containing a single stereocenter (Scheme 1, A). [START_REF] James | Highly Enantioselective Construction of Sterically Hindered α-Allyl-α-Aryl Lactones via Palladium-Catalyzed Decarboxylative Asymmetric Allylic Alkylation[END_REF] Unfortunately, high enantiomeric ratios were found for lactones bearing hindered aryl groups. At the same time the synthesis of enantioenriched lactones bearing adjacent quaternary and tertiary stereocenters was achieved 4 through the use of iridium catalysis 5 (Scheme 1, B). Nonetheless, the broad applicability of this strategy has not been demonstrated to date. As frequently observed in the last two decades organocatalysis is a fruitful complementary approach to organometallic catalysis. Organocatalyzed AAA, mediated by the Lewis base activation of Morita-Baylis-Hillman (MBH) adducts, [START_REF] Liu | Organocatalytic asymmetric transformations of modified Morita-Baylis-Hillman adducts[END_REF] to access single quaternary stereocenter [START_REF] Tan | Phosphine-Catalyzed Asymmetric Allylic Alkylation of Achiral MBH Carbonates with 3,3′-Bisindolines: Enantioselective Construction of Quaternary Stereogenic Centers[END_REF] or adjacent quaternary and tertiary stereocenters, 8 mainly involved stabilized prochiral nucleophiles. However, recently, the synthesis of 𝛾-butyrolactones bearing a single quaternary stereocenter by organocatalyzed AAA has been shown to be possible through the design of a specific class of Lewis base (LB) (Scheme 1, C). 9 This demonstrates that LB1 was able to control the addition of the prochiral faces of the lactone enolate on the chiral allyl-pyridinium intermediate by non-covalent interactions. The access to chiral 𝛾-butyrolactones bearing adjacent tertiary and quaternary stereocenters requires additional levels of control which is well illustrated by the need of two different metals (Pd vs Ir) in metal catalyzed AAA. In the present work, we use such Lewis base to catalyze AAA of the less studied 𝛼- aryl-𝛾-butyrolactones scaffold and efficiently fix selectivity issues (i.e. regio-, diastereo-, and enantioselectivities) required for the construction of adjacent tertiary and quaternary stereocenters (scheme 1, D). Scheme 1. Access to 𝛼-aryl-𝛼'-allyl-𝛾-butyrolactones using AAA

The benchmark study was carried out using lactone 1 as a model substrate for 𝛼-aryl-𝛾-butyrolactone and using 1.2 equivalent of MBH carbonate 2 (Scheme 2). Preliminary investigations using DMAP as a Lewis base highlighted that the solvent has a pronounced influence on the regioselectivity of the reaction. Indeed, when the reaction was conducted in DMF racemic linear product 3 was mainly formed (branched/linear ratio of 5/95) and isolated (Scheme 2, A). Interestingly, a reverse regioselectivity was observed when nonpolar solvents were involved culminating with the absence of the linear product in the crude reaction mixture when toluene was used. Good diastereoisomeric ratio (dr 14:86) for the desired branched product was observed and the major diastereoisomer 4 was isolated in high yield (79%) (Scheme 2, B). The relative configuration of created stereocenters in compound 4 was unambiguously determined by single crystal X-ray analysis. We further observed that a high regioselectivity in favor of the desired branched product (b:l 95:5) was maintained in toluene when 5 mol% of LB1 was used as catalyst (Scheme 2, C). Noteworthy, in these reaction conditions, a good level of diastereoselectivity is maintained but with an opposite trend. The major diastereoisomer 5 was isolated in 40% yield and with an excellent 98:2 enantiomeric ratio. Further extensive exploration of various reaction conditions including solvents, stoichiometry of reactants and catalysts as well as catalyst structure was performed (see the ESI for details). Eventually, optimized reaction conditions involving the use of 10 mol% of LB2 as catalyst and 2-methyltetrahydrofuran as solvent highlighted a virtual total regioselectivity (b:l>97:3) and a good diastereoselectivity (dr 84:16) (Scheme 2, D). Thus lactone 5 was isolated in a good yield (79%) and an excellent enantiomeric ratio (er 98:2). Scheme 2. Regioselectivity, diastereoselectivity and enantioselectivity of organocatalyzed AAA with 𝛼-aryl-𝛾-butyrolactone 1

With these optimized conditions in hand, we next explored the scope of the reaction (Scheme 3). MBH carbonates possessing diverse electronic properties were examined using lactone 1 as nucleophile partner in optimized reaction conditions (scheme 3, A). In all cases total conversion is reached in less than 8 h and a virtually total regioselectivity is observed (b:l>97:3). The MBH-carbonate bearing phenyl ring substituted either by electron donating or electron withdrawing groups gave good diastereoisomeric ratios (dr 80:20 to 94:6). The major diastereoisomer was isolated in all cases and robustness of the methodology was consolidated by experiments on 1 or 2 mmol scales.

Starting from electron MBH-carbonates bearing 4-CH3O and 4-CH3-phenyl substitution, lactones 6 and 7 were obtained in good yields (63% and 69%) and excellent enantiomeric ratios (98:2 and 99:1). Notably use of deficient phenyl ring MBHderivatives didn't compromise enantioselectivities of the AAA. Indeed lactones 8-10 were obtained in good yields (63-67%) and in high enantiomeric ratios (er 96:4 to 99:1). MBH carbonates embedding various meta substituents on the aryl group can be efficiently engaged in diastereoselective AAA (dr 66:33 to 85:15) and the major diastereoisomers (11 to 15) were isolated in good yields (up to 74 %) and good enantiomeric ratios (from 94:6 to 98:2). The absolute configuration of the created stereocenters were unambiguously determined by single crystal X-ray analysis of compounds 8 and 11. Finally, chiral lactones 16-18 bearing ortho and disubstituted phenyl can be obtained in high enantiomeric ratios (up to >99:1) through a moderate to highly diastereoselective AAA (dr up to 94:6). We next explored the reactivity of 𝛼-aryl-𝛾-butyrolactones bearing other heteroaromatic ring. To our delight lactone bearing 𝛼-benzothienyl ring is a highly competent substrate for AAA which proceeded in high regioselectivity (no trace of linear product) and high diastereoselectivity (dr 95:5) (scheme 3, B). In this way lactone 19 was obtained in good yield and excellent enantiomeric ratio (99:1) on a 2 mmol scale experiment. The absolute configuration of lactone 19 was attributed by X-ray analysis. Unless specified, the reactions were carried out with 0.25 mmol of lactone, 0.55 mmol of MBH-carbonate and 0.025 mmol of catalyst LB2 in 2-MeTHF (2 mL). b b:l and dr were determined by 1 H NMR of the crude reaction mixture. The b:l ratio was >97:3 (linear product not detected in the 1 H NMR spectra) when not mentioned. er was determined by chiral HPLC analysis. Absolute configuration was assigned by single crystal X-ray analysis for compounds 8, 11, 19, 27 and 28 and attributed by analogy for others. c Reaction carried out on 1 mmol scale. d Reaction carried out on 2 mmol scale. e yield and er after recrystallization. f er before recrystallization.

Gratefully, the AAA of 𝛼-benzothienyl-𝛾-butyrolactones with other MBH derivatives proceeded in equally high regioselectivities and stereoselectivities as demonstrated by the procurement of enantioenriched lactones 20-22 (er up to >99:1). Lactones embedding unprotected indole rings are a particularly interesting class of compounds for further applications of the methodology to the synthesis of biologically active compounds. The AAA also proceeded in virtually total regioselectivity and high diastereoselectivity affording the corresponding lactones 23 and 24 in high yields (69% and 84%) and enantioselectivities (95:5 and 93:7) (scheme 3, C). Good diastereoselectivities were also observed when 𝛼-thienyl-𝛾-butywere used and lactone 25 was obtained in an excellent enantioselectivity (er 99:1) (Scheme 3, D). The simple 𝛼phenyl-𝛾-butyrolactones as well as the one embedding an electron rich substituent at the para position and ortho-substituted-𝛼-phenyl-𝛾-lactones highlighted a poor reactivity in this process and no significant amount of the desired branched product can be isolated. However, substrates containing halogen atom at the para position afforded the corresponding branched major diastereoisomers (26-29) in moderate to good yields (55% to 76%) and high enantiomeric ratios (95:5 to 97:3) (Scheme 3, E). Nevertheless, and contrary to the class of substrates previously described, the presence of linear product was observed in some cases. The single X-ray analysis of compounds 27 and 28 confirmed that the stereoinduction of the AAA is identical to the one previously observed for the lactones substituted by heteroaromatic rings. The lactones bearing a meta substituted phenyl ring were less favorable substrates as a significant amount of a linear product (b:l up to 75:25) was observed in the crude reaction mixture. However, the reaction proceeded in good diastereoselectivities. The major diastereoisomers 30-34 were thus isolated in moderate yields (37 % to 61 %) and good enantiomeric ratios (86:14 to 96:4). The current process also occurred with the 𝛼naphthyl lactone group, providing enantioenriched 35 and 36 in moderate and excellent enantiomeric ratios (89:11 and 99:1). An important aspect of the present methodology is that it allows the construction of a quaternary stereocenter in the benzylic position of heteroaromatic compounds and the functionality introduced by the AAA process can thus be further exploited to access quickly to polycyclic chiral compounds. To illustrate this, indoyl lactones 23 and 24 were converted in polycyclic chiral compounds using a catalytic amount of DBU in toluene at room temperature (Scheme 4). The corresponding lactones 37 and 38 were isolated in good yields (88 and 83 %), and as expected, this transformation occurred with no depletion of enantioenrichment. Scheme 4. Synthetic useful transformation: access to polycyclic indoyl compounds In summary, we have developed an organocatalyzed AAA which allowed the direct functionalization of 𝛼-phenyl-𝛾-butyrolactones. This work demonstrates that this reaction proceeds in high regio-, diastereo-, and enantioselectivities affording chiral compounds with adjacent quaternary and tertiary stereocenters. Contrasting with previously described methodologies, a wide range of diastereoisomerically pure and highly enantioenriched lactones bearing heteroaromatic rings have been isolated in good yields. The expected mechanism of this AAA most probably occurs through an SN2′/ SN2′ mechanism. However, the stereoelectronic features of the expected allyl-pyridinium intermediate and its interaction with the lactone enolate in the transition state entailing a high stereoselectivity could not be easily established and are under investigation in our group. The high functionality obtained can be exploited notably for the synthesis of chiral polycyclic compounds. Noteworthy, the created structures embed a 1,5hexadiene scaffold where one 𝝅 bond is part of an aromatic ring, which provides the opportunity to investigate the elusive aromatic Cope reaction. [START_REF] Bos | Iridium-catalysed asymmetric allylic alkylation of benzofuran γ-lactones followed by heteroaromatic Cope rearrangement: study of an unusual reaction sequence[END_REF][START_REF] Tomiczek | Aromatic Cope rearrangements[END_REF] Investigation on this understudied reactivity is ongoing in our laboratory and will be reported in due course.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website. Experimental procedures, characterization data, copies of NMR spectra and HPLC chromatograms of all new compounds and Xray crystallographic structures of (±)-

✓Scheme 3 .

 3 Scheme 3. Scope of organocatalyzed AAA of 𝛼-aryl-𝛾-butyrolactone a,b

  4 CCDC 2176403, 8 CCDC 2176404, 11 CCDC 2176405, 19 CCDC 2176406, 27 CCDC 2176407, 28 CCDC 2176408. (PDF)

	H 3 CO 2 C	R	O	O
	H N	O	O	toluene, rt DBU (10 mol%)	O N	R
	R=H 23 er 93:7		R=H 37 88% er 92:8
	R=Br 24 er 93:7		R=Br 38 83% er 94:6

ACKNOWLEDGMENT

We gratefully thank the Université de Reims Champagne-Ardenne for financial support (MM). The EU-program FEDER is acknowledged for the facilities of the Analytical Platform (PlAneT CPER project). We also gratefully acknowledged A. Martinez (URCA, ICMR) for her help with NMR analyses and A. Vallée (URCA, ICMR) for his assistance with HPLC and HRMS.

AUTHOR INFORMATION

Corresponding Author

Emmanuel Riguet -Université de Reims Champagne-Ardenne, CNRS, Institut de Chimie Moléculaire de Reims (ICMR) UMR 7312, 51087 Reims France. Email: emmanuel.riguet@univ-reims.fr

Author Contributions

E.R. proposed the project and performed preliminary study. E.R. and F.G. supervised the project and outlined a general strategy. M.M. conducted the experimental study, refined the approach, collected, and analyzed the data. M.F. performed the preliminary experiments on regioselectivity and diastereoselectivity. C.K collected and refined the X-ray diffraction data. E.R. and F.G. wrote the manuscript. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.