Guilherme Picheth

Sophie Houvenagel

Camille Dejean

Olivier Couture

Rilton Alves De Freitas

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

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

Echogenicity enhancement by end-fluorinated polylactide perfluorohexane nanocapsules: Towards ultrasound-activable nanosystems

Keywords: Nanocapsules, perfluorohexane, fluorinated polymers, ultrasound imaging, focused ultrasound, fluorous interactions; cell viability

Introduction

Ultrasound contrast agents (UCAs) are efficient intravascular echo-enhancers currently indispensable to safe and accurate diagnosis of many diseases, including kidney cists [START_REF] Chang | Management of Indeterminate Cystic Kidney Lesions: Review of Contrast-enhanced Ultrasound as a Diagnostic Tool[END_REF], acute myocardial ischemia [START_REF] Kaul | Microvasculature in Acute Myocardial Ischemia: Part I. Evolving Concepts in Pathophysiology[END_REF] and solid tumors [START_REF] Quaia | Characterization of Focal Liver Lesions with Contrast-specific US Modes and a Sulfur Hexafluoride-filled Microbubble Contrast Agent: Diagnostic Performance and Confidence[END_REF]. All the commercially available UCAs, such as Definity ® and Sonovue ® , are microbubbles constituted by a perfluorinated gaseous-core stabilized by a monolayer of phospholipids [START_REF] Borden | Lateral Phase Separation in Lipid-Coated Microbubbles[END_REF]. Unfortunately, the imaging and therapeutic applications of such materials are severely limited by [START_REF] Chang | Management of Indeterminate Cystic Kidney Lesions: Review of Contrast-enhanced Ultrasound as a Diagnostic Tool[END_REF] the fast-diffusing gas component, responsible for a relatively short distribution half-life in the bloodstream (~60s after bolus injection) and an elimination half-life of about 6 minutes [5,[START_REF] Schneider | Characteristics of SonoVue(TM)[END_REF], and (2) their inherent micrometer size range that prevents extravasation into solid tumors.

To overcome these limitations, research has focused on nanosystems that can be more stable and are able to diffuse beyond the vascular compartment. Since gas nanobubbles are difficult to stabilize [START_REF] Leroy | Investigating the Existence of Bulk Nanobubbles with Ultrasound[END_REF], the strategy has consisted in encapsulating liquid perfluorocarbons (PFCs) into nanoemulsions using surfactants or in polymer nanocapsules [START_REF] Cosco | Perfluorocarbon-loaded micro and nanosystems for medical imaging: A state of the art[END_REF]. Although interesting, this approach leads to a significant reduction of the echogenicity. Indeed, as the scattering cross section of a particle is defined according to equation 1, a reduction of size and the replacement of the gas core by a liquid one, induce a decrease of the scattering cross section and subsequently of echogenicity. , Eq. 1

where k is the wavenumber, R is the radius of the particle, κ d and κ the compressibilities of respectively the particle and the medium, ρ d and ρ the densities of respectively the particle and the medium [START_REF] Dejong | Principles and Recent Developments in Ultrasound Contrast Agents[END_REF].

To provide strong and long-lasting ultrasonic echoes, liquid PFCs of low boiling point, such as perfluorohexane (PFH) or perfluoropentane, have been selected for their ability to experience a liquid-to-gas transition once exposed to high acoustic pressures [START_REF] Duncanson | Microfluidic Fabrication of Perfluorohexane-Shelled Double Emulsions for Controlled Loading and Acoustic-Triggered Release of Hydrophilic Agents[END_REF]. During this phenomenonknown as acoustic drop vaporization (ADV)the PFC vapor phase undergoes oscillations around an equilibrium radius, which enhances the ultrasound scattering intensity of nanometric systems by several orders of magnitude [START_REF] Kripfgans | Acoustic droplet vaporization for therapeutic and diagnostic applications[END_REF][START_REF] Nami | Nanoparticles with Multiple Perfluorocarbons for Controllable Ultrasonically Induced Phase Shifting[END_REF][START_REF] Rapoport | Ultrasound-mediated tumor imaging and nanotherapy using drug loaded, block copolymer stabilized perfluorocarbon nanoemulsions[END_REF][START_REF] Sheeran | Design of ultrasonically-activatable nanoparticles using low boiling point perfluorocarbons[END_REF][START_REF] Shpak | Acoustic droplet vaporization is initiated by superharmonic focusing[END_REF][START_REF] Guédra | A model for acoustic vaporization of encapsulated droplets[END_REF]. An advantage of nanocapsules over microbubbles is their ability to co-encapsulate a drug for further therapeutic use [START_REF] Boissenot | Paclitaxel-loaded PEGylated nanocapsules of perfluorooctyl bromide as theranostic agents[END_REF]. As previously reported, PFC-containing nanocapsules can preserve their integrity and initial diameter after intravenous administration and passively accumulate in tumor tissues through the enhanced permeability and retention effect (EPR) [START_REF] Boissenot | Paclitaxel-loaded PEGylated nanocapsules of perfluorooctyl bromide as theranostic agents[END_REF][START_REF] Maeda | Vascular permeability in cancer and infection as related to macromolecular drug delivery, with emphasis on the EPR effect for tumor-selective drug targeting[END_REF][START_REF] Diou | Long-circulating perfluorooctyl bromide nanocapsules for tumor imaging by 19FMRI[END_REF][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] provided this effect occurs in patients [START_REF] Danhier | To exploit the tumor microenvironment: Since the EPR effect fails in the clinic, what is the future of nanomedicine?[END_REF]. After reaching tumor tissue, the release of a co-encapsulated drug might be triggered by focused ultrasound (FUS), causing local PFC cavitation/vaporization and may be followed by capsule shell rupture with subsequent drug release [START_REF] Singh | Phase transitions of nanoemulsions using ultrasound: Experimental observations[END_REF]. This strategy is currently investigated to deliver higher drug concentrations in the tumor vicinity and increase the chemotherapy efficacy while avoiding unwanted toxicity to healthy cells [START_REF] Clark | CRLX101 nanoparticles localize in human tumors and not in adjacent, nonneoplastic tissue after intravenous dosing[END_REF]. As FUS is clinically employed to ablate and eradicate tumor cells, particularly in prostate [START_REF] Uchida | Improved Outcomes with Advancements in High Intensity Focused Ultrasound Devices for the Treatment of Localized Prostate Cancer[END_REF] and hepatocellular carcinomas [START_REF] You | Nanoparticleenhanced synergistic HIFU ablation and transarterial chemoembolization for efficient cancer therapy[END_REF], it represents an ideal trigger for drug delivery because it is a non-invasive technique, safe to adjacent tissues and provides a precise spatiotemporal control over the thermal and mechanical energy dissipation [START_REF] Bezagu | High spatiotemporal control of spontaneous reactions using ultrasound-triggered composite droplets[END_REF].

Before pushing forward these theranostic applications, the efficient encapsulation of low boiling point PFCs is required. Contrary to high boiling point PFCs such as perfluorooctylbromide, the entrapment of low boiling point PFCs such as PFH into polymeric nanosystems is a challenging process due to their fluorophilic character and high vapor pressure [START_REF] Pisani | Surfactant dependent morphology of polymeric capsules of perfluorooctyl bromide: Influence of polymer adsorption at the dichloromethane-water interface[END_REF][START_REF] Mousnier | Influence of polymer end-chemistry on the morphology of perfluorohexane polymeric microcapsules intended as ultrasound contrast agents[END_REF]. Perfluorinated compounds are usually immiscible with hydrophilic or hydrophobic solvents due to favored interactions between fluorinated domains [START_REF] Koda | Fluorinated Microgels in Star Polymers: From In-Core Dynamics to Fluorous Encapsulation[END_REF].

Accordingly, perfluorinated liquids tend to phase-separate, resulting in low encapsulation efficienciesfrom 3 to 9% in nano-sized formulations [START_REF] Srinivas | Customizable, multifunctional fluorocarbon nanoparticles for quantitative in vivo imaging using F-19 MRI and optical imaging[END_REF][START_REF] Li | Phase-transition contrast nanocapsules triggered by low-intensity ultrasound[END_REF] which represents a limitation for ultrasound imaging and ultrasound-triggered drug delivery. To promote better PFH encapsulation into nanocapsules, we have synthesized polylactide (PLA) polymers terminated by linear fluorinated end-groups of distinct lengths. The presence of the fluorinated moiety is expected to increase PFH encapsulation efficiency by playing on fluorous-fluorous interactions as it was observed for another PFC: perfluorooctyl bromide [START_REF] Houvenagel | End-chain fluorination of polyesters favors perfluorooctyl bromide encapsulation into echogenic PEGylated nanocapsules[END_REF]. Polymer synthesis, nanocapsule formulation and characterization are reported. Finally, the nanocapsule echogenicity and ability to be destroyed by focused ultrasound is evaluated.

Materials and Methods

Materials

D,L-lactide was purchased from Polysciences (Germany) and perfluorohexane (purity>98%) was acquired from Alfa Aesar (Germany). 1-hexanol from Acros Organics (Belgium), 2,2,3,3,4,4-heptafluoro-1-butanol, 1H,1H perfluoro-1-heptanol, 1H,1H perfluoro-1-nonanol, 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). Stannous octoate, dry toluene, D 2 O, sodium cholate, trifluoroacetic acid (TFA) and polyvinyl alcohol (PVA, 30-70kDa, 87-90% hydrolyzed) were provided by Sigma-Aldrich (France). Deuterated solvents (CDCl3 and acetone-d) were purchased from Eurisotop (France), Cell culture reagents such as DMEM (Dulbecco's modified Eagle's medium), RPMI 1640 (Roswell Park Memorial Institute medium), FBS (Fetal Bovine Serum), trypsin-EDTA solution and PBS (Ca 2+ and Mg 2+ free phosphate buffer) were purchased from Sigma Aldrich (France). The ultrapure water was produced by a Millipore Synergy 185 apparatus coupled with a RiOs5 TM (Millipore, France) with a resistivity of 18.2 MΩ.cm. 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 non-fluorinated (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 | Synthesis, 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, 1.5g) and corresponding initiator (0.075 mmol) -1-hexanol for PLA-C 6 H 13 , 2,2,3,3,4,4-heptafluoro-1butanol for PLA-C 3 F 7 , 1H,1H perfluoro-1-heptanol for PLA-C 6 F 13 , 1H,1H perfluoro-1nonanol for PLA-C 8 F 17 , 1H,1H perfluoro-1-dodecanol for PLA-C 11 F 23 or 1H,1Hp-1tetradecanol 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 0.5h 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 (5mL), whereas the fluorinated polymers were dissolved in acetone (20 mL) and precipitated again in ultrapure water (150 mL). The samples were freeze-dried for 24h 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 19 F-NMR spectra were obtained on a Bruker Advance 400 MHz in CDCl 3 at room temperature.

Differential Scanning Calorimetry (DSC)

All polymers were sealed in aluminum pans (50 μL) and placed in a DSC apparatus (DSC Q1000, TA Instruments). All analyses were conducted under nitrogen flow (20 mL.min -1 ) in a temperature range between 20 and 70°C at a heat and cooling rate of 20°C.min -1 . The glass transition temperature was determined during the second heating run.

Nanocapsules Formulation

Nanocapsules (NCs) were prepared by the emulsion-evaporation technique, as previously described with modifications [START_REF] Diou | Long-circulating perfluorooctyl bromide nanocapsules for tumor imaging by 19FMRI[END_REF]. Briefly, 50 mg of polymer was dissolved in 2 mL of chloroform in a 50 mL glass flask that 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 in a vortex for 1 min. The mixture was then submitted to probe sonication (Branson digital sonifier, France) at 30% of maximal power for 1 min over ice. The solvent was evaporated by magnetic stirring at 300 RPM during 4h in a thermostated bath at 20°C. The samples were then filtered on 0.45 µm PVDF filters (Whatman) and incubated with PVA 1% w/w for 5 days at 4°C. Finally, they were centrifuged at 27.240 g (Optima LE-80K Ultracentrifuge Beckman Coulter, France) during 1h at 4°C and the pellet was resuspended in 5 mL of ultrapure water to yield a final polymer concentration of 25 mg.mL -1 .

Size and ζ-potential

The hydrodynamic diameter, polydispersity index (PdI) and zeta potential of nanocapsules were measured using a Zetasizer Nano ZS (Malvern, France) at 20°C. All suspensions were diluted 1:10 in ultrapure water for size measurements and 1 mM NaCl for zeta potential measurements.

Electron Microscopy

Scanning electron microscopy was performed using a Merlin 6040 (Carl Zeiss, Germany) operating at 3 kV. The nanocapsules were deposited on carbon conductive double-sided tape (Euro-medex, France) and dried at room temperature. Afterwards, they were coated with a palladium-platinium layer of about 3 nm using a Cressington sputtercoater 208HR with a rotary-planetary-tilt stage, fitted with an MTM-20 thickness controller.

Transmission electron microscopy (TEM) images were obtained in a JEOL 1400 (Jeol Ltd, USA) operating at 80 kV. All nanocapsules suspensions were diluted to a final concentration of 1 mg/mL polymer in ultrapure water and a 5 μL droplet was deposited on a glow discharged carbon-coated 200 mesh grid. Negative staining was performed using 2% phosphotungstic acid (w/v). All images were acquired with an Orius camera (Gatan Inc, USA).

Cryo-Transmission Electron Microscopy (Cryo-TEM) images were performed using a JEOL 1400 TEM/STEM operating at 120 kV. NC suspensions at 50 mg.mL -1 polymer were deposited (5 μL) on a glow discharged carbon grid (Lacey 200 mesh), automatically blotted for 5 s and plunged into liquid ethane at -175°C by a grid plunge freezer (Leica EM GP). The samples were kept in liquid nitrogen until analysis. Next, they were transferred to a pre-cooled cryo-TEM holder (Gatan 914) and observed under 10 pA/cm 2 of luminosity, 2 s of exposition and -2 μm of defocus. All images were acquired with a US1000XP camera (Gatan Inc, USA).

PFH encapsulation efficiency

For PFH quantification, 500 μL of NC suspension freshly prepared was transferred to NMR tubes loaded with a stem coaxial insert containing TFA in D 2 O (12 µmol.mL -1 ) as external standard [START_REF] Houvenagel | End-chain fluorination of polyesters favors perfluorooctyl bromide encapsulation into echogenic PEGylated nanocapsules[END_REF][START_REF] Henderson | Quantitative NMR Spectroscopy Using Coaxial Inserts Containing a Reference Standard: Purity Determinations for Military Nerve Agents[END_REF].Freeze-dried samples were also analyzed: 1 mL of freshly prepared nanocapsule suspension was freeze-dried during 24h employing an Alpha-1-2 LD apparatus (Christ, France). Afterwards, they were hermetically sealed with a rubber cap and stored at -20°C until analysis. A total volume of 1 mL of chloroform then 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 1000 RPM. The organic solution was collected in a cold room at 4°C and introduced into an NMR tube loaded with the stem coaxial insert containing TFA in D 2 O (12 µmol.mL -1 ). All spectra were obtained by a Bruker Avance 300 (400 MHz) operating at 4°C. The total amount of encapsulated PFH was determined based on a PFH calibration curve. For each NC 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.

Cell viability

The cytotoxicity of all NCs was evaluated in two distinct cell lines obtained from ATCC (USA). Human endothelial umbilical vein cells (HUVEC) were cultivated in DMEM supplemented with 50 U.mL -1 of penicillin, 50 U.mL -1 of streptomycin and 10% FBS. Murine macrophage-monocytes (J774.A1cell line) were cultivated in RPMI 1640 medium supplemented with 50 U.mL -1 of penicillin, 50 U.mL -1 of streptomycin and 10% heat inactivated FBS. All cells were cultivated at 37°C at 5% CO 2 in a humidified atmosphere; they were splitted twice a week by trypsinisation for HUVEC and scraping for J774.A1. To assess the in vitro cytotoxicity, the MTT assay that evaluates the mitochondrial activity was employed [START_REF] Gerlier | Use of MTT colorimetric assay to measure cell activation[END_REF]. A total volume of 100 µL of the cells suspension was seeded into 96 well plates (TPP, Switzerland) at a concentration of 8x10 3 cells/well or 2x10 3 cells/well for 24 and 72h of incubation, respectively. The cells were pre-incubated for 24h and 100 µL of a serial dilution of NCs (1.0x10 -2 to 10 mg.mL -1 ) was added to the medium. After 24 or 72h of incubation, 20 µL of MTT solution at 5mg/mL(3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) (Sigma, France) was added to each well. After 1hof incubation, the medium was then aspired and replaced by 200 µL of mL dimethylsulfoxide (ACS grade, BioBasic Inc, France). The absorbance was measured with a microplate reader (LAB Systems Original Multiscan MS, Finland) at 570 nm and the cell viability was calculated according to Eq. 2, where [A] control corresponds to the absorbance of untreated control cells:

Eq. 2

In vitro Ultrasound Characterization

Ultrasound images were acquired in a degassed water bath maintained at 37°C equipped with acoustic absorbing rubber. All NC were previously degassed and 0.8 mL were transferred to a silicone tube to a final 50 mg.mL -1 suspension. Suspensions of Sonovue ® 0.1%, PVA 0.7% and nanoparticles (NP) produced with PLA-C 8 F 17 (without PFH)

were used as controls. In vitro ultrasound imaging experiments were performed using an ultrafast programmable ultrasound scanner (Aixplorer, Supersonic Imagine, Aix-En-Provence, France). The scanner was equipped with a 5 MHz linear array transducer (128 elements, 0.2 mm pitch) able to emit and accumulate a series of plane waves. The US focus was placed at the center of the silicone tube relative to the transducer at 3 cm depth.

The pulse repetition frequencies used in this experiment was 50 Hz. The images were obtained using a plane-wave B-mode sequence [START_REF] Couture | Ultrafast Imaging of Ultrasound Contrast Agents[END_REF] at 0.1 MPa peak negative pressure.

Conventional focused B-mode (0.1 MPa) and contrast specific mode (~0.2 MPa, executed by pulse inversion and amplitude modulation) images were initially performed for each sample independently. Imaging and monitoring pulses were 1 cycle long at 5 MHz. Finally, a disruption pulse was emitted (~5 MPa peak negative pressure, 100 cycles long, which corresponds to 20 µs) and the imaging process was repeated, with 65 B-mode images for the total 140 ms pulse sequence. Plane-wave images were acquired at a frame rate of 100

Hz and all data was transferred to a computer for analysis at Matlab (Mathworks, Natick, MA, USA). The backscattered intensity of the samples was obtained by averaging the power of the first and last image of the video in the region-of-interest (the silicone tube).

The resulting intensities were subtracted by the backscattered intensity obtained with the tube filled with Milli-Q water.

Insert Figure 1 here

Focused Ultrasound

Focused ultrasound experiments were performed to assess the ability to destroy NCs. 100 µL of 5 mg.mL -1 NC suspension were transferred to a 96 well microplate (OptiCell ® ) that was placed in a support positioned at the center of the chamber. A 2.25 MHz transducer (Imasonics, Voray-sur-l'Ognon, France) was focused (38 mm f/d=1) separately at each microplate well and single bursts of 5 cycles were transmitted by a waveform generator and amplified to 12.3 MPa peak-negative pressure by a radio frequency amplifier [START_REF] Bezagu | High spatiotemporal control of spontaneous reactions using ultrasound-triggered composite droplets[END_REF].

Results and discussion

Synthesis and characterization of fluorinated polymers

Polylactide polymers functionalized with either alkyl or fluorinated end-groups of five distinct lengths were synthesized by ring opening polymerization of D,L-lactide as described previously [START_REF] Houvenagel | End-chain fluorination of polyesters favors perfluorooctyl bromide encapsulation into echogenic PEGylated nanocapsules[END_REF]. The end-groups are composed of linear carbon chains substituted by fluorine or hydrogen atoms; henceforth, the synthesized polymers were designated according to the terminal group chemistry and length of fluorinated unit as PLA-C 6 H 13 , PLA- As shown in Table 1, a relative good agreement between the experimental molar masses determined by SEC and the molar masses targeted at 20 000 g.mol -1 for all the derivatives was observed (Supplementary infromation Figure S3). The M n was also determined by 1 H-NMR analysis on the basis of the integration ratio of peak at 5.23 ppm (methine proton of lactide) and 4.55 -4.74 ppm (methylene protons adjacent to the CF chain) (Figure 2A). Slightly lower M n were calculated than using SEC, around 18 000 g.mol - 1 , which may be attributed to the difference of polymer conformation with poly(methyl methacrylate) standards.

The end-modified polymers were also characterized by DSC. They all displayed the same glass-transition temperature (T g ), from 50 to 53ºC, similar to unmodified PLA polymers of similar molar mass (Table 1) [START_REF] Giuntoli | Fluoro-functionalized PLA polymers as potential water-repellent coating materials for protection of stone[END_REF][START_REF] Chen | Preparation and characterization of biodegradable PLA polymeric blends[END_REF]. Hence, the small molecular weight endgroup did not impact the T g of high molar mass polylactide.

Insert Figure 2 here Insert Table 1 here

PFH Nanocapsules

Fully fluorinated liquid perfluorocarbons usually display high vapor pressure values -29.1 kPa at 25°C for PFHand restricted solubility in organic solvents due to their fluorophilic character [START_REF] West | CO2-Induced Miscibility of Fluorous and Organic Solvents for Recycling Homogeneous Catalysts[END_REF]. As a result, they tend either to phase separate or to evaporate during the formulation of nano or microcapsules by solvent emulsion-evaporation, reducing the PFC encapsulation efficacy [START_REF] Pisani | Surfactant dependent morphology of polymeric capsules of perfluorooctyl bromide: Influence of polymer adsorption at the dichloromethane-water interface[END_REF][START_REF] Mousnier | Influence of polymer end-chemistry on the morphology of perfluorohexane polymeric microcapsules intended as ultrasound contrast agents[END_REF]. Therefore, fluorinated end-groups with distinct lengths were introduced into the PLA structure to promote "fluorous -fluorous" interactions between the polymer and liquid perfluorocarbons and enhance PFH encapsulation into nanocapsules. This strategy was shown efficient recently for a high-boiling point PFC:

perfluorooctyl bromide [START_REF] Houvenagel | End-chain fluorination of polyesters favors perfluorooctyl bromide encapsulation into echogenic PEGylated nanocapsules[END_REF]. The features of NCs formulated with fluorinated polymers, such as morphology, size, surface charge and PFH encapsulation efficiency were compared with samples produced with the non-fluorinated polymer PLA-C 6 H 13 .

Initial characterization demonstrated that all NC were spherical with smooth surfaces as shown by TEM and SEM images (Figure 3). The Z-average size was around 150 nmin agreement with electron microscopy observationswith a negative ζ-potential around -8 mV and a low polydispersity below 0.15, regardless of end-group chemistry or fluorinated length as displayed in Table 2.

Insert Table 2 here Insert Figure 3 Some capsules also presented a predominant lighter core or bright spots within the perfluorocarbon phase (white arrows in Figure 4). Such areas might arise from the freezing process: as PFH retracts when it solidifies, low PFH densities regions appear in the capsules with probable coexistence of solid PFH (dark) with gaseous PFH (light). After exposure to the electron beam radiation, partial melting of the solid PFH occurs and light regions becomes darker (Supplementary Information, Figure S4). Those observations are consistent with Lattin et al. that reported the increased frequency of bright nanoemulsion droplets for the lower boiling point perfluoropentane (Bp=29°C) in comparison with perfluorohexane (Bp=56°C) and perfluoroheptane (Bp=82°C) [START_REF] Lattin | Formation of eLiposomes as a drug delivery vehicle[END_REF] and from our own observations with PFOB [START_REF] Houvenagel | End-chain fluorination of polyesters favors perfluorooctyl bromide encapsulation into echogenic PEGylated nanocapsules[END_REF][START_REF] Diou | PEGylated nanocapsules of perfluorooctyl bromide: Mechanism of formation, influence of polymer concentration on morphology and mechanical properties[END_REF].

Insert Figure 4 here

The total encapsulated PFH was then quantified by 19 F-NMR for NC suspensions either freshly prepared or after freeze-drying. The samples prepared with the nonfluorinated polymer (PLA-C 6 H 13 ) exhibited an encapsulation efficiency of 9.2±0.1% for freshly prepared suspensions and 4.0±0.5% after freeze drying, in agreement with results obtained for nanocapsules formulated with regular PLGA polymers [START_REF] Srinivas | Customizable, multifunctional fluorocarbon nanoparticles for quantitative in vivo imaging using F-19 MRI and optical imaging[END_REF]. All NC formulated with F-polymers showed a 2-fold increase in the total PFH encapsulation compared to PLA-C 6 H 13 as exhibited in Figure 5. A maximum encapsulation efficiency of 15±1.0% for suspensions and a 9.5±0.8% plateau for freeze-dried samples was reached, although without significant difference as a function of fluorinated chain length. Obviously, the freezedrying process induced the loss of non-encapsulated PFH droplets, as previously observed for PFOB NCs [START_REF] Diou | PEGylated nanocapsules of perfluorooctyl bromide: Mechanism of formation, influence of polymer concentration on morphology and mechanical properties[END_REF], although no free PFH could be observed by cryo-TEM. These results suggest a preferential interaction between fluorinated polymers and PFH, independently of the fluorinated end-chain length, reducing the PFH loss during solvent evaporation and increasing the final encapsulation efficiency. Nonetheless, the PFH solubility and volatility still prevents higher encapsulation. Comparatively, mPEG-polycaprolactone polymers containing terminal C 3 F 7 groups were also successful in enhancing of PFH entrapment into NCs around ~15% as reported by Li et al. [START_REF] Li | Phase-transition contrast nanocapsules triggered by low-intensity ultrasound[END_REF].

Insert Figure 5 here

In vitro cytotoxicity of NCs

The use of fluorinated-compounds for biomedical applications is usually limited because of their persistence and consequent toxicity in the organism [START_REF] Riess | Highly fluorinated amphiphilic molecules and self-assemblies with biomedical potential[END_REF]. Indeed, the stable C-F bond hinders an effective enzymatic or metabolic degradation that results in low excretion profiles, especially for linear fluorinated compounds longer than C 7 F 15 [START_REF] Zaggia | Recent advances on synthesis of potentially non-bioaccumulable fluorinated surfactants[END_REF][START_REF] Barmentlo | Acute and chronic toxicity of short chained perfluoroalkyl substances to Daphnia magna[END_REF]. To assess the in vitro impact of fluorinated end-groups with distinct lengths on cellular viability, NCs formulated with fluorinated polymers were incubated with two distinct cell lines, human umbilical vein endothelial (HUVEC) and murine macrophage-like (J774.A1) during 24h or 72h. The MTT assays showed that NCs did not inhibit HUVEC growth after 24h or 72h, independently of NC concentration, end-group chemistry or fluorinated end-group lengths (Figure 6). For J774.A1, an inhibition down to 75% was observed for 10 mg/mL NCs after 24h of exposure. After 72h exposure, cell viability decreased down to 75 and 50% of controls at NC concentrations of 4 and 10 mg/mL, respectively (Figure 6). For both cell lines, no specific toxicity was induced due to the presence of the fluorinated groups. One should note that the reduction of cell viability of J774.A1 cells was only observed for very high NC concentrations. In addition, the distinct profiles for the two cell lines arise from the ability of J774.A1 to internalize faster and to a higher extent NCs than HUVEC [START_REF] Reul | Near infrared labeling of PLGA for in vivo imaging of nanoparticles[END_REF].

Although only a limited in vitro cytotoxicity was observed, additional in vivo studies are required to investigate the effects resulting from the prolonged exposure to NCs.

Insert Figure 6 here

In vitro Ultrasound imaging

The imaging ability of NCs was evaluated in vitro using a plane-wave B-mode imaging system based on compounded plane-wave ultrasound transmissions [START_REF] Couture | Ultrasound contrast plane wave imaging[END_REF]. All images were obtained with fixed 50 mg.mL -1 concentrations of NC at 37°C and compared to the background gray-scale level produced by ultrapure water. At this temperature, the PFH vapor pressure increases to 48.6 kPaagainst 29.1 kPa at 25°Cand potentially favors a gas-phase nucleation. Ultrasound images obtained in the conventional focused B-mode displayed a significant contrast enhancement (p<0.001) arising from NC formulated with fluorinated polymers (from 1.5 x10 4 until 5.5x10 4 arbitrary units, a.u.) compared to those produced with PLA-C 6 H 13 (0.5x10 4 a.u.) (Figure 7). Moreover, significant differences were observed between fluorinated samples (p<0.001). Additionally, the fluorinated NCs generated contrast specific echoes, enhancing the backscattering intensities from 10-fold to 40-fold higher than PLA-C 6 H 13 . Again, significant differences were observed between fluorinated samples except for PLA-C 11 F 23 and PLA-C 13 F 27 which are not statistically different. For both ultrasound-imaging modalities, all samples exhibited long-lasting echogenicity for more than 10 minutes. As expected, Sonovue ® greatly increased the ultrasound contrast compared to all NCs (from 12-fold to 35-fold) in the conventional focused B-mode and contrast specific mode due to its micrometer size and its gaseous core. In absence of PFH, the PLA-C 8 F 17 nanoparticles (NP in Figure 7) and PVA solution did not yield any significant signal enhancement.

Insert Figure 7 here

The acoustic enhancement observed for samples formulated with fluorinated polymers indicates that the total amount of PFH is a key parameter required to modulate the echogenicity of NCs, given these possess similar sizes. Interestingly, polymers containing longer F-units, such as PLA-C 11 F 23 and PLA-C 13 F 27 , exhibited higher backscattering compared to shorter or non-fluorinated NCs. Given NC sizes are similar, such variation of echogenicity with F-length might be related to an increase of NC density that further affects the scattering cross section of the NCs as described in the introduction [START_REF] Dejong | Principles and Recent Developments in Ultrasound Contrast Agents[END_REF]. The fluorinated initiator density increases from 1.6 g.cm -3 to 1.73 g.cm -3 for C 3 F 7 to C 13 F 27 (as provided by the supplier) whereas the density of PLA is close to the one of PLGA (1.28 g.cm -3 ) [START_REF] Astafyeva | Properties of theranostic nanoparticles determined in suspension by ultrasonic spectroscopy[END_REF]. In addition, as we expect fluorinated moieties to position at the PFH/polymer interface, they might provide nuclei for PFH cavitation/bubble nucleation [START_REF] Choi | Large-scale Generation of Patterned Bubble Arrays on Printed Bi-functional Boiling Surfaces[END_REF].

The presence of these bubbles could also explain the harmonic response of fluorinated NCs as no contrast specific signal can be observed for non-fluorinated ones (obtained with PLA-C 6 H 13 ) or with nanoparticles (NP, Figure 7).

At higher acoustic pressures (peak negative pressure ~5 MPa), a signal enhancement is observed after 20 ms for all NCs formulated with fluorinated polymers, most probably indicating that the shell was disrupted from a certain population of capsules (as illustrated for PLA-C 8 F 17 in Figure 8). For these samples, the PFH is possibly completely vaporized causing the polymeric shell to shatter and allow the gas-phase to freely vibrate, which momentarily increases the echo. This transitory higher echogenicity then decreases back to the initial echogenicity as the gas bubble is dissolved or destroyed in a similar profile as observed for microbubbles [START_REF] Couture | Ultrasound contrast plane wave imaging[END_REF].

Insert Figure 8 here All samples displayed a predominant population of spherical and intact NCs with core-shell morphology (Figure 9 top). However, a distinct population of capsules that exhibited deformed structures according to polymer end-chemistry were also visualized:

samples produced with PLA-C 6 H 13 exhibited some buckled NCs, suggesting that a partial PFH vaporization occurred and produced only a localized distortion of the polymeric shell.

For NCs prepared with fluorinated polymers -PLA-C 3 F 7 , PLA-C 8 F 17 and PLA-C 13 F 27more intense shell deformations led to a complete morphological change that culminated, in many cases, in capsule disruption and observation of debris, some of them at the micrometric size range (white arrows) (Figure 9 bottom). It is important to emphasize that the focus of the ultrasonic beam is small (less than 1 μL or 1mm 3 ) compared to the total volume of the samples, a factor that contributed to reduce the number of viewable debris during cryo-TEM.. The presence of PFH inside several capsules indicates that longer FUS exposition time intervals are still required for homogeneous NC disruption.

Insert Figure 9 here

Comparatively, corresponding samples were submitted to heating at 60°C for 10 minutes in an oil bathabove the PFH boiling point (56°C). The results showed no morphological discrepancy or size variation compared to the control group (Supplementary information, Figures S5 andS6), thus suggesting that the ultrasound mechanical effects are required in addition to the thermal ones to promote a more effective vaporization as previously reported [START_REF] Novell | Focused ultrasound influence on calcein-loaded thermosensitive stealth liposomes[END_REF]. Additionally, as the temperature in focal area of FUS might become higher than 65°C within 1s [START_REF] You | Nanoparticleenhanced synergistic HIFU ablation and transarterial chemoembolization for efficient cancer therapy[END_REF], the polymer shell might be above its T g of 52°C (Table 2)therefore making polymer shell deformation easier.

Conclusion

We have successfully synthesized polylactide polymers containing fluorinated end-groups of different lengths, from C 3 F 7 until C 13 F 27 that were formulated into nanocapsules containing a perfluorohexane core. The employment of fluorinated polymers increased by 2-fold the encapsulation efficiency of perfluorohexane into nanocapsules, although no effect of fluorine chain length was observed. Fluorination of the polymers did not induce any specific in vitro cytotoxicity towards HUVEC and J774.A1 cell lines. Nanocapsules formulated with fluorinated polymers enhanced their acoustic response compared to PLA-C 6 H 13 in both fundamental and harmonic ultrasound imaging modalities. An increased echogenicity and harmonic response was observed as the fluorinated chain-length increased, probably due to an increase of density and promotion of bubble nucleation.

When submitted to focused ultrasound, both intact and exploded nanocapsules could be observed, also with end-group dependency, indicating that the perfluorocarbon was partly vaporized. These results pave the way to theranostic perfluorohexane nanocapsules coencapsulating a drug for precision delivery using focused ultrasound.

Tables and Figures captions

Table 1. Number-average molar masses (Mn) obtained by SEC/1H-NMR, dispersity and glasstransition temperature (Tg) of all synthesized polymers. Table 2. Z-average size, polydispersity index and ζ-potential for nanocapsules formulated with fluorinated and non-fluorinated polymers. Data are presented as mean±SD (n=3).

  Echogenicity enhancement by end-fluorinated polylactide perfluorohexane nanocapsules: towards ultrasound-activable nanosystems Guilherme Picheth a,d , Sophie Houvenagel a , Camille Dejean b , Olivier Couture c , Rilton Alves de Freitas d , Laurence Moine a* , Nicolas Tsapis a* a Institut Galien Paris-Sud, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 92296 Châtenay-Malabry, France. b BioCIS, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 92296 Châtenay-Malabry, France. c Institut Langevin, ESPCI Paris, CNRS (UMR 7587), INSERM (U979), Paris, France d Biopol, Chemistry Department, Federal University of Paraná, 81531-980. Curitiba, PR, Brazil.

C 3 F 7 ,

 37 PLA-C 6 F 13 , PLA-C 8 F 17 , PLA-C 11 F 23 and PLA-C 13 F 27 . All synthesis products were initially characterized by the presence of the lactide signals at 1.60 and 5.23 ppm and the new ester linkage performed with the fluorinated or alkylated alcohol initiators by 1 H-NMR (Figure 2A). As previously observed by Böhmer et al., all the fluorinated polymers exhibited a significant shift of the -CH 2 O-signal compared with the correspondent initiator from 4.1 ppm to 4.55 and 4.74 ppm (Supplementary information, Figures S1 and S2), indicating that the chemical environment of those groups were severely influenced by the PLA backbone as well as by the adjacent CF 2 units [38]. Accordingly, the -CH 2 O-intensity was splitted into two signals with multiplicity of two triplets due to the strong coupling with both fluorine atoms and the geminal proton in the same order of magnitude (~13 Hz), which confirmed the synthesis of fluorinated PLA polymers. Also, the fluorinated derivatives exhibited a shift of the -CF 2 CH 2signals while other CF 2 and CF 3 (-78.9 to -79.0 ppm) resonances were similar to each specific precursor as displayed in the 19 F spectra (Figure 2B). The formation of the non-fluorinated derivative, PLA-C 6 H 13 , was verified by the -CH 2 O-signal shift from 3.60 to 4.14 ppm of the initiator.

C 13 F 27 .

 1327 here Encapsulated PFH was effectively visualized by Cryo-TEM images, revealing spherical core-shell morphologies constituted by thin polymeric layers surrounding the electron-dense perfluorocarbon as shown in Figure4for PLA-C 6 H 13 , PLA-C 3 F 7 , PLA-C 8 F 17 and PLA-All samples displayed capsules containing distinct amounts of PFH, exhibiting dark cores with a wide intensity range and also presented apparently unloaded polymeric nanoparticles, an indication of inhomogeneous encapsulation. From these images, no morphological distinction among polymer end-group chemistry or F-length was observed.

3. 5

 5 PFH vaporization by focused ultrasoundPFH-loaded NCs present an interesting potential to release chemotherapeutics inside solid tumors after local accumulation driven by the EPR effect, provided they are PEGylated and can long circulate in the blood stream. Their great advantage resides on the acoustic-sensitivity of the low boiling point PFH that can be remotely induced to vaporize by focused ultrasound (FUS) to trigger the release of co-encapsulated drug locally. To evaluate the ability of PFH to undergo acoustic drop vaporization under FUS, NCs were exposed to FUS acoustic pulses and analyzed morphologically by cryo-TEM.

Figure 1 :

 1 Figure 1: Schematic representation (A) and real view (B) of the US set-up.

Figure 2 .

 2 Figure 2. 1H-NMR spectra of all synthesized polymers with enlargement of the corresponding lactide and ester linkage signals (A). Magnified CF3 and CF2 regions in the 19F spectra for PLA-C3F7, PLA-C6F13, PLA-C8F17, PLA-C11F23 and PLA-C13F27 (B). The 1H and 19F peak assignments are indicated in the insert. All samples were dissolved in CDCl3.

Figure 3 .

 3 Figure 3. Transmission (left) and scanning electron microscopy images (right) for nanocapsules formulated with fluorinated and non-fluorinated polymers.

Figure 4 .

 4 Figure 4. Cryo-TEM images of nanocapsules formulated with fluorinated PLA-C3F7, PLA-C8F17 and PLA-C13F27 as well as non-fluorinated PLA-C6H13 polymers. The white arrows show the bright regions within the nanocapsules.

Figure 5 .

 5 Figure 5. PFH encapsulation efficiency for NCs freshly prepared suspension (red bars) or submitted to freeze-drying (blue bars).

Figure 6 .

 6 Figure 6. Cell viability as a function of NC concentration for HUVEC (top) and J774.A1 (bottom) at 24h (left) and 72h (right). All results are presented as mean ± SD (N=2).

Figure 7 .

 7 Figure 7. Ultrasound backscattered intensity at the conventional focused B-mode (black symbols) and contrast specific (red symbols) modalities for PVA, nanoparticles of PLA-C 8 F 17 (NP) and all formulated nanocapsules. The error bars represent the ultrasound signal variability between the echo-pulses for each sample (about 5% error). Differences between NC formulated with fluorinated polymers and those produced with PLA-C 6 H 13 were statistically significant in both modes. Moreover, significant differences were observed

Figure 8 .

 8 Figure 8. Sequential ultrasonic images of PLA-C8F17 nanocapsules prior (-200 ms) and after exposure to FUS (from 20 to 1000 ms). The average amplitude signal is presented for each image (bottom). The scale bar corresponds to 1 mm.

Figure 9 .

 9 Figure 9. Cryo-TEM images of nanocapsules after focused ultrasound exposure. Intact/spherical (top) and buckled or debris of nanocapsules (bottom) were observed for all samples. All scale bars represent 100 nm.

PLA-C 6 H 13 :

  H-NMR [400 MHz, CDCl 3 , 25°C] δ H 5.10-5.28 (PLA-CHCH 3 COO-), 1.52-1.61 (-CCH 3 ), 0.88-0.92 (-CH 2 CH 3 ).

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 :

	δ H 5.10-5.28 (PLA-CHCH 3 COO-), 4.50-4.70 (-
	OCH 2 CF 2 -), 1.52-1.61 (-CCH 3 ). 19 F-NMR [200 MHz, CDCl 3 , 25°C] PLA-C 3 F 7 : δ F -78.9 to -
	79.05 (-CF 3 ), -118.5 to -119 (-CH 2 CF 2 -) and -125.7 to 125.8 (-CF 2 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 : 19

	F-NMR [400 MHz, CDCl 3 , 25°C]: δ F -78.8 to -
	79.05 (-CF 3 ), -117.6 to -117.9 (-CH 2 CF 2 ), -120.0 to -120.6 (-CF 2 CF 2 CF 3 ), -120.7 to -121.2 (-
	CH 2 CF 2 CF 2 ), -121.2 to -121.6 (-CH 2 CF 2 CF 2 CF 2 ) and -124.0 to -124.5 (-CF 2 CF 3 ).

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.