Zhengxing Qin

Ludovic Pinard

Mohammed Amine Benghalem

T Jean Daou

Georgian Melinte

Ovidiu Ersen

Shunsuke Asahina

Jean-Pierre Gilson

Valentin Valtchev

Preparation of Single Crystals "House-of-Cards"like ZSM-5 and Their Performance in Ethanol-to-Hydrocarbons Conversion

The present study reports the unbiased chemical etching of micron-sized ZSM-5 crystals with an NH4F solution resulting in house-of-cards-like single crystals containing large rectangular cavities surrounded by thin (15 -30 nm), flat and highly crystalline walls. The formation of such house-of-cards-like architecture is a result of the preferential extraction of mis-oriented nanocrystalline domains followed by the uniform dissolution of the remaining part of the crystal.

The characteristic features of NH4F-treated zeolites are the retention of framework composition (Si/Al) and Brønsted acidity, high crystallinity, a moderate increase in external surface area and increased accessibility to their active sites. Such a combination produces zeolitic catalysts with superior performances (activity, stability and coke resistance) in the ethanol-to-hydrocarbons conversion. The physicochemical properties of this newly engineered zeolite are compared with a hierarchical zeolite obtained by caustic leaching and zeolite nano-sheets synthesized with a bifunctional template, a di-quaternary ammonium-type surfactant.

Introduction

Zeolites revolutionized oil refining and petrochemistry due to their excellent chemical and (hydro)thermal stability, unique microporous structures of molecular dimensions and tunable acidity located in a shape-selective environment [START_REF] Martínez | Inorganic molecular sieves: Preparation, modification and industrial application in catalytic processes[END_REF][START_REF] Vermeiren | Impact of Zeolites on the Petroleum and Petrochemical Industry[END_REF][START_REF] Vogt | Fluid catalytic cracking: recent developments on the grand old lady of zeolite catalysis[END_REF]. With an increasing demand for highly active and stable catalysts for existing and emerging processes, recent work with zeolites focused on morphology tailoring either during their synthesis (bottom-up approach) or by post-synthesis modifications (top-down approach), the so-called zeolite crystal engineering [START_REF] Tosheva | Synthesis, Crystallization Mechanism, and Applications[END_REF][START_REF] Valtchev | Porous Nanosized Particles: Preparation, Properties, and Applications[END_REF][START_REF] Fan | Hierarchical nanofabrication of microporous crystals with ordered mesoporosity[END_REF][START_REF] Awala | Template-free nanosized faujasite-type zeolites[END_REF][START_REF] Valtchev | Tailored crystalline microporous materials by post-synthesis modification[END_REF].

Transport limitations in their micropore channels are probably the most serious drawback of zeolite catalysts [START_REF] Hartmann | Catalytic test reactions for the evaluation of hierarchical zeolites[END_REF]. Decreasing zeolite crystal size is a straightforward approach to minimize their impact. Advances in the understanding of zeolite nucleation/crystal growth allowed the preparation of nanocrystals of many industrially important zeolites [START_REF] Smaihi | Investigation of the Crystallization Stages of LTA-Type Zeolite by Complementary Characterization Techniques[END_REF][START_REF] Valtchev | Preparation of regular macroporous structures built of intergrown silicalite-1 nanocrystals[END_REF]. However, the number of zeolite crystals synthesized with nanosized dimensions is still limited. Zeolite materials with nano-dimensions in a particular crystallographic direction were also prepared [START_REF] Tsapatsis | 2-dimensional zeolites[END_REF]. These two-dimensional microporous materials, referred to as layered-or sheet-like zeolites, offer the advantage of extended external surface areas and enhanced diffusion in a particular crystallographic direction. They were prepared, for instance, by Corma and co-workers by exfoliating layered MWW-type zeolite precursors [START_REF] Corma | Delaminated zeolite precursors as selective acidic catalysts[END_REF]. The exfoliation approach is however limited to a few zeolites since it works with layered precursors with weaker bonds in a particular crystal direction. Ryoo et al. successfully synthesized a series of crystalline molecular sieves (ZSM-5 and zeolite Beta) with tunable mesostructures and nanosheet or nanosponge morphologies using amphiphilic organosilanes or multi-quaternary ammonium surfactants as bifunctional templates [START_REF] Choi | Amphiphilic organosilane-directed synthesis of crystalline zeolite with tunable mesoporosity[END_REF][START_REF] Choi | Stable single-unit-cell nanosheets of zeolite MFI as active and long-lived catalysts[END_REF][START_REF] Na | Directing Zeolite Structures into Hierarchically Nanoporous Architectures[END_REF]. A low-cost approach to zeolite nanosheets forming "house-ofcards" structures was reported by Tsapatsis et al. [START_REF] Zhang | Synthesis of Self-Pillared Zeolite Nanosheets by Repetitive Branching[END_REF]. They used a template promoting the repetitive branching of orthogonally connected nanosheets. Such intergrowths generate structures with a mesoporous network of 2-7 nm. The resulting polycrystalline hierarchical materials offer the advantages of higher accessibility for bulky molecules, shorter diffusion pathlengths in the microporosity and a larger external surface area. At present such advanced materials were only obtained by a bottom-up approach. To the best of our knowledge, the preparation of house-ofcards by a top-down approach has not yet been reported. A post-synthesis approach to house-ofcards zeolites would provide significant advantages such as i) applicability to a large number of zeolites; ii) easy transformation of single crystals in house-of-cards materials; iii) cost and environmentally benign scaling-up to produce zeolites.

Extraction of framework cations by chemical etching has been used since the first commercial applications of zeolites (eg. hydrocracking, fluid catalytic cracking [FCC], isomerization…) to alleviate these ever-present mass transport limitations. For instance, high-temperature (> 600°C) steaming extracts aluminum from zeolite frameworks and redistributes it in extra-framework positions; while the overall Si/Al is unchanged, the framework Si/Al increases and rearrangement of framework Si generates a secondary mesoporosity [START_REF] Van Donk | Characterization, and Impact of Mesopores in Zeolite Catalysts[END_REF]. Recently, caustic leaching [START_REF] Young | Hydrocarbon conversion process and catalyst comprising a crystalline aluminosilicate leached with sodium hydroxide[END_REF][START_REF] Gilson | Process for isomerizing hydrocarbons[END_REF] was extensively revisited, better understood and shown to generate secondary mesopores in high silica zeolites [START_REF] Milina | Mesopore quality determines the lifetime of hierarchically structured zeolite catalysts[END_REF]. Chemical etching is an efficient and straightforward method to improve intra-crystalline transport and reduce diffusion barriers. However, the preferential or biased extraction of a particular framework cation (Al or Si) leads to a dissolution profile, strongly dependent on the zeolite composition and therefore difficult to control.

Recently, an unbiased chemical etching was developed using NH4F solutions [START_REF] Valtchev | High Energy Ion Irradiation-Induced Ordered Macropores in Zeolite Crystals[END_REF][START_REF] Qin | Mesoporous zeolites by fluoride etching[END_REF][START_REF] Qin | The Mosaic Structure of Zeolite Crystals[END_REF][START_REF] Qin | Opening the Cages of Faujasite-Type Zeolite[END_REF][START_REF] Valtchev | Method for the preparation of synthetic crystalline zeolite materials with enhanced total pore volume[END_REF].

Concentrated ammonium fluoride solutions were shown to dissolve Si and Al from zeolite frameworks at equal rates. While leaving the zeolite composition (Si/Al) unchanged, this approach dissolves preferentially defects in zeolite crystals, such as interfaces between twin crystals, grain boundaries, lattice defects… This unbiased fluoride extraction is HF-free; moreover, the biased (NH4)2SiF6 processing of zeolites is currently used in the commercial production of modified (dealumination and subsequent healing by silication) FAU zeolites (LZ-210 from UOP LLC. [START_REF] Breck | Silicon substituted zeolite compositions and process for preparing same[END_REF][START_REF] Skeels | Proceedings of the Sixth International Zeolite Conference[END_REF])

The objective of the present work is to develop a universal top-down approach to produce single crystals house-of-cards-like zeolite catalysts. It is based on the recently discovered mosaic structure of zeolite crystals, i.e., the presence of rectangular nanocrystalline domains with welldefined grain boundaries [START_REF] Qin | The Mosaic Structure of Zeolite Crystals[END_REF]. Such domains were observed in MFI [START_REF] Qin | The Mosaic Structure of Zeolite Crystals[END_REF], FER [START_REF] Chen | In situ and post-synthesis control of physicochemical properties of FER-type crystals[END_REF], FAU [START_REF] Ajot | Formation of Secondary Pores in Zeolites During Dealumination: Influence of The Crystallographic Structure and Of the Si/Al Ratio[END_REF], and appear to be ubiquitous. They are preferentially dissolved when an unbiased chemical etching is applied to zeolite crystals, leaving behind rectangular cavities several tens of nanometers wide. The remaining part of the crystals dissolves with a similar rate of dissolution following the morphology of extracted nanocrystalline domains, which is employed in the present study to form a house-of-cards-like material by post-synthesis etching. This is illustrated by comparing the unbiased NH4F etching of a parent (ZP) ZSM-5 zeolite, ZF, a Si biased caustic leaching of the same parent, ZB [START_REF] Lakiss | On the remarkable resistance to coke formation of nanometer-sized and hierarchical MFI zeolites during ethanol to hydrocarbons transformation[END_REF], and zeolite nano-sheets (ZNS) prepared with a di-quaternary ammonium-type surfactant acting as a bifunctional structure directing agent [START_REF] Boltz | MFI-type zeolite nanosheets for gas-phase aromatics chlorination: a strategy to overcome mass transfer limitations[END_REF]. The Ethanolto-hydrocarbons (ETH) conversion was used as a model reaction to evaluate the catalytic potential of these zeolites and relate it to their physicochemical properties.

Results and discussion

Physicochemical characteristics of the zeolites

XRD

The samples preparation is provided in the supporting information. The XRD patterns of ZP (commercially available zeolite ZSM-5, the parent), ZB (a biased caustic etching of ZP), ZF (an unbiased NH4F etched derivative), and ZNS (a bottom-up ZSM-5 nanosheets preparation) are displayed in Figure 1. ZP shows sharp and narrow peaks characteristic of a highly crystalline micron-sized MFI-type material. The XRD peaks of ZF are equally narrow and well-defined, indicating that the NH4F etching does not impact crystallinity. ZB shows much lower peak intensities than its parent. Some of the peaks are missing in the XRD pattern of ZNS, as expected for such type of material [START_REF] Boltz | MFI-type zeolite nanosheets for gas-phase aromatics chlorination: a strategy to overcome mass transfer limitations[END_REF].

Figure 1

The XRD patterns of the parent zeolite (ZP), its NH4F (ZF) and caustic (ZB) etched derivatives, and a nano-sheet zeolite (ZNS).

N2 Physisorption

The physicochemical properties of all zeolites are summarized in Table 1. The parent micronsized (ZP) crystals display a type I isotherm (Figure S1) characteristic of a purely microporous zeolite [START_REF] Thommes | Physisorption of gases, with special reference to the evaluation of surface area and pore size distribution (IUPAC Technical Report)[END_REF]. ZF and ZB zeolites exhibit a second uptake and a hysteresis loop at high relative pressure revealing the presence of mesopores. The micropore volume of ZF is fully retained compared to its parent and its external surface area is lower than its caustic leached counterpart, ZB; ZB has however lost some of its micropore volume, in agreement with the XRD data. The zeolite nano-sheets, ZNS, display a Type I isotherm at low p/p 0 combined with a type IV(a) at high p/p 0 . The presence of a hysteresis loop in the relative pressure range 0.4 < p/p 0 < 1 is typical for such lamellar materials due to the stacking of nanosheets. ZNS also exhibits a lower micropore volume and larger external surface area. The mesopores distribution for all samples is presented in Figure 2. No mesopores are observed in the parent micron-sized crystals, ZP. ZF exhibits a large pore size distribution between ca 10 nm and more than 100 nm. The ZB pore size distribution is also large, but within the mesopore range, i.e., 2 to 50 nm [START_REF] Thommes | Physisorption of gases, with special reference to the evaluation of surface area and pore size distribution (IUPAC Technical Report)[END_REF]. The pore size distribution of ZNS, with a maximum of around 6 nm, is much sharper compared with ZB and ZF.

All three hierarchical zeolites have a mesopore volume higher than 0.3 cm 3 g -1 (Table 1). However, ZF shows only a slight increase in external surface area compared to its parent.

Conversely, ZB and ZNS both develop external surfaces exceeding 200 m 2 g -1 . We attribute these differences to the types of pores formed by NH4F and caustic leaching and the stacking of nanosheets, respectively. The dissolution in fluoride medium results in the formation of large meso-and macropores (Figure 2), which exhibit low specific surface area (Table 1). This can be further interpreted based on a rough estimation of the relation between the surface (S), the pore diameter (d) and the pore volume (V) using d = 4V/S. In contrast, both ZB and ZNS contain a substantial amount of small mesopores with size below 10 nm (Figure 2). Accordingly, a substantial increase in the external surface area is observed (Table 1). etched derivatives and the nano-sheet zeolite (ZNS).

Morphological analysis

The morphology and secondary porosity of the zeolites are described by combining scanning (SEM) and transmission electron microscopy (TEM). NH4F etching transforms the micron-sized ZSM-5 crystals (Figure 3a, insert) into a foam-like material with thin crystalline walls (Figure 3a,b). The macro-morphological features of the crystals are retained. However, the crystalline body is fully transformed into a mosaic of rectangular cavities, large enough to be observed with a scanning electron microscope (Figure 3a,b). TEM provides further insights into the structure and morphology of the NH4F etched zeolite. Figure 3c shows a representative TEM image of such an NH4F etched ZSM-5 crystal. Figure 3e-g show slices through the electron tomography reconstruction of a selected zeolite grain. The slices highlight the high porosity of chemically treated zeolite but also the remained crystalline pore walls (Figure 3d) with a thickness ranging from 10 to 30 nm. The tomography shows that the NH4F etched zeolite contains two types of secondary pores: i) large interconnected rectangular-shaped, ii) smaller ones probably formed later on during the etching. 3D models indicate that even the vast majority of the smaller mesopores are connected to the secondary porous network, indicating that pore connectivity approaches 100 % and an overall meso-macroporosity of around 47%. This excellent connectivity between the various pore networks is an important, albeit often overlooked, feature in hierarchical zeolites [START_REF] Kortunov | The role of mesopores in intracrystalline transport in USY zeolite: PFG NMR diffusion study on various length scales[END_REF]. The porosity could even be higher as some of the surface pores (Figure S2) were excluded from the quantification. The 3D geometry of the porous network and their walls resemble the "house-of-cards" architecture. A schematic presentation of the remaining part of the crystals with the vast sytem of rectangular, interconnected cavities is presented in Figure S3. It is remarkable that such a significant secondary porosity increase in ZF is achieved without loss of intrinsic microporosity and acidity (Table 1) as well as crystallinity (Figure 1, Figure 3d). Zeolite dissolution process is strongly influenced by the zeolite framework composition and crystal zoning in the case of selective chemical extraction of a framework cation [START_REF] Groen | Creation of Hollow Zeolite Architectures by Controlled Desilication of Al-Zoned ZSM-5 Crystals[END_REF][START_REF] Mei | Regular HZSM-5 microboxes prepared via a mild alkaline treatment[END_REF][START_REF] Danilina | Where Are the Active Sites in Zeolites? Origin of Aluminum Zoning in ZSM-5[END_REF]. The NH4F did not show any sensibility to the framework ratio, including in the case of very low silica zeolites [START_REF] Qin | Opening the Cages of Faujasite-Type Zeolite[END_REF][START_REF] Valtchev | Method for the preparation of synthetic crystalline zeolite materials with enhanced total pore volume[END_REF]. Indeed the ZF sample shows a Si/Al ratio very similar to its parent (Table 1).

For this reason, we consider that the exceptional resistance of the periferic parts of the crystals to the NH4F etching (Figure 3a, Figure S4, S5) is an important observation. In order to get a deeper insight into this peculiar "selected-area" dissolution behavior, a comparative study was conducted by combined use of high and low voltage SEM. The electron beam penetrates deep into the sample at high accelerating voltage. Accordingly, the signal will carry information from deeper layers of the sample. In contrast, low voltage high resolution SEM provides only the surface information thanks to the much lower landing voltage [START_REF] Suga | Recent progress in scanning electron microscopy for the characterization of fine structural details of nano materials[END_REF]. Therefore, as the intensity of the generated secondary electrons varies depending on the magnitude of the accelerating voltage, variations in the roughness of specimen surface and the density of the substance can be detected.

As can be seen in Figure S6, the crystal surface of ZP shows a uniform contrast under both high and low voltage observation models. The 5 min NH4F-treated sample also shows a uniform contrast under the low voltage observation model (Figure S7a). In the case of the high voltage model, however, obvious dark areas occur on the b crystal face of the NH4F-treated sample (Figure S7b). This contrast difference is not related to crystal surface etching (i.e. surface roughness), since only a few seperated holes can be observed on the b crystal face (Figure S8e).

This crystal surface still shows a similar smouth morphology as the untreated sample, with the growth steps clearly distinguishable (Figure S8b,e). In addition, the difference in surface contrast is not an individual but a general phenomenon, as a similar inhomegeneous distribution of the phase contrast was also observed in case of the 10 and 20 min NH4F-treated samples (Figure S9). The surface contrast is not so different in the case of the longer time (i.e. 30 and 60 min) treated samples (Figure S10). In the later case the original crystal surface are either deeply etched, or detached as a result of the substantial dissolution of the inner part of zeolite crystals (Figure S11).

Based on these intensive SEM observations, we attribute the difference in surface contrast to the difference in density in different parts of zeolite crystals. Namely, the dissolution of zeolite crystals in NH4F solution is spacially inhomogeneous in the macro sense. In general, the inner part of zeolite crystals are preferentially dissolved. The gravimetric measurement of the weight loss of zeolite with the extension of NH4F etching was also conducted (Figure S12). The continuous decrease of the slop of the weight loss curve shows unambiguously that the dissolution of the ZP crystals is relatively faster at the initial stage and the dissolution rate slows down with increasing etching time (Figure S12). We further attribute the faster dissolution of the inner part to the fast growth rate at the early stage of crystal growth, and the slower dissolution of the crystal periphery to the slow growth rate at the end of the crystallization process where a few defects are formed. Thus the defect-poor parts of the crystals are resistant to the etching.

This result is additional evidence showing that the dissolution starts preferentially from framework defects when unbiased etching solution is used.

The relation between the crystallographic structure of the zeolite crystals and the etching direction shown in Figure S4 is confirmed by the high-resolution analysis of some similar grains.

Figure S13 shows a typical high-resolution TEM image and the corresponding FFT pattern which illustrate that the preferentially etched facets are on {010} face, as revealed also by the low-magnification TEM images of the various analyzed zeolite crystals. It should be underlined that the empty cages resulting from the crystal dissolution are orianted along the crystallographic axes. This is a proof for the connection between the growing process and dissolution behaviour of the crystals. The high-resolution image shown in Figure S14 also sustains this important finding.

We attribute both the spacially inhomegeneous dissolution and the facet-dependent etching behavior to the growth mechanism. The fluoride etching is sensitive solely to the zones of structural stress and deffect concentartion (Figure S8d) which are more vulnerable to chemical attack [START_REF] Qin | The Mosaic Structure of Zeolite Crystals[END_REF][START_REF] Wang | Hollow TS-1 crystals formed via a dissolution-recrystallization process[END_REF][START_REF] Qin | Chemical Equilibrium Controlled Etching of MFI-Type Zeolite and Its Influence on Zeolite Structure, Acidity, and Catalytic Activity[END_REF]. Thus the dissolution reflects the particularities of the growth process. Layer type growth is characteristic of most of the zeolitic materials [START_REF] Anderson | Growth models in microporous materials[END_REF][START_REF] Meza | Controlling Relative Fundamental Crystal Growth Rates in Silicalite: AFM Observation[END_REF][START_REF] Lupulescu | In Situ Imaging of Silicalite-1 Surface Growth Reveals the Mechanism of Crystallization[END_REF][START_REF] De Yoreo | Crystallization by particle attachment in synthetic, biogenic, and geologic environments[END_REF]. The detailed mechanism of MFI growth was revealed recently by in situ AFM microscopy [START_REF] Lupulescu | In Situ Imaging of Silicalite-1 Surface Growth Reveals the Mechanism of Crystallization[END_REF]. It was observed that the MFI growth involves two simultaneous phenomenon -oriented attachment of nanoparticle and mono-or low-weight silica species. The nanodomains integrated into the growing crystal contain well defined grain boundaries and thus are rapidly disolved by NH4F etching (Figure S8c, f), leaving behind rectangular cages [START_REF] Qin | The Mosaic Structure of Zeolite Crystals[END_REF]. These rectangular cages are surrounded by crystalline material, which is a result of the uniform growth by means of low weight silica species. The rate of dissolution of this more stable and uniform part of the crystal is lower (Figure S12). Judging by the straight and flat crystalline walls surrounding the cages the dissolution can be described as "layer-by-layer" process. The results of the present study show that the mechanism of dissolution does not change with the time of treatment and thus a house-of-cards-like material can be synthesized. A fundamental feature of this material is that each "house-of-cards" is a single crystal thanks to the preferential removal of defect-rich zones (Figure 3c). Electron diffraction is a very appropriate technique for the assessment of the crystallinity of various types of specimens at the nanometer level. As shown in Figure S15, the ZSM-5 grains give rise to a unique set of well-defined diffraction spots, allowing us to assign to these crystals a single crystalline structure. In contrast to the interpenetrated "house-of-cards" with myriad intergrown plane prepared by a bottom-up approach, the single crystal house-of-cards-like material reported here offers highly crystalline with a few frameworks defects left.

In order to shed light in the dissolution process and the general applicability of the method, we have employed a series of industrial ZSM-5 sample (see Supporting information). These samples exhibit different morphology and particle size (Figure S16), Si/Al ratio (Table S1) and level of aggregation. The samples were NH4F etched under similar conditions as the time of treatment was varied between 5 and 50 min. The product yield ploted against the etching time is presented in Figure S12. As can be seen, the dissolution rate depends on the crystal size, the larger the crystal size, the lower the dissolurion rate is. We relate this result with the accomplishement of the growth process. As discussed, the well shaped crystals with terminated crystal faces show higher resistnace to the NH4F etching. On the other side, all the trend lines show an initially faster dissolution rate and then converge, with an apparently lower dissolution rate, to a similar product yield value under the applied experimental conditions. This result shows that no matter of basic zeolite crystal characteristics, the mechanism of dissolution is identical and the final product is similar (Figure 3, Figure S17).

The silicon selective alkaline etching also leads to a hierarchical zeolite, ZB (Figure 4a,b). It is a highly porous material with large variation of mesopore size and pore distribution [START_REF] Lakiss | On the remarkable resistance to coke formation of nanometer-sized and hierarchical MFI zeolites during ethanol to hydrocarbons transformation[END_REF][START_REF] Groen | Creation of Hollow Zeolite Architectures by Controlled Desilication of Al-Zoned ZSM-5 Crystals[END_REF].

Similarly to NH4F etching, the crystal morphology is retained (Figure 4a). However, a closer look reveals a grain-like morphology (Figure 4b), the result of preferential Si extraction and redeposition of most of the extracted Al on the etched surface. Such a biased Si etching changes the zeolite framework composition and acidity (Table 1).

ZNS displays a flower-like morphology composed of interpenetrating plate-like nanosheets (Figure 4c,d). The overall thickness of the lamellar stacking of nanosheets is 20-60 nm (Figure 4e). TEM reveals that the stacking "plate" is composed of alternating 2 nm-thick aluminosilicate nanosheets and 2.8 nm micellar layers (Figure 4f). This well-defined mesoporosity is inherently related to the properties of the structure-directing agent used.

IR spectroscopy

Chemical analysis of ZP and ZNS indicates Si/Al ratios of 21 and 37, respectively. A negligible change in the Si/Al ratio after NH4F etching is observed, while caustic etching produces a material with a Si/Al ratio of 9 (Table 1). These results are expected for unbiased (NH4F) and biased (NaOH) etchings, respectively [START_REF] Qin | Mesoporous zeolites by fluoride etching[END_REF]. IR spectroscopy of adsorbed pyridine highlights further these conclusions as ZB has a lower concentration of Brønsted acid sites and a substantial increase of Lewis acid sites compared to its parent ZP and its NH4F etched derivative ZF (Table 1). ZF Brønsted acidity does not change significantly, as excepted. ZNS has a lower Brønsted acid sites concentration, due to a higher Si/Al ratio and the presence of numerous silanols, vide-infra [START_REF] De Yoreo | Crystallization by particle attachment in synthetic, biogenic, and geologic environments[END_REF]. The pristine surface of all samples can also be probed by FTIR spectroscopy, namely their ν(OH) region where isolated silanols (3747 cm -1 ) located on the external surface and bridged hydroxyls, i.e. Brønsted acid sites (3613 cm -1 ) are clearly observed (Figure 5). ZP shows such bands and a broad absorption extending from 3500 to 3000 cm -1 (Figure 5a), ascribed to Hbonded internal silanol nests [START_REF] Lønstad Bleken | Probing the surface of nanosheet H-ZSM-5 with FTIR spectroscopy[END_REF]. The ZF spectra does not show such a broad band, and the baseline is almost flat, indicating that a substantial part of the silanols is eliminated during NH4F etching. Again, this is the result of a preferential dissolution of highly defective zones in the crystals, a typical characteristic of NH4F etching [START_REF] Qin | The Mosaic Structure of Zeolite Crystals[END_REF][START_REF] Qin | Chemical Equilibrium Controlled Etching of MFI-Type Zeolite and Its Influence on Zeolite Structure, Acidity, and Catalytic Activity[END_REF]. In ZF, the intensity of the 3747 cm -1 band increases compared to its parent ZP, indicative of an increased external surface area (Table 1). For ZB, the intensity of the 3747 cm -1 band further increases while 3613 cm -1 band decreases noticeably. The IR spectra of ZNS is dominated by external surface silanols at 3743 cm -1 (Figure 5a) while a very small amount of Brønsted acid sites is detected (Figure 5b). The above results indicate that a ZSM-5 house-of-cards-like architecture, ZF, can be prepared by a top-down unbiased NH4F etching of ZSM-5. Single zeolite crystals are converted in a mosaic of connected rectangular cavities separated by nanometer thick crystalline walls. Such walls are thicker than the house-of-cards obtained by a bottom-up approach, ZNS (Figure 3e-g and Figure 4e, f). However, the aspect ratios between the wall thickness and mesopore space of both ZF and ZNS are similar. As mentioned, the formation of rectangular cavities is a consequence of the unbiased dissolution of highly defect zones and small crystalline domains mostly presented in zeolite crystals. Thus, an essential advantage of the top-down approach is that the "house" is a single crystal almost free of defect zones. A decrease of Lewis acid sites is observed, again a consequence of the preferential dissolution of defective portions of the crystals (Table 1) while the number of Brønsted acid sites is almost unchanged. The resulting house-ofcards-like material comprises relatively large meso-and macro-pores with a flat surface. Another essential feature of the house-of-cards-like material is the moderate increase in external surface area and retention of micropore volume compared to the caustic leached ZSM-5. Nanosheet-like ZSM-5 also shows relatively low micropore volume. ZF differs substantially from the caustic leached zeolite produced from the same parent (ZB) and the nanosheet ZSM-5 (ZNS). In contrast, the preferential extraction of Si and redeposition of Al in the case of ZB results in a considerable increase of the external surface area at the expense of the micropore volume, dramatic changes in the chemical composition and the acidic properties of the zeolite (Table 1). Nanosheet ZSM-5 also exhibits a huge external surface area and reduced micropore volume. It should be noted the very low Brønsted acidity of this material (Table 1) and the vast presence of surface silanols in this material (Figure 5). This series of catalysts with well distinct physicochemical properties offers the possibility a structure-property relationship to be established.

Ethanol-to-hydrocarbons (ETH) conversion on ZB, ZF, and ZNS zeolite catalysts

ETH, like the methanol-to-hydrocarbons (MTH) process, is a key step in the transformation of biomass, coal, natural gas, heavy oils into olefins (major building blocks in petrochemistry) and higher hydrocarbons (gasoline fuel) [START_REF] Derouane | Elucidation of the mechanism of conversion of methanol and ethanol to hydrocarbons on a new type of synthetic zeolite[END_REF]. The performance of all these four zeolites is evaluated in this reaction as already discussed previously [START_REF] Lakiss | On the remarkable resistance to coke formation of nanometer-sized and hierarchical MFI zeolites during ethanol to hydrocarbons transformation[END_REF]. Ethanol is converted into water and light olefins as well as heavier hydrocarbons (3 to 18 carbon atoms). Small amounts of methane and ethane are detected but no CO and CO2. The yields are presented as molar % in the organic fraction (Figures 6 and7). While ethanol dehydration can take place on weak acid sites, oligomerization, cyclization and hydrogen transfer reactions require strong acid sites as found in zeolites. On all our catalysts, ethanol conversion is initially complete and decreases slowly with time-on-stream (Figure S18a). Therefore, the catalyst deactivation is related to the time evolution of the C3+ yield. On the parent zeolite (Zp), the initial yield of C3+ is only 35 % (extrapolated value at zero conversion) and decreases rapidly becoming negligible after only 20 h (Figure 6). On ZB, the initial C3+ yield is slightly less than 100% (Figure 6), but the catalyst deactivates rapidly and yields almost no C3+ after 90 h on stream. On ZNS, the initial C3+ yield is lower than ZB but quite high considering its much lower Brønsted acid site concentration. However, its deactivation rate is much lower than ZB: after 90 h on stream, the C3+ yield decreases to ca. 20 mol.% while it was almost zero on ZB. The direct synthesis of a hierarchical zeolite using a complex surfactant is an efficient, albeit expensive, way of minimizing deactivation. ZF, although not the catalyst with the best initial C3+ yield retains it much longer as it deactivates much slower than ZB and ZNS (Figure 6); after 90 h of TOS, it still produces a substantial C3+ yield, ca. 60 mol.%.

The unbiased NH4F etching of a micrometer-sized ZSM-5 zeolite leads to a diffusion path length similar to that of nanosized one. Indeed, the stability of ZF is similar, or even higher for longer reaction time, to that on a commercial zeolite with a crystal size of 45 nm [START_REF] Lakiss | On the remarkable resistance to coke formation of nanometer-sized and hierarchical MFI zeolites during ethanol to hydrocarbons transformation[END_REF]. See also Figure S19 in the present work. The superior stability of ZF on a nanoscale zeolite can be related to both a high intracrystalline mesoporous volume (0.31 cm 3 .g -1 ) and quasi-perfect connectivity with the microporous network, a consequence of single crystal nature of house-of-cards-like material. As ZF is devoid of defects, contrariwise on ZNS, the immobilization and growth of coke precursors are mitigated. Therefore the catalyst is more stable. On the other hand, as the caustic leaching divides by only four the diffusion path length as shown by the desorption experiments [START_REF] Meunier | Influence of crystal size and probe molecule on diffusion in hierarchical ZSM-5 zeolites prepared by desilication[END_REF], the gain in stability is rather limited.

The molar yields of the paraffins (P), olefins (O), naphthenes (N), and aromatics (A), as well as the paraffins to aromatics molar ratio (P/A), are reported as a function of the C3+ yield and time-on-stream in Figure 7 and Figure S18b-f, respectively. As far as product distribution is concerned ZP, ZNS and ZP give the same product selectivity for the same C3+ yield, meaning that the reactions involved are identical, even during the deactivation and regardless of the acid properties of the three catalysts. It is worth to note that a difference exists only on ZB with a yield in O3+. Yields in N and O3+ pass through a high yielding maximum in C3+, while those in P and A increase exponentially. Regardless of C3+ yield the paraffins to aromatics molar ratio (P/A) is ca. 3 (Figure 7e), indicative of hydrogen transfer between naphthene and olefins (Scheme 1). The initial low value of P/A ratio (open symbol in figure 7e) is due to coke formation.

Scheme 1.

The hydrogen transfer reaction between naphthene and olefins.

In sharp contrast, ZF shows considerably higher aromatic and naphthene yields than ZB and ZNS, while its paraffin yield is low (Figure 7a,c,d). Apparently, hydrogen transfer reactions are substantially suppressed in this catalyst. Its P/A is lower than 0.5 (Figure 7e), implying that on the NH4F treated zeolite, aromatics are formed by a monomolecular protolytic dehydrogenation.

Lercher and coworkers [START_REF] Feller | On the mechanism of catalyzed isobutane/butene alkylation by zeolites[END_REF] have shown that a high ratio of Brønsted acid sites to Lewis acid sites (BAS/LAS) minimizes the alkenes concentration and causes high hydride transfer (HT)

activity. Yet on ZF catalyst, HT is limited although the house-of-cards-like catalyst possesses the highest BAS/LAS ratio. Lercher et al. [START_REF] Narbeshuber | Dehydrogenation of Light Alkanes over Zeolites[END_REF] also demonstrated that the rate determining step for dehydrogenation activity of alkane on H-ZSM-5 is represented by olefin desorption from the catalyst surface which is promoted by a short diffusion path length. It is worth mentioning that the products selectivity of ZF is very close to nanometer-sized ZSM-5 zeolite (Figure S20), confirming that the diffusion path length is limited to a few nanometers on both zeolites. The coke content, the evolution of the number of acid sites (Brønsted and Lewis) and pore volumes accessible to nitrogen after more than 48 h of reaction are gathered in Table 2. The coke content is around 13 wt % on ZP after 48 h of reaction. NH4F etching has no impact on the coke level, while on the alkaline leached ZB catalyst, coke grows continuously to reach a level almost twice as high as its parent. On ZNS, although its Brønsted acid site concentration is 4 times lower than on ZP, coke content is quite similar. The residual acidity (Brønsted and Lewis), after deactivation, is very low for all samples. An important decrease of micropore volume occurs on all zeolites. On ZNS which is the least acidic catalyst, the loss of microporosity is only 25%. The coked ZF and ZNS catalysts, despite a near total loss of acidity, keep converting ethanol to C3+ hydrocarbons (60 %, and 20 %, respectively, (Figure 6)); ethanol transformation occurring probably by pore mouth catalysis on hybrid active sites (immobilized carbocations) by a carbon pool mechanism [START_REF] Lakiss | On the remarkable resistance to coke formation of nanometer-sized and hierarchical MFI zeolites during ethanol to hydrocarbons transformation[END_REF]. Differences in the activity and stability between ZP, ZB, ZF, and ZNS are related to changes in the carbon pool composition. The composition of the coke trapped in the zeolite micropores, i.e., internal coke, is determined by mineralizing the zeolite and subsequently analyzing the organic phase after a liquid-liquid extraction with CH2Cl2 [START_REF] Magnoux | New Technique for the Characterization of Carbonaceous Compounds Responsible for Zeolite Deactivation[END_REF]. Coke molecules can be lumped in four families according to their number of aromatic rings: alkylbenzenes (I), alkyl naphthalenes (II), alkyl-phenanthrenes or -anthracenes (III) and alkylpyrenes (IV). The sizes of all compounds trapped in the micropores vary between the dimensions of the pore openings (0.55 nm) and channel intersections (~1 nm) of H-ZSM-5. The coke on ZP and ZB contain representatives of all families. A simpler and lighter coke distribution is found on ZF and ZNS, where families I and III dominate (Figure S21); such a coke composition is similar to that reported elsewhere on nanosized zeolite [START_REF] Lakiss | On the remarkable resistance to coke formation of nanometer-sized and hierarchical MFI zeolites during ethanol to hydrocarbons transformation[END_REF]. The average molar mass of coke trapped in the micropores, calculated from these compositions, is 200 g mol -1 for ZP and ZB, and 180 g mol -1 for ZF and ZNS.

Taking this average molar mass of 200 g mol -1 for the coke molecules trapped inside the micropores (ca. 13 wt % on the ZP catalysts), its concentration is ca. 645 mol g -1 , corresponding to the amount of Brønsted acid sites on the fresh zeolite. Coking is therefore mainly a site poisoning effect, further confirmed by a complete disappearance of the bridged OH groups (Figure S22). Assuming that on all hierarchical zeolites, one coke molecule poisons only one protonic site, the coke trapped within the micropores and that located in the mesopores can be estimated. The results are reported in the bar chart on Figure 8. The most significant accumulations of coke on the mesopores are for the caustic leached micron-sized zeolite (ZB) and the nanosheet zeolite (ZNS), reaching up to 55% and ca. 75% of the total coke, on the other hand, it is limited to 16% on the catalyst free of defect (ZF) and as expected, almost zero on the parent zeolite. The accumulation of coke in mesopores is likely related to the quality of the crystals. The increased number of silanols and Lewis acid sites on ZB and ZNS are prone to trap desorbed products [START_REF] Barbera | Structure-deactivation relationship for ZSM-5 catalysts governed by framework defects[END_REF][START_REF] Thibault-Starzyk | 2D-COS IR study of coking in xylene isomerisation on H-MFI zeolite[END_REF][START_REF] Lee | Cooperative effects of zeolite mesoporosity and defect sites on the amount and location of coke formation and its consequence in deactivation[END_REF] as confirmed by the disappearance of silanol groups on spent catalysts (Figure S22). Taking a coke density of 1.1 g cm -3 , the volume of coke in the micropores corresponds to the loss of micropore volume measured by nitrogen adsorption (Figure 8). These calculated values are very close to the experimental data, highlighting the relevance of this procedure to discriminate between internal and external coke contents. In addition, the coke light-off performances of the spent catalysts are compared in Table 2 where temperatures required to combust 50% (T50) and 90% (T90) of the coke are reported. T50

and T90 are both lower on ZB than on ZP (-35 °C and -25 °C respectively ) and much lower on ZNS and ZF (-55 °C). The differences are related to a higher accessibility (difusion) of oxygen to the coke molecules trapped in the micropores. The house-of-cards-like zeolite, already the best catalyst (cumulative performance due to its time stability) is as good as a nanosheet one for regeneration.

The ETH reaction illustrates the superior stability and selectivity of ZF, a house-of-cards-like ZSM-5 catalyst, although other hierarchical zeolites, ZB and ZNS possess higher external surface area and mesopore volume. The nano-sheet zeolite ZNS with a higher Si/Al ratio of 37 and lower acid site density displays a remarkably high initial C3 + yield and was once expected to be a superior candidate for ETH reactions [START_REF] Madeira | Ethanol transformation into hydrocarbons on ZSM-5 zeolites: Influence of Si/Al ratio on catalytic performances and deactivation rate. Study of the radical species role[END_REF] but lacks stability as shown in this study. The ZF catalyst outperforms both ZB and ZNS in the present study, but the ZNS hierarchization procedure brings superior performances than a micron-sized zeolite, ZP (Figure 6). Zeolite acidity and mesoporosity cannot alone fully explain the differences in catalytic performances discussed above. Based on our detailed comparison of the hierarchical zeolites, we propose that, in addition to mesopore size, shape, uptake capacity and connectivity, the quality of the hierarchical zeolites (micropore accessibility, absence of defects [silanol nests], Lewis acidity) also play important roles in the ETH reaction. The higher catalytic performances of NH4F leached zeolites could also be related to their ability to dehydrogenate naphthenic compounds (Figure 7); this monomolecular reaction requires less space than the bimolecular hydrogen transfer occurring on the caustic leached zeolite and nano-sheet zeolite. The absence of defects combined with a small diffusion pathlength inside the microporosity by retarding the transformation of the desired active carbon pool to unwanted coke, slows catalyst deactivation. The presence of numerous silanol groups, on the other hand, traps coke precursors on the surface, promoting a rapid catalyst deactivation by a fast accumulation of aromatic compounds. Indeed, as coke formation is a genuine shape selective reaction, the availability of more space favors the production of larger molecules, coke, i.e. an inactive carbon pool. These results highlight that, in addition to the loosely defined "mesopore quality" [START_REF] Milina | Mesopore quality determines the lifetime of hierarchically structured zeolite catalysts[END_REF], the surface quality of the remaining microporous crystal domains determines the catalytic performance of hierarchically structured zeolite crystals.

Conclusion

Single house-of-cards-like ZSM-5 crystals were prepared by a top-down approach using a novel unbiased chemical etching with an NH4F solution. Such an etching extracts Si and Al at similar rates and preferentially removes less-stable defect zones in the crystals. The process is therefore controlled by the number of intergrowths, the structural stress and the nanocrystalline domains with well-defined grain boundaries, i.e. the history of the zeolite synthesis. Such a unique dissolution behavior is inherently related to the growth process where simultaneous monomer-by-monomer addition and oriented attachment take place [START_REF] Meza | Controlling Relative Fundamental Crystal Growth Rates in Silicalite: AFM Observation[END_REF]. The unbiased NH4F dissolution leads to the preferential extraction of misoriented and more vulnerable to chemical attack nanocrystalline domain followed by progressive etching of the remaining part of the crystals, which is more resistant due to the limited number of structural defects. This dissolution proceeds by a layer-by-layer mechanism following the rectangular features of previously extracted nanodomains, thus the well shaped cavities grown in size and connect each other providing a house-of-cards-like morphology.

Such a hierarchical material is analogous to a collection of nanosized high quality crystals with the same framework composition and Brønsted acidity of their parent. The high accessibility and short diffusion pathlength offered by the house-of-cards-like morphology is therefore combined with the intact native microporosity and acidity of their parent zeolite and does not require expensive and one-off structure directing agents. The optimization of structural and chemical parameters results in an excellent catalyst easier to regenerate, as demonstrated in the challenging (deactivation prone) ethanol-to-hydrocarbon reaction. Indeed, regenerating deactivated catalysts by controlled coke combustion often leads to, often overlooked, irreversible changes due to the operating conditions (such an exothermic reaction produces steam near the zeolite active sites and can lead to structural and chemical damages such as dealumination) of this step. The performance of such single crystals house-of-cards-like catalysts is so far second to none as it maintains its performance over longer periods of time and appears easier to regenerate, which even outperforms the nanometer-sized zeolites (Figure S19). Noting that we have previously concluded, based on the samples studied, that the reduction of crystal size is the most efficient way to increase the performance of zeolites for applications such as alcohols to hydrocarbons, and hence nano-zeolites are preferred [START_REF] Lakiss | On the remarkable resistance to coke formation of nanometer-sized and hierarchical MFI zeolites during ethanol to hydrocarbons transformation[END_REF].

This study highlights that hierarchical zeolitic catalysts with superior performances can be designed not only by promoting high accessibility and short diffusion to and from their active sites, but also by retaining the crystallinity and acidity of their parents while keeping a minimum level of defects. Since the presence of crystalline domains with well-defined grain boundaries is a ubiquitous in zeolite materials, the preparation of single crystal house-of cards can be extended to other zeolite types. Further insights in the hierarchization process of zeolites, either bottom-up or top-down (this work) hold much potential to design ever bettter zeolite catalysts for current and forthcoming applications [START_REF] Ennaert | Potential and Challenges of Zeolite Chemistry in the Catalytic Conversion of Biomass[END_REF].

Supporting Information. Experimental details, the nitrogen physisorption isotherms, the combined 3D models of zeolite body and pores, the additional SEM and TEM images and a graphical representation of the house-of-cards-like architecture, the additional data of the Ethanol-to-Hydrocarbons reaction, coke composition by GC analysis, and the IR spectra of the coked zeolite samples.

Corresponding Author *valentin.valtchev@ensicaen.fr 1 2

Figure 2 .

 2 Figure 2. BJH pore size distribution of the parent zeolite (ZP), its NH4F (ZF) and caustic (ZB)

Figure 3 .

 3 Figure 3. Low (a) and high (b) resolution SEM images of ZF sample. Representative low (c) and

Figure 4 .

 4 Figure 4. Low (a) and high (b) magnification of SEM micrographs of caustic etched ZSM-5

Figure 5 .

 5 Figure 5. IR spectra of ZP, ZF, ZB, and ZNS in the 2600-3800 cm -1 ν(OH) region (a) and zoom on the ν(OH) (3550-3770 cm -1 ) region (b).

Figure 6 .

 6 Figure 6. Molar yields of C3+ hydrocarbons as a function of time-on-stream during the

Figure 7 .

 7 Figure 7. Ethanol transformation at 623 K and 3.0 MPa on ZP, ZF, ZB and ZNS zeolite: (a) molar

Figure 8 .

 8 Figure 8. Estimation of the coke distribution between micropores and mesopores on ZP, ZF, ZB

Table 1 .

 1 Physicochemical characteristics of the series of ZSM-5 samples. BET surface area. c t-plot. d Vmeso = Vtotal -Vmic (Vtotal: the volume absorbed at p/p 0 = 0.99).

			SBET b	Sext c	Vmic c	Vmeso d	BPy e	LPy e
	Samples	Si/Al a	m 2 g -1	m 2 g -1 cm 3 g -1 cm 3 g -1 µmol g -1 µmol g -1
	ZP	21	377	9	0.18	0.02	617	91
	ZF	22	395	54	0.17	0.31	582	51
	ZB	9	494	206	0.13	0.36	495	282
	ZNS	37	504	266	0.11	0.40	162	98

a ICP. b e The Brønsted (BPy) and Lewis (LPy) acidity were determined by IR analysis of pyridine sorption.

Table 2 :

 2 Coke content, T50 and T90 (temperatures required to remove, by combustion, 50% and 90% of coke respectively), pore volumes and acidity of ZP, ZF, ZB and ZNS zeolite spent catalysts.

		Time	Coke	T50	T90	Vmic a	Vmeso b	BPy c	LPy c
	Samples
		h	wt%	°C	°C	cm 3 g -1 cm 3 g -1 µmol.g -1 µmol g -1
	ZP	48	12.9	615	705	0.01	0.02	4	5
	ZF	90	12.5	560	670	0.05	0.13	1	10
	ZB	110	20	580	680	0.03	0.36	2	14
	ZNS	90	10.5	560	665	0.08	0.17	0	14
	a t-plot. b Vtotal -Vmic (Vtotal: the volume absorbed at p/p 0 = 0.99). c The Brønsted acidity (BPy) and
	Lewis acidity (LPy) determined by pyridine sorption.

ACKNOWLEDGMENT

Zhengxing Qin acknowledges funding support from NSFC 21706285, China University of Petroleum (East China) starting funding, and the Fundamental Research Funds for the Central Universities (18CX02013A). ZQ, JPG and VV acknowledge funding from the French-Sino International Laboratory (LIA) "Zeolites".


Riham Gharib

Jouda Mediouni

Ben Jemâa

Catherine Charcosset

Sophie Fourmentin

Professor, Hélène Greige-Gerges
email: greigegeorges@yahoo.com

Retention of eucalyptol, a natural volatile insecticide, in delivery systems based on hydroxypropyl-ß-cyclodextrin and liposomes

Keywords: conventional liposomes, drug-in-cyclodextrin-in-liposomes, eucalyptol, freeze-drying, hydroxypropyl-ß-cyclodextrin conventional liposome

CL); cyclodextrin (CD); 1,6-diphenylhexatriene (DPH); encapsulation efficiency (EE); eucalyptol (Euc); drug-in-cyclodextrin-inliposome

Introduction

Eucalyptol (Euc) or 1,8-cineole, is a natural monoterpene. It is a major compound of many plant essential oils and is mainly extracted from Eucalyptus globulus and Rosmarinus officinalis essential oils [1] . Batish et al. [2] pointed out that 1,8-cineole supports the pesticidal activity of Eucalyptus oils. Similarly, Scriven and Meloan [3] reported that cineole act as a natural repellent against Periplaneta americana. Moreover, Corbet et al., [4] proved that cineole is a mosquito larvicide. Additionally, Euc is also used as a fumigant agent against insect-pest of stored grains [5] . Considering the repellent index, it was reported that Eucalyptos kruseana oil showed the highest repellency as compared to other essential oils [6] . In human, Euc has been used to treat lung inflammation and respiratory diseases and is found as active principle in a commercial medicinal product "Soledum ® Kapseln" contained pure isolated monoterpene 1,8-cineol [1] , and it has been demonstrated that Euc showed neuroprotective effects in an ischemic stroke model [7] .

To our knowledge there is no data concerning the stability study of eucalyptol under light and at various temperatures. The product is recommended to be stored at 4ºC and in a dark bottle. Generally, chemical reactions are accelerated with increasing heat. Some essential oil components including eucalyptol are known to easily convert into each other by oxidation, isomerization, cyclization, or dehydrogenation reactions, triggered either enzymatically or chemically [8] . The chemical oxidation of Euc has been carried out by strong oxidizing reagents to give oxo compounds (3-oxo-1,8-cineole, 5-oxo-1,8-cineole and 3,5-dioxo-1,8-cineole) [9] . The sites of oxidation are the carbon 3 and/or 5. The encapsulation of Euc can be considered as a potential solution to overcome the drawbacks related to its physico-chemical properties.

Based on the drug-in-cyclodextrin-in-liposomes (DCL) delivery system proposed by

McCormack & Gregoriadis [10] , we developed many formulations loading essential oil compounds to protect them from degradation, to achieve their controlled release, and to facilitate their handling through preparation in freeze-dried forms [11,12,13] . The DCL system, combining the relative advantages of both carriers, improved the encapsulation of many bioactive compounds such as trans-anethole [11] , eugenol [14], estragole [13] , quercetin [15] , nerolidol [16] , and reduced their release compared to conventional liposomes [11, 13] .

Cyclodextrins (CDs) are cyclic oligosaccharides consisted of glucopyranose units. They are non-hygroscopic substances, homogeneous and crystalline [17] . CDs have a form of a truncated cone with an internal hydrophobic cavity and an external hydrophilic surface. This particular structure confers to CDs the ability to form inclusion complexes with a large number of hydrophobic guest molecules [18] . Among numerous CDs, hydroxypropyl-ß-cyclodextrin (HP-β-CD) was shown to be very effective for Euc encapsulation [18] . Liposomes are microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane. They are usually made of natural, biodegradable, non-toxic and nonimmunogenic lipid molecules [19] . In aqueous media, the phospholipids can slowly become oxidized or hydrolyzed [20] and this could induce liposomes membrane destabilization and leakage of the encapsulated drug. Freeze-drying liposomes increased their shelf-life [21] ; however during freeze-drying the disruption of liposome membrane may occur leading to vesicles aggregation [20] ; for that cryoprotecting agents such as HP-ß-CD are generally used to stabilize liposome structures during freeze-drying [12] .

In this study, ethanol injection method was applied using hydrogenated soybean phospholipid and cholesterol to prepare conventional liposomes (CL) and DCL. These liposomes were characterized for their size, polydispersity index, zeta potential and morphology before and after freeze-drying. To obtain reproducible and efficient insecticidal effects, the homogeneity and the stability of liposome formulations should be ensured. For that, the particle size distribution value of the batches was determined. Zeta potential is a parameter used to evaluate the physical stability of liposomal formulations.

It characterizes the particles surface charge and gives an indication about repulsive forces between particles, thus allowing predicting stability of colloidal dispersions [22] . The loading rate of Euc was determined after its HPLC analysis. The release of Euc from the inclusion complex, CL and DCL was studied by multiple headspace extraction at 60ºC. Hence, the fluorescence anisotropy of 1,6-diphenylhexatriene (DPH) inserted in the lipid membranes of CLs and DCLs was studied before and after freeze-drying at 25 and 37°C.

Materials and methods

Materials

Hydrogenated phospholipon 90H (90% soybean phosphatidylcholine, 4% lysophosphatidylcholine, 2% triglycerides, 2% water, 0.5% ethanol, 1% iodine) was supplied by Lipoid GmbH (Ludwigshafen, Germany). HP-ß-CD-oral grade (MS=0.85) was obtained from Roquette (Lestrem, France), eugenol, absolute ethanol, cholesterol and methanol-HPLC grade were from Sigma-Aldrich (Germany) and Euc was purchased from Sigma-Aldrich (USA). Water was purified on a Milli-Q system obtained from a Millipore® synergy system (Millipore, Billerica, Massachusetts, USA).

Preparation of HP-ß-CD/Euc inclusion complex

Excess amount of Euc (7.712 mg) was added to 5 mL HP-ß-CD solutions (0, 10, 25, 50, and 100 mM). The mixtures were shaken at 25 °C for 24 h then filtered through a 0.45 μm membrane filter. The HPLC method described below was used to determine the concentrations of Euc in the filtrates.

The encapsulation efficiency was calculated using the following equation:

where m Euc exp is the mass of Euc experimentally determined by HPLC in the inclusion complex solution and m Euc i is the mass of Euc initially used to prepare the inclusion complex.

Preparation of HP-ß-CD/Euc inclusion complex for DCL preparations

HP-ß-CD (50 mM, 74 mg/mL) was dissolved in ultrapure water and the required amount of Euc was added to obtain HP-ß-CD:Euc molar ratio of 5:1. HP-ß-CD/Euc inclusion complex was prepared as described above and the solution was used for DCL preparations.

Preparation of liposomes by ethanol injection method

The liposomes were prepared by the ethanol injection method using Phospholipon 90H (10 mg/mL) and cholesterol (5 mg/mL) according to the protocol described previously [11] . Different formulations were prepared: 1) blank-CL; 2) blank-DCL, where HP-ß-CD was dissolved in the aqueous phase at a concentration of 50 mM; 3) Euc-loaded liposomes (Euc-CL), where Euc was added in the organic phase at a concentration of 2.5 mg/mL; 4) HP-ß-CD/Euc inclusion complex-loaded liposomes (Euc-DCL), where HP-ß-CD/Euc inclusion complex obtained at molar ratio of 5:1 was used as an aqueous phase.

Each batch was prepared in triplicate and underwent characterization as described below.

Freeze-drying

Freshly prepared liposomes (5 mL) were freeze-dried according to the protocol described in our previous study [12] , where HP-ß-CD was used as a cryoprotector agent at concentrations of 25 and 50 mM for CL and 50 mM for DCL formulations. The obtained powders were then reconstituted with ultra-pure water to the original volume (5 mL) prior to characterization and further analysis.

HP-ß-CD/Euc inclusion complex was also freeze-dried and studied for its ability to retain Euc as described below.

Liposome characterization

Dynamic Light Scattering Analysis

Malvern Zetasizer Nanoseries (Zetasizer Nano ZS; Malvern Instruments Ltd, France) was used to determine the mean size, polydispersity index and zeta potential values of liposome batches [12] .

Morphological characterization by transmission electron microscopy

Blank-DCL and Euc-DCL before and after freeze-drying were imaged with transmission electron microscope (TEM) (CM 120; Philips, Eindhoven, Netherlands) according to the protocol described by Gharib et al [11] .

Determination of encapsulation efficiency and loading rate of Euc

The EE of Euc into liposomes was calculated after determination of the concentrations of free and total Euc present in the liposome suspensions by HPLC. Aliquots were removed from the liposomal suspension to determine the total Euc concentration ([Euc] Tot ). The liposomal suspension underwent ultracentrifugation (Optima TM Ultracentrifuge, Beckman Coulter, USA) at 170000g for 1 h at 4 °C. Aliquots were removed from the supernatant to determine the concentration of free Euc ([Euc] F ). The EE of Euc was calculated as follows:

where [Euc] Tot and [Euc] F corresponds to the concentration of total and free Euc, respectively.

The LR of Euc was calculated by the following equation:

(3)

The LR after freeze-drying was calculated using the following equation: where m Initial for Euc-DCL is the initial mass of Euc used to prepare CD/Euc inclusion complex. For Euc-CL, m initial is the initial mass of Euc added to the organic phase during liposome preparation. m reconstituted liposomes is the mass of Euc totally found in the liposomal suspension after freeze-drying and reconstitution of liposomes.

HPLC method

The concentration of Euc was determined by HPLC. Stock standard solutions of Euc (1 mg/mL) and of the internal standard, eugenol (1 mg/mL) were prepared in methanol.

Aliquots were removed from the Euc stock solution and diluted in methanol to obtain final concentrations of Euc ranging from 100 to 1000 µg/mL. The diluted solution of eugenol (1 µg/mL) was prepared in methanol. Two hundred µL of each filtrate were added to 200 µL of eugenol (1 µg/mL) and 400 µL of methanol. The samples were sonicated for 10 min at room temperature. The samples were analyzed by HPLC (Agilent Technologie 1200 series) using an analytical column C18 15 cm x 4.6 mm, 5 µm, (Agilent Technologies). The mobile phase was a mixture of methanol and water (70/30).

The flow rate was fixed at 1 mL/min and the detection was set at 206 nm.

The retention times of eugenol and Euc were 4.3 and 7.3 min, respectively.

Fluorescence anisotropy measurements

The effect of Euc, HP-ß-CD and freeze-drying process on the membrane fluidity of vesicles (blank-CL, blank-DCL, Euc-CL, Euc-DCL), the steady state fluorescence polarization measurements were determined using a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies). DPH was dissolved in tetrahydrofuran (4.30 10 -3 mol/L) then an aliquot was added to the vesicles suspension to obtain DPH:phospholipid molar ratio of 1:500. The measurements were conducted following the protocol described previously [23] .

Release of Euc

Multiple headspace extraction (MHE) coupled to gas chromatography was used to study the release of Euc form freeze-dried CL and DCL as well as from reconstituted vesicles after 6 months of storage at 4ºC. Freeze-dried powders of Euc-CL, Euc-DCL (10 mg), reconstituted Euc-CL, Euc-DCL in 10 mL water, inclusion complex solution prepared at a molar ratio HP-ß-CD/Euc of 5:1, and Euc dissolved in water (0.2 mg/mL) were placed in 22 ml sealed vials. The protocol described in a previous study [13] was used.

After equilibrium, the release of Euc at time t was calculated as follows: (6) Where, A t and A 1 corresponds to the area of the chromatographic peak of Euc at time t and at the first extraction, respectively. The plot of ln (A t /A 1 ) as a function of time followed a first-order release kinetics. The release rate constant K was calculated using the following equation:

Ln (A t /A 1 ) = -Kt + b (7)

Loading capacity determination

The amount of Euc in freeze-dried forms of CL and DCL was determined after 6 months of storage at 4ºC using MHE. The concentration of Euc was determined according to the protocol developed by Kolb & Ettre [START_REF] Kolb | Static Headspace-Gas Chromatography: Theory andPractice[END_REF] and used in our previous studies [13] .

Loading capacity (LC) of Euc is expressed as mg of encapsulated Euc per gram of liposomes.

(8)

Statistical analysis

Statistical analysis was performed using the Student's t-test. P values equal or less than 0.05 were considered statistically significant.

Results and discussion

Determination of the optimal HP-ß-CD/Euc inclusion complex

Table 1 presents the encapsulation efficiency of Euc using various HP-β-CD concentrations. EE CD values were calculated using equation 1. It was found that a HP-β-CD concentration of 50 mM was necessary to dissolve the total amount of Euc. Same result was found for anethole, another essential oil component [12] . HP-β-CD:Euc of 5:1 molar ratio was used subsequently to prepare DCL formulations.

Liposomes characterization before and after freeze-drying

Size, polydispersity index and zeta potential values

CL and DCL liposomal suspension were prepared by the ethanol injection method using hydrogenated phospholipids (Phospholipon 90H) and cholesterol. Table 2 summarizes the size, polydispersity index and zeta potential values of vesicles before and after freezedrying.

Before freeze-drying, the size of blank-DCL, Euc-CL and Euc-DCL showed no significant difference compared to blank-CL, since similar range of size (179 to 201 nm) was obtained. These results demonstrated that the presence of HP-ß-CD and/or Euc did not affect the liposome particle size. Same results were reported for encapsulation of anethole [11,12] , estragole [13] and clove essential oil [14] in liposomes composed of hydrogenated phospholipids. HP-ß-CD and essential oil components are able to affect the size of liposomes composed of unsaturated phospholipids [START_REF] Hammoud | [END_REF] . Besides, polydispersity index values for all batches were less than 0.2, suggesting that liposomes suspensions were homogenous (Table 2). The zeta potential values for the various formulations were negative ranging between (-2 to -9 mV) suggesting that the presence of HP-ß-CD or Euc did not affect the zeta potential values.

In a previous work, we optimized the freeze-drying of hydrogenated liposomes using HPß-CD as cryoprotectant for CL and DCLs [12] . The results showed that dispersing the pellet of CL in HP-ß-CD solution (25 or 50 mM), that of DCL in water or in HP-ß-CD solution (10 to 100 mM) protect the structures during freeze-drying. For DCL pellet, HPß-CD solution was used at the same CD concentration present in the internal aqueous phase of vesicles. The Euc-DCL formulation was prepared using HP-ß-CD/Euc molar ratio of 5:1 where HP-ß-CD (50 mM) and Euc (10 mM) were used to prepare the inclusion complex. Thus HP-ß-CD (50 mM) was used as a cryoprotector for DCL formulations.

After freeze-drying, the size, pdI and zeta potential values were maintained proving that the freeze-drying process was well optimized and could be used for a large number of bioactive compounds loaded CL or DCL (Table 2).

Morphology

The TEM images of phospholipon 90H liposomes obtained before and after freeze-drying showed the formation of nanometer-sized vesicles. Figure 1 (A-B) and(C-D) presented the images of blank-DCL and Euc-DCL obtained before and after freeze-drying, respectively. The vesicles appeared to be oligolamellar and spherical in shape. Similar morphology was found for DCLs loading anethole [12,26] , estragole [13] and eugenol [14] .

The sizes of the vesicles were well below those determined by DLS technique. In this work, the number of TEM images taken for each formulation was not enough to determine the mean particle size for each batch; moreover some factors may affect the results obtained by DLS such as temperature and the viscosity of the sample.

Encapsulation efficiency

The encapsulation efficiency (EE) and the loading rate (LR) of Euc-loaded liposomes (CL and DCL) before and after freeze-drying were calculated using equations (2-4) and the values are listed in Table 3. The concentration of Euc before freeze-drying in Euc-CL was not detected by HPLC analysis. Same results were obtained for estragole [13] . The Henry's law constant, previously determined by static headspace-gas chromatography at 30ºC were of 0.01 and 0.03 for Euc and estragole, respectively. It has been reported that these molecules exhibited higher volatility compared to other essential oil compounds; the Henry's law constant for isoeugenol, pulegone, terpineol, and thymol at 30ºC were of 0.005, 0.005, 0.001 and 0.003 respectively. The Henry's law constant was increased to 0.05, 0.07, 0.008, 0.009, 0.003 and 0.009 for Euc, estargole, isoeugenol, pulegone, terpineol, and thymol respectively upon increasing the temperature from 30 to 60 °C [START_REF] Hammoud | [END_REF] .

The chromatogram obtained for eucalyptol at 60ºC did not shown any additional peak or presence of a degradation product during the time of the analysis (8 min). The volatilization of Euc during the preparation of liposomes at 55ºC can explain the dramatic loss of this volatile agent.

The Euc-DCL system improved the encapsulation of Euc with EE value 78.24 ± 2.33%, and the LR value was 8.59 ± 1.61%. Hence, DCL improved the encapsulation of different drugs including volatile ones like trans-anethole [12] , estragole [13] . Some non-volatile compounds like betamethasone [27] , celcoxib [28] and curcumin [29] were also better encapsulated in DCL compared to conventional liposomes.

The LR of Euc significantly decreased after freeze-drying to 0.81 ± 0.22 for Euc-DCL (Table 3). Also, a remarkable anethole loss from DCL was obtained during freeze-drying process [12] . The vacuum pressure of 100 μbar provokes probably anethole and Euc loss.

For that, further experiments should be performed to optimize the freeze-drying of DCL loading highly volatile compounds.

Fluorescence anisotropy

Table 4 showed the DPH anisotropy values obtained at 25 and 37 ºC for blank-CL, blank-DCL, Euc-CL and Euc-DCL before and after freeze-drying.

The membrane fluidity increased with the temperature in accordance with literature [23] .

Compared to blank-CL, the presence of Euc or the inclusion complex HP-ß-CD/Euc did not affect the DPH anisotropy values at 25 and 37 ºC. The result obtained for Euc-CL has been expected since Euc was not detected in Euc-loaded CL by HPLC. Gharib, et al [30] demonstrated that the presence of monoterpenes (pulegone, terpineol and eucalyptol) at high concentration of monoterpene to phospholipid molar ratios (10%) decreased in a concentration dependent manner the anisotropy value of DPPC membrane at 28, 41 and 50 ºC. Besides, the presence of HP-ß-CD as cryoprotectant did not affect the membrane fluidity, since no changes of the DPH anisotropy values were observed before and after freeze-drying for all batches (Table 4).

Release studies

The Euc release experiments from freeze-dried HP-ß-CD/Euc inclusion complex, Euc-CL and Euc-DCL were conducted at 60°C using the MHE method after 6 months of storage at 4ºC. Figure 2 showed the Euc peak areas of the external standard (0.2 ppm of Euc in water), inclusion complex HP-ß-CD/Euc, Euc-CL and Euc-DCL (in powder or reconstituted in water). The amount of Euc in powders (CL and DCL) was very low compared to reconstituted liposomes suggesting the high retention of Euc in powder samples. Thus, the powders were dissolved in 10 mL water and placed in sealed vials and analyzed by MHE. Compared to CL, the encapsulation of Euc was better in DCL, while the inclusion complex showed the higher ability to retain Euc (Figure 2). Reconstituted batches were then considered for the release study and for the determination of the LC m Euc /m powder of Euc.

The remaining percentage of Euc as free drug or when encapsulated in lipid vesicles are illustrated in Figure 3. Surprisingly, the remaining percentage of Euc was lower in CL (58.5%) than in free Euc (78.2%). Thus, CL prepared in our conditions failed to be an effective delivery system for Euc. For the other formulations, the remaining percentage of Euc increased in the order: free Euc (78.2%) > Euc-DCL (82.5%) > HP-ß-CD:Euc (85%). Thus the Euc of eucalyptol in DCL system and HP-ß-CD:Euc inclusion complex improve its release compared to free Euc.

The release rate constant was calculated according to equation 7 and the values are listed in Table 5. The retention of Euc was improved by 1.6 and 1.3 times for HP-ß-CD/Euc inclusion complex and Euc-DCL compared to free Euc (0.2 ppm).

Determination of loading capacity of Euc

The LC values of Euc in HP-ß-CD/Euc inclusion complex, Euc-CL and Euc-DCL, after 6 months of storage at 4ºC, were determined using equation 8 and presented in Table 6. As expected, a small amount of Euc was obtained in Euc-CL with LC value 0.0155μg of Euc/mg of powder, since Euc was not detected by HPLC method. However, Hammoud et al [START_REF] Hammoud | [END_REF] studied the encapsulation of Euc in liposomes composed of unsaturated soybean phospholipid (Lipoid S100) and the results showed that CL loading Euc had a low loading capacity value of Euc (1.1 μg of drug/mg of total organic components). The encapsulation of Euc in DCL system was 38 times greater than CL formulations with LC values of 0.6 μg of Euc/mg of powder (Table 6). However, the higher LC value of Euc was obtained with HP-ß-CD/Euc inclusion complex with 4.5 μg of Euc/mg of powder.

These results suggest that DCL system improve the encapsulation of Euc compared to CL. The inclusion complex HP-ß-CD/Euc showed the best ability to retain high amount of Euc.

Conclusion

In this paper, we demonstrated a successful freeze-drying of Euc loaded Phospholipon 90H liposomes where the characteristics of hydrogenated-liposomes (size, pdI, zeta potential and morphology) were maintained after freeze-drying using HP-ß-CD as cryoprotectant for CL and DCL systems. HP-ß-CD/Euc and Euc-DCL controlled drug release and displayed a good stability after 6 months of storage at 4 ºC in powder form.

These results revealed that freeze-dried CD/drug inclusion complex and DCL reduced the volatilization of Euc and improved its retention. This work suggests that the encapsulated Euc could be considered as a suitable approach for the insecticide application.

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: The Euc peak areas of the external standard (0.2 ppm of Euc in water),

Figure 3 :

 3 Figure 3: The percentage of remaining Euc from Euc external standard (0.2 ppm of

Table 1 :

 1 The encapsulation efficiency of Euc in HP-ß-CD/Euc inclusion complex using different HP-ß-CD concentrations.

	[HP-ß-CD] mM	EE CD (%)
	0	72.72 ± 10.16
	10	71.51 ± 6.95
	25	92.78 ± 5.37
	50	98.66 ± 6.20
	100	99.08 ± 2.2

Table 2 :

 2 Size, polydispersity index, zeta potential values for fresh and reconstituted Euc-CL and Euc-DCL from Phospholipon 90H before and after freeze-drying.The values obtained after lyophilization were compared to those before lyophilization.

	Before freeze-drying

Table 3 :

 3 Encapsulation efficiency of Euc for fresh CL and DCL suspensions and loading rate of Euc in fresh and reconstituted CL and DCL before and after freeze-drying

		Before freeze-drying	After freeze-drying
		Fresh liposomes	Reconstituted liposomes
		EE (%)	LR I (%)	LR fd (%)
	Euc-CL	ND	ND	ND
	Euc-DCL 50	78.24 ± 2.33	8.59 ± 1.61	0.81 ± 0.22*
	ND: not detected
	 indicates a value of P < 0.05 statistically significant.

Table 4 :

 4 DPH fluorescence anisotropy measurements for blank-CL, blank-DCL, Euc-CL and Euc-DCL made from Phospholipon 90H and cholesterol at 25 and 37ºC before and after freeze-drying.Values are expressed as the means of three repetitions ± SD; n.d.: not determined P<0.05 compared to those before freeze-drying

		Before lyophilization	After lyophilization
		25ºC	37ºC	25ºC	37ºC
	Blank-CL	0.22 ± 0.0	0.17 ± 0.0	n.d.	n.d.
	Blank DCL	0.21 ± 0.0	0.17 ± 0.0	0.22 ± 0.00	0.17 ± 0
	Euc-CL	0.22 ± 0.0	0.17 ± 0.0	n.d.	n.d.
	Euc-DCL	0.21 ± 0.0	0.18 ± 0.0	0.22 ± 0.01	0.17 ± 0

Table 5 :

 5 Release rate constants of free Euc, HP-ß-CD/Euc inclusion complex, Euc-CL and

	Euc-DCL formulations
		Release rate constant K
		(min -1 )
	Euc 0.2 ppm	0.011
	Euc-CL reconstituted	0.021
	Euc-DCL reconstituted	0.008
	HP-ß-CD:Euc complex	0.007

Table 6 :

 6 Loading capacity of Euc in HP-ß-CD/Euc inclusion complex and in Euc-CL and Euc-DCL carrier systems after 6 months of storage at 4ºC.

		LC (μg of Euc/mg of powder)
	HP-ß-CD:Euc	4.5
	Euc-CL	0.0155
	Euc-DCL	0.6

Acknowledgements

The research was supported by the Research Funding Program at the Lebanese University (2018-2020) and the "Agence Universitaire de la Francophonie", projet PCSI (2018-2020).

Conflicts of interest

We confirm that there are no known conflicts of interest associated with this publication.