Claire Albert

Nicolas Huang
email: n.huangnicolas.huang@u-psud.fr

Nicolas Tsapis

Sandrine Geiger

Véronique Rosilio

Ghozlene Mekhloufi

David Chapron

Baptiste Robin

Mohamed Beladjine

Valérie Nicolas

Elias Fattal

Florence Agnely

Bare and Sterically Stabilized PLGA Nanoparticles for the Stabilization of Pickering Emulsions

Keywords: Pickering emulsion, PLGA, nanoparticles, stability, multiple emulsion, microstructure, interfacial properties

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Introduction

Emulsions are today widely used in food, pharmaceutical and cosmetic applications, since they allow the encapsulation of an active ingredient in the dispersed phase, to protect it from degradation and to preserve its activity in a sustained manner. [START_REF] Marti-Mestres | Emulsions in Health Care Applications-An Overview[END_REF] However, emulsions are thermodynamically unstable systems requiring the use of stabilizers for their fabrication and long-term stability. [START_REF] Tadros | Emulsion Formation and Stability[END_REF] Until now, emulsions have been mostly stabilized using synthetic surfactants, [START_REF] Scherlund | Stabilization of a Thermosetting Emulsion System Using Ionic and Nonionic Surfactants[END_REF] which raises direct or indirect toxicity and environmental issues. [START_REF] Cserháti | Biological Activity and Environmental Impact of Anionic Surfactants[END_REF] In particular, in topical long-term treatment, skin irritation is often observed. [START_REF] Branco | Long-Term Repetitive Sodium Lauryl Sulfate-Induced Irritation of the Skin: An in Vivo Study[END_REF][START_REF] Lémery | Skin Toxicity of Surfactants: Structure/Toxicity Relationships[END_REF] New stabilization approaches have been developed such as the use of solid particles instead of surfactants. [START_REF] Aveyard | Emulsions Stabilised Solely by Colloidal Particles[END_REF][START_REF] Chevalier | Emulsions Stabilized with Solid Nanoparticles: Pickering Emulsions[END_REF][START_REF] Schmitt | Colloidal Particles as Liquid Dispersion Stabilizer: Pickering Emulsions and Materials Thereof[END_REF] Such emulsions, also called Pickering emulsions, display a very good stability (sometimes up to several years) thanks to their high resistance to coalescence. 10,[START_REF] Ramsden | Separation of Solids in the Surface-Layers of Solutions and "Suspensions" (Observations on Surface-Membranes, Bubbles, Emulsions, and Mechanical Coagulation). --Preliminary Account[END_REF] Many types of particles (mineral or organic) can be used to prepare Pickering emulsions. [START_REF] Yang | An Overview of Pickering Emulsions: Solid-Particle Materials, Classification, Morphology, and Applications[END_REF] However, most of the emulsions thus produced are not biocompatible and/or not biodegradable, due to the nature of the particles or oil used, which is a crucial issue for cosmetic or pharmaceutical applications.

In this respect, poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) appear as very promising systems. Indeed, PLGA is a well-known biodegradable and biocompatible copolymer currently used in drug delivery to prepare NPs whose degradation and drug release rates can be tuned by varying the PLGA molecular weight or its lactide:glycolide ratio. [START_REF] Makadia | Poly Lactic-Co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier[END_REF] Such NPs can be obtained by different processes such as nanoprecipitation or emulsion-evaporation that most often require the use of a stabilizing polymer. PLGA-PVA NPs, i.e. PLGA NPs sterically stabilized by poly(vinyl alcohol) (PVA), a non-toxic stabilizing polymer, 14 have already been largely described. [START_REF] Astete | Synthesis and Characterization of PLGA Nanoparticles[END_REF] However, Pickering emulsions stabilized by this type of NPs have only been reported thrice. [START_REF] Whitby | Poly(lactic-Co-Glycolic Acid) as a Particulate Emulsifier[END_REF][START_REF] Qi | Systematic Studies of Pickering Emulsions Stabilized by Uniform-Sized PLGA Particles: Preparation and Stabilization Mechanism[END_REF][START_REF] Deschamps | Pickering-Emulsion for Liver Trans-Arterial Chemo-Embolization with Oxaliplatin[END_REF] In these reported studies, though biocompatible oils were tested (isopropyl myristate, sunflower oil or lipiodol, an iodized and radio-opaque oil), thorough physico-chemical characterizations were either conducted with non-biocompatible model oils such as dodecane or octanol, or were lacking. Beside, only few descriptions of the preparation of stabilizer-free PLGA NPs were reported in the literature. [START_REF] Stolnik | The Colloidal Properties of Surfactant-Free Biodegradable Nanospheres from poly(B-Malic Acid-Co-Benzyl Malate)s and Poly(lactic Acid-Co-Glycolide)[END_REF][START_REF] Grabowski | Surface Coating Mediates the Toxicity of Polymeric Nanoparticles towards Human-like Macrophages[END_REF][START_REF] Gyulai | Interaction of Poly(lactic-Co -Glycolic Acid) Nanoparticles at Fluid Interfaces[END_REF] To the best of our knowledge, these bare PLGA NPs obtained by nanoprecipitation without any other polymer or surfactant were never used to formulate Pickering emulsions. However, both bare PLGA NPs and PLGA-PVA NPs are interesting for potential pharmaceutical or cosmetic applications as a very low toxicity was observed on THP-1 derived macrophages [START_REF] Grabowski | Surface Coating Mediates the Toxicity of Polymeric Nanoparticles towards Human-like Macrophages[END_REF] with PLGA NPs and PLGA-PVA NPs, and on different lung cells [START_REF] Mura; Fattal | Influence of Surface Charge on the Potential Toxicity of PLGA Nanoparticles towards Calu-3 Cells[END_REF][START_REF] Grabowski | Toxicity of Surface-Modified PLGA Nanoparticles toward Lung Alveolar Epithelial Cells[END_REF] with PLGA-PVA NPs.

In this context, the main objective of this study was to formulate Pickering emulsions using bare PLGA NPs and PLGA-PVA NPs and to evaluate their stability, microstructure and interfacial properties. To specifically assess the influence of PVA, the two types of NPs were prepared with similar sizes, around 200 nm. In a first step, NPs were characterized in terms of size, surface properties, hydrophobicity and mechanical properties. Then, Pickering emulsions were formulated with both types of NPs using Miglyol 812 N as the oil phase. This oil composed of medium-chain triglycerides was selected as it is widely used in cosmetics and in topical, oral or parenteral pharmaceutical preparations (emulsions, solutions or suspensions). 14 It has numerous advantages such as absorption enhancement, stability against oxidation, non-toxicity, non-irritating and non-inhibition of skin respiration. 14 We compared the macroscopic, microscopic and interfacial structures of the resulting Pickering emulsions, as well as their stability over 55 days. A thorough physicochemical study was performed to clarify the mechanisms of stabilization with both types of NPs. The interface between the oil and aqueous phases was characterized by confocal microscopy, interfacial tension and interfacial rheology.

The NP hydrophobicity was evaluated by contact angle measurements. Such characterizations are particularly interesting both from application but also from fundamental standpoints. Indeed, most previous studies dealing with the adsorption of core-shell particles at an oil-water interface were performed with a chemically grafted corona of polymer [START_REF] Isa | Adsorption of Core-Shell Nanoparticles at Liquid-liquid Interfaces[END_REF][START_REF] Garbin | Nanoparticles at Fluid Interfaces: Exploiting Capping Ligands to Control Adsorption, Stability and Dynamics[END_REF] whereas, here, the stabilizing chains of PVA are adsorbed at the particle surface.

Experimental section

Chemicals

Poly(lactide-co-glycolide) (PLGA, 75:25 Resomer© RG756, ester ending, inherent viscosity at 25 °C and 0.1% in CHCl3: 0.7-1.0 dL/g according to the supplier) was purchased from Boehringer-Ingelheim (Germany). PLGA-rhodamine B conjugate (MW ≈ 30,000 g/mol) was provided by Akina Inc. (USA). Poly(vinyl alcohol) (PVA, molecular weight 30,000-70,000 g/mol, 87-90% hydrolyzed according to the supplier), and D-trehalose (from saccharomyces cerevisiae, ≥ 99%) were obtained from Sigma Aldrich (France). Miglyol (812 N), a medium chain triglyceride oil, was purchased from Cremer Oleo GmbH & Co. (Germany).

Iodine (I 2 ) and potassium iodide (KI) used to prepare the iodine solution were purchased respectively from Touzart et Matignon (France) and VWR PROLABO (France). Boric acid and calcein came from Sigma (France). Oregon Green was provided by Invitrogen (France). All the solvents (acetonitrile, acetone and dichloromethane) were provided at the highest grade by Carlo Erba (Milan, Italy). Water was purified using a water system ultrapure MilliQ Direct Type 1 (Millipore, resistivity of 18.2 MΩ.cm, France).

NPs preparation

PLGA-PVA NPs stabilized by using poly(vinyl alcohol) (PVA) were prepared according to the previously described emulsion-evaporation method. [START_REF] Mura; Fattal | Influence of Surface Charge on the Potential Toxicity of PLGA Nanoparticles towards Calu-3 Cells[END_REF] PLGA NPs were prepared, without any other polymer or surfactant, according to the nanoprecipitation method previously reported by Grabowski et al. [START_REF] Grabowski | Surface Coating Mediates the Toxicity of Polymeric Nanoparticles towards Human-like Macrophages[END_REF] The preparation methods are briefly described in Supporting Information 1.

Fluorescently labeled NPs were prepared by replacing the total quantity of PLGA by a mixture of PLGA and a PLGA-rhodamine conjugate at a ratio of 90/10 (w/w) for PLGA-PVArhod NPs and of 99.9/0.1 (w/w) for PLGA-rhod NPs.

NP characterization

The size distribution and zeta potential of NPs were measured with a Zetasizer (Nano ZS 90, Malvern Instruments, France, 633 nm He-Ne laser, Smoluchowski equation) operating at 25 °C with a 173° scattering angle. The NP suspension was diluted in MilliQ water for size distribution measurements and in a 1 mM NaCl aqueous solution for zeta potential measurements, until reaching an attenuator value of 6. For each sample, three measurements were performed.

NP hydrophobicity was determined using contact angle measurements. A layer of NPs was formed by drying (during 15 h at 25 °C, protected against dust) a 45 µL drop of NP suspension in water at 25 mg/mL deposited on a freshly cleaved mica surface. Contact angle measurements were performed at 20 °C using the sessile drop method with a pendant drop tensiometer (Tracker, Teclis, France). A 5 µL drop of liquid (water or Miglyol) was gently deposited at the colloidal layer surface. The contact angle was then measured by drop shape analysis over time with the apparatus software (Windrop). The presented results are the average of at least 10 measurements.

Emulsion preparation

Emulsions were prepared with an aqueous phase/oil phase ratio of 90/10 (w/w) using an Ultraturrax (IKA T10 basic) at 20,000 rpm for 2 min with a 5G-stainless steel dispersing tool for the emulsification step. The oil phase was Miglyol and different aqueous phases (solutions or NP suspensions) were studied: water as a reference, a PVA solution at 2 mg/mL, a trehalose solution at 150 mg/mL, a PLGA NP suspension at a concentration of 25 mg/mL in water or in a PVA solution (2 mg/mL), a suspension of previously lyophilized PLGA-PVA NPs at a concentration of 25 mg/mL in water and a suspension of non-lyophilized PLGA-PVA NPs at a concentration of 25 mg/mL in water or in a trehalose aqueous solution (150 mg/mL).

Emulsion characterization

Stability

The emulsion stability during storage at 25 °C and protected from light was evaluated over 55 days by monitoring the destabilization phenomena using a Turbiscan Classic MA 2000 apparatus (Formulation, Toulouse, France). This technique is based on multiple light scattering: a near-infrared light source scans the sample from bottom to top. Backscattered and transmitted intensities versus sample height were obtained at different days (D0, D1, D2, D3, D7, D12, D20, D35 and D55), thus allowing the detection of destabilization phenomena (creaming, sedimentation, flocculation and coalescence), even at an early stage (invisible to the eye). [START_REF] Mengual | Multiple Light Scattering Measurement for Concentrated Emulsion and Suspension Instability Analysis[END_REF] In the meantime, macroscopic observations were performed by taking pictures of emulsions in Turbiscan glass tubes. The droplet size distributions were determined by laser granulometry on a Mastersizer S apparatus (Malvern Instrument, Orsay, France). Aliquots of the emulsion layer were diluted with the appropriate volume of MilliQ water (in order to obtain an obscuration between 5 and 10%). Measurements were performed at least in triplicate, using the Fraunhofer optical model, on different emulsions at D0, D1 and D55. Results are presented in percentage of the volume distribution.

Microscopic structure

The microscopic structure of the emulsions was observed using a confocal scanning laser microscope (inverted Leica TCS SP8-gated STED, Germany) equipped with a WLL laser (488 and 563 nm excitation wavelengths) using a HC PL APO CS2 63x/1.40 oil immersion objective lens. 20 µL were taken from the emulsion layer for these experiments. To prevent the deformation of emulsion droplets, the sample was placed in a curved glass slip with a cover glass slide on the top. The NPs were labeled in red with rhodamine as previously described (see section 2.2). The aqueous phase was labeled in green by re-suspension of the lyophilized PLGA-PVA NPs in a 0.08 mg/mL solution of Oregon green instead of MilliQ water or by the addition of 100 µL of calcein solution at 0.4 mg/mL to the PLGA NP suspension. Red fluorescence was observed with a 600-710 nm filter under a 563 nm laser illumination. Green fluorescence was observed with a 500-535 nm filter under a 488 nm laser illumination. A hybrid detector under a gated mode (0.3-6.5 ns) was used in order to avoid reflection. The green and the red fluorescence emissions were collected under a sequential mode. The pinhole was set at 1.0 Airy unit.

Interfacial analysis

To determine the interfacial organization and rheological behavior of the oil/water interface of the emulsions, the interfacial tension γ, the absolute value of the complex dilatational modulus |E * | as well as the interfacial elastic E' and viscous E" moduli were measured using the pendant drop tensiometer. A drop of the aqueous phase was formed (with an 18 G, straight needle and a 500 µL syringe) into Miglyol. Sinusoidal oscillations were applied to the drop volume with a 60 s period and a variation of 10% volume at 25 °C corresponding to the linear regime. The oscillations were started 1800 s after the drop formation to allow the initial structuration of the interface without any disturbance due to the oscillations. The results presented are the average ± standard deviation of at least three measurements.

Results and discussion

NP formulation and characterization

Bare PLGA NPs and PLGA-PVA NPs were prepared. Their properties were first thoroughly assessed and compared. As shown in Table 1, all NPs exhibited narrow size distributions as all PdI values were below 0.1. [START_REF]Particle Size Analysis -Dynamic Light Scattering (DLS) ISO 22412[END_REF] PLGA NPs were 170 nm in hydrodynamic diameter, and slightly smaller than PLGA-PVA NPs. The difference in size between the two types of NPs might be explained by the use of two different methods of preparation and by the presence of the hydrated PVA layer on PLGA-PVA NP surface, which should increase the hydrodynamic diameter. The presence of PVA on the surface could also explain the differences in zeta potential measurements: -35 mV for PLGA NPs and -4 mV for PLGA-PVA NPs. Very likely, the negative charges of PLGA, due to its carboxylic end groups, are screened by PVA, a neutral polymer, resulting in the reduction of zeta potential values. Indeed, the amount of PVA associated to PLGA-PVA NPs was quantified at 8% (w/w of PLGA) of PVA in PLGA-PVA NPs (see Supporting Information 2) and these PVA chains were found to be at the NP surface (see Supporting Information 3). PLGA-PVA NPs were lyophilized using trehalose as a cryoprotectant without any significant change in their size and zeta potential (Table 1). Lyophilization is useful for NP storage following their preparation. However, it was impossible to preserve these properties when lyophilizing PLGA NPs despite the presence of trehalose because of the aggregation of NPs, even after resuspension.

NPs

Assuming a molecular weight M = 50,000 g/mol for PVA chains and a density of 1,3 g/cm [START_REF] Scherlund | Stabilization of a Thermosetting Emulsion System Using Ionic and Nonionic Surfactants[END_REF] for NPs), [START_REF] Vauthier | Measurement of the Density of Polymeric Nanoparticulate Drug Carriers by Isopycnic Centrifugation[END_REF] it was possible to estimate the average area occupied by a PVA chain at the surface of the PLGA-PVA NPs. The obtained value (≈ 22 nm 2 ) was in good agreement with the one reported by Gyulai and Kiss 21 (≈ 20 nm 2 ) for a Pluronic F27 chain at the surface of PLGA NPs . The radius of gyration, R g , of a PVA coil in water (good solvent conditions) was calculated from the Flory equation with N the number of segments in the PVA chain and the size of the PVA segment (0.37 nm). [START_REF] Jin | Organic-Inorganic Composite Membranes for Molecular Separation[END_REF] As R g (≈ 10 nm) was higher than the average distance between PVA chains at the particle suface (≈ 5 nm), the corona of PVA around the PLGA-PVA NPs was expected to be dense in water and its thickness to be higher than 10 nm.

The Young's moduli of the NPs were assessed by AFM measurements (see Supporting Information 4). In the light of these results, no significant deformation of the core of the PLGA-PVA and of the PLGA NPs at the oil/water interface was expected (Young's moduli ≈ 8 GPa and ≈ 4 GPa, respectively), contrarily to what was observed with nanogels (Young's modulus between ≈ 1 and 35 MPa). [START_REF] Schmitt | Colloidal Particles as Liquid Dispersion Stabilizer: Pickering Emulsions and Materials Thereof[END_REF] A similar assumption was made by Gyulai and Kiss with bare PLGA NPs and PLGA NPs sterically stabilized by Pluronic F127. [START_REF] Gyulai | Interaction of Poly(lactic-Co -Glycolic Acid) Nanoparticles at Fluid Interfaces[END_REF] NP hydrophobicity was evaluated by measuring the contact angle of a drop of liquid (water or Miglyol) deposited in the air on top of a NP layer adsorbed onto a mica substrate (Table 2). As seen in AFM pictures, the NP layers covered the substrate as a dense particle layer with a low roughness, similar for PLGA NPs and the non-lyophilized PLGA-PVA NPs (see Supporting Information 4). The contact angle value was recorded 0.4 s after droplet deposition, to avoid any imbibition effects (see Supporting Information 5). The NPs were soluble neither in water nor in Miglyol and kept their integrity, although PLGA chains were not chemically crosslinked. Both types of NPs presented a lower contact angle with Miglyol than with water: the colloidal layers were more hydrophobic than hydrophilic. With water, the contact angle for PLGA-PVA NPs (≈ 82 °) was lower than for PLGA NPs (≈ 120 °): the PLGA-PVA NP layer was slightly more hydrophilic than the PLGA NP layer. This difference of hydrophilicity is due to the presence of PVA at the surface of PLGA-PVA NPs, since PVA is a more hydrophilic polymer than PLGA. [START_REF] Sahoo | Residual Polyvinyl Alcohol Associated with poly(D,L-Lactide-Co-Glycolide) Nanoparticles Affects Their Physical Properties and Cellular Uptake[END_REF] Surprisingly, the contact angles with Miglyol were identical for PLGA NPs and PLGA-PVA NPs (≈ 17 °): PVA had no influence on the wettability of the NPs by Miglyol. An interpretation of this phenomenon is given in section 3.5.2.

Contact angle (°)

This physicochemical characterization showed that the NPs were in the same size range but were different in terms of zeta potentials, Young's moduli and hydrophobicity. This difference was mostly attributed to the PVA at the PLGA-PVA NP surface. Emulsions stabilized by one or the other type of NPs were then formulated and compared.

Emulsion formulation

Emulsions at a 90/10 aqueous phase/oil phase (w/w) ratio were formulated with Miglyol as the oil phase and with, as the aqueous phase, a suspension in water at 25 mg/mL of either lyophilized PLGA-PVA NPs (Figure 1a) or PLGA NPs (Figure 1b). This particular aqueous phase/oil phase ratio was chosen in order to have a sufficient amount of NPs to stabilize the oil/water interface. Both resulting emulsions were very different in their macroscopic aspect.

After a creaming step, the emulsion stabilized with PLGA-PVA NPs exhibited a thin emulsion layer (≈ 7 mm) on top of the sample with a cloudy aqueous phase, due to an excess of NPs. The emulsion stabilized with PLGA NPs exhibited a significantly thicker emulsion layer (≈ 21 mm) on top of a clear aqueous phase. This difference was consistent with the previous observation that PLGA-PVA NPs were more hydrophilic than PLGA NPs (see section 3.1). The oil had no inherent stabilizing properties as the mixture of Miglyol and water only, used as a control, phase separated within minutes (Figure 1c). The stability of these three emulsions, stored at 25 °C without light over 55 days, was then evaluated with a Turbiscan apparatus.

Emulsion stability study

The evolution of the Turbiscan curves with time provided information on the stability or the destabilization phenomena such as creaming or sedimentation and coalescenceflocculation. [START_REF] Mengual | Multiple Light Scattering Measurement for Concentrated Emulsion and Suspension Instability Analysis[END_REF][START_REF] Liu | Turbiscan Lab® Expert Analysis of the Biological Demulsification of a Water-in-Oil Emulsion by Two Biodemulsifiers[END_REF] Examples of Turbiscan curves obtained for the emulsion stabilized by lyophilized PLGA-PVA NPs are given in Supporting Information 6. From the Turbiscan curves of each emulsion combined with the macroscopic observations performed at the same time, it was possible to construct a scheme summarizing the evolution of the emulsion layer for the different formulations (Figure 2a). The creaming step observed for each emulsion would not be a problem for a pharmaceutical or a cosmetic application provided that the droplet size remained constant.

Indeed, if there is no evolution of the droplet size, creaming is a reversible phenomenon which can be bypassed by shaking the sample before use. 32 Turbiscan analysis (Figure 2a). Moreover, NP lyophilization did not influence emulsion stabilization: no difference in stability and in macroscopic structure was noticed between the emulsions stabilized either by lyophilized PLGA-PVA NPs or by non-lyophilized PLGA-PVA NPs (Supporting Information 7). Consequently, the emulsions for studies hereunder were prepared using lyophilized PLGA-PVA NPs. For the emulsion stabilized with PLGA NPs (Figure 2c), no significant change in the droplets size was noticed over time: after the creaming stage, no evolution of the emulsion was observed for 55 days at least.

Microstructure of the emulsions

The emulsions stabilized by bare PLGA NPs or by lyophilized PLGA-PVA NPs appeared very different in their macroscopic structure and stability: the presence of PVA adsorbed on the NPs strongly modified the emulsions obtained. Therefore, to better understand these differences, their microscopic structure was studied by confocal scanning laser microscopy. In the confocal microscopy images (Figure 3a andb), NPs appear in red (rhodamine), the aqueous phase in green (Oregon green or calcein) and the Miglyol phase in black. The emulsion stabilized with lyophilized PLGA-PVA NPs was a simple oil in water (O/W) emulsion as the oil formed droplets in the aqueous phase (Figure 3a). NPs surrounded the oil droplets, confirming that NPs adsorbed at the interface to stabilize the emulsion. NPs were also present in excess in the aqueous phase as previously observed at the macroscopic scale (Figure 1a). The graph giving the fluorescence intensity collected in both channels (green and red) (Figure 3c), measured along the white oblique line of Figure 3a, supports these observations. Indeed, two peaks were detected at the edge of the droplet in the red channel, corresponding to the NPs adsorbed at the interface. The fluorescence intensity in the red channel was not null in the aqueous phase of the emulsion layer due to the presence of NPs, whereas the fluorescence intensity in both channels, red and green, was null inside the droplet and not null outside, as expected for simple O/W emulsions. All these results were consistent with the macroscopic observations.

In contrast, the microscopic structure of the emulsion stabilized by PLGA NPs was completely different. Figure 3b shows the presence of both multiple W/O/W and simple O/W droplets. All the droplets were surrounded by NPs and there were some NP aggregates in the aqueous phase of the emulsion layer. The type of multiple droplets was determined by the fluorescence intensity graph (Figure 3d) measured along the white oblique line on Figure 3b. The two peaks in the red channel at the edge of the internal droplet corresponded to the NPs adsorbed at the interface, whereas the peak in the green channel confirmed the presence of water inside the inner droplets. Thus, the multiple droplets were water in oil in water (W/O/W).

The presence of multiple droplets is remarkable as usually, to obtain a multiple Pickering emulsion, two emulsification steps are required, using two types of NPs. The more hydrophilic ones stabilize the O/W droplets and the more hydrophobic ones stabilize the W/O ones. [START_REF] Aveyard | Emulsions Stabilised Solely by Colloidal Particles[END_REF][START_REF] Binks | Ellipsometric Study of Monodisperse Silica Particles at an Oil-Water Interface[END_REF][START_REF] Arditty | Materials Based on Solid-Stabilized Emulsions[END_REF][START_REF] Gao | Dual Nanocomposite Multihollow Polymer Microspheres Prepared by Suspension Polymerization Based on a Multiple Pickering Emulsion[END_REF][START_REF] Zou | Surfactant-Free Multiple Pickering Emulsions Stabilized by Combining Hydrophobic and Hydrophilic Nanoparticles[END_REF][START_REF] Rymaruk | Bespoke Contrast-Matched Diblock Copolymer Nanoparticles Enable the Rational Design of Highly Transparent Pickering Double Emulsions[END_REF] Here, a stable multiple emulsion was obtained using a single emulsification step and only a single type of NPs. Clearly, all the droplets were surrounded by NPs, confirming that the PLGA NPs were able to adsorb at the interface of both droplets: O/W and W/O. Since the first description of Pickering multiple emulsions obtained in a single step with silica NPs by Binks & Rodrigues in 2003, [START_REF] Binks | Types of Phase Inversion of Silica Particle Stabilized Emulsions Containing Triglyceride Oil[END_REF] several types of NPs were used: silica NPs, [START_REF] Binks | Phase Inversion of Particle-Stabilised Perfume Oil-water Emulsions: Experiment and Theory[END_REF][START_REF] White | Inversion of Particle-Stabilized Emulsions of Partially Miscible Liquids by Mild Drying of Modified Silica Particles[END_REF][START_REF] Binks | Silica Particle-Stabilized Emulsions of Silicone Oil and Water: Aspects of Emulsification[END_REF] poly(dodecyl acrylate-co-acrylic acid) NPs, [START_REF] Zhu | One-Step Formation of Multiple Pickering Emulsions Stabilized by Self-Assembled Poly(dodecyl Acrylate-Co-Acrylic Acid) Nanoparticles[END_REF] boehmite alumina powder, [START_REF] Chen | Influence of the Particle Type on the Rheological Behavior of Pickering Emulsions[END_REF] graphene oxide or silver NPs coated graphene oxide, [START_REF] He | Factors That Affect Pickering Emulsions Stabilized by Graphene Oxide[END_REF][START_REF] Tang | Factors That Affect the Stability, Type and Morphology of Pickering Emulsion Stabilized by Silver Nanoparticles/Graphene Oxide Nanocomposites[END_REF] silicone microbowls, [START_REF] Nonomura | Multiple Pickering Emulsions Stabilized by Microbowls[END_REF] PDMA-PMMA coated latex [START_REF] Read | Effect of Varying the Oil Phase on the Behavior of pH-Responsive Latex-Based Emulsifiers: Demulsification versus Transitional Phase Inversion[END_REF] and styrene/acid acrylic Janus particles. [START_REF] Tu | One-Step Encapsulation and Triggered Release Based on Janus Particle-Stabilized Multiple Emulsions[END_REF] The mechanism for the formation of multiple Pickering emulsions obtained in a single step using one type of NPs only was not clearly defined. In these previous studies, four main hypotheses were proposed to explain the formation of a multiple emulsion in a single step using only one type of NPs: (1) a three-phase contact angle at the oil, NPs and water interface around 90°, so that the NPs can adsorb both at the oil/water and the water/oil interfaces; [START_REF] Zhu | One-Step Formation of Multiple Pickering Emulsions Stabilized by Self-Assembled Poly(dodecyl Acrylate-Co-Acrylic Acid) Nanoparticles[END_REF][START_REF] Chen | Influence of the Particle Type on the Rheological Behavior of Pickering Emulsions[END_REF] (2) a variation of hydrophobicity inherent to the NPs, so that the most hydrophobic ones would adsorb at the water/oil interface while the most hydrophilic ones would adsorb at the oil/water interface; [START_REF] Tang | Factors That Affect the Stability, Type and Morphology of Pickering Emulsion Stabilized by Silver Nanoparticles/Graphene Oxide Nanocomposites[END_REF] (3) a variation of hydrophobicity among the NPs due to their interactions with the liquid they first establish contact; 40,46,49 (4) the proximity to the phase inversion: just before becoming the dispersed phase, the continuous phase would form some droplets in the drops of the other liquid. [START_REF] White | Inversion of Particle-Stabilized Emulsions of Partially Miscible Liquids by Mild Drying of Modified Silica Particles[END_REF][START_REF] Nonomura | Multiple Pickering Emulsions Stabilized by Microbowls[END_REF][START_REF] Clegg | One-Step Production of Multiple Emulsions: Microfluidic, Polymer-Stabilized and Particle-Stabilized Approaches[END_REF][START_REF] Groeneweg | On the Mechanism of the Inversion of Emulsions[END_REF] Binks et al. [START_REF] Binks | Phase Inversion of Particle-Stabilised Perfume Oil-water Emulsions: Experiment and Theory[END_REF] proposed that the multiple emulsion formation would be due simultaneously to a three-phase contact angle around 90° and to the proximity with the phase inversion (hypotheses 1 and 4). He et al. [START_REF] He | Factors That Affect Pickering Emulsions Stabilized by Graphene Oxide[END_REF] also showed that parameters such as particles concentration, oil/water ratio, salt concentration or pH had an influence on the type of the emulsion (simple or multiple). The mechanism of the formation of such double emulsions is thus complex and is not fully elucidated yet. In the present study, the third hypothesis was unlikely to occur as PLGA NPs were all initially dispersed in water. Besides, a very low oil fraction could not explain the presence of multiple droplets, as multiple emulsions were also observed at significantly higher oil ratios. However, it was shown that when increasing the oil ratio, the number of multiple droplets increased until reaching the phase inversion (Supporting Information 8). Thus, for our system, among the hypotheses discussed above, the formation of multiple droplets might involve a three-phase contact angle around 90°, an inherent variation of hydrophobicity among the NPs and/or the proximity with the phase inversion.

The presence of multiple droplets in the emulsion stabilized by PLGA NPs could explain the higher emulsion layer observed compared to the simple O/W emulsion stabilized with lyophilized PLGA-PVA NPs. Indeed, with multiple droplets, a higher amount of water participated in the emulsion layer, since some water was also entrapped as small droplets within the oil globules.

Thus, the macroscopic and microscopic structures and the stability of the two emulsions were very different, even though they were prepared with the same oil phase, the same aqueous phase/oil phase ratio of 90/10 (w/w) and the same quantities of NPs in the aqueous phase. The only difference was the type of PLGA NPs (PLGA-PVA NPs or bare PLGA NPs). To further elucidate these differences, an interfacial study was conducted.

Interfacial analysis

The interfacial properties of the NPs at the oil/water interface were investigated with the oscillating pendant drop method. The interfacial tension (γ) (Figure 4a) as well as the interfacial elastic (E') (Figure 4b) and viscous (E") (Figure 4c) moduli were recorded over 12 hours. For this purpose, a drop of aqueous phase was formed in Miglyol. The values of γ, E', E" and |E * | (absolute value of the complex dilatational modulus) at 12 hours for all the samples are summarized in Supporting Information 9. All NP suspensions were prepared at a PLGA concentration of 25 mg/mL. The concentration of the NP suspensions was 25 mg/mL. For some systems, error bars are too small to be visible.

When the aqueous phase was a suspension of PLGA NPs in water, a slight decrease of the interfacial tension was noticed compared to the control (water) (Figure 4a). This is consistent with the work of Gyulai and Kiss 21 who reported the thermodynamically favorable adsorption and the surface activity of bare PLGA NPs at air/water and octane/water interfaces both with a drop tensiometer and a Langmuir trough. On the other hand, when the aqueous phase was a suspension of PLGA-PVA NPs in water (lyophilized or not), the interfacial tension was significantly lowered compared to the interfacial tension of the water-Miglyol interface without any stabilizer (Figure 4a). We investigated the adsorption kinetics of the NPs at the interface according to a model developed by Bizmark et al. [START_REF] Bizmark | Effects of Ionic Strength on the Colloidal Stability and Interfacial Assembly of Hydrophobic Ethyl Cellulose Nanoparticles[END_REF] and also used by Nelson et al. [START_REF] Nelson | A Multiscale Approach to the Adsorption of Core-shell Nanoparticles at Fluid Interfaces[END_REF] (see Supporting Information 10). According to this model, we found that the adsorption energy barrier (~10-15 ) was negligible compared to the adsorption energy of a single NP (~10 3 -10 4 ).

Consequently, the interface could be considered as barrier-free. The NP adsorption kinetics was mainly governed by diffusion and the NPs were irreversibly adsorbed at the interface, as the energy necessary for desorption ranged from 10 3 to 10 4 in order of magnitude (Supporting Information 10).

Trehalose influence on the interfacial tension

The interfacial tension for a suspension of non-lyophilized PLGA-PVA NPs in water (without trehalose) was significantly higher than for the suspension of lyophilized PLGA-PVA NPs water (with 150 mg/mL trehalose used as a cryoprotectant) (14.4 ± 0.4 mN/m versus 8.1 ± 0.3 mN/m at 12 h) (Figure 4a). On the other hand, the interfacial tension of the suspension of non-lyophilized PLGA-PVA NPs in a solution of trehalose at 150 mg/mL (corresponding to the concentration of trehalose in the lyophilized NP suspension) was the same as the one obtained with lyophilized PLGA-PVA NPs in water (with 150 mg/mL trehalose). This means that the difference was not due to the lyophilization step but to the presence of trehalose in solution.

Arnett et al. [START_REF] Arnett | No Phospholipid Monolayer-Sugar Interactions[END_REF] reported that some reputedly pure carbohydrates (trehalose, galactose and sucrose)

were contaminated by surfactants. The purity of our trehalose sample was verified by a foaming technique that demonstrated the presence of surface active impurities. These impurities were not successfully removed from the solution. However, this was not a crucial issue in our study, considering that the emulsion prepared with trehalose alone (150 mg/mL) had a poor stability (Supporting Information 7). Moreover, the interfacial tension for a suspension of non-lyophilized PLGA-PVA NPs in water (without trehalose) was lower than that for a suspension of PLGA NPs in water underlining the crucial role played by PVA in the interfacial tension lowering.

Influence of the PVA chains sterically stabilizing the NPs on the interfacial tension

As deduced from the quantification of PVA in NPs (see Supporting Information 2), 2 mg of PVA were adsorbed at the surface of 25 mg of PLGA-PVA NPs. We also observed that a PVA solution at 2 mg/mL led to a pronounced lowering of the interfacial tension (Figure 4a). This was not surprising, considering that PVA is known for its surfactant properties. [START_REF] Blehn | Polyvinyl Alcohol as an Emulsifying Agent[END_REF] It should be noted that no PVA aggregate was present in these solutions, as all the PVA aqueous solutions prepared in this study were at a concentration lower than the critical aggregation concentration (CAC) of the PVA (9.2 mg/mL) (Supporting Information 11).

To get a better insight into the effect of PVA sterically stabilizing the NPs, a bare PLGA NP suspension at 25 mg/mL was mixed with a solution of PVA at 2 mg/mL. A decrease of the interfacial tension was observed compared to the PLGA NP suspension in water (Figure 4a). Finally, at the same concentration, PVA adsorbed onto PLGA-PVA NPs induced a more significant decrease in the interfacial tension than the mixture of free PVA and PLGA NPs (Figure 4a). More PVA chains were able to adsorb at the interface when they were previously adsorbed at the NP surface than when they were free in the aqueous phase in presence of PLGA NPs. This could be explained by a partial desorption of PVA from the particle surface when in contact with the oil phase (Figure 5b). Indeed, as previously observed (see section 3.1 Table 2),

the PVA adsorbed at the NP surface made the surface more hydrophilic than PLGA. Interactions of PVA with the oil phase were thus less favorable than those of PLGA with the same phase. Our results suggest that PVA chains might partially detach from the NP surface at the water/Miglyol interface, and consequently favor the adsorption of the NPs at the interface. Once partially detached, these PVA chains or segments could adsorb at the oil/water interface in a more extended conformation and lower the interfacial tension. [START_REF] Isa | Core-shell Nanoparticle Monolayers at Planar Liquidliquid Interfaces: Effects of Polymer Architecture on the Interface Microstructure[END_REF] PVA chains at the NP surface and directly in contact with Miglyol would contract. Indeed, the radius of gyration of a PVA coil (≈ 10 nm in water as seen previously) was ≈ 2 nm in Miglyol (in a poor solvent, a compact conformation of the coil is obtained and ). This value is below the average distance between PVA chains at the particle suface (≈ 5 nm) calculated previously. The thickeness of the corona in contact with Miglyol should thus be in the order of magnitude of .

Besides, due to the contraction of the PVA chains at the particle surface when in contact with Miglyol, PLGA segments which are more hydrophobic were more exposed towards the oil phase.

This would also explain why PVA did not affect the contact angle with Miglyol, whereas it did affect the contact angle with water (see section 3.1. Table 2). Figure 5d displays the asymmetrical conformation of one PLGA-PVA NP at the oil/water interface. Such asymmetrical conformation is similar to the one of core-shell NPs obtained with grafted polymers at liquid-liquid interfaces. [START_REF] Isa | Adsorption of Core-Shell Nanoparticles at Liquid-liquid Interfaces[END_REF] Thus, PVA and, to a lesser extent, trehalose contributed to the differences in interfacial tension observed between lyophilized PLGA-PVA NPs and PLGA NPs.

Interfacial rheology

The elastic and viscous moduli of the different systems described above are shown in NPs. This is also very different from oil/water interfaces covered with proteins, 56 e.g. βlactoglubulin (a small globular protein), which are very elastic (|E * | and E' ≈ 30 mN/m at equilibrium for β-lactoglubulin covering a Miglyol/water interface). [START_REF] Ali | Effect of High Pressure Homogenization on the Structure and the Interfacial and Emulsifying Properties of β-Lactoglobulin[END_REF] Nelson et al. reported that even an interface that is highly covered by NPs could remain fluid. [START_REF] Nelson | A Multiscale Approach to the Adsorption of Core-shell Nanoparticles at Fluid Interfaces[END_REF] Thus, mobility of the NPs at the interface could explain that the NPs had no influence on the interfacial rheological properties.

This mobility at the interface is governed by the interfacial diffusion coefficient. Rearrangement timescales for a displacement of one NP diameter were estimated between 10 -2 to 50 s, depending on interfacial diffusion coefficients (whose values could drop to 30% to 0.01% of their bulk values in oil or water). [START_REF] Nelson | A Multiscale Approach to the Adsorption of Core-shell Nanoparticles at Fluid Interfaces[END_REF] The rearrangement timescales could be compared to the droplet oscillation period of 60 s. The absence of NP influence on interfacial rheological properties in our systems suggested a quick interfacial rearrangement, and thus a limited decrease of the interfacial diffusion coefficient compared to the bulk diffusion coefficient. Fluidity of the interface could possibly be attributed to the presence of a polymer shell surrounding the NPs, [START_REF] Nelson | A Multiscale Approach to the Adsorption of Core-shell Nanoparticles at Fluid Interfaces[END_REF] but, in our study, such mechanical properties also occured for bare PLGA NP without polymer shell. Indeed, because of their surface charge, PLGA NPs could act as electrostatic dipoles at the interface. [START_REF] Dani | Hydrodynamics of Particles at an Oil-Water Interface[END_REF] Resulting electrostatic repulsions between NPs might prevent dynamic arrest and ensure NP mobility in absence of a steric stabilizer.

NP desorption phenomena could also explain the occurrence of a fluid interface, and such observations were reported in interface compression experiments. Though NP desorption is seemingly in contradiction with the principle of irreversibility of adsorption of the NPs at the interface, [START_REF] Binks | Interfacial Structure of Solid-Stabilised Emulsions Studied by Scanning Electron Microscopy[END_REF][START_REF] Binks | Colloidal Particles at Liquid Interfaces[END_REF][START_REF] Pieranski | Two-Dimensional Interfacial Colloidal Crystals[END_REF] a substantial compression (e.g., ≈ 35% decrease of the droplet area) [START_REF] Garbin | Forced Desorption of Nanoparticles from an Oil-Water Interface[END_REF] could bring enough energy to force NPs to desorb from the interface. [START_REF] Garbin | Nanoparticles at Fluid Interfaces: Exploiting Capping Ligands to Control Adsorption, Stability and Dynamics[END_REF][START_REF] Nelson | A Multiscale Approach to the Adsorption of Core-shell Nanoparticles at Fluid Interfaces[END_REF][START_REF] Garbin | Forced Desorption of Nanoparticles from an Oil-Water Interface[END_REF][START_REF] Zell | Adsorption Energies of Poly(ethylene Oxide)-Based Surfactants and Nanoparticles on an Air-Water Surface[END_REF] In our case, viscoelasticity was assessed through oscillatory variations of the droplet volume, i.e., through a series of sinusoidal expansion and compression of the droplet area. However, droplet area variations were small (± 10%) and the energy necessary for desorption was high (Supporting Information 10).

Moreover, surface pressure vs droplet area during oscillations were reversible through expansion/compression cycles and did not reach any plateau, invalidating the NP desorption hypothesis.

Conclusion

Pickering emulsions using biodegradable and biocompatible components were successfully

Figure 1 .

 1 Figure 1. Images at D7 of emulsions containing 10% (w/w) of Miglyol and a) 90% (w/w) of

PFigure 2 .

 2 Figure 2. a) Scheme summarizing the evolution of the emulsion layer as determined by

Figure 3 :

 3 Figure 3: a) and b) Confocal microscopy images of emulsions (green channel: aqueous phase;

  A -P V A N P S tr e h a lo s e n o ly o p h 2 m g /m L ly o p h iliz e d P L G A -P V A N P s W a te r P L G A N P s P L G A N P s + P V A n o n -ly o p h iliz e d P L G A -P V A N P s T r e h a lo s e 1 5 0 m g /m L n o n -ly o p h iliz e d P L G A -P V A N P s + tre h a lo s e

Figure 4 .

 4 Figure 4. a) Interfacial tensions γ b) interfacial elastic moduli E' and c) interfacial viscous

  When bare PLGA NPs and free PVA are mixed in the same solution, free PVA chains adsorb more rapidly than PLGA NPs and lower the interfacial tension. Indeed, Stokes-Einstein diffusion coefficients D for PVA chains in water and for non-lyophilized PLGA-PVA NPs could be estimated from PVA radius of gyration in water ( ~ 10 nm; see section 3.1) and from the NP radius (see details in Supporting Information 10). For PVA in water, D = 3.7 × 10 -11 m 2 /s and for bare PLGA NPs, D = 2.5 × 10 -12 m 2 /s. Nevertheless, the interfacial tension decrease was more limited compared to the PVA solution at 2 mg/mL. A lower amount of PVA chains was able to adsorb at the oil/water interface in presence of PLGA NPs than when only PVA chains were present in the aqueous phase, since adsorbed PLGA NPs limited the adsorption or reconfiguration of PVA chains at the interface (see the schematic depictions of Fig 5a and 5c).

Figure 5 .

 5 Figure 5. Schematic depiction of the interface between Miglyol and aqueous phase containing a)

Figure

  Figure 4b and 4c. Regardless of the type of NPs adsorbed (PLGA NPs, PLGA-PVA NPs with or

  prepared using Miglyol as the oil, and bare PLGA NPs as stabilizers. They were compared with emulsions stabilized with PLGA-PVA NPs. Emulsions prepared with bare PLGA NPs displayed a better stability over 55 days. Interestingly, these emulsions exhibited very different macroscopic structures in terms of emulsion layer thickness and NP excess in the lower aqueous phase. They also displayed very different microscopic structures. Surprisingly, the co-existence of multiple W/O/W droplets and simple O/W droplets was obtained with bare PLGA NPs after a single step of emulsification, whereas PLGA-PVA NPs led to simple O/W emulsions. Not only PVA chains led to a more hydrophilic surface as demonstrated by contact angle measurements, but they also lowered the interfacial tension. It is likely that PVA chains could partially desorb from the NPs to adsorb at the oil/water interface. This emphasizes the key role of the sterically stabilizing polymer present at the NP surface in the stabilization mechanism of Pickering emulsions. However, the rheological behavior of the interface was unaffected by the presence of NPs or their composition. Although further analysis is needed, this rheological behavior could be explained by the ability of NPs to move and quickly reorganize at the interface upon dilatation and compression. Moreover, the determination of the three-phase contact angle performed at oil/water interface on individual NPs (e.g. with FreSCA cryo-SEM)[START_REF] Isa | Measuring Single-Nanoparticle Wetting Properties by Freeze-Fracture Shadow-Casting Cryo-Scanning Electron Microscopy[END_REF] could also provide valuable information to further explain the microstructure of the emulsions. Finally, this system offers the possibility to control the structure of an emulsion by the nature of the NPs that stabilize it. Such Pickering emulsions using PLGA NPs appear as very promising systems for pharmaceutical and cosmetic applications and could be extended to other biocompatible oils.ASSOCIATED CONTENTSupportingInformation 1: NP preparation Supporting Information 2: Amount of PVA adsorbed on the surface of the NPs Supporting Information 3: Characterization of PLGA-PVA NP surface Supporting Information 4: AFM imaging of the layers of NPs Supporting Information 5: NP hydrophobicity measurements Supporting Information 6: Examples of Turbiscan curves obtained for the emulsion stabilized by lyophilized PLGA-PVA NPs

Table 1 .

 1 Physico-chemical properties of NPs.

		Hydrodynamic diameter (nm)	PdI	Zeta potential (mV)
	PLGA	170 ± 5	0.04	-35 ± 2
	PLGA-PVA	215 ± 10	0.06	-4 ± 1
	lyophilized PLGA-PVA	220 ± 15	0.08	-4 ± 1

Table 2 .

 2 Contact angles measured on a NP colloidal layer on a mica substrate at t = 0.4 s (number of independent measurements: n = 10 at least).

	layer	liquid	Water	Miglyol
	Without NPs		< 0.2	38 ± 6
	PLGA NPs		120 ± 5	18 ± 2
	PLGA-PVA NPs	82 ± 7	17 ± 2

ABBREVIATIONS AFM, atomic force microscope; CAC, critical aggregation concentration; D, day or days; |E*|, absolute value of the complex dilatational modulus; E', interfacial elastic modulus; E'', interfacial viscous modulus; Miglyol, Miglyol 812 N; NPs, nanoparticles; O/W, oil in water; PdI, Polydispersity Index; PLGA: poly(lactic-co-glycolic) acid; PVA, poly(vinyl alcohol); Rhod, rhodamine; W/O, water in oil; W/O/W, water in oil in water

Authors are grateful to Dr Bertrand Fournier for his help on computer solving of adsorption energy equations and to reviewers of the manuscript for constructive comments that have helped improving the quality of the article.

Table of Contents Graphic


Jean-Nicolas Dumez

Basile Vuichoud

Daniele Mammoli

Aurélien Bornet

Arthur C Pinon

Gabriele Stevanato

Benno Meier

Geoffrey Bodenhausen

Sami Jannin

Malcolm H Levitt

Dynamic Nuclear Polarization of Long-Lived Nuclear Spin States in Methyl Groups

2

We have induced hyperpolarized long-lived states in compounds containing [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C-bearing methyl groups by dynamic nuclear polarization (DNP) at cryogenic temperatures, followed by dissolution with a warm solvent. The hyperpolarized methyl long-lived states give rise to enhanced antiphase [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C NMR signals in solution, which often persist for times much longer than the [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C and 1 H spin-lattice relaxation times under the same conditions. The DNP-induced effects are similar to quantum-rotor-induced-polarization (QRIP), but are observed in a wider range of compounds, since they do not depend critically on the height of the rotational barrier. We interpret our observations with a model in which nuclear-Zeeman and methyl-tunnelling reservoirs adopt an approximately uniform temperature, under DNP conditions. The generation of hyperpolarized NMR signals which persist for relatively long times in a range of methylbearing substances may be important for applications such as investigations of metabolism, enzymatic reactions, protein-ligand binding, drug screening, and molecular imaging.

TOC GRAPHICS

Nuclear magnetic resonance (NMR) is a highly versatile and informative analytical technique, with applications ranging from anatomic imaging to atomic-scale analysis of molecular structure and dynamics. The main limitation of NMR is arguably its low sensitivity, which originates in the small population differences between spin energy levels in thermal equilibrium, at even the highest static magnetic fields available. A range of hyperpolarization methods is available for increasing the NMR sensitivity by boosting the polarization. In solution, large enhancements are generated by Dissolution Dynamic Nuclear Polarization (d-DNP) and Parahydrogen Induced Polarization (PHIP). [START_REF] Ardenkjaer-Larsen | Increase in Signal-to-Noise Ratio of > 10,000 Times in Liquid-State Nmr[END_REF][START_REF] Adams | Reversible Interactions with Para-Hydrogen Enhance Nmr Sensitivity by Polarization Transfer[END_REF][START_REF] Bowers | Para-Hydrogen and Synthesis Allow Dramatically Enhanced Nuclear Alignment[END_REF] The main drawback of these methods is that once generated, the hyperpolarization decays on a timescale governed by the longitudinal relaxation time, T 1 , which is an irreversible process.

Long-lived nuclear spin states (LLS) offer promise for alleviating this limitation. LLS are defined as nuclear spin configurations that relax much more slowly than longitudinal magnetization. [START_REF] Carravetta | Beyond the T-1 Limit: Singlet Nuclear Spin States in Low Magnetic Fields[END_REF][START_REF] Carravetta | Long-Lived Nuclear Spin States in High-Field Solution Nmr[END_REF][START_REF] Levitt | Singlet Nuclear Magnetic Resonance[END_REF] LLS exist for groups of two spins or more, when the effects of fluctuating spin interactions are cancelled or reduced for symmetry reasons. The prototypical long-lived state, also known as singlet order, is the population difference between the singlet and triplet manifolds in an ensemble of spin-1/2 pairs. Potential applications of the LLS concept include drug screening, molecular imaging and reaction monitoring. [START_REF] Pileio | Recycling and Imaging of Nuclear Singlet Hyperpolarization[END_REF][START_REF] Bornet | Long-Lived States of Magnetically Equivalent Spins Populated by Dissolution-Dnp and Revealed by Enzymatic Reactions[END_REF][START_REF] Buratto | Drug Screening Boosted by Hyperpolarized Long-Lived States in Nmr[END_REF] LLS have been used as a carrier of hyperpolarisation. [START_REF] Vasos | Long-Lived States to Sustain Hyperpolarized Magnetization[END_REF][START_REF] Warren | Increasing Hyperpolarized Spin Lifetimes through True Singlet Eigenstates[END_REF][START_REF] Franzoni | Long-Lived 1h Singlet Spin States Originating from Para-Hydrogen in Cs-Symmetric Molecules Stored for Minutes in High Magnetic Fields[END_REF][START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF][START_REF] Theis | Direct and Cost-Efficient Hyperpolarization of Long-Lived Nuclear Spin States on Universal N-15(2)-Diazirine Molecular Tags[END_REF][START_REF] Eills | Singlet Order Conversion and Parahydrogen-Induced Hyperpolarization of C-13 Nuclei in near-Equivalent Spin Systems[END_REF] Molecular scaffolds have been designed to minimise the relaxation rates of singlet order, with reported lifetimes of over an hour. [START_REF] Stevanato | A Nuclear Singlet Lifetime of More Than One Hour in Room-Temperature Solution[END_REF] Such record lifetimes, however, concern chemical motifs that are not commonly found.

Long-lived nuclear spin states are also known to exist in methyl, CH 3 groups, [START_REF] Dumez | Theory of Long-Lived Nuclear Spin States in Methyl Groups and Quantum-Rotor Induced Polarisation[END_REF][START_REF] Meier | Long-Lived Nuclear Spin States in Methyl Groups and Quantum-Rotor-Induced Polarization[END_REF] which are ubiquitous in organic chemistry and biochemistry. Methyl LLS correspond to a population imbalance between manifolds of states of different spin permutation symmetry, conventionally Page 3 of 25

ACS Paragon Plus Environment

The Journal of Physical Chemistry Letters denoted as A and E manifolds; we refer to the relevant population difference as an A/E imbalance (AEI). Such a population imbalance is protected from dominant relaxation mechanisms by rapid methyl rotation, leading to long lifetimes in solution.

Methyl LLS are implicated in the phenomenon of quantum-rotor-induced polarization (QRIP), in which strong antiphase [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C signals are observed when certain compounds are cooled to cryogenic temperatures, rapidly dissolved in a hot solvent, and observed by solution NMR at room temperature. [START_REF] Icker | Unexpected Multiplet Patterns Induced by the Haupt-Effect[END_REF][START_REF] Icker | Experimental Boundaries of the Quantum Rotor Induced Polarization (Qrip) in Liquid State Nmr[END_REF] QRIP has been observed for compounds such as γ-picoline (4methylpyridine) for which the methyl rotation encounters an unusually low rotational barrier, leading to a significant tunnelling splitting of ~ 6 K between the A and E manifolds in the cryogenic solid state. For such special cases, a significant AEI may be established simply by equilibrating the sample at a temperature below 10 K, without any paramagnetic agents or microwaves. This population imbalance builds up within tens of minutes to a value that is significantly larger than the Zeeman polarization at room temperature. The AEI is maintained to a significant degree through the dissolution process, leading to the generation of a hyperpolarized methyl LLS in the room temperature solution, and hence enhanced antiphase [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C signals through cross-relaxation. [START_REF] Dumez | Theory of Long-Lived Nuclear Spin States in Methyl Groups and Quantum-Rotor Induced Polarisation[END_REF] In this paper, we show that the requirement of low rotational barriers (or, equivalently, large tunnelling splittings) required for QRIP at liquid-helium temperature may be alleviated by using dynamic nuclear polarization (DNP). This opens up the practical use of methyl AEI and in turn long-lived states to arbitrary methyl-bearing molecules. We report observations of this effect for compounds containing methyl groups in several molecular environments. The interplay between quantum-rotor and DNP effects is illustrated by simple energy-level diagrams. Consider a [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C-bearing methyl group in a solid. The A 3 X spin system consists of three protons and one carbon-13, with 2 4 = 16 energy levels. Provided that the three protons are magnetically equivalent, i.e., the rapid methyl rotation averages the chemical shift anisotropy of the three protons and their dipolar couplings with external spins, the energy eigenstates are given by symmetry-adapted combinations of these energy levels, which transform as irreducible representations of the group C3(M), with 8 combinations of symmetry A, and 8 of symmetry E (see ref. [START_REF] Dumez | Theory of Long-Lived Nuclear Spin States in Methyl Groups and Quantum-Rotor Induced Polarisation[END_REF] for a detailed description). In general, the A and E levels are split by the 1 H and [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C Zeeman splittings, while the E levels are raised in energy with respect to the A levels by the tunnelling splitting. This splitting is associated with the overlap of the spatial wave-functions of the methyl protons, and depends strongly on the methyl rotational barrier. [START_REF] Horsewill | Quantum Tunnelling Aspects of Methyl Group Rotation Studied by Nmr[END_REF] Two extreme cases may be identified: in the case of a large rotational barrier, the tunnelling splitting is very small, and the energy level structure is dominated by the Zeeman splittings (Fig. 1a-b). In the case of a small rotational barrier, on the other hand, the tunnelling splitting may be much larger than the Zeeman splitting (Fig. 1c-d). In extreme cases such as 4-methylpyridine (γ-picoline), the tunnelling splitting is ~126 GHz, which is more than 2 orders of magnitude larger than the 1 H nuclear Zeeman splitting in accessible magnetic fields.

In the case of a very large tunnelling splitting (Fig. 1c-d), a large and positive AEI may be established by allowing the sample to reach thermal equilibrium at a temperature of a few Kelvin by cooling with liquid He. This resulting positive AEI is substantially maintained through the dissolution process, leading to a hyperpolarized methyl LLS that gives rise to enhanced hyperpolarized [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C signals through cross-relaxation. This is the origin of QRIP. [START_REF] Dumez | Theory of Long-Lived Nuclear Spin States in Methyl Groups and Quantum-Rotor Induced Polarisation[END_REF][START_REF] Meier | Long-Lived Nuclear Spin States in Methyl Groups and Quantum-Rotor-Induced Polarization[END_REF][START_REF] Icker | Unexpected Multiplet Patterns Induced by the Haupt-Effect[END_REF][START_REF] Icker | Experimental Boundaries of the Quantum Rotor Induced Polarization (Qrip) in Liquid State Nmr[END_REF] However, in the case of a high rotational barrier and a small tunnelling splitting (Fig. 1a-b), cooling by liquid He leads to a small or absent AEI. Conventional QRIP experiments fail for such systems unless the sample temperature is lowered well below 1 K, where equilibration of the sample may take prohibitively long times.

DNP may be used to establish Zeeman population imbalances of arbitrary signs and far from thermal equilibrium values. [START_REF] Wenckebach | Essentials of Dynamic Nuclear Polarization[END_REF] This has been used to generate hyperpolarized long-lived states in pairs of spins with I = ½. [START_REF] Bornet | Long-Lived States of Magnetically Equivalent Spins Populated by Dissolution-Dnp and Revealed by Enzymatic Reactions[END_REF][START_REF] Tayler | Direct Enhancement of Nuclear Singlet Order by Dynamic Nuclear Polarization[END_REF][START_REF] Mammoli | Hyperpolarized Para-Ethanol[END_REF] Similarly, in the case of methyl groups, we show that DNP can give rise to significant AEI, by preferentially populating one manifold with respect to the other, even when these groups are not shifted with respect to each other by a large tunnelling splitting.

As in the QRIP effect, the AEI in the solid state translates into a hyperpolarized LLS in solution, which in turn gives rise to hyperpolarized antiphase [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C signals in solution. Related effects have been observed for materials containing CD 3 groups. [START_REF] Jhajharia | Communication: Dissolution Dnp Reveals a Long-Lived Deuterium Spin State Imbalance in Methyl Groups[END_REF] Fig. 2 Experimental procedure for the dissolution-NMR experiments. After polarization in the solid-state, the sample is dissolved and transferred to the high-resolution liquid-state NMR system; the evolution of the magnetic field and the temperature is shown in a. For the acquisition, in b, the following sequence of event is looped: T 00 filter, build-up delay T B , 90° pulse, acquisition. In c, the T 00 filter is looped N filter times, and a series of spectra is acquired with The experimental procedure used to demonstrate this effect is sketched in Fig. 2. The species of interest are prepared in a glass-forming solvent (100 µL of D 2 O:glycerol-d 8 1:1 with 50 mM TEMPOL), cooled to a temperature of ~1.2 K in a field of 6.7 T and irradiated with frequencymodulated microwaves, [START_REF] Jannin | High Field Dynamic Nuclear Polarization at 6.7 T: Carbon-13 Polarization above 70% within 20[END_REF][START_REF] Bornet | Microwave Frequency Modulation to Enhance Dissolution Dynamic Nuclear Polarization[END_REF][START_REF] Bornet | Optimizing Dissolution Dynamic Nuclear Polarization[END_REF] with a frequency slightly displaced from the centre of the electron spin resonance line. The polarization and the AEI were allowed to build up for 20 minutes. The Zeeman polarization was found to be P( 1 H) ~50% for positive DNP (microwave frequency at f µwaves = 187.8 GHz, below the centre of the electron spin resonance line) and P( 1 H) ~ -50% for negative DNP (microwave frequency at f µwaves = 188.3 GHz, above the centre of the electron spin resonance line).

Once the polarization and AEI are established, the dissolution-NMR experiment is carried out.

The DNP sample is i) dissolved with D 2 O (5 mL heated to ca. 420 K at a pressure of 1 MPa) in 700 ms, then ii) pushed in 4.5 s with a pressure of 0.6 MPa He gas to a home-built injector in a 11.7 T magnet, and iii) finally injected in ca. 2 s in a 5-mm sample tube (complete sequence 7.2 s). The TEMPOL concentration in the final sample is 1 mM. The AEI is NMR silent, but "bleeds" by cross-relaxation into spin-state population differences that give rise to an antiphase 13 C multiplet upon application of a radiofrequency pulse on the [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C channel [START_REF] Dumez | Theory of Long-Lived Nuclear Spin States in Methyl Groups and Quantum-Rotor Induced Polarisation[END_REF][START_REF] Meier | Long-Lived Nuclear Spin States in Methyl Groups and Quantum-Rotor-Induced Polarization[END_REF] . Fig. 2b shows the pulse sequence used to identify methyl LLS and measure relaxation rates. After injection of the sample in the high-resolution NMR system, a series of [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C spectra is obtained, with a repetition time of 5 s. Before each acquisition, a combination of RF pulses and gradients known as "T 00 filter" is used to suppress signals arising from any components of the spin density matrix other than the AEI (the notation "T 00 " filter reflects the suppression of density operator terms that do not transform as spherical tensor operators of rank 0 and component index 0) [START_REF] Tayler | Accessing Long-Lived Nuclear Spin Order by Isotope-Induced Symmetry Breaking[END_REF] . The spin system is then left to evolve for a relaxation delay, during which the AEI converts partially to population differences across observable transitions. A strong, non-selective 90° excitation pulse is applied on the [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C channel before acquisition.

In the case of 2-13 C-acetate, 3-13 C-pyruvate and 13 CH 3 -methionine, this procedure leads to an enhanced antiphase [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C multiplet, as shown in Fig. 3a-c, indicating that a significant AEI is generated by dynamic nuclear polarization; no such signal is observed for 3-13 C-alanine (not shown). In the case of 2-13 C-acetate, small antiphase signals are also observed when no microwave irradiation is applied under cryogenic conditions (Fig. 3g); no such signals are observed for the three other compounds (Fig. 3h-i). Changing the microwave irradiation frequency to the opposite side of the electron spin resonance line inverts the sign of the antiphase 13 C signals for 2-13 C-acetate, but not for [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] CH 3 -methionine and 3-13 C-pyruvate (Fig. 3d-f). Fig. 3 13 C 1D spectra of the methyl signal of 2-13 C-acetate (left), 3-13 C-pyruvate (middle) and Me- [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C-methionine (right) at 11.7 T and 298 K. Spectra a to i are obtained using the scheme shown in Fig. 2b. For Me- [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C-methionine, the first spectrum is shown. For 2-13 C-acetate, the 10th spectrum is shown; for 3-13 C-pyruvate, the 4th spectrum is shown; earlier spectra are contaminated by residual magnetization. The polarization step in the solid state used either positive DNP (a-c), negative DNP (d-f), or no DNP (g-i). Thermal equilibrium spectra acquired with 4 scans are shown in j-l. The vertical scale is independent for each molecule but is consistent for all the spectra of a given molecule.

A full explanation of these observations is beyond the scope of this preliminary report.

Nevertheless, the salient features may be rationalised by considering Boltzmann population distributions within the energy level diagrams of Fig 1, assuming for simplicity that DNP establishes a common temperature across the entire manifold of nuclear Zeeman and tunnelling quantum levels. For brevity, we refer to this common temperature as "spin temperature", bearing in mind that the associated quantum system involves tunnelling energy as well as Zeeman energy. This assumption is plausible in the current case, since prior observations have shown that the thermal contact between the Zeeman system and the lattice, and the tunnelling system and the lattice, are both very weak at cryogenic temperatures, relative to the Zeemantunnelling contact. [START_REF] Horsewill | Quantum Tunnelling Aspects of Methyl Group Rotation Studied by Nmr[END_REF] Nevertheless, the interpretation given here is merely qualitative, and encounters obvious problems in some cases, for example when negative temperatures are invoked. Although a negative temperature is a valid concept for finite spin systems, which have a finite set of quantum levels, the concept of negative temperature is inherently flawed for tunnelling levels, which are unbounded at high energy. [START_REF] Abragam | Nuclear Magnetism: Order and Disorder[END_REF] A fuller understanding will require a more detailed analysis of the quantum dynamics, supported by further experiments, as is the case for conventional DNP processes. [START_REF] Hovav | Theoretical Aspects of Dynamic Nuclear Polarization in the Solid State -the Solid Effect[END_REF][START_REF] Hovav | Theoretical Aspects of Dynamic Nuclear Polarization in the Solid State -the Cross Effect[END_REF][START_REF] Hovav | Theoretical Aspects of Dynamic Nuclear Polarization in the Solid State -Spin Temperature and Thermal Mixing[END_REF][START_REF] Hovav | Theoretical Aspects of Dynamic Nuclear Polarization in the Solid State: The Influence of High Radical Concentrations on the Solid Effect and Cross Effect Mechanisms[END_REF][START_REF] Shimon | Dynamic Nuclear Polarization in the Solid State: A Transition between the Cross Effect and the Solid Effect[END_REF][START_REF] Caracciolo | Evidence of Spin-Temperature in Dynamic Nuclear Polarization: An Exact Computation of the Epr Spectrum[END_REF][START_REF] Serra | Relevance of Electron Spin Dissipative Processes to Dynamic Nuclear Polarization Via Thermal Mixing[END_REF] There is extensive prior literature on the interaction of electron spins and methyl tunnelling splittings. [START_REF] Clough | Dynamic Nuclear Polarization by the Cooling of Tunnelling Methyl Groups[END_REF][START_REF] Clough | Tunnelling Resonances in Proton Spin-Lattice Relaxation[END_REF][START_REF] Freed | Quantum Effects of Methyl-Group Rotations in Magnetic Resonance -Esr Splittings and Linewidths[END_REF][START_REF] Horsewill | Dynamic Tunneling Polarization as a Quantum Rotor Analogue of Dynamic Nuclear Polarization and the Nmr Solid Effect[END_REF] Page 10 of 25

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Hence, either a very low positive spin temperature (in which only the lowest energy level is significantly populated, Fig. 1a) or a very low negative spin temperature (so that only the highest energy level is significantly populated, Fig. 1b) both give rise to excess population in the A manifold, compared to the E manifold. We therefore expect that in such systems the sign of the LLS after dissolution, and hence that of the antiphase [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C signals, is independent of the sign of DNP. As shown in Fig. 3c and3f, this is observed for 13 CH 3 -methionine and in 3b and 3e for 3-13 C-pyruvate.

Different behaviour is anticipated when the tunnelling splitting is large compared to the Zeeman splitting (Fig. 1c-d). In this case, the lowest energy level belongs to the A manifold, while the highest energy level belongs to the E manifold. Hence, very low positive or negative spin temperatures are expected to give rise to A/E population imbalances of opposite sign. We therefore expect that in such systems, a change in sign of DNP changes the sign of the LLS after dissolution, and hence that of the antiphase [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C signals. As shown in Fig. 3a and3d this is observed for 2-13 C-acetate. The reduced intensity of the observed signals may be associated with a breakdown of the spin temperature hypothesis for negative DNP involving unbounded tunnelling levels, as discussed above. The existence of a relatively large tunnelling splitting for 2-13 C-acetate is consistent with the observation of a small QRIP effect in the absence of microwave irradiation (Fig. 3g). The relaxation rate constant of the LLS after dissolution to the liquid state may be obtained from 1D [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C spectra obtained with the pulse sequence shown in Fig. 2b. Experimental spectra of 2-13 C-acetate are shown in Fig. 4a. Note the long persistence of these enhanced signals, with relaxation times in the range of T LLS = 50 s, relative to the Zeeman relaxation times for 2-13 Cacetate, which are T 1 ( 13 C) = 13.5 s and T 1 ( 1 H) = 5.5 s. Table 1 summarises the relaxation time constants of the LLS, together with longitudinal relaxation time constants for 1 H and [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C magnetization. In all cases where antiphase multiplets were detected, T LLS is found to be longer than T 1 ( 1 H). For 3-13 C-pyruvate and 13 CH 3 -methionine, however, the relaxation time T LLS of the A/E imbalance is found to be comparable to T 1 ( 13 C). The AEI is nevertheless long lived, since it involves [START_REF] Ardenkjaer-Larsen | Increase in Signal-to-Noise Ratio of > 10,000 Times in Liquid-State Nmr[END_REF] H spin order that relaxes more slowly than 1 H magnetization, but it does not extend the timescales that may be probed with [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C magnetization. On the other hand, for acetate T LLS is found to be larger than both T 1 ( 1 H) and T 1 ( 13 C). The long lifetime of the LLS for 2-13 C-acetate in solution may be explained by the low rotational barrier for the methyl group in this compound, which gives rise to a short correlation time for methyl rotation in solution [START_REF] Dumez | Theory of Long-Lived Nuclear Spin States in Methyl Groups and Quantum-Rotor Induced Polarisation[END_REF][START_REF] Meier | Long-Lived Nuclear Spin States in Methyl Groups and Quantum-Rotor-Induced Polarization[END_REF] . At this point, it is not known whether the radical content of the dissolved solution significantly influences the decay of the methyl long-lived states. Paramagnetic effects on the relaxation of 2-spin long-lived states are generally weaker than for conventional magnetization. [START_REF] Tayler | Paramagnetic Relaxation of Nuclear Singlet States[END_REF] The absence of any hyperpolarized antiphase [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C multiplet in our dissolution-NMR experiments on 3-13 C-alanine may be due to the fact that the methyl group is bound to an sp 3 carbon, a configuration which is known to result in a large rotational barrier. [START_REF] Prager | Rotational Tunneling and Neutron Spectroscopy: A Compilation[END_REF] Strong hindering of methyl rotation may destroy the long-lived polarization effects, for at least two main reasons.

Table 1 Relaxation time constants (in

Firstly, the symmetry-adapted basis states, with their A and E symmetry labels, may not be accurate energy eigenstates for frozen methyl groups in the solid state. It is therefore debatable as to whether the AEI is established in the solid state in this case; Secondly, as shown by theory 17- 18 , a large value for the rotational correlation time τ R of the methyl group is associated with a Page 13 of 25

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The Journal of Physical Chemistry Letters short AEI decay time constant in solution. Hence, in 3-13 C-alanine, the methyl AEI, even if it is generated by DNP in the solid state, may be too short-lived to survive the dissolution, transfer and injection process. Further experiments are needed to resolve these issues.

Fig. 5 Intensities of the components of the hyperpolarized antiphase signal in a dissolution-NMR experiment on 2-13 C-acetate at 11.7 T and 298 K, with positive DNP. The acquisition was performed using the pulse sequence in Fig. 2c. The time axis starts with the last T 00 filter.

Figure 5 shows the build-up and decay of the four antiphase components of the [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C multiplet of 2-13 C-acetate, monitored with a series of experiments with small-tip-angle pulses, using a train of T 00 filters after dissolution (see Fig. 2c). The build-up occurs on a time scale that is comparable with T 1 ( 1 H). These trajectories are similar to those obtained in the QRIP observations on γ-picoline, which have been thoroughly analysed in terms of 1 H- [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C dipolar and CSA cross-relaxation effects for the case of a rapidly rotating methyl group. 17 The enhancements of the anti-phase signals shown in Fig. 3a-c are reported in Table 2. The enhancements are significantly smaller than those achieved by direct polarization in dissolution-DNP experiments. This is mainly because observable population differences across the [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C transitions are only progressively released from the LLS by cross-relaxation processes. This may be seen either as a limitation (smaller enhancements) or as an advantage (repeated observations) of methyl LLS as carriers of hyperpolarized spin order. The enhanced Zeeman magnetization generated in conventional dissolution DNP experiments is completely destroyed when a single 90° pulse is applied to generate observable NMR signals. This is not the case for the experiments described here, since the observation pulses do not influence the methyl LLS, allowing repeated observations by "harvesting" the LLS in small fractions. Table 2 also shows figures for the enhancements obtained when the entire time series of signals is integrated. This cumulative enhancement provides qualitative information on the degree of hyperpolarization stored in and released by the A/E imbalance (note that if N consecutive spectra in the time series were summed, the increase in signal to noise ratio (SNR) would be given by the cumulative enhancement divided by √ܰ. Since the antiphase magnetisation builds up on a time-scale comparable to T 1 , the SNR could be optimised with Ernst-Angle-type excitation [START_REF] Ernst | Principles of Nuclear Magnetic Resonance in One and Two Dimensions[END_REF] ).

In summary, we have demonstrated that long-lived nuclear spin state imbalances in methyl groups may be generated by dynamic nuclear polarization. The signs of the resulting hyperpolarized antiphase [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] 13 C offset (Hz) [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C offset (Hz) [START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C offset (Hz)

Fig. 1

 1 Fig. 1 Schematic energy-level diagram for a methyl group, at liquid-helium temperature, with a

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Fig. 4

 4 Fig.4 Selection of[START_REF] Roy | Long-Lived States to Sustain Sabre Hyperpolarised Magnetisation[END_REF] C 1D spectra of the methyl signal of 2-13 C-acetate from the time series

  C signals are explained by invoking the spin-temperature hypothesis, taking into account the distribution of energy levels. The relaxation time constants of methyl LLSs are found to be strongly dependent on the molecular environment, but are often longer than the 1 H or 13 C spin-lattice relaxation times constants. Methyl long-lived states may provide a new class of relaxation-based probes to characterize molecular dynamics, and serve as carriers of hyperpolarized spin order, facilitating applications to NMR investigations of biochemistry and metabolism, and in molecular imaging.

  e a f t e r d i s s o l u t i o n ( s )

Table 2

 2 DNP-induced enhancements in dissolution NMR experiments of methyl-13 C-molecules.

The enhancements are expressed as ratios of the area of n th component in the hyperpolarized quartet to that of the corresponding component in the thermal equilibrium spectrum. The

Page 14 of 25 ACS Paragon Plus Environment The Journal of Physical Chemistry Letters components

  of the multiplets are numbered from left to right. The enhancement for 1 scan is given for the spectrum shown in Fig.3a-c. The summed enhancements are given for 20, 4 and 10 consecutive scans for 2-13 C-acetate, Me-13 C-methionine and 3-13 C-pyruvate, respectively

		enhancements (for 1 scan)	summed enhancements
	substance	1	2	3	4	1	2	3	4
	2-13 C-acetate	62	-20	11	-47	570	-154	115	-410
	3-13 C -pyruvate	274	-110	48	-244	1020	-380	191	-867
	13 CH 3 -methionine	172	-119	21	-150	281	-179	44	-233

ACKNOWLEDGMENT

The authors thank Lyndon Emsley for making means available. The research leading to these results has received funding from COST (CA15209), Royal Society CNRS (grant code IE140525), EPSRC-UK (grant codes EP/N002482/1, EP/P009980/1), the European Research Council under the European Union's Horizon 2020 research and innovation programme (ERC Grant Agreement n 714519 / HP4all), the ERC grant "Dilute para-water", the EPFL, the Swiss National Science Foundation, and Bruker BioSpin SA Switzerland.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interests.

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Introduction

N-Heterocyclic carbenes (NHCs) are neutral ligands defined as singlet carbenes, in which the divalent carbenic center, possessing 6 valence electrons, is connected directly to at least one nitrogen atom within the heterocycle. [1] The ease of synthesis and versatility of NHCs have contributed to their recognition as ubiquitous ligands in organometallic chemistry as evidenced by their wide use ranging from homogeneous catalysis to material sciences. [2] More recently, several groups pointed out the importance of N-heterocyclic carbene ligands as new structures for the development of metallodrugs. [3,[START_REF] Kowalski | For general review on metallodrugs, see: (a)[END_REF] Indeed, the high stability of the corresponding complexes and the ease of derivatization of the NHC-ligands make them suitable candidates for drug development. [5] In addition to their antimicrobial properties, NHC complexes have been highlighted as promising agents for anticancer applications. For this purpose, several metals have been studied, especially gold, silver, copper, palladium and platinum, displaying antitumor activities through various mechanisms that differ from the cisplatin used in clinics. [START_REF] Hindi | For reviews on the medicinal properties of metal-NHC complexes[END_REF] In 2010, Marinetti and coworkers demonstrated the efficiency of trans-configured platinum complexes of general formula (NHC)PtX2(L) (with L = amine) against cancer cell lines. [3b] Cytotoxicity was investigated on leukemia cells and lung cancer cells and showed that most complexes exhibit similar or higher activities than cisplatin 7 against both cell lines.

Further investigations on cisplatin resistant cells from human ovarian cancers showed better activities than cisplatin and oxaliplatin. Altogether, these results demonstrate the potency of trans-Pt(II)-NHC complexes as anticancer agents. Indeed, despite the fact that trans-platinum complexes have for long time been overlooked due to the low activity observed with transplatin, [8] recent work in the area showed the ability of trans-Pt-NHC complexes to induce cytotoxic activities (even on cisplatin resistant cells), which may indicate a different mechanism of action than cisplatin.

Soon after, Marinetti [3b] and others [9,10,11] investigated the effect on cell activity of several Nheterocyclic carbene platinum complexes. These studies established that both the nature of the NHC backbone and the amine that is trans-coordinated to the carbene might influence the cytotoxic activity of the corresponding Pt complex. In the search for more potent species, we investigated the effect of the nature of the N-benzyl substituent onto the carbene, while keeping all substituents identical (i.e. iodide and pyridine ligands). Indeed, Tacke and collaborators already pointed out in early 2010 that benzyl-substitution on Ag, Au, Cu or Ru NHC complexes could positively influence their biological activities. [12] We report here that little change on the benzyl moiety (i.e. introducing p-NO2 or p-Br for example) can significantly enhance the cytotoxicity of the complexes.

Result and discussion

All imidazolium salts were prepared using standard and well-established synthetic procedures. 13 All platinum complexes were then prepared by reaction of the imidazolium salt in the presence of platinum dichloride, excess NaI and K2CO3 in dry pyridine at 100°C overnight, under nitrogen atmosphere (Scheme 1). The (NHC)PtI2(pyridine) complexes were purified by flash chromatography on silica with pentane/dichloromethane as eluent. Figure 1 displays the molecular structures of all complexes. The 4-position of the benzyl moiety was functionalized with either bromo, nitro or formyl substituents, leading to complexes 2, 3 and 4, respectively. The platinum complex 5 contains polyethylene glycol chains that may increase the solubility of the system in aqueous media. Complexes 6 and 7 were substituted with 9-methylanthracenyl or 1-methylpyrenyl moieties. Compound 8 is a trinuclear platinum complex whereas complexes 9 and 10 have identical substituents on both nitrogen atoms (i.e. 4-nitrobenzyl and 9-methylanthracenyl, respectively). Isolated yields are ranging from ca. 85-90 % (complexes 2, 3) to ca. 20 % (complexes 4, 5 and 8). The yield for Pt complex 4 is found surprisingly low (19 %). This result may be due to the presence of the benzaldehyde moiety that may react with the free carbene (benzoin-type reactivity). Starting from PtCl2(COD) as precursor, compound 4 was obtained in 80 % yield. The greater solubility of this platinum source may allow the quick trapping of the in situ formed carbene, thus preventing side reactions due to the presence of the aldehyde moiety.

Scheme 1. General synthesis of the platinum pyridine NHC complexes. The formation of the (NHC)Pt-pyridine complexes was confirmed by the typical appearance of a signal at δ 135-143 ppm in the 13 C NMR spectrum, which was assigned to the carbenic carbon, and also by the absence of the resonance peak from the 2H-imidazolium proton in the 1 H NMR spectrum. The molecular structures of compounds 3 and 7 were determined by Xray diffraction studies (Figures 2 and3). The platinum center displays a classical square planar geometry where the pyridine is located trans to the carbene. The NHC-platinum bond lengths are 1.974(3) Å and 1.969( 6) Å, whereas the metal-pyridine bond lengths are 2.089 Å in both cases. 3b,14 The platinum complexes were evaluated for their IC50 values on a panel of different cancer cell lines (MCF7, HCT116, PC3, OV3 and MRC5) and non-cancer cell line (EPC) and the results are enlisted in Table 1. Cisplatin was used as reference, and displayed significantly lower activity compared to all complexes, except for 5 and 10. [START_REF]Cisplatin was used as reference for the studies; however due to the instability of cisplatin in DMSO, the latter was used as stock solution in water[END_REF] The introduction of a bromo or formyl group in 4-position of the benzyl is not found to affect the overall activities of the compounds (compare compound 1 with 2 and 4). Surprisingly, the nitrobenzyl-containing complex 3 presented excellent cytotoxicity with IC50 value below 5 nM toward both HCT116 and MRC5 cancer cells. The presence of an anthracenyl or pyrenyl moiety also lead to promising results with IC50 ranging from 0.165 µM to 0.01 µM, whereas introduction of PEG chains onto the compound (complex 5) induced a significant drop of the activity. Trinuclear platinum complex 8 also displayed a moderate activity compared to mononuclear species. [START_REF] Chtchigrovsky | [END_REF] Finally, the bis-nitrobenzyl complex 9 was found less active than the mono-nitrobenzyl complex 3 and bis-anthracenyl derivative 10 was inactive, which may be due to its low solubility. The enhanced activities may be related to a synergistic effect since the most active complexes are combining NHC Pt complexes with a benzylic moiety that is able to intercalate into DNA base pairs. [17] Compound Table 1. Half inhibitory concentrations IC50 (in µM) of the compounds against human cancer and non-cancer cell lines (after 72h of incubation). [a]

Conclusion

In summary, a series of platinum complexes bearing N-heterocyclic carbene ligands functionalized by various benzyl moieties has been synthesized and characterized. The complexes show a high stability both in solid state and in solution. The compounds have demonstrated variable in vitro activities, which was found to be dependent on the substituents on the benzyl moiety. Nonetheless, they usually show cytotoxic activities significantly higher than that of cisplatin on 6 different cell lines. Low IC50 values up to 0.005 µM were obtained when a nitrobenzyl was introduced onto the NHC ligand. Further studies will focus on mechanistic investigations to gain more insights into the therapeutic potential of these compounds.

Figure 1 .

 1 Figure 1. Molecular structure of the platinum pyridine NHC complexes and complexation yield.

Figure 2 .

 2 Figure 2. Molecular structure of complex 3. Selected bond distances (Å) and angles (deg): C(1)-Pt(1), 1.974(3); Pt(1)-N(4), 2.089(3); Pt(1)-I(1), 2.5887(3); Pt(1)-I(2), 2.5979(3); N(2)-C(1)-N(1), 105.2(3); N(4)-Pt(1)-I(1), 89.70(7); N(4)-Pt(1)-I(2), 90.69(7); I(1)-Pt(1)-I(2), 178.436(9); C(1)-Pt(1)N(4), 178.85(12); C(12)-N(4)-C(1)-N(2), -43.0(3); I(1)-I(2)-C(1)-N(4), -1.9(4).

Figure 3 .

 3 Figure 3. Molecular structure of the Pt complex 7. Selected bond distances (Å) and angles (deg): C(1)-Pt(1), 1.969(6); Pt(1)-N(3), 2.089(5); Pt(1)-I(1), 2.5936(6); Pt(1)-I(2), 2.5863(6); N(1)-C(1)-N(2), 104.5(5); N(3)-Pt(1)-C(1), 177.8(3); I(1)-Pt(1)-I(2), 179.46(2); C(26)-N(3)-C(1)-N(1), 24.30(10); I(1)-I(2)-C(1)-N(3), 1.15(11).

Acknowledgments

The authors gratefully acknowledge the Ministère de l'Enseignement Supérieur et de la Recherche for Ph.D. grants to G. D. and M. B. This work was also supported by La Ligue contre le Cancer -Région Grand Est and University of Strasbourg/CNRS-Program IDEX Interdisciplinaire. Biological evaluations of cell proliferation inhibition have been performed at the Ciblothèque Cellulaire ICSN (Gif sur Yvette, France).