Odile Diou

Nicolas Tsapis

Céline Giraudeau

Julien Valette

Claire Gueutin

Fanchon Bourasset

Sandrine Zanna

Christine Vauthier

Elias Fattal

Long-circulating perfluorooctyl bromide nanocapsules for tumor imaging by 19FMRI

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

Introduction

From the 1970s, perfluorocarbons (PFCs) have been considered of great interest for clinical applications. They were initially developed as gas/oxygen carriers for lung ventilation or as blood substitutes [1][2]. Their high fluorine content, chemical inertness and variable physico-chemical properties, such as molecular weight, density or boiling point, widen their field of applications.

Their potential as contrast agents is now well established as illustrated by their use in many different imaging modalities [3]. Mattrey and al. injected perfluorooctyl bromide (PFOB) in rabbits to detect liver-abscesses with Computed Tomography [4]. Perfluorohexyl bromide and PFOB helped to diagnose small-bowel obstruction during X-Ray examination [5]. Angiogenesis of breast cancer tumor was evidenced by ultrasonography with nanodroplets of perfluoropentane (T b =28°C), which convert to highly echogenic nanobubbles upon heating to physiological temperatures [6]. Finally, Perfluoro-15-crown-5-ether (PFCE) emulsions were used for Magnetic Resonance Imaging either to label immunotherapeutic cells and follow their migration after administration or to visualize and quantify the development of neovasculature on atherosclerotic plaques [7][8].

The benefits of fluorine in MRI compared to proton-based imaging are numerous. 19 F has a spin ½ nucleus and a gyromagnetic ratio very close to the proton. Thus, for a given magnetic field value, the spin precesses at almost the same frequency, yielding a high sensitivity and allowing the use of proton spectrometer to detect fluorine. Moreover 19 F is found only in trace amounts in biological tissues, meaning that there is no endogenous background signal and that the signature of an exogenous fluorinated contrast agent is unique and specific. The choice of PFCs for 19 F MRI is crucial. PFOB is one of the most interesting because it is a FDA-approved dense liquid with a low diffusion coefficient into blood, increasing its residence time in the circulation as compared to other PFCs. Nevertheless, as observed for perfluorodecalin and perfluorohexane, PFOB has non equivalent fluorine nuclei, which induce a multi peak resonance spectrum leading to chemical shift artefacts with replicated images [9], and signal loss during echo time due to spin coupling.

The recent design of a specific 19 F MRI multi spin echo (MSE) sequence allowed using PFOB as an MRI constrast agent while eliminating much of the restrictions associated with non-equivalent coupled nuclei [10].

Although most PFOB-based 19 F MRI contrast agents consist in emulsions [11][12], we have focused on recently developed polymeric nanocapsules of poly(lactide-co-glycolide) (PLGA), encapsulating PFOB [13][14]. The biodegradable polymeric shell ensures a good stability of the system, along with mechanical strength [15][16] as compared to emulsions. In addition, polymer chemistry offers versatility of chemical moieties, which can be further functionalized with targeted ligands [17] and a loading compartment is made available in the shell for any lipophilic drug [18][19]. Preliminary in vitro 19 F MRI experiments revealed that encapsulation of PFOB within PLGA nanocapsules does not modify its contrast properties and may provide a significant contrast enhancement at 9. 4T[14].

The in vivo use of PLGA/PFOB nanocapsules is however limited by their rather hydrophobic surface. After intravenous administration, PLGA nanocapsules undergo opsonization followed by rapid elimination by the mononuclear phagocyte system (MPS). Capsules end up mostly in the liver and the spleen and passive tumor targeting, based on the enhanced permeation and retention effect [20] cannot be achieved. To increase the plasmatic half life of nanocapsules, different strategies have been considered including surface modification of nanoparticles either with polysaccharides (dextran, heparin, chitosan) [21][22] or polyethylene glycol (PEG) chains [23].

The hydrophilic shell located on nanoparticle surface prevents opsonization by steric repulsion of blood proteins. PEG is FDA approved and PEGylation may be obtained by different methods such as physical adsorption, covalent grafting on preformed nanoparticles [24] or direct formulation using PEG-copolymers [25]. In the case of PLGA/PFOB capsules, decoration of the shell by PEGylated phospholipids was considered [3,[26][27] . This last strategy revealed deceitful. Since capsule mechanism of formation is exquisitely sensitive to interfacial phenomena [13] it was difficult to yield a good PEG coverage. The best PEG coverage obtained by this method expressed as the minimum surface occupied by one PEG chain, was 100nm². As PEG chains have a large range of motion, they may adopt a mushroom configuration or leave free spaces between each other allowing blood protein adsorption. In any case, the phospholipid strategy revealed unfavorable to reduce uptake by the MPS [3].

Our study focuses on the use of PLGA-PEG 5000 diblock copolymer instead of PLGA to formulate PFOB nanocapsules. PFOB encapsulation within a polymer shell is exquisitely sensitive to the interfacial properties of both the polymer and the surfactant chosen, therefore requiring optimisation [13]. The encapsulation of PFOB will be optimized by comparing two formulation pathways: nanoprecipitation and emulsion-evaporation. The physico-chemical properties of the PEGylated nanocapsules will be assessed to validate their potential stealthiness after intravenous administration. Finally plain and PEGylated nanocapsules accumulation in liver, spleen and tumor will be measured by 19 F MRI to evidence the PEGylation efficacy.

Materials and methods

Materials

Methylene chloride (CH 2 Cl 2 ) RPE-ACS 99.5% and Acetone 99.8% were provided by Carlo Erba Reactifs (France). Sulfuric Acid ACS Reagent 95-98% and CDCl 3 (99.96 atom % D) were provided by Sigma Aldrich (France). Poly(lactide-co-glycolide) Resomer RG502 (intrinsic viscosity 0.16-0.24 dl/g ; M n =9 500-15 000 g/mol) and poly(ethylene glycol)-poly(DL-lactide-co-glycolide) 50:50 Resomer RGP d 50105 (intrinsic viscosity 0.72 dl/g ; PLGA M n =45 000 g/mol containing 10 wt% of PEG M n =5 000 g/mol) were obtained from Boehringer-Ingelheim (Germany). Poly(vinyl alcohol) (PVA) (M W =30 000-70 000g/mol, 89% hydrolyzed) and sodium cholate (SC) were purchased from Sigma-Aldrich.

Perfluorooctyl bromide (PFOB) and Perfluoro-15-crown-5-ether (PFCE) were purchased from Fluorochem (UK). Water was purified using a RIOS/Synergy system from Millipore (France).

Sample Preparation

Nanocapsules were prepared either by nanoprecipitation or emulsion-evaporation. In both cases, the polymer (100 mg) was dissolved into the organic phase along with 60 µL of PFOB and placed in a thermostated bath maintained at 20 °C. For nanoprecipitation, the aqueous phase (20 mL) was added quickly to the organic phase (acetone/CH 2 Cl 2 : 9.4 mL/0.6mL) with a syringe and needle, or opposite. For emulsion-evaporation, the organic phase (4 mL CH 2 Cl 2 ) was emulsified into 20 mL of 1.5% sodium cholate (w/v) aqueous solution using a vortex for 1min and then a vibrating metallic tip (IBP7677, Ultrasons, Annemasse, France) at 180V, for 1 min over ice. Solvents were then evaporated by magnetic stirring at 300 rpm for about 3 h (for emulsion-evaporation) to overnight (for nanoprecipitation) in a thermostated bath (20 °C). Plain and PEGylated nanocapsules were prepared respectively with PLGA and PLGA-PEG copolymers. For plain nanocapsules, an incubation with PVA (1% w/v) for 5 days at 4°C is necessary, prior to the purification step, in order to replace sodium cholate by PVA and prevent further aggregation. Both types of nanocapsules were then washed by ultracentrifugation for 1 hour, at T=4°C and at 6 860 g, 27 440g for PLGA and PLGA-PEG nanocapsules respectively (Optima LE-80K Ultracentrifuge Beckman Coulter). The pellet was finally resuspended in water.

Size and Zeta potential

The hydrodynamic diameter (d H ) and polydispersity index (PDI) of the nanocapsules, were measured by quasi elastic light scattering, using a Zetasizer Nano ZS instrument (Malvern, France). Suspensions were diluted in water filtered over a 0.22 µm membrane. Measurements were performed in triplicate at 20°C, at an angle of 173° to avoid multiple scattering. The counting time was set at 60s. Zeta potential measurements were carried out with the same instrument, at 25°C, in 1 mM NaCl.

Transmission Electron Microscopy (TEM)

Transmission electron microscopy was performed at CCME (Orsay, France) using a Philips EM208 operating at 80 kV. Suspensions of nanocapsules (1 mg/mL) were deposited on copper grids covered with a formwar film (400 mesh) for 2 min. The excess solution was blotted off using filter paper and grids were air dried before observation. Images were acquired using a high-resolution camera, Advantage HR3/ 12GO4 (AMT-Hamamatsu).

Scanning Electron Microscopy (SEM)

Scanning electron microscopy was performed using a LEO 1530 (LEO Electron Microscopy Inc, Thornwood, NY) operating between 1 and 3 kV with a filament current of about 0.5 mA. Washed and freeze dried nanocapsules were deposited on carbon conductive double-sided tape (Euromedex, France). They were coated with two palladium-platinum layers of ca. 1 nm using a Cressington sputter-coater 208HR with a rotary-planetary-tilt stage, equipped with an MTM-20 thickness controller. An In-lens detector has been used for imaging.

Determination of PFOB encapsulation efficacy by 19 F NMR

Fresh unpurified capsules were freeze dried for 24-48 h using an Alpha 1-2 LD plus freeze-dryer (Christ). Lyophilisates were then dissolved into CDCl 3 containing perfluoro-15-crown-5-ether (PFCE) as an internal standard ([PFCE]=0.76mmol/L). The amount of PFOB NMR PFOB n was determined after integration of the peak at -81ppm corresponding to the CF 3 group and normalization by the area of the PFCE peak at -89.5ppm. Encapsulation efficiency ɳ encaps was calculated as follows:

Sodium cholate determination by spectrophotometry

The method used for sodium cholate assay was originally proposed by Mosbach and al. (1954) and then used by . Concentrated solutions were prepared by dissolving 25 to 125mg of sodium cholate up to 10mL of water. Aliquots of 40µL of each solution were pipetted accurately into a 20mL volumetric flask, which is completed with freshly prepared 65% w/v sulfuric acid solution. Absorbance of the solution was measured at 320nm using a UV-Vis Spectrophotometer (Shimadzu UV-2101PC). Absorbance as a function of concentration of sodium cholate in sulfuric acid solution was plotted as a calibration curve over a concentration range from 5.0 to 25.0 µg/mL (y=33.225x+0.0101, R²=0.9999).

A 25µL sample was taken from just-prepared nanocapsules suspension or supernatant of washed nanocapsules and placed into a 20mL volumetric flask. Final volume was completed with the 65% w/v sulfuric acid solution. Absorbance was measured at 320nm. The amount of sodium cholate was calculated using the calibration curve mentioned above. Experiment was done in triplicate for each sample.

To validate the accuracy of the method, a protocol derived from standard addition method, was used. Various volumes (from 5 to 13 µL) of suspension and 20 µL of the standard solution of sodium cholate ([SC]=5 µg/mL) were mixed into a 10 mL volumetric flask, completed with the 65% w/v sulfuric acid solution. Absorbance was plotted versus total concentration of sodium cholate (y=33.021x+0.0087, n=3 for PLGA and y=34.508x+0.0144 for PLGA-PEG). Assuming that only sodium cholate absorbs at 320 nm, the relative error on the attenuation coefficient is Δε/ε 0 =0.61% (with PLGA samples) and Δε/ε 0 =3.86% (with PLGA-PEG samples). It can be admitted that nanocapsules alone do not absorb at the considered wavelength.

Stability of nanocapsules over time

After purification, nanocapsules were dispersed in PBS and incubated at 37°C under stirring. Size variation of nanocapsules was followed with time by dynamic light scattering, as previously described. Simultaneously, aliquots of 2mL were taken out, freeze-dried and redispersed in CDCl 3 .

The volume of PFOB remaining encapsulated was evaluated by 19 F NMR.

X-ray Photoelectron Spectroscopy

X-ray photoelectron spectroscopy (XPS) was used to determine the surface composition of NPs.

Powder of raw PLGA and PLGA-PEG polymers and lyophilisates of washed nanoparticles were deposited on the grid. A Thermo Electron Escalab 250 spectrometer with a monochromated radiation (1486.6 eV) was used. For all samples, a survey spectrum was recorded over a binding energy range of 0 to 1100 eV, the analyzer pass energy was 100 eV. In all cases the survey spectra only revealed the presence of oxygen (O1s 533 eV) and carbon (C1s 285 eV). C1s core was analysed in more details from 280 to 295 eV and pass energy of 20 eV. The spectrometer was calibrated against Au 4f7/2 at 84.1 eV. The photoelectron take-off angle (angle of the surface with the direction in which the photoelectrons are analyzed) was 90°. Curve fitting of the spectra was performed with the Thermo Electron Avantage software.

Complement Activation

The complement activation of PEGylated and non PEGylated nanocapsules was evidenced by evaluating the conversion of C3 into C3a and C3b by a 2-D immunoelectrophoresis method, as described by others [21,30]. Human serum was obtained after calcifying plasma from healthy donors, and stored at -80 °C until use. Nanocapsule suspensions were incubated, under gentle agitation, for 1 h at 37°C with 50 µL human serum and 50 µL Veronal Buffer, containing 0.15mM calcium chloride and 0.5mM magnesium chloride (VBS 2+ ). To ensure a valid comparison of the different suspensions, concentrations in nanocapsules were adjusted to obtain an equal surface area of S =1000 cm 2 and 2000 cm 2 in 100µL. After incubation, 7 µL of each sample were subjected to a first electrophoresis on 1% agarose gel in tricine buffer (pH =8.6). The migration was achieved using Pharmacia LKR Multiphor (600 V, 8.8mA, and 5W) for 1.5 hours. The second-dimension electrophoresis was carried out for 18 hours, on Gelbond films in 1% agarose gel plates containing a polyclonal antibody to human C3 (Complement C3 antiserum rose in goat, Sigma, France), recognizing C3 as well as C3a and C3b. The films were finally dried and stained with Coomassie blue to reveal the presence of the proteins, which have reacted with the antibody (Sigma). The complement activation factor (CAF) was then calculated from the ratio of the total area of the peaks attributed to C3a and C3b over the sum of the areas of the peaks attributed to C3a+b and to C3. This ratio was then normalized on a scale ranging from 0 to 100 in which 100 indicated total activation, obtained with poly(isobutylcyanoacrylate) nanoparticles [31] and 0 the spontaneous activation, measured in absence of nanocapsules.

In vivo accumulation study

Six-week-old females Foxn1nu (nu/nu) mice weighing from 20 to 22 g were purchased from Harlan (France). Animal procedures were in accordance with the recommendations of the EEC (86/609/CEE) and the French National Committee (decree 87/848) for the care and use of laboratory animals.

Animals were anesthetized with ketamine and domitor. The 19 F Imaging was performed on a 7-T small-animal MRI scanner (Bruker, Ettlingen, Germany) using a double-tuned, 1 H/ 19 F 3.2-cmdiameter, linear, birdcage coil, built at Neurospin (CEA, Saclay, France). Anatomic 1 H images were recorded at the beginning of the experiment to localize the organs. The multi spin echo imaging sequence (TE = 15.5 ms, TR = 4000 ms, 60 echoes one 16mm-thick slices, 0.63×0.94 mm 2 in-plane resolution) was used for 19

Results and Discussion

Formulation process

PLGA/PFOB nanocapsules were previously prepared by an emulsion-evaporation process which allowed modulating capsule diameter and thickness by simply varying the sonication power, the sodium cholate (SC) concentration or the PLGA/PFOB proportions [14,32]. Another process has been fully reviewed in the literature to produce polymeric capsules: solvent displacement method, often called nanoprecipitation. Since it is a spontaneous process, the energy input is reduced. Besides, the use of surfactants may be optional and purification issues may be avoided. Considering these advantages and the amphiphilic nature of PLGA-PEG, nanoprecipitation was considered as an alternative to produce PLGA-PEG/PFOB nanocapsules. The mixture of acetone and methylene chloride (9.6mL/0.4mL) was chosen to ensure miscibility with water and full solubility of PFOB as well as PLGA-PEG. Sodium cholate (SC) was optionally added to the aqueous phase in order to compare the two processes in the very same conditions. The different formulations characteristics such as hydrodynamic diameter (d H ), polydispersity index (PDI) and encapsulation efficiency of PFOB ( encaps ) are presented in Table 1.

The order of addition of each phase was investigated. It is shown that when the solvent is poured in the water phase, nanoparticles of around 120nm with high PDI values are obtained: 0.31 and 0.37 with and without SC respectively. The high polydispersity values can be explained by the very quick diffusion of the organic phase into the aqueous one followed by rapid and turbulent capsule formation, as already observed [33]. In addition, the PFOB encapsulation efficiency is high:  encaps = 73 ± 2% and 70 ± 1% with and without SC respectively. Nevertheless, the nanoparticles recovery is low (48 and 41%), as seen by weighing freeze dried samples before and after filtration (0.45µm).

Optical microscopy reveals the presence of microcapsules of 2-6µm. In the opposite way, adding the aqueous phase to the solvent leads to slightly larger but less polydisperse objects. In this case, suspensions are turbid which is surprising since suspensions of nanocapsules are usually very transparent as PFOB refractive index is 1.30 (25°C) close to the one of water [34]. Nanoparticles and nanocapsules are probably formed all together, which is also evidenced by the low encapsulation efficiencies:  encaps = 30 ± 3% and 31 ± 2%, respectively with and without SC.

Influence of sodium cholate on nanocapsule characteristics was also determined. This surfactant seems to have a little influence on size and polydispersity of the objects, however it controls their morphology. In absence of SC, acorn morphologies, in which a hemisphere of liquid PFOB coexists with a hemisphere of solid PLGA-PEG, are observed by optical microscopy. Actually, PLGA-PEG is a poor stabilizer for the methylene chloride-water interface compared to sodium cholate. The interfacial tension is lowered from 28.2 mN/m to 17.5 mN/m in presence of 25 mg/mL copolymer, while it reaches 8.1mN/m, when the aqueous phase contains 1.5 w/v % of SC [13,35].

To conclude, none of the studied nanoprecipitation conditions fulfils the requirements for the production of PLGA-PEG nanocapsules in terms of size (d H ~150nm), polydispersity (PDI<0.20) and PFOB encapsulation efficiency. Emulsion-evaporation conditions seem to be more satisfying, considering Table 2. With PLGA-PEG, the objects are around 120nm, have a low PDI (0.18) and very high encapsulation efficiency (85 ± 4%), suggesting that the majority of the objects corresponds to nanocapsules. When PLGA is used, capsules are almost of the same size d H =130nm but are more polydisperse, PDI=0.24, and encapsulate a reduced quantity of PFOB:  encaps =63 ± 7%. Independently of the polymer used, the encapsulation efficacy of PFOB is much higher as compared with other studies: Srinivas et al. only succeeded to encapsulate 8% of PFOB in PLGA using the very same process [9]. This difference most probably arises from the use of PVA as surfactant instead of SC. The relevance to formulate PLGA-PEG by an emulsion-evaporation process is thus confirmed in order to produce PEGylated nanocapsules and will be used in the following of the article.

Morphology

PLGA and PLGA-PEG nanocapsules were observed by Scanning Electron Microscopy. Images show spherical objects with sizes in good agreement with the hydrodynamic diameter measured by dynamic light scattering (Figure 1). In addition, smooth surfaces are observed for both nanocapsules.

For PLGA-PEG nanocapsules, this result contrasts with previous experiments showing that microcapsules obtained by emulsion-evaporation present spongy/porous surfaces [35]. The porous morphology was arising from the necessity to create surface to accommodate all PEG chains in the aqueous environment. Since nanocapsules possess a smooth surface, one can assume that the specific surface of nanocapsules is large enough for all PEG chains to be exposed to the aqueous environment.

Morphological analysis was completed by Transmission Electronic Microscopy imaging, which confirms that the core-shell structure is preserved with PLGA-PEG. The PFOB core appears in grey, whereas the polymeric shell seems darker. The intrinsic contrast between PFOB and PLGA-PEG has already been observed for PLGA nanocapsules. However, the shell of the PLGA-PEG nanocapsules appears more diffuse than the one for PLGA, considering previously published pictures [32]. The PEG chains are fully hydrated, lowering the contrast between the shell and water, leading to a blurry surface (Figure 1).

Nanocapsule purification

Sodium cholate, a bile salt is obviously tolerable by intestinal cells when given orally but is reported to destabilize cell membranes, and cause hemolysis in the blood at concentration of 1 to 20mM [36][37]. An intravenous administration of nanocapsules requires ensuring SC removal. PLGA and PLGA-PEG nanocapsule suspensions were purified by ultracentrifugation to remove SC. The concentration of SC in native suspension and supernatant were measured by UV-spectrophotometry in H 2 SO 4 at 320nm (Table 3). In water, since SC has no chromophore, the molecule cannot be quantified by spectrophotometry. However, when dissolved in concentrated sulfuric acid solution, dehydration of the alcohol functions occurs, giving alkene functions. Thanks to the double bonds, quantification by spectrophotometry is made possible.

Sodium cholate was efficiently removed by this method since 99.45 ± 1% (PLGA-PEG) and 99.55 ± 1% (PLGA) of the initial amount in suspension, is recovered in the supernatant. The remaining SC,

[SC]≤0.15 mM, can be neglected. It is very close to the detection limit of UV spectrophotometry, around 10 -4 M, 10 times lower than reported haemolytic dose [36][37].

After purification, PLGA-PEG nanocapsules preserve both their size and low polydispersity (Table 2).

No aggregation occurs during the centrifugation, probably thanks to steric repulsions of PEG chains.

After purification, PLGA-PEG nanocapsules zeta potential decreases from -15.6mV to -29.3mV, since surface charges are no longer screened by the concentrated sodium cholate. Indeed, when the same initial amount of SC is added to the purified formulation, the zeta potential recovers almost perfectly its initial value (ζ=-15.9 mV). For PLGA nanocapsules, size increases from 130.4 ± 3 nm to 175.1 ± 0.2 nm and the zeta potential becomes neutral (ζ=-0.02mV) due to the presence of residual PVA chains on nanocapsule surface, as already observed [3,38]. Strong interaction by hydrogen bonding between PVA and PLGA are favoured during incubation.

Stability

PLGA copolymers are known for their biodegradability and biocompatibility. The degradation is characterized by random hydrolytic scission of the polyester backbone. The higher the glycolide content, the faster the degradation. For PLGA 50:50, final products: lactic and glycolic acids are detected from the first day in the medium [39]. In this case, in vitro degradation of PLGA-PEG nanocapsules was assessed by following the size and PFOB content variations in PBS buffer (pH=7.4) at 37°C (Figure 2). At the beginning of the study, nanocapsules are slightly bigger than reported in table 2, d H =154 ± 2 nm. This increase in size can be explained by a higher swelling of the shell due to formation of carboxylic acid and hydroxyl groups among PLGA in PBS. Then the size remains almost constant for 120h. Small variations of the PFOB concentration occur during the first 48h. Between 48h and 120h, drop of the PFOB content and appearance of aggregates in the suspensions, evidence the disintegration of the nanocapsules. Considering the relative loss of PFOB, it can be estimated that only around 11% of nanocapsules are destroyed after 120h, the remaining ones preserve their sizes.

Nanocapsule PEGylation

To ascertain the presence of PEG moieties on PEGylated nanocapsule surface, XPS experiments were performed. The high resolution C1s region was recorded and peak fitted for the raw polymers PLGA and PLGA-PEG, as references and for nanoparticles of both types. Some of the parameters were set during peak fitting. First the binding energy positions of peaks were located according to the literature [38,40]. Secondly, the intensities of ester and methine peaks were strained to be equal, since PLGA 50:50 comprises both functions in the same proportion [41]. PLGA spectra indicates 3 distinct peaks namely aliphatic carbon (C-C) at 285 eV, methine carbon (O=C-C*-O) at 287.0 eV and ester carbon (O=C-O) at 289.2 eV. Meanwhile 4 C1s peaks appear on the PLGA-PEG spectra, the additional peak resulting from an ether group (C-O) at 286.2 eV, characteristic of PEG (Figure 3). The insertion of this fourth peak slightly shifts the peak binding energies of methine and ester carbons to higher energies, respectively 287.3 eV and 289.4 eV. We obtained excellent agreement between experimental and curve fit data for the raw PLGA-PEG polymer. For the PLGA-PEG nanoparticles, the half-height width of peaks is larger, enlightening the amorphous state of the polymeric shell. Table 4 summarizes atomic composition for the C1s peak fitting results. Considering the chemical formula of the used PLGA 346 -PEG 114 , the theoretical value of the ratio between atomic percentages of ether carbon and ether and ester carbons taken together, is 24.8%. The experimental value is 26.2%. The small difference may be explained by the rather high polymolecularity of the copolymer. This ratio is increased to 38.1% for the PLGA-PEG nanoparticles. Ether functions enrichment reveals that PEG chains are surface oriented in the case of nanoparticles [42]. This is probably occurring during the emulsion-evaporation process. As the PLGA-PEG reaches its solubility limit in methylene chloride, it migrates to the interface and exposes the hydrophilic PEG moieties toward the aqueous phase [35].

Assuming all PEG chains are present at nanocapsule surface, the available surface for each PEG chain, S PEG , can be evaluated as follows:

where m NC is the weight of nanocapsules obtained at the end of the process (m NC =140mg), N PEG is the total number of PEG chains in the final formulation, easily calculated from m NC and  encaps =85%, S sp NC is the specific surface area of nanocapsules estimated from d H , the hydrodynamic diameter of nanocapsule (d H =120nm) and ρ, the density of nanocapsule, measured by densimetry, ρ =1.5g/cm 3 . Finally, one PEG chain occupies an area of around 5nm² at nanocapsule surface. This value is very close to 2nm², which is considered as the optimal surface to ensure minimal adsorption of opsonins [43].

Evaluation of complement activation

The complement system ensures the innate immune response, which consists in non-specific recognition of foreign bodies (i.e. opsonisation). Complement activation cascade is complex and consists in a chain of bio-chemical reactions [44][45]. One triggering factor is the binding and the conformational changes of the plasma protein C3. The native form is then cleaved into two fragments: C3a and C3b. 2D immunoelectrophoresis was used to compare the relative amount of these three species after incubation of PEGylated and plain nanocapsules in serum. The capacity of the different nanocapsules to trigger complement activation was evaluated. The influence of the surface area (A/ 1000 and B/ 2000cm²) was studied by changing the concentration of nanocapsules in

PEG NC sp NC PEG N S m S   H NC sp d S
6  the incubated serum. The four gels obtained (Figure 4) show the presence of two peaks. The higher one, on the left side corresponds to the native C3 protein, while the large double peak on the right side reveals the activation of the protein with production of the two smaller fragments: C3a and C3b.

At low surface area (1000cm²), the two types of nanocapsules induce a similar and weak activation of the complement activation, evidenced on the electrophoregram by a larger C3 peak as compared with C3a and C3b. One obtains a CAF value of 19 ± 3% for PLGA and an even lower one for PLGA-PEG: CAF=14 ± 3%. Since the CAF difference is of the same order of magnitude as the experimental error, around 5%, the two types of nanocapsules cannot really be distinguished [31]. The residual PVA coating on PLGA nanocapsules might protect plasma protein adsorption by steric repulsion [46]. At higher surface area (2000cm²), the difference between PLGA and PLGA-PEG nanocapsules becomes clearer. On one hand, PLGA-PEG activation remains very low (CAF=11 ± 2%). On the other hand, for PLGA, the right peak increases noticeably and the activation is moderate (CAF value is 32 ± 2%).

Low activation of PEGylated nanocapsules, even at very high concentration (S spé ~2000 cm² C NC = 66.7 mg/mL) is noticeable. From a complement activation point of view, PEG coverage is more efficient than PVA coating. The hydrophilic chains probably adopt a brush conformation and prevent serum protein adsorption more efficiently. Since the in vitro evaluation of complement activation is considered to be predictive of the in vivo fate of nanocapsules after intravenous administration [47]: PLGA-PEG nanocapsules should therefore be more stealth than PLGA ones.

In Vivo 19 F Magnetic Resonance Imaging

Kinetic study

In vivo 19 F MR imaging was performed in four mice after intravenous injection of PLGA and PLGA-PEG nanocapsules. After 4 hours, both types of nanocapsules are mainly accumulated in the spleen and liver (data not shown). Since the chosen MRI sequence does not allow any observation of the vascular compartment, the 19 F signal only arises from the immobilized nanocapsules in the tissues [11]. Liver and spleen uptake kinetics were studied in more details for the first 90 minutes after intravenous injection.

For the spleen, kinetic profiles for PLGA and PLGA-PEG nanocapsules do not follow any characteristic trend and interanimal variability is high (data not shown). This probably arises from the complex mechanism of spleen capture and the numerous functions of the organ (erythrocyte conditioning and maintenance, platelet storage, immune functions...) which can interfere with each other. In any case, after 90 minutes the fractions of nanocapsules accumulated are 5.3±0.2% and 5.7±1.7% for nanocapsules and in agreement with those found for poloxamer coated polystyrene or PHDCA-PEG nanoparticles of the same size [25,48]. Splenic uptake is often compared to a filtering or a sieving process, particularly effective to remove poorly opsonized antigens, therefore it does not depend on the nanoparticles surface properties [49].

For the liver, the kinetic profiles present less interanimal variability. The fraction of nanocapsules accumulated in the liver increases continuously as a function of time until it reaches a plateau value of 12.5% of injected dose for PLGA capsules. For PLGA-PEG capsules, accumulation increases until 16.1% of the injected dose after 90 minutes but the plateau is probably not reached yet (Figure 5). A two-compartment model was chosen for experimental data fitting, meaning that nanocapsules are transferred from the bloodstream to the liver compartment. The time dependency of the nanocapsule concentration (via PFOB concentration) in the organ is given by the following formula:

The fitted values of C 0 liver and k liver are reported in table 5. R values are higher than 0.94 for the 4 mice, confirming the accuracy of the fits.

The half-life absorption constant k liver is calculated as:

liver k t / ) 2

  

PLGA-PEG nanocapsules, fractions found in the organs 7 hours after injection are higher than those found 90 minutes after injection: 6.8 ± 1.9% in the spleen and 18.9 ± 1.5% in the liver. Nanocapsule elimination from the liver and spleen is compensated by their continuous uptake (Figure 6).

Tumor accumulation confirms these results. For PLGA nanocapsules, the 19 F signal in the tumor does not exceed the background noise arising from the rest of the viscera. The detection limit was evaluated to be C PFOB =0.04mM for 35 minutes of acquisition. For PLGA-PEG nanocapsules the intensity is weak in the tumor but the concentration of PFOB could be quantified: C PFOB = 0.19 ± 0.02 mM (Figure 7), corresponding to about 1% of the injected dose (n=2), of the same order of what is usually observed [53][54]. Altogether, these results prove the efficacy of PEGylation to increase the residence time of nanocapsules in the blood stream, allowing them to reach tumors by EPR effect.

Conclusion

Encapsulation of PFOB with PEGylated nanocapsules of PLGA-PEG has been optimized by the solvent emulsion-evaporation method, leading to nanocapsules of about 120nm with a high yield of encapsulation of PFOB around 80%. The PEG coverage seems rather dense allowing low complement activation in vitro. 19 F-MRI was used to follow the liver and spleen accumulation and revealed that PEGylated nanocapsules were circulating longer than their plain counterparts (t 1/2 PLGA =13.6min and t 1/2 PLGA-PEG =23.9min). This enhanced circulation time was further confirmed by the ability to detect implanted tumors 7 hours after PEGylated nanocapsule intravenous injection. These PEGylated nanocapsules appear promising for detecting tumors using the EPR effect using 19 F-MRI. Future studies will focus on nanocapsule accumulation as a function of tumor growth stage and the imaging time post-injection.

Tables and Figures

  masses of the components introduced in the organic phase, m NC corresponds to the mass of capsules recovered after freeze-drying and M PFOB is the molecular weight of PFOB (498.96 g/mol).

  F imaging. It takes into account NMR properties of PFOB to maximize sensitivity [10]. Images were acquired every 4.5 minutes after intravenous infusion of the nanocapsules in the tail vein (200µL C PFOB =120mM and 55mM respectively for plain and PEGylated nanocapsules). An external reference was placed close to the right flank of the mouse for signal calibration. Concentration of PFOB was measured as a function of time in the liver and spleen for 1.5 hours, multiplied by the volume of organs (V liver =1mL and V spleen =150µL) and normalised by the injected dose. In vivo tumor imaging Murine colon carcinoma cell line CT26.WT was purchased from ATCC (Molsheim, France). Four-weekold females Fox1nu (nu/nu) mice weighing around 20g were purchased from Harlan. The colon tumor model was established by injecting 3.10 5 cells (in 100 µL cell culture medium) subcutaneously into the right flank of the animal. Twelve days after tumor inoculation (V tumor >150mm 3 ), PLGA and PLGA-PEG nanocapsules were injected in the tail vein (200µL, C PFOB =82.5mM). 7 hours after the injection, the mouse was anesthetized with i.p. injection of ketamine and domitor and placed inside the coil. After acquisition of anatomical 1 H images, 19 F acquisitions were performed with the previously described MSE MRI sequence. Total acquisition time was 35min. Concentration of PFOB was measured in the liver, spleen and tumor multiplied by the volume of organs and normalised by the injected dose.

Figure 1 :

 1 Figure 1: Top: SEM images of PLGA (left) and PLGA-PEG (right) nanocapsules Bottom: TEM images of PLGA-PEG nanocapsules with intrinsic negative contrast of the polymer. The insert is a zoom on one capsule (scale bar 100nm). The PFOB core is surrounded by a cloudy shell.

Figure 2 :

 2 Figure 2: Evolution of size and PFOB concentration during the incubation of PLGA-PEG nanocapsules in PBS buffer at 37°C.

Figure 3 :

 3 Figure 3 : High resolution XPS spectra of C1s region for the raw PLGA-PEG polymer (left) and the corresponding nanoparticles (right). Red dashed line corresponds to experimental data whereas black full lines represent the fits.

Figure 4 :

 4 Figure 4 : Profiles of complement activation for PLGA and PLGA-PEG nanocapsules as revealed by 2D electrophoresis of C3 antigen and their corresponding CAF values. Concentrations of nanocapsules were chosen to obtain a surface area of A/ 1000 cm² and B/ 2000 cm 2 . CAF=C3 a+b /(C3+C3 a+b ).

Figure 5 :

 5 Figure 5 : Evolution of the PFOB concentration in the liver, as measured by 19 F MRI during the first 90 minutes following the IV administration of PLGA or PLGA-PEG nanocapsules.

Figure 6 :

 6 Figure 6: Cumulative PFOB accumulation in the liver and spleen 1.5 hours (left) and 7 hours (right) after nanocapsule injection.

Figure 7 :

 7 Figure 7 : 19 F MR image superimposed with anatomical 1 H MR image of a tail-head longitudinal cross section of a mouse 7 hours after intravenous injection of PLGA-PEG nanocapsules for a tumor of 720mm 3 (white dotted circles).

Table 1 :

 1 Characteristics of nanocapsules obtained by nanoprecipitation (n=2).

		% SC (w /v)	d H (nm)	PDI	 encaps (%)
	Solvent in water	0	122 ± 3	0.37	70 ± 1
		1.5	120 ± 2	0.31	73 ± 2
	Water in solvent	0	145 ± 0.1	0.16	31 ± 2
		1.5	178	0.19	30 ± 3

Table 2 :

 2 Characteristics of nanocapsules obtained by the emulsion evaporation process and after the purification step (n=3).

			[SC] (mM)
	Suspension	PLGA-PEG	29.01 ± 1.28
		PLGA	28.94 ± 0.28
	Supernatant	PLGA-PEG	29.15 ± 1.02
		PLGA	28.80 ± 0.12

Table 3 :

 3 SC Concentration, calculated from the measured absorbance at 320nm, in the just prepared suspensions of PLGA and PLGA-PEG nanocapsules and in their supernatants collected after ultracentrifugation. Data correspond to mean ±SD, n=3.

			Atomic % of C1s, high resolution
		Aliphatic	Ether	Methine	Ester
		C-C	C-O	O=C-C*-O	O=C-O
	PLGA	16.60 (285 eV)	0	41.70 (287.0 eV) 41.70 (289.2 eV)
	PLGA NPs	19.75 (285 eV)	0	40.42 (287.0 eV) 39.83 (289.2 eV)
	PLGA-PEG	29.69 (285 eV)	10.54 (286.2 eV) 30.03 (287.3 eV) 29.73 (289.4 eV)
	PLGA-PEG NPs	12.55 (285 eV)	20.61 (286.2 eV) 33.42 (287.3 eV) 33.42 (289.4 eV)

Table 4 :

 4 Relative atomic percentages from the area under the curves of fitted peaks, whose positions in binding energy appear in the brackets.

Acknowledgements

Authors acknowledge financial support from Fondation de l'Avenir (ET9-545). Authors would like to thank M-F Trichet for access to the SEM facility (ICMPE, Thiais, France), D Jaillard for TEM experiments, E. Morvan for her help with 19 F NMR spectroscopy, and N. Mignet and J. Seguin for providing fruitful advice for tumor growth. Our laboratory is a member of the Laboratory of Excellence LERMIT supported by a grant from ANR (ANR-10-LABX-33).

PLGA

PLGA-PEG C 0 liver (Fraction of injected dose) 12.4 ± 2.2% 16.1 ± 3.1% k liver (min -1 ) 0.051 ± 0.008 0.029 ± 0.0008

Table 5 : Fitted kinetic parameters for PLGA and PLGA-PEG nanocapsules in the liver (n=2).


Nathalie Ménard

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

Cécile Poirier

Thomas Arnauld
email: thomas.arnauld@fr.netgrs.com

Laurence Moine

François Lefoulon

Jean-Manuel Péan

Elias Fattal

Drug solubilization and in vitro toxicity evaluation of lipoamino acid surfactants

Keywords: amino acid, lipid, surfactant, micelle, self-assembly, drug solubilization

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

Introduction

A large number of anticancer drugs are poorly water-soluble and belong to classes II and IV of the Biopharmaceutics Classification System (BCS) [START_REF] Amidon | A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability[END_REF]. A large body of formulation is required to get these drugs administered through the intravenous route due to their poor solubility in water. Various formulations have been considered such as liposomes, nanoparticles or co-solvents [START_REF] Zhang | Development and characterization of a novel Cremophor® EL free liposome-based paclitaxel (LEP-ETU) formulation[END_REF][START_REF] Cortes | Nanoparticle albumin-bound (nab(TM))-paclitaxel: improving efficacy and tolerability by targeted drug delivery in metastatic breast cancer[END_REF][START_REF] Miyako | Solubility enhancement of hydrophobic compounds by cosolvents: Role of solute hydrophobicity on the solubilization effect[END_REF], all leading to the presence of organic solvents residues. To avoid toxic effects arising from organic solvents, micellar drug delivery systems can be used [START_REF] Torchilin | Structure and design of polymeric surfactant-based drug delivery systems[END_REF][START_REF] Lukyanov | Micelles from lipid derivatives of water-soluble polymers as delivery systems for poorly soluble drugs[END_REF]. These nanostructures are obtained following a selfassembling process of the surfactants and are able to solubilize hydrophobic drugs in their core. The use of natural surfactants is of increasing interest in drug solubilization. Many studies focus their attention on the synthesis of amino acid-derived surfactants. These novel surfactants possess interesting properties such as a lower toxicity [START_REF] Holmberg | Natural surfactants[END_REF][START_REF] Benavides | Assessment of the potential irritation and photoirritation of novel amino acid-based surfactants by in vitro methods as alternative to the animal tests[END_REF]Sanchez et al., 2007a;Sanchez et al., 2007b) and a higher biodegradability [START_REF] Infante | Non-conventional surfactants from amino acids and glycolipids: Structure, preparation and properties[END_REF][START_REF] Infante | Amino acid-based surfactants[END_REF]. Lysine amino acid was previously used in surfactant design [START_REF] Infante | Non-conventional surfactants from amino acids and glycolipids: Structure, preparation and properties[END_REF]Sanchez et al., 2007a). In particular, Vives et al. studied anionic surfactants derived from lysine varying their hydrophobic chain length from C6 to C10. Shortest-chain compounds (C6) were found less haemolytic and less irritating than longer ones [START_REF] Vives | Hemolytic Action of Anionic Surfactants of the Diacyl Lysine Type[END_REF].

In the present study, to improve water solubility of new drug candidates, innovative surfactants were designed and evaluated. They were composed of the same hydrophilic moiety that derives from lysine: nitrilo triacetic acid (NTA) and various fatty acids moieties. Indeed, fatty acids were already used as hydrophobic chains of surfactants linked to different hydrophilic moieties. Palma et al. have conjugated L-ascorbic acid to decanoic acid as hydrophobic part. This surfactant was able to self-assemble into water. In addition, micelles of ascorbyl-decanoate significantly enhanced the solubility of hydrophobic molecules (phenacetin, danthron, and griseofulvin) [START_REF] Palma | Drugs solubilization in ascorbyl-decanoate micellar solutions[END_REF]. For this reason, to obtain novel surfactant structures with potent solubilization properties, lipid hydrophobic moieties were linked to NTA via an amide bond. The lipid moieties were composed of 19 to 25 carbon atoms. In addition, their structures were saturated or contained unsaturations consisting in double bonds or diacetylenic groups. Their self-assembling properties, in vitro toxicity and ability to solubilize an insoluble drug were compared to polysorbate 80, a model solubilizer. This study will allow to evaluate the influence of the hydrophobic moiety on surfactant properties.

Materials and methods

Materials

Polysorbate 80 was obtained from Seppic. 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC, HCl) was obtained from Iris Biotech GmbH. Pyrene, triton X-100, 1,3dicyclohexylcarbodiimide (DCC), palladium on carbon 5 % (w/w), phosphate buffer saline (PBS) and N-hydroxysuccinimide (NHS) were provided by Sigma-Aldrich. Dimethyl sulfoxide (DMSO), trifuoroacetic acid (TFA), EtOAc, HCl and dichloromethane (CH 2 Cl 2 ) were purchased from Carlo Erba. Water was purified using a RIOS/synergy system from Millipore. Acetonitrile gradient grade for liquid chromatography, ammonia solution 25 % for analysis, dimethyl formamide (DMF), methanol for HPLC and triethylamine were obtained from Merck. Ammonium formate and bis(carboxymethyl)lysine (NTA) were provided by Fluka analytical.

NMR and mass spectrometry experiments

NMR spectra were recorded in deuterated DMSO at 300 K on a Bruker AvANCE-600 spectrometer operating at 600.13 MHz for the 1 H and a Bruker AvANCE-400 spectrometer operating at 400.24 MHz for the 1 H. The spectrometer was equipped with a 5-mm Z gradient inversion probe head. The parameters used were 10200 Hz and 7200 Hz spectral width for 600 and 400 spectrometer respectively. 64 scans were accumulated using Bruker standard sequence. Chemical shifts (CSs) were given using an external standard reference (Tetramethylsilane, TMS). Mass spectrometry experiments were recorded with a Synapt HDMS (Waters) using a negative electrospray mode.

Surfactant synthesis

Synthesis of (3S)-3-[bis(carboxymethyl)amino]-6-(nonadecanoylamino)hexanoic acid

(nonadecanoyl-NTA / C19:0-NTA) Nonadecanoic acid (29.3 g, 98.2 mmol, 1 eq) and N-hydroxysuccinimide (16.9 g, 147.3 mmol, 1.5 eq) were dissolved in CH 2 Cl 2 (700 mL) followed by the addition of EDC, HCl (28.6 g, 147.3 mmol, 1.5 eq) (Table 1, Fig. 1). The mixture was placed under magnetic stirring at room temperature for 24 h and then diluted in water (700 mL). Organic and aqueous phases were then separated. The aqueous phase was extracted with CH 2 Cl 2 (3 x 250 mL). Organic phases were collected, washed with brine (250 mL), dried with Na 2 SO 4 , filtered and concentrated under reduced pressure. The product (nonadecanoyl-NHS) was finally obtained as a white powder after recrystallisation from hot EtOAc (700 mL).

Nonadecanoyl-NHS (3.5 g, 9.17 mmol, 1 eq), bis(carboxymethyl)lysine (NTA) (2.4 g, 9.17 mmol, 1 eq) and triethylamine (9 mL, 64.2 mmol, 7 eq) were dissolved in DMF (50 mL) under magnetic stirring at room temperature for 48 h. Solvent was then evaporated under reduced pressure. The residue was taken in water (150 mL) and acidified with HCl 1N until precipitation occured. The obtained suspension was stirred at room temperature for 1 h and filtered to yield the product as a white powder after drying under vacuum (4.17 g, 7.68 mmol, 84 %). The product was analysed with 1 H NMR and mass spectrometry for purity and chemical structure.

Nonadecanoyl-NTA (C19:0-NTA) (C 29 H 54 N 2 O 7 ): 1 H NMR (400 MHz, DMSO-d 6 ): δ 0.85 (3H, t), 1. 23-1.65 (40H, m), 2.01 (2H, t), 3.00 (2H, d), 3.30-3.52 (12H, m), 7.68 ( 1H, t). [(5Z,8Z,11Z,14Z)-icosa-5,8,11,14tetraenoylamino]hexanoic acid (arachidonoyl-NTA / C20:4-NTA) Bis(carboxymethyl)lysine (NTA) (0.7 g, 2.51 mmol, 1 eq) was dissolved in DMF (70.3 mL) and triethylamine (2.2 mL, 15.7 mmol, 6.3 eq) (Table 1, Fig. 2). Arachidonoyl-NHS (1 g, 2.49 mmol, 1 eq) was dissolved in DMF (34.7 mL) under magnetic stirring and nitrogen flow at room temperature. Then arachidonoyl-NHS solution was added to the bis(carboxymethyl)lysine (NTA) solution. The mixture was protected from light under magnetic stirring and nitrogen flow at 100°C for 40 h. The reaction was monitored using Ultra-Fast Liquid Chromatography (UFLC) at 210 nm. DMF was then evaporated under reduced pressure. The residue was dissolved in a mixture of acetonitrile and water (50/50 v/v). After filtration through a membrane filter of 0.45 µm pore size (polysulfone), the solution was injected onto a preparative reversed phase column chromatography. A 300 g bed of grafted silica (Kromasil, AkzoNobel) was used as stationnary phase. Eluting conditions were as follow: 0-15 min 100 % A, 15-35 min gradient from 100 % A to 100 % B then 35-45 min 100 % B. A, being purified water with 0.2 % (v/v) TFA and B CH 3 CN with 0.2 % (v/v) TFA. The expected product was collected after 31 min elution time, concentrated under pressure and freeze-dried (0.77 g, 1.4 mmol, 55 %). The product was analysed with 1 H NMR and mass spectrometry for purity and chemical structure.

Synthesis of (3S)-3-[bis(carboxymethyl)amino]-6-

Arachidonoyl-NTA (C20:4-NTA) (C 30 H 48 N 2 O 7 ): 1 H NMR (600 MHz, DMSO-d 6 ): δ 0.85 (3H, t), 1.20-1.40 (10H, m), 1.52 (2H, qt), 1.45-1.65 (2H, m), 2.00 (4H, q), 2.03 (2H, t), 2.75-2.79
(4H, m), 2.80 (2H, t), 2.98 (2H, q), 3.32 (1H, t), 3.44 (2H, d), 3.50 (2H, d), 5.25-5.40 (8H, m), 7.71 (1H, t), 12-13 (3H, vbs).

MS (ESI-) : m/z : 547.3 [M-H] -, 569.3 [M+Na-2H] -, 585.3 [M+K-2H] - 2.3.3 Synthesis of (3S)-3-[bis(carboxymethyl)amino]-6-(pentacosa-10,12-diynoylamino) hexanoic acid (10,12-pentacosadiynoyl-NTA / C25:4-NTA)
The synthesis of this surfactant was previously described by Ogier et al. In their study, the new drug carrier system was based on self-assembly and polymerization of polydiacetylenic surfactants. The authors also studied the solubilization capacity of non-polymerized analogues [START_REF] Ogier | Enhanced drug loading in polymerized micellar cargo[END_REF].

Synthesis of (3S)-3-[bis(carboxymethyl)amino]-6-(pentacosanoylamino)hexanoic acid

(pentacosanoyl-NTA / C25:0-NTA) 10,12-pentacosadiynoyl-NTA (C25:4-NTA, 0.5 g, 0.81 mmol, 1 eq) was dissolved in methanol with 250 mg of palladium on carbon 5 % (w/w) at room temperature (Fig. 3).

Hydrogenation was performed in a Paar apparatus under H 2 atmosphere (3.5 atm) for 16 h.

The mixture was then filtered through a nylon membrane filter and concentrated under reduced pressure. The residue was then solubilized in CH 2 Cl 2 and filtered. After CH 2 Cl 2 evaporation, the expected product was obtained as a white powder (0.01 g, 0.016 mmol, 2 %) Taking into account the low yield after purification, it was hypothesized that a large quantity of the surfactant was still chelated with paladium on carbon [START_REF] Richard | Supramolecular self-assembly of lipid derivatives on carbon nanotubes[END_REF]. Residual Pd on carbon was added to a mixture of water (25 mL), NaOH 1N (25 mL) and toluene (25 mL). After sonication, the organic layer was removed and replaced by CH 2 Cl 2 . The solution was then filtered through a membrane filter of 0.45 µm pore size. A yellowish liquid was obtained after filtration and freeze-dried. The residue obtained after lyophilization was solubilized in water and HCl 1N in large excess until precipitation occured. The precipitate was filtered and freeze-dried to remove the remaining water (0.3 g, 0.48 mmol, 59 %). The product was analysed with 1 H NMR and mass spectrometry for purity and chemical structure. 12-13 (3H, vbs).

Pentacosanoyl-NTA (C25:0-NTA) (C 35 H 66 N 2 O 7 ): 1 H NMR (400 MHz, DMSO-d 6 ): δ 0.85 (3H, m), 1.23 (40H, s), 1.30-1.70 (10H, m), 2.01 (2H, t), 2.99 (2H, q), 3.42 (2H, d), 3.49 (2H, d), 7.67 (1H, t),
MS (ESI-) : m/z : 625.5 [M-H] -, 647.5 [M+Na-2H] -

Molecular modelling

Surfactants 3-D structures were estimated in vacuo and treated at pH 7.5. Lower energy conformers and packing parameters were calculated using Volsurf+ software (1.0.6) (Molecular Discovery, London). From the Israelachvili packing parameter P, it is possible to predict the shape of the aggregate [START_REF] Israelachvili | A model for the packing of lipids in bilayer membranes[END_REF]. The molecular packing parameter is defined as P = v 0 /(al 0 ) where v 0 and l 0 are the volume and the length of the surfactant tail respectively and a is the area of the surfactant head group. The P values for prediction of spherical, cylindrical and lamellar aggregates are respectively for P ≤ 0.33, 0.33 ≤ P ≤ 0.5 and 0.5 ≤ P ≤ 1.

Fluorescence spectroscopy: CMC determination

Pyrene was used as a fluorescent hydrophobic probe to determine the critical micellar concentration (CMC) of surfactants. Pyrene would preferentially partition into hydrophobic cores of micelles with a concurrent change of the photophysical properties. Experiments were performed using a LS50B spectrofluorimeter (PerkinElmer). A pyrene stock solution (2 x 10 -4 M) was prepared in acetone. A 10 μL aliquot of this solution was introduced into empty vials and the solvent was evaporated at 37°C for 1 h. After evaporation, vials were filled with 1 mL of surfactant solution at a fixed concentration and gently stirred overnight at room temperature to ensure pyrene incorporation into micelles. Experiments were performed in triplicate. Samples were protected from light throughout the experiment. Samples were excited at λ = 335 nm and pyrene emission spectra were recorded from 370 to 400 nm wavelength. Both excitation and emission slit widths were set at 2.5 nm. Pyrene emission spectra presented vibronic peaks at λ 1 = 372 nm (intensity I 1 ) and λ 3 = 392 nm (intensity I 3 ).

The fluorescence intensity ratio (I 1 /I 3 ) was calculated and plotted as a function of surfactant concentration. Experimentally, the CMC was determined from the inflection point of the plot of the fluorescence intensity ratio versus surfactant concentration (semi-log plot). Using linear regression, the equations describing the two linear parts of the plot were established. The CMC was then obtained from the intersection of these two lines.

In vitro toxicity assays

Human Umbilical Vein Endothelial Cells (HUVEC) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing L-glutamine and a high glucose concentration (4,5 g/L), supplemented with 10 % Fetal Bovine Serum (FBS) and antibiotics (5 IU/mL penicillin and 50 IU/mL streptomycin). They were maintained as an adherent culture and were grown as a monolayer in a humidified incubator, with 5 % CO 2 , at 37°C in Petri dishes. DMEM and FBS were purchased from Lonza.

Cell viability assay

Surfactant toxicity was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyl tetrazolium bromide (MTT) assay. This colorimetric test measures the mitochondrial dehydrogenase cell activity, an indicator of cell viability. This assay is based on the reduction, by living cells, of the tetrazolium salt, MTT, which forms a blue formazan product. HUVEC were seeded onto 96 well plates with 200 µL medium per well at a density of 40000 cells/well for 24 h. Then, 10 µL of surfactant solution at the desired concentrations were added into each well in triplicate and plates were incubated for 72 h in a humidified incubator, with 5 % CO 2 , at 37°C. After incubation, 20 µL of MTT solution (5 mg/mL in PBS) were added into each well, except for negative control. Cells were further incubated for 2.5 h. After this incubation time, MTT containing media were removed and replaced with 200 µL of dimethylsulfoxide in order to dissolve the blue formazan product. MTT absorbance was measured at 570 nm wavelength with a microplate reader (Metertech Σ 960, Fisher Bioblock, France). Absorbance value was proportional to the number of living cells. The IC 50 was calculated as the sample concentration which inhibits growth of 50 % of cells relative to the control cells according to Unger et al. [START_REF] Unger | Branched polyesters based on poly[vinyl-3-(dialkylamino)alkylcarbamate-co-vinyl acetate-co-vinyl alcohol]-graft-poly(d,l-lactide-co-glycolide): Effects of polymer structure on cytotoxicity[END_REF]. IC 50 was calculated using the sigmoidal fitting function from Origin ® v 8.0 (OriginLab, Northampton, USA) when possible.

Membrane damage assay

Lactate dehydrogenase (LDH) is a cytosolic enzyme. During the experiment, a release of LDH in the extracellular medium indicates an alteration of the cell membrane. LDH catalyzes the oxidation of lactate to pyruvate with the parallel reduction of NAD + into nicotinamide adenine dinucleotide plus hydrogen (NADH). In turn, NADH reacts with an assay reagent (tetrazolium salt) to yield a red formazan product. LDH escaping from the cytoplasm compartment to the extracellular medium was measured through the formation of the red formazan product in medium, measured at 492 nm using a UV/VIS spectrophotomer. As a comparison, total release of LDH was measured after solubilization of HUVEC membranes with triton X-100 1 % (w/v) after an incubation time of 4 h. The fraction of LDH release was then used as an index of cellular membrane integrity: where 'LDH in sample' is LDH released in external medium after treatment with surfactants, 'LDH 0 %' is LDH present in culture medium and 'LDH 100 %' is LDH released after treatment with 1 % (w/v) triton X-100 corresponding to 100 % cell lysis. The activity of LDH was determined using a LDH kit (Promega).

Haemolytic assay

Blood samples (4 mL) were collected in citrated tubes (0.105 M) (BD Vacutainer ® ) from Wistar rats (250-350 g, Charles River Laboratories) by cardiac puncture, under anesthesia with sodium pentobarbital (50 mg/kg). Blood was then centrifuged (2000 rpm, 10 min) with a JOUAN MR1812 centrifuge to isolate red blood cells (RBCs). The erythrocyte suspension was washed twice and dispersed in isotonic PBS buffer (123.3 mM NaCl, 22.2 mM Na 2 HPO 4 and 5.5 mM KH 2 PO 4 , 300 mOsmol/L) at pH 7.4. All surfactant solutions were prepared in PBS buffer in order to obtain a pH close to normal blood pH (pH 7.4). Surfactant solutions (V = 100 µL) were incubated and stirred with RBCs suspension (V = 50 µL) at 37°C for 1 h. Instead of surfactants, minimum (0 %) and maximum (100 %) haemolysis were determined in PBS (V = 100 µL) and in triton X-100, 1 % (w/v) (V = 100 µL), respectively. Each sample was prepared in triplicate. Then, 2 mL of cold PBS were added to each sample to stop haemolysis, except for the 100 % haemolysis standard. For this specific standard, 2 mL of purified water were added. The unlysed RBCs were removed by centrifugation (2000 rpm for 10 min), and the supernatant was analyzed for haemoglobin release by UV/VIS spectroscopy at 450 nm wavelength (Shimadzu). Haemolysis percentage was determined from the equation below and plotted as a function of surfactant concentration. HC 50 , the concentration where 50 % of RBCs are lysed, was determined for each surfactant. The haemolysis percentage was calculated by the equation: where 'Abs', 'Abs 0 ', and 'Abs 100 ' were sample absorbance, control with PBS and control in the presence of haemolytic dose of triton X-100, respectively.

Drug solubilization

Surfactant solutions were prepared in purified water whose pH was adjusted to 12 with NaOH 1N. Then HCl 1N and 0.1N were added to accurately adjust the pH to 7.4 (C > CMC). An insoluble drug synthesized by Technologie SERVIER (MW: 861.5 g/mol, solubility: in water < 0.15 µg/mL) was used as a model insoluble drug. This anticancer drug is an antagonist of Bcl-2 receptors, which is soluble in methanol and acetonitrile at 8.5 mg/mL and 1.0 mg/mL, respectively. The drug was added at 20 mg/mL in the surfactant solution and magnetic stirring was applied during 24 h. Filtration of the insoluble part of the drug was then performed through a membrane filter of 0.45 µm pore size (polysulfone). The filtrate was diluted in acetonitrile/purified water (50/50, V/V). The amount of solubilized drug was quantified by HPLC system with UV detection at 250 nm. Drug loading was calculated dividing the mass of the solubilized drug by the sum of the surfactant mass (10 mg/mL) and the solubilized drug mass.

Results and discussion

Surfactant synthesis and solubility

All surfactants were synthesized and isolated with good purity as described above. Their chemical structure was evidenced using 1 H NMR spectra and mass spectrometry analysis.

NTA-based surfactants water-solubility was pH-dependent due to their anionic polar head. A required amount of NaOH 1N was therefore added to ensure a complete dissolution of each surfactant at pH 12. Then HCl 1N and 0.1N were added to accurately adjust the pH to 7.4. Surprisingly, C25:0-NTA exhibited a pronounced gelation effect increasing proportionally with surfactant concentration. This gelation occurred after surfactant solubilization in basic conditions and the gel was preserved at pH 7.4. Due to the monomeric structure of C25:0-NTA, this phenomenon could be explained by the formation of cylindrical worm-like micelles in solution [START_REF] Walker | Scattering from polymer-like micelles[END_REF][START_REF] Berret | Controlling electrostatic co-assembly using ion-containing copolymers: From surfactants to nanoparticles[END_REF]. Gelification most probably occurs by entanglement of wormlike micelles as their concentration increases. To strengthen this hypothesis, a molecular modelling study was performed.

Molecular modelling

Lower energy conformers of surfactants were determined and plotted in Table 2 for easier comparison. Both saturated surfactants, composed of lipid tail containing 19 or 25 carbon atoms, possess extended hydrophobic moieties. Because it contains a diacetylenic group, C25:4-NTA conformer presents a bend that confers a lower flexibility to the lipid chain without affecting its linear conformation. By contrast, C20:4-NTA exhibits a clear different behaviour with its lipid moiety totally folded on itself due to the presence of four double bonds. This leads to a non-linear conformation. Regarding the packing parameter [START_REF] Israelachvili | A model for the packing of lipids in bilayer membranes[END_REF], short hydrophobic chain surfactants, C19:0-NTA and C20:4-NTA, have geometric packing parameter values lower than 0.33 (Table 2). Taking into account geometric parameters of the polar and apolar moieties of the surfactant, the value of the packing parameter indicates the trend of both surfactants to self-assemble into spherical aggregates. C25:0-NTA and C25:4-NTA geometric packing parameter values are rather close to 0.5, which is characteristic of cylindrical or lamellar structures. Since alkanes chains composed of 25 carbon atoms are bulkier than the shorter ones, these surfactants need to have a very large headgroup to form a spherical aggregate in order to balance the effect of the bulky hydrophobic tail [START_REF] El Moujahid | Comparative structural study of aqueous fluorinated non-ionic micelles[END_REF]. For C25:0-NTA, the calculated packing parameter is consistent with the hypothesis of cylindrical micelles, forming an entangled gel at high concentration.

Surfactant self-assembling capacity: pyrene fluorescence measurements

Pyrene fluorescence measurements were performed and fluorescence intensitiy ratios (I 1 /I 3 ) were plotted as a function of surfactant concentration (semi-log plot, Fig. 4). The I 1 /I 3 ratio is initially steady with low surfactant concentrations, proving that pyrene is in a hydrophilic environment. Then a significant decrease of I 1 /I 3 ratio is observed indicating the formation of hydrophobic domains where pyrene is solubilized, a signature of surfactant micellization.

CMC values were determined and reported in Table 3 for easier comparison. Polysorbate 80 CMC value is 14 µg/mL, consistent with data from literature [START_REF] Wan | The limiting solubilizing capacity of some nonionic surfactants[END_REF]. C19:0-NTA, C25:0-NTA and C25:4-NTA CMC values were found of the same order of magnitude: 12, 40 and 24 µg/mL, respectively. The slight difference of CMC between C25:0-NTA and C25:4-NTA probably arises from interactions between neighbouring diacetylenic groups of the latter, leading to a lower CMC. The CMC of C20:4-NTA is an order of magnitude larger with a value of 430 µg/mL, most probably due to the presence of unsaturations which induce a weaker hydrophobicity of the acyl chain [START_REF] Chen | Surface activities of monoacyl trehaloses in aqueous solution[END_REF]. In addition, these results are in agreement with the study of Brito et al., who evidenced that CMC was higher for unsaturated compounds than for saturated ones. The presence of unsaturations creates larger chain packing constraints in the micellar core, due to a smaller number of conformational degrees of freedom. This does not favour micellization hence increasing CMC [START_REF] Brito | Enhanced interfacial properties of novel amino acid-derived surfactants: Effects of headgroup chemistry and of alkyl chain length and unsaturation[END_REF]. All surfactants tested were able to spontaneously self-assemble into water, with low CMC values in the same range as polysorbate 80 for 3 of them.

In vitro toxicity evaluation of surfactants

Cell viability assay

HUVEC viability was then assessed (Fig. 5, Table 3). A significant decrease of HUVEC viability was observed with C19:0-NTA concentrations higher than its CMC value. It is likely that C19:0-NTA micelles interact with HUVEC membrane and thus caused a decrease of cell viability. With an IC 50 of 0.02 mg/mL, C19:0-NTA was more cytotoxic than polysorbate 80 (IC 50 = 0.25 mg/mL) despite their similar CMC values. This was probably due to the higher hydrophobicity of C19:0-NTA, in comparison with polysorbate 80. Surprisingly, C25:0-NTA didn't decrease cell viability within the concentration range tested. The diffusion rate of surfactant monomers and micelles went down in the particular case of C25:0-NTA, due to gelation. Therefore, we hypothesized that gelation prevents their interaction with HUVEC, as observed by the absence of toxicity of this surfactant. A decrease of cell viability was observed with C25:4-NTA concentrations higher than its CMC value, leading to an IC 50 of 0.04 mg/mL. As a comparison, C20:4-NTA was slightly less toxic than polysorbate 80 with an IC 50 of 0.32 mg/mL. C25:0-NTA and C25:4-NTA do not seem to interact with HUVEC in the same way. Saturated surfactant, C25:0-NTA, was well tolerated whereas the unsaturated one, C25:4-NTA, decreased cell viability dramatically. These results evidenced that the presence of unsaturations in hydrophobic backbone influenced cytotoxicity. In the case of C25:4-NTA, the diacetylenic group prevents gelation, micelles are therefore able to diffuse rapidly and interact with HUVEC.

Membrane damage assay

Toxicity towards cell membrane was evaluated using LDH assay (Fig. 6, Table 3). The toxic effect of C19:0-NTA towards cell membrane was slightly lower than with polysorbate 80 with 50 % membrane lysis for concentrations of 0.2 and 0.13 mg/mL respectively. The assay evidences that C19:0-NTA is able to destabilize and disrupt HUVEC membranes probably by insertion of the hydrophobic chain, into the cell membrane [START_REF] Nogueira | The role of counterions in the membranedisruptive properties of pH-sensitive lysine-based surfactants[END_REF]. As a comparison, C25:0-NTA is well tolerated and does not induce cell damage: no IC 50 could be determined, probably because of its gelation properties. The network probably slows down the diffusion of surfactant monomers and thus limits their interaction with HUVEC membranes. Both surfactants composed of unsaturated hydrophobic chains, C20:4-NTA and C25:4-NTA were less toxic than polysorbate 80, with IC 50 values of 0.68 and 0.64 mg/mL respectively (Fig. 6, Table 3). The presence of unsaturations in their hydrophobic moieties decreases surfactant interactions with cell membrane. In the case of C20:4 hydrophobic part, unsaturations lead to an important steric hindrance which prevents its incorporation within the cell membrane. C25-based surfactants can be compared in terms of cytotoxicity: the unsaturated surfactant C25:4-NTA is toxic towards cell membranes whereas in the case of the saturated one, C25:0-NTA was not toxic, probably due to its gelling properties.

To conclude, membrane toxicity is clearly related to the length of surfactant hydrophobic moiety: small and saturated hydrophobic chains (C19:0) interact strongly with cell membranes whereas longer saturated chains (C25:0) do not incorporate into cell membrane. Furthermore, C20:4-NTA results demonstrate that steric hindrance prevents cell membrane interactions and thus cytotoxic effects.

Haemolytic assay

Haemolytic activity of surfactants was evaluated using rat RBCs. Haemolysis percentage was plotted as a function of surfactant concentration (semi-log plot). For C19:0-NTA and C25:0-NTA, results evidence that 50 % haemolysis occurs within the same concentration range, with HC 50 values of 0.15 and 0.13 mg/mL, respectively (Fig. 7 and Table 3). These surfactants are more haemolytic than polysorbate 80, which HC 50 value is 0.76 mg/mL. The length of the hydrophobic moiety, C19:0 versus C25:0, does not seem to influence surfactant haemolytic activity. C25:4-NTA is also more haemolytic than polysorbate 80 with an HC 50 of 0.25 mg/mL, in the same range as C19:0-NTA and C25:0-NTA. Surprisingly, a dramatic improvement was observed with C20:4-NTA which showed a 5-fold increase of the HC 50 value compared to polysorbate 80 (3.7 mg/mL). As observed for LDH release, the presence of four Z-unsaturations in the hydrophobic moiety of C20:4-NTA, leads to an important steric hindrance that prevents the insertion of the hydrophobic moiety within RBC membrane. The other surfactants which hydrophobic moieties are rather linear, can easily insert in RBCs membrane, causing haemolysis.

Drug solubilization capacity

Finally, drug solubilization was evaluated on a poorly soluble drug (< 0.15 µg/mL) (Fig. 8 and Table 4). C20:4-NTA was far less efficient than polysorbate 80 as solubilizing agent with solubility of 0.27 mg/mL (x 2000), corresponding to a drug loading of 3.5 % (w/w) only. As observed, on cell studies, the steric hindrance of C20:4-NTA prevents interactions with other hydrophobic compounds and therefore drug solubilization. Three surfactants out of four were able to increase drug solubility more efficiently than polysorbate 80 which only led to the solubility of 3.16 mg/mL (x 20000), corresponding to a drug loading of 24 % (w/w) (Table 4). C19:0-NTA, C25:0-NTA and C25:4-NTA increased drug solubility to concentrations higher than 7 mg/mL (x 47000 to x 57000) with drug loading of 41 %, 43 % and 46 % (w/w) respectively (Table 4). C25:0-NTA seems slightly more efficient than C19:0-NTA in solubilizing the drug which tends to indicate that a longer chain favours solubilization. Surprisingly, C25:4-NTA exhibits better solubilization efficiency than C25:0-NTA. The diacetylenic group, present in C25:4-NTA chemical structure seems to be favourable to drug solubilization.

Conclusion

Lysine based-surfactants synthesized in this study self-assemble into water with low CMC values ranging from 0.012 to 0.430 mg/mL. C19:0-NTA and C25:4-NTA were found to be more cytotoxic than polysorbate 80, whereas gelling properties of C25:0-NTA led to a low cytotoxicity. In addition, C20:4-NTA also exhibits a low cytotoxicity due to the presence of Z-unsaturations in its hydrophobic moiety. All surfactants exert a lower membrane toxicity than polysorbate 80. Surfactants with linear and flexible hydrophobic moieties lead to haemolysis at lower concentrations than polysorbate 80. On the other hand, C20:4-NTA, possesses a lower haemolytic activity due to the steric hindrance of its lipidic tail that prevents its insertion into RBC membranes. C19:0-NTA, C25:0-NTA and C25:4-NTA, are more efficient solubilizers, than polysorbate 80, due to the high flexibility and linear conformation of their hydrophobic moieties that favours interaction with the hydrophobic drug. They constitute promising drug delivery systems to solubilize anticancer drugs, via their selfassembling ability into spherical, cylindrical or lamellar structures. Overall, it appears that selection of a hydrophobic moiety will have to be done considering the affinity for the cell membrane leading to cytotoxicity and the strength of the hydrophobic interaction with the drug leading to an improved solubility. The best compromise will have to be found and will be tested in vivo in due time.

Acknowledgements

Authors would like to thank A. Petit and F. Munari for 1 H RMN experiments, for structural determination, W. Luijten, T. Renaud and M. Ginières for mass spectrometry analysis and P.

Vayer for molecular modelling (Technologie SERVIER), V. Marsaud for help in cell culture, V. Rosilio and V. Faivre for fruitful discussions (UMR CNRS 8612). This work was financially supported by ANRT from the Ministère de l'enseignement supérieur et de la recherche and Technologie SERVIER.