Clémentine Puech
email: c.puech@univ-st-etienne.fr

Sophie Hodin

Valérie Forest

Zhiguo He

Patrick Mismetti

Xavier Delavenne

Nathalie Perek

Assessment of HBEC-5i endothelial cell line cultivated in astrocyte conditioned medium as a human blood-brain barrier model for ABC drug transport studies

Keywords: Blood-Brain Barrier; ABC transporters, efflux ratio, human in vitro models, astrocyte conditioned medium

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

Introduction

The Blood-Brain Barrier (BBB) is a protective barrier between the blood and the Central Nervous System (CNS) [START_REF] Eigenmann | Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies[END_REF]. The BBB is composed of endothelial cells, pericytes, astrocytes, and neurons, the whole forming the neurovascular unit. Endothelial cells are the main components of the BBB because they restrict and control the passage of molecules inside and outside the brain. Barrier property of endothelial cells is mainly provided by tight junction proteins (TJs), and ATP Binding Cassette (ABC) transporters [START_REF] Abbott | Blood-brain barrier structure and function and the challenges for CNS drug delivery[END_REF].

ABC transporters are a large family of transporters, the most involved in BBB are Pglycoprotein protein (P-gp), Breast Cancer Resistance Protein (BCRP) and Multidrug

Resistance associated Proteins (MRPs). They are responsible for active efflux of therapeutic agents. It has been estimated that less than 2% of small molecule drugs are able to cross the BBB [START_REF] Eigenmann | Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies[END_REF][START_REF] Pardridge | The Blood-Brain Barrier: Bottleneck in Brain Drug Development[END_REF][START_REF] Ronaldson | Targeting transporters: promoting blood-brain barrier repair in response to oxidative stress injury[END_REF]. The prediction of drug passage through BBB and involvement of ABC transporters represent major issues in drug development.

The evaluation of drug transport through the BBB mediated by ABC transporters requires an in vitro human barrier model which must fulfil different criteria to be considered as a functional and adequate model for drug research development. These criteria include: the permeability to several compounds, the presence of TJs, and the expression of functional ABC transporters with bidirectional studies and efflux ratio (ER) measurement (Food and drug administration, 2012; [START_REF] Puech | The expected characteristics of an in vitro human Blood Brain Barrier model derived from cell lines, for studying how ABC transporters influence drug permeability[END_REF]. This latter is possible only with a monolayer of endothelial cells cultivated on a filter. Currently, no standard human BBB model to study interactions of drugs with ABC transporters correspond entirely to these criteria. There is a need to develop models that form an adequate monolayer on filter.

Several human cell lines were investigated in the literature i.e. TY08 [START_REF] Sano | Establishment of a new conditionally immortalized human brain microvascular endothelial cell line retaining an in vivo blood-brain barrier function[END_REF], TY10 [START_REF] Eigenmann | Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies[END_REF], HCEC [START_REF] Muruganandam | Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier[END_REF], HMEC-1 [START_REF] Ades | HMEC-1: establishment of an immortalized human microvascular endothelial cell line[END_REF], BB19 [START_REF] Prudhomme | Studies of Plasmodium falciparum cytoadherence using immortalized human brain capillary endothelial cells[END_REF], HBMEC [START_REF] Eigenmann | Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies[END_REF], hCMEC/D3 [START_REF] Weksler | Blood-brain barrier-specific properties of a human adult brain endothelial cell line[END_REF]. In the literature, the major human endothelial cell lines that have been well characterized are human Cerebro Microvascular Endothelial Cells (hCMEC/D3) [START_REF] Weksler | The hCMEC/D3 cell line as a model of the human blood brain barrier[END_REF] and Human Brain Microvascular Endothelial Cells (hBMEC) (Eigenmann et al., 2016b). However, these models are grown in a medium containing a cocktail of adjuvants (Eigenmann et al., 2016b;[START_REF] Kulczar | Development of a direct contact astrocyte-human cerebral microvessel endothelial cells blood-brain barrier coculture model[END_REF][START_REF] Weksler | The hCMEC/D3 cell line as a model of the human blood brain barrier[END_REF] and it is known that adjuvants have an impact on the expression of ABC transporters [START_REF] Eisenblätter | A new multidrug resistance protein at the blood-brain barrier[END_REF][START_REF] Maines | Evaluation of the role of Pglycoprotein in the uptake of paroxetine, clozapine, phenytoin and carbamazapine by bovine retinal endothelial cells[END_REF][START_REF] Von Wedel-Parlow | Regulation of major efflux transporters under inflammatory conditions at the blood-brain barrier in vitro[END_REF].

In this work, we have chosen another human brain endothelial cell line: HBEC-5i [START_REF] Jiang | HIV-1 Transactivator Protein Induces ZO-1 and Neprilysin Dysfunction in Brain Endothelial Cells via the Ras Signaling Pathway[END_REF] that can grow in monolayer on inserts without adjuvants. Furthermore, this cell line was cultivated in presence of human astrocyte medium as the barrier properties are mostly induced and maintained by the close links between brain endothelial cells and astrocytes [START_REF] Abbott | Astrocyte-endothelial interactions at the blood-brain barrier[END_REF][START_REF] Janzer | Astrocytes induce blood-brain barrier properties in endothelial cells[END_REF]. In the literature, it has been described that the soluble factor released by the astrocytes can induce modifications in the expression of functionality of the P-gp [START_REF] Abbott | Astrocyte-endothelial interactions at the blood-brain barrier[END_REF][START_REF] Berezowski | Endothelial Cells and Astrocytes: A Concerto en Duo in Ischemic Pathophysiology[END_REF][START_REF] Dohgu | Brain pericytes contribute to the induction and up-regulation of blood-brain barrier functions through transforming growth factor-beta production[END_REF]. We have compared the monoculture in standard medium with a model with astrocyte conditioned medium. ABC transporters functionality was evaluated with accumulation and bidirectional studies as well as ER (efflux ratio) parameter with fluorescent selective substrates and inhibitors. HBEC-5i monolayer presents an adequate BBB model that can be routinely used to evaluate drug interactions with ABC transporters.

Material and methods

2.1.Chemicals and reagents

Cell culture inserts for 24-well plates (High Density of pore, 0.4 μm pore diameter size, translucent poly-terephthalate ethylene (PET) membrane were purchased from Becton-Dickinson (Franklin Lakes, USA). EVOM ® voltohmmeter system was purchased from World LGC standard (Molsheim, France). Normal growth supplement (NGS) was obtained from Millipore (Burlington, USA).

All other reagents were purchased from Sigma-Aldrich (St Quentin Fallavier, France).

2.2.In vitro BBB model

Cell culture

The model was composed of HBEC-5i endothelial cells (from ATCC-Manassas, VA, USA) and the Human Astrocyte cell line (HA from ScienCell Research Laboratories, Carlsbad, CA, USA).

Initially, HBEC-5i were cultured in Dulbecco's Modified Eagle Medium: nutrient Mixture F-12 (DMEM-F12 HAM), supplemented with 10% FBS, 40 µg/mL endothelial cell growth supplement (ECGS), and 1% antibiotic antimycotic solution and HA were grown in AM Medium containing 2% FBS, 1% AGS and 1% penicillin-streptomycin solution according to manufacturer's instructions. For the drug transport studies, the two cell lines were cultured in a DMEM-F12 HAM medium without ECGS.

A monoculture model with endothelial cell line was performed, and cooperation with astrocytes was also evaluated with astrocytes conditioned medium. Endothelial cells were seeded at a density of 2x10 5 cells/wells on translucent PET culture inserts with a 0.33 cm² growth area and high-density of 0.4 µm pore size. For the conditioned medium, a monoculture of HBEC-5i was cultivated with astrocyte conditioned medium at the basolateral side of the filter. For each condition, the medium was renewed every two days.

Barrier properties

The apparent permeability (Papp) was assessed using two hydrophilic fluorescent molecules: sodium fluorescein (Na-Fl MW = 376 Da) or fluorescein isothyocyanate Dextran (Dextran-FITC MW= 4 kDa). Hank's balanced salt sodium (HBSS) transporter buffer supplemented with 10 mM HEPES containing 10µg/mL of Na-Fl or dextran-FITC was loaded onto the apical side of the insert and incubated at 37°C for 1 hour. Fluorescence was measured with a fluorescence spectrophotometer (Fluoroskan Ascent TM , Thermo Fischer scientific, France) with 485 nm excitation and 530 nm emission wavelengths. The permeability for all molecules was computed using the following formula:

𝑃 𝑎𝑝𝑝 = 𝑉 𝑟 𝐶 0 × 1 𝑆 × 𝐶 1 𝑡 (Formula 1)
Where Papp is the apparent permeability, Vr is the volume of medium in the receiver chamber, C0 is the concentration of fluorescent compound in the donor chamber at T0, S is the surface area of the monolayer, C1 is the concentration of fluorescent molecule in the receiver chamber after 1 hour of incubation and t is the incubation time. The results are expressed in cm.s -1 [START_REF] Hodin | In Vitro Comparison of the Role of P-Glycoprotein and Breast Cancer Resistance Protein on Direct Oral Anticoagulants Disposition[END_REF][START_REF] Puech | The expected characteristics of an in vitro human Blood Brain Barrier model derived from cell lines, for studying how ABC transporters influence drug permeability[END_REF]. The evaluation of a passage of pharmacological hydrophilic molecule, the caffeine (MW=194 Da) was performed in the same way. The quantification was carried out in liquid chromatography-mass spectrometry (LC-MS: Waters, Saint-Quentin-en-Yvelines, France).

Trans-Endothelial Electrical Resistance (TEER) was recorded using an EVOM ® resistance meter with STX-2 electrode to characterize the formation of a tight endothelial cell monolayer. The measurement of a blank filter, i.e. without cells, was performed and the signal was subtracted from that recorded for the filter with cells. Then the resulting value was reported to the membrane area to obtain the TEER measurement in Ω.cm².

Proteins expression

Whole cell ELISA

HBEC-5i cells were fixed with 4% paraformaldehyde and permeabilized with 0.1%

Triton-100-X in PBS containing 1% Bovine Serum Albumin (BSA). Phenotypic marker expression and ABC transporters proteins were evaluated with primary polyclonal antibodies at the optimal concentration between 2-6µg/ml for all antibodies. Secondary IgG biotinylated antibody was added. Then, streptavidin was added during 20 min. 3, 3', 5, 5'tetramethylbenzidine (TMB) substrate was added in each well. At the end, the reaction was stopped by addition of HCl 1N. The color reaction was measured with a spectrophotometer (Multiskan TM RC, Thermo Fisher Scientific, France) at 450 nm. Results were normalized with the total protein content assay, according to the manufacturer's instructions.

Immunofluorescence assays

The immunofluorescence method had been previously described [START_REF] Forest | Optimization of immunostaining on flat-mounted human corneas[END_REF][START_REF] He | Optimization of immunolocalization of cell cycle proteins in human corneal endothelial cells[END_REF]. Briefly, inserts were fixed in glacial methanol (pre-stored at -20°C) for 6 minutes (for TJs) or 1% PFA (for ABC transporters) at RT for 30 min. PFA-fixed cells were permeabilized with 1% Triton X-100 for 5 min at RT. The non-specific binding sites were then blocked for 30 min at 37°C in blocking buffer that was 2% goat serum and 2% BSA in PBS.

The primary antibodies were diluted at 1/200 in the blocking buffer and incubated for 1 hour at 37 °C. The secondary antibodies were diluted at 1/500 in the blocking buffer and incubated for 45 min at 37 °C. Nuclei were finally counterstained with DAPI diluted 1/1000 in PBS at room temperature for 30 min. The inserts were finally placed on glass slides, submerged with Vectashield Mounting Medium (H1000, Vector Laboratories,USA), and covered with glass coverslip. The staining of all biomarkers had been imaged using an epifluorescence microscope IX81 (Olympus, Tokyo, Japan) equipped with the CellSens imaging software (Olympus, Munster, Germany).

ABC transporter functional studies

Accumulation studies

The functionality of the P-gp, MRPs and BCRP ABC transporter was evaluated at the cellular level by targeted accumulation studies based on three fluorescent substrates, BCECF

(2 µM), rhodamine 123 (10 µM) and Hoechst 33342 (20 µM), as broad spectrum substrate of MRPs and substrates of P-gp and BCRP or selective substrate of BCRP, respectively.

Cells were pre-treated for 15 min at 37°C with a range of inhibitors such as verapamil (100 µM), probenecid (100 µM), cyclosporin A (100 µM) and Ko143 (10 µM). The fluorescent probes were then diluted independently in each inhibitor solution prior to further incubation for 1 h at 37°C. Finally, the cells were washed with PBS at 4°C and lysed at room temperature during 30 min in a solution of sodium dodecyl sulfate containing 1% of 10 mM sodium borate.

Fluorescence in lysed cells was measured with a fluorescence spectrophotometer with 485 nm excitation and 530 nm emission wavelengths for BCECF and rhodamine 123. For Hoechst 33342, the fluorescence was measured with a fluorescence at 355/460 nm. The result was normalized by micro BCA total protein assay according to the manufacturer's instructions.

Bidirectional studies and efflux ratio (ER) measurements

We investigated the permeability of both therapeutic compounds and selective substrates of ABC transporters using a standardized bi-directional transport protocol according to FDA guidelines (Food and drug administration, 2012). Rhodamine 123 (10 µM) was evaluated as a P-gp and BCRP substrate with verapamil and Ko143 as selective inhibitors.

BCECF at 10µM was assessed as a broad spectrum MRPs substrate with probenecid as a general MRPs inhibitor. Rivaroxaban (10 µM) was evaluated as P-gp and BCRP substrate with verapamil and Ko143. Methotrexate (100 µM) was assessed as a MRP and BCRP substrate with probenecid as a general MRPs inhibitor and Ko143 as inhibitor of BCRP.

The experiment consisted of two mono-directional assays of trans-endothelial passage, with the tested drug initially added either in the apical compartment to obtain the apical to basolateral permeability (Papp AB) or in the basolateral compartment to obtain the basolateralto-apical permeability (Papp BA) with the Formula 1

To determine whether the tested compounds underwent active efflux by ABCs transporters, the ER of each compound was computed according to the following formula:

ER = 𝑃 𝑎𝑝𝑝 (𝐵 → 𝐴)/𝑃 𝑎𝑝𝑝 (𝐴 → 𝐵) Formula 2
The standard deviation of the ER being calculated according to the formula proposed by Gnoth et al. [START_REF] Gnoth | In vitro and in vivo Pglycoprotein transport characteristics of rivaroxaban[END_REF]. According to FDA guidelines an ER>2 indicates that the drug is an efflux transporter substrate. This ER must be decreased with selective inhibitor. The inhibitor must halve of the ER or make it less than 2 (Food and drug administration, 2012).

Percent of ER inhibition was computed using the Formula 3 [START_REF] Hodin | In Vitro Comparison of the Role of P-Glycoprotein and Breast Cancer Resistance Protein on Direct Oral Anticoagulants Disposition[END_REF]:

ER inhibition(%) = 1 - 𝐸𝑅 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟 𝐸𝑅 × 100 𝐅𝐨𝐫𝐦𝐮𝐥𝐚 𝟑
.

Liquid chromatographymass spectrometry analysis

Rivaroxaban, methotrexate and caffeine were quantified using an Aquity UPLC system coupled with a Xevo TQ-S triple quadrupole mass spectrometer (Waters, Saint-Quentin-en-Yvelines, France). Positive ionization condition was used for analysis of rivaroxaban (m/z 436.07→231.11), methotrexate (m/z 455.12→308.17), and caffeine (m/z 195.09→138.01).

The internal standard (IS) for each drug were [ 13 C6]-rivaroxaban (m/z 442.09→144.95), [ 13 C,

2 H3]-methotrexate (m/z 458.12→311.17), [ 3 H3]-caffeine (m/z 204.16→144.1) respectively.

Samples (50 µL) were extracted with 500 µL of IS in methanol. The mobile phase comprised a mixture of water containing 0.1% formic acid (FA) and acetonitrile containing 0.1% FA. An eluate gradient was applied to a BEH C18 column (50 mm × 2.1 mm × 2.7 µm) (Waters, Saint-Quentin-en-Yvelines, France). For the permeability calculation, the peak area ratios of the drugs and their respective IS were used as C0 and C1.

Statistical Analysis

Graphpad software was used for data analysis and graphic outputs. Data were expressed as the mean ± standard deviation. Statistical analyses were performed using Graphpad software (Graphpad software Incorporation, San Diego, USA). A Mann-Whiney test was used. Treated samples were compared to untreated control. The differences were considered statistically significant when p-value<0.05.

Results

Development and characterization of the model

To develop the model, the first step was to adapt endothelial and astrocytic cells in the same culture medium without adjuvant. The expression of two phenotypic markers, GFAP (Glial Fibrillary Acidic Protein) for astrocytes and VE-cadherin for endothelial cells were performed and were stable along the culture experiments (data not shown).

In a second step, we compared two endothelial cells monoculture conditions: a standard culture medium (HBEC-5i alone) and astrocytes conditioned medium (HBEC-5i with HA conditioned medium). TEER gradually increased to reach a steady state plateau at day 19 which was maintained during two days for HBEC-5i alone. The steady state plateau was reached at day 14 and maintained during five days for HBEC-5i with HA conditioned medium (Table 1).

In parallel, permeability studies were realized with two molecules: Na-Fl (376 Da) and dextran-FITC (4 kDa). The lowest Papp values were obtained for the HBEC-5i with HA conditioned medium (Table 1). The Papp value for the dextran-FITC was lower than the Papp value for Na-Fl, in agreement with the molecular weight of the compounds. All these results indicated that HBEC-5i with HA conditioned medium allowed to have a higher tightness of the barrier compared to the HBEC-5i alone.

HBEC5i cells were able to form a monolayer on insert and to express TJs proteins: ZO-1 and claudin-5 as represented in figure 1 AB.

Expression of ABC transporters

The expression of ABC transporters was evaluated with semi-quantitative protein expression by whole cell ELISA assay. We verified whether, astrocytes conditioned medium was able to induce any modification of the ABC transporters expression (figure 2 ABCD). Pgp, MRP-1, MRP-2 and BCRP had the same expression in the two culture media. In this experiment, we also used cells cultivated during 7 days with hydrocortisone as a positive control, as reported in the literature to obtain a tight model [START_REF] Kulczar | Development of a direct contact astrocyte-human cerebral microvessel endothelial cells blood-brain barrier coculture model[END_REF]. The hydrocortisone increased P-gp expression 1.8 fold and BCRP expression 1.3 fold on our HBEC-5i model. On the contrary the MRPs expression was not modified by hydrocortisone treatment.

These results support the idea that adjuvants have to be avoided to study drug interactions with ABC transporter as the main adjuvant reported in the literature is hydrocortisone.

The expression of P-gp, BCRP, MRP-1 and MRP-2 were also assessed in monolayer inserts of HBEC-5i with immunostaining (figure 2EFGH).

Functionality of ABC transporters

Accumulation studies

The functionality of the ABC transporters was assessed at the cellular level in standard and conditioned media conditions by means of accumulation assays. These experiments were based on the use of three fluorescent substrates BCECF (MRPs), rhodamine 123 (P-gp) and

Hoechst 33342 (BCRP) incubated with or without inhibitors: verapamil, probenecid, Ko143 against P-gp, MRPs and BCRP respectively. In addition, cyclosporin A, a large spectrum inhibitor has been used (figure 3ABC). In all experiments, we compared the two culture conditions.

The functionality of ABC transporters on HBEC-5i endothelial cells was similar when cells were cultured either in standard medium or in astrocytes medium. The addition of cyclosporine A induced an increase of the accumulation of all fluorescent substrates, which is in agreement with the large spectrum inhibition of cyclosporin A. For example, the addition of cyclosporin

A induced an accumulation of Hoechst 33342 three-fold higher than in control cells, whatever the culture condition (figure 3A).

The dual use of substrates and inhibitors allowed to demonstrate the P-gp, MRPs and BCRP transporters functionalities in our model. For instance, the accumulation of BCECF in cells treated with probenecid increased to 286 ± 34 % for the standard medium and 276 ± 24% for the conditioned medium (figure 3C).

Bidirectional permeability studies

Bidirectional permeability measurement that lead to ER allow to complete the evaluation of the ABC transporters functionality in a barrier model. The Hoechst fluorophore could not be used for bidirectional studies because this fluorescent molecule can be trapped in the nucleus to stain DNA of endothelial cells.

We noticed that the presence of HA conditioned medium allowed a better functionality of ABC transporters with higher ER (figure 4).

The ER of rhodamine 123 was 2.3 fold higher in the HBEC-5i cultured in the presence of HA conditioned medium than in HBEC-5i cultured alone. In the HA conditioned medium, the use of Ko143 and verapamil decreased ER value by 49% and by 65% respectively (figure 4AB).

The ER of BCECF was 2.8 fold higher in HBEC-5i with the HA conditioned medium than in HBEC-5i alone. Inhibition with probenecid allowed a decrease of 52 % in HBEC-5i

with the HA conditioned medium (Figure 4 C).

Taken together, these results indicate that in our model, ABC transporters are functional.

In a last part, and as reported by figure 5, we investigated the permeability profile of three pharmaceutical agents: rivaroxaban, methotrexate and caffeine in HBEC-5i with the HA conditioned medium.

Caffeine is a hydrophilic molecule, which crosses the barrier by a para-cellular pathway.

We found a permeability value of 67.0 ± 4.4 x 10 -6 cm.s -1 . This value is higher than the value for the Na-Fl and the dextran-FITC (figure 5A). These results are in agreement with the lowest molecular weight of this molecule compared to the others.

We evaluated the bidirectional transport of two drugs known to be substrates of ABC transporters. The rivaroxaban was released outside the cells forming the barrier with an efflux ratio at 10.1 ± 1.2 (figure 5B). The use of verapamil and Ko143 drastically inhibited the efflux of rivaroxaban by 79 % and 91% respectively. These results indicated that in our model, rivaroxaban is transported by BCRP and P-gp.

The methotrexate was released outside cells with an efflux ratio at 3.1 ± 0.2 (figure 5C).

The Ko143 and the probenecid diminished the ER below 2. The use of probenecid and Ko143 inhibited the efflux of rivaroxaban by 59 % and 69% respectively. These results confirmed that in our model, methotrexate is transported by MRPs and BCRP. These results show that in our model ABC transporters can transport pharmaceutical molecules.

Discussion

The transport of drugs from the blood to the brain mainly depends on their properties and interaction with ABC transporters [START_REF] Bernacki | Physiology and pharmacological role of the blood-brain barrier[END_REF]. To study the ABC transporters at the BBB, it is necessary to develop a suitable barrier model based on brain endothelial cells grown as monoculture on inserts [START_REF] Puech | The expected characteristics of an in vitro human Blood Brain Barrier model derived from cell lines, for studying how ABC transporters influence drug permeability[END_REF].

To that purpose, we developed a model composed of HBEC-5i cells cultivated either in standard medium or in the presence of HA conditioned medium. We first characterized the tightness of our model. When HBEC-5i cells were cultured with the HA conditioned medium it took 14 days to the model to reach stability (maximal TEER) which was maintained for 5 days. In comparison, the model cultivated with standard medium allowed the maximal TEER value after19 days and was stable only during 2 days. Similarly, the other models of human BBB found in the literature, show a short stability, about 2 days only [START_REF] Biemans | Limitations of the hCMEC/D3 cell line as a model for Aβ clearance by the human blood-brain barrier[END_REF]Eigenmann et al., 2016b[START_REF] Eigenmann | Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies[END_REF][START_REF] Weksler | The hCMEC/D3 cell line as a model of the human blood brain barrier[END_REF]. We also observed that HBEC-5i cells with HA conditioned medium exhibited a better permeability for Na-Fl than the hCMEC/D3 cells commonly used in the literature (5.7 x 10 -6 cm.s -1 and 13 x 10 -6 cm.s -1 respectively) [START_REF] Eigenmann | Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies[END_REF]. HBEC-5i cells expressed ZO-1 and claudin-5 the two essential TJs involved as reported in the literature with hCMEC/D3 [START_REF] Eigenmann | Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies[END_REF][START_REF] Kulczar | Development of a direct contact astrocyte-human cerebral microvessel endothelial cells blood-brain barrier coculture model[END_REF][START_REF] Poller | The human brain endothelial cell line hCMEC/D3 as a human bloodbrain barrier model for drug transport studies[END_REF][START_REF] Weksler | Blood-brain barrier-specific properties of a human adult brain endothelial cell line[END_REF]. We highlighted with immunofluorescent staining that our model exhibited the same characteristic membrane distribution of TJs for brain vascular endothelial cells as hCMEC/D3 [START_REF] Adriani | A 3D neurovascular microfluidic model consisting of neurons, astrocytes and cerebral endothelial cells as a blood-brain barrier[END_REF][START_REF] Sajja | Impact of altered glycaemia on blood-brain barrier endothelium: An in vitro study using the hCMEC/D3 cell line[END_REF]. The expression of tight junctions in our model limited the passage of small molecules in the paracellular pathways, it was correlated with the result of the Papp for dextran-FITC and Na-Fl. The results obtained showed that astrocytes cooperate with brain endothelial cells and are necessary in the acquisition of a better tightness of a BBB [START_REF] Abbott | Astrocyte-endothelial interactions at the blood-brain barrier[END_REF][START_REF] Dehouck | An easier, reproducible, and mass-production method to study the blood-brain barrier in vitro[END_REF][START_REF] Kulczar | Development of a direct contact astrocyte-human cerebral microvessel endothelial cells blood-brain barrier coculture model[END_REF]. Most of co-culture models found in the literature allow to observe the essential role of astrocytes on the tightness and permeability, i.e. the physical barrier properties [START_REF] Janzer | Astrocytes induce blood-brain barrier properties in endothelial cells[END_REF][START_REF] Neuhaus | The pivotal role of astrocytes in an in vitro stroke model of the blood-brain barrier[END_REF]. More precisely, it has been shown that the cocultures with astrocytes induce a higher tightness of the BBB composed either of the HCMEC/D3 or HBMEC cell lines [START_REF] Hatherell | Development of a three-dimensional, all-human in vitro model of the blood-brain barrier using mono-, co-, and tri-cultivation Transwell models[END_REF][START_REF] Kulczar | Development of a direct contact astrocyte-human cerebral microvessel endothelial cells blood-brain barrier coculture model[END_REF][START_REF] Kuo | Effect of human astrocytes on the characteristics of human brainmicrovascular endothelial cells in the blood-brain barrier[END_REF].

In a second part, we demonstrated that our HBEC-5i model expressed P-gp, MRP1, 2 and BCRP which are the most representative human in vivo ABC transporters [START_REF] Abbott | Blood-brain barrier structure and function and the challenges for CNS drug delivery[END_REF].

Our experiments with hydrocortisone reinforced the need to not use this compound in culture medium as previously discussed [START_REF] Eisenblätter | A new multidrug resistance protein at the blood-brain barrier[END_REF][START_REF] Maines | Evaluation of the role of Pglycoprotein in the uptake of paroxetine, clozapine, phenytoin and carbamazapine by bovine retinal endothelial cells[END_REF][START_REF] Von Wedel-Parlow | Regulation of major efflux transporters under inflammatory conditions at the blood-brain barrier in vitro[END_REF]. We have also demonstrated the functionality of ABC transporters with accumulation studies with specific substrates as described in the literature [START_REF] Bachmeier | Drug Efflux Transport Properties of 2′,7′-Bis(2carboxyethyl)-5(6)-carboxyfluorescein Acetoxymethyl Ester (BCECF-AM) and Its Fluorescent Free Acid, BCECF[END_REF][START_REF] Kodama | Characteristic Analysis of Intestinal Transport in Enterocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells[END_REF][START_REF] Poller | The human brain endothelial cell line hCMEC/D3 as a human bloodbrain barrier model for drug transport studies[END_REF]. As expected, we showed that all the ABC transporters in our model were present and functional as for hCMEC/D3 and HBMEC [START_REF] Kuo | Expression of P-glycoprotein and multidrug resistance-associated protein on human brain-microvascular endothelial cells with electromagnetic stimulation[END_REF][START_REF] Weksler | The hCMEC/D3 cell line as a model of the human blood brain barrier[END_REF].

In a third part, we performed bilateral studies, to determine ER in our model. This parameter completely determines the interaction between the drugs and ABC transporter function (Food and drug administration, 2012;[START_REF] Hubatsch | Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers[END_REF][START_REF] Kikuchi | In vitro P-glycoprotein efflux ratio can predict the in vivo brain penetration regardless of biopharmaceutics drug disposition classification system class[END_REF]. To date, only few studies on human BBB cell lines have evaluated this parameter (Eigenmann et al., 2016a;[START_REF] Jähne | Pharmacokinetics and In Vitro Blood-Brain Barrier Screening of the Plant-Derived Alkaloid Tryptanthrin[END_REF][START_REF] Ma | Transport of ginkgolides with different lipophilicities based on an hCMEC/D3 cell monolayer as a blood-brain barrier cell model[END_REF]. In our model, ER parameter was higher in HBEC-5i with the HA conditioned medium than HBEC-5i alone. This result is the first to demonstrate on a BBB model that astrocyte cooperation is necessary in ABC transporters bilateral studies.

There are only few studies on the effect of the astrocytes on metabolic properties of the BBB involving ABC transporters expression. In hCMEC/D3 a contact co-culture model with human astrocytes, has shown a lower accumulation in rhodamine 123, a fluorescent substrate of P-gp, suggesting that the level of this ABC transporter is higher compared to the mono-culture condition [START_REF] Kulczar | Development of a direct contact astrocyte-human cerebral microvessel endothelial cells blood-brain barrier coculture model[END_REF].

The co-culture contact model prevents the bilateral studies, because there are two monolayers on both sides of the insert [START_REF] Puech | The expected characteristics of an in vitro human Blood Brain Barrier model derived from cell lines, for studying how ABC transporters influence drug permeability[END_REF]. This configuration can induce an under or overestimation of the involvement of the ABC transporters in the drug transport studies. The noncontact co-culture configuration can be an alternative to the drawback of the co-culture contact, with the assessment of ER on a monolayer of endothelial cells (Eigenmann et al., 2016a).

In a last part, we evaluated the capacity of our model to discriminate pharmacological molecules in agreement with their properties. The result obtained for the caffeine is in agreement with the result obtained in the literature in the HBMEC cell line (Eigenmann et al., 2016b). In our model and in accordance with the literature the rivaroxaban was transported by BRCP and P-gp [START_REF] Gnoth | In vitro and in vivo Pglycoprotein transport characteristics of rivaroxaban[END_REF][START_REF] Hodin | In Vitro Comparison of the Role of P-Glycoprotein and Breast Cancer Resistance Protein on Direct Oral Anticoagulants Disposition[END_REF]. The value of the ER for methotrexate was in agreement with the presence of MRPs at the basolateral and apical membrane of the endothelial cells. Methotrexate was transported by MRPs, its active efflux by one MRP transporter in the apical compartment could be balanced by the active efflux in the basolateral compartment by other MRPs [START_REF] Wolf | Expression of resistance markers to methotrexate predicts clinical improvement in patients with rheumatoid arthritis[END_REF]. We have clearly demonstrated that our BBB model can evaluate interaction or not with ABC transporters.

To conclude, we have developed a suitable human BBB model composed of endothelial cell line HBEC-5i. This model offers several advantages like easy to use and can be used routinely in a laboratory to screen drug interactions. In addition, our model presents many advantages which are not found on the other BBB models available in the literature. For instance, a stability during five days which may allow to screen long term effects of therapeutic agents. Furthermore, the culture is performed without adjuvant and gives the possibility to realize ER with pharmacological molecules. Our model can be used to predict the permeability of therapeutic molecules across the BBB. Table 1: Main characteristics of tightness of our model. We have tested two configurations: the HBEC-5i alone and HBEC-5i + HA conditioned medium. Mean TEER and apparent permeability for the Na-Fl and Dextran-FITC 4kDa values over time for human endothelial cell line HBEC-5i on 24 well tissue culture inserts are reported. "Days" corresponds to the time necessary to obtain mean max TEER, and "stability" corresponds to the number of the days during which mean max TEER was maintained. Results are expressed as mean ± SD, n= 3, N=3.

Figure 1 :

 1 Figure 1: Immunofluorescent staining of ZO-1 (A) and Claudin-5 (B) tight junction proteins. Tight junctions proteins are stained in green, nuclei in blue (DAPI). Scale bar= 50µm (AB).

Figure 2 :

 2 Figure 2: Protein expression of the BRCP (A), P-gp (B), MRP-1 (C), MRP2 (D) by Whole Cell ELISA in different conditions: HBEC-5i alone, HBEC-5i with HA conditioned medium medium (+ HA conditioned medium) or with hydrocortisone. Results are expressed as mean ± SD, n= 6.***p-value<0.005 compared to standard medium as determined by a Mann-Whitney test. Immunofluorescence staining of BCRP (E), P-gp (F), MRP-1 (G), MRP-2 (F) in the HBEC-5i cell line. Scale bar= 50µm.

Figure 3 :

 3 Figure 3: Intracellular accumulation of Hoescht 33342 (A), rhodamine 123 (B) and BCECF (C) inside HBEC-5i cells treated with verapamil, probenecid, Ko143 and cyclosporin A after 60 min of incubation. Two models were evaluated: HBEC-5i alone and HBEC-5i with HA conditioned medium. N= 6 for each condition. Intracellular accumulation was normalized to the whole cell content. Data are expressed as the mean ±SD, n= 6. ** p-value < 0.01; ***p-value<0.005 compared to untreated control as determined by a Mann-Whitney test.

Figure 4 :

 4 Figure 4: Bidirectional permeability of rhodamine 123 (Rhodamine) (AB) and BCECF (C) through the HBEC-5i model after 1 hour in HBEC-5i alone or HBEC-5i with HA conditioned medium medium (HA CM). Selective inhibitors used were: Ko143, verapamil and probenecid. Results are expressed as mean ± SD, n=3.

Figure 5 :

 5 Figure 5: Evaluation of permeability of pharmacological molecules through the HBEC-5i model. Para-cellular permeability of caffeine and fluorescent small molecules (A). Bidirectional permeability of rivaroxaban (riva) (B) and methotrexate (Mtx)(C) through the HBEC-5i model after 1 hour. Selective inhibitors used were: Ko143, verapamil and probenecid. Results are expressed as mean ± SD, n=3 for each experiment.

  Precision Instruments with STX-2 electrode (Sarasota, USA). Twenty-five culture flasks and companion plates were purchased from Dominique Dutscher (Strasbourg, France).

	Rabbit polyclonal anti-vascular endothelial cadherin (VE-cadherin-ab33168), rabbit polyclonal
	anti-Zonula Occulens-1 (ZO-1ab59720) and rabbit polyclonal anti-P-gp (ab129450) antibodies
	for immunofluorescence (IF) and ELISA were purchased from Abcam (Cambridge, United
	Kingdom). Rabbit polyclonal anti-BCRP(sc-25822), rabbit polyclonal anti-MRP-1(sc-13960),
	rabbit polyclonal anti-MRP-2 (sc-20766), and rabbit polyclonal anti-claudin 5 (sc-28670)
	antibodies for IF or ELISA and mouse monoclonal anti-rabbit IgG-biotin for cell ELISA were
	purchased from Santa Cruz Biotechnology (Dallas, Texas, U.S.A). Hoechst 33342, rhodamine
	123 and secondary antibodies for IF: goat anti-rabbit antibodies (A-21127Alexa Fluor TM 555),
	4', 6-diamidino-2-phenylindole (DAPI) for microscopy and Coomassie plus TM protein assay
	and superFrost TM microscope slide were obtained from Thermo Fischer Scientific (Waltham,
	USA).
	Astrocyte Medium (AM), Astrocyte Growth Supplement (AGS), Fetal Bovine Serum (FBS)
	and penicillin-streptomycin solution for astrocytes were purchased from CliniSciences
	(Nanterre, France). Rivaroxaban, [ 13 C6]-rivaroxaban, caffeine, methotrexate and [ 13 C, 2 H3]-
	methotrexate were obtained from Alsachim (Illkirch, France). Caffeine-D9 was purchased from

Conflict of interest statement

The authors declare that they have no conflict of interest.


Alain Phommahaxay

Gaëlle Lissorgues

Lionel Rousseau

Tarik Bourouina

Pierre Nicole

SURFACE CONDITIONING EFFECT ON VACUUM MICROELECTRONICS COMPONENTS FABRICATED BY DEEP REACTIVE ION ETCHING

Advances in material processing such as silicon micromachining are opening the way to vacuum microelectronics. Two-dimensional vacuum components can be fabricated using the microsystems processes. We developed such devices using a single metal layer and silicon micromachining by DRIE. The latter technological step has significant impact on the characteristics of the vacuum components. This paper presents a brief summary of electron emission possibilities and the design leading to the fabrication of a lateral field emission diode. First measurement results and the aging of the devices are also discussed.

INTRODUCTION

The development of silicon micromachining led to the development of a variety of MEMS devices. High Q resonators are for instance useful in selective filtering or sensor applications. However they need to be operated at low pressures to minimize viscous damping. On the other hand, vacuum electronics components can now be fabricated with similar processes. Associations of MEMS devices with vacuum electronics are thus possible and can enable new functions.

Using a single mask fabrication process, our work enables the rapid prototyping of vacuum microcomponents. The first prototypes, including lateral diodes and triodes, have been produced with a low-cost aluminum process, consisting of one metal layer, one lithography step and silicon micromachining. The effects of the process optimization on the characteristics will be assessed. Early measurement results and first aging study will show some drift of their performances over time. New applications such as the real time monitoring of the vacuum level in various MEMS devices could use this effect. Different configurations using more suitable and stable field emitter materials such as tungsten will be studied in the future.

ELECTRON EMISSION AND VACUUM MICROELECTRONICS

Despite the progress made in solid-state electronics, vacuum tube-based components are still employed but only in very specific applications. Indeed some of their performances are still unattained by solid-state devices [START_REF] Zhu | Vacuum Microelectronics[END_REF]. Systems sustaining high powers and frequencies still use vacuum tubes. Indeed, the scattering probability of electrons traveling in vacuum is very low, leading to a quasi ballistic transport. Devices with cut-off frequencies in the range of tens of gigahertz are feasible with such technologies. Moreover their radiation hardness and power handling make them indispensable in space and harsh environment applications.

Thermionic Emission Figure 1 : Thermionic Emission Principle

Before the emergence of solid state electronics after the invention of the semiconductor transistors by Bardeen, Brattain and Shockley in the 1950s, most of the circuits were using vacuum tubes [START_REF] Brinkman | A history of the invention of the transistor and where it will lead us[END_REF]. Since then, vacuum tubes disappeared from most applications mainly due to the difficulty to integrate them as their semiconductor counterparts. Indeed, vacuum tubes were using the thermo-ionic principle, shown in Figure 1 The typical operating temperature of such tubes is thus above 800°C, making high integration density difficult. The advances in micro-and nano-fabrication techniques have led to a renewed interest in vacuum electronics. Indeed thanks to micromachining technology, their integration on silicon substrate is now possible, opening the way to vacuum microelectronics [START_REF] Zhu | Vacuum Microelectronics[END_REF]. Micrometer and sub-micrometer-spaced electrodes can now be fabricated thanks to the dimensions achievable by lithography. High electric fields can be obtained by applying relatively small voltages, allowing electron tunneling and/or field emission. Such devices can furthermore be integrated with MEMS devices [START_REF] Yamashita | An RF-MEMS device with a lateral field-emission detector[END_REF]- [START_REF] Liu | Characterization of a high-sensitivity micromachined tunneling accelerometer with micro-g resolution[END_REF] allowing the design of relatively new functions.

Field Emission Figure 2 : Field Emission Principle

Contrary to thermionic emission, field emission does not require to heat a cathode. Indeed, it is based on the principle represented in Figure 2. Electrons tunnel from the Fermi level into vacuum under high electric field. Most part of the energy brought into the system is thus used to extract electrons, increasing the emission efficiency compared to the thermionic principle.

Field emission is principally governed by the Fowler-Nordheim theory, which uses the so-called triangular barrier approximation. The current density equation at 0°K is given by [START_REF] Zhu | Vacuum Microelectronics[END_REF]:

[ ] 2 3 2 4 ( ) exp 2 4 3 µ q J F F m F µ φ π φ φ ⎛ ⎞ ≈ - ⎜ ⎟ + ⎝ ⎠
where F is the electric field, µ is the Fermi level, ħ is the Plank constant, q is the charge and m is the particle mass. In various works [START_REF] Lim | In-situ vacuum sealed lateral FEAs with low turn-on voltage and high transconductance[END_REF]- [START_REF] Ding | Silicon field emission arrays with atomically sharp tips: turn-on voltage and the effect of tip radius distribution[END_REF], this equation is simplified and leads to the following one:

( ) 2 ( ) exp / FN FN J F a F b F ≈ -
where a FN and b FN are two constants depending on φ only if µ≈ φ , which is true for metals. For a given electric field, the current density only depends on the material properties. The electrical field and the materials are the two parameters that can be tuned in order to increase the electron emission for a given geometry.

In fact F=βV, high electrical field values can be obtained by increasing either the voltage V or the field enhancement factor β, which is preferable for low voltage operation. This factor β only depends on the geometry. The sharpest the tip is, the greater is this factor. The distance between electrodes can be reduced but it should be kept in mind that electrical breakdown can occur under intense fields [START_REF] Torres | Electric field breakdown at micrometre separations[END_REF].

New materials such as carbon nanotubes or diamondlike carbon either increase β or decrease φ. They allow the emission of electron with good uniformity and rather high current up to 1µA per tip. They are for example used in Field Emission Displays [START_REF] Li | Field emission characteristic of screen-printed carbon nanotube cathode[END_REF]. For a given geometry, the field enhancement factor can be tuned during the fabrication process. For a twodimensional device fabricated on silicon, the Deep Reactive Ion Etching step would allow the tuning of this factor β, therefore changing the characteristics of the devices. Early electrical measurements were planned to take place at atmospheric pressure, therefore low current levels were expected. Then, 20 parallel planar diodes as in Figure 3 were designed in order to increase the total emission current. The electrodes were spaced with 2µm-wide airgap. The periodicity of the diode structure was chosen to minimize the self screening effect that can occur when two cathodes are too close to each other. A schematic process flow is illustrated in Figure 4. Only one photomask combined with one metal layer are needed to achieve the fabrication of a lateral field emission diode. First of all, a thick thermal oxide was grown on the top of the silicon wafer. The early prototypes were fabricated with a 4000 Å SiO2 layer on a P-type substrate. The metal was then deposited using either an electron beam evaporator or by sputtering as shown in Figure 4 (a). A 1 µm-thick aluminum film was used on our first devices. A hard mask was then formed by standard lithography and by etching the aluminum layer in chlorine plasma at step (b). The thermal oxide was then removed by fluorine anisotropic plasma etching through the mask as in Figure 4 (c). Deep Reactive Ion Etching, DRIE of silicon was then performed to release the emitter tip using an Alcatel 601E reactor. Varying process parameters allowed the control of the silicon etching profile. In fact, the length of the lateral etching and its deepness were easily controlled by alternating etching and passivation steps of the so-called Bosch process. The photoresist was finally stripped by O2 plasma at step (d).

VACUUM MICRODIODES DESIGN

As this structure tends to bend due to the residual stress of the silicon oxide layer, a misalignment between the two electrodes can happen. To avoid this, a High Resistivity Silicon (HRS) wafer can be used. Indeed the SiO2 layer purpose is to ensure a good insulation between the metallic electrodes. This can also be done with HRS, and it simplifies the process since neither thermal oxidation nor oxide etching are needed. This alternative process using HRS substrates can be very fast and offers prototypes in a matter of hours. The silicon oxide removal by plasma etching was indeed the slowest step of the process. Optical characterizations were performed on the first prototypes. An interferometric optical profiler was used to control the silicon etching depth, while a scanning electron microscope was used to visualize the emitter profile and tip apex, as shown in Figure 5. Even though a standard lithography was used on the first samples, a 100nm-apex was found, which theoretically helps to enhance the electrical field.

CHARACTERIZATIONS

Optical Characterization

Electrical Characterization

Preliminary measurements at atmospheric pressure were performed on the first prototypes. The on-wafer test setup

Alain Phommahaxay, Gaëlle Lissorgues, Lionel Rousseau, Tarik Bourouina, Pierre Nicole Surface Conditioning Effect on Vacuum Microelectronics Components Fabricated by DRIE illustrated in Figure 6 was composed of two electrical probes connected to the anode and the cathode. A voltage was applied through a Hewlett-Packard 4140B picoamperemeter.

Figure 6 : Electrical Test Setup

Surface Conditioning Influence

Six months after their fabrication, some devices were characterized and then cleaned by acetone and orthophosphoric acid to remove the oxide layer formed on the aluminum surface. A very noisy response was recorded before the cleaning procedure. Current peeks during the measurement disappeared after the surface reconditioning as shown in Figure 7. However a smaller current value was detected, suggesting that impurities and protrusions can participate to the emission process. The device composed of 20 parallel diodes with 2 micrometers-spaced electrodes showed a turn-on voltage of 25V and a current of 0.3µA when biased at 100V in atmospheric pressure before cleaning. The surface reconditioning increased the turn-on voltage to about 70V and reduced the current to 0.15µA at 100V. The devices are thus sensitive to the emitter surface chemistry and state.

Impurities deposited on non-encapsulated emitters can participate to the conduction, thus increasing the current but also instabilities given the high electric field between the anode and the cathode. Gases can also modify the emitter surface chemistry and the real work function. Oxygen found in the atmosphere can oxidize the surface of the conductor, consequently adding a barrier to the electron emission. Low work function materials are unfortunately more reactive chemically. Field emission devices must therefore be encapsulated in an inert environment such as nitrogen or even vacuum.

PACKAGING MONITORING

Pressures below 10 -4 mbar are typically required to ensure good lifetimes and operating conditions in vacuum microelectronics [START_REF] Zhu | Vacuum Microelectronics[END_REF]. The devices can still be operated at higher pressures but electrons would then collide with residual gases, possibly creating heavy ions, which would then bombard the emitters.

Such sensitivity to the pressure can allow the design of real-time monitoring structures inside micromachined vacuum cavities. The pressure level inside the cavity can be measured through a change of quality factor in MEMS resonating structures [START_REF] Sparks | Reliable vacuum packaging using nanogetters and glass frit bonding[END_REF]- [START_REF] Choa | Reliability of vacuum packaged MEMS gyroscopes[END_REF] or a frequency drift [START_REF] Kaajakari | Stability of wafer level vacuum encapsulated single-crystal silicon resonators[END_REF]. Field emitters would allow such monitoring without any mechanical structures. Indeed, the electron scattering probability and the surface chemistry are influenced by the gas type and pressure inside the cavity, thus modulating the current. New applications of our vacuum microelectronics devices could then be studied.

CONCLUSION

First prototypes of lateral vacuum microdiodes were fabricated and characterized at atmospheric pressure. The aging of the structures was studied. Changes in the current characteristics are mainly due to impurities and surface chemistry modifications. Such sensitivity to the environment can further be exploited in applications like real time vacuum monitoring in MEMS devices. Further experiments by encapsulating field emitters using either aluminum or other materials in an inert gas or in vacuum will be conducted in the near future. However this latter solution requires the use of getter materials, gas absorbers activated at high temperatures, to insure a low pressure level. Indeed, desorption will occur, various gases would be then trapped inside the cavity and increase the overall pressure.

Current (A) After cleaning ressure Voltage (V)

,

  K 1 and K 2 constants.

Figure 3 :

 3 Figure 3 : Vacuum Diodes Test Structure

Figure 4 :

 4 Figure 4 : Main Fabrication Process Steps

Figure 5 :

 5 Figure 5 : SEM Picture of a Diode Structure Fabricated with Various DRIE Parameters

Figure 7 :

 7 Figure 7 : I(V) Curve of 20 Parallel Vacuum Diodes Before and After Surface Reconditioning

I

  (V) Characteristic for 20 parallel diodes with 2µm spaced electrodes at atmospheric p Before cleaning

  , to generate a current flow. Electrons at the Fermi level have enough thermal energy to be extracted into vacuum. The

	Alain Phommahaxay, Gaëlle Lissorgues, Lionel Rousseau, Tarik Bourouina, Pierre Nicole
	Surface Conditioning Effect on Vacuum Microelectronics Components Fabricated by DRIE
	corresponding current density is given by the Richardson-
	Dushmann equation [5]:
	-² kT J AT e φ =
	where A is Richardson's constant, T is the temperature in Kelvin, φ is the metal work function and k is the
	Boltzmann constant.

Alain Phommahaxay, Gaëlle Lissorgues, Lionel Rousseau, Tarik Bourouina, Pierre Nicole Surface Conditioning Effect on Vacuum Microelectronics Components Fabricated by DRIE

AKNOWLEDGEMENT

This work is supported by the French Ministry of Defence (DGA) and Thales Airborne Systems. The authors would like to thank the ESIEE SMM team for their support and help during the fabrication process.