Mathilde Lorscheider

Nicolas Tsapis

Rosana Simón-Vázquez

Nicolas Guiblin

Noureddine Ghermani

Franceline Reynaud

Romain Canioni

Sonia Abreu

Pierre Chaminade

Elias Fattal

Rosana Simon-Vaquez

Nanoscale Lipophilic Prodrugs of Dexamethasone with Enhanced Pharmacokinetics

Keywords: Prodrug, Nanoparticle, Drug loading, Pharmacokinetics, Biodistribution

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

Table of Content Graphic 1 INTRODUCTION

Glucocorticoids (GCs) have a long history as anti-inflammatory and immunosuppressive drugs but their use is limited due to their side effects which become critical during long term therapy. [START_REF] Mcdonough | The Epidemiology of Glucocorticoid-Associated Adverse Events[END_REF][START_REF] Saag | Low Dose Long-Term Corticosteroid Therapy in Rheumatoid Arthritis: An Analysis of Serious Adverse Events[END_REF] To improve GCs pharmacokinetics and increase their concentrations in inflamed therapeutic sites, the use of PEGylated nanocarriers has been considered because of their prolonged circulation in the blood stream [START_REF] Allen | The Use of Glycolipids and Hydrophilic Polymers in Avoiding Rapid Uptake of Liposomes by the Mononuclear Phagocyte System[END_REF] and their passive diffusion through highly permeable vasculature of inflamed tissues. [START_REF] Metselaar | Complete Remission of Experimental Arthritis by Joint Targeting of Glucocorticoids with Long-Circulating Liposomes[END_REF] However, in the case of GCs and many other drugs, nanocarriers suffer from several drawbacks such as the low drug loading, the difficulty to entrap hydrophobic drugs [START_REF] Gómez-Gaete | Encapsulation of Dexamethasone into Biodegradable Polymeric Nanoparticles[END_REF][START_REF] Gómez-Gaete | Dexamethasone Acetate Encapsulation into Trojan Particles[END_REF] and the premature burst release [START_REF] Gómez-Gaete | Encapsulation of Dexamethasone into Biodegradable Polymeric Nanoparticles[END_REF][START_REF] Allen | Liposomal Drug Delivery Systems: From Concept to Clinical Applications[END_REF][START_REF] Allen | Serum-Induced Leakage of Liposome Contents[END_REF] that severely hinders their clinical translation. Indeed, most physically entrapped active molecules present a drug loading comprised between 0.1 and 10%. [START_REF] Barenholz | Relevancy of Drug Loading to Liposomal Formulation Therapeutic Efficacy[END_REF] This low proportion of active drug per nanocarrier directly leads to the administration of high quantities of potentially harmful excipients such as surfactants, stabilizers (poloxamers, poly(vinyl alcohol), tween 80…), lipids or polymers. Stability of the nanosuspension may also be an issue for hydrophobic drugs. Indeed, over time, the entrapped drug is susceptible to be released in the media and crystallization is often observed due to Ostwald ripening. [START_REF] Gómez-Gaete | Encapsulation of Dexamethasone into Biodegradable Polymeric Nanoparticles[END_REF][START_REF] Gómez-Gaete | Dexamethasone Acetate Encapsulation into Trojan Particles[END_REF][START_REF] Teeranachaideekul | Development of Ascorbyl Palmitate Nanocrystals Applying the Nanosuspension Technology[END_REF][START_REF] Boissenot | Paclitaxel-Loaded PEGylated Nanocapsules of Perfluorooctyl Bromide as Theranostic Agents[END_REF] In addition, the physical entrapment is often associated with a premature drug release once the formulation is injected intravenously [START_REF] Scherphof | Disintegration of Phosphatidylcholine Liposomes in Plasma As a Result of Interaction With High-Density Lipoproteins[END_REF] . Tsotas et al.

demonstrated the influence of lipid composition of liposomes on dexamethasone (DXM) entrapment efficiency but never reached above 0.5 DXM/lipid molar ratio. [START_REF] Tsotas | Dexamethasone Incorporating Liposomes: Effect of Lipid Composition on Drug Trapping Efficiency and Vesicle Stability[END_REF] Entrapment of dexamethasone or dexamethasone acetate into poly(lactide-co-glycolide) nanoparticles led to a low drug loading of 0.2 and 0.6% w/w respectively. [START_REF] Gómez-Gaete | Encapsulation of Dexamethasone into Biodegradable Polymeric Nanoparticles[END_REF][START_REF] Gómez-Gaete | Dexamethasone Acetate Encapsulation into Trojan Particles[END_REF] In addition, drug crystallization was observed inducing the destabilization of the nanosuspension and the burst release was very important. [START_REF] Gómez-Gaete | Encapsulation of Dexamethasone into Biodegradable Polymeric Nanoparticles[END_REF][START_REF] Gómez-Gaete | Dexamethasone Acetate Encapsulation into Trojan Particles[END_REF] More recent approaches to overcome the low loading efficiency issue consisted in encapsulating within colloidal nanocarriers lipophilic prodrugs from GCs such as palmitate derivatives. [START_REF] Daull | A Preliminary Evaluation of Dexamethasone Palmitate Emulsion: A Novel Intravitreal Sustained Delivery of Corticosteroid for Treatment of Macular Edema[END_REF][START_REF] Wijagkanalan | Enhanced Anti-Inflammation of Inhaled Dexamethasone Palmitate Using Mannosylated Liposomes in an Endotoxin-Induced Lung Inflammation Model[END_REF][START_REF] Hong Kee | Liposomes with Anti-Inflammatory Steroid Prednisolone Palmitate[END_REF][START_REF] Yokoyama | Limethason as a Lipid Microsphere Preparation: An Overview[END_REF][START_REF] Kim | Uniformity of Drug Payload and Its Effect on Stability of Solid Lipid Nanoparticles Containing an Ester Prodrug[END_REF] For instance, prednisolone palmitate or dexamethasone palmitate have been incorporated into liposomes with a high entrapment efficiency. [START_REF] Hong Kee | Liposomes with Anti-Inflammatory Steroid Prednisolone Palmitate[END_REF][START_REF] Benameur | Liposome-Incorporated Dexamethasone Palmitate : Chemical and Physical Properties[END_REF] The presence of strong hydrophobic interactions between the alkyl chain of the prodrug and those of lipids forming liposomes membrane can explain the stable retention of the prodrug in the nanocarrier. [START_REF] Teshima | Prednisolone Retention in Integrated Liposomes by Chemical Approach and Pharmaceutical Approach[END_REF] A lipid nanoemulsion containing dexamethasone palmitate (DXP), Limethason® or Lipotalon®, is currently on the market in Japan and Germany, indicated for rheumatoid arthritis and osteoarthritis via intra-articular injection. [START_REF] Bias | Sustained-Release Dexamethasone Palmitate -Pharmacokinetics and Efficacy in Patients with Activated Inflammatory Osteoarthritis of the Knee[END_REF][START_REF] Mizushima | Tissue Distribution and Anti-Inflammatory Activity of Corticosteroids Incorporated in Lipid Emulsion[END_REF] Although being a crucial parameter, the burst release issue was not approached here. Therefore, to improve drug loading and limit burst release, nanocarriers consisting in solely prodrugs have been proposed. [START_REF] Gaudin | Squalenoyl Adenosine Nanoparticles Provide Neuroprotection after Stroke and Spinal Cord Injury[END_REF][START_REF] Maksimenko | A Unique Squalenoylated and Nonpegylated Doxorubicin Nanomedicine with Systemic Long-Circulating Properties and Anticancer Activity[END_REF][START_REF] Quan | Nanomedicines for Inflammatory Arthritis: Head-to-Head Comparison of Glucocorticoid-Containing Polymers, Micelles, and Liposomes[END_REF][START_REF] Leng | Development and Comparison of Intramuscularly Long-Acting Paliperidone Palmitate Nanosuspensions with Different Particle Size[END_REF] In the present report, a novel approach was used to formulate DXP directly into nanoparticles using a biocompatible and biodegradable lipid, Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000)] (DSPE-PEG 2000 ) as crystallization inhibitor and colloidal stabilizer.

Nanoprecipitation and emulsion-evaporation processes were tested and compared in terms of nanoparticle size, polydispersity index, surface charge, stability, DSPE-PEG 2000, and DXP entrapment efficiency and internal morphology. The optimal formulation obtained thanks to the emulsionevaporation process with DXP/DSPE-PEG 2000 concentrations of 5/2.5mg/mL was characterized for in vitro cytotoxicity and anti-inflammatory efficiency on activated macrophages. In vivo studies on mice were conducted to determine the pharmacokinetics and biodistribution showing enhanced pharmacokinetics and modified tissue distribution of the nanoparticle due to the dysopsonization effect of PEG.

MATERIALS AND METHODS

Materials

Dexamethasone palmitate (DXP) was purchased from Interchim (France). DSPE-PEG 2000 ( was purchased from Fagron (The Netherlands). MilliQ water was purified using a RIOS system from Merck-Millipore (France). Chloroform HPLC-grade was obtained from Carlo Erba Reagents (France), methanol and acetonitrile HPLC-grade were purchased from VWR Chemicals (France). All experiments were performed in amber vials due to the photosensitivity of dexamethasone palmitate and DSPE-PEG 2000

Nanoparticle preparation

Nanoparticles were formulated either by an emulsion-evaporation or a nanoprecipitation process to obtain seven targeted final concentrations of dexamethasone palmitate with the following DXP/DSPE-PEG 2000 ratios: 1.25/0.625; 2.5/1.25; 5/2.5; 5/5; 5/7.5; 7.5/2.5; 10/5 in mg/mL For emulsion-evaporation, 10mL of milliQ water was prechilled at 4°C. The desired amounts of DXP (12.5; 25; 50; 75 or 100mg) and DSPE-PEG 2000 (6.25; 12.5; 25; 50 or 75mg) were dissolved in 1mL chloroform. The organic phase was then injected into the water phase thanks to a solventcompatible syringe (Injekt®, B.Braun) and a 20Gx2 3/4 needle. The mixture was then pre-emulsified by vortexing for 30 seconds before being placed under ultrasonication during 2min at an amplitude of 40% in an ice bath. The organic phase evaporation was performed under reduced pressure using a rotary evaporator (Büchi). After full evaporation of the solvent, the suspension volume was completed to 10 mL with Milli-Q water in a volumetric flask and nanoparticles were stored at 4°C protected from light.

For nanoprecipitation, DSPE-PEG 2000 (6.25; 12.5; 25; 50 or 75mg) was dissolved in 5mL milliQ water at 60°C. DXP (12.5; 25; 50; 75 or 100mg) was dissolved in 1mL acetone and then slowly injected into the aqueous phase under magnetic stirring. After 5min stirring, the preparation was evaporated under reduced pressure using a rotary evaporator. After full evaporation of the solvent, the suspension volume was completed to 10mL with Milli-Q water in a volumetric flask and nanoparticles were stored at 4°C protected from light. Osmolality was measured on a freezing point depression automatic micro osmometer (Type 13/13DR autocal, Roebling, Germany).

Size and zeta potential measurement

Size and zeta potential were determined with a Zetasizer Nano-ZS from Malvern Instrument (UK), based on quasi-elastic light scattering. The measurement was performed in triplicate at an angle of 173° at 25°C. Hydrodynamic diameter (d H ), polydispersity index (PdI) and zeta potential were recorded on 1/10 diluted samples in milliQ water for size measurements or in NaCl 1mM for zeta potential measurements.

Transmission electron microscopy (TEM)

Transmission electron microscopy was performed at I2BC (CNRS, Gif-sur-Yvette, France). A volume of 5L of the nanoparticle suspension at 5mg/mL DXP was deposited for 1 minute on 400 mesh formwar-coated copper grids. Negative staining was performed by addition of a drop of uranyl acetate at 2% w/w for 30 seconds. Excess solution was removed and grids were left to dry before observation. The observations were carried out on a JEOL JEM-1400 microscope at an acceleration voltage of 80KV. Images were acquired using an Orius camera (Gatan Inc, USA).

X-ray powder diffraction

X-ray powder diffraction (XRPD) measurements were performed using a Rigaku rotating copper anode automated diffractometer operating at 50kV and 200mA using Cu Kα radiation. Nanoparticles suspensions were concentrated with Amicon 100kDa, the concentrate was gently introduced into quartz capillaries before sealing them. The intensity of the diffracted X-ray beam was measured as a function of the theta angle (1 to 60°).

Entrapment efficiency

After DXP nanoparticles (DXP-NPs) preparation, quantification of encapsulated DXP and DSPE-PEG 2000 was performed with HPLC coupled to an evaporative light-scattering detector (ELSD, Eurosep, Cergy, France) and a UV detector (785A, Applied Biosystems). DXP was detected with both detectors and DSPE-PEG 2000 was only detected with ELSD detector. UV detection was performed at 240nm wavelength and ELSD detection settings were a nebulization temperature of 35°C and an evaporation temperature of 45°C. An autosampler (Series 200, Perkin Elmer) was employed connected to a Perkin Elmer pump and both detectors UV and ELSD. The SymmetryShieldTM RP18 column (5 µm, 250×4.6 mm; Waters, France) was maintained at room temperature, the mobile phase was composed of MeOH:ACN:Ammonium acetate (pH 4.00, 200mM) 70:20:10 added with 0.043% acetic acid and 0.104% trimethylamine, flow rate was set at 1mL/min.

To separate DXP-NPs form non-encapsulated molecules, ultracentrifugation was performed at 40000rpm (=109760g) during 4h at 4°C (Beckman Coulter Optima LE-80K ultracentrifuge, 70-1Ti rotor). The supernatant was delicately removed to avoid resuspension of the pellet and freeze-dried for 24h (Alpha 1-2 LD Plus, Bioblock, France) after freezing in liquid nitrogen (-196°C). Lyophilisates were dissolved in methanol to reach suitable concentrations within the range of the calibration curve. A volume of 30µL of samples dissolved in MeOH was injected and analyzed during 12min.

Retention times were 7min and 9min for DXP and DSPE-PEG 2000, respectively. The concentration ranges of the calibration curves were 12.5-750µg/mL for DXP and 37.5-375µg/mL for DSPE-PEG 2000 . ELSD detection calibration curves followed a power law model, equations and correlation coefficients were y=219.71x 1.2458 , R²=0.9959 for DXP and y=144.12x 1.2714 , R²=0.9991 for DSPE-PEG 2000 , respectively. UV calibration curve of DXP followed a linear model, y=486.2x+4.6095, R²=0.9987.

Entrapment efficiency was calculated as the DXP or DSPE-PEG 2000 amount encapsulated compared to the initial amount weighted for the DXP-NPs preparation. Drug loading results correspond to the proportion equivalent DXM amount encapsulated compared to the total mass of the DXP-NPs.

Surface PEG density

PEG density at the surface of DXP-NPs 5mg/mL prepared through nanoprecipitation or emulsionevaporation was quantified using data presented thereafter. DXP-NPs density was calculated as an average between ratios of  DXP =1.12g/cm 3 and  DSPE-PEG2000 =1g/cm [START_REF] Allen | The Use of Glycolipids and Hydrophilic Polymers in Avoiding Rapid Uptake of Liposomes by the Mononuclear Phagocyte System[END_REF] Using these data, the surface area (S sp , specific surface) of the suspension depends on (nanoparticle density) and d H (nanoparticle average diameter) and can be calculated as (Equation 1). Equation 1S sp = Thus, the available surface (S available ) in a suspension can be calculated using Equation 2.

Equation 2 S available = where m DXP and m DSPE-PEG are the actual masses of DXP and DSPE-PEG 2000 found in a formulation.

The number of DSPE-PEG 2000 in the suspension can be calculated using Equation 3Equation 3 N DSPE-PEG = where N A is the Avogadro number Using these three values, it is possible to deduce the surface available per PEG chain on the surface of the nanoparticles (Equation 4). Equation 4Surface PEG =

The PEG density is expressed as surface area (nm 2 ) per PEG chain.

Cell culture

The murine macrophage cell line RAW 264.7 obtained from ATCC (USA) was cultured in 75 cm 2 flask with 15 mL of DMEM supplemented with 10% FBS, 10 000 unit/mL of penicillin and 10 mg/mL of streptomycin, in a humidified incubator at 37°C supplied with 5% CO 2 . Cells were split twice per week at 1/10 ratio using a scraper to detach them and were used between passages 3 to 20 after thawing.

Cell viability

The influence of the DXP-NPs on cell viability was assessed using the MTT (3-[4,5-dimethylthiazol-2yl]-3,5-diphenyl tetrazolium bromide) colorimetric assay [START_REF] Mosmann | Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays[END_REF] that evaluates mitochondrial activity. Cells were seeded in 96-well plates (TTP, Zurich, Switzerland) at a density of 8x10 3 cells/well in 100 µL of culture medium, and were left overnight in the incubator. DXP-NPs were diluted in culture medium at various concentrations (37.5-450µg/mL DXP), 100µL of theses dilution were added on cells, the culture medium was used as a negative control. DXP-NPs alone in the culture medium, without cells, were also tested to check their potential interference with the MTT assay. Plates were incubated for 24 hours and the yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) was added at a final concentration of 500 µg/mL and incubated for another hour. The MTT is reduced by metabolically active cells to form the purple formazan crystals. After the formation of the crystals, the medium was discarded and replaced by 200 µL of dimethyl sulfoxide (DMSO) to dissolve crystals and the absorbance was measured at 570nm. The formula used to calculate cell viability was the following: (Abs: absorbance)

 100

Following the same protocol, dexamethasone sodium phosphate (DSP) was tested as a positive control. Serial dilutions in culture medium were performed ranging from 41 to 657µg/mL DSP. To compare DXP-NPs and DSP, tested concentrations were calculated as dexamethasone base equivalents (eq.DXM), corresponding to the active drug. All measurements were performed in triplicate.

Cytokine quantification

For the cytokine quantification, RAW 264.7 cells were seeded in 24-well plates at a cell density of 4x10 4 cells/well in culture medium and were incubated for 48 hours until 80% confluency. Then, the medium was replaced by fresh medium alone or fresh medium with LPS at 0.1 µg/mL to induce inflammation, and plates were incubated another 3 hours. Afterwards, nanoparticles produced by emulsion-evaporation (5/2.5 mg/mL DXP/DSPE-PEG 2000 ) at three concentrations diluted in culture medium: 1, 10 and 100µg/mL of DXP and free dexamethasone phosphate (DSP) at 82µg/mL in culture medium, which correspond to 100µg/mL DXP considering molecular weight ratio, were added. Culture medium alone was used as negative control and LPS 0.1 µg/mL as a positive control.

After 24 hours of incubation with the treatments, cell supernatants were collected and frozen at -20°C until analysis was performed. Cells were detached and counted. Mouse inflammatory cytokines TNFα, MCP-1, IL-10, and IL-6 were quantified using a Cytometric Beads Array (CBA) detection kit (BD Biosciences, USA). In each test tube, 50µL of mouse inflammation capture bead suspension was added, completed with either 50µL of standards solution (20-5000pg/mL) or 50µL of supernatants samples. Phycoerythrin (PE) detection reagent was added 50µl to each tube and incubation during 2h at room temperature was performed. Samples were washed with 1mL wash buffer provided in the kit and tubes were centrifuged (200g, 5min) to recover the pellet. 300µL of wash buffer was added to resuspend the pellet and samples were quantified with the BD Accuri C6 Cytometer (BD Biosciences, USA). Cytokines results were analyzed with the FACP Array™ Software and were obtained as pg/mL concentrations. All measurements were performed in triplicate. Statistical analysis was performed with two-way ANOVA followed by Tukey's post-test using GraphPad Prism.

In vivo studies

In vivo experimental procedures using DBA/1OlaHsd mice were approved by the ethical committee

No 026 and by the French ministry of education and research (Accepted protocol No 2842-2015110914248481_v5). The number of animals was determined according to the power analysis method [START_REF] Altman | Guidelines for the Design and Statistical Analysis of Experiments Using Laboratory Animals[END_REF] . 9-12-week-old male DBA/1OlaHsd mice were purchased from Envigo (UK) and let for one week after shipping for adaptation before starting experiments. Mice were kept in a separate animal room under climate-controlled conditions with a 12h light/dark cycle, housed in polystyrene cages containing wood shavings and fed standard rodent chow and water ad libitum. Mouse colonies were screened and determined to be pathogen-free.

Pharmacokinetics and biodistribution

Nanoparticles prepared by emulsion-evaporation 5/2.5 mg/mL (DXP/DSPE-PEG 2000 ) as described above were half-diluted in PBS to obtain a final concentration of 2.5mg/mL DXP, which corresponds to an equivalent of 1.55mg/mL DXM. A control of dexamethasone disodium phosphate (DSP) in PBS was prepared to obtain a final concentration of 1.55mg/mL (eq. DXM). DBA/1OlaHsd male mice aged 10-12 weeks were divided into two groups. One group was administered with the nanoparticle suspension and the other received the control. The administration was performed by IV injection in the tail vein at 12mg/kg (eq. DXM), an intermediate dose in the range found in the literature for efficacy in mice (1-60mg/kg) [START_REF] Quan | Nanomedicines for Inflammatory Arthritis: Head-to-Head Comparison of Glucocorticoid-Containing Polymers, Micelles, and Liposomes[END_REF][START_REF] Quan | The Evaluation of the Therapeutic Efficacy and Side Effects of a Macromolecular Dexamethasone Prodrug in the Collagen-Induced Arthritis Mouse Model[END_REF] . For each group, 10 pharmacokinetics time points were selected with 7 mice per time point. Blood sampling was achieved by terminal cardiac puncture using a 25G needle while mice were previously deeply anesthetized with a lethal dose of pentobarbital.

Immediately after sampling, blood was centrifuged to recover plasma which was stored at -80°C and extracted to quantify DXP and DXM as described after. For each time point, 5 mice out of 7

underwent organ sampling after a cardiac puncture. Liver, spleen, kidneys, and lungs were removed and stored at -80°C for further dosing. PK analysis was performed using the PK solver plugin from Excel.

DXP and DXM quantification in plasma

To quantify DXP and DXM in plasma, an extraction process was developed. 100L of plasma samples were introduced into centrifugation tubes and 100L of the internal standards (IS) mixture, testosterone decanoate (TestD) for DXP and dexamethasone acetate (DXA) for DXM, both at 4g/mL in acetonitrile were added and vortexed for 30 seconds. 3mL of a 9/1 chloroform/methanol (v/v) mixture was added and tubes were vortexed vigorously for 3 minutes to precipitate proteins.

Centrifugation was performed at 3500rpm (=1690g) for 30min (ST16R centrifuge, rotor TX-400, Thermo Scientific, France). The supernatant organic phase was recovered and evaporated using an evaporator under nitrogen gas flow (Sample concentrator, Stuart, UK). Dried samples were dissolved into 200L acetonitrile and DXP or DXM were quantified by HPLC-UV. Each sample was analyzed twice, once with the DXP quantification method, second with the DXM quantification method.

A Waters 717 Plus autosampler chromatographic system was employed equipped with a Waters Regarding short time points (10min, 20min), DXP and DXM concentrations were above the linearity range of the calibration curve. A second calibration curve for each molecule was determined with 50L plasma completed to 100L with milliQ water. Following extraction steps were the same as previously described. Calibration curves "50L plasma" of DXP and DXM were linear, respectively in the range 50-800g/mL (R²=0.9808, y=0.2229x-3.2128) and 20-400g/mL (R²=0.9986, y=0.7178x-0.059).

DXP and DXM quantification in organs

Organs were homogenized in PBS using a micro-pestle coupled with a turbine at 2038rpm during the time needed to obtain a liquid preparation, approximately 5min. DXM and DXP were extracted from the homogenate using the same method than plasma extraction, beginning with 100µg of homogenate for liver and kidneys, 25µg for spleen and lungs completed up to 100µg with milliQ water. 100µL of internal standard mixture TestD and DXA at 4µg/mL was added and vortexed 30sec, followed by the addition of 3mL of 9/1 chloroform/methanol (v/v), vortexed for 3min and centrifuged in the same conditions as above. The organic phase was evaporated and 200µL acetonitrile was added and mixed during 1.5min. This final sample was analyzed by HPLC-UV, using the same conditions as plasma samples.

RESULTS & DISCUSSION

Formulation process

Two different formulation processes were tested to produce DXP nanoparticles coated with phospholipids coupled to PEG. As DXP is highly hydrophobic and known to be prone to crystallization in aqueous suspension [START_REF] Doi | Physicochemical Properties of Dexamethasone Palmitate, a High Fatty Acid Ester of an Anti-Inflammatory Drug: Polymorphism and Crystal Structure[END_REF] , DSPE-PEG 2000 was added to the prodrug to limit its crystallization and to inhibit Ostwald ripening. Indeed, Ostwald ripening due to the high DXP proportion was already described for DXP microemulsions [START_REF] Lu | Nanoparticles Containing Anti-Inflammatory Agents as Chemotherapy Adjuvants: Optimization and in Vitro Characterization[END_REF] and for palmitate derivatives such as ascorbyl palmitate nanocrystals. [START_REF] Teeranachaideekul | Encapsulation of Ascorbyl Palmitate in Nanostructured Lipid Carriers (NLC)-Effects of Formulation Parameters on Physicochemical Stability[END_REF] DSPE-PEG 2000 is an excipient mainly used in the formulation of clinically applied liposomes and in solid-lipid nanoparticles. [START_REF] Wang | Application of Poly(Ethylene Glycol)-Distearoylphosphatidylethanolamine (PEG-DSPE) Block Copolymers and Their Derivatives as Nanomaterials in Drug Delivery[END_REF] To choose the process best suited for further IV administration of GCs nanoparticles, several DXP/DSPE-PEG 2000 ratios were selected with a fixed DXP initial concentration of 5mg/mL. Both processes led to the formation of nanoparticles. When applying nanoprecipitation, the hydrodynamic diameter did not vary, regardless of the DXP/DSPE-PEG 2000 ratio with nanoparticles around 230-250nm (Figure 1a). For emulsion-evaporation, smaller nanoparticles whose diameter was in the range of 120-130nm were obtained, independently of the DXP/DSPE-PEG 2000 ratio (Figure 1a). This difference can be explained when examining more closely the process parameters. During nanoprecipitation, DXP was dissolved in acetone and DSPE-PEG 2000 was solubilized in water at 60°C. Heating the aqueous phase at 60°C eases the dissolution of the PEGylated lipid and promotes the diffusion of acetone in water during the injection step to favor the fast formation of nanoparticles. On the contrary, heating the aqueous phase during emulsionevaporation could lead to the fast evaporation of chloroform during the emulsion step with ultrasonication, resulting in the destabilization of the primary emulsion and no DXP-NPs formation.

For this reason, we opted to dissolve at room temperature DSPE-PEG 2000 with DXP in chloroform for the emulsion-evaporation process. Thereby, the presence of DXP and DSPE-PEG 2000 together in chloroform favors their interaction and leads to the formation of smaller nanoparticles. The smaller size of emulsion-evaporation DXP-NPs may also arise from a reduced interfacial tension of the chloroform-water interface due to the presence of the PEGylated lipid.

To determine the impact of DXP concentration on nanoparticle size, four DXP concentrations were then tested 1.25, 2.5, 5 and 10mg/mL for both formulation processes with a constant 2/1 DXP/DSPE-PEG 2000 ratio. Figure 1b clearly shows that d H increases as DXP concentration increases using the nanoprecipitation process. Very polydisperse nanoparticles of 300nm at a DXP concentration of 10mg/mL were obtained. In the case of nanoprecipitation, nanoparticle size can be adjusted by tuning the DXP initial concentration. On the contrary, the emulsion-evaporation process led to the formation of monodisperse nanoparticles around 130nm, with a slight increase of size up to 170nm, for the highest concentration tested.

Our rationale was to design a nanoparticle suspension with high drug concentration and low concentration of excipients for IV administration, the DXP/DSPE-PEG 2000 5/2.5mg/mL conditions were therefore selected for remaining experiments. For both preparation methods, formulations were characterized and compared in terms of stability, entrapment efficiency, drug loading, and DSPE-PEG coating.

Figure 1. a. Impact of the DXP/DSPE-PEG 2000 weight ratio on the size of the obtained nanoparticles (constant final DXP concentration of 5mg/mL). The ratios 3/1, 2/1, 1/1 and 2/3 were tested through nanoprecipitation (red circles) and emulsion-evaporation (blue squares) processes. b. Evaluation of the impact of DXP concentration in both nanoprecipitation (red circles) and emulsion-evaporation (blue squares) processes with a constant 2/1 DXP/DSPE-PEG 2000 ratio.

Size, zeta potential and stability over time

After preparation of DXP/DSPE-PEG 5/2.5mg/mL nanoparticles by either nanoprecipitation or emulsion-evaporation, their size, polydispersity index (PdI) and zeta potential were followed during 37 days to confirm the stability of the suspension (Figure 2). Formulation storage at 4°C and room temperature were compared. Emulsion-evaporation-prepared DXP-NPs stored at 4°C were clearly more stable in terms of size, PdI and zeta potential. Under 4°C storage, nanoparticle hydrodynamic diameter (d H ) was kept around 130±3nm (Figure 2a) up to 37 days after preparation. Under the same storage conditions, the zeta potential and the PdI were stable around -50 to -60 mV and 0.2, respectively. On the contrary, at room temperature, size increased after 4 days and on day 14, a clear destabilization of the nanosuspension appeared, characterized by a size increase of 40%, associated with a rise of polydispersity and zeta potential (Figure 2a). On day 21 and later, no size and zeta potential measurements could be performed for samples stored at room temperature due to precipitation resulting from the formation of DXP crystals within the precipitate as evidenced by optical microscopy (Figure S1). By contrast, the nanoprecipitation process yielded stable nanoparticles which mean diameter remained around 230nm at 4°C and presented size increase at room temperature on day 37 due to their destabilization (figure 2b). DXP-NPs polydispersity index was kept below 0.2 highlighting a rather monodisperse population. The zeta potential was also stable ranging from -50 to -60mV. This high negative surface charge was able to promote electrostatic repulsion between nanoparticles and prevents aggregation and destabilization of the preparation.

These nanoparticles prepared by either emulsion-evaporation or nanoprecipitation techniques, with high concentrations of DXP/DSPE-PEG 2000 5/2.5mg/mL, present a clear advantage being long term stable at 4°C without the need of purification steps or freeze-drying. This characteristic is an important point in terms of ease of production.

Morphology and internal structure of the nanoparticles

TEM observations were consistent with dynamic light scattering measurements (Figure 3). Indeed, emulsion-evaporation process led to small spherical nanoparticles, with higher polydispersity than those obtained by nanoprecipitation. The mean diameter of the emulsion-evaporation-prepared nanoparticles was lower than 200nm. Figure 3b presents monodisperse and spherical DXP-NPs prepared by nanoprecipitation. Their hydrodynamic diameter above 200nm confirmed DLS studies.

No crystals or crystalline structure could be observed on these images. DXP-NPs suspensions were analyzed by X-ray diffraction to determine their internal structure. Using both formulation processes, the resulting DXP-NPs were analyzed the day of preparation and 3 weeks after storage at 4°C. They were compared to crystalline DXP form obtained from recrystallization in acetone that gives needleshaped crystals [START_REF] Benameur | Liposome-Incorporated Dexamethasone Palmitate : Chemical and Physical Properties[END_REF][START_REF] Doi | Physicochemical Properties of Dexamethasone Palmitate, a High Fatty Acid Ester of an Anti-Inflammatory Drug: Polymorphism and Crystal Structure[END_REF] . The diffractogram of recrystallized DXP after acetone evaporation is typical of a crystalline structure with many diffraction peaks (Figure 4 bottom) as described by Doi et al . [START_REF] Doi | Physicochemical Properties of Dexamethasone Palmitate, a High Fatty Acid Ester of an Anti-Inflammatory Drug: Polymorphism and Crystal Structure[END_REF]

Entrapment efficiency and drug loading

During nanoparticles preparation, DSPE-PEG 2000 is expected to associate strongly with DXP thanks to hydrophobic interactions between the distearoyl chains of DSPE-PEG 2000 and the palmitate chain of DXP. Entrapment efficiency was determined by quantifying, on one hand, the amount of DSPE-PEG 2000 associated to DXP-NPs, and on the other hand, the amount of DXP in DXP-NPs as it is related to the potential amount of DXM that could be delivered. A simple method of HPLC-ELSD that can separate and detect both components in a single injection on the same chromatogram was developed. After separation by ultracentrifugation, non-encapsulated DXP and DSPE-PEG 2000 in the supernatant were quantified and their amount in NPs was calculated indirectly. The proportion of encapsulated DSPE-PEG 2000 presented variations according to the formulation process (Figure 5a).

Nanoprecipitation led to 20% of encapsulated DSPE-PEG 2000 whereas 53% was encapsulated thanks to emulsion-evaporation process (p < 0.05, unpaired t-test). The difference of DSPE-PEG 2000 entrapment efficiency between the two processes, nanoprecipitation 20%w/w and emulsionevaporation 53%w/w, could be linked to DSPE-PEG 2000 solvent affinity (acetone or chloroform) and its impact on hydrophobic interaction between lipids and DXP occurring during nanoparticles preparation. Considering the amounts of encapsulated DXP and DSPE-PEG 2000 , the DXP prodrug loading and the corresponding active drug DXM loading can be calculated (Figure 5b).

Nanoprecipitated nanoparticles present a high DXP loading (91%w/w), corresponding to an equivalent of 56% of DXM of the DXP-NPs total mass. Emulsion-evaporation leads to 78%w/w DXP loading corresponding to an equivalent of 48%w/w DXM loading, meaning that half of the total mass of DXP-NPs is composed of the active drug. When comparing to literature, these drug loadings are very high. Indeed, the formulation of DXP in the lipid emulsion Limethason® exhibits a low drug loading of 4%w/w DXP/lipids resulting in an equivalent DXM loading of 2.5% w/w. We suspect that this low drug loading was chosen due to the possible issues of crystallization and stability of this formulation at higher DXP concentrations. Here, despite the differences of DSPE-PEG 2000 encapsulated by both methods, 98% of the initial amount of DXP was detected in DXP-NPs with no significant differences (p > 0.05, unpaired t-test). Irrespective of the formulation process and thanks to the presence of palmitate chain on dexamethasone, the highest possible drug entrapment efficiency was reached. Because nanoparticle formulation process clearly impacts on the PEGylated lipid number in nanoparticles, we have calculated the area occupied by each PEG chain on nanoparticle surface in order to evaluate PEG density. To calculate this density, we need to hypothesize that 100% of PEG chains are situated on the surface of the nanoparticles and not in the matrix/core. Since PEG is hydrophilic and DSPE hydrophobic, it seems realistic that most of it stand at the surface of nanoparticles, with the DSPE moiety inserted in the matrix while the PEG chain is exposed in contact with water.

For emulsion-evaporation, data used for PEG density calculation are =1.09g/cm 3 , the average diameter of the nanoparticles is d=130nm and the percentages of encapsulated DXP=98% and encapsulated DSPE-PEG 2000 =53% for a DXP/DSPE-PEG 2000 5/2.5 mg/mL suspension. Using these data, the surface available per PEG chain on the surface of the nanoparticles formed by emulsionevaporation is therefore 0.93nm 2 /PEG. For nanoprecipitation process, after injecting correct values:

=1.11g/cm 3 , d=230nm, one finds that the available surface per PEG chain is 1.18nm²/PEG. The emulsion-evaporation process was therefore considered as gold standard formulation method with small and stable nanoparticles, high drug loading and high PEG density, favorable to good opsonin repulsion. Indeed, to allow passive diffusion through high permeable vessels, nanoparticle size range of 50-400nm is preferred with better accumulation when size is below 200nm. [START_REF] Yuan | Vascular Permeability in a Human Tumor Xenograft : Molecular Size Dependence and Cutoff Size Advances in Brief Vascular Permeability in a Human Tumor Xenograft : Molecular Size Dependence and Cutoff Size1[END_REF][START_REF] Li | Size and Shape Effects on Receptor-Mediated Endocytosis of Nanoparticles[END_REF][START_REF] Verma | Effect of Surface Properties on Nanoparticle-Cell Interactions[END_REF][START_REF] Ahsan | Targeting to Macrophages : Role of Physicochemical Properties of Particulate Carriers -Liposomes and Microspheres -on the Phagocytosis by Macrophages[END_REF] Regardless of the formulation technique, DXP-NPs presented suitable characteristics for this type of indication.

However, nanoparticles obtained by the emulsion-evaporation process, using a DXP concentration of 5mg/mL, led to smaller and monodisperse nanoparticles that could be used in the treatment of inflammation diseases. Moreover, for IV administration, a nanoparticle suspension with a high concentration of active substance will be preferably chosen in order to inject a volume as small as possible. NP characterization suggests that DXP-NPs prepared by emulsion-evaporation method are optimally producing small and stable nanoparticles with high drug loading and high PEG density.

Cytotoxicity and anti-inflammatory activity

A range of DXP-NPs concentrations from 37.5µg/mL up to 450µg/mL (corresponding to 23.3-280µg/mL eq.DXM) was tested and compared to the water-soluble free drug, dexamethasone sodium phosphate (DSP) on macrophages. According to ISO guideline for MTT assay, potential cytotoxicity is defined when cell viability decreases below 70% of the control. [START_REF]Biological Evaluation of Medical Devices-Part 5: Tests for in Vitro Cytotoxicity[END_REF] Figure 6 clearly shows DXP-NPs presented no cytotoxicity up to DXM equivalent concentration of 280µg/mL, whereas no decrease in cell viability was observed for DSP in the concentration range tested. In addition, 280µg/mL (eq.DXM) is a rather high concentration that is unlikely to be reached during in vivo studies after IV injection. To ensure that the anti-inflammatory activity of DXM was preserved after formulation, the release of pro-inflammatory cytokines (MCP-1, IL-10, IL-6, TNFα) by LPS-activated Raw 264.7 macrophages into the cell culture medium was quantified after their exposure to DXP-NPs at different concentrations (1; 10; 100µg/mL DXP) or free DSP at 82µg/mL, corresponding to 100µg/mL of DXP. A decrease of cytokines concentration was clearly observed resulting from the anti-inflammatory effect of DXP-NPs (Figure 7). The MCP-1 chemokine (Figure 7a) was strongly and significantly reduced by DXP-NPs in the presence of LPS. Regardless of DXP concentration in nanoparticles, no dose-effect was observed, meaning that the lower concentration of DXP-NPs (1µg/mL) was sufficient to yield the expected antiinflammatory effect. The same conclusions hold for the secretion of IL-10 (Figure 7b). Indeed, a significant reduction of IL-10 level was observed after treatment with DXP nanoparticles, regardless of DXP concentrations. Concerning IL-6 release (Figure 7c), results were more balanced. No significant reduction was detected for DXP nanoparticles and free dexamethasone, but a downward trend can be observed when comparing positive control and DXP-NPs activity. This result can be related to the usual classification of IL-6 as a simultaneous pro-and anti-inflammatory cytokine.

Interestingly, DXP concentration in nanoparticles has an impact on TNFα release (Figure 7d). Indeed, only 100µg/mL DXP in nanoparticles could reduce significantly the release of this important proinflammatory cytokine, whereas no effect was detected with free dexamethasone phosphate at the equivalent concentration.

A control study was performed to ensure that DXP-NPs do not induce inflammation by themselves (Figure S2). All these results confirmed that the use of a prodrug and its formulation into nanoparticles does not affect the therapeutic activity of the drug. DXM is most probably released from DXP-NPs due to the presence of esterases in the cell culture medium, as it was observed in plasma [START_REF] Lorscheider | Dexamethasone Palmitate Nanoparticles: An Efficient Treatment for Rheumatoid Arthritis[END_REF] or even in macrophage lysosomal compartments. Based on these promising results, in vivo studies were performed.

Pharmacokinetics and biodistribution

To obtain a better understanding of the behavior of DXP-NPs after IV injection, their pharmacokinetics and biodistribution were determined. Dexamethasone palmitate is a prodrug and dexamethasone base (DXM) is the active drug released from DXP after ester bond hydrolysis.

Thereby, DXM and DXP were quantified from mice plasma after DXP-NPs were intravenously injected. DXP-NPs were compared to control DSP, water-soluble prodrug of DXM (Figure 8a). Ten minutes after injection, DXM from DSP control solution was obviously eliminated very fast, reaching a plateau around 10% of the injected dose up to 48h. DXP plasmatic concentration decreases slowly until 8h after injection. After 8h, no DXP was detected in mice plasma. The prodrug is transformed into DXM, which plasmatic concentration presents a slow decrease for 18h. The main pharmacokinetic parameters were calculated according to a non-compartmental model (Figure 8b).

The area under the curve (AUC 0-∞ ) of DXM from nanoparticles 126400µmol*min/L, whereas DXM from control was 93600µmol*min/L meaning the active DXM molecule exposure in the body is 1.35 times higher compared to injection of free dexamethasone. When comparing data up to 4h, the AUC (0-240) is 3.3 fold higher for DXM from nanoparticles. At T max , 10min for both, C max of DXM from nanoparticles, 327µmol/L (or 128.5µg/mL) is 5.8 times higher than the control, C max = 56µmol/L (or 22.1µg/mL). Volumes of distribution (Vd) reveal DXP-NPs were less distributed in the organs and stay longer in the blood, with Vd for DXM from nanoparticles 1.8-fold lower than the control. However, elimination half-life and mean residence time (MRT) are higher for the control DSP, probably due to the plasmatic plateau observed. These data support the fact that DXP-NPs lead to a higher exposure after IV injection compared to the free drug, due to the enhanced pharmacokinetics properties of DXP-NPs. Nevertheless, DXP act as a prodrug, encapsulated into NPs and seems to be rapidly hydrolyzed to release free DXM in the blood. Figure 9 presents the biodistribution of DXP-NPs and DSP control, both injected intravenously at 12mg/kg (eq.DXM), in liver, spleen, kidneys, and lungs. Among the four organs, the liver presents the highest accumulation of DXM from nanoparticles with an increase up to 18% of the injected dose 40 min after injection followed by a slow decrease until 48h. However, DXP and DXM liver concentrations from nanoparticles were clearly lower than the control DSP for the first-time points after injection. Less than 2% of the injected dose of drug or prodrug was detected in the kidneys. This presence is linked to the elimination of the molecules through urinary excretion. Very small quantities, lower than 1%, were detected in the lungs. After statistical analysis, a significant difference was found between DXM from DSP and DXM from DXP-NPs on the whole data in the spleen, whereas values in the liver, kidneys, and lungs were not significantly different (unpaired ttest). Pharmacokinetics results are correlated with biodistribution evaluation. In the liver, the small amounts of DXP detected indicate that PEGylation of nanoparticles allows a reduction of their opsonization, helping them to escape from the immune system recognition in the bloodstream. The presence of DXM from DXP-NPs and from DSP in the liver is linked to the intrinsic hepatic accumulation of DXM. [START_REF] Begg | The Pharmacokinetics of Corticosteroid Agents[END_REF] On the other hand, DXP spleen concentrations from the nanoparticles were significantly higher compared to DSP. Even though DXP in spleen represents less than 2% of the injected dose, its highest concentration in this organ could be explained by the presence of the PEG at the surface of the nanoparticles. [START_REF] Peracchia | Stealth(®) PEGylated Polycyanoacrylate Nanoparticles for Intravenous Administration and Splenic Targeting[END_REF] Indeed, the spleen is less accessible than liver due to its low vascularization, however, the modified pharmacokinetics properties of DXP-NPs induce more accumulation in this organ compared to free drug. Very low DXP quantities were observed in the lungs proving no crystallization or aggregation of the particles occurred after IV injection. DXP-NPs stability in the blood is therefore attested.

CONCLUSIONS

We have succeeded to formulate a hydrophobic prodrug of dexamethasone into PEGylated solid lipid nanoparticles using two different formulation processes. Either nanoprecipitation or emulsionevaporation leads to the formation of stable nanoparticles. Nanoparticle size can be tuned between 130 and 300nm depending on the process, the prodrug/lipid ratio, and the prodrug concentration. A very high prodrug entrapment efficiency of 98% w/w was reached for both processes, corresponding to a very high equivalent DXM loading around 50% w/w. In terms of PEGylated lipid association, emulsion-evaporation method enables 53% of DSPE-PEG 2000 entrapment whereas only 20% was obtained by nanoprecipitation. Since DXP-NPs obtained by emulsion-evaporation were smaller in size and allowed to obtain very dense PEG brushes on their surface with an available surface of 0.93nm 2 /PEG chain, they were considered the perfect candidates for further in vitro and in vivo evaluation. After IV injection of DXP-NPs, the release of DXM in plasma was clearly controlled up to 18h compared to the free drug rapidly eliminated from plasma after administration. These prodrug nanoparticles represent a promising treatment for chronic inflammatory diseases as they possess all the prerequisites to accumulate in inflamed tissues after extravasation.

  1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-(methoxy(polyethylene glycol)-2000) (ammonium salt)) was obtained from Avanti Polar Lipids, Inc. (USA). Dexamethasone base (DXM) and dexamethasone acetate (DXA) were provided from Chemos GmbH (Germany). Testosterone decanoate (TesD) was obtained from Interchim (France). Dexamethasone disodium phosphate (DSP)

  according to the entrapment efficiency of each component, the average diameter of the nanoparticles, the molar mass of DSPE-PEG 2000 is M DSPE-PEG =2805.54g/mol. Masses of DXP and DSPE-PEG 2000 in NPs were respectively 50mg and 25mg with the respective percentages of encapsulated DXP and DSPE-PEG 2000 depending on the technique used.

  1525 binary HPLC pump, a Waters 2487 dual λ absorbance UV detector, and a Breeze software. The analysis was performed at 240nm wavelength using a SymmetryShieldTM RP18 column (5 µm, 250×4.6 mm; Waters, Saint-Quentin-en-Yvelines, France). The column temperature was maintained at 40°C. The mobile phase was composed by a mixture of acetonitrile and milliQ water: 85/15 v/v for DXP and 35/65 v/v for DXM. The mobile phase flow rate was 1.2mL/min, the injection volume was 50L and the run time was 30min. Retention times were 24min and 9min for DXP and DXM, respectively, 21min for TestD (IS of DXP) and 26min for DXA (IS of DXM). Calibration curves of DXP and DXM were linear, respectively in the range 0.5-100g/mL (R²=0.9997, y=0.2199x-0.0165, LOQ=0.5µg/mL, LOQ=0.16µg/mL) and 0.1-100g/mL (R²=0.9974, y=1.056x+0.1445, LOQ=0.1µg/mL, LOQ=0.033µg/mL).

  . By contrast, almost no diffraction peaks can be observed for DXP-NPs either freshly prepared or 3 weeks old, regardless of the preparation technique (Figure4top). The diffractogram of DXP-NPs presents a "bump" which is typical of the presence of an unorganized amorphous system. Results clearly show that neither DXP nor DSPE-PEG 2000 present a crystalline organization. In addition, the amorphous nanoparticles appear stable upon storage at 4°C, a clear advantage for further clinical use. Therefore whatever the process of formulation, DXP-NPs remain amorphous and stable under storage at 4°C as no crystal is observed microscopically or detected by X-ray diffraction. As demonstrated for other excipients such as Pluronic F127[START_REF] Vetter | Slowing the Growth Rate of Ibuprofen Crystals Using the Polymeric Additive Pluronic F127[END_REF] and HPMC[START_REF] Warren | Using Polymeric Precipitation Inhibitors to Improve the Absorption of Poorly Water-Soluble Drugs : A Mechanistic Basis for Utility[END_REF] , the presence of DSPE-PEG 2000 seems to slow down the nucleation and crystal growth of DXP in the suspension. During the formation of DXP-NPs, the DXP palmitate chain interacts with the stearate chains of DSPE-PEG 2000 thanks to strong hydrophobic interactions introducing some disorder. This strong interaction might also be responsible for the limitation of Ostwald ripening. Moreover, the hydrophilic PEG chains exposed at nanoparticle surface improve stability by steric hindrance.

Figure 2 .

 2 Figure 2. Size, PdI and zeta potential stability of DXP/DSPE-PEG 2000 5/2.5 mg/mL nanoparticles, at room temperature (RT) or 4°C. a. emulsion-evaporation, b. nanoprecipitation. Results are presented as mean±SEM (n=5 at least).

Figure 3 .

 3 Figure 3. DXP/DSPE-PEG 2000 5/2.5mg/mL nanoparticles imaged by TEM after negative staining. a. emulsion-evaporation and b. nanoprecipitation process.

Figure 4 .

 4 Figure 4. X-ray diffractogram of DXP-NPs obtained directly after their formulation (day 0, emulsionevaporation (red) and nanoprecipitation (purple)), or after 3 weeks storage at 4°C (emulsionevaporation (blue) and nanoprecipitation (orange)), and DXP crystals (green).

Figure 5 .

 5 Figure 5. a. DXP (red) and DSPE-PEG (blue) entrapment efficiency in DXP-NPs prepared by nanoprecipitation or emulsion-evaporation. b. Corresponding DXP (red) and equivalent DXM (light red) loading for both formulation processes. Results are presented as mean±SEM (n=7).

Figure 6 .

 6 Figure 6. Raw 264.7 macrophages viability after 24h exposure to DXP-NPs prepared by emulsionevaporation (green circles) or DSP solution (blue squares) at different concentrations. Concentrations are expressed as equivalent DXM. Results are presented as mean±SEM (n=3).

Figure 7 .

 7 Figure 7. Cytokine production by Raw 264.7 macrophages without LPS induction (C-) or with LPS induction 0.1µg/mL (C+), DXP nanoparticles at 1µg/mL DXP (LPS+DXP1), 10µg/mL (LPS+DXP10), 100µg/mL (LPS+DXP100) or DSP solution at 82µg/mL corresponding to 100µg/mL of DXP (LPS+DSP82). Four cytokines were evaluated a) MCP-1, b) IL-10, c) IL-6 and d) TNF-α. Results are presented as mean±SEM (n=3) for all groups. Statistical analysis was performed with two-way ANOVA followed by Tukey's post test using GraphPad Prism. * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 indicates differences with positive control (C+).

Figure 8 .

 8 Figure 8. a. Pharmacokinetics of DXP and DXM expressed as molar plasmatic concentration. Plasma concentrations of DXP (green) and DXM (red) released from DXP nanoparticles and DXM (blue) released from DSP solution after IV injection of 12mg/kg (eq.DXM) on males DBA/1 healthy mice. Results are presented as mean±SEM (n=7). PK curves were significantly different up to 480min (6 hours) (* p < 0.05, unpaired t-test). b. Corresponding pharmacokinetic parameters.

Figure 9 .

 9 Figure 9. Biodistribution of DXP-NPs (DXP in green, DXM in red) and DSP solution (DXM in blue) after 12mg/kg (eq.DXM) injected dose on male DBA/1 mice. Quantification was performed in a) liver (not significant (n.s.) between DXM from DSP and DXM from NPs, unpaired t-test), b) kidney (n.s. between DXM from DSP and DXM from NPs, unpaired t-test), c) spleen (p< 0.05 between DXM from DSP and DXM from NPs, unpaired t-test) and d) lungs (n.s. between DXM from DSP and DXM from NPs, unpaired t-test). Results are presented as mean±SEM (n=5).

ACKNOWLEDGMENTS

The present work has benefited also from the core facilities of Imagerie-Gif, (http://www.i2bc.parissaclay.fr), member of IBiSA (http://www.ibisa.net), supported by "France-BioImaging" (ANR-10-INBS-04-01), and the Labex "Saclay Plant Science" (ANR-11-IDEX-0003-02). Institut Galien Paris-Sud is a member of the Laboratory of Excellence LERMIT supported by a grant from ANR (ANR-10-LABX-33).

Authors would like to acknowledge the precious help of S. Denis for cell culture, Ivana Stolfa for HPLC-ELSD settings and quantification, Claire Gueutin for HPLC-UV and Henri Benech for pharmacokinetics analysis.

ASSOCIATED CONTENT

Supporting information

Additional figures as noted in the text: DXP crystals apparition after nanoparticles storage at room temperature during more than 37 days (S1); Cytokine production by Raw 264.7 macrophages with or without LPS induction after exposure to DXP nanoparticles (S2).

AUTHOR INFORMATION Corresponding Author

*Phone: +33146835568. Fax: +33146835946. E-mail: elias.fattal@u-psud.fr ORCID Elias Fattal: 0000-0002-3194-961X

Notes

The authors declare no competing financial interest.


Guilherme F Picheth

Laurence Moine
email: laurence.moine@u-psud.fr

Sophie Houvenagel

Leociley Menezes

Guilherme L Sassaki

Camille Dejean

Nicolas Huang

Rilton Alves De Freitas

R A Menezes

Nicolas Tsapis
email: nicolas.tsapis@u-psud.fr

Impact of Polylactide Fluorinated End-Group Lengths and Their Dynamics on Perfluorohexane Microcapsule Morphology

Keywords: Perfluorocarbon microcapsules, perfluorohexane, fluorinated polymers, focused ultrasound, end-group dynamics, fluorous interaction

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

Introduction

Fully and partly fluorinated compounds are widely explored in the chemical industry and academic research for their inherent immiscibility with hydrophobic and lipophobic solvents. This so-called "fluorophilic" character is responsible for low or "gas like" intermolecular attractions which confer to such molecules inertness and biocompatibility, features that attracted special attention for a wide range of biomedical applications 1,[START_REF] Krafft | Physical Chemistry, and Uses of Molecular Fluorocarbon-Hydrocarbon Diblocks, Triblocks, and Related Compounds-Unique "Apblar" Components for Self-Assembled Colloid and Interface Engineering[END_REF] . In particular, liquid perfluorocarbons (PFCs), such as perfluorohexane (PFH) or perfluorooctyl bromide (PFOB) are described to be minimally absorbed, eliminated from the lungs by evaporation and did not lead to any significant histological, cellular or biochemical disturbance [START_REF] Shaffer | Analysis of Perfluorochemical Elimination from the Respiratory System[END_REF] . Therefore, these PFCs are intensively investigated for their potential to act as intravascular contrast agents [START_REF] Cosco | Perfluorocarbon-Loaded Micro and Nanosystems for Medical Imaging: A State of the Art[END_REF] .

Recent reports describe the use of capsules containing PFOB to provide in vivo [START_REF] Houvenagel | End-Chain Fluorination of Polyesters Favors Perfluorooctyl Bromide Encapsulation into Echogenic PEGylated Nanocapsules[END_REF] F-MRI or ultrasound detection and also passively deliver chemotherapeutics to cancer cells [START_REF] Pisani | Perfluorooctyl Bromide Polymeric Capsules as Dual Contrast Agents for Ultrasonography and Magnetic Resonance Imaging[END_REF][START_REF] Jafari | High-Frequency (20 to 40 MHz) Acoustic Response of Liquid-Filled Nanocapsules[END_REF][START_REF] Diou | Long-Circulating Perfluorooctyl Bromide Nanocapsules for Tumor Imaging by (FMRI)-F-19[END_REF][START_REF] Boissenot | Paclitaxel-Loaded PEGylated Nanocapsules of Perfluorooctyl Bromide as Theranostic Agents[END_REF][START_REF] Boissenot | Ultrasound-Induced Mild Hyperthermia Improves the Anticancer Efficacy of Both Taxol® and Paclitaxel-Loaded Nanocapsules[END_REF] . Similar formulations use PFH as an active core for its lower boiling point, as it favors liquid-to-gas transition by acoustic and thermal stimuli, allowing real-time ultrasonic imaging as well as localized drug-release [START_REF] Singh | Phase Transitions of Nanoemulsions Using Ultrasound: Experimental Observations[END_REF][START_REF] Li | Phase-Transition Contrast Nanocapsules Triggered by Low-Intensity Ultrasound[END_REF][START_REF] You | Nanoparticle-Enhanced Synergistic HIFU Ablation and Transarterial Chemoembolization for Efficient Cancer Therapy[END_REF] . Nonetheless, the entrapment of fully fluorinated PFCs, such as PFH, within a polymeric shell is proving to be difficult due to their dual lipophobic and hydrophobic character and their trend to easily evaporate during formulation [START_REF] Picheth | Echogenicity Enhancement by End-Fluorinated Polylactide Perfluorohexane Nanocapsules: Towards Ultrasound-Activable Nanosystems[END_REF] . Several groups have turned to the use of fluorinated surfactants or lipids to improve PFCs encapsulation [START_REF] Astafyeva | Perfluorocarbon Nanodroplets Stabilized by Fluorinated Surfactants: Characterization and Potentiality as Theranostic Agents[END_REF][START_REF] Fernandes | Synthesis of Stable Multifunctional Perfluorocarbon Nanoemulsions for Cancer Therapy and Imaging[END_REF][START_REF] Oda | Development of Fluorous Lipid-Based Nanobubbles for Efficiently Containing Perfluoropropane[END_REF] . Literature also reports the functionalization of polymer with fluorinated end-groups to favor interactions with PFCs [START_REF] Li | Phase-Transition Contrast Nanocapsules Triggered by Low-Intensity Ultrasound[END_REF][START_REF] Picheth | Echogenicity Enhancement by End-Fluorinated Polylactide Perfluorohexane Nanocapsules: Towards Ultrasound-Activable Nanosystems[END_REF][START_REF] Koda | Fluorinated Microgel Star Polymers as Fluorous Nanocapsules for the Encapsulation and Release of Perfluorinated Compounds[END_REF][START_REF] Houvenagel | End-Chain Fluorination of Polyesters Favors Perfluorooctyl Bromide Encapsulation into Echogenic PEGylated Nanocapsules[END_REF][START_REF] Houvenagel | Comb-Like Fluorophilic-Lipophilic-Hydrophilic Polymers for Nanocapsules as Ultrasound Contrast Agents[END_REF][START_REF] Kaberov | Novel Triphilic Block Copolymers Based on Poly(2-Methyl-2-Oxazoline)-Block -poly(2-Octyl-2-Oxazoline) with Different Terminal Perfluoroalkyl Fragments: Synthesis and Self-Assembly Behaviour[END_REF][START_REF] Kaberov | Fluorinated 2-Alkyl-2-Oxazolines of High Reactivity: Spacer-Length-Induced Acceleration for Cationic Ring-Opening Polymerization As a Basis for Triphilic Block Copolymer Synthesis[END_REF][START_REF] Riabtseva | Structural Characterization of Nanoparticles Formed by Fluorinated Poly(2-Oxazoline)-Based Polyphiles[END_REF][START_REF] Kaberov | Fluorophilic-Lipophilic-Hydrophilic Poly(2-Oxazoline) Block Copolymers as MRI Contrast Agents: From Synthesis to Self-Assembly[END_REF] . It was previously demonstrated that a wide range of fluorinated end-groups linked to the PLA chainfrom C 3 F 7 to C 13 F 27increased the entrapment of PFCs into nanocapsules, resulting in more efficient acoustic responses [START_REF] Picheth | Echogenicity Enhancement by End-Fluorinated Polylactide Perfluorohexane Nanocapsules: Towards Ultrasound-Activable Nanosystems[END_REF][START_REF] Houvenagel | End-Chain Fluorination of Polyesters Favors Perfluorooctyl Bromide Encapsulation into Echogenic PEGylated Nanocapsules[END_REF] . However, no significant distinction regarding the morphology or PFC entrapment was observed by increasing the length of the end-fluorinated groups in nanoformulations.

The main objective of this study is to investigate deeper the influence and extent of fluorous interactions between fluorinated end-groups of PLA-C x F 2x+1 and PFH on the final morphology of microcapsules (MC) as a function of fluorinated end-group length. In particular, we examine the role of the solvent in which the polymer was dissolved on the strength of fluorous interactions by comparing chloroform and dichloromethane, two solvents often used to formulate polymer particles and capsules [START_REF] Horváth | Fluorous Biphase Chemistry[END_REF][START_REF] Koda | Amphiphilic/Fluorous Random Copolymers as a New Class of Non-Cytotoxic Polymeric Materials for Protein Conjugation[END_REF] . To fully explore the impact of fluorinated end-group chain length and solvent, end-group dynamics and PFH entrapment efficiency were evaluated by NMR and MC morphology was assessed by confocal microscopy. This study provides information for the morphological design of new ultrasound activable systems formulated with fluorinated PLA polymers.

Materials and Methods

Materials

D,L-lactide was purchased from Polysciences (Germany) and perfluorohexane 98+% was acquired from Alfa Aesar (Germany). 1-hexanol was obtained from Acros Organics (Belgium), 2,2,3,3,4,4-heptafluoro-1-butanol, 1H,1H perfluoro-1-heptanol, 1H,1H perfluoro-1nonanol, 1H,1H perfluoro-1-dodecanol and 1H,1H perfluoro-1-tetradecanol were acquired from Fluorochem (United Kingdom). Acetone, tetrahydrofuran (THF) and dichloromethane were purchased from Carlo Erba Reactifs (France), chloroform and diethyl ether from VWR (France). Nile red, stannous octoate, sodium cholate, trifluoroacetic acid (TFA), deuterated chloroform and deuterated dichloromethane were provided by Sigma-Aldrich (France). The ultrapure water was produced by a RIOS/Milli-Q system (Millipore, France), with a resistivity of 18.2 MΩcm. The NMR sample tubes and coaxial inserts were obtained from CortecNet (France).

Polymer Synthesis

All fluorinated (PLA-C 3 F 7 , PLA-C 6 F 13 , PLA-C 8 F 17 , PLA-C 11 F 23 and PLA-C 13 F 27 ) and nonfluorinated (PLA-C 6 H 13 ) derivatives of polylactide polymers were synthesized by ring opening polymerization (ROP) with the presence of stannous octoate as catalyst [START_REF] Singh | Characterization and Surface Properties of Poly(Lactic Acid)-Perfluoropolyether Block Copolymers[END_REF][START_REF] Giuntoli | Fluoro-Functionalized PLA Polymers as Potential Water-Repellent Coating Materials for Protection of Stone[END_REF] . All glassware and stir bars were flame-dried and cooled under argon flow. Briefly, in a 10 mL schlenck tube equipped with a magnetic stir-bar, the D,L-lactide (10.4 mmol, 1.5 g) and corresponding initiator (0.075 mmol) -1-hexanol for PLA-C 6 H 13 , 2,2,3,3,4,4-heptafluoro-1-butanol for PLA-

C 3 F 7 , 1H,1H perfluoro-1-heptanol for PLA-C 6 F 13 , 1H,1H perfluoro-1-nonanol for PLA-C 8 F 17 ,
1H,1H perfluoro-1-dodecanol for PLA-C 11 F 23 or 1H,1H perfluoro-1-tetradecanol for PLA-C 13 F 27were added to the flask under argon flow. The tube was sealed with a rubber cap and a stannous octoate solution (0.05 mmol, 20 mg) dissolved in 2 mL of dried toluene was added through the septum. The tube was purged with argon for 0.5 h and the polymerization reaction was conducted with continuous stirring at 130 °C for 55 minutes in an oil bath under argon flow. The reaction was quenched by immersing the flask in a cold water bath.

Afterwards, the solvent was evaporated under reduced pressure for 1h and the material was dissolved in 5 mL of chloroform. The product was purified by precipitation as previously described [START_REF] Diou | RGD Decoration of PEGylated Polyester Nanocapsules of Perfluorooctyl Bromide for Tumor Imaging: Influence of Pre or Post-Functionalization on Capsule Morphology[END_REF] : all polymers were precipitated into cold diethyl ether (80 mL), next, PLA-C 6 H 13 was dissolved into THF (5 mL), whereas the fluorinated polymers were dissolved in acetone (20 mL) and precipitated again in ultrapure water (150 mL). The samples were freeze-dried for 24 h and a white powder was obtained. Lactide conversion ≥95% ( 1 H-NMR). 1

Characterization

All polymers were characterized by size exclusion chromatography (SEC) in chloroform at 30 °C with a flow rate of 1 mL.min -1 by two columns (PL-gel 5µm MIXED-D 300 x 7.5 mm) calibrated against a curve based on poly(methyl methracrylate) standards (PMMA, Polymer Laboratories, Varian Inc.) The system was coupled to a refractive index detector (Spectrasystem RI-150, Thermo Electro Corp.). 1 H and [START_REF] Houvenagel | End-Chain Fluorination of Polyesters Favors Perfluorooctyl Bromide Encapsulation into Echogenic PEGylated Nanocapsules[END_REF] F-NMR spectra were obtained on a Bruker AVANCE III HD 400 MHz in CDCl 3 at 25ºC.

Interfacial surface tension

The interfacial surface tension was performed in a Tracker tensiometer (Teclis, France). PFH was previously filtrated by a column of aluminum oxide. A constant 2 µL PFH or sodium cholate drop was formed by a syringe and a G20 stainless steel needle inside an optical glass cuvette containing fixed polymer concentrations of 25 mg.mL -1 in chloroform or dichloromethane previously filtered by 0.22 µm (Millipore). The temperature was kept at 20 °C and the interfacial surface tension was determined from the drop profile employing the Laplace equation and the forces balance between capillarity and gravity.

19 F-NMR T 1 and T 2 Relaxation

19 F Spin-lattice and spin-spin relaxation measurements were performed at 25 ºC in CDCl 3 or CD 2 Cl 2 with fixed polymer concentrations of 25 mg.mL -1 . Samples containing 15 µL of PFH were also evaluated. The samples were not degassed. All experiments were performed on a Bruker AVANCE III HD NMR spectrometer operating at 9.4 T, equipped with a 5-mm multinuclear inverse detection probe, observing [START_REF] Houvenagel | End-Chain Fluorination of Polyesters Favors Perfluorooctyl Bromide Encapsulation into Echogenic PEGylated Nanocapsules[END_REF] F and 1 H nuclei at 376.49 and 400.13 MHz, respectively. [START_REF] Houvenagel | End-Chain Fluorination of Polyesters Favors Perfluorooctyl Bromide Encapsulation into Echogenic PEGylated Nanocapsules[END_REF] F T 1 was determined by the inversion-recovery pulse sequence (T 1 IR). The delay (τ) between the inversion and read-pulse was varied along 16 values (1 to 22 s). The total number of scans was 32, spectral width = 100 ppm and relaxation delay = 22 s. [START_REF] Houvenagel | End-Chain Fluorination of Polyesters Favors Perfluorooctyl Bromide Encapsulation into Echogenic PEGylated Nanocapsules[END_REF] F T 2 values were measured by the PROJECT-CPMG pulse sequence along 15 values of τ (0.1 to 2 ms) [START_REF] Aguilar | Spin Echo NMR Spectra without J Modulation[END_REF] . The number of scans was 128, spectral width = 100 ppm and relaxation delay = 10 s. Relaxation times were obtained by nonlinear least-squares fitting of a mono-exponential function with r 2 ≥0.998 for all samples.

Microcapsules Formulation

Microcapsules were prepared by the emulsion-evaporation technique, as previously described [START_REF] Mousnier | Influence of Polymer End-Chemistry on the Morphology of Perfluorohexane Polymeric Microcapsules Intended as Ultrasound Contrast Agents[END_REF] , with minor modifications. Briefly, the desired amount of polymer was dissolved into 2 mL of chloroform or dichloromethane in a 50 mL glass flask. Afterwards, 50 µL of nile red (55 µg.mL -1 ) was added and the flask was sealed with a rubber cap. Next, 30 µL of PFH was introduced through the septum with constant stirring and the media was emulsified with 10 mL of sodium cholate 1.5% (w/w) at 4 °C. The mixture was submitted to high-speed homogenization at 8.000 RPM using an Ultra-Turrax T25 (IKA) coupled with an SN25-10G dispersing tool in an ice bath for 1 minute. The solvent was removed by rotary evaporation (Büchi) with a 30 °C water bath and recirculation chiller at -10 °C. A vacuum gradient from 400 to 30 mbar in 7 steps of 5 minutes was employed. Afterwards, the total volume of each sample was completed to 10 mL with ultrapure water.

Confocal Microscopy

Microcapsules were washed twice with ultrapure water by centrifugation and resuspended in glycerol to minimize motion. All samples were observed in a Leica TCS SP5 confocal scanning microscope (Leica) with a 1 mW helium neon laser operating at excitation of 551 nm and 636 nm of emission, Plan Achromat 63X objective (NA 1.40 oil immersion) and pinhole diameter at 71 nm. For diameter analysis, the measurements were performed in the equatorial plane of each capsule as previously described [START_REF] Mousnier | Influence of Polymer End-Chemistry on the Morphology of Perfluorohexane Polymeric Microcapsules Intended as Ultrasound Contrast Agents[END_REF] .

Scanning Electron Microscopy

Scanning electron microscopy was performed in a Merlin 6040 (Carl Zeiss, Germany) operating at 3 kV. All microcapsules were washed twice with ultrapure water by centrifugation to remove the excess of sodium cholate, deposited on carbon conductive double-sided tape (Euro-medex, France) and dried at room temperature. Afterwards, they were coated with a palladium-platinum layer of about 3 nm using a Cressington sputtercoater 208HR with a rotary-planetary-tilt stage, fitted with an MTM-20 thickness controller.

PFH encapsulation efficiency by 19 F-NMR spectroscopy

For PFH quantification, 1 mL of microcapsules was freeze-dried during 24 h employing an Alpha-1-2 LD apparatus (Christ). The flasks were hermetically sealed with a rubber cap and stored at -20 °C until analysis. The PFH encapsulation efficiency was determined as described by Diou et al. [START_REF] Diou | RGD Decoration of PEGylated Polyester Nanocapsules of Perfluorooctyl Bromide for Tumor Imaging: Influence of Pre or Post-Functionalization on Capsule Morphology[END_REF] . A total volume of 1 mL of chloroform was introduced into the sealed tube by a needle, the suspension was vortexed (5 cycles of 30 seconds) and centrifuged at 0 °C for 10 minutes at 5000 rpm (Jouan CR4I centrifuge, Thermo Electron Corporation). The organic solution was collected in a cold room at 4 °C and introduced into an NMR tube that was afterwards loaded with a stem coaxial insert containing TFA in D 2 O (12 µmol.mL -1 ) as external standard [START_REF] Henderson | Quantitative NMR Spectroscopy Using Coaxial Inserts Containing a Reference Standard: Purity Determinations for Military Nerve Agents[END_REF] . All spectra were obtained at 4 °C on a Bruker Avance 300 (400 MHz) spectrometer. The total amount of encapsulated PFH was determined based on a PFH calibration curve. For each MC sample, the integration of the TFA peak at -76.5 ppm was determined as 3 and the resulting integration of the PFH CF 3 at -81.2 ppm was used to calculate the PFH concentration in the NMR tube based to the equation of the calibration curve.

Statistical analysis

The statistical analysis was performed using Statistica™ 8.0 software (StatSoft Inc., Tulsa, USA). The data was analyzed by two-sided Student's t-test and a statistical significance was set at the level of p<0.05.

Results and Discussion

Polymers synthesis and characterization

Six end-group fluorinated or alkylated polymers were synthesized by ring opening polymerization of D,L-lactide with appropriate initiator (hexanol or fluorinated alcohols) as previously described [START_REF] Picheth | Echogenicity Enhancement by End-Fluorinated Polylactide Perfluorohexane Nanocapsules: Towards Ultrasound-Activable Nanosystems[END_REF][START_REF] Houvenagel | End-Chain Fluorination of Polyesters Favors Perfluorooctyl Bromide Encapsulation into Echogenic PEGylated Nanocapsules[END_REF][START_REF] Giuntoli | Fluoro-Functionalized PLA Polymers as Potential Water-Repellent Coating Materials for Protection of Stone[END_REF][START_REF] Böhmer | Preparation of Monodisperse Polymer Particles and Capsules by Ink-Jet Printing[END_REF] (reaction yield > 85%). The polylactide main chain is constituted by ~140 repetitive units containing different lengths of end fluorinated-groups: polymers are designated according to the terminal group chemistry and length of fluorinated units as PLA-C 6 H 13 , PLA-C 3 F 7 , PLA-C 6 F 13 , PLA-C 8 F 17 , PLA-C 11 F 23 and PLA-C 13 F 27 . They were characterized by 1 H and [START_REF] Houvenagel | End-Chain Fluorination of Polyesters Favors Perfluorooctyl Bromide Encapsulation into Echogenic PEGylated Nanocapsules[END_REF] F-NMR and exhibited a molecular weight around 18000-21000 g.mol -1 as assessed by NMR, with a narrow dispersity (Supporting information Figure S1 and Table S1).

Polymers dynamics

To explore the role and extent of fluorous interactions between the different PLA-C x F 2x+1 and PFH during the formation of microcapsules, we have investigated interfacial properties, dynamics in solution as well as morphological features generated by each of these polymers.

As fluorine recognition and compartmentalization are strictly dependent on the solvent's character, the behavior and impact of polymer end-group chemistry was studied in two distinct solvents usually employed in the formulation of MC, namely, chloroform and dichloromethane [START_REF] Horváth | Fluorous Biphase Chemistry[END_REF][START_REF] Koda | Amphiphilic/Fluorous Random Copolymers as a New Class of Non-Cytotoxic Polymeric Materials for Protein Conjugation[END_REF] .

The interfacial activity of the polymers was determined at the solvent/PFH and solvent/sodium cholate (SC) interfaces by interfacial surface tension measurements (Table 1). At the solvent/SC interface, interfacial tension values were between 7-8 mN.m -1 in CHCl 3 or CH 2 Cl 2 , for all polymers, suggesting no specific solvent or end-group contribution to the interfacial activity. At the PFH/solvent interface, polymers dissolved in CHCl 3 exhibited an interfacial tension average value of 3.0±0.05 mN.m -1 whereas 6.0±0.1 mN.m -1 was measured for polymers dissolved in CH 2 Cl 2 , indicating that CHCl 3 favors PFH spreading. Nonetheless, none of the polymers was able to decrease the interfacial tension compared to the solvent itself regardless the end-group chemistry or the length of the fluorinated end-group. The lack of adsorption at the PFH/solvent interface might be explained by the mass ratio difference between the relatively small fluorinated blocks compared to the PLA main chain ), a conformational rearrangement based on the higher solubility of the polymeric chains within the solvents may hide the fluorinated groups inside the PLA coil, hindering an effective interaction with the PFH phase [START_REF] Houvenagel | End-Chain Fluorination of Polyesters Favors Perfluorooctyl Bromide Encapsulation into Echogenic PEGylated Nanocapsules[END_REF] .

Although no interfacial activity was observed at the PFH/solvent interface during static measurements, the solvent-evaporation process is dynamic and associated with transitory interfacial instabilities due to system disorganization [START_REF] Foster | Giant Biocompatible and Biodegradable PEG-PMCL Vesicles and Microcapsules by Solvent Evaporation from Double Emulsion Droplets[END_REF] . Such constant rearrangement of water and organic solvent molecules may promote the formation of fluorinated domains favorable for PFH encapsulation [START_REF] Berger | Organic Fluorine Compounds: A Great Opportunity for Enhanced Materials Properties[END_REF] . Indeed, many studies carried on mixed systems, containing both fluorocarbons and organic solvents, showed the tendency of perfluoralkyl moieties to self-associate and generate fluorous compartments. This aggregation in rich fluorous domains is generally associated to a reduced mobility of fluorinated groups [START_REF] Koda | Amphiphilic/Fluorous Random Copolymers as a New Class of Non-Cytotoxic Polymeric Materials for Protein Conjugation[END_REF][START_REF] Ojogun | Fluorophilicity of Alkyl and Polyfluoroalkyl Nicotinic Acid Ester Prodrugs[END_REF][START_REF] Shen | Blends of Polystyrene and Poly(n-Butyl Methacrylate) Mediated by Perfluorocarbon End Groups[END_REF] .

To assess the behavior of the fluorinated chains in a more dynamic media containing PFH - T 2 analysis revealed that the CF 3 resonance mobility decreased strongly for polymers functionalized with longer fluorinated units in the order C 3 F 7 >C 8 F 17 >C 13 F 27 for both solvents (Table 2). Such reduction in the local fluorine dynamics according to chain length is related to the strong electronegativity of the C-F bond that generates linear and rigid chain conformations, reducing the segmental mobility as the chain length increases [START_REF] Kadayakkara | F Spin-lattice Relaxation of Perfluoropolyethers: Dependence on Temperature and Magnetic Field Strength[END_REF] .

Nonetheless, samples dissolved in CDCl 3 showed smaller relaxation values compared to their counterparts prepared in CD 2 Cl 2 : this effect is clearly illustrated by the CF 3 resonance of PLA-C 3 F 7 , with T 2 of 540±50 ms in CDCl 3 and 1530±150 ms in CD 2 Cl 2. Such results were also confirmed by T 1 measurements (Supporting information Table S2). These significant changes in relaxation times indicate that chloroform is not a good solvent for the fluorinated end-groups and drives self-association of [START_REF] Houvenagel | End-Chain Fluorination of Polyesters Favors Perfluorooctyl Bromide Encapsulation into Echogenic PEGylated Nanocapsules[END_REF] F containing units. This lower mobility denotes a polymer rearrangement in presence of PFH evidencing an increased tendency for the fluorinated segments to associate with each other (Figure 2). According to those results, the introduction of PFH was able to reduce the local endfluorinated group mobility of PLA-C 3 F 7 in CDCl 3 . As PFH is well dissolved in chloroform, its contact with the fluorinated units may lead to the formation of fluorophilic domains, a process that might also depends on the length of the fluorinated group. This scenario, however, illustrates the system's behavior only in stable organic solutions; during the solventevaporation process in which MCs are formulated, transitory and thermodynamically unstable interfaces are created. Therefore, the ability of each fluorinated derivative to better stabilize and retain PFH as a liquid core was further studied in MCs formulations.

Indeed, in

Solvent Influence on MC formulation

The impact of the fluorinated end-groups on the final morphology of microcapsules was then studied for all synthesized polymers. All microcapsules were initially formulated with fixed amounts of polymer (50 mg) that were dissolved either in chloroform or dichloromethane and subsequently submitted to the same solvent-evaporation process. The encapsulation efficiency (EE) of PFH was quantified in freeze-dried samples by [START_REF] Houvenagel | End-Chain Fluorination of Polyesters Favors Perfluorooctyl Bromide Encapsulation into Echogenic PEGylated Nanocapsules[END_REF] F-NMR and the size of all MCs was measured by confocal microscopy as listed in Table 3. As determined by [START_REF] Houvenagel | End-Chain Fluorination of Polyesters Favors Perfluorooctyl Bromide Encapsulation into Echogenic PEGylated Nanocapsules[END_REF] F NMR, the non-fluorinated polymer, PLA-C 6 H 13 , presented an encapsulation efficiency of 5.5±0.1% when prepared in CHCl 3 . The use of fluorinated polymers led to a 2-fold increase in PFH encapsulation efficiency, reaching 11±3.0% without any significant distinction as a function of the fluorinated end-group length (Table 3). The reduced end-group mobility of fluorinated polymers in CDCl 3 containing PFH was translated into higher encapsulation efficiencies. Altogether, these results evidence the ability of fluorinated polymers to interact with the fluorocarbon phase and enhance its stabilization inside the organic phase, as previously reported for nanocapsules [START_REF] Picheth | Echogenicity Enhancement by End-Fluorinated Polylactide Perfluorohexane Nanocapsules: Towards Ultrasound-Activable Nanosystems[END_REF] . For microcapsules prepared in CH 2 Cl 2 , no trend was observed for the encapsulation efficiency, probably due the absence of specific interactions between PFH and the polymer (Table 2).

Nonetheless, PLA or PLGA polymers are able to encapsulate higher amounts of PFH into MCs (~40%) [START_REF] Mousnier | Influence of Polymer End-Chemistry on the Morphology of Perfluorohexane Polymeric Microcapsules Intended as Ultrasound Contrast Agents[END_REF] , the discrepancy with our results may be related to the fast process of solvent evaporation employed here, whereas Mousnier et al. have evaporated the organic solvent slowly at ambient pressure [START_REF] Mousnier | Influence of Polymer End-Chemistry on the Morphology of Perfluorohexane Polymeric Microcapsules Intended as Ultrasound Contrast Agents[END_REF] .

Although all samples were predominately smooth as shown by SEM in Figures 3 and4 (bottom), a certain amount of rough shelled MCs were also observed in all images.

According to Chung, et al., this morphological variation might be related to the fast evaporation process used, which promoted an uneven polymer concentration gradient towards the interface [START_REF] Chung | Effects of the Rate of Solvent Evaporation on the Characteristics of Drug Loaded PLLA and PDLLA Microspheres[END_REF] . Also, a partial PFH vaporization during the solvent evaporation step probably reflected the reduced extent of interactions between the end-groups and PFH.

Nonetheless, such samples were still able to alter the nucleation pattern, inducing the PFH to accumulate towards the drop's periphery, also suggesting the fluorinated segregation effect.

The obtained decentered morphologies are, however, favored by the solvent's lower boiling point (39.6 °C for CH 2 Cl 2 vs. 61.2 °C for CHCl 3 ) that accelerates the onset of polymer precipitation.

In chloroform, the shell of polymers with longer fluorinated groups precipitates before the complete fluorocarbon nucleation restraining PFH diffusion and causes multiple PFH cores dispersed inside the rigid polymeric phase. As disclosed by T 2 results, the PFH is able to interact more efficiently with the fluorinated end-groups in this solvent. Thus, such structure is probably attributable to formation of F-rich domains driven by the end-group polymer arrangement to minimize contact with the surrounding polymer. This effect indicates that longer fluorinated units interact more importantly with PFH.

Influence of polymer concentration on MC morphology

To further evaluate the end-group chemistry and length influence on microcapsules structure,

Conclusion

We have successfully synthesized and characterized poly(lactide) end-group fluorinated polymers for improved fluorous interaction with PFH. The presence of fluorinated end-groups provided more favorable interfaces with perfluorohexane in organic solution of chloroform with fluorinated end-group length dependency, as revealed by spin-spin relaxation measurements. The formation of fluorophilic environment is reflected with improved perfluorohexane encapsulation efficiencies into microcapsules and by modifying the final morphology, particularly in the case of longer fluorinated end-groups and important polymer masses.

Acknowledgements

This

(C 3

 3 to C 13 for fluorinated block vs. 140 repetitive units for PLA) and the conformation assumed by the polymer in solution. Indeed, as the PLA polymers are well solvated in CHCl 3 and CH 2 Cl 2 (Hildebrand solubility parameter ( ) of 20.8 MPa 0.5 for unmodified PLA, 18.7 MPa 0.5 for CHCl 3 and 20.2 MPa 0.5 for CH 2 Cl 2 33

a 1 .

 1 situation similar to what happens prior to solvent evaporationthe end-group mobility of three fluorinated polymers, PLA-C 3 F 7 , PLA-C 8 F 17 and PLA-C 13 F 27 , was analyzed by fluorine NMR spectroscopy employing spin-lattice (T 1 ) and spin-spin (T 2 ) relaxation measurements at fixed polymer concentration of 25 mg.mL -1 and 15 µL of PFH (corresponding to the formulation conditions). Accordingly, the CF 3 group dynamics in each solvent was probed in presence and absence of PFH in the solvent. Unfortunately, some of the PFH resonances overlapped with the CF 3 polymer signals for PLA-C 8 F 17 and PLA-C 13 F 27 in both solvents. Only PLA-C 3 F 7 exhibited well segregated resonance from those of PFH because of the different chemical environments, which permitted a resonance shift towards lower magnetic fields, not overlapping with PFH as displayed in Figure The nomenclature and 19 F-NMR peak labeling consists alphabetically with the position of the fluorinated resonance respective to the PLA chain[START_REF] Marchione | Complete Multinuclear Magnetic Resonance Characterization of a Set of Polyfluorinated Acids and Alcohols[END_REF][START_REF] Twum | Characterization of the Chain-Ends and Branching Structures in Polyvinylidene Fluoride with Multidimensional NMR[END_REF] .

Figure 1 .

 1 Figure 1. Magnified CF 3 and CF 2 regions of the 19 F-NMR spectra for PLA-C 3 F 7 , PLA-C 8 F 17 and PLA-C 13 F 27 in CDCl 3 . The insets (left) exhibit the CF 3 resonance in a mixed system of polymer and perfluorohexane. 19 F peak assignment is indicated in the top of each image.

  CHCl 3 , addition of PFH further decreases the solubility of the fluorinated segments which promotes dipolar interactions between the fluorinated end-group and PFH. These more efficient interactions in CHCl 3 suggest formation of F-rich domains where the reduced mobility of the fluorinated segment leads to shorter T 2 values.

Figure 2 .

 2 Figure 2. T 2 decay profiles for the CF 3 resonance of PLA-C 3 F 7 in CDCl 3 (A) and CD 2 Cl 2 (B) without (open circles) and with perfluorohexane (dark circles).

(

  performed at 30 °C) may have caused a volumetric expansion of the MCs and, as the temperature dropped, a subsequent shrinkage altered the continuous spreading of the polymer at the interface as previously reported by Lebeveda et al. for PFH containing microspheres 42 .

Figure 3 .

 3 Figure 3. Confocal (top) and scanning electron microscopy (bottom) images of

Figure 4 .

 4 Figure 4. Confocal (top) and scanning electron microscopy (bottom) images of

Figure 5 .

 5 Figure 5. Schematic representation of the perfluorohexane phase-separation and nucleation

6 .

 6 samples PLA-C 6 H 13 , PLA-C 3 F 7 , PLA-C 8 F 17 and PLA-C 13 F 27 were prepared only in chloroform using different polymer masses (12.5, 25, 50 and 100 mg) and constant PFH volume of 30 µL. Samples prepared with 12.5 mg presented core-shell structures with thin and smooth polymer layers that were predominant for all polymers, independently of end-group chemistry or length as shown in Figure The increase of polymer mass to 25 mg altered only the morphology of MCs having the longest fluorinated group: PLA-C 13 F 27 by formation of multinucleated capsules (42±10%) as displayed by the white arrow in Figure 6. The percentage of multiple PFH cores for PLA-C 13 F 27 became more obvious for 50 and 100 mg,in which the total amount of multinucleated MCs increased from 60±5% to 91±3%, respectively. The same effect but at a lower extent was observed for PLA-C 8 F 17 , as thinshelled MCs are observed in the formulations containing 25 mg and multinucleated morphologies increased from 10±2% to 56±7% for 50 and 100 mg, respectively. A similar behavior was observed for MC formulated with the polymer containing the shortest fluorine end-group, PLA-C 3 F 7 , as core-shell structures predominated until a polymer mass of 100 mg, in which a total of 45±5% of multinucleated MC were found. In contrast, MC formulated with the non-fluorinated sample, PLA-C 6 H 13 , exhibited core-shell morphologies for all of the tested polymer masses.

Figure 6 .

 6 Figure 6. Confocal microscopy images of microcapsules formulated in chloroform with non-

1 H

 1 work has been supported by the Région Ile-de-France in the framework of DIM Nano-K and the French National Research Agency (ANR) under grant ANR-10-NANO-06 as part of the "Investissements d'Avenir" program. Institut Galien Paris-Sud is a member of the Laboratory of Excellence LERMIT supported by a grant from ANR (ANR-10-LABX-33). We are also thankful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the financial support, to Valerie Nicolas from MIPSIT for helping with confocal experiments, to Prof. Jaísa Soares as well as Prof. Jens Dittmer for their help with NMR relaxation experiments and to Carine Van-Heijenoort, Christina Sizun and Marc-André Delsuc for fruitful discussions. and 19 F-NMR spectra of all synthesized polymers in CDCl3. SEC chromatogram of all polymers in chloroform at 30°C. Number-average molar masses (Mn) obtained by SEC/1H-NMR, dispersity and glasstransition temperature (Tg) of all polymers. Spin-lattice relaxation (T1) values of correspondent 19 F resonances for samples dissolved in CDCl3 and CD2Cl2 with and without perfluorohexane.

PLA-C 3 F 7 , PLA-C 6 F 13 , PLA-C 8 F 17 , PLA-C 11 F 23 and PLA-C 13 F 27 :

	H-NMR
	[400 MHz, CDCl 3 , 25°C] PLA-C 6 H 13 : δ H 5.10-5.28 (PLA-CHCH 3 COO-), 1.52-1.61 (PLA-
	CHCH 3 COO-), 0.88-0.92 (-CH 2 CH 3 ). δ H 5.10-5.28 (PLA-CHCH 3 COO-), 4.50-4.70 (-OCH 2 CF 2 -), 1.52-1.61 (PLA-
	CHCH 3 COO-). 19 F-NMR [400 MHz, CDCl 3 , 25°C] PLA-C 3 F 7 : δ F -79.0 (-CF 3 ), -118.7 (-
	OCH 2 CF 2 -) and -125.7 (-CF 2 CF 3 );

PLA-C 6 F 13 , PLA-C 8 F 17 , PLA-C 11 F 23 and PLA-C 13 F 27 : δ F - 78

  .9 (-CF 3 ), -117.7 (-OCH 2 CF 2 -), -120.0 (-(CF 2 ) n CF 2 CF 3 ), -120.7 (-OCH 2 CF 2 CF 2 CF 2 -), -121.4 (-OCH 2 CF 2 CF 2 -) and -124.2 (-CF 2 CF 3 ).

Table 1 .

 1 Interfacial tensions at the perfluorohexane/solvent and alkylated or fluorinated polymer/sodium cholate 1.5% (w/v) interfaces. All polymers were dissolved at 25 mg.mL -1 in chloroform or dichloromethane. All results are expressed in mN.m -1 .

			Chloroform	Dichloromethane
		PFH/solvent Solvent/SC 1.5% PFH/solvent Solvent/SC 1.5%
	Solvent	3.2±0.3	7.5±0.3	6.0±0.3	8.1±0.2
	PLA-C 6 H 13	3.0±0.5	7.5±0.2	6.2±0.5	8.0±0.2
	PLA-C 3 F 7	3.0±0.3	7.0±0.1	6.1±0.7	7.5±0.3
	PLA-C 6 F 13	3.0±0.5	7.1±0.5	6.2±0.4	7.7±0.5
	PLA-C 8 F 17	3.1±0.4	7.1±0.2	5.9±0.4	7.7±0.4
	PLA-C 11 F 23	3.0±0.3	7.2±0.3	5.9±0.3	7.8±0.2
	PLA-C 13 F 27	3.1±0.4	7.4±0.1	6.1±0.4	7.6±0.1
	PFH-SC (1.5% w/v) interfacial tension was 22.5±0.2 mN.m	-1

Table 2 .

 2 [START_REF] Houvenagel | End-Chain Fluorination of Polyesters Favors Perfluorooctyl Bromide Encapsulation into Echogenic PEGylated Nanocapsules[END_REF] F spin-spin relaxation (T 2 ) values of CF 3 resonances for samples dissolved in deuterated chloroform or dichloromethane in presence of absence of perfluorohexane. All polymers were dissolved at fixed concentration of 25 mg.mL -1 (Experiment performed in duplicate, n=2).

	Sample	T 2 (ms)	T 2 (ms)
		CDCl 3 CDCl 3 +PFH	CD 2 Cl 2	CD 2 Cl 2 +PFH
	PLA-C 3 F 7	540±50	81±15	1530±150	1710±250
	PLA-C 8 F 17 210±20	---	1263±165	---
	PLA-C 13 F 27 76±10	---	790±50	---

Afterwards, PFH was added to each polymer solution to simulate the system behavior just before emulsification with sodium cholate. In CD 2 Cl 2 , T 2 varied from 1530±150 ms to 1710±250 ms in presence of PFH, showing that the end-group dynamics is not significantly affected by PFH addition in the media (p = 0.475). However, in CDCl 3 , the CF 3 signal of PLA-C 3 F 7 showed a significant T 2 decrease with PFH, from 540±50 ms to 81±15 ms (p <0.05).

Table 3 .

 3 Encapsulation efficiency and size of microcapsules formulated with 50 mg of polymer prepared in chloroform and dichloromethane.

	Sample		Chloroform	Dichloromethane
		Size (µm)*	PFH EE (%)	Size (µm)*	PFH EE (%)
	PLA-C 6 H 13	4.2±1.0	5.5±0.2	4.8±0.8	12.3±0.2
	PLA-C 3 F 7	4.4±0.6	10.1±0.2	4.7±0.8	9.1±0.1
	PLA-C 6 F 13	4.8±1.0	12.7±0.2	4.6±1.0	13.1±1.3
	PLA-C 8 F 17	4.6±0.8	10.3±0.5	4.9±1.2	11.1±0.2
	PLA-C 11 F 23	5.2±1.4	11.1±1.4	4.8±0.9	7.1±0.1
	PLA-C 13 F 27	5.2±1.0	11.4±1.5	5.3±0.6	10.0±0.7

*All measurements correspond to observation and analysis of at least 100 microcapsules per each group.