Emilie Roger
email: emilie.roger@univ-angers.fr

Jean-Christophe Gimel

Conor Bensley

Andrey S Klymchenko

Jean-Pierre Benoit

Lipid nanocapsules maintain full integrity after crossing a human intestinal epithelium model

Keywords: Nanocapsules, Caco-2, intestinal transport, FRET, integrity

Lipid nanocapsules (LNCs) have demonstrated great potential for the oral delivery of drugs having very limited oral bioavailability (BCS class II, III and IV molecules). It has been shown previously that orally-administered LNCs can permeate through mucus, increase drug absorption by the epithelial tissue, and finally, increase drug bioavailability. However, even if transport mechanisms through mucus and the intestinal barrier have already been clarified, the preservation of particle integrity is still not known. The aim of the present work is to study in vitro the fate of LNCs after their transportation across an intestinal epithelium model (Caco-2 cell model). For this, two complementary techniques were employed: Förster Resonance Energy Transfer (FRET) and Nanoparticle Tracking Analysis (NTA). Results showed, after 2 hours, the presence of nanoparticles in the basolateral side of the cell layer and a measurable FRET signal. This provides very good evidence for the transcellular intact crossing of the nanocarriers.

Introduction

The oral route is the most common route for drug delivery. However, oral bioavailability is influenced by drug solubility and permeability. For drugs belonging to the Biopharmaceutics Classification System class II (high permeability, low solubility), class III (low permeability, high solubility), and class IV (low permeability, low solubility) [START_REF] Amidon | A Theoretical Basis for a Biopharmaceutic Drug Classification: The Correlation of in Vitro Drug Product Dissolution and in Vivo Bioavailability[END_REF], drug encapsulation in nanocarriers provides an alternative solution to enhance bioavailability. Indeed, knowledge of the relationship between physico-chemical characteristics of nanocarriers (size and nature of wall material, surface charge, ligands...) and the gastrointestinal physiology (acidic pH, digestive enzymes of gastrointestinal media, mucus and intestinal barrier), allowed drug bioavailability [START_REF] Roger | Biopharmaceutical parameters to consider in order to alter the fate of nanocarriers after oral delivery[END_REF] to be enhanced. In the literature, various nanocarriers (liposomes, micelles, polymeric and lipid-based nanoparticles) are described for the oral delivery of chemotherapeutics, proteins, peptides, vaccines, etc. [START_REF] Plapied | Fate of polymeric nanocarriers for oral drug delivery[END_REF] Lipid nanocapsules, developed by our group, have also proved to be very interesting for oral administration [START_REF] Huynh | Lipid nanocapsules: A new platform for nanomedicine[END_REF]. These nanoparticles have a size range from 20 to 100nm and are prepared by a well-known low-energy emulsification process: the phase-inversion temperature method [START_REF] Heurtault | Lipid nanocapsules, preparation method and use as medicine[END_REF][START_REF] Heurtault | A novel phase inversionbased process for the preparation of lipid nanocarriers[END_REF][START_REF] Heurtault | The influence of lipid nanocapsule composition on their size distribution[END_REF].

They have already shown promising properties for the oral delivery of paclitaxel [START_REF] Peltier | Enhanced oral paclitaxel bioavailability after administration of paclitaxel-loaded lipid nanocapsules[END_REF][START_REF] Groo | In vivo evaluation of paclitaxel-loaded lipid nanocapsules after intravenous and oral administration on resistant tumor[END_REF], Sn38 [START_REF] Roger | Development and characterization of a novel lipid nanocapsule formulation of Sn38 for oral administration[END_REF], fondaparinux [11] and miltefosine [12]. Indeed, LNCs have demonstrated in vitro stability in simulated gastrointestinal media [13]. They have also shown their stability and diffusion in intestinal mucus [14]. Furthermore, in a previous study by Roger et al. [15], it was shown that LNCs were taken up by Caco-2 cells mainly via active endocytic and more particularly via clathrin-dependent and caveolae-dependent transport mechanisms; this active transport was not size-dependent. Comparing the incubation of paclitaxel-LNCs at 4°C and 37°C, an important decrease of paclitaxel permeability at 4°C was observed (more than 10-fold for the larger LNCs)

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A N U S C R I P T 4 and so, very few passive transport was evidenced. Moreover, tight junctions were not disrupted by LNCs and consequently paracellular transport was not possible. Finally, using transmission electron microscopy (TEM), nano-objects were observed on the basolateral side of the Caco-2 cell monolayers when LNCs were applied on the apical side, which probably allow the formulation of a transcytosis hypothesis [15]. Additionally, these nanocarriers have demonstrated a direct effect of P-gp on their endocytosis [16]. In view of these results, LNCs have the ability to enhance the bioavailability of class II and IV drugs [17].

Besides, another challenge of drug delivery via the oral route is to target pharmacological receptors after absorption, like nanocarriers injected via the intravenous route (I.V.). For this purpose, it is important to design nanocarriers that are able to be absorbed while keeping their integrity, and thus able to behave as circulating nanocarriers. Nevertheless, even if the mechanisms of transporting nanocarriers through the intestinal barrier have been well characterised, the fate of these nanocarriers after oral delivery is still unknown [START_REF] Plapied | Fate of polymeric nanocarriers for oral drug delivery[END_REF]. This lack can be explained by the difficulty to find relevant methods to confirm the real integrity of the carrier after absorption [START_REF] Niño-Pariente | Relevant Physicochemical Descriptors of "Soft Nanomedicines" to Bypass Biological Barriers[END_REF]. Indeed, many physico-chemical characterisation techniques such as dynamic light scattering (size and charge analysis), microscopy (TEM, AFM, confocal) and spectroscopy (IR, UV, FTIR, NMR) characterise the overall carrier structure or its individual components but not its integrity -i.e. is the cargo still inside the carrier? In this way, new tools are needed to determine how nanocarriers are absorbed and eliminated.

FRET is the most common fluorescence spectroscopy technique used to monitor the proximity at the nanoscale level [START_REF] Niño-Pariente | Relevant Physicochemical Descriptors of "Soft Nanomedicines" to Bypass Biological Barriers[END_REF]. It is based on the interactions between spatially-close (2 to 10 nm) donor and acceptor dye molecules. FRET occurs when the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor. The excitation energy of the donor is

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transferred to the acceptor whose subsequent emission can be detected. FRET is highly sensitive to donor-acceptor distances and consequently it indicates a preserved nanoscale environment. This technique is widely used to study biological phenomena such as interactions between proteins or proteins and nucleic acids, conformational changes, lipid membrane dynamics, hostpathogenic interactions, etc. [START_REF] Sahoo | Forster resonance energy transfer -A spectroscopic nanoruler: Principle and applications[END_REF]. More recently, FRET has been used to study the integrity of nanocarriers in vitro in different media, after cell internalisation, or in vivo after I.V. administration [START_REF] Bouchaala | Integrity of lipid nanocarriers in bloodstream and tumor quantified by near-infrared ratiometric FRET imaging in living mice[END_REF][START_REF] Klymchenko | Highly lipophilic fluorescent dyes in nano-emulsions: towards bright nonleaking nano-droplets[END_REF][START_REF] Lainé | Conventional versus stealth lipid nanoparticles: Formulation and in vivo fate prediction through FRET monitoring[END_REF][START_REF] Lu | Stability of self-assembled polymeric micelles in serum[END_REF][START_REF] Miteva | Tuning PEGylation of mixed micelles to overcome[END_REF][START_REF] Quadir | PEG -Polypeptide Block Copolymers as pH-Responsive Endosome-Solubilizing Drug Nanocarriers[END_REF][START_REF] Morton | FRET-enabled biological characterization of polymeric micelles[END_REF][START_REF] Gravier | FRET imaging approaches for in vitro and in vivo characterization of synthetic lipid nanoparticles[END_REF][START_REF] Lee | FRET imaging reveals different cellular entry routes of self-assembled and disulfide bonded polymeric micelles[END_REF][START_REF] Nguyen | On the interaction of alginate-based core-shell nanocarriers with keratinocytes in vitro[END_REF]. Importantly, it enables quantification of nanocarrier integrity in vitro and in vivo [START_REF] Bouchaala | Integrity of lipid nanocarriers in bloodstream and tumor quantified by near-infrared ratiometric FRET imaging in living mice[END_REF]. Only 3 studies involving FRET and oral administration have been reported. Firstly, Groo et al. [14] demonstrated the integrity of LNCs, using 1,1′-dioctadecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate (DiI) and 1,1′-dioctadecyl-3,3,3′,3′tetramethylindodicarbocyanine perchlorate (DiD) as FRET pairs, after contact with mucus. Lu et al. [START_REF] Lu | FRET-based dual-emission and pH-responsive nanocarriers for enhanced delivery of protein across intestinal epithelial cell barrier[END_REF] used fluorescein isothiocyanate (FITC) and Rhodamine B pairs to monitor the assembly of nanocomplexes while pH evolves in the intestine. More recently, Li et al. [START_REF] Li | Size-exclusive effect of nanostructured lipid carriers on oral drug delivery[END_REF] developed a FRET assay with DiO and DiI pairs to evaluate nanostructured lipid carrier (NLC) structural integrity in cell uptake; they observed a FRET signal in Caco-2 cells, and they speculated that NLCs could maintain their structural integrity after crossing intestinal cells, but they did not prove it. To our knowledge, no published work has clearly highlighted the integrity of nanocarriers after their transport through the intestinal barrier.

In order to use the FRET technique to study nanocarrier transport across enterocytes with a Transwell ® set-up, it is important firstly to reach an efficient encapsulation load for both dyes leading to acceptable FRET efficiency [START_REF] Preus | Advances in Quantitative FRET-Based Methods for Studying Nucleic Acids[END_REF] and, secondly, to measure with high sensitivity the FRET signal, since a high level of dilution is expected in the basolateral compartment.

Moreover, to avoid the immediate partitioning of dye from the carrier to subcellular lipid

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compartments, which could decrease FRET efficiency even if nanocarriers are still intact; it is crucial to achieve very stable dye encapsulation with no significant leakage. According to Bastiat et al. [START_REF] Bastiat | A new tool to ensure the fluorescent dye labeling stability of nanocarriers: A real challenge for fluorescence imaging[END_REF], loading lipophilic carbocyanine dyes (DiO, DiI and DiD) is recommended since, unlike other dyes, they remain inside the nanocapsules in the presence of lipid acceptor compartments.

Furthermore, to increase the loading of carbocyanine dyes into the lipidic core of the LNCs and to obtain a significant FRET signal, a new concept of counterion-based dye was introduced by Kilin et al. [START_REF] Kilin | Counterion-enhanced cyanine dye loading into lipid nano-droplets for single-particle tracking in zebrafish[END_REF]. They substituted the perchlorate counterion for the bulky and highly hydrophobic tetraphenyl borate (TPB) which improves dye solubility in oil and consequently the dye loading capacity (Figure 1).

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A In this context, we encapsulated two lipophilic cyanine dyes (DiD and DiI) bearing TPB counterions inside 50 nm LNCs. We optimised dye loading to achieve an efficient FRET signal.

Then, the transport of the obtained ultrabright FRET-loaded LNCs was studied with a wellestablished intestinal epithelium model (Caco-2 cells) [START_REF] Artursson | Caco-2 monolayers in experimental and theoretical predictions of drug transport[END_REF][START_REF] Balimane | A critique of cell culture models for intestinal permeability[END_REF]. To complete this approach, NTA was also performed to measure the size distribution of nanoparticles and their concentration. The objective of the present study is to investigate the fate of LNCs after crossing the intestinal barrier, focusing on nanoparticle integrity.

Materials and Methods

Materials

All the chemicals and solvents for synthesis were from Sigma-Aldrich (Saint-Quentin Fallavier, France). Captex ® 8000 (tricaprylin) was a gift from Abitec Corp. (Colombus, Ohio, USA). Lipoid ® S75-3 (soybean lecithin at 70% of phosphatidylcholine and 10% of phosphatidylethanolamine) and Kolliphor ® HS15 (mixture of free polyethylene glycol 660 and polyethylene glycol 660 hydroxystearate) were purchased from Lipoïd GmbH (Ludwigshafen, Germany) and BASF (Ludwigshafen, Germany), respectively. NaCl was purchased from Prolabo VWR International (Fontenay-sous-Bois, France). Ultrapure water was obtained from a Milli-Q ® Advantage A10 System (Merck Millipore, Darmstadt, Germany). Culture reagents were obtained from Sigma (St.Louis, USA) and Lonza (Verviers, Belgium).

Preparation of DiI-TPB and DiD-TPB

DiI-TPB was synthesized as described elsewhere [START_REF] Kilin | Counterion-enhanced cyanine dye loading into lipid nano-droplets for single-particle tracking in zebrafish[END_REF]. To synthetize DiD-TPB, 100 mg of 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine perchlorate (DiD, Life Technologies,

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USA) was mixed with 0.71 g (20 mol eq.) of sodium tetraphenylborate in ethyl acetate, which dissolved both salts easily. The formation of the desired salt was confirmed by thin-layer chromatography, where the product moved much faster than the starting DiD perchlorate (dichloromethane/methanol, 95/5). After solvent evaporation, the product (DiD-TPB) was purified by column chromatography (dichloromethane/methanol, 95/5).

Formulation and characterisation of lipid nanocapsules (LNCs)

LNC formulation was based on a phase-inversion process and has been thoroughly described [START_REF] Heurtault | Lipid nanocapsules, preparation method and use as medicine[END_REF][START_REF] Heurtault | A novel phase inversionbased process for the preparation of lipid nanocarriers[END_REF]. Briefly, the fluorescent dyes were solubilised in Captex ® 8000. Then, 150 mg of Captex ® 8000 (oil phase), 225.0 mg of water, 9.1 mg of NaCl (aqueous phase), 8.4 mg of Lipoid® S75-3 and 125 mg of Kolliphor ® HS15 (surfactants) were precisely weighed. After this, three cycles of heating and cooling between 70 and 90°C were carried out and the suspension of LNCs was obtained by adding 625 μL of water. LNC suspensions were filtered (0.22 μm) before use.

Formulation and characterisation of nanoemulsions (NEs)

Nanoemulsions were prepared by spontaneous nano-emulsification. The fluorescent dyes were solubilised in Captex ® 8000. Then, 105 mg of Captex ® 8000 and 160 mg of Kolliphor ® HS15 and 8.4 mg of Lipoid ® S75-3 were weighed and homogenised under magnetic stirring at 90°C.

Nano-emulsions were formed by adding 860 μL of cold water.

Nanoparticle Tracking Analysis (NTA) measurements

The concentration and the size distribution of particles were determined using a NS300

NanoSight (Malvern Instrument, Worcesterchire, UK) with a low-volume flow cell plate and a 405 nm laser. The nanoparticle suspensions were put directly into the chamber using a 1 mL syringe installed on a syringe pump. We used the NTA analytical software, version 3.2, to analyse video images. The measurements were made at room temperature, and each video

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sequence was captured over 60 s. Each analysis was replicated five times for each sample.

Concentrations inferior to 5 particles/frame were considered as unreliable, and corresponding samples were considered as being without particles. LNC and NE suspensions were diluted in ultrapure water by factor 6,000,000 ( v / v ) before analysis. Apical and basolateral media were diluted in ultrapure water by factor 50,000 and 10 ( v / v ), respectively, before analysis. We haven't done measurements using the fluorescence mode of the NTA as the measured fluorescence of a NP varies depending on its position with respect to the focal plane.

Caco-2 cell culture: model of gastrointestinal barrier

The human colon adenocarcinoma cell line (Caco-2) was obtained from the American Type Culture Collection (Manassas, USA) and used between passages 26 and 30. Cells were cultured in Dulbecco's modified Eagle medium (D-MEM, high glucose) supplemented with 15% ( v / v ) foetal bovine serum, 1% ( v / v ) non-essential amino acids, 1% ( v / v ) sodium pyruvate, and 1% antibiotic solution (110 4 UI/mL penicillin, 10mg/mL streptomycin, 25 μg/mL amphotericin B) in a humidified incubator 5% CO 2 /95% air atmosphere at 37°C. Cells were plated on a 75 cm 2 flask at a density of 110 6 cells/flask and were then harvested at 80% confluence with trypsin-EDTA and seeded onto polycarbonate membrane filters (0.4 μm pore size, 1.12 cm 2 growth area) inside Transwell ® cell culture chambers (Corning Costar, Cambridge, MA) at a density of 0.110 6 cells/insert. The culture medium (0.5 mL per insert and 1.5 mL per well) was replaced every two days for the first two weeks and everyday thereafter.

Nanoparticle transport across Caco-2

The integrity of lipid nanocarriers was estimated using Förster Resonance Energy transfer (FRET) between two encapsulated dyes, 0.5% of DiI-TPB (with respect to Captex ® 8000) as energy donor and 0.5% of DiD-TPB as energy acceptor were used. The transports of NEs, LNCs and HBSS (control) were studied in the apical to basolateral direction on Caco-2 cells in quadruplicate. Nanoparticle (LNC and NE) suspensions were diluted 100 times in HBSS and 1%

of HEPES from the original formulation and were applied on all the apical side. Experiments were started by adding 0.5 mL of test solution at the apical side and 1.5 mL of HBSS / HEPES at the basolateral side. After 2 h of incubation at 37°C, samples were taken from apical and basolateral sides (n=4) and the nanocarrier integrity was determined by FRET measurements coupled to NTA.

Fluorescence spectroscopy

Fluorescence spectra were recorded on a FluoroMax ® -4 spectrophotometer (HORIBA Jobin Yvon Inc., New Jersey, USA). Fluorescence emission spectra were recorded at room temperature with 520 nm excitation wavelengths, with an excitation slit width of 3 nm, an emission slit width of 2 nm and an integration time of 0.5 s. Emission spectra were collected from 530 to 720 nm with a 1 nm increment. They were corrected for the lamp source fluctuations and the wavelength-dependent response of the detector.

The semi-quantitative parameter of FRET efficiency was calculated according to the following equation PR=A/(A+D), where A and D are the maximum of fluorescence intensity of the acceptor (674 nm) and donor (570 nm), respectively.

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Results and discussion

As seen previously, the LNC platform represents one type of nanoparticle with promising use for oral delivery. They are formulated with Generally Recognized as Safe (GRAS) excipients by a phase-inversion temperature (PIT) process, a low-energy nano-emulsification method [START_REF] Heurtault | A novel phase inversionbased process for the preparation of lipid nanocarriers[END_REF]. LNCs are composed of an oily core (Captex ® 8000: a medium-chain triglyceride) surrounded by a mixed layer of lecithin (Lipoid ® S-75) and a PEGylated surfactant (Kolliphor ® HS15) [START_REF] Heurtault | A novel phase inversionbased process for the preparation of lipid nanocarriers[END_REF].

Different in vitro and in vivo experiments have demonstrated the ability of LNCs to diffuse across mucus and to enhance drug intestinal permeability as well as to improve paclitaxel bioavailability [START_REF] Groo | In vivo evaluation of paclitaxel-loaded lipid nanocapsules after intravenous and oral administration on resistant tumor[END_REF]15,17]. Nevertheless, a better understanding of the fate of these LNCs for oral drug delivery should be investigated in order to determine how the carriers are absorbed before planning thorough biodistribution studies.

As a comparison, we also tested nanoemulsions (NEs), a system close to LNCs. They are also formulated by a low-energy method with the same excipients but are obtained by a spontaneous emulsification mechanism [START_REF] Anton | The universality of low-energy nano-emulsification[END_REF]. Compared to LNCs, NEs use a different formulation pathway that never goes through the microemulsion step as with the PIT method [START_REF] Anton | Design and production of nanoparticles formulated from nano-emulsion templates-A review[END_REF][START_REF] Anton | Nano-emulsions and nanocapsules by the PIT method: An investigation on the role of the temperature cycling on the emulsion phase inversion[END_REF]. Both systems were applied on the apical side of a Caco-2 cell monolayer and NEs were considered as a control. In the following we mainly focused on LNC behaviour.

The use of FRET to assess LNC integrity has been successfully employed for in vitro stability in mucus [14] and in plasma [START_REF] Lainé | Conventional versus stealth lipid nanoparticles: Formulation and in vivo fate prediction through FRET monitoring[END_REF] as well as for in vivo biodistribution after intravenous administration [START_REF] Lainé | Conventional versus stealth lipid nanoparticles: Formulation and in vivo fate prediction through FRET monitoring[END_REF][START_REF] Gravier | FRET imaging approaches for in vitro and in vivo characterization of synthetic lipid nanoparticles[END_REF]. Consequently, carbocyanine dyes are more appropriate for FRET use. More recently, Kilin et al.

[34] introduced the substitution of the perchlorate counterion by a bulky and hydrophobic tetraphenylborate one to increase dye solubility in oil. Based on these works, we formulated various LNCs with DiI-TPB and/or DiD-TPB that showed solubility in Captex ® above 3 % w/w.

Firstly, single dye-loaded LNCs were formulated and the fluorescent spectra were measured.

Figure 2 presents excitation and emission spectra of both individually-loaded dyes. An overlay of the emission spectrum of DiI-TPB with the excitation spectrum of DiD-TPB was observed (see the hatched patterns in Figure 2a). Accordingly, a FRET signal could be expected with DiI-TPB as a donor and DiD-TPB as an acceptor. The maximum of the excitation spectrum of DiI is 548 nm but, in order to minimise DiD-TPB direct excitation, a wavelength of 520 nm was preferred as the excitation level for the FRET signal, i.e. using 520 nm instead of 548 nm reduced by a factor of 4 the emission intensity of DiD-TPB from direct excitation (supplementary Figure 1S).

Nevertheless, even with excitation at 520 nm, a low-intensity emission peak of DiD-TPB LNCs at 674 nm was still noticeable (see the red-dashed line in Figure 3) as well as a residual contribution of the donor itself (see the blue-dashed line in Figure 3). The emission spectrum of a mixture of DiI-TPB LNCs and DiD-TPB LNCs appeared as a simple sum of the individual emission spectra (see the full-black line in Figure 3), which is a relatively low contribution level

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13 of the acceptor (DiD-TPB) peak at 674 nm. In contrast, when DiI-TPB and DiD-TPB were both encapsulated inside the same LNCs, the so-called FRET-LNCs, a significant FRET signal occurred: a significant increase of the acceptor (DiD-TPB) peak at 674 nm was observed together with a decrease at 568 nm of the donor (DiI-TPB) (see the full-red line in Figure 3).

This phenomenon was specific of a fluorescent energy transfer between the two dyes due to their close confinement in the LNCs. Fluorescence spectra of FRET-LNCs, with an increasing amount of DiI-TPB and DiD-TPB, are represented in Figure 4A. As the concentration of both dyes increased (in a mass ratio 1:1), an increase in the acceptor emission band was clearly seen, whereas the donor's band was saturated. This could be formally highlighted by calculating the relative FRET efficiency, also called the proximity ratio (PR), that characterises the efficacy of the transfer and that is closely related to the dye proximity [START_REF] Sahoo | Forster resonance energy transfer -A spectroscopic nanoruler: Principle and applications[END_REF][START_REF] Preus | Advances in Quantitative FRET-Based Methods for Studying Nucleic Acids[END_REF][START_REF] Müller | Pulsed interleaved excitation[END_REF]. It was calculated as the ratio of the acceptor intensity at the peak (A) to the total intensity (A+D) where D was the intensity at the donor peak, upon donor excitation (at 520 nm): PR=A/(A+D). This value is reported in the inset of Figure 4A with various dye concentrations from 0.1% to 0.5% w/w of Captex  . As expected, the most efficient PR was obtained for the highest concentration tested i.e. 0.5%. But as the addition of carbocyanine dyes could modify the LNC structure and its zeta potential [14], 0.5% w/w corresponded to a good compromise between a good brightness level and no significant change in the zeta potential (a weakly-negative zeta potential was obtained corresponding to blank LNC values [15]). Moreover, the FRET signal was disrupted when the LNCs were destroyed in organic solvents (Methanol/THF, DMSO, and dioxane) [START_REF] Bouchaala | Integrity of lipid nanocarriers in bloodstream and tumor quantified by near-infrared ratiometric FRET imaging in living mice[END_REF][START_REF] Lainé | Conventional versus stealth lipid nanoparticles: Formulation and in vivo fate prediction through FRET monitoring[END_REF]. The donor fluorescence was no longer transferred to the acceptor and an emission spectrum close to that of an LNC mixture was recovered (Figure 4B), as well as small shifts in peak positions due resolved but its trajectory is recorded and a diffusion coefficient, thus a hydrodynamic diameter, is extracted from each track. This provides information about the number particle size distribution and the particle concentration. This technique has also been used to study the timedependent effect of biological conditions on nanoparticles [START_REF] Niño-Pariente | Relevant Physicochemical Descriptors of "Soft Nanomedicines" to Bypass Biological Barriers[END_REF]. We found a very narrow size distribution of FRET-LNCs with a number average diameter of 55.8 nm (supplementary Figure 2S). The measured concentration was around 1.210 15 particles/ml. (the oil component of the LNC core). For both dyes, a slight red shift of their maximum intensity emission (from 568 nm to 572 nm for DiI-TPB, and from 674 nm to 682 nm for DiD-TPB) was noticed in pure Captex ® . This was characteristic of a change in the dye environment. Therefore, it can be assumed that, in LNCs, dyes were also localised at the core/shell interface and not only in the bulk of the oily core. Finally, as we did the counterion substitution, we had to check that dye-TPB encapsulated inside LNCs behaved like perchlorate ones, and could not partition with another lipidic compartment-like cell membranes [START_REF] Gravier | FRET imaging approaches for in vitro and in vivo characterization of synthetic lipid nanoparticles[END_REF] which could lead to a FRET signal loss even if the LNCs stayed intact. For this, we investigated the encapsulation stability of dye-loaded LNCs after mixing them with pure Captex ® oil. No leakage phenomenon was evidenced; FRET efficiency was preserved as well as the size of LNCs even if a small particle concentration decrease was observed (Table 1). This lipid transfer experiment was described by Bastiat et al.

[33] and authors explained the absence of leakage of lipophilic carbocyanines by their highly hydrophobic stearyl chain. The presence of TPB counterions rendered carbocyanine dyes even

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A N U S C R I P T 18 more lipophilic. Thus, FRET-LNCs exhibited relevant properties to assess their integrity.

Finally, a FRET-LNC suspension at 0.5wt % of each dye, at a concentration of 0.910 15 particles/mL, was diluted in water and a fluorescence spectrum was measured for each dilution.

As represented in Figure 5, peak fluorescence intensities D (at 568 nm) and A (674 nm) were plotted as a function of the LNC concentration. Both curves were fitted on a double logarithmic scale with a linear function D = 10 -6.644 C LNC for the donor and A = 10 -6,237 C LNC for the acceptor (see both straight lines in Figure 5). Based on these calibration curves, fluorescence intensities at 568 nm and 674 nm obtained in the following could be correlated to a concentration of particles.

Moreover, for each dilution a proximity ratio (PR) was determined and a value of 0.718±0.117 (n=21 dilutions, average ± S.D) was obtained whatever the concentration. A similar calibration curve in terms of Captex ® concentration was obtained with NEs (supplementary Figure 3S) but with a PR value of 0.512±0.033 (n=19 dilutions, average ± S.D) and a size distribution much more polydispersed (supplementary Figure 2S). This large size distribution prevented the establishment of a relevant calibration curve expressed in terms of nanoparticle concentration. To carry out quantitative particle analysis on the basolateral side of the Caco-2 cell monolayer, the fluorescence spectrum at  ex =520 nm was measured. Firstly, the PR was checked for a qualitative measurement of overall LNC integrity, then the concentration of intact particles was estimated with the equations given above and finally compared with the concentration given by NTA. It has to be noted that nanoparticles detected by the NTA technique were not necessarily Before performing transport experiments in Transwell ® systems, the stability of FRET-LNCs and FRET-NEs was assessed by incubating them at a 100-fold dilution in HBSS at 37°C, conditions used for in vitro studies. Firstly, no significant change was observed in the proximity ratio value nor in the particle size after 2 hours, the incubation time used for transport experiments (Table 2). Moreover, no variation in the concentration of particles calculated either with the fluorescence calibration curve at 674 nm or determined directly by NTA was observed.

Consequently, one could conclude that both FRET-LNCs and FRET-NEs had very good stability at 37°C and could be used safely for in vitro experiments. Even so, the concentration of FRET-LNC particles obtained by fluorescence calculation or by NTA showed a slight difference (0.610 15 particles/mL versus 0.910 15 particles/mL, respectively at the initial time for LNC). This difference was still present after 2 hours of incubation and could be explained by the presence of dust particles in the HBSS medium or in the water used for the dilutions needed to perform NTA (even though HBSS and dilution water were filtrated through a 0.22 µm filter). These particles were counted by the NTA technique but did not contribute to the fluorescence signal which explains the discrepancy observed.

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A The in vitro transport across Caco-2 cells was then performed in quadruplicate to take into account the variability of the biological activity from one well to another one. The basolateral medium was analysed after 2 hours of incubation with FRET-LNCs, FRET-NEs or HBSS (controls) in the apical side. TEER values were checked before and after the incubation; no significant TEER decrease and typical values between 300 and 450 Ω.cm 2 were obtained confirming that no paracellular transport was possible. As summarized in Table 3 and Figures 6 and7, in control HBSS no particles were observed on the apical side nor on the basolateral side.

With control FRET-NE particles, a number average size above 87 nm were found with a Proximity Ratio value of 0.549 in the apical side, but neither particles nor a FRET signal were detected on the basolateral side. This result suggested an absence of the transport of intact NEs across Caco-2 cells. However, using FRET-LNCs particles, fluorescence spectra performed in the basolateral side showed clearly a FRET signal with an average PR value close to 0.75 even if

A C C E P T E D M

A N U S C R I P T 22 absolute peak intensity varies from 1 to 6 (see Figure 7 and Table 3). Moreover, NTA measurements showed the presence of particles with an average size ranging from 66 to 72 nm (a movie showing the particles can be found in supplementary information). Therefore, particles that appeared on the basolateral side were intact LNCs, with a slightly larger diameter than could be explained by some surface decoration resulting from their transcellular passage well to another on were perfectly correlated. This corresponds to around 0.3% of the initial quantity of LNCs. Moreover, the same analysis has shown that 99.3±0,1% (n=4, average±S.D.)

were still on the apical side. So for the first time it was demonstrated that intact particles were transported. The low amount of transcytosis could be related to the short time exposure (only 2 hours). It should be stressed that this study was performed with the conventional Caco-2 cell model. Even if a good correlation between the in vitro permeability of the drug and their in vivo bioavailability was established [START_REF] Lennernas | Comparison between active and passive drug transport in human intestinal epithelial (Caco-2) cells in vitro and human jejunum in vivo[END_REF][START_REF] Le-Thi-Thu | Prediction of Caco-2 Cell Permeability Using Bilinear Indices and Multiple Linear Regression[END_REF], this model was not optimised for studying intestinal absorption (absence of mucus layer and M-cells or physiological medium). Moreover, the behaviour of LNCs could be affected by physico-chemical properties such as particle size, surface potential and surface functionalisation. For example, we demonstrated [15], in spite of a predominant active endocytic mechanism for the transport of LNCs, a size-dependent passive transport of LNCs (increasing with decreasing size). Moreover, better diffusion across a mucus layer of LNCs was shown with positive or neutral zeta potentials [START_REF] Groo | Development of 2D and 3D mucus models and their interactions with mucuspenetrating paclitaxel-loaded lipid nanocapsules[END_REF]. Therefore, the next step of this work will be to perform an in vivo study (pharmacokinetics) and assess the impact of particle surface and size.

Conclusions

In conclusion, very stable LNCs with high dye content, no leakage, and able to produce a very efficient FRET signal, were formulated. Using a high-sensitivity spectrofluorometer, a calibration curve down to very low LNC concentrations (less than 10 10 particles/mL) with a FRET detection threshold even lower, was established. Fluorescence measurements were systematically supplemented by NTA experiments to measure particle sizes and concentrations.

Using a Caco2 cell monolayer, it was clearly demonstrated that LNCs were able to be transported intact by transcytosis. This was not the case for FRET-NEs. LNC concentrations on

7 Figure 1 .

 71 Figure 1. Chemical structures of DiI and DiD dyes with perchlorate and tetraphenylborate

  After 21 days in culture, cell monolayers were used for the following assays. Before experiments, cell monolayers were washed twice with Hank's buffered salt solution (HBSS) for 15 min at 37 °C. The transepithelial electrical resistance (TEER) of monolayers was checked before and after each experiment by using a Millicell®-ER-2 A C C E P T E D M A N U S C R I P T 10 system (Millipore Corporation, Billerica, MA). Only cell monolayers with TEER values over 250 Ω.cm 2 were used.

  Nevertheless, no work to date has demonstrated the potential of LNCs to cross the intestinal barrier in an intact form; only the presence of nano-objects on the basolateral side has been evidenced by TEM when LNCs were applied on the apical side of enterocytes [15], but no proof of integrity was established. Consequently, in order to achieve this, it is important toA C C E P T E D M A N U S C R I P T12 have a highly-efficient FRET signal combined with a high-detection capability of the fluorescence emission. For this, dyes had to be closely confined and a high dye loading was requested without any dye precipitation or release. Simonsson et al.[START_REF] Simonsson | Inter-nanocarrier and nanocarrier-to-cell transfer assays demonstrate the risk of an immediate unloading of dye from labeled lipid nanocapsules[END_REF] studied the potential transfer of various liphophilic dyes from the LNC core to favourable lipidic environments present in the cell. They demonstrated in vitro that no transfer of lipophilic carbocyanine dyes (DiI, DiD, DiO) occurred whereas a rapid transfer of Nile Red was observed from LNCs to cells.

  . The resulting values of PR collapse from 0.72 to below 0.22 making PR a reliable indicator of LNC integrity.

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. A) Excitation spectra and emission spectra of dye-loaded LNCs (0.5% w/w of Captex  ). The dashed-blue line (DiI-TPB) and dashed-red line (DiD-TPB) are excitation spectra

Figure 2

 2 Figure 2 displays emission spectra of DiI-TPB and DID-TPB inside LNCs and in pure Captex ®

Figure 5 .

 5 Figure 5. Evolution of the peak fluorescence intensities of the donor (blue circles,  = 568 nm)

  result from the metabolic activity of Caco2 cells. Only the combination of both techniques allowed us to state that intact LNCs were present in the basolateral compartment.

(

  supplementary Figure. 4S). The crossing of intact nanoparticles through the transcytosis mechanism has been already mentioned by several authors [15,[START_REF] Cartiera | The uptake and intracellular fate of PLGA nanoparticles in epithelial cells[END_REF][START_REF] Beloqui | Mechanism of transport of saquinavir-loaded nanostructured lipid carriers across the intestinal barrier[END_REF][START_REF] Zhang | The characteristics and mechanism of simvastatin loaded lipid nanoparticles to increase oral bioavailability in rats[END_REF]. Until now, the existence of nano-objects on the basolateral side of Caco-2 monolayers has only been proven by TEM[15,[START_REF] Du | Transferrin receptor specific nanocarriers conjugated with functional 7peptide for oral drug delivery[END_REF] but no evidence has been provided about their origin. More recently, Hu et al.[START_REF] Hu | Evidence does not support absorption of intact solid lipid nanoparticles via oral delivery[END_REF], using confocal microscopy, did not succeed to observe any fluorescence on the basolateral side in the presence of solid lipid nanoparticles (SLNs) or simulated mixed micelles (SMMs). Working with loaded near-infrared dyes with quenching properties upon contact with water, they concluded that both SLNs and SMMs could not be transported intact across Caco-2 cell monolayers.

Fig. 6 .Fig. 7 .

 67 Fig. 6. Emission spectra of the apical side solution of Caco-2 cells at time zero. The black line

  were successfully measured and, after 2 hours, around 0.3% of the LNCs initially present on the apical side had been transported. These unambiguous results open the way to future in vivo works in order to establish the pharmacokinetics and biodistribution of FRET-LNCs after their oral administration. By consequence, in a near future, standard procedures to measure the FRET signal from LNCs in blood and in relevant organs will be developed.A C C E P T E D MA N U S C R I P T

Table 1 :

 1 FRET efficiency and particle tracking analysis of LNCs during the lipid transfer experiment.

		Proximity	number average	Concentration of particles
		Ratio	Size (nm)	(particles/ml)
	Before the lipid transfer experiment	0.783	55.8	1.210 15
	After the lipid transfer experiment	0.781	60.6	0.810 15

Table 2 .

 2 Stability of FRET-LNCs and FRET-NEs in biological conditions: proximity ratio, concentration of particles (calculated with the equation obtained at 674 nm and determined by NTA) and size of particles (N.A for not applicable).

				Concentration of particles
				(particles/mL)	Number average
	LNCs NEs	Time (h) 0 2 0 2	Proximity Ratio 0.737 0.735 0.564 0.564	M Calculated by fluorescence at 674 nm 0.610 15 0.610 15 N.A A N U S C R I P T Determined by NTA 0.910 15 0.810 15 0.510 15 N.A 0.510 15	Size (nm) 67.1 72.8 83.0 75.1
		C C E P T E D
						21

Table 3 .

 3 FRET signal, proximity ratio, size of particles, concentration of particles (calculated with the equation obtained at 674 nm and determined by NTA) of apical and basolateral media of Caco-2 cells incubated with NEs, LNCs or HBSS for 2h at 37°C (basolateral side n=4; data are showed as mean±SD).

					Concentration of particles
					(particles/ml)	number average
		Time (h)	FRET signal	Proximity Ratio	Calculated by	Determined	Size (nm)
					fluorescence	by NTA
					at 674 nm
	HBSS	0 (apical side)	No	/	/	/	/
		2 (basolateral side)	No	/	/	/	/
	NEs	0 (apical side)	Yes	0.549	N.A	0.910 13	87.1
		2 (basolateral side)	Yes	/	/	not detected	not detected
	LNCs	0 (apical side)	Yes	0.743	6.710 12	1.210 13	55.9
		2 (basolateral side)	No	0.748±0.050	6.2±3.810 9	4.12.610 9	68.83.0

ACKNOWLEDGMENTS

We thank R. Bouchaala and I. Shulov for their assistance in the preparation of dye samples.

[12] M.M. Eissa, R.M. El-Moslemany, A.A.


Lilia Zaourar
email: lilia.zaourar@lip6.fr

Yann Kieffer
email: yann.kieffer@grenoble-inp.fr

Chouki Aktouf
email: chouki.aktouf@defactotech.com

A global optimization for scan chain insertion at the RT-level

Keywords: Design For Test, high-level testing, optimization

We present a new method for scan chain ordering specifically tailored for RTL-scan and its unique challenges.

I. INTRODUCTION

A current trend is to raise the level of design methodologies, in order to handle more abstract and more concise structures, increasing designing efficiency. This trend is not always followed in the field of IC testing. Although several proposals have been made to raise full-scan at a higher-level [START_REF] Roy | RTL based scan bist[END_REF] [2] [START_REF] Huang | Synthesis of scan chains for netlist descriptions at RT-level[END_REF], the industry is not ready to take that jump.

Obstacles to raising scan insertion at the RT-level are as follows. First, fault models talk about gate-level entities; so inserting logic to detect such faults at higher-level is counterintuitive. But it is feasible as demonstrated in [START_REF] Roy | RTL based scan bist[END_REF], [START_REF] Aktouf | Inserting scan at the behavorial level[END_REF] and [START_REF] Huang | Synthesis of scan chains for netlist descriptions at RT-level[END_REF]. An example of RTL code modification to include scan is given in Fig. 1 (additional code is presented in boldface).

Second, scan insertion at RTL means that scan logic and functional logic are intermingled and undistinguished by usual synthesis tools: to them, the whole tested designs looks "just functional". This is a strength of RTL-scan: logic common to both functional and test modes can be optimized away. But this means that no stitching reoptimization can be done during or after synthesis. Whereas gate-level scan takes benefit from low-level information available only late in the flow for scan stitching reoptimization [START_REF] Hirech | A new approach to scan chain reordering using physical design information[END_REF], [START_REF] Gupta | Routing aware scan chain ordering[END_REF], [START_REF] Gupta | Layoutaware scan chain synthesis for improved path delay fault coverage[END_REF], the usual proposal for RTL scan is to do it before logic synthesis. It is a one-step process, independent of what's before and after; in particular, there is no possibility of improvement through iterations: it has to be done right in just one pass.

Third, scan chain ordering is critical to the successful insertion of scan. A bad ordering can seriously impede placement and routing, increasing congestion and area.

It is to the combination of obtacles 2 and 3-finding a good ordering before logic synthesis, long before any placement information is known-that this paper tries to answer to. We use a graph model for the estimation of the proximity of FFs in the placed design in order to compute a good stitching ordering. Before we present our model, we explain why we use total wirelength as a measure for the quality of scan chain orderings. The parameters that are seriously impacted by the stitching ordering are performance (i.e. timing closure), congestion during routing, power consumption in both functional and test modes. We use the total wirelength of the design as a measure of the quality of a stitching ordering.

III. PROXIMITY GRAPH

We have defined our objective as minimizing the total wirelength of the scanned design, through the computation of a good scan chain ordering.

Since no placement information is known at scan-insertion time, one has to anticipate what will happen during the placement and routing steps.

To this end, we introduce the proximity graph (P-graph) model of the design. The P-graph has as vertices the FFs of the design, and edges correspond to (shortest) paths between FFs. Those paths can use wires either in the direction of information transmission or in the reverse direction. They cannot go through another FF. Furthermore, the number of gates traversed is limited to a fixed threshold t. The edges of the graph are labeled with the length of the underlying path, where the length is counted as the number of gates traversed by the path.

Fig. 2 gives an example design together with the corresponding P-graph. Since there is no canonical mapping from RTL to FFs and gates, a kind of synthesis has to be done to generate a P-graph from RTL code. More details are given in section V. Note that although RTL scan insertion does include a lightweight synthesis step, the RTL scan tool works completely independently from the logic synthesis tool, which comes later in the flow.

1 2 1 3 4 1 2 1 D Q D Q D Q D Q 1 2 3 4

IV. STITCHING ORDERING OPTIMIZATION

Although the P-graph is not a complete graph (i.e. some pairs of vertices are not connected), it is possible to reduce the scan chain ordering optimization problem to the Traveling Salesman's Problem. Note that connections in the scan chain can happen outside the P-graph; indeed, the P-graph may very well not contain a Hamilton path.

For scan chain optimization, a classical TSP heuristic (a 2-approximation algorithm) is adapted to our problem.

V. IMPLEMENTATION

Our method for computing scan chain orderings for RTL scan insertion has been implemented in an experimental version of DeFacTo Technologies' HiDFT-SIGNOFF R . The mapping from RTL code to a generic netlist is done by a commercially available software library; P-graph generation and TSP optimization have been implemented using the Boost Graph Library. The threshold value t (defined in section III) is fixed to 4.

VI. NUMERICAL RESULTS

Table I gives actual wirelength values (after placement & routing) for 11 ITC'99 benchmarks and 3 Opencore Verilog designs. Gate-level scan values (column GL-S) are from Magma's Talus integrated flow, as well as logic synthesis, placement and routing. RTL-scan values (column RTL-S) are from our enriched version of HiDFT-SIGNOFF R . The slack column shows the overhead for RTL scan. It is negative except for designs with less than 250 FFs, and when positive, doesn't exceed 6%.

Insertion times are also given in the last two columns. The times are split into two components: P-graph computation and TSP solution. Note that most of the time is spent in establishing the proximity-graph. The longest overall time is for b19 with more than 6000 FFs, at less than 6 minutes.

VII. CONCLUSION

We presented both a metric for the quality of scan chain ordering, and a method to compute good stitching orderings. The method has been implemented and validated on medium-sized designs, showing reasonable computation times, and overall improved wirelengths over Magma's Talus' gate-level scan module.

Perspectives of this work are a more thorough validation of the method; further optimization by taking into account more local factors; combining the method with powerreducing methods for test-mode; and extending the method to the setting of partial scan.

Figure 1 :

 1 Figure 1: Example scanned VHDL process

Figure 2 :

 2 Figure 2: (a) Design example; (b) Corresponding P-graph.

Table I :

 I Wirelengths and insertion times

		#FF	Wirelength	Slack	Insertion
					(%)	time (ms)
	Design		GL-S	RTL-S		P-graph	TSP
			ITC 99 Benchmarks (VHDL)
	b09	28	2.06	1.63	-20.59	<1	<1
	b10	17	1.94	2.06	5.97	10	<1
	b11	31	4.77	5.03	5.38	20	10
	b12	121	12.6	12.92	2.59	200	20
	b13	53	3.39	3.32	-2.25	20	<1
	b14	245	63.64	64.2	0.96	3s	110
	b15	449	126.3	122	-3.39	6s	320
	b17	1415	395.6	370.4	-6.37	30s	3s
	b18	3320	1187.2	922.6	-22.29	3m	10s
	b19	6642	2329.6	1858.2	-20.24	5m	20s
			Opencore designs (Verilog)
	Simple-spi	132	11.5	10.4	-8.95	100	20
	Biquad	204	34.1	31.3	-8.14	350	80
	Ac97	2289	247.7	238.5	-3.71	30s	8s


Process-Centred Approaches for Model-Driven E n g i n e e r i n g( P M D E )-F i r s tE d i t i o n

Reda Bendraou 1 , Redouane Lbath 2 , Bernard Coulette 2 , and Marie-Pierre Gervais 3 1 LIP6, Université Pierre & Marie Curie, France reda.bendraou@lip6.fr 2 Université Pierre de Toulouse le Mirail, France Lbath@univ-tlse2.fr, Bernard.Coulette@univ-tlse2.fr 3 LIP6, Université Paris Ouest, France marie-pierre.gervais@lip6.fr

Abstract. On one hand, the process engineering community has developed a large background on how to specify, to execute and to improve processes. On the other hand, the model-driven engineering community brings new techniques for easing the construction of languages and applications, editors and compilers. The goal of the PMDE workshop is to bring together experts from both communities to discuss the complementarity of these domains and how they can be combined together for a better software productivity and reliability. The workshop will invite contributions from both academia and the industry and will present the emerging research topics with their main challenges.

Introduction

Despite the benefits brought by the Model-Driven Engineering approach, the complexity of today's applications is still hard to master. Building complex and trustworthy software systems in the shortest time-to-market remains the challenging objective that competitive companies are facing constantly. A more challenging objective for these companies is to be able to formalize their development processes in order to analyze them, to simulate and execute them, and to reason about their possible improvement.

The PMDE Workshop aims to gather researchers and industrial practitioners working in the field of Model-Based Engineering, and more particularly on the use of processes to improve software reliability and productivity.

The PMDE workshop will invite papers presenting research results or work-inprogress in all areas of process-based approaches for model-driven engineering, including:

-Modelling software and systems processes for model-driven engineering -Domain-Specific Languages (DSL) for modelling software and systems processes

Transformation-based process modelling: structural and behavioural aspects

  We ask for papers in PDF format and not exceeding 10 pages in the LNCS style. The paper should present original research work related to the abovecited topics and should not be published or submitted simultaneously to other workshops, conferences or journals. Industrial papers are welcome. Each paper will be blindly reviewed by three members of the program committee.

	-MDE Process patterns: modelling and application
	-MDE Process patterns for reuse: definition, search methods, application of
	patterns
	-Process models refactoring and composition
	-Process lines and MDE
	-Verification of process models
	-MDE process enactment and simulation
	-MDE Process resource management
	-Process metrics for model-driven engineering
	-Management of distributed MDE processes
	-MDE process evolution: static evolution, process models refactoring, dy-
	namic process evolution, process deviations management
	-Process-centred MDE tools
	-Description of case studies based on MDE processes, experimentations on
	real projects, empirical studies
	2 Submissions and Selection Process
	3 Program committee
	-Colin Atkinson (University of Mannheim, Mannheim, Germany)
	-Behzad Bordbar (University of Birmingham, UK)
	-Jacky Estublier (University of Grenoble, France)
	-Christian Haerdt (EADS, Germany)
	-Jason Xabier Mansell (TECNALIA -ICT/European Software Institute, Spain)
	-Larrucea Uriarte Xabier (TECNALIA -ICT/European Software Institute,
	Spain)
	-Leon J. Osterweil (University of Massachusetts, USA)
	-Richard Paige (University of York, UK)
	-Kakade Rupesh (General Motors Technical Centre, India)
	-Garousi Vahid (University of Calgary Alberta, Canada)
	4W o r k s h o p O r g a n i z e r s
	1

-Bendraou Reda (LIP6, France) -Lbath Redouane (IRIT, France) -Coulette Bernard (IRIT, France) -Gervais Marie-Pierre (LIP6, France)