Directional Threading and Sliding of a Dissymmetrical Foldamer

Helix on Dissymmetrical Axles

Xiang Wang, [a] Quan Gan, [a] Barbara Wicher, [b] Yann Ferrand,* [a] and Ivan Huc* [a,c] Abstract: We have investigated the self-assembly of a dissymmetrical aromatic oligoamide helix on linear amido-carbamate rods. A dissymmetric sequence bearing two differentiated ends is able to wrap around dissymmetric dumbbell guest molecules. Structural and thermodynamic investigations allowed us to decipher the mode of binding of the helix that can bind specifically to the amide and carbamate groups of the rod. In parallel kinetic studies of threading and sliding of the helix along linear axles were also monitored by 1 H NMR. Results show that threading of a dissymmetrical host can be kinetically biased by the nature of the guest terminus allowing a preferential sense of sliding of the helix. The study presented below further demonstrates the valuable potential of foldaxanes to combine designed molecular recognition patterns with fine control of selfassembly kinetics to conceive complex supramolecular events.

Inspired by the molecular biomachinery, chemists have long tried to reproduce the controlled directional translations observed, for example, in the motion of DNA polymerases along DNA templates or of kinesin proteins along microtubules. Abundant literature thus exists on the translation of macrocycles on rod-like molecular axles. [1] Miniature artificial walkers have also been described. [2,3] Translation directionality can be imposed when changing the preferred position of macrocycles on a molecular rod by means of an external stimulus. Biased Brownian motion then allows the system to relax to its new preferred state leading to a net flux from one station to the other. [4] Reverting the preferred position back to its original state leads to a reversal of translation and shuttling is observed. [5] Besides the directionality of the motion, the relative inherent orientation of the axle and the component that slides along it is an intriguing aspect that has less often been addressed. [6][7][8] This stems from the fact that most macrocycles that have been used as molecular shuttles have a symmetrical structure. In contrast dissymmetrical macrocycles, e.g. cone-shaped calixarenes [6] cylodextrins [7] and others, [8] may thread onto a dissymmetrical axle. The lack of symmetry allows for the definition of a front and a rear of the shuttle, and also of frontward and backward motions. By extension, one could imagine chemical transformations that would occur exclusively at the rear and favor forward motion, for example via a ratcheting mechanism induced by processive catalysis (i.e. the installation of stoppers on the rod), or via a self-propelling mechanism. [9] As preliminary steps towards this goal, we herein present a dissymmetrical helix-rod host-guest system with a very strong thermodynamic preference for a particular relative helix/rod orientation (Figure 1a). We also show that kinetic bias in threading the rod into the helix cavity allows for the implementation of the directional motion of an oriented helix along an oriented rod.

We have previously introduced multi-turn helical aromatic oligoamide foldamers that form stable pseudo-rotaxane complexes, also termed foldaxanes, around rod-like guests. [10,11] Helices based on 7-amino-8-fluoro-2-quinolinecarboxylic acid oligoamides such as 1 possess a tubular cavity that can accommodate ,-alkanediamine-derived dicarbamate guests and slide long them. Threading and sliding of these helices along guests are much faster than their direct unwinding and rewinding, allowing for the design of kinetically favored supramolecular pathways depending on whether bulk too large to pass through the helix cavity is on the way or not. [12] Solution and solid state data demonstrated that helix-rod association is mainly driven by hydrogen bonds between carbamate carbonyl groups on the rods and 2,6-pyridinedicarboxamide protons on the helix carbamate cleft (CC in Figure 1b). This interaction should in principle make it possible to bind rods with other carbonyl-containing groups than carbamates. However, oligomers such as 1 do not bind to diamide rods or to ,-alkanediol-derived dicarbamate rods, i.e. when the orientation of the carbamates has been reverted. A closer look at earlier crystal structures [11a] pointed to secondary hydrogen bonding between the terminal pivaloyl amide proton and the carbamate sp3 oxygen atom as being responsible for this selectivity (Figure 1c). This observation hinted at the possibility to replace the foldamer terminal amide NH donor by a hydrogen acceptor and change guest selectivity, eventually giving access to dissymmetrical guest binding.

The AC segment was designed for this purpose (Figure 1b). For this, the terminal pyridine ring of CC was mutated into a naphthyridine [13] unit with the hope to create repulsive electrostatic interactions with the carbamate sp3 oxygen atom of the rod (Figure 1d). By analogy with 1, sequence 2 comprising a CC and an AC terminal segments was thus designed and synthesized (see the supporting information). To ascertain our hypothesis, dumbbell shaped guests 3-9 including both amide and carbamate groups were also prepared (Figure 1e). Titrations between single helix 2 and rods 3-9 were monitored by 1 H NMR in CDCl3 and, in some cases, revealed the emergence of a single new species in slow exchange on the NMR time scale typical of foldaxane formation (Figures S12-S17). As previously observed for binding studies between 1 and dicarbamate rods, [14] high foldaxane stability with 2 requires a match between the helix and the rod lengths (Figure 1e). The highest affinity constant was obtained for rod 5 bearing seven methylene units between its two binding anchors (Ka = 2.8 10 4 M -1 ).

Because of the dumbbell shape of the guests, host-guest complex formation requires the winding of the helix around the rod and this process is slow (Figure 1a). Monitoring of the binding of 5 by 2 at 323 K (i.e. under slight heating) revealed the progressive disappearance of the empty host, the concomitant emergence of the thermodynamically favored complex and also the transient appearance of another species (Figure 2a-2d). This is consistent with the initial formation of both a matching and a mismatching complex followed by the progressive disappearance of the less stable mismatching complex. At equilibrium, the minor species has disappeared showing quantitative bias as far as NMR can detect. Remarkably, the initial proportion of the mismatching complex is small (less than 6 %), indicating that this species is not only less stable, i.e. that it has a larger dissociation rate constant, but also that it has a slower formation rate. Thus, upon winding around the rod, the helix finds a faster pathway towards the most stable complex.

Structural information on 25 and in particular its assignment to a matching complex was gathered using X-ray crystallography. Single crystals were obtained by slow diffusion of n-hexane into a chloroform solution. The structure revealed that the single helix can fully wrap around the alkyl segment of the rod whose diphenyl groups protrude from both apertures of the foldamer cavity (Figure 2e,2f). The structure fully validates the initial hypothesis that the newly designed AC binds to the amide group whereas the CC hydrogen bonds to the carbamate of the guest (Figure 2g,2h). These favorable interactions and the repulsions that are expected to occur in the mismatching complex are strong enough to achieve quantitative selectivity in solution.

Foldaxane formation was then investigated with single station rods 10 and 11 possessing a bulky stopper at one end only (Figure 3a). This design allows for the unhindered threading of the other end of each axle into the helix at rates much faster than the winding of the helix onto binding stations (Figure 3b). Threading is actually so fast that an oligoethylene glycol chain (PEG750, M.W. = 750 g/mol) had to be introduced and the samples cooled to 273 K to slow it down and observe intermediates before equilibrium is reached. Rods 10 and 11 were used to study a possible preferred direction of threading. They both have a methoxy-terminated PEG chain and differ mainly in the position of amide and carbamate groups: the respective matching and mismatching complexes on these two rods have opposite orientations. For both rods, the two complexes are easily distinguished by 1 H NMR. In the case of 10, some mismatching complex was detected but, even at very early stages of the threading, its proportion remained small (< 20%). At equilibrium the mismatching complex had disappeared (Figure 3d-3g). This indicates that threading through the CC extremity of the helix, i.e. threading conducive to the matching complex, has been kinetically favored. In contrast, threading of 11 first produces a majority of mismatching complex that eventually quantitatively converts to the matching complex (Figure 3h-3k), confirming the faster threading through the CC extremity, even when it is conducive to a mismatching complex. Additional rods with various termini were prepared (compounds 13-16 in the supporting information). Monitoring foldaxane formation kinetics hints to threading being inherently favored at the CC terminus regardless of the rod functionality (Figures S22-S30). The rate limiting step in the threading process may involve favorable conformations of the helix, desolvation and interactions at the helix entrance. However, there is no information on which of these parameter is critical and why threading may be favored at the CC terminus.

We then prepared 12 to achieve a fully controlled oriented threading. This new rod bears two identical -and -binding stations (Figure 3a) positioned on both sides of a PEG600 spacer. A short n-propyl segment before the -station allows very fast threading/unthreading, while reaching the  station should be slower. Upon mixing 12 with 2, NMR showed the immediate and quasi quantitative formation of a new species that corresponds to threading of 2 on the -station of 12 (212,Figure 3m). No mismatching complex was observed on the -station presumably because equilibrium was reached before the first NMR measurement. At this early stage, only traces of matching 212could be detected (Figure 3m). Because matching/mismatching equilibrium is reached on the -station before translation motion to the -station occurs, this translation proceeds quantitatively towards the matching 212complex. Of note is the fact that this translation is significantly slower than the formation of e.g. 210 (Figure 3n, Figure S30-S31). This slow translation was ascribed to the dissociation of the stable matching 212 complex. At equilibrium, the proportions of 212and212 were similar due to the identical structures of -and -binding stations (Figure 3o). The formation of 212was thus eventually achieved without any detectable level of any mismatching complex, i.e. through a fully oriented process.

In summary, this study demonstrates that helical foldamers can be used to design selective molecular recognition patterns of rod-like guests and combine them with fine control of selfassembly kinetics to promote directional sliding processes in which both the rod and the helix have a defined orientation. We showed that a biased threading of a dissymmetrical helix can be programmed by varying the structure of the axle. We also demonstrated that the kinetic segregation of the helix threading and unthreading from its sliding further along the axle made possible a full control over the helix orientation on the rod. Precise anisotropic arrangements as those shown could give access to (poly)foldaxanes with potential use in processive catalysis or transport. Progress in these directions are currently being made in our laborotary and will be reported in due course.

Figure 1 .

 1 Figure 1. Host-guest components and assembly. (a) Schematic representation of the complex formation between a dissymmetric single helix and a dissymmetric dumbbell guest. (b) Formula of 1 and 2. Binding mode of a carbamate in a carbamate cleft(c) or in an amide cleft (red). The latter is destabilized by electrostatic repulsion (green double arrow). (e) Formula of dumbbell guests 3-9 and their association constants (Ka) with 2 at 323K.

Figure 2 .

 2 Figure 2. Excerpts of 400 MHz 1 H NMR spectra (323K in CDCl3) showing the amide resonances of 2 at 1 mM (a) and in the presence of 3 equiv. of 5 after: (b) 5 min; (c) 60 min. and (d) at equilibrium. Amide signals of the free single helix 2, matching and mismatching complexes 25 are marked with empty diamond, empty circles and black crosses, respectively. (e,f) Side views of 25 solid state structure analyzed by single crystal X-ray crystallography. In (e) the helix and the dumbbell rod are shown in CPK representation. In (f) the helix and the rod are shown as tube representation. The latter is covered by a transparent isosurface representing its volume. (g,h) Views from above and below showing the binding modes between AC and CC and the amide and carbamate groups of the rods shown in red and blue colors, respectively. In (e-h) CC and AC and the central helical segment are colored in blue, red and grey, respectively. Side chains (OiBu groups) and included solvent molecules have been removed for clarity.

Figure 3 .

 3 Figure 3. (a) Formula of rods 10-12. Schematic representations of: (b) the biased threading of a dissymmetrical single helix along a dissymmetrical rod and (c) the threading followed by the unidirectional sliding of a dissymmetrical single helix along a double station rod. Excerpts from the 700 MHz 1 H NMR spectra (CDCl3)showing the amide resonances of 2 at 1 mM alone(d,h,l), and in the presence of: 10 (3 equiv., 273K) after 7 min. (e); 30 min (f); and at equilibrium (g); 11 (3 equiv. , 273K) after 7 min. (i); 30 min. (j); and at equilibrium (k); 12 (3 equiv. at 283K) after 7 min (m); 120 min (n); and at equilibrium (o). Signals of the free double helix, the matching and mismatching complexes on the single station rods are marked with empty diamonds, empty circles and black crosses, respectively. Amide signals of the matching complex at -station and -station, are marked with empty and black circles, respectively.

Acknowledgements

This work was supported by the China Scholarship Council (predoctoral fellowship to X.W.), the Conseil Régional d'Aquitaine (Q.G.) and by grant ANR-17-CE07-0014. The authors thank Brice Kauffmann (IECB-UMS 3033) for his help during data collection and resolution of the crystal structures. this work has benefited from the facilities and expertises of the Biophysical and Structural Chemistry platform (BPCS) at IECB, CNRS UMS3033, Inserm US001, Bordeaux University.

Keywords: foldamer • molecular recognition • foldaxane • directional motion • molecular shuttle


Anita Chvatal

Rita Ambrus

Petra Party

Gábor Katona

Orsolya Jójárt-Laczkovich

Piroska Szabó-Révész

Elias Fattal

Nicolas Tsapis

Formulation and comparison of spray dried non-porous and large porous particles containing meloxicam for pulmonary drug delivery

Keywords: Meloxicam, Spray Drying, Dry Powder Inhalation, Large Porous Particles, Andersen Cascade Impactor AB -Ammonium Bicarbonate API -Active Pharmaceutical Ingredient CF -Cystic Fibrosis COPD -Chronic Obstructive Pulmonary Disease D[0.5] -Median

Introduction

In the past decades, carrier-free dry powder inhalers (DPIs) have shown promises for the local pulmonary treatment of chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). In carrier-free formulations, active pharmaceutical ingredients (APIs) and excipients are formulated together into the inhalable DPI form without using large carriers such as lactose [START_REF] Hoppentocht | Technological and practical challenges of dry powder inhalers and formulations[END_REF].

For pulmonary disorders with increased inflammation, the use of non-steroidal antiinflammatory drugs (NSAIDs), such as ibuprofen, diclofenac, gained the attention of research studies [START_REF] Szabó-Révész | Modifying the physicochemical properties of NSAIDs for nasal and pulmonary administration[END_REF]. Among NSAIDs, meloxicam (MX) is a low water-soluble cyclooxygenase enzyme-2 selective inhibitor, which has anti-inflammatory and antioxidant effects. Due to these effects, MX could be of interest for pulmonary administration to locally treat CF, COPD or any other lung inflammation [START_REF] Tsubouchi | Meloxicam inhibits the growth of non-small cell lung cancer[END_REF][START_REF] Arafa | Anti-fibrotic effect of meloxicam in a murine lung fibrosis model[END_REF].

So far, literature data reported preparation of MX containing inhalable microparticles by presuspension spray drying method. Pomázi et al. described the preparation MX particles suspended in an aqueous solution of excipients (mannitol, poly-vinyl-alcohol, polyvinylpyrrolidone and leucine) which was then spray dried at 130 °C to produce inhalable microparticles with enhanced aerodynamic properties [START_REF] Pomázi | Analysis of co-spray-dried meloxicam-mannitol systems containing crystalline microcomposites[END_REF][START_REF] Pomázi | Effect of polymers for aerolization properties of mannitol-based microcomposites containing meloxicam[END_REF][START_REF] Pomázi | Physicochemical stability and aerosolization performance of mannitol-based microcomposites[END_REF]. Due to the preparation method, MX was in crystalline form in the formulations. Another option for the inhalable microcomposite preparation is the use of MX potassium salt form and additional excipients (Poly-vinyl alcohol, leucine) [START_REF] Chvatal | Structural and aerodynamic evaluation of microcomposites containing meloxicam potassium[END_REF]. Due to the water solubility of the MX salt [START_REF] Mezei | Manufacture of high-purity meloxicam via its novel potassium salt monohydrate Org[END_REF], an easier feed stock preparation and spray drying conditions could be developed, leading to amorphous MX [START_REF] Chvatal | Structural and aerodynamic evaluation of microcomposites containing meloxicam potassium[END_REF].

The newest DPI researches report about several high potency carrier-free formulations prepared by spray drying. Co-spray drying has been widely used in the DPI production because it is scalable and offers an easily controlled particle formulation. Besides using this process for the micronization of the API, it can also lead to good aerodynamic properties for products [START_REF] Healy | Dry powders for oral inhalation free of lactose carrier particles[END_REF]. Innovative formulations by spray drying can be produced to create special structure (e.g. porous particles) and appropriate morphology of the API both for carrier-based and for carrier-free formulations [START_REF] Tsapis | Spray-drying from fundamentals to pulmonary administration of therapeutic molecules[END_REF].

Carrier-free formulation aimed to reduce the intrinsic cohesion of the particles, increase dispersion and delivery from the inhaler. Special excipients are used (for e.g. amino acids, polymers, bulking agents, phospholipids) in the particle design phase to implement also low density and enhanced aerosolization of the particles [START_REF] Healy | Dry powders for oral inhalation free of lactose carrier particles[END_REF].

Among widely used excipients to increase the aerosolization behaviour of the particles are leucine (LEU) and its analogues such as tri-Leucine [START_REF] Lechuga-Ballesteros | Trileucine Improves Aerosol Performance and Stability of Spray-Dried Powders for Inhalation[END_REF][START_REF] Vehring | Pharmaceutical particle engineering via spray drying[END_REF]. LEU was shown to promote good aerosolization of the particles by decreasing the surface energy (cohesive and adhesive forces) between particles as it is accumulates at particle surface [START_REF] Raula | Investigations on particle surface characteristics vs. dispersion behaviour of l-leucine coated carrier-free inhalable powders[END_REF]. It was also demonstrated that the LEU (above 20%, w/w content) may recrystallizes during the spray drying covering the surface of the particle and protecting it against moisture [START_REF] Li | L-Leucine as an excipient against moisture on in vitro aerosolization performances of highly hygroscopic spray-dried powders[END_REF].

Biodegradable polymers, such as sodium hyaluronate (HA), are also used to enhance the aerosolization of the API. HA can be used in the pulmonary drug delivery as a drug targeting, due to its mucoadhesive characteristics, absorption promoter and structure stabilizing effect [START_REF] Martinelli | Engineered sodium hyaluronate respirable dry powders for pulmonary drug delivery Int[END_REF]. HA also increases the viscosity of the primary solution to obtain large droplets for spray drying and thus larger particle sizes.

The aerodynamic performance of the DPIs also depends on the density of the powder. Particles with low density have better flowability and can be delivered to the lung easier with higher deposition [START_REF] Bosquillon | Influence of formulation excipients and physical characteristics of inhalation dry powders on their aerosolization performance[END_REF]). An exact density value is not clearly established, but most of the studies consider the DPI "low density" from a tap density around 0.4-0.1 g/cm 3 or lower [START_REF] Ogienko | Large porous particles for respiratory drug delivery. Glycine-based formulations[END_REF][START_REF] Ógáin | Particle engineering of materials for oral inhalation by dry powder inhalers. I-Particles of sugar excipients (trehalose and raffinose) for protein delivery[END_REF][START_REF] Watts | Respirable Low-Density Microparticles Formed In Situ from Aerosolized Brittle Matrices[END_REF]. Large porous particles (LPPs) can be obtained using porogen agents (e.g. volatile liquids, ammonium carbonate or bicarbonate) in the primary solutions or emulsions [START_REF] Gervelas | Direct lung delivery of a dry powder formulation of DTPA with improved aerosolization properties: Effect on lung and systemic decorporation of plutonium[END_REF]. The porogen evaporates quicker than the dispersion phase is drying, leaving holes in the interior of the larger sized particles [START_REF] N'guessan | Dexamethasone palmitate large porous particles: A controlled release formulation for lung delivery of corticosteroids[END_REF][START_REF] Pham | Formulation of pyrazinamide-loaded large porous particles for the pulmonary route: Avoiding crystal growth using excipients[END_REF]. The formulation of stable structured LPPs with the appropriate excipient-API concentration and production properties may be the challenge of the pharmaceutical technology.

We aimed to produce LPPs from MX aqueous solution by forming an MX salt at high pH. For this, we have optimized the spray drying procedure, by varying the solution excipient concentration. The planned LPPs with particle size larger than 5 µm and tap density lower than 0.25 g/cm 3 should be endowed with enhanced aerosolization properties [START_REF] Cruz | Formulation and in vivo evaluation of sodium alendronate spray-dried microparticles intended for lung delivery[END_REF]. The efficacies of the prepared LPPs were compared with non-porous formulations by their spray drying yield, physical chemical properties, morphology and in vitro aerodynamic behaviour. To the best of our knowledge, no other studies on the formulation of inhalable LPPs containing MX from solution exist.

Materials and methods

Materials

Meloxicam (MX) was obtained from Egis Plc (Hungary). L-Leucine (LEU) and ammonium bicarbonate (AB) were purchased from Sigma-Aldrich (France) and sodium hyaluronate (HA) from Acros Organics (Belgium). Sodium hydroxide was obtained from Sigma-Aldrich (France). Water was purified using a RIOS/MilliQ system from Millipore (France).

Preparation of the spray dried microparticles

MX exhibits a pH dependent solubility: in buffers at pH 7.4-7.7 MX demonstrates almost 43 fold solubility (1.74±0.2 mg/mL, 37 °C) at this pH than in distilled water (0.04±0.01 mg/mL, 37 °C) [START_REF] Horváth | Effect of solubility enhancement on nasal absorption of meloxicam[END_REF]. Due to this characteristic, MX was dissolved at room temperature (18-20 °C) at higher pH, stabilized with 1 M sodium hydroxide aqueous solution, to prepare a stock solution for the spray drying. First, MX was dissolved in the high pH water (8.00±0.1 pH, stirring for 2 hours, 600 rpm) at a constant concentration of 1.5 mg/mL for each formulation. Secondly, in the MX solution, LEU was dissolved (15 min, 600 rpm) to reach a final concentration of 0.75 mg/mL. In some cases, HA was added to finally yield a HA concentration of 0.15, 0.30 or 0.45 mg/mL (HA solutions were prepared the day before spray drying, stirring for 24 hours, 400 rpm). These solutions containing MX, MX+LEU or MX+LEU+HA were used for the formulation of non-porous particles. For LPP formulations, different concentrations of AB were added (0.5, 1.5 and 2.0 mg/mL) which functions as porogen agent as it decomposes into CO 2 and H 2 O upon heating. AB was dissolved 5 min before the spray drying (stirring for 2 min, 200 rpm) to minimize its decomposition prior to spray drying. The inhalable microparticles were produced by spray drying using a Büchi B-290 spray dryer equipped with a 0.7 mm two-fluid nozzle (Büchi, Switzerland). Drying properties were the followings: 200 °C inlet temperature, 9 mL/min feed pump rate (30%), 100% aspirator rate, and 414 L/h gas flow rate.

The spray drying yield was calculated as a percentage by dividing the mass of the powder collected from the container by the initial mass of solids in the solution prepared for drying. Prepared powders were stored in desiccator containing cobalt crystals to decrease moisture uptake (at 23±1 °C).

Identification of spray dried API

To investigate the characteristics of the API in the formulations, Raman spectroscopy was applied using Thermo Fisher DXR Dispersive Raman with CCD camera (Thermo Fisher Sco. Inc., Waltham, MA, USA). Following parameters were used during measurements: laser diode operating at a wavelength of 780 nm; the applied laser power was 6-24 mW at 25 µm slit aperture size on a 2 µm spot size; spectra were collected with 6 sec exposure time of 20 scanning in the spectral range of 3300-200 cm -1 . Reference sample, MX-recrystallized, was obtained with dissolving MX with the same method as MX solutions were prepared for spray drying. 1.5 mg/mL MX was dissolved in 8±0.1 pH sodium hydroxide aqueous solution (stirring for 2 hours, 600 rpm) and dried out at 40 °C, for 24 hours to study the changes during dissolution.

The actual API content (%) after spray drying was measured by dissolving 1.0-1.1 mg formulation in 25 ml of methanol:pH 7.4 phosphate buffer (60:40%, v/v), which solution was used for the aerodynamic assessment also. Solutions were mixed for 10 min, 600 rpm and API content was quantified by UV/Vis spectrophotometry (ATI-UNICAM UV/VIS Spectrophotometer, Cambridge, UK) at 362 nm wavelength. Each sample was measured in triplicate. The calibration curve y= 0.00465x, R 2 =0.9994 was used for quantification. Values LOD (0.037 µg/mL) and LOQ (0.124 µg/mL) were also established.

Morphology

Scanning electron microscopy (SEM) (Hitachi S4700, Hitachi Scientific Ltd., Tokyo, Japan) was used to characterize the morphology of the spray dried formulations, applying 10 kV high voltage and 1.3-13.0 mPa air pressure. A high vacuum evaporator and argon atmosphere was used to sputter-coat samples with gold-palladium in order to make them conductive (Bio-Rad SC 502, VG Microtech, Uckfield, UK).

Particle size distribution

The geometric diameter of the particles was established using laser diffraction (Mastersizer 2000 equipped with a Sirocco dry disperser, Malvern Instruments, France). For the measurements a micro size plate was used at a dispersing pressure of 2 bars. Each sample was measured in triplicate. The particle size distribution was characterized by the D[0.1] (10% of the volume distribution is below this value), D[0.5] (the volume median diameter is the diameter where 50% of the distribution is above and 50% is below) and D[0.9] (90% of the volume distribution is below this value) values. The size distribution Span was calculated according to Eq. 1. A high Span value denotes a broad particle size distribution. The higher the Span value, the broader the particle size distribution is [START_REF] Li | Interparticle van der Waals force in powder flowability and compactibility[END_REF]. All samples were measured in triplicate.

(Eq. 1)

Density measurements

The density of the formulations was measured using a 5 mL cylinder, filled with 2-4 mL powder (for bulk density) and tapped 1000 times (for tap density) using a tapping apparatus (Pharma test PT-TD1) (European Pharmacopeia 9 th edition). All samples were measured in triplicate.

Structural analyses

To establish the crystalline or amorphous character of the spray dried samples, X-ray powder diffraction (XRPD) spectra were recorded with a BRUKER D8 Advance X-ray diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) system with Cu Kα1 radiation (λ = 1.5406 Å) over the interval 3-40°. Measurement conditions were as follows: target, Cu; filter, Ni; voltage, 40 kV; current, 40 mA; time constant, 0.1 s; angular step 0.010°.

Aerodynamic assessment

The aerosolization efficacy of the spray dried formulations was assessed in vitro, using an Andersen cascade impactor -Apparatus D, (European Pharmacopeia 9 th edition), Copley Scientific, Switzerland). The inhalation flow rate was set at 30±1 L/min (High-capacity Pump Model HCP5, Critical Flow Controller Model TPK, Copley Scientific Ltd., Nottingham, UK). The inhalation time was 4 s for one inhalation simulation. Breezhaler® single dose devices were used, with size 3 capsules (transparent, Capsugel) filled with 2-2.5 mg of powder (constantly for each formulation). The inhaler was actuated twice for each capsule. The plates of the impactor were coated with 1 % w/v mixture of Span 85 and cyclohexane, to allow for the attachment of floating particles. After actuation, the inhalation device, the capsules, the induction port, the collection plates and the filter were washed with methanol:pH 7.4 phosphate buffer (60:40 %, v/v) to collect and dissolve the deposited API. The collected MX was quantified by UV/Vis spectrophotometry (ATI-UNICAM UV/VIS Spectrophotometer, Cambridge, UK) at 362 nm wavelengths. Aerodynamic properties (Fine particle fraction -FPF and Mass median aerodynamic diameter -MMAD) were calculated from a plot of the cumulative percentage undersize of API on log probability scale against the effective cut-off aerodynamic diameter [START_REF] Colombo | Inhalation drug delivery: techniques and products[END_REF]. The emitted fraction (EF) was expressed as the percentage of the API found in the cascade impactor (except the API found in the capsules and device) after inhalation relative to the total loaded dose.

All the samples were measured at the 1 st week after the spray drying and after 10 weeks of storage. Samples were stored at room temperature (23±1 °C), in a separate desiccator containing cobalt crystals to assess for their stability. Each sample was measured in triplicate. Statistical analyses were taken for the stability assessment using t-test calculation, 0.05 significance level and one-tailed hypothesis.

Results and discussion

Formulations were prepared according to Table I, by fixing MX concentration and varying the content in LEU and HA. In addition, non-porous particles (no AB added) were compared to LPPs prepared with AB. The Raman spectrum of MX-raw exhibits characteristic bands at 1155, 1309, 1540 and 1595 cm -1 (Fig. 1.). MX-recrystallized shows difference at the 1390 cm -1 compared to MX-raw where this band is missing. However, there is no difference in the spectra of the spray dried and the recrystallized samples. These Raman spectra are similar to the spectra of MX sodium salt according to Bio-Rad Laboratories database (Bio-Rad Laboratories, Inc. SpectraBase; Meloxicam sodium). Based on this result it was established that during dissolution of MX (at pH 8±0.1 in sodium hydroxide aqueous solution) MX sodium salt form was obtained. From the SEM pictures, one can observe that the excipient free sample (A: MX-SD) exhibits a slightly rough surface and spherical-like shape (Fig. 3.). The same surface morphology was obtained with the spray drying of meloxicam potassium salt from aqueous solution [START_REF] Chvatal | Structural and aerodynamic evaluation of microcomposites containing meloxicam potassium[END_REF]. In other studies, the spray drying of meloxicam from aqueous suspension resulted in smooth surface particles with around 19 µm size [START_REF] Pomázi | Analysis of co-spray-dried meloxicam-mannitol systems containing crystalline microcomposites[END_REF]. The presence of LEU increased the roughness of the surface and resulted in irregular-shaped particles (B: MX/LEU, C: MX/LEU/HA 0.3 , D: MX/LEU/HA 0.15 ). The shape modification can be explained by the crystallization of LEU on droplet surface forming an external shell which collapses during the spray drying process [START_REF] Vehring | Pharmaceutical particle engineering via spray drying[END_REF]. The surface accumulation of LEU and the resulting rough surface may reduce the adhesion between particles and the attachment to capsule or inhalation device walls, leading to higher EF or FPF values (Aquino et al, 2012;[START_REF] Mangal | Relationship between surface concentration of L-leucine and bulk powder properties in spray dried formulations[END_REF]. With 0.15-0.30 mg/mL HA content (C: MX/LEU/HA 0.15 and D: MX/LEU/HA 0.3 ) the particles kept similar wrinkled surfaces and the polymer has no visible impact on the morphology. However, literature data report that sodium hyaluronate could promote surface roughness of spray dried salbutamol sulphate particles [START_REF] Li | Inhaled hyaluronic acid microparticles extended pulmonary retention and suppressed systemic exposure of a shortacting bronchodilator[END_REF]. LPPs exhibit significantly different morphology than non-porous formulations proving the essential role of the porogen agent AB. The SEM pictures further demonstrated the porogen effect of AB, which was an essential excipient for LPP formulation. Samples MX/LEU/HA 0.15 /AB 1.5 (E), MX/LEU/HA 0.15 /AB 2 (F), MX/LEU/HA 0.3 /AB 1.5 (G) and MX/LEU/HA 0.3 /AB 2 (H) present spherical shaped large sized particles. However the surface roughness of LPPs was much lower, slight wrinkles can be detected on the surface of the spherical particles. This change in shape could arise from the gas formation from AB during the drying procedure, which blows up the wrinkled structure (Fig. 3). This effect of AB is a well described in literature [START_REF] Cruz | Formulation and in vivo evaluation of sodium alendronate spray-dried microparticles intended for lung delivery[END_REF][START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF][START_REF] Gervelas | Direct lung delivery of a dry powder formulation of DTPA with improved aerosolization properties: Effect on lung and systemic decorporation of plutonium[END_REF]. Some of the broken shells demonstrate the internal hollow structure which reflects the porosity and low density.

Size distribution

Despite all the powders were prepared using similar drying parameters, the final particle size distributions were different (Table II.). The geometric diameter of spray dried MX was around 3 µm, nearly the same as the sample prepared with LEU (MX-SD 3.2 µm and MX/LEU 3.4 µm). Incorporating 0.75 mg/mL LEU in the formulation did not increase the geometric size of the MX containing particles as reported with other MX formulations using 0.6 g/L or higher LEU concentrations [START_REF] Pham | Formulation of pyrazinamide-loaded large porous particles for the pulmonary route: Avoiding crystal growth using excipients[END_REF][START_REF] Chvatal | Structural and aerodynamic evaluation of microcomposites containing meloxicam potassium[END_REF]. Studies with different active ingredients also showed no effect of leucine concentration on particle size up to 20% w/w (Aquino et al, 2012) or only a low effect up to 15% w/w [START_REF] Mangal | Relationship between surface concentration of L-leucine and bulk powder properties in spray dried formulations[END_REF]. We demonstrated that the particle size is slightly increasing with an increasing amount of HA: from 3.4 µm up to 4.3 µm with 0.30 mg/mL HA. In case of LPPs the geometric size was also increased significantly with the higher HA concentration (from 5.0 µm to 5.6 µm with 0.15 and 0.30 mg/mL HA concentrations respectively). This increase probably arises from an increase of the viscosity of the spray dried solution leading to larger droplets and thus resulting in larger particle size. Li et al. also demonstrated that increased sodium hyaluronate concentration increases the particle size of co-spray dried salbutamol sulphate formulations from 3.8 µm up to 4.8 µm (0.20 and 0.35% w/w HA concentrations respectively) [START_REF] Li | Inhaled hyaluronic acid microparticles extended pulmonary retention and suppressed systemic exposure of a shortacting bronchodilator[END_REF]. With addition of AB to the LEU and HA combination, we obtained the aimed larger geometric diameter (larger than 4.9 µm) due to the formation of pores (blowing effect) of AB, as AB decomposes into water and gas during the drying process [START_REF] Cruz | Formulation and in vivo evaluation of sodium alendronate spray-dried microparticles intended for lung delivery[END_REF][START_REF] Pham | Pulmonary delivery of pyrazinamide-loaded large porous particles[END_REF]. No evident effect on the geometric size was detected comparing 1.5 and 2.0 mg/mL AB concentrations. Both non-porous particles and LPPs have narrow size distribution with Span ≤2.0. No significant differences were detected between the geometric diameter of the particles measured at 1 st week and 10 weeks after of storage.

Density of the spray dried microparticles

As expected, non-porous particles have higher density than LPPs. Comparing the tap and bulk density, it can be established, that the difference between tap and bulk density of LPPs is lower than the difference between the same parameters for non-porous particles. This lower tap-bulk density differences indicates the better rearrangement of LPPs upon tapping. Briefly, from the density of the spray dried MX (MX-SD) and the formulation with 0.75 mg/mL of LEU (MX/LEU) it is clear that the presence of LEU increased the tap density from 0.36 up to 0.45 g/cm 3 (Table II.). However, the bulk densities of these two formulations were similar (0.23 and 0.27 g/cm 3 ). This can be explained with the rough surface LEU-containing particles: particles with rough surface or wrinkled shape can pack more efficiently and result in higher tap density [START_REF] Simon | Development of a novel dry powder inhalation formulation for the delivery of rivastigmine hydrogen tartrate[END_REF]. Our results were similar with previous observations where leucine inclusion up to 15% (w/w) led to higher bulk density powders while up to 20% (w/w) produced powders with similar or slightly lower bulk density (Aquino et al, 2012).

Results show that the HA content has no relevant influence on the density of the powders. Despite the fact that HA is increasing the viscosity of solutions, it has no significant effect on the density of dried particles. Without AB all the samples containing HA (MX/LEU/HA 0.15 and MX/LEU/HA 0.3 ) had a tap density around 0.42-0.47 g/cm 3 . Low density of LPPs is related to the AB content which leads to the formation of a porous structure as it decomposes upon drying. The 2.0 and 1.5 mg/mL AB content have almost the same density decreasing effect and all the following samples (MX/LEU/HA 0.15 /AB 1.5 , MX/LEU/HA 0.15 /AB 2 , MX/LEU/HA 0.3 /AB 1.5 and MX/LEU/HA 0.15 /AB 2 ) reach tap density <0.19 g/cm 3 .

Table II. Compositions of the non-porous and large porous particles, prepared with the optimized spray drying conditions (200 °C inlet temperature, 100% aspirator capacity, 9 mL/min feed pump rate, and 414 L/h gas flow rate), the spray drying characteristics (API content and spray drying yield), size distribution (D[0.5] = Median geometric diameter and Span) and density properties (bulk and tap density). Data are represented as mean ±S.D., n=3. MX/LEU/HA 0.15 ) and LPPs (E: MX/LEU/HA 0.3 /AB 1.5 , F: MX/LEU/HA 0.15 /AB 2 , G: MX/LEU/HA 0.15 /AB 1.5 , H: MX/LEU/HA 0.3 /AB 2 ).

Sample names

X-Ray powder diffraction was then used to characterize the crystalline state of MX after the spray drying process (Fig. 4.). The raw MX has characteristic peaks with the highest intensities at 6. 6°, 11.4°, 13.1º, 13.5°, 15.1º, 18.7°, 19.3°, 25.9° and 26.4º 2-theta peaks indicating its crystalline structure [START_REF] Aytekin | Optical properties of Meloxicam in the far-infrared spectral region[END_REF]. The fact that the characteristic peaks of crystalline MX are missing from the diffractogram of the spray dried sample (MX-SD) indicates that the raw material becomes amorphous when spray drying without excipients (Fig. 4. and Supplementary material S1). However, characteristic peaks of MX at 6.6°, 11.4°, 13°, 13.6° and 19.2° 2-theta appear in the formulations containing LEU (Supplementary Material S1), indicating a low level of crystallinity. We detected the same characteristic peaks of LEU that literature data reported [START_REF] Najafabadi | The effect on vehicle on physical properties and aerolization behaviour of disodium cromoglycate microparticles spray dried alone or with L-leucine[END_REF]Li et al., 20016;[START_REF] Chvatal | Structural and aerodynamic evaluation of microcomposites containing meloxicam potassium[END_REF]. The characteristic peak 5.9° 2-theta of LEU could be observed on the spectra of the formulations but broader and with much lower intensity. These low intensity peaks demonstrate the low crystallinity of LEU after spray drying. The presence of HA has no effect on the diffractogram of the samples.

Aerodynamic properties

The aerodynamic properties (EF, FPF and MMAD) of powders were tested at 1 st week and 10 weeks after the spray drying. No significant difference can be detected in the EF, FPF and MMAD of the samples comparing the properties at 1 st week and after 10 weeks storage (desiccator, 23±1 °C). The aerodynamic properties of the spray dried formulations remain unchanged. An effect of excipients was observed for the non-porous formulations: those prepared with LEU have significantly higher EF than MX-SD. LEU increases the EF from ≤53.6% to ≥57.1%. LEU not just minimized the cohesion between particles, but also reduces the attachment to capsule wall during inhalation which resulted in higher EF values (Aquino et al, 2012;[START_REF] Mangal | Relationship between surface concentration of L-leucine and bulk powder properties in spray dried formulations[END_REF]. However, contrary to other results showing that surface roughness lead to an increased FPF for LEU containing corrugated particles [START_REF] Chew | How Much Particle Surface Corrugation Is Sufficient to Improve Aerosol Performance of Powders?[END_REF], we did not detect the FPF increasing effect of LEU. For the non-porous particles, the FPF remained in the 35-41% range independently of the excipients.

There are significant differences in the aerodynamic properties of LPPs and non-porous particles. LPPs result in higher EF and FPF than non-porous formulations (Table III).

Briefly, the EF of the non-porous particles is not exceeding 62.1%, while LPPs EF are above 76.1% in all cases. The high EF of LPPs is related to the good flowability of particles and low adhesion to the capsule and device walls. It can be demonstrated, that the used AB concentrations have a relevant effect on the aerodynamic behaviour of LPPs. LPPs have significantly lower tap density (comparing to non-porous particles) which resulted improved lung deposition (FPF 54.5-65.8%). Bosquillon et al also established the same correlation between density and aerodynamics: the lower the tap density (0.04-0.25 g/cm 3 ), the higher the FPF [START_REF] Bosquillon | Influence of formulation excipients and physical characteristics of inhalation dry powders on their aerosolization performance[END_REF]. LPP formulations prepared with 1.5 mg/mL AB concentrations have lower FPF (≤57.4%) than formulations containing 2.0 mg/mL AB (≥59.7%). The increased AB concentration (2.0 mg/mL) resulted in the highest FPF with 65.8% in case of MX/LEU/HA 0.15 /AB 2 (measured at the 1 st week). Despite the significant effect on the FPF, AB concentration has no significant effect on the EF of the LPPs.

Comparing the LPP formulations, it can be concluded that HA content has no significant effect on the EF or FPF. The presented low density LPPs (≤0.19 g/cm 3 ) have better aerosolization properties (EF≥76.1% and FPF≥54.5%) and can reach the lower airways more easily than the smaller but denser non-porous particles (EF≤62.1% and FPF≤41.2%). Figure 5 demonstrates the relevance of the large geometric diameter (>5 µm) and low density (<0.25 g/cm 3 ) LPPs for lung delivery (Fig. 5.). LPPs and non-porous particles result in the same MMAD values (average 2.55 µm) with no significant differences. However, LPPs have larger geometric diameter (≤4.9 µm) than non-porous formulations. In case of non-porous formulations the increasing D[0.5] resulted in larger MMADs. In case of LPPs the MMAD is not increasing with the D[0.5]. Different effects were observed comparing the excipient concentrations just in case of LPPs. LPPs with 2.0 mg/mL AB concentration exhibit lower MMAD (2.41-2.32 µm) than those with 1.5 mg/mL (2.57-2.74 µm). The higher amount of porogen affected positively the flowability of the particles thus resulted in lower aerodynamic diameter. By contrast, there is no significant difference between the MMAD of 0.15 and 0.30 mg/mL HA concentrations.

Conclusions

In the present study, two types of carrier-free DPIs were produced and compared: non-porous and LPP formulations of MX. We produced inhalable microparticles from MX aqueous solution with pH adjustment at pH 8±0.1, to facilitate the particle formulation process with in situ water soluble salt formation. Inhalable microparticles were produced with optimized spray drying conditions: 200 °C inlet temperature, 100% aspirator rate, 9 mL/min pump rate, and 414 L/h gas flow rate. LPPs with particle size larger than 4.9 µm and tap density lower than 0.19 g/cm 3 were obtained using 1.5 mg/mL MX, with 0.75 mg/mL LEU, 0.15 and 0.3 mg/mL HA; 1.5 and 2.0 mg/mL AB (samples MX/LEU/HA 0.3 /AB 1.5 , MX/LEU/HA 0.15 /AB 2 , MX/LEU/HA 0.15 /AB 1.5 and MX/LEU/HA 0.3 /AB 2 ). The aerosolization efficacies of the prepared LLPs were compared with the non-porous particles by testing the in vitro aerodynamical behaviour (with Andersen Cascade Impactor). The study was carried out using 30 L/min to simulate the low inhalation flow rate characteristic of lung inflammation diseases. LPPs show EF above 77% and FPF above 54%, both exceeding the non-porous particles values. The aerodynamical properties remain unchanged after 10 weeks of storage for all formulations. The concentration of AB, a porogen agent used in the primary solutions is a critical parameter of the LPP formulation. The best considered LPPs (with the highest spray drying yield >66% and FPF reaching 69%) were the ones containing 2 mg/mL AB as porogen beside the appropriate concentration of LEU and HA (samples MX/LEU/HA 0.3 /AB 2 and MX/LEU/HA 0.15 /AB 2 ). The presented aerodynamic properties have no significant difference measured after 1 and 10 weeks of storage. Thanks to the good aerosolization properties, the presented LPPs containing MX may offer an effective local treatment for lung inflammation diseases and should be tested in vivo in a near future.

3. 1 Fig. 1 .

 11 Fig. 1. Raman spectra of the raw MX (MX-raw), MX dissolved in sodium hydroxide at 1.5 mg/mL and dried out at 40 °C, 24 hours (MX-recrystallized) and spray dried MX (MX-SD).

Fig. 2 .Fig. 3 .

 23 Fig. 2. Comparison of the Raman spectra of the spray dried MX (MX-SD) and one of the LPP formulations (MX/LEU/HA 0.3 /AB 2 ).Spray dried MX (MX-SD) and formulations with excipients (Fig.2. MX/LEU/HA 0.3 /AB 2 LPP formulation presented) exhibit the same spectra as MX sodium salt with characteristic Raman bands at 1390 and 1595 cm -1 , indicating MX sodium salt form is present in spray dried formulations.

Fig. 4 .

 4 Fig. 4. XRPD spectra: The crystalline raw MX (MX-raw), and LEU (LEU-raw) are compared with the spray dried sample (A), non-porous particles (B: MX/LEU, C: MX/LEU/HA 0.3 , D: MX/LEU/HA 0.15 ) and LPPs (E: MX/LEU/HA 0.3 /AB 1.5 , F: MX/LEU/HA 0.15 /AB 2 , G: MX/LEU/HA 0.15 /AB 1.5 , H: MX/LEU/HA 0.3 /AB 2 ).

Fig. 5 .

 5 Fig. 5. Comparison of the aerodynamic and geometric diameter (MMAD = mass median aerodynamic diameter and D[0.5] = median geometric diameter) of the spray dried formulations. Data show the low density LPPs (E: MX/LEU/HA 0.3 /AB 1.5 , F: MX/LEU/HA 0.15 /AB 1.5 , G: MX/LEU/HA 0.3 /AB 2 and H: MX/LEU/HA 0.15 /AB 2 ) have the same or smaller MMAD than smaller but denser non-porous particles (A: MX-SD, B: MX/LEU, C: MX/LEU/HA 0.3 and D: MX/LEU/HA 0.15 ). Data are represented as mean ± S.D., n=3.

Table I .

 I Composition of the solutions prepared for spray drying (mg/mL) and the compositions of the spray dried products (%). MX=meloxicam, LEU=leucine, HA=sodium hyaluronate and AB=ammonium bicarbonate.

	Sample names	MX	LEU	HA	AB*
	Non-porous particles
	MX-SD	1.5 mg/mL (100%)	-	-	-
	MX/LEU	1.5 mg/mL (66.7%)	0.75 mg/mL (33.3%)	-	-
	MX/LEU/HA 0.3	1.5 mg/mL (58.8%)	0.75 mg/mL (29.4%)	0.30 mg/mL (11.8%)	-
	MX/LEU/HA 0.15	1.5 mg/mL (62.5%)	0.75 mg/mL (31.2%)	0.15 mg/mL (6.3%)	-
	Large porous particles
	MX/LEU/HA 0.3 /AB 1.5	1.5 mg/mL (58.8%)	0.75 mg/mL (29.4%)	0.30 mg/mL (11.8%)	1.5
	MX/LEU/HA 0.15 /AB 1.5	1.5 mg/mL (62.5%)	0.75 mg/mL (31.2%)	0.15 mg/mL (6.3%)	1.5
	MX/LEU/HA 0.3 /AB 2	1.5 mg/mL (58.8%)	0.75 mg/mL (29.4%)	0.30 mg/mL (11.8%)	2.0
	MX/LEU/HA 0.15 /AB 2	1.5 mg/mL (62.5%)	0.75 mg/mL (31.2%)	0.15 mg/mL (6.3%)	2.0
	*AB is not present in the dried samples as it decomposes into CO 2 and H 2 O during spray
	drying.

Table III .

 III Comparison of the aerodynamic properties of the non-porous and large porous particles tested at 1 st week and 10 weeks after spray drying simulating a 30 L/min inhalation flow rate. EF = emitted fraction, FPF = fine particle fraction. Data are represented as mean ±S.D., n=3.

	Sample names	1 st week EF (%) FPF (%)*	10 th week EF (%) FPF (%)*
	Non-porous particles
	MX-SD	53.6±10.5	38.6±4.7	50.4±6.7	37.9±4.8
	MX/LEU	61.7±4.1	41.2±3.3	59.9±8.1	39.7±1.7
	MX/LEU/HA 0.3	57.1±1.8	36.2±4.2	61.5±3.8	36.8±3.1
	MX/LEU/HA 0.15	59.9±1.7	35.6±2.7	62.1±2.9	35.9±2.6
	Large porous particles
	MX/LEU/HA 0.3 /AB 1.5	79.5±4.2	54.5±2.1	79.4±2.3	55.9±2.3
	MX/LEU/HA 0.15 /AB 1.5	77.5±6.8	57.4±5.6	76.1±9.9	54.5±10.1
	MX/LEU/HA 0.3 /AB 2	79.5±5.5	59.7±4.0	85.4±3.2	60.5±1.6
	MX/LEU/HA 0.15 /AB 2	82.8±6.8	65.8±3.2	82.3±8.3	63.0±4.5
	*FPF (<5 µm) was expressed with reference to the loaded dose.

Acknowledgements

This work was supported by UNKP-17-3-I-SZTE-UNKP-17-3-I-SZTE New National Excellence Program of the Ministry of Human Capacities. This project was completed as part of the SimInhale COST Action MP1404 -Short Time Scientific Missions (STSM) between Univ. Paris-Sud, Institut Galien Paris-Sud and the University of Szeged, Faculty of Pharmacy, Institute of Pharmaceutical Technology and Regulatory Affairs.

Supplementary Material

Figure S1: Zoom on the diffractograms of the spray dried samples (A), non-porous particles (B: MX/LEU, C: MX/LEU/HA 0.3 , D: MX/LEU/HA 0.15 ) and LPPs (E: MX/LEU/HA 0.3 /AB 1.5 , F: MX/LEU/HA 0.15 /AB 2 , G: MX/LEU/HA 0.15 /AB 1.5 , H: MX/LEU/HA 0.3 /AB 2 ).


Alexandre Bordat

Imen Nancy Soliman

Ben Chraït

Katia Manerlax

Najet Yagoubi

Tanguy Boissenot

Julien Nicolas
email: julien.nicolas@u-psud.fr

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

Keywords: thermoresponsive, polymer, nanoparticles, UCST, doxorubicin, hyperthermia

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.

The crucial role of macromolecular engineering, drug encapsulation and dilution on the thermoresponsiveness of UCST diblock copolymer nanoparticles used for hyperthermia

1

The crucial role of macromolecular engineering, drug encapsulation and dilution on the thermoresponsiveness of UCST diblock copolymer nanoparticles used for hyperthermia Alexandre Bordat 1 , Nancy Soliman 1 , Imen Ben Chraït 1 , Katia Manerlax 2 , Najet Yagoubi 2 , Tanguy Boissenot 1 , Julien Nicolas 1, *, Nicolas Tsapis 1, *

Introduction

Nanomedicine holds great promise in dealing with the shortcomings of fragile and/or poorly soluble anticancer drug formulations, such as longer circulation time [START_REF] Owens | Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles[END_REF] and fewer side effects for the healthy tissues. [START_REF] O'brien | Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX TM /Doxil®) versus conventional doxorubicin for first-line treatment of metastatic breast cancer[END_REF] Newer generations of nanoparticles aim at going one step further by allowing full control of the spatial and temporal release of the drug. This is made possible by, for instance, employing stimulus-responsive starting material to build the drug carrier. [START_REF] Mura | Stimuli-responsive nanocarriers for drug delivery[END_REF] Temperature is an easy-to-use trigger that can be exploited through mild hyperthermia, corresponding to an elevation of tissue temperature from 37 °C to 43 °C. [START_REF] Boissenot | Ultrasound-triggered drug delivery for cancer treatment using drug delivery systems: From theoretical considerations to practical applications[END_REF] Clinicians dispose of a wide array of tools to locally heat tissue, such as ultrasound, [START_REF] Kruse | Spatial and Temporal-Controlled Tissue Heating on a Modified Clinical Ultrasound Scanner for Generating Mild Hyperthermia in Tumors[END_REF] microwave, [START_REF] Johnson | Automatic Temperature Controller for Multielement Array Hyperthermia Systems[END_REF] radiofrequency [START_REF] Nagata | Clinical results of radiofrequency hyperthermia for malignant liver tumors[END_REF] and infrared [START_REF] Hoffmann | Principles and working mechanisms of water-filtered infrared-A (wIRA) in relation to wound healing[END_REF] energy sources. Mild hyperthermia has a biological effect on the tumor microenvironment as the endothelial tissue becomes more permeable [START_REF] Kong | Hyperthermia Enables Tumor-specific Nanoparticle Delivery: Effect of Particle Size[END_REF][START_REF] Kong | Characterization of the Effect of Hyperthermia on Nanoparticle Extravasation from Tumor Vasculature[END_REF] and immune response is enhanced. [START_REF] Calderwood | How is the immune response affected by hyperthermia and heat shock proteins?[END_REF][START_REF] Hildebrandt | The cellular and molecular basis of hyperthermia[END_REF] Thus, the prospect of using mild-hyperthermia with tailored nanomedicine is enthralling and should lead to better disease management. [START_REF] Dunne | Thermosensitive nanomedicines could revolutionize thermal therapy in oncology[END_REF] To the best of our knowledge, only one thermoresponsive formulation of doxorubicin (Dox) exists in clinical stages of development. It is in the form of thermosensitive liposomes that are currently undergoing phase 3 of clinical trials, under the name of ThermoDox® by Celsion. [START_REF] Ta | Thermosensitive liposomes for localized delivery and triggered release of chemotherapy[END_REF] ThermoDox® is intended to work as a companion for radiofrequency tumor ablation. It is administered intravenously while the tumor is being ablated by radiofrequency. The generated heat triggers the local release of Dox that in turn exerts its effect on the remaining tumor cells that were not fully ablated by radiofrequency. Although the formulation is in clinical trials, there are still some limitations regarding this system as liposomes are fragile and have poor drug retention. [START_REF] Sandström | Effects of lipid segregation and lysolipid dissociation on drug release from thermosensitive liposomes[END_REF][START_REF] Banno | The functional roles of poly(ethylene glycol)-lipid and lysolipid in the drug retention and release from lysolipid-containing thermosensitive liposomes in vitro and in vivo[END_REF] In contrast, polymer nanoparticles may provide better drug retention and in terms of polymer chemistry, there is still no thermoresponsive polymer nanoparticle formulation undergoing clinical trials. Lower critical solution temperature (LCST) thermoreponsive polymers have been used to produce thermoresponsive nanocarriers for several decades, [START_REF] Cammas | Thermo-responsive polymer nanoparticles with a core-shell micelle structure as site-specific drug carriers[END_REF] but yet without successful translation to the clinic. [START_REF] Karimi | Temperature-Responsive Smart Nanocarriers for Delivery of Therapeutic Agents: Applications and Recent Advances[END_REF] These polymers are soluble in water below the LCST and insoluble above. On the other hand, upper critical solution temperature (UCST) polymers are less widely investigated while their thermoresponsiveness appears more intuitive as they are fully soluble above the UCST and insoluble below. An example of such polymer is poly(acrylamide-coacrylonitrile) (P(AAm-co-AN)), [START_REF] Seuring | First Example of a Universal and Cost-Effective Approach : Polymers with Tunable Upper Critical Solution Temperature in Water and Electrolyte Solution[END_REF] a UCST-type copolymer that owes its thermoresponsiveness to the reversible hydrogen bonds made between the copolymer chains below the UCST and the surrounding water molecules above the UCST. In terms of polymer synthesis, P(AAm-co-AN) can be easily synthesized by free-radical polymerization [START_REF] Seuring | First Example of a Universal and Cost-Effective Approach : Polymers with Tunable Upper Critical Solution Temperature in Water and Electrolyte Solution[END_REF][START_REF] Käfer | LCST and UCST in One: Double Thermoresponsive Behavior of Block Copolymers of Poly(ethylene glycol) and Poly(acrylamide-co-acrylonitrile)[END_REF][START_REF] Hou | Understanding the UCST-type transition of P(AAm-co-AN) in H 2 O and D 2 O: dramatic effects of solvent isotopes[END_REF] or reversible deactivation radical polymerization techniques such as reversible addition fragmentation chain transfer (RAFT)

polymerization. [START_REF] Zhang | Diverse Thermoresponsive Behaviors of Uncharged UCST Block Copolymer Micelles in Physiological Medium[END_REF][START_REF] Huang | Self-assembled UCST-type micelles as potential drug carriers for cancer therapeutics[END_REF][START_REF] Asadujjaman | Dielectric analysis of the upper critical solution temperature behaviour of a poly(acrylamide-co-acrylonitrile) copolymer system in water[END_REF][START_REF] Asadujjaman | Phase transition and aggregation behaviour of an UCST-type copolymer poly(acrylamide-co-acrylonitrile) in water: effect of acrylonitrile content, concentration in solution, copolymer chain length and presence of electrolyte[END_REF][START_REF] Yang | NIR-controlled morphology transformation and pulsatile drug delivery based on multifunctional phototheranostic nanoparticles for photoacoustic imaging-guided photothermal-chemotherapy[END_REF] The copolymer obtained by free-radical polymerization has been grafted

with poly(ethylene glycol) (PEG) on the acrylamide monomers to formulate thermoresponsive nanoparticles efficiently responding to mild-hyperthermia for in vivo tumor treatment. [START_REF] Li | Antitumor Drug Delivery Modulated by A Polymeric Micelle with an Upper Critical Solution Temperature[END_REF] Here, we design a different synthetic route to yield a well-defined, amphiphilic block copolymer. A poly(acrylamide-co-acrylonitrile)-b-poly[(oligo(ethylene glycol) methyl ether methacrylate)]

(P(AAm-co-AN)-b-POEGMA) diblock copolymer was synthesized by RAFT polymerization of OEGMA from a P(AAm-co-AN) UCST-type macro-RAFT agent. This strategy allowed control on the size of both the thermoresponsive block and the hydrophilic block. Copolymers with different Mn and AN content were synthesized to optimize the cloud point (Tt-UCST) for mild hyperthermia. These copolymers were then formulated into nanoparticles and the influence of the Tt-UCST on the physicochemical properties of the colloidal suspension was investigated. We also evaluated the effect of Dox encapsulation on nanoparticle thermoresponsiveness and characteristics, as the influence of drug encapsulation is often overlooked despite its crucial impact in the development of thermosensitive nanoscale drug delivery systems. Finally, Dox-loaded nanoparticle cytotoxicity was assessed in vitro on ovarian cancer cells with or without application of hyperthermia.

Materials and methods

Materials

Acrylamide (AAm) (Sigma-Aldrich) was recrystallized in chloroform, acrylonitrile (AN) (Sigma-Aldrich) was purified by passing through a column filled with activated, basic, Brockman I aluminum oxide. Azobisisobutyronitrile (Sigma-Aldrich) was recrystallized in ethanol.

Oligo(ethylene glycol) methyl ether methacrylate (OEGMA) (Mn ~ 300 g.mol -1 , Sigma-Aldrich), 2cyano-2-propyl benzodithioate (CPDB) (Sigma-Aldrich), doxorubicin hydrochloride (Dox.HCl) (Carbosynth), and triethylamine (TEA) (Sigma-Aldrich) were used as received. RPMI-1640 cell culture medium and bovine insulin were purchased from Sigma-Aldrich. Water was purified using a

MilliQ Reference system from Merck-Millipore. Solvents were of HPLC analytical grade and were provided by Carlo Erba (Italy). For NMR measurements, d6-DMSO was provided by Eurisotop (France).

Fourier transform infrared (FTIR) spectroscopy for AN quantification

KBr pellets containing the polymer samples were prepared for transmission FTIR analysis using FTIR Spectrum One (PerkinElmer) by mixing polymer samples with KBr at 5 wt.% and running 5 scans at 4 cm -1 resolution per sample. For mol.% AN determination, a calibration curve was obtained by mixing PAAm and PAN (both from Sigma-Aldrich) at determined ratios and by calculating the peak area ratio between the carbonyl peak (C=O at 1659 cm -1 ) and the nitrile peak (C≡N at 2242 cm -1 ) (Supplementary Information, Fig. S7). Experiments were performed in

Nuclear magnetic resonance (NMR) spectroscopy

and the broad peak between 1.40 and 2.09 ppm assigned to the protons of the methylene group in all monomer units (noted g in Supplementary Information, Fig. S1), that allowed determination of the total content in AAm and AN. By using the measured mol.% AN using method 2.2., the Mn was then determined. For P(AAm-co-AN)-b-POEGMA copolymers, integration of the broad peak between 6.25 and 7.75 ppm corresponding to the NH2 protons of the acrylamide, was compared to the integration of the peak between 3.95 and 4.18 ppm corresponding to 2 OEGMA protons (Supplementary Information, Fig. S1).

Size exclusion chromatography (SEC)

SEC was performed at 50 °C with two columns from PSS Gram (GPC column 300 x 8 mm; bead diameter 10 µm; molar mass range 500 -10 6 g.mol -1 ) and a triple detection system (Viscotek TDA/GPCmax from Malvern) with a differential refractive index detector, low and right-angle light scattering detectors and a differential viscometer detector. The eluent was dimethyl sulfoxide (DMSO) at a flow rate of 0.7 mL.min -1 . The system was calibrated using a narrow pullulan standard and each polymer sample was injected at 5 different injection volumes for the determination of the dn/dc parameter. This allowed the determination of the number-average molar mass (Mn), the weight-average molar mass (Mw) and the dispersity (Ð = Mw/Mn).

Suspension turbidity for Tt-UCST and Tt-LCST determination

Light transmittance (%) of samples was measured using a Lambda 25 UV/VIS spectrometer equipped with a PTP 1+1 Peltier system for temperature control (PerkinElmer). Samples were prepared at 4.5 mg.mL -1 , spectrometer wavelength was set at 600 nm, temperature ramp was at 0.5 °C.min -1 and data acquisition was performed every 30 s. The transition temperature Tt-UCST is defined as the point at which the transmittance reaches a maximum plateau for UCST behavior. For LCST behavior Tt-LCST corresponds to the point at which the transmittance reaches a minimum plateau.

Dynamic light scattering (DLS) and zeta potential

Nanoparticle intensity-averaged diameters (Dz) and zeta potentials (ζ) were measured by dynamic light scattering (DLS) with a Nano ZS from Malvern (173° scattering angle) at a temperature of 20 °C. The surface charge of the nanoparticles was investigated by ζ-potential (mV) measurement at 20 °C after dilution with 1 mM NaCl, using the Smoluchowski equation. Experiments were performed in triplicate.

2.7.

UV spectroscopy for doxorubicin quantification 3 mL of the suspension were centrifuged at 20,000 g for 1 h at 4 °C (Optima LE-80K Ultracentrifuge Beckman Coulter). The supernatant was discarded, and the pellet was dissolved in 5 mL of DMSO.

The dissolved samples were put in 96-well plates before quantification at 490 nm by a plate reader.

Calibration curve was determined by preparing a solution of Dox at 6 mgmL -1 and TEA (35 µL) in DMSO and subsequent dilution to a range between 6 and 200 µg.mL -1 of Dox. Drug loading (DL, wt.%) and encapsulation efficiency (EE, wt.%) were calculated using the following formulas:

DL = EE =
Experiments were performed in triplicate.

Transmission electron microscopy (TEM)

TEM images were obtained using transmission electron microscope of 80 kV Jeol 1400 (Imagif, I2BC, CNRS, Gif-sur-Yvette, France). Briefly, each suspension was diluted ten-fold in water. Then, 3 μL of this dilution were placed on a glow-discharged copper grid covered with formvar-carbon film.

The excess solution was blotted off using a filter paper. Images were acquired using an Orius camera (Gatan Inc, USA).

Synthesis of P(AAm-co-AN)

The overall procedure was adapted from the work of Zhang et al. [START_REF] Zhang | Diverse Thermoresponsive Behaviors of Uncharged UCST Block Copolymer Micelles in Physiological Medium[END_REF] S2). The mixture was purged with argon for 15 min under vigorous stirring before being placed in a 70 °C-oil bath for 24 h under stirring. After the reaction, the copolymer was precipitated twice in methanol (20 times the volume of the mixture). The copolymer was further solubilized in DMSO and placed in a 3.5 kD Spectra/Por 3 dialysis bag for dialysis against water for 3 days at room temperature (20°C), with dialysis water changed twice per day. The dialysate was then freeze-dried to yield P(AAm-co-AN) as a red hard solid paste. Spectra/Por 3 dialysis bag for dialysis against water for 24 h, with dialysis water changed three times. The dialysate was then freeze-dried to yield the diblock copolymer as a white-reddish soft solid paste.

Synthesis of P(AAm

Unloaded nanoparticle formulation

Preparation of thermoresponsive copolymer nanoparticles was carried out using the nanoprecipitation technique. 50 mg of copolymer were dissolved in 1 mL of DMSO. The solution was added at 150 µL/min using a syringe pump (Harvard Apparatus Pump 11 Pico Plus Elite) to 10 mL of water (or PBS 10mM) under vigorous stirring. To remove DMSO, the suspension was put into a 3.5 kD Spectra/Por 3 dialysis bag for dialysis against water (or PBS) for 24 h, with dialysis medium changed twice.

Doxorubicin-loaded nanoparticle formulation

The protocol was adapted from Huang et al. [START_REF] Huang | Self-assembled UCST-type micelles as potential drug carriers for cancer therapeutics[END_REF] and is described as follows: 50 mg of copolymer were dissolved in 500 µL of DMSO. Dox.HCl (5 mg, 8.62 µmol) was dissolved in 500 µL of DMSO prior to addition of 35 µL (251 µmol) of TEA and the solution was stirred for 7 h shaded from light at room temperature. The copolymer and Dox solutions were then mixed, and the following steps were the same as in 2.11. in water only, with the solutions shaded from light. For the drug encapsulation curve, the amount of initial Dox.HCl varied from 1 to 5 mg.

Cell culture

Nanoparticle cytotoxicity was evaluated on the human ovarian carcinoma cell line OVCAR-3 obtained from ATCC (USA). OVCAR-3 cells were cultured in RPMI-1640 medium supplemented with 100 U/mL penicillin, 20% heat inactivated FBS, and 0.01 mg.mL -1 bovine insulin. The cell line was maintained at 37 °C and 5% CO2 in a humidified atmosphere and cells were split twice weekly. The cell viability was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were seeded in 100 μL of culture medium (8 x 10 3 cells/well for 72 h incubation) in 96 well plates (TPP, Switzerland) and pre-incubated for 24 h. 100 μL of a serial dilution of nanoparticle suspension was then added to the medium. After 72 h of incubation, 20 μL of MTT solution (5 mg.mL -1 in PBS) was added to each well. After 4 h incubation, the culture medium was gently aspirated and replaced by 200 μL DMSO (ACS grade, VWR, France) to dissolve the formazan crystals. The absorbance of the solubilized dye, which correlates with the number of living cells, was measured with a microplate reader (LAB Systems Original Multiscan MS, Finland) at 570 nm. The percentage of viable cells in each well was calculated as the absorbance ratio between nanoparticle-treated and untreated control cells. For hyperthermia studies, the protocol was the same except cells were incubated at 43 °C for 30 min, either 6 h or 24 h after nanoparticle addition to the wells. After 30 min, the cells were put back in the incubator at 37 °C until they had been exposed for a total of 72 h. Experiments were performed in triplicate. Data was fitted to a Hill slope model with four parameters using GraphPad Prism 7 which allowed determination of the IC50 for the Dox-containing treatments. The different IC50 values were compared using a one-way ANOVA test with GraphPad Prism 7.

Results and discussion

Thermoresponsive copolymer synthesis and UCST behavior

Thermoresponsive poly(acrylamide-co-acrylonitrile) (P(AAm-co-AN)) copolymers with different AN contents were synthesized to evaluate the dependency of the UCST behavior relative to the AN content (Fig. 1, T1-T8, Table 1). Among the different reversible deactivation radical polymerization techniques, RAFT polymerization was chosen for its simplicity, its ability to control such monomers, together with its capability to form well-defined block copolymers by efficient chain-extension from macro-RAFT agents (Fig. 1). [START_REF] Chong | More versatile route to block copolymers and other polymers of complex architecture by living radical polymerization: the RAFT process[END_REF][START_REF] Moad | Living free radical polymerization with reversible addition -fragmentation chain transfer (the life of RAFT)[END_REF][START_REF] Tang | RAFT polymerization of acrylonitrile and preparation of block copolymers using 2-cyanoethyl dithiobenzoate as the transfer agent[END_REF][START_REF] Thomas | Kinetics and molecular weight control of the polymerization of acrylamide via RAFT[END_REF] AN content was determined by infrared spectroscopy against a pre-determined calibration curve (Supporting Information, Fig. S7). AN content was in good correlation with the theoretical value, yet the technique was not completely robust as values for T4

and T8 had an important standard deviation and it proved difficult to get a good measurement for T3. After synthesis of the P(AAm-co-AN) block, our approach consisted in performing its chainextension by polymerizing oligo(ethylene glycol) methyl ether methacrylate (OEGMA, Mn ~300 g.mol -1 ) under RAFT control to yield the corresponding P(AAm-co-AN)-b-POEGMA diblock copolymer comprising a thermoresponsive UCST-type block and a hydrophilic one (Fig. 1, P1 -P8, Table 2). Importantly, the hydrophilic POEGMA block acts as a biocompatible and protein repellent corona to prolong the blood circulation time of the nanoparticles (the so-called "stealth" feature). [START_REF] Otsuka | PEGylated nanoparticles for biological and pharmaceutical applications[END_REF][START_REF] Veronese | Branched and linear poly(ethylene glycol): Influence of the polymer structure on enzymological, pharmacokinetic, and immunological properties of protein conjugates[END_REF] The Mn of the different copolymers was measured both by 1 H-NMR and a triple detection SEC (that allows absolute Mn values to be determined). Even though SEC is the most used method for Mn determination, 1 H-NMR is appealing as integration of chain-end protons allows precise Mn measurement (irrespectively of the nature of the polymer), providing the Mn is rather low. In our case, since we have synthesized relatively large copolymers (Mn,SEC =20-63 kg.mol -1 ), some discrepancies were observed between, Mn,SEC and Mn,NMR (Fig. 1, Table 1 and2). Overall, welldefined copolymers were obtained with dispersity values below 1.2 for both the thermoresponsive block and the diblock copolymers. Copolymers P1 to P7 were soluble in DMSO or in hot water (above their Tt, up to 80 °C), whereas P8 was only soluble in DMSO. These results indicate that ~40 mol.% AN is too high for hyperthermia as it yielded a water-insoluble copolymer with an out-ofrange UCST value (> 100 °C). S3).

The influence of PEGylation on the thermoresponsiveness of the P(AAm-co-AN) block was then investigated. Addition of the POEGMA block had no impact on the Tt-UCST, as shown with T3 and P3a -P3c (Fig. 2a) until a certain chain length where POEGMA exhibited a LCST [START_REF] Lutz | Polymerization of oligo(ethylene glycol) (meth)acrylates: Toward new generations of smart biocompatible materials[END_REF] behavior that was influenced by its Mn (Fig. 2b). Despite the increasing Mn of the hydrophilic POEGMA blocks added to T3, from 4,900 g.mol -1 to 22,400 g.mol -1 , it did not prevent the diblock copolymers to retain their thermoresponsiveness. This is true even for P7, having a Mn composed 50% by the thermoresponsive block and 50% by the hydrophilic block, which retained its UCST properties.

Importantly, the Tt-LCST values of T3-based thermosensitive diblock copolymers were all ≥ 70 °C, which is much higher than mild-hyperthermia (43 °C) and should not hamper the mildhyperthermia-mediated drug release. chromatograms are presented in Supplementary Information (Fig. S3).

Since these copolymers are intended for use in a biological setting, we have evaluated the influence of the presence of electrolytes on their UCST behavior in water and in PBS. For P2, we observed that the copolymer in water did not exhibit a marked hysteresis and the heating and cooling Tt-UCST were nearly identical (Supplementary Information Fig. S4a). The effect of ionic strength and pH was then assessed by measuring the Tt-UCST in PBS. The hysteresis is here more pronounced compared to that obtained in water (Supplementary Information Fig. S4b). When comparing the heating and cooling curves in water and PBS, Tt-UCST remained constant at 47 °C in both media, but the thermoresponsiveness onset for the heating curve occurred earlier in PBS (30 °C) than in water ( 35°C) (Supplementary Information Fig S4c andS4d). The slightly different behavior between PBS and water might be due to a change in the hydration state of the copolymer chains. With higher ionic strength, the copolymer is more readily solubilized. Nevertheless, since the thermoresponsiveness of the copolymer relies on reversible hydrogen bonds between the different copolymer chains, this property is maintained even at higher ionic strength, which represents a strong advantage for use in a biological context as compared with charged or zwitterionic UCST-type (co)polymers. [START_REF] Seuring | Polymers with upper critical solution temperature in aqueous solution[END_REF] The influence of the AN content on thermoresponsiveness was then assessed directly on the P(AAm-co-AN)-b-POEGMA diblock copolymers (P1 to P8). The Tt-UCST of the copolymers increased with increasing AN content (Fig. 3a and3b). The trend was not clearly defined due to the IR measurements of the AN content in the polymers being not precise enough. For 40 mol.% AN (P8), the copolymer was insoluble in water at any temperature. While the copolymers had different Mn values ranging between 25 and 82 kg.mol -1 , this parameter did not appear to have as much impact as the AN content.

Nanoparticle formulation

As the different copolymers were only soluble in DMSO, which is a water-miscible solvent, the nanoparticles were thus prepared by the nanoprecipitation technique. [START_REF] Fessi | Nanocapsule formation by interfacial polymer deposition following solvent displacement[END_REF] The average diameters and polydispersity indexes of the obtained nanoparticles in water followed a decreasing trend as the Tt-UCST increased (Fig. 3c). This can be attributed to the different hydration state of the copolymers depending on the temperature of the solution. All formulations were prepared at room temperature (circa 23 °C) and measured at 20 °C. For the copolymer having a Tt-UCST at 30 °C (P1), the DLS measurements were not appropriate as the obtained suspension was on the micron size range. While Tt-UCST of P1 was measured at 30 °C, the onset temperature is lower, close to 20 °C. The copolymer chains were thus hydrated and the nanoparticles swollen. As the Tt-UCST increased, so did the hydrophobicity of the copolymer, which in turn hindered the interactions between water molecules and the copolymer chains thus yielding more compact nanoparticles. The trend reached a lower point at 154 nm with 0.09 as a PdI for P8.

Formulation of the thermoresponsive copolymers into nanoparticles in different aqueous media modified their physico-chemical properties. For instance, P6 led to nanoparticles with a greater average diameter in PBS than in water (Supplementary Information Fig. S5a), revealing a higher hydration state in such a medium. This result is well-correlated with the UCST behavior as the temperature onset for thermoresponsiveness was lower in PBS than in water. DLS measurements were also carried out at 37 °C for P2 nanoparticles formulated in water (Supplementary Information Fig. S5b). The swelling behavior was observed here once again, as the copolymer chains preferentially interacted with water molecules at higher temperature. This result was indicative of the solubilization mechanism of the nanoparticles as they appeared to swell to bigger sizes before eventually becoming fully soluble at a temperature higher than the Tt-UCST. As for the morphology of the different nanoparticles, TEM images suggested a significant influence of the copolymer's Tt-UCST (Fig. 4). Indeed, for a high Tt-UCST (P7), the nano-objects appeared to be well-defined spheres whereas at a lower Tt-UCST (P2), the suspension was not homogeneous, and the nano-objects seemed to be fused together in ill-defined shapes. A compromise was made in terms of selection of the polymer for further drug encapsulation studies, as P2 offered the best choice in terms of Tt-UCST but formed particles larger than 300 nm. The subsequent studies were carried out with P2.

Impact of doxorubicin loading on the nanoparticle properties

Doxorubicin hydrochloride is a water-soluble anticancer drug widely used to treat a variety of cancers. Here, Dox was rendered hydrophobic by letting it react with triethylamine (weak base). [START_REF] Huang | Self-assembled UCST-type micelles as potential drug carriers for cancer therapeutics[END_REF][START_REF] Li | Antitumor Drug Delivery Modulated by A Polymeric Micelle with an Upper Critical Solution Temperature[END_REF] It will therefore be encapsulated in the P(AAm-co-AN) hydrophobic core of nanoparticles. We studied the impact of Dox loading on the physico-chemical properties of the nanoparticles. Note that nanoparticles were formulated in water, as formulation in PBS resulted in precipitation of Dox.

In The drug loading of Dox showed a linear increase as a function of the initial amount of Dox up to 3.3 mg of initial Dox where the DL value stabilized at around 4 wt.% (Fig. 5a). Interestingly, this maximum DL value was in agreement with other studies where Dox was encapsulated in regular non-thermoresponsive polymer nanoparticles. [START_REF] Betancourt | Doxorubicin-loaded PLGA nanoparticles by nanoprecipitation: preparation, characterization and in vitro evaluation[END_REF][START_REF] Park | PEGylated PLGA nanoparticles for the improved delivery of doxorubicin, Nanomedicine Nanotechnology[END_REF] Above this maximum value, the copolymer matrix was saturated with Dox and was not able to incorporate more drug. This effect was clearly supported by looking at the encapsulation efficiency, which increased from 30 to 50 wt.% as the initial Dox mass increased, and then decreased after saturation of the copolymer matrix (Fig. 5b).

Encapsulation of Dox also modified the size and surface charge properties of the nanoparticles as shown in Fig. 5c and5d. The size followed a decreasing trend as encapsulated Dox increased, and the surface zeta-potential (ζ) became significantly more negative in the presence of Dox compared to unloaded nanoparticles, likely because of the deprotonated state of Dox. The change in surface charge was also indicative of the encapsulation of the drug in the polymer matrix. As for the size decrease of the nanoparticles, it may be due to a preferential interaction between Dox and the copolymer chains, where Dox acted as a hydrophobic nucleus facilitating the formation of more compact and dense nanoparticles. Upon increasing the amount of encapsulated Dox in the nanoparticles, the Tt-UCST increased both for the heating curve (Fig. 6a) and the cooling curve (Fig. 6b). The maximum transmittance value of the nanoparticles containing Dox did not reach 100% due to the presence of Dox that absorbed part of the incoming light even at a high wavelength (600 nm). The maximum transmittance value decreased with increasing the Dox content. The change in Tt-UCST for the heating and cooling curves showed an increase of the hysteresis of the system with increasing the encapsulated Dox content (Fig. 6b). In agreement with our hypothesis, the hysteresis might be due to the strong interaction between Dox and the copolymer chains, resulting in more energy needed to disrupt the Doxcopolymer interactions and fully solubilize the copolymer chains in water. The cooling Tt-UCST values, while higher than that obtained for unloaded nanoparticles, remained constant for the different amounts of encapsulated Dox. This observation can be attributed to the drug release: once Dox was freed from the nanoparticles, the Tt-UCST came back to a value closer to that of the unloaded nanoparticles.

Thermoresponsive copolymers exhibited a phase diagram in aqueous medium that displayed a concentration-dependent cloud point. This aspect must be elucidated to verify if the thermoresponsive properties of the nanoparticle formulation at 4.5 mg.mL -1 of copolymer remained similar upon dilution, which is an important feature for the applicability.Dilution decreased the Tt-UCST of the suspensions even in the presence of Dox (Fig. 6c andd). While the thermoresponsiveness was retained, a shift of the Tt-UCST towards lower values was observed; from 47 °C down to 40 °C in the case of unloaded nanoparticles (Fig. 6c). Not only did the Tt-UCST decrease but also the onset temperature; from 30 °C to below 20 °C. For Dox-loaded nanoparticles, this phenomenon was still observed (Fig. 6d). For the cooling Tt-UCST values, they were equal to 51, 42 and 37 °C for undiluted, 4 times diluted and 10 times diluted suspensions, respectively. This change of Tt-UCST upon dilution raised questions regarding the applicability of this system for clinical use.

Indeed, once administered to the patient intravenously, the formulation undergoes an important dilution, from a 500 mL perfusion bag to a 5-liter blood volume. This issue may be tackled by heating the tumor at mild-hyperthermia prior to the administration of the formulation, to minimize the chance of uncontrolled drug release.

Upon Dox loading, the overall shape of the nanoparticles visible by TEM did not change conversely to the size which appeared to be smaller (Supplementary Information, Fig. S6), which is in agreement with DLS measurements. Traditional polymer nanoparticles made from amphiphilic diblock copolymers, such as PLA-b-PEG, exhibit a well-defined spherical shape. Solubility of P(AAmco-AN)-b-POEGMA in water is temperature-and concentration-dependent unlike PLA-b-PEG. Thus, our hypothesis is that in observation conditions, as nanoparticles are diluted to allow observation, some copolymer chains were solubilized and allowed merging of different nanoparticles to form illdefined objects.

Cell culture

The aim of the cell culture studies was to show an enhanced activity of Dox encapsulated in nanoparticles under mild hyperthermia conditions. OVCAR-3 cells were chosen as they are Doxsensitive and represent a good cancer model for localized hyperthermia. [START_REF] Wang | High-intensity focused ultrasound compared with irradiation for ovarian castration in premenopausal females with hormone receptor-positive breast cancer after radical mastectomy[END_REF][START_REF] He | The Interaction between a Drug and Ultrasound in Sonochemotherapy against Ovarian Cancers[END_REF] The mitochondrial activity results showed that unloaded nanoparticles were not toxic up to 500 µg.mL -1 , whereas 60 % of mitochondrial activity was retained at 1 mg.mL -1 (Fig. 7a). This result was encouraging as it showed that the thermoresponsive copolymer used is rather well-tolerated making it a good candidate for drug delivery applications.

Mild hyperthermia was applied for 30 min, at 6 h or 24 h post-treatment. The trend showed that cytotoxicity was greater when hyperthermia was applied 24 h post-treatment rather than 6 h post-treatment (Fig. 7). This trend was verified in the case of free Dox and for Dox-loaded nanoparticles although not statistically significant. IC50 for free Dox was at 0.8 mM, whereas the IC50 for Doxloaded nanoparticles was at 7.2 mM (Fig. 7d). The increase of the IC50 was due to the encapsulation of the drug in the nanoparticles, where it is rendered less accessible to the cells. Dox induced its anticancer activity inside the cells' nucleus where it intercalates between complementary DNA strands, inhibiting the progression of topoisomerase II and further blocking proper transcription and replication. [START_REF] Bodley | DNA Topoisomerase II-mediated Interaction of Doxorubicin and Daunorubicin Congeners with DNA[END_REF] This mechanism of action thus requires Dox to reach the cells' nucleus and not only reside in the cytosol for it to exert its anticancer activity. In the results shown here, the 5-fold difference between free Dox and encapsulated Dox is significant and might be due to the strong affinity between the copolymer and Dox mentioned earlier in the manuscript. Indeed, if Dox has a strong affinity with the copolymer, it may not readily be released from the nanoparticles and reach the cell's nucleus, as it might be retained by the copolymer, which has a large molar mass not allowing proper diffusion to the cell's nucleus. However, upon hyperthermia, the polymer chains should be solubilized, allowing Dox to freely diffuse away from the copolymer. As our results under hyperthermia showed that Tt-UCST of Dox-loaded nanoparticles decreased upon dilution, we expected to see a difference between unheated and heated nanoparticles. While there was a visible trend, we were not able to obtain the same activity, i.e. same IC50, as the free Dox when treating with Dox-loaded nanoparticles with hyperthermia. Several aspects of the system can be improved to address some of the limitations presented in this study. Regarding the dilution affecting the UCST, a possible solution would be to add a hydrophobic block to the polymer that would act as a solid foundation upon which the nanoparticles could be built. This would force the thermoresponsive copolymer chains to remain close to each other at any dilution. On the contrary, this solution may also force the drug to be primarily encapsulated in the hydrophobic polymer chains, thus having potentially less drug release upon heating. Nevertheless, this methodology is already extensively used for LCST-type drug delivery systems and would be interesting to test for UCST-type drug delivery systems. The nature of the encapsulated anticancer drug can also be changed to select a molecule whose anticancer mechanism takes place inside the cell's cytosol as opposed to its nucleus. For instance, taxane derivatives such as paclitaxel are good candidates as they disrupt the microtubules' stability in the cytosol. In terms of physico-chemistry, these drugs are already hydrophobic, simplifying the preparation of the drug-loaded nanoparticles.

Conclusion

The aim of this study was to formulate thermoresponsive nanoparticles from P(AAm-co-AN), a hydrogen bonding-based UCST copolymer building block. We successfully synthesized UCST-type P(AAm-co-AN)-b-POEGMA diblock copolymer by a two-step RAFT polymerization and successfully tuned the thermoresponsiveness by varying the amount of AN in the copolymer. The copolymer was formulated into nanoparticles by the nanoprecipitation technique into stable nanoparticles of different sizes and dispersity governed by the Tt-UCST of the copolymer: the higher the Tt-UCST, the smaller the size. Encapsulation of Dox was achieved and modified the properties of the nanoparticles in a way never reported before. Surprisingly, the higher the amount of encapsulated Dox, the higher the Tt-UCST of the nanoparticles. In vitro, unloaded nanoparticles were not toxic up to 500 µg.mL -1 . However, the effect of hyperthermia on cell viability was not significant probably due to the modification of Tt-UCST of the nanoparticles upon dilution. The system can still be improved by changing the encapsulated drug or adding a hydrophobic polymer block to yield a better-defined suspension less sensitive to dilution effects. Taken together, these results highlighted the crucial role of the drug encapsulation and dilution on the expected beneficial effect of the thermoresponsiveness. Therefore, it seems important to carefully study those parameters for any kind of thermosensitive nanoscale drug delivery systems.

  -co-AN)-b-POEGMA P(AAm-co-AN) was used as a macro-RAFT agent to polymerize OEGMA and yield a P(AAm-co-AN)b-POEGMA diblock copolymer. An example of synthesis as follows: P(AAm-co-AN) (T2) (200 mg, 6.25 µmol), OEGMA (75 mg, 250 µmol), AIBN (1.7 mg, 10.4 µmol) and DMSO (4.5 mL) were charged in a 20 mL-vial. The mixture was purged with argon for 15 min under vigorous stirring before being placed in a 70 °C-oil bath for 5 h under stirring. The mixture was directly put into a 3.5 kD

Figure 1 :

 1 Figure 1: Synthesis of a series of UCST thermoresponsive poly(acrylamide-co-acrylonitrile), P(AAm-co-AN), copolymers (T1 -T8, Table 1) used as macro-RAFT agents to yield poly(acrylamide-co-acrylonitrile)-b-poly[(oligo(ethylene glycol) methyl ether methacrylate)] (P(AAm-co-AN)-b-POEGMA) diblock copolymers (P1 -P8, Table2). Table1: AN content

Figure 2 :

 2 Figure 2: (a) Thermoresponsive properties (transmittance curves, 4.5 mg.mL -1 in water) of P(AAmco-AN) T3 and P(AAm-co-AN)-b-POEGMA diblock copolymers of different Mn P3a to P3c. (b) Tt-UCST and Tt-LCST values of the synthesized copolymers. Mn was calculated by 1 H-NMR, Tt-LCST is the temperature at which the transmittance reaches a minimum plateau, n.m. : not measurable. SEC

Figure 3 :

 3 Figure 3: Heating transmittance curves of the different polymers at 4.5 mg.mL -1 in PBS (a) and Tt-UCST plotted against the measured AN mol.% content (b). Intensity-averaged diameter (Dz) and polydispersity index (PdI) in water measured by DLS of P(AAm-co-AN)-b-POEGMA diblock copolymers nanoparticles as a function of the Tt-UCST (c). The tags on each data point correspond to the polydispersity index (PdI). The line at 154 nm represents the Dz of nanoparticles obtained with the non-thermoresponsive polymer P8 (PdI = 0.05). For each sample n ≥ 3.

Figure 4 :

 4 Figure 4: TEM images of P2 unloaded nanoparticles (left), P7 unloaded nanoparticles (right).

Figure 5 :

 5 Figure 5: Physico-chemical properties of Dox loaded NPs formulated with P2 at 4.5 mg.mL -1 polymer concentration in water. (a) Drug loading content (DL) and (b) encapsulation efficiency (EE) as a function of initial mass of doxorubicin. (c) Dz and (d) ζ-potential values as a function of encapsulated Dox (n ≥ 2).

Figure 6 :

 6 Figure 6: Doxorubicin encapsulation impact on Tt-UCST of formulated nanoparticles from P2 at 4.5 mg.mL -1 in water. (a) Heating transmittance curves with various amounts of encapsulated Dox. (b) Heating and cooling Tt-UCST values depending on the amount of encapsulated Dox. Cooling curves in

H y p e r t h e r m i a a f t e r 2 4 hN o h y p e r t h e r m i a H y p e r t h e r m i a a f t e r 6 H y p e r t h e r m i a a f t e r 2 4 hN o h y p e r t h e r m i a H y p e r t h e r m i a a f t e r 6 H y p e r t h e r m i a a f t e r 2 4 hN o h y p e r t h e r m i a H y p e r t h e r m i a a f t e r 6 Figure 7 :

 4646467 Figure 7: Mitochondrial activity of OVCAR-3 cells in the presence of unloaded P2 nanoparticles (a), free Dox (b), or Dox-loaded P2 nanoparticles (c) ± hyperthermia (43 °C). For unloaded nanoparticles, dashed line indicates 80% mitochondrial activity, for others dashed line indicates the IC50 (d).

  Proportions were changed according to the desired polymer composition. A typical synthesis (T2) is as follows: AIBN (2.3 mg, 0.014 mmol), CPDB (15.7 mg, 0.071 mmol), AAm (3.324 g, 46.76 mmol), AN (0.918 g, 17.29 mmol)

and DMSO (16.01 mL) were charged in a 50 mL-round-bottom flask. For other copolymer compositions, see details in Supplementary Information (Table

Table 2 :

 2 Mn-NMR calculated by1 H NMR in d6-DMSO. Mn-SEC determined by triple detection SEC in DMSO. Ð is polymer dispersity. Tt determined by UV-vis temperature ramp at 4.5 mg.mL -1 in PBS. ins. : insoluble in water at any temperature. SEC chromatograms of P1-P8 are presented in Supplementary Information (Fig.

). Table

1

: AN content determined by IR spectroscopy, value given with SD. Mn-NMR calculated by 1 H NMR in d6-DMSO. *: sample was difficult to process into KBr pellets and measured value was out of calibration range. ins.: insoluble in water at any temperature.

H NMR spectroscopy was performed in 5 mm diameter tubes in d6-DMSO at 70 °C for 128 scans on a Bruker Avance 3 HD 400 spectrometer at 400 MHz. NMR determination of the Mn of P(AAm-co-AN) copolymers was achieved by integrating the triplet between 7.88 and 7.98 ppm corresponding

to 2 aromatic protons from the CPDB end-chain (noted a in Supplementary Information, Fig.S1)

Acknowledgements

Authors acknowledge the support of la Ligue contre le Cancer for Alexandre Bordat's Ph.D. fellowship, Cancéropôle IDF Emergence and Fondation ARC for funding. The present work has benefited also from the core facilities of Imagerie-Gif, (http://www.i2bc.paris-saclay.fr), member of IBiSA (http://www.ibisa.net), supported by "France-BioImaging" (ANR-10-INBS-04-01), and the Labex "Saclay Plant Science" (ANR-11-IDEX-0003-02). Institut Galien Paris-Sud is a member of the Laboratory of Excellence LERMIT supported by a grant from ANR (ANR-10-LABX-33). Authors would like to acknowledge the precious help of S. Denis for cell culture and C. Dejean for NMR.

Supplementary Information

Polymer reaction mixture compositions are presented as well as NMR spectra, SEC chromatograms, some transmittance curves, IR spectrum and IR calibration curve.