F Pedraza
email: fpedraza@univ-lr.fr.fr

M Mollard

B Rannou

B Bouchaud

J Balmain

G Bonnet

OXIDATION RESISTANCE OF THERMAL BARRIER COATINGS BASED ON HOLLOW ALUMINA PARTICLES

Keywords: slurry aluminide, superalloys, isothermal oxidation, cyclic oxidation

Thermal barrier coatings were synthesized in a single step process from a slurry containing Al microspheres onto different Ni-based superalloys. Upon growth of the coating a top coat of hollow alumina spheres linked to an aluminium diffused coating through an alumina TGO formed. The isothermal and cyclic oxidation tests at different temperatures (900 till 1100°C) up to 1000h or 1500 cycles revealed progressive growth of different thermal oxides depending on the substrate composition.

Faster degradation of the coatings occurred in the titanium-rich substrates (e.g. IN-738LC and PWA1483) compared to the titanium-poor ones . By comparing with conventional low activity aluminide coatings, it appeared that the incorporation of alloying elements (notably Ti and Ta) to the diffused layers upon the high activity slurry coating process is responsible for such fastest degradation.

INTRODUCTION

The use of thermal barrier coatings (TBCs) in the high pressure turbine components of aeroengines made of nickel based superalloys is compulsory nowadays to withstand the very high surface temperatures of the corrosive gas released from the combustion chamber. This system is often based on a top ceramic coat of yttria stabilized zirconia (YSZ) made by electron beam physical vapour deposition (EB-PVD) with columnar structure to comply with thermal cycling, an intermediate -Al2O3 thermally grown oxide (TGO) to attach the YSZ and to provide oxidation protection and an underlying -NiPtAl diffusion coating that acts as an aluminium reservoir [1]. New low-cost and environmentally friendly coatings were recently proposed in the European project "Particoat" [2] in which a water-based slurry containing Al microparticles was sprayed and annealed [3] onto pure nickel [4] and different nickel-based superalloys [5] or steels [6]. The isothermal oxidation behaviour of these coatings onto a pure Ni [7] and a René N5 superalloy [8] indicated faster oxidation kinetics and faster -NiAl'-Ni3Al transformation than in conventional low activity -NiAl although both systems grew duplex NiAl2O4/Al2O3 scales. In contrast, the presence of the hollow alumina top coat and the precipitates segregated at the thermally grown oxide limited the appearance of rumpling compared to the conventional coatings. Nevertheless, the incorporation of alloying elements from the substrate to the coating (e.g. titanium, tantalum, chromium…) may affect the corrosion/oxidation resistance of such coatings. Indeed, as pointed out by Evans et al. the bond coat is the most crucial component of the TBC system as its chemistry and microstructure influence durability through the structure and morphology of the TGO upon oxidation [9]. For instance, titanium grew non protective TiO2 on nanocrystalline IN-738 superalloy [10] whereas interspersed tantalum rich oxide particles (CrTaO4/NiTa2O6) developed in the top spinel ((Ni(Cr,Al,Co)2O4) scale on PWA-1484 superalloy [11]. It therefore appears that further understanding in this field is required for these new single step thermal barrier coatings sintered from a slurry. For such purpose, the increasing amounts of alloying elements in the superalloys and their incorporation to the coatings, hence into the oxide layers were investigated in this work. The evolution of the diffusion coatings in terms of -NiAl'-Ni3Al and growth of topological closepacked phases (TCPs) will be also considered to shed light on the durability of these new slurry coatings.

EXPERIMENTAL

The slurry TBCs were obtained from a 1/10 PVA/H2O solution to which 45 mass% 5µm-sized Al microparticles were added [3]. After spray onto four nickel-based superalloys with different composition and crystal structure (Table 1) and dry in air, the slurries were annealed at 700°C/2h and additional 1100°C/2h in Ar(g). The traces of O2 (2 vpm) and H2O (5 vpm) in Ar were shown to be sufficient to grow the top coat and the TGO composed of hollow and plain α-Al2O3, respectively [4].

The slurry TBCs were compared with industrially manufactured low activity out-of-pack (70Cr-30Al donor, NH4F activator, treated at 1080°C for 6h in vacuum) and high activity pack cemented (60Cr-40Al donor, NaCl activator, Al2O3 filler treated at 860°C for 6h in argon and subsequently annealed at 1120°C for 2h in vacuum). Short (100h) isothermal tests were performed in a Setaram TGA92 thermobalance while for the long term (500 and 1000h) tests a Pyrox furnace was employed. In both cases, purging with Ar(g) was let in the chamber till the oxidizing temperatures (900, 1000, 1100°C) were reached and synthetic air replaced Ar(g). For the cyclic oxidation tests at 1000 and 1100°C ambient air was employed in a Delta Thermique furnace. The thermal cycles corresponded to a hot dwell of 45 min after which the samples were quenched by air flow for 15 min to reach surface temperatures of 40(±10)°C. Note however that the oxidation temperatures depended on the limit operating temperature of the substrates as gathered in Table 2. X-ray diffraction (XRD, Bruker AXS D8, KαCu=0.15406 nm), Raman spectroscopy (Jobin Yvon Horiba LabRam HR, λ=632.817 nm), light microscopy (Leica M165C), scanning electron microscopy with a coupled energy dispersive energy spectrometer (SEM/EDS, FEI-Quanta 200F/EDAX and JEOL5410/Oxford) and Electron probe microanalysis (EPMA, JEOL JXA-1800) allowed to characterize the degradation of the coatings in the as-deposited condition and after oxidation.

RESULTS AND DISCUSSION

3.1.-Coatings

All the slurry coatings displayed the same structure as the one given in Figure 1 with a top coat of hollow alumina spheres, a very thin thermally grown alumina scale and a bottom aluminium diffused layer. In the backscattered electron mode of SEM and by EPMA some nickel was revealed at the top coat. A schematic illustration gathering the major mechanisms of formation of these coatings is included in Figure 2. Such mechanisms were thoroughly described in previous works [4,12,13]. Once the slurry containing the passivated Al microparticles is dried and annealing at sufficiently high temperatures starts, the phase transformation of the alumina shell surrounding the Al particles results in cracks through which Al diffuses out and wets the Ni-based substrate [4]. Then, Ni dissolves in molten Al [12] and the subsequent greatly exothermic reactions between Al and Ni bring about the synthesis of the Al-rich nickel aluminides by a self-propagating combustion mechanism [13]. Once all molten Al is consumed, solid state interdiffusion occurs, in particular upon the final temperature step of the synthesis process. However, the roughness of the interface between the top coat and the diffused layer increased with greater alloying element content in the substrate (Figure 3). Galetz et al. [13] proposed that molten Al builds a network between the Al particles at the top and the substrate surface resulting in a "stalagmite" microstructure that is more pronounced when using greater particles [14].

Here, the segregation of precipitates at the surface of the substrate seemed to limit the contact between Ni and Al, thus reducing the local temperature released from the great exothermal Ni-Al reactions. As a result, molten Al cannot homogeneously hit the substrate when compared to the absence of precipitates. In addition, and although different microstructures of the diffused layers were obtained depending on the initial composition and crystal structure of the Ni-based superalloys investigated, they all exhibited a typical microstructure of high activity aluminides also observed by Montero et al.

on similar CM-247, IN-738 and René 80 superalloy substrates than ours [5]. In yet unpublished results, the EPMA X-ray maps of the slurry coatings revealed increasing partition of Ti and Ta to the TGO/diffusion coating interface and of Cr to the top of the diffusion layer with increasing content in the substrate alloy, e.g. more Ti segregated in PWA-1483 (4.1 mass %Ti) than in CM-247 (0.7 mass %Ti). The precipitates in the pack cemented coatings distributed very similarly to the slurry ones while the out-of-pack barely contained any precipitate as was also the case for slurry on pure Ni [4].

3.2-Isothermal oxidation

The parabolic rate constants calculated using the complete law approach [15] were plotted in the Arrhenius diagram derived from a bulk -NiAl by Brumm and Grabke [16] in Figure 4. It appeared that the slurry coatings onto pure nickel grew transition -Al2O3 for the first 100h regardless of the oxidation temperature. In contrast, the coated superalloys exhibited an evolution from  to -Al2O3 with increasing temperature in agreement with the well-known third element effect that Cr procures to develop -Al2O3 scales rapidly [17]. However, one must bear in mind that the greatest kinetics of the slurry coatings can be related to further oxidation of remnants of metallic Al in the spheres after the coating elaboration process [7]. Further extension to 500 and 1000h of oxidation displayed less differences in terms of specific mass gain compared to a low activity -NiAl. A similar slurry coating onto CM-247LC superalloy oxidised by Montero et al. at 1000°C for 1000h displayed in contrast much greater mass gains than a conventional high activity pack cemented coating but the mass gains were reduced by previous blasting of the top coat [18]. It shall be noted however that the top coat partially detached depending on the substrate (coating) composition (Table 3) likely during the cooling step ( 50°C/min). This hypothesis was confirmed from the X-ray patterns (Table 3), where the incorporation of titanium and tantalum oxides became more relevant with oxidation time in all the systems. Additional tungsten, hafnium and spinel oxides appeared on N5, CM and PWA substrates. The EDS elemental maps in the cross sections confirmed the XRD results. Cr mainly partitioned to the interdiffusion zone (IDZ) [19] and therefore could not contribute to the formation of Cr2O3. In contrast, Ta and Ti were found in both at the bond coat/TGO interface and within the IDZ. Such partition was previously reported by Bouchaud et al. for Ta [20] and by Pfening and coll. for Ti [21] and can be related to the relatively high diffusion coefficient of both refractory elements in these coating and alloy systems [22]. It was also noted that upon transformation of the -NiAl'-Ni3Al, dissolution of the precipitates of refractory elements occurred with temperature and time because of their limited solubilities in the -NiAl phase [e.g. 22]. This is clearly demonstrated in Figure 5, where the low activity out-of-pack bond coat transformed less -NiAl phase than the high activity slurry one and therefore refractory-rich oxides precipitated in the duplex oxide scale with NiAl2O4 on top of a -Al2O3 grown in both coating systems. However, and in spite of further transformation of -NiAl into the L12 (or martensitic L10) '-Ni3Al quoted to induce rumpling [23,24] in the slurry coatings than in the conventional CVD ones, the latter displayed a more rumpled surface. It therefore appeared that the presence of the refractory oxide precipitates within the scale and the top coat of hollow alumina particles allowed to pin the TGO and the underlying bond. As summarised in Table 4, Al diffusion into the substrate brought about the precipitation of TCPs, whose extent and composition depended on temperature and time and they can lead to a degradation of the mechanical properties upon mechanical loading [19]. The greater the temperature and time, the thicker the extent of the TCPs area except for CM-247LC since the precipitates seemed to solubilize in the /' matrix. Additional needle-like precipitates in this area can also ascribed to AlN [25,26] with N from the substrate diffusing outwardly [27,28]. Similar TCPs were also found in the low activity out-of-pack aluminides after oxidation in terms of morphology and extension in a DS superalloy having a composition very alike to that of CM-247LC [20].

3.1.-Cyclic oxidation

Figure 6 displays the mass gains per surface unit of René N5 and PWA 1483 aluminized using the conventional low activity and the slurry methods upon cyclic oxidation at 1100°C in air. Compared to the low activity aluminide, some spallation compensated by further mass gains occurred in the slurry René N5. In contrast, the slurry PWA 1483 underwent significant mass losses in less than 100 cycles.

Since the major mass loss can be attributed to the top coat of hollow spheres, it appears that quite extended areas became naked (see inset in Fig. 5). The naked surface displayed a dark yellow colour indicative of TiO2 and the presence of this oxide was confirmed by Raman spectroscopy and XRD.

Therefore, it can be considered TiO2 as the cause of such spallation. Indeed, by decreasing the oxidation temperature to 1000°C (Figure 7), the top coat remained adherent until 1500 cycles of oxidation although some low intensity peaks of TiO2 were detected together with those of the major -Al2O3 and NiAl2O4 spinel oxide phases.

The evolution of the coating microstructure with cycling was similar to the one observed under isothermal regime. However, the -NiAl'-Ni3Al transformation was comparatively less extended while the rumpling phenomenon was greater than in isothermal regime. Although the rumpling phenomenon is often reported to result from the volume change induced upon the ' phase transformation [24,29], additional factors such as the differences of thermal expansion coefficients between the scale and the coating [30] and the coating and the substrate [START_REF] Touloukian | Thermophysical Properties of Matter,Thermal Expansion of Nonmetallic Solids[END_REF] in addition to the lateral growth of the scale [START_REF] Chen | [END_REF] shall be also considered. Here, the precipitate-free aluminide coatings obtained by out-of-pack rumpled more than the slurry ones (Figure 8) and therefore confirms the pinning effect of both the precipitates in the coatings that limit the plastic deformation like in a composite material and of the top coat since the expansion and contraction of hollow spheres likely limit the introduction of additional stresses to the TGO.

As result of interdiffusion, the composition of such TGO also evolved with the number of cycles at temperature. In particular, and as with the isothermal regime, Ta and Ti diffused outwardly and incorporate in the TGO like in the examples shown in Figure 9 for the X-ray elemental maps of the slurry aluminised René N5 after 1000 cycles of oxidation at 1100°C and in Table 4 with the phases identified by XRD after different cycles of oxidation at 1000°C of the slurry, out-of-pack and pack cemented aluminides in PWA-1483. In the former, the maps clearly revealed major segregation on top of the Al-rich TGO likely resulting in mixed CrTaO4/NiTa2O6 or simple TaO2 oxides [11]. In the latter, TiO2 was unambiguously identified from 500 cycles onwards in the high activity (slurry and pack) aluminides in addition to the major contribution of α-Al2O3 but was absent in the precipitate-free low activity out-of-pack aluminide. Such TiO2 segregation was quoted to result from the very coating process itself [8] but additional outward diffusion could occur upon the increased β-NiAl  γ'-Ni3Al transformation of the high activity aluminides since Ti partitions to Ni3Al [START_REF] Reed | Superalloys, Fundamentals and applications[END_REF]. In spite of a greater relative intensity of TiO2 with respect the NiAl2O4 spinel all the scales (and top coat) appeared adherent even after 1500 cycles.

In addition, the extent of the TCPs area also increased with temperature and time in a similar fashion than in the isothermal regime (Table 4). The thickness of the TCP extension was very alike in both high activity coatings but was slightly thicker in the out-of-pack ones probably from their greater initial Al content. Nevertheless, these results are very much in agreement with those proposed in the open literature for different aluminised nickel-based superalloy substrates [e.g. 24] and demonstrate the overall similar degradation mechanisms of these new slurry coatings to the conventional out-of-pack and pack aluminides.

CONCLUSIONS

The slurry coatings grown through a high activity process underwent relatively faster oxidation kinetics than bulk -NiAl materials at the different temperatures investigated (900, 1000 and 1100°C).

Although remnants of metallic Al may contribute to the overall mass gain, the formation of titanium and tantalum oxides very likely enhanced growth of the oxide scales that were mainly based on a top NiAl2O4 and a bottom -Al2O3. These detrimental oxides grew with increasing oxidation temperature in particular in the Ti and Ta-rich alloys. However, Cr remained trapped in the TCP phases and did not influence the oxidation kinetics. Although the oxidation kinetics and the -NiAl'-Ni3Al transformation were enhanced in the high activity slurry coatings, the refractory-rich precipitates at the hollow spheres top coat/diffusion layer interface and the presence of the top coat seemed to limit rumpling compared to the conventional low activity -NiAl ones. Nevertheless, extensive growth of TiO2 appears to be the major cause of spallation as demonstrated in the cyclic oxidation tests and therefore the operating temperature for the high activity coatings rich in Ti shall not surpass 1000°C.

Figure 1 .

 1 Figure 1.-Slurry TBC coatings investigated. SEM top surface with hollow alumina particles (a), detail of the sintered hollow microparticles (b) and the corresponding cross section with the top coat and the diffused layer over the superalloy substrate (c). EPMA maps of (d) oxygen, (e) aluminium and (f) nickel to reveal the intermediate thermally grown oxide.

Figure 2 .-

 2 Figure 2.-Schematic illustration of the mechanisms of formation of the slurry TBC coatings. (a) The passivated Al microspheres lay on the substrate after spray deposition and drying. (b) With increasing temperature Al diffuses into the substrate. The outermost layers of Al microspheres supply Al to the underlying layers. Ni starts to diffuse outwardly or to solubilise into molten Al. Thickening of the oxide shells occurs. (c) At higher temperatures, the alumina shells transform into the stable  phase. Al is consumed in the core which leaves behind a hollow structure. Ni diffuses preferentially to Al to form the -NiAl bond coat.

Figure 3 .

 3 Figure 3.-SEM cross sections of the as a function of superalloy substrate (a) and (d) René N5 or CM-247, (b) and (e) PWA-1483, (c) and (f) IN-738LC. Note the rough interface between the hollow particles and the diffusion layer at the top and the different microstructures of the aluminium diffused layers.

Figure 4 .

 4 Figure 4.-Arrhenius plot of the kp values determined from the oxidation kinetics and comparison with the kp values from a pure -NiAl [16]. The data for pure Ni are taken fromRef.[7].

Figure 5 .

 5 Figure 5.-Evolution of the microstructures in CM-247LC aluminised by (a) and (c) conventional low activity out-of-pack and by (b) and (d) high activity slurry after oxidation at 1100°C in air for 500 h (a) and (b) and for 1000h (c) and (d).

Figure 6 .

 6 Figure 6.-Evolution of the mass gains per surface unit of the aluminised René N5 and PWA-1483 with cyclic oxidation at 1100°C in air. Note the great spallation of the slurry aluminised PWA-1483 compared to René N5.

Figure 7 .Figure 8 .

 78 Figure 7.-Evolution of the mass gains per surface unit of the PWA-1483 uncoated and aluminised with different methods with cyclic oxidation at 1000°C in air.

Figure 9 .

 9 Figure 9.-EPMA X-ray maps of different elements and the SEM cross section of the slurry coated René N5 coated after cyclic oxidation at 1100°C in air for 1000 cycles. Note the great partition of Ta to the top of the oxide scale.

  2 O 3 / Ta x O y / AlTaO 4 / WO 2 α-Al 2 O 3 / NiAl 2 O 4 / Mo x O y / TiO 2 / HfO 2

Table 1 . Nominal composition (mass%) of the nickel-based superalloys investigated.

 1

		Ni	Cr	Co	Mo	W	Ta	Al	Ti	Other
	René N5	61.6	7	8	2	5	7	6.2		0.2 Hf, 3.0 Re
	CM-247LC	61.7	8.1	9.2	0.5	9.5	3.2	5.6	0.7	1.4 Hf
	PWA-1483	61.2	12.2	9	1	3.8	5	3.6	4.1
	IN-738LC	61	16	8.5	1.7	2.6	1.7	3.4	3.4	0.9Fe, 0.3 Si

Table 2 .-Temperatures of oxidation of the different substrates as a function of the regime.

 2

	Temperature (°C)	Isothermal	Cyclic
	900	CM-247, PWA-1483, IN-738LC	--
	1000	CM-247, PWA-1483	PWA-1483
	1100	CM-247, René N5	PWA-1483, René N5

Table 3 .-Oxide phases identified by XRD and their adherence (visual observations) to the respective coating.

 3

	Oxidation		Substrate
	time (h)	René N5	CM 247	PWA 1483	INCO 738
			Oxidation at 900°C
			adherent	adherent
	100	X	α-Al 2 O 3 / Ta x O y / TiO 2 / HfO 2	α-Al 2 O 3 / TiO 2	X
			adherent	adherent	adherent
	500	X	α-Al 2 O 3 / Ta x O y / TiO 2	α-Al 2 O 3 / TiO 2	α-Al 2 O 3 / TiO 2
			adherent	adherent	Adherent
	1000	X	α-Al 2 O 3 / Ta x O y / TiO 2 / HfO 2	α-Al 2 O 3 / NiAl 2 O 4 / TiO 2	α-Al 2 O 3 / TiO 2
			Oxidation at 1000°C
			adherent	adherent
	100	X	α-Al 2 O 3 / Ta x O y / TiO 2 /	α-Al 2 O 3 / NiAl 2 O 4 /	X
			HfO 2	TiO 2

Table 4 .-Thickness (± 2 µm)of TCP extension as a function of oxidation regime, temperature, and time of the slurry coatings on different substrates.

 4

				900°C
		Isothermal		Cyclic
		500 h	1000 h	500	1000	1500
	CM-247LC	10	16	x	x	x
	PWA-1483	19	28	x	x	x
	IN-738LC	18	23	x	x	x
				1000°C
		Isothermal		Cyclic
		500 h	1000 h	500	1000	1500
	CM-247LC	12	24	x	x	x
	PWA-1483	21	36	21	24	29
				1100°C
		Isothermal		Cyclic
		500 h	1000 h	500	1000	1500
	CM-247LC	6	12	x	x	x
	René N5	45	62	39	44	60

Table 5 .-Oxide phases identified by XRD for the different aluminide coatings on PWA- 1483 after cyclic oxidation at 1000°C in air. (NB: the symbols > and >> indicate, respectively, greater or much greater relative intensity of the phase in the patterns).

 5

Acknowledgements

The DECHEMA Forschung Institut (Germany) is gratefully acknowledged for the EPMA analyses. SR Technics Airfoil Services (Ireland) and Turbocoating (Italy) kindly provided, respectively, the low activity out-of-pack and the pack cemented coatings. This study was performed under the programme PARTICOAT FP7-NMP-2007-LARGE-1-CP-IP-211329-2 (2008-2012) funded by the European Union.


Frederic Tewes
email: ftewes@univ-poitiers.fr

Oliviero L Gobbo

Carsten Ehrhardt

Anne Marie Healy
email: healyam@tcd.ie

Amorphous calcium carbonate based-microparticles for peptide pulmonary delivery

Keywords: Amorphous calcium carbonate, composite microparticle, pulmonary inhalation, biomimetic process, peptide Amorphous calcium carbonate, composite microparticle, pulmonary inhalation, biomimetic process, peptide

Amorphous calcium carbonate (ACC) is known to interact with proteins, for example in biogenic ACC, to form stable amorphous phases. The control of amorphous:crystalline and inorganic:organic ratios in inhalable calcium carbonate microparticles may enable particle properties to be adapted to suit the requirements of dry powders for pulmonary delivery by oral inhalation. For example, an amorphous phase can immobilise and stabilise polypeptides in their native structure and amorphous and crystalline phases have different mechanical properties. Therefore, inhalable composite microparticles made of inorganic (i.e. calcium carbonate and calcium formate) and organic (i.e. hyaluronan (HA)) amorphous and crystalline phases were investigated for peptide and protein pulmonary aerosol delivery. The crystalline:amorphous ratio and polymorphic form of the inorganic component was altered by changing the microparticle drying rate and by changing the ammonium carbonate and HA initial concentration. The bioactivity of the model peptide, salmon calcitonin (sCT), coprocessed with alpha-1-antitrypsin (AAT), a model protein with peptidase inhibitor activity was maintained during processing and the microparticles had excellent aerodynamic properties, making them suitable for pulmonary aerosol delivery. The bioavailability of sCT after aerosol delivery as sCT and AAT-loaded composite microparticles to rats was 4-times higher than that of sCT solution.

Introduction

For peptides, polypeptides and proteins the lung is an attractive route into the bloodstream, owing to the high and thin surface area of the alveolar epithelium and due to lower metabolic activity compared to the gastrointestinal tract. [START_REF] Patton | Breathing life into protein drugs[END_REF][START_REF] Patton | Inhaling medicines: delivering drugs to the body through the lungs[END_REF] Peptide deposition within the lung can be controlled by the development of peptide-loaded microparticles with suitable aerodynamic properties that allow them reach the distal lungs, where the absorption capacity is higher. When developing a polypeptide formulation it is mandatory to prevent its degradation and unfolding. This protection can be provided by the presence of excipient(s) in an amorphous phase that immobilise and isolate peptide molecules in their native structure. [START_REF] Yu | Amorphous pharmaceutical solids: preparation, characterization and stabilization[END_REF] Ideally, in this vitrification-based stabilisation strategy, both polypeptide and excipient(s) are amorphous and miscible, forming a single amorphous phase, which should have a high glass transition temperature (Tg) in order to be stable at room temperature. According to Gordon and Taylor [START_REF] Gordon | Ideal copolymers and the second-order transitions of synthetic rubbers. i. non-crystalline copolymers[END_REF] the Tg of this composite glass will be intermediate to the Tg of the polypeptide and the excipient. Also, the Tg of the excipient should be high in order to increase the Tg of the composite glass. In addition to vitrification, peptide-excipient interactions are important for polypeptide stabilisation. [START_REF] Arakawa | Factors affecting short-term and long-term stabilities of proteins[END_REF] These interactions should replace the hydrogen-bonds formed by water at the surface of polypeptide molecule, preserving their native structure. Consequently, the presence of excipients forming an amorphous phase with the polypeptide in the formulations can be very important to stabilise these molecules. [START_REF] Yu | Amorphous pharmaceutical solids: preparation, characterization and stabilization[END_REF] Amorphous calcium carbonate (ACC) is an amorphous mineral phase which is known to interact with proteins and peptides, for example in biogenic ACC, to form stable amorphous phases. [START_REF] Aizenberg | Stabilization of amorphous calcium carbonate by specialized macromolecules in biological and synthetic precipitates[END_REF][START_REF] Raz | The transient phase of amorphous calcium carbonate in sea urchin larval spicules: the involvement of proteins and magnesium ions in its formation and stabilization[END_REF][START_REF] Addadi | Taking advantage of disorder: Amorphous calcium carbonate and its roles in biomineralization[END_REF][START_REF] Raz | Stable amorphous calcium carbonate is the main component of the calcium storage structures of the crustacean Orchestia cavimana[END_REF] No information about the Tg of ACC is found in the literature. An estimation of the Tg of dry materials can be made from the melting temperature of its crystalline phase since studies have shown that the Tg is around 0.7-times the melting temperature. [START_REF] Kerc | Thermal analysis of glassy pharmaceuticals[END_REF][START_REF] Johnson | Bulk amorphous metal-An emerging engineering material[END_REF] Vaterite, the more metastable crystalline calcium carbonate (CaCO3) polymorph, converts to calcite upon heating at transition temperatures of 320-460°C, depending on the method of preparation. [START_REF] Baitalow | Thermochemical Investigations of Calcium Carbonate Phase Transitions I. Thermal activated vaterite-calcite transition[END_REF][START_REF] Turnbull | A thermochemical study of vaterite[END_REF][START_REF] Wolf | Thermophysical Investigations of the Polymorphous Phases of Calcium Carbonate[END_REF] If we assume, as a minimum, that the Tg of ACC is 0.7-times the phase transition temperature of vaterite to calcite, the Tg of ACC would be around 224°C, which is higher than the Tg of most excipients used to stabilise amorphous pharmaceutical solids. Therefore, having potentially a high Tg and being able to interact with proteins, we hypothesise that ACC could be used to stabilise peptides in the formulation of inhalable particles. Qi et al. [START_REF] Qi | ATP-Stabilized Amorphous Calcium Carbonate Nanospheres and Their Application in Protein Adsorption[END_REF][START_REF] Qi | Microwave Hydrothermal Transformation of Amorphous Calcium Carbonate Nanospheres and Application in Protein Adsorption[END_REF] similarly proposed ACC nanospheres for protein drug delivery.

Additionally, it has been shown that ACC can coexist with one of the CaCO3 crystalline forms to produce material with advantageous specific properties. [START_REF] Aizenberg | Stabilization of amorphous calcium carbonate by specialized macromolecules in biological and synthetic precipitates[END_REF][START_REF] Aizenberg | Factors involved in the formation of amorphous and crystalline calcium carbonate: A study of an ascidian skeleton[END_REF][START_REF] Aizenberg | Coexistence of amorphous and crystalline calcium carbonate in skeletal tissues[END_REF][START_REF] Politi | Sea urchin spine calcite forms via a transient amorphous calcium carbonate phase[END_REF][START_REF] Grunenfelder | Crustacean-Derived Biomimetic Components and Nanostructured Composites[END_REF] Biominerals such as seashells, [START_REF] Aizenberg | Stabilization of amorphous calcium carbonate by specialized macromolecules in biological and synthetic precipitates[END_REF][START_REF] Aizenberg | Factors involved in the formation of amorphous and crystalline calcium carbonate: A study of an ascidian skeleton[END_REF] eggshells [START_REF] Lakshminarayanan | Formation of Transient Amorphous Calcium Carbonate Precursor in Quail Eggshell Mineralization: An In Vitro Study[END_REF] and bones [START_REF] Aizenberg | Coexistence of amorphous and crystalline calcium carbonate in skeletal tissues[END_REF][START_REF] Termine | Amorphous/crystalline interrelationships in bone mineral[END_REF][START_REF] Mahamid | Amorphous calcium phosphate is a major component of the forming fin bones of zebrafish: Indications for an amorphous precursor phase[END_REF] are examples of hybrid materials composed of biopolymer and amorphous and crystalline minerals having high mechanical strength and light weight. Amorphous materials have the advantage of being isotropic and elastic, with no preferred growth directions so they can be more easily shaped. [START_REF] Addadi | Taking advantage of disorder: Amorphous calcium carbonate and its roles in biomineralization[END_REF][START_REF] Grunenfelder | Crustacean-Derived Biomimetic Components and Nanostructured Composites[END_REF][START_REF] Bentov | Stabilization of amorphous calcium carbonate by phosphate rich organic matrix proteins and by single phosphoamino acids[END_REF] On the other hand, crystalline CaCO3 are less soluble but harder and more stable than their amorphous counterpart. [START_REF] Politi | Sea urchin spine calcite forms via a transient amorphous calcium carbonate phase[END_REF] The combination of the two phases, amorphous and crystalline, may offer some advantageous properties to an inhalable powder. For example, materials with different proportions of amorphous and various polymorph crystalline phases could lead to different aerodynamic properties as well as different solubilities and dissolution rates.

Complex CaCO3-based structures are created by invertebrates by using the highly soluble ACC and a biopolymer controlling the CaCO3 polymorphs conversion. [START_REF] Aizenberg | Factors involved in the formation of amorphous and crystalline calcium carbonate: A study of an ascidian skeleton[END_REF][START_REF] Akiva-Tal | In situ molecular NMR picture of bioavailable calcium stabilized as amorphous CaCO3 biomineral in crayfish gastroliths[END_REF][START_REF] Aizenberg | Direct fabrication of large micropatterned single crystals[END_REF][START_REF] Walsh | Fabrication of hollow porous shells of calcium carbonate from self-organizing media[END_REF][START_REF] Pouget | The initial stages of template-controlled CaCO3 formation revealed by Cryo-TEM[END_REF][START_REF] Politi | Transformation mechanism of amorphous calcium carbonate into calcite in the sea urchin larval spicule[END_REF][START_REF] Radha | Transformation and crystallization energetics of synthetic and biogenic amorphous calcium carbonate[END_REF] One way for the biopolymer to influence the shape of the mineral is by chemical interaction. [START_REF] Bassett | Stabilization of Amorphous Calcium Carbonate with Nanofibrillar Biopolymers[END_REF] Hyaluronate (HA) polysaccharide could be used to facilitate the particle formation. In fact, negatively charged carboxylic groups from HA could be used to interact with Ca 2+ and control the nucleation of CaCO3. [START_REF] Arias | Polysaccharides and Proteoglycans in Calcium Carbonate-based Biomineralization[END_REF] Furthermore, HA is a ubiquitous anionic polysaccharide that is part of the extracellular lung matrix and has already been investigated as an inhalable therapeutic agent for lung diseases. [START_REF] Turino | Hyaluronan in Respiratory Injury and Repair[END_REF][START_REF] Ros | Hyaluronic Acid Improves the Tolerability of Hypertonic Saline in the Chronic Treatment of Cystic Fibrosis Patients: A Multicenter, Randomized, Controlled Clinical Trial[END_REF][START_REF] Huang | High MW hyaluronan inhibits smoke inhalation-induced lung injury and improves survival[END_REF] For example, inhaled HA prevents the side effects of hypertonic saline inhalation alone, which is used to treat bronchiolitis and patients with cystic fibrosis. [START_REF] Ros | Hyaluronic Acid Improves the Tolerability of Hypertonic Saline in the Chronic Treatment of Cystic Fibrosis Patients: A Multicenter, Randomized, Controlled Clinical Trial[END_REF][START_REF] Nenna | Seven percent hypertonic saline-0.1% hyaluronic acid in infants with mild-to-moderate bronchiolitis[END_REF] We hypothesise that inhalable microparticles could be made of ACC, crystalline CaCO3 and biopolymer (HA), enabling their properties to be adapted in terms of aerodynamic characteristics, drug release profile, mechanical strength and protein stability. In particles of this type, the properties could be tuned by controlling the organic:inorganic materials ratio and by controlling the solid state nature of the inorganic components. Therefore, we investigated the possibility of pulmonary delivery of salmon calcitonin (sCT) and alpha-1-antitrypsin (AAT), chosen as a model polypeptide and model protein, respectively, by way of inhalable composite microparticles made of HA and CaCO3. [START_REF] Monk | Inhaled Alpha 1-Antitrypsin: Gauging Patient Interest in a New Treatment[END_REF]

Results

The spray drying process developed produced spherical hollow particles (shells) with wall thickness around 80 nm (Figure 1). The properties of these particles were tuned by altering three parameters: the spray drying inlet temperature, the concentration of HA and the concentration of ammonium carbonate ((NH4)2CO3) in the spray dried feed solutions (Table 1).

Processing temperature effect

Changing the spray drying inlet temperature (Tinlet) in the 100 °C -140 °C range did not alter the particle morphology (Figure 1) or particle size distribution (Table 1). The formulations had a volume median geometric diameter (D50) around 4.6 ± 0.4 µm and high specific surface area values (16.5 to 19.6 m 2 g -1 ). The calcium concentration in the particles was not modified but the true density of the particle changed with the Tinlet (Table 1), indicating a change in their composition or in their solid state. The solid state of the powders was first analysed by XRD (Figure 2). Powders formulated at a Tinlet of 100 °C showed just a diffuse halo on their XRD pattern, characteristic of XRD amorphous material (Figure 2A). An increase in Tinlet from 110 °C up to 140 °C led to the appearance of low intensity diffraction peaks ascribed to crystalline calcium formate (Ca(HCOO)2). [START_REF] Ridha | Post-combustion CO2 capture by formic acid-modified CaO-based sorbents[END_REF] The presence of Ca(HCOO)2 resulted from the reaction between calcium hydroxide Ca(OH)2 and formic acid (HCOOH) initially present in the solutions. Fourier transform-infrared (FTIR) spectroscopy was used to investigate the chemical nature of the particles (Figure 3A). All spectra presented absorption bands typical of carbonate and carboxylate. [START_REF] Al-Hosney | Heterogeneous uptake and reactivity of formic acid on calcium carbonate particles: a Knudsen cell reactor, FTIR and SEM study[END_REF][START_REF] Vagenas | Quantitative analysis of synthetic calcium carbonate polymorphs using FT-IR spectroscopy[END_REF] The absence of the carbonate absorption bands at 700, 713 and at 745 cm -1 40 in the spectra indicates the absence of detectable anhydrous crystalline CaCO3 polymorphs. However, ACC spectra are characterised by a broad band of the carbonate bending at 866 cm -1 and a split in the asymmetric stretch of the carbonate ion at 1417 and 1488 cm -1 . [START_REF] Raz | Stable amorphous calcium carbonate is the main component of the calcium storage structures of the crustacean Orchestia cavimana[END_REF][START_REF] Politi | Sea urchin spine calcite forms via a transient amorphous calcium carbonate phase[END_REF][START_REF] Bassett | Stabilization of Amorphous Calcium Carbonate with Nanofibrillar Biopolymers[END_REF][START_REF] Meiron | Solubility and Bioavailability of Stabilized Amorphous Calcium Carbonate[END_REF][START_REF] Foran | Biogenic Fish-gut Calcium Carbonate is a Stable Amorphous Phase in the Gilt-head Seabream, Sparus aurata[END_REF][START_REF] Ihli | Dehydration and crystallization of amorphous calcium carbonate in solution and in air[END_REF] Carbonate absorption bands recorded for the microparticles formulated at Tinlet of 100 °C have maximal values typical for ACC, confirming the XRD data. An increase in Tinlet resulted in a reduction in the intensity of bands typical of ACC. When processed at Tinlet = 140 °C, the 866 cm -1 band was split, with a sharper peak at 877 cm -1 indicative of the possible presence of some crystalline calcite or vaterite in addition to the ACC. [START_REF] Foran | Biogenic Fish-gut Calcium Carbonate is a Stable Amorphous Phase in the Gilt-head Seabream, Sparus aurata[END_REF] The bands at 1591 cm -1 and 792 cm -1 can be attributed to the asymmetric stretch and the bending of the carboxyl group of calcium formate. [START_REF] Al-Hosney | Heterogeneous uptake and reactivity of formic acid on calcium carbonate particles: a Knudsen cell reactor, FTIR and SEM study[END_REF][START_REF] Leyva | Thermal Decomposition of Copper (II) Calcium (II) Formate[END_REF][START_REF] Wilson | Formate incorporation in the structure of Cadeficient apatite: Rietveld structure refinement[END_REF] The relative intensity of these bands increased with an increase in Tinlet, suggesting an increase in calcium formate concentration in the particles.

A B

For amorphous solids, the interactions with water are fundamental aspects to be studied because they can affect both dissolution properties and physical stability. [START_REF] Puri | Wettability and surface chemistry of crystalline and amorphous forms of a poorly water soluble drug[END_REF] The water sorption-desorption isotherm of composite microparticles formulated at 120 °C (temp2) showed water mass increase of 10 % (w/w) with an increase in RH up to 70 %, followed by a mass decrease for the 70 to 80 % RH step (Figure 4A). A decrease in mass with increasing RH for amorphous substances corresponds to crystallisation of the material. [START_REF] Bianco | Bulk, surface properties and water uptake mechanisms of salt/acid amorphous composite systems[END_REF][START_REF] Tewes | Evaluation of HPβCD-PEG microparticles for salmon calcitonin administration via pulmonary delivery[END_REF][START_REF] Tewes | Development and characterisation of soluble polymeric particles for pulmonary peptide delivery[END_REF] XRD diffractograms of the formulation temp2 measured after the DVS experiment (Figure 4B) showed new diffraction peaks characteristic of the vaterite CaCO3 polymorph [START_REF] Matahwa | Calcium carbonate crystallization in the presence of modified polysaccharides and linear polymeric additives[END_REF][START_REF] Wang | Biomimetic nucleation and growth of hydrophobic vaterite nanoparticles with oleic acid in a methanol solution[END_REF] and an increase in the intensity of the peak corresponding to calcium formate. Therefore, the crystallisation which occurred during water sorption corresponds to further crystallisation of calcium formate and to the crystallisation of the ACC to vaterite. The second water sorption-desorption cycle did not show any sign of crystallisation or polymorphic change.

Ammonium carbonate concentration effect

Changing (NH4)2CO3 feed solution concentration did not alter particle morphology as observed by SEM (supporting information Figure S4), but changed their physicochemical properties (Table 1). The increase in (NH4)2CO3 from 1.2 g L -1 to 4.8 g L -1 increased 2-fold the specific surface area and increased the calcium concentration in the particles from 38.7 ± 2.6 % to 65.2 ± 3.1 %, respectively.

True density changed variably with the (NH4)2CO3 concentration but increased markedly to 2.26 ± 0.06 g cm -3 for 6 g L -1 of (NH4)2CO3. This density is higher than that of calcium formate (2.009 g cm - 3 ), and closer to the density of pure CaCO3 (vaterite 2.645 g cm -3 , calcite 2.711 g cm -3 ). [START_REF] Saharay | Dehydration-induced amorphous phases of calcium carbonate[END_REF] Analysis of the effect of (NH4)2CO3 concentration on the particles properties by XRD (Figure 2B) and ATR-FTIR (Figure 3B) showed solid state changes. For a concentration of 1.2 g L -1 in (NH4)2CO3 only the XRD Bragg peaks specific to calcium formate were observed. An increase in (NH4)2CO3 concentration in the solutions higher than 1.2 g L -1 induced the formation of vaterite (Figure 3B). From 2.4 g L -1 to 6 g L -1 only Bragg peaks characteristic of vaterite were observed.

FTIR spectra of particles formulated at low (NH4)2CO3 concentration (Figure 3B) were characterised by a broad band of the carbonate bending at 866 cm -1 , typical of ACC. [START_REF] Foran | Biogenic Fish-gut Calcium Carbonate is a Stable Amorphous Phase in the Gilt-head Seabream, Sparus aurata[END_REF] An increase in (NH4)2CO3 concentration in the spray dried feed solution higher than 1.2 g L -1 induced a shift of this band to 876 cm -1 , characteristic of calcite or vaterite. [START_REF] Wang | Biomimetic nucleation and growth of hydrophobic vaterite nanoparticles with oleic acid in a methanol solution[END_REF] The bands split at 1414 and 1484 cm -1 , which is characteristic of the asymmetric stretch of the carbonate ion [START_REF] Raz | Stable amorphous calcium carbonate is the main component of the calcium storage structures of the crustacean Orchestia cavimana[END_REF][START_REF] Politi | Sea urchin spine calcite forms via a transient amorphous calcium carbonate phase[END_REF][START_REF] Meiron | Solubility and Bioavailability of Stabilized Amorphous Calcium Carbonate[END_REF][START_REF] Foran | Biogenic Fish-gut Calcium Carbonate is a Stable Amorphous Phase in the Gilt-head Seabream, Sparus aurata[END_REF] , increased with the (NH4)2CO3 concentration increase, while the asymmetric stretch at 1592 cm -1 and the bending at 793 cm -1 of the carboxyl ion as calcium formate [START_REF] Al-Hosney | Heterogeneous uptake and reactivity of formic acid on calcium carbonate particles: a Knudsen cell reactor, FTIR and SEM study[END_REF] decreased in intensity, confirming the decrease in calcium formate concentration in the particles suggested by XRD.

Hyaluronan concentration effect

The major change induced by the increase in HA concentration was an increase in particle size, for HA concentrations higher than 0.2 g L -1 , and a decrease in particle density and calcium concentration (Table 1). The increase in particle size could be attributed to the increase in the feed solution viscosity, producing larger droplets in the spray dryer. Particle morphology observed by SEM was similar for the various HA concentrations (Supporting Information Figure S5).

The intensities of the diffraction peaks on the XRD patterns were low, suggesting that the formulations were mostly amorphous (Figure 2C). However, an increase in HA concentration from 0.1 g L -1 to 0.6 g L -1 changed the solid state nature of the material. For HA concentrations ranging from 0.1 g L -1 to 0.4 g L -1 only diffraction peaks characteristic of calcium formate were observed. The intensity of these peaks decreased with an increase in HA concentration, suggesting a decrease in concentration or in crystallinity of calcium formate in the particles. At a HA concentration of 0.6 g L -1 , a diffraction peak at 29.4 (2q), specific to the CaCO3 polymorph, calcite, was observed. [START_REF] Matahwa | Calcium carbonate crystallization in the presence of modified polysaccharides and linear polymeric additives[END_REF][START_REF] Wang | Biomimetic nucleation and growth of hydrophobic vaterite nanoparticles with oleic acid in a methanol solution[END_REF] The major change observed on FTIR spectra (Supporting Information Figure S2) induced by an increase in HA concentration was a decrease in intensity of the calcium formate carboxyl band at 793 cm -1 , [START_REF] Al-Hosney | Heterogeneous uptake and reactivity of formic acid on calcium carbonate particles: a Knudsen cell reactor, FTIR and SEM study[END_REF] suggesting that the increase in HA concentration inhibited the formation of calcium formate in the particles. [START_REF] Pitarresi | Composite Nanoparticles Based on Hyaluronic Acid Chemically Cross-Linked with α,β-Polyaspartylhydrazide[END_REF] In this study, sCT was chosen as a model peptide which is sensitive to pulmonary peptidase, limiting its pulmonary bioavailability (F%). To achieve higher F%, sCT was co-administrated with AAT to provide protection from lung peptidases. AAT is a 52-kDa protein produced by the liver, with its main physiological function being the inactivation of proteolytic enzymes in pulmonary tissue. Inhaled AAT has been shown to be suitable for the treatment of AAT deficiency and is currently in clinical trials. [START_REF] Monk | Inhaled Alpha 1-Antitrypsin: Gauging Patient Interest in a New Treatment[END_REF] However, AAT has not yet been used to protect inhaled peptides against degradation and could be an interesting new approach.

Salmon calcitonin-loaded microparticles

sCT and sCT-AAT-loaded microparticles were formulated by adding sCT or sCT and AAT to the Carb2 formulation, which was principally composed of amorphous materials (Table 1). This formulation was chosen, not only because of its amorphous composition, but also based on the high specific surface area and low D50, suggesting good aerodynamic properties. The addition of sCT alone (loading 24.6 ± 2.4% (w/w)) or AAT and sCT (loading 9.0 ± 0.8% (w/w/)) in the same proportion in the composite microparticles totally changed their morphology as observed by SEM (Figure 5). Blank particles were hollow shells, but became crimpled basket-shaped particles in the presence of sCT. This new morphology, with rough wrinkled surface should enhance the particle aerodynamic properties by increasing the particle shape factor. [START_REF] Healy | Dry powders for oral inhalation free of lactose carrier particles[END_REF] Again, all formulation XRD patterns had very low intensity diffraction peaks, revealing the low degree of crystallinity in the particles. The only residual diffraction peaks recorded for the blank particles indicated the presence of vaterite (Figure 2D). The sCT-loaded particles XRD pattern showed only low intensity peaks specific to calcium formate. The loading of both sCT and AAT made the formulations completely XRD amorphous. Analysis by FTIR showed that the incorporation of sCT in the formulation increased the intensity of the bands assigned to calcium formate and decreased the intensity of the band characteristic of ACC (Supporting Information Figure S2). The combined addition of sCT and AAT resulted in an increase in intensity of the FTIR band characteristic of ACC, which was consistent with the XRD results.

As was the case for the blank particles, the water sorption-desorption isotherm of sCT-AAT-loaded microparticles (supplementary information Figure S6) showed mass increase with an increase in RH up to 70%, followed by a mass decrease for the 70 to 80% RH step. The XRD patterns of sCT and sCT-AAT-loaded formulations recorded after a full water sorption-desorption isotherm showed diffraction peaks specific to vaterite and calcium formate of higher intensity than before the RH change cycle. Thus, as for the blank particles, two crystallisation processes occurred at 80% of RH, the crystallisation of vaterite from ACC and the increase in crystalline calcium formate.

Aerodynamic analysis of the sCT-AAT-loaded microparticles showed optimal properties for lung inhalation when using a dry powder inhaler (Supporting information Figure S7). In order to reach the pulmonary alveoli, particles must have an aerodynamic diameter in the 1 to 5 µm range. [START_REF] Healy | Dry powders for oral inhalation free of lactose carrier particles[END_REF] sCT-AATloaded particles emitted from the dry powder inhaler had a mass median aerodynamic diameter of 2.89 ± 0.23 µm with a geometric standard deviation of 1.82 ± 0.16. The fine particle fraction, the fraction of particles that are capable of penetrating and depositing within the lungs, expressed as per the emitted recovered dose, was 64.1 ± 7.1 %. sCT release from sCT or sCT-AAT-loaded microparticles was complete after 30 min in PBS pH 7.4

(Figure 6A). Both formulations showed the same fast release profile. In the presence of trypsin in the release buffer, sCT was rapidly degraded, leading to a reduction in intact sCT released from sCTloaded particles, with the rate of degradation increasing with the trypsin concentration (Figure 6B). In the presence of 10 U (BAEE units) of trypsin in the buffer, all the sCT released from the sCT-loaded particles was degraded after 2 h. For sCT release from sCT-AAT-loaded microparticles, higher proteolytic stability was observed and 85 % of the sCT initially loaded in the particles was detected in the buffer containing 10U of trypsin after 2 h. These results suggest that AAT proteins present in the particles were still active and able to protect sCT from degradation. sCT displayed nonlinear pulmonary pharmacokinetics (PK) in rat and increasing the sCT dose by nebulising sCT solutions of increasing concentration changed the sCT bioavailability (Figure 7). At a low dose (50 µg kg -1 ) pulmonary sCT bioavailability from a nebulised solution was low (4 %) and increased with increasing dose to plateau at around 23 % for a dose of 200 and 400 µg kg -1 . Also, increasing the sCT dose increased Tmax from 5 min for a dose of 50 µg kg -1 up to 15 min for a dose of 200 µg kg -1 . The non-linear sCT PK following pulmonary administration was not due to a change in sCT plasma elimination, as i.v. sCT PK was linear in the same dose range (50 -400 µg kg -1 ).

Likewise, sCT demonstrated linear PK following i.v. administration of 1, 5 and 10 µg doses in rats. [START_REF] Lee | Oral delivery of salmon calcitonin[END_REF] However, sCT PK after i.v. administration in dogs were non-linear over a higher dose range (1-1000 µg). [START_REF] Lee | Oral delivery of salmon calcitonin[END_REF] Average sCT plasma concentration-versus-time profiles were different after pulmonary administration of a solution of sCT alone (100 µg kg -1 ) or when combined with AAT (Figure 8A). For sCT alone, sCT lung absorption was fast, with a Tmax of 4.2 ± 1.1 min, followed by a fast elimination leading to a bioavailability (F) of 11.9 ± 3.9 %. In the presence of AAT, sCT bioavailability was not significantly changed. However, Tmax was shifted to 30 ± 0.1 min and the systemic absorption was slowed down in comparison to the pure sCT solution, as described by the lower absorption constant Ka = 0.128 ± 0.002 min -1 (Table 2).

Administration of sCT as composite microparticles (without AAT) increased only the absorption Tmax

but other all pharmacokinetic parameters were identical to those obtained with pure sCT solutions.

This increase in Tmax should be related to the time for sCT release from the particles. With the addition of AAT to the particles, most of the pharmacokinetic parameters were significantly changed. Cmax, Tmax and AUC of sCT plasma concentrations-versus-time profiles were increased, leading to a 4-times increased sCT bioavailability compared to sCT solution (Figure 8B).

Discussion

In many living entities, solid CaCO3 is shaped into different structures to adapt and fulfil different functions. [START_REF] Politi | Sea urchin spine calcite forms via a transient amorphous calcium carbonate phase[END_REF] Eggshell and bones are examples of CaCO3-made structures which are light and have good mechanical resistance. In this study, we attempt to use these properties to formulate microparticles for pulmonary peptide delivery. We showed that shell-like inorganic-organic inhalable microparticles could be made of CaCO3, calcium formate and biopolymer (HA) with properties matching the pharmaceutical requirements of an aerosolisable powder. In this process, the microparticle properties were tuned by different means, i.e. by altering the drying temperature, the (NH4)2CO3 and HA concentration in the spray dried feed solutions.

Increasing the spray drying temperature, which increases the rate and extent of drying [START_REF] Vehring | Particle formation in spray drying[END_REF] had several effects on the particles properties. First, it favoured the formation of calcium formate (Ca(HCOO)2)

over the formation of CaCO3. Second, it impacted on the solid state of the particles' inorganic component. Calcium formate was present in the particles in the crystalline and/or amorphous state. To our knowledge, this is the first report of amorphous calcium formate. Spray drying is a process which often produces amorphous pharmaceuticals. The ratio between amorphous and crystalline calcium formate depended on the spray drying temperature. At low drying temperature (100 °C) the particles were fully amorphous. An increase in temperature up to 140 °C led to the increased formation of crystalline calcium formate phase. CaCO3 was XRD amorphous at all processing temperatures used.

However, for the particles produced at 140 °C, FTIR experiments suggested the presence, in low proportions, of a calcium carbonate phase which was more organised than ACC. CaCO3 can solidify as three anhydrous crystalline polymorphs (calcite, aragonite, vaterite), two hydrates (hexahydrate and monohydrate) and ACC, which is the most thermodynamically unstable. [START_REF] Akiva-Tal | In situ molecular NMR picture of bioavailable calcium stabilized as amorphous CaCO3 biomineral in crayfish gastroliths[END_REF] Stable ACC can be obtained by the addition of different excipients, such as polymers, to the solid phase [START_REF] Aizenberg | Stabilization of amorphous calcium carbonate by specialized macromolecules in biological and synthetic precipitates[END_REF][START_REF] Pouget | The initial stages of template-controlled CaCO3 formation revealed by Cryo-TEM[END_REF][START_REF] Ihli | The Effect of Additives on Amorphous Calcium Carbonate (ACC): Janus Behavior in Solution and the Solid State[END_REF] and by controlling the amount of water present in the solid phase (hydrated ACC). This last phenomenon was mainly observed with ACC produced in living organisms. [START_REF] Addadi | Taking advantage of disorder: Amorphous calcium carbonate and its roles in biomineralization[END_REF] Water present in CaCO3 lowers the energy barrier towards the solidification of hydrated ACC compared with the anhydrous crystalline forms. [START_REF] Ihli | Dehydration and crystallization of amorphous calcium carbonate in solution and in air[END_REF][START_REF] Raiteri | Water Is the Key to Nonclassical Nucleation of Amorphous Calcium Carbonate[END_REF] Also, maintaining this hydration could be important for stability of the ACC, as crystallisation induced by heat is thought to involve dehydration of hydrated ACC. [START_REF] Radha | Transformation and crystallization energetics of synthetic and biogenic amorphous calcium carbonate[END_REF][START_REF] Ihli | Dehydration and crystallization of amorphous calcium carbonate in solution and in air[END_REF][START_REF] Saharay | Dehydration-induced amorphous phases of calcium carbonate[END_REF][START_REF] Saharay | Onset of Orientational Order in Amorphous Calcium Carbonate (ACC) upon Dehydration[END_REF][START_REF] Rodriguez-Blanco | The kinetics and mechanisms of amorphous calcium carbonate (ACC) crystallization to calcite, viavaterite[END_REF][START_REF] Ogino | The formation and transformation mechanism of calcium carbonate in water[END_REF][START_REF] Ogino | The rate and mechanism of polymorphic transformation of calcium carbonate in water[END_REF] Likewise, ACC crystallisation can be retarded by providing a barrier to water loss. [START_REF] Ihli | Dehydration and crystallization of amorphous calcium carbonate in solution and in air[END_REF] In a pharmaceutical context, water is usually considered to be a plasticizer, decreasing the Tg of an amorphous solid and increasing the local molecular mobility and physical instability. It is generally recognized that exposure to water has to be avoided in order to obtain stable amorphous pharmaceutical solids; thus the behaviour of ACC appears to be an exception.

At ambient temperature, as for the polymorphic transformation of crystalline CaCO3, ACC crystallisation is triggered through local superficial ACC dissolution in surface water and local crystallisation to form nucleation clusters. [START_REF] Ihli | Dehydration and crystallization of amorphous calcium carbonate in solution and in air[END_REF] Surface water is thus important for this dissolutioncrystallisation process to occur. For example, ACC washed with ethanol to remove surface water or ACC prepared by freeze drying that has no surface water shows extended stability. [START_REF] Sand | Binding of ethanol on calcite: the role of the OH bond and its relevance to biomineralization[END_REF][START_REF] Ihli | Freeze-drying yields stable and pure amorphous calcium carbonate (ACC)[END_REF] The conversion of the ACC into the vaterite crystalline phase observed when the relative humidity was increased above 70% should be related to the local dissolution of ACC in adsorbed and absorbed water.

Generally, polymorphs formed after ACC crystallization at low temperature (14 to 30 °C) are vaterite and calcite. [START_REF] Ogino | The formation and transformation mechanism of calcium carbonate in water[END_REF] Recent work suggests that vaterite is often the first phase to form during crystallisation of pure ACC. [START_REF] Saharay | Onset of Orientational Order in Amorphous Calcium Carbonate (ACC) upon Dehydration[END_REF][START_REF] Rodriguez-Blanco | The kinetics and mechanisms of amorphous calcium carbonate (ACC) crystallization to calcite, viavaterite[END_REF] However, it is not a general result and depends on how the ACC is made. [START_REF] Gebauer | Proto-Calcite and Proto-Vaterite in Amorphous Calcium Carbonates[END_REF] Aqueous solubility of hydrates is generally lower than the anhydrous form. [START_REF] Pudipeddi | Trends in solubility of polymorphs[END_REF] The dissolution-crystallisation process needed to crystallise CaCO3 could be favoured for anhydrous compared to hydrated ACC.

Thus, avoiding the complete dehydration of CaCO3 during the solidification step, by adjusting the Tinlet, seems important to formulate stable ACC by spray drying. Controlling the spray drying temperature, water evaporation rate and residual amount of water in the final particle could be a way to form stable amorphous/crystalline composite particles. CaCO3 may not be exclusively involved in this crystallization process and this could happen for calcium formate also.

Changing HA and (NH4)2CO3 concentrations had several consequences on the nature of the particles' inorganic component. First, increasing HA and (NH4)2CO3 concentrations facilitated the formation of CaCO3 over calcium formate. Second, HA and (NH4)2CO3 concentrations influenced the solid state nature of the CaCO3. High HA concentration induced the formation of calcite, the stable CaCO3 polymorph. Negatively charged carboxylic groups of HA polymers are assumed to be responsible for nucleation of this particular CaCO3 polymorph by interacting with calcium ions. [START_REF] Butler | Calcium Carbonate Crystallization in the Presence of Biopolymers[END_REF] In fact, HA was previously shown to facilitate and control the formation and shape of calcite. [START_REF] Arias | Polysaccharides and Proteoglycans in Calcium Carbonate-based Biomineralization[END_REF] Also, it has been shown that CaCO3 polymorphic transformations proceeded by the dissolution of the metastable phases and the growth of calcite, and that the rate-determining step was the growth of calcite. [START_REF] Ogino | The rate and mechanism of polymorphic transformation of calcium carbonate in water[END_REF] An increase in HA concentration results in an increase in solution viscosity and slows down the mobility of the Ca 2+ and CO3 2-ions. This reduction of the ions' mobility should favor the thermodynamic over the kinetic control of the crystallisation process, leading to the formation of the stable calcite. In fact, an increasing proportion of calcite over vaterite was previously observed for CaCO3 growth in solution for solutions with viscosities above 1.6 mPa s. [START_REF] Dickinson | Switching between kinetic and thermodynamic control: calcium carbonate growth in the presence of a simple alcohol[END_REF] Increasing (NH4)2CO3 concentration facilitates the formation of vaterite phase. Changing (NH4)2CO3 feed solution concentration had a direct effect on carbonate ion concentration. A previous study showed that the carbonate ion concentration had a kinetic control on the growth of CaCO3, facilitating the formation of vaterite as the carbonate ion concentration increased for a given calcium ion concentration. [START_REF] Dickinson | Controlling the kinetic versus thermodynamic crystallisation of calcium carbonate[END_REF] The control of the solid state nature and composition of the inorganic part of the particles could enable their physicochemical properties to be adapted to best suit the requirements of pharmaceutical dry powders for inhalation. This new process produced particles especially well-suited for pulmonary drug delivery. In order to reach the pulmonary alveoli, particles must have an aerodynamic-equivalent diameter (da) in the 1-5 µm range. [START_REF] Healy | Dry powders for oral inhalation free of lactose carrier particles[END_REF] da is linked to the volume-equivalent geometric diameter (dg) of the particles by the particle shape, evaluated by the particle shape factor (χ), and by the particle density. [START_REF] Shekunov | Particle Size Analysis in Pharmaceutics: Principles, Methods and Applications[END_REF][START_REF] Decarlo | Particle Morphology and Density Characterization by Combined Mobility and Aerodynamic Diameter Measurements. Part 1: Theory[END_REF] χ is defined as the ratio of the drag force on a particle to the drag force on a volumeequivalent sphere at the same velocity. [START_REF] Decarlo | Particle Morphology and Density Characterization by Combined Mobility and Aerodynamic Diameter Measurements. Part 1: Theory[END_REF] In order to decrease da of geometrically large particles below 5 µm, several studies focused on enhancing the particle porosity and specific surface area to increase χ and decrease ρp. [START_REF] Tewes | Evaluation of HPβCD-PEG microparticles for salmon calcitonin administration via pulmonary delivery[END_REF][START_REF] Tewes | Development and characterisation of soluble polymeric particles for pulmonary peptide delivery[END_REF][START_REF] Tsapis | Trojan particles: Large porous carriers of nanoparticles for drug delivery[END_REF][START_REF] Tewes | Steroid/mucokinetic hybrid nanoporous microparticles for pulmonary drug delivery[END_REF][START_REF] Amaro | Optimisation of spray drying process conditions for sugar nanoporous microparticles (NPMPs) intended for inhalation[END_REF] All blank particles produced in the current study were shell-like and had high specific surface areas compared to solid spherical particles of the same size. [START_REF] Amaro | Optimisation of spray drying process conditions for sugar nanoporous microparticles (NPMPs) intended for inhalation[END_REF] The incorporation of sCT in the formulation completely changed the particle morphology to wrinkled particles. Particle morphology produced by spray drying can be explained by the material's Peclet number (Pe). [START_REF] Tsapis | Trojan particles: Large porous carriers of nanoparticles for drug delivery[END_REF] The Peclet number compares the diffusion rate of the solute and the evaporation rate of the receding droplet surface. For materials having a Pe larger than 1, e.g. moving slowly in the liquid, the sprayed droplet surface moves faster than the dissolved or suspended materials, resulting in their accumulation and solidification at the surface. The resulting particles can be hollow spheres if the shell becomes rigid quickly, as happened for the blank particles. If the surface is too soft, it can buckle or fold on the internal and external void space to form a wrinkled or dimpled morphology, as was observed for the particles containing sCT. sCT appears to make the particle wall softer, leading to the formation of wrinkled particles. In fact sCT has a Tg around 147 C 75 , which should be lower than the Tg of ACC and make the particle wall softer. Similarly, it was previously observed that the addition of sCT to spherical particles made of polyethylene glycol (PEG) and polyvinylpyrrolidone reduced the degree of crystallinity in the particles and made them wrinkled. [START_REF] Tewes | Development and characterisation of soluble polymeric particles for pulmonary peptide delivery[END_REF] The incorporation sCT and AAT in the formulation favoured the formation of ACC and resulted in the production of completely amorphous particles, that could be important to stabilize these molecules and prevent any structural damage during processing and storage. [START_REF] Yu | Amorphous pharmaceutical solids: preparation, characterization and stabilization[END_REF] After landing in the lung, the microparticles release sCT that is then absorbed or eliminated from the lung by clearance mechanisms.

sCT bioavailability may be improved by either increasing the sCT absorption rate or reducing the effect of clearance mechanisms. Depending on the region of the lung that the particle reaches, the lung clearance mechanisms are different. In the deep peripheral lung the main clearance mechanisms for sCT are proteases activity [START_REF] Garcia-Verdugo | Lung protease/anti-protease network and modulation of mucus production and surfactant activity[END_REF] and macrophage phagocytosis. The release of sCT from the particles was fast and complete within 30 min. ACC is the most soluble form of CaCO3 and dissolves quickly, which should be responsible for the fast sCT release. This fast release is beneficial, as it can prevent sCT macrophage degradation after particle phagocytosis and allows for a high sCT concentration gradient across the lung-blood barrier, facilitating sCT absorption.

sCT displayed non-linear pulmonary PK, leading to an increase in sCT bioavailability with an increase in sCT dose. This non-linear PK obtained after pulmonary nebulisation of sCT solution could involve the saturation of sCT clearance mechanisms. In fact, if above a certain sCT concentration, the elimination rate tends to reach a maximal value and there is no further increase in the elimination rate when sCT concentration increases, then an increase in sCT concentration in the lung must increase its bioavailability. However, from a sCT dose of 200 µg kg -1 and above, sCT bioavailability plateaued at around 23 % and Tmax increased with the sCT dose, suggesting the slow-down of the sCT absorption and the action of other slower clearance mechanisms, resulting in a bioavailability of 100% not being achieved. Likewise, the increase in Tmax and the limitation of sCT bioavailability to 18.5 ± 3.2 % in the presence of AAT, after nebulisation of sCT and AAT solutions, could be explained by the saturation of sCT absorption mechanisms and the action of clearance mechanisms other than proteases.

Thus, AAT nebulised solution in the lung increased sCT bioavailability by reducing sCT enzymatic degradation. However, sCT bioavailability is limited by its absorption rate drop at high concentration.

In the same way, pulmonary delivered PEGylated sCT had 4-times increased bioavailability due to higher proteolytic resistances and partly due to reduction of elimination rate in plasma, but reached only 50% bioavailability. [START_REF] Youn | Improved intrapulmonary delivery of site-specific PEGylated salmon calcitonin: Optimization by PEG size selection[END_REF] This limitation could also be due to a slowing down of the sCT absorption rate with increasing sCT concentration in the lung and the concomitant action of other elimination mechanisms. sCT administered as sCT-AAT particles had higher bioavailability than sCT delivered as solution with or without AAT but had a similar absorption rate. This suggests that the AAT-loaded composite particles were able to further extend the protecting action of AAT in the lung to clearance mechanisms other than protease.

Conclusions

We have succeeded in developing a novel formulation approach to prepare shell-like composite microparticles made of selected polymorphs of calcium carbonate (amorphous, calcite and vaterite), calcium formate and hyaluronan. In this new process, different levels of control could be tuned to adapt the microparticle properties. The first level is the organic biopolymer/mineral (CaCO3 and Ca(HCOO)2) ratio. The second level of control is the solid nature of the mineral component, crystalline/amorphous ratio and polymorphic properties. These inorganic/organic hybrid microparticles were able to preserve peptide and protein activities and deliver them to the lung. The combination of alpha-1-antitrypsin associated with composite particles was able to improve sCT bioavailability 4-fold.

Figure 1 :

 1 Figure 1: SEM micrographs of Ca/HA composite microparticles formulated at different spray drying inlet temperatures (Tinlet).

Figure 2 :

 2 Figure 2: XRD patterns of Ca/HA composite particle: A) Formulated at different temperatures, B) Formulated at different concentrations of (NH4)2CO3, C) Formulated at different concentrations of HA, D) Loaded with sCT and AAT. (F) label is for calcium formate, (V) label is for vaterite CaCO3 polymorph and (C) is for calcite CaCO3 polymorph.

Figure 3 :

 3 Figure 3: FTIR spectra of Ca/HA composite microparticle A) Formulated at different temperatures, B) Formulated at different concentrations of (NH4)2CO3.

Figure 4 :

 4 Figure 4: A) Water sorption isotherm (25 °C) of Ca/HA composite microparticles formulated at 120 °C (temp2). B) XRD patterns recorded before and after DVS experiment. (F) label is for calcium formate and (V) label is for vaterite CaCO3 polymorph.

Figure 5 :

 5 Figure 5: SEM micrographs of Ca/HA composite particles loaded with sCT (A) and sCT and  (B).

Figure 6 :

 6 Figure 6: sCT release from sCT and sCT-AAT-loaded microparticles in the absence (A) or in the presence (B) of trypsin in the PBS pH 7.4.

Figure 7 :

 7 Figure 7: sCT bioavailability (F) after pulmonary administration of various sCT dose by nebulising 100 µL of sCT solutions at different concentration.

Figure 8 :

 8 Figure 8: sCT plasma concentration-versus-time profiles after: A) sCT solution nebulisation B) sCT-loaded composite microparticles inhalation (mean ± SD). Dose 100 µg Kg -1 .

Table 1 : Composition and physical properties of Ca/HA composite particles

 1

			Formulation parameters		Results
	Code	Ca(OH) 2 (g L -1 )	HA (g L -1 )	(NH 4 ) 2 CO 3 (g L -1 )	inlet/outlet temp (C)	D 50 (µm)	specific surface area (m 2 g -1 )	True density (g cm -3 )	Ca loading % (Assay)
	temp1	0.8	0.2	1.2	100/39	4.65 ± 0.5	17.8 ± 1.2	1.25 ± 0.02 36.8 ± 2.3
	temp2	0.8	0.2	1.2	110/44	4.55 ± 0.4	16.5 ± 0.8	1.06 ± 0.07 38.7 ± 2.6
	temp3	0.8	0.2	1.2	120/50	4.74 ± 0.3	19.6 ± 0.8	1.37 ±0.01 34.3 ± 2.9
	temp4	0.8	0.2	1.2	140/68	4.67 ± 0.4	19.3 ± 0.6	2.06 ± 0.01 37.3 ± 3.2
	Carb1	0.8	0.2	1.2	120/50	4.55 ± 0.4	16.5 ± 0.6	1.06 ± 0.07 38.7 ± 2.6
	Carb2	0.8	0.2	2.4	120/50	5.48 ± 0.7	30.1 ± 1.0	1.74 ± 0.03 57.3 ± 4.6
	Carb3	0.8	0.2	4.8	120/50	7.07 ± 0.5	28.0 ± 0.9	1.49 ± 0.06 65.2 ± 3.1
	Carb4	0.8	0.2	6	120/50	5.34 ± 0.3	23.6 ± 0.8	2.26 ± 0.06 53.5 ± 4.0
	HA1	0.8	0.1	1.2	120/50	5.36 ± 0.4	18.7 ± 0.6	2.35 ± 0.05 48.9 ± 2.9
	HA2	0.8	0.2	1.2	120/50	4.74 ± 0.3	19.6 ± 0.8	1.37 ±0.01 34.3 ± 2.9
	HA3	0.8	0.4	1.2	120/50	7.45 ± 0.5	17.7 ± 0.7	1.10 ± 0.05 34.6 ± 3.5
	HA4	0.8	0.6	1.2	120/50	10.20 ± 0.6	15.7 ± 0.8	1.21 ± 0.10 32.0 ± 4.1
	test12	0.8	0.4	4.8	120/50	9.55 ± 0.8	25.5 ± 0.9	1.55 ± 0.02 44.3 ±2.7

2 q degree sCT-AAT CaCO3 sCT CaCO3 Blank particle

	F 16.0	F 26.3
		V 24.9	V 27.1	V 32.8	V 44.0

Table 2 : Pharmacokinetic parameters of the sCT plasma concentration versus time profiles. (mean ± SD (n = 2-7)); * P < 0.05, indicating a significant difference between the mean values of the PK parameters compare to the one obtained for sCT solution pulmonary administration. AUC  is the area under the sCT plasma concentration versus time curve. Tmax is the time of peak sCT plasma concentration. Cmax is the maximal sCT plasma concentration obtained at Tmax. ka is the first order absorption rate constant. F (the bioavailability) is the fraction of the dose which can be absorbed

 2

		sCT solution	sCT AAT solution	sCT composite microparticles	sCT-AAT composite microparticles
	AUC ∞ (min ng mL -1 )	1493 ± 526	2319 ± 397	1705 ± 549	4907 ± 3085*
	Tmax (min)	4.2 ± 1.1	30.0 ± 0.1	16.6 ± 4.7	23.3 ± 4.7*
	Cmax (ng mL-1)	24.1 ± 4.5	31.4 ± 1.3	18.0 ± 6.2	46.8 ± 30.3*
	K a (min -1 )	0.476 ± 0.152	0.128 ± 0.002*	0.188 ± 0.027*	0.155 ± 0.025*
	F(%)	11.9 ± 3.9	18.5 ± 3.2	13.6 ± 5.4	39.2 ± 24.6*

Acknowledgements

This publication has emanated from research conducted with the financial support of Science Foundation Ireland (SFI) under Grant Numbers 07/SRC/B1158 and SFI/12/RC/2275