Pascal Clerc

Pauline Jeanjean

Nicolas Hallali

Michel Gougeon

Bernard Pipy

Julian Carrey

Daniel Fourmy

Véronique Gigoux
email: veronique.gigoux@insermfr

Daniel Founny

Targeted Magnetic Intra-Lysosomal Hyperthermia produces lysosomal reactive oxygen species and causes Caspase-1 dependent cell death

Introduction

Cancer is a leading cause of death with millions of new people di agnosed with cancer every year. One ma jor reason that limits outcome and drug response of anti cancer therapies is multidrug resistance (1). However, reoent studies have shown that cancer cells resistant to tra ditional therapies can be sensitive to agents which induce lysosomal cell death through lysosome membrane pa-meabilization (LMP) (2). In fact, LMP can be triggered by a wide variety of stimuli including death ligands, oxidative stress and treatment by lysosomotropic agents (3). In some circumstances (i.e. treatment by lysosomotropic agents), LMP appears to be an early event triggering apoptosis whereas in others (i.e. tre atment by death ligands), LMP results from the activation of apop tosis signaling pathways and contributes to the amplification of death signais.

Alternatively to these above agents, we (4) and others (5,6) have reoently proposed that Magnetic Intra Lysosomal Hyperthermia (termed MILH), induced by application of a high frequency altemating magnetic field (AMF) to cells containing magnetic nanoparticles (MNPs) in their lysosomes, is an effective way to specifi cally trigger lysosomal cell death in cancer cells [START_REF] Fourmy | Targeted nanoscale magnetic hyperthermia: challenges and potentials of peptide-based targeting[END_REF]. This is the result of the heat released by MNPs when exposed to AMF. Moreover, MNPs offer the potential to be driven into lysosomes of tumor cells by grafting them with ligands or antibodies that recognize receptors overexpressed in tumors. Subsequently to their activ ation, receptors are intemalized together with ligand grafted MNPs and traffic to lysosomes [START_REF] Sanchez | Targeting a G-proteincoupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death[END_REF][START_REF] Domenech | Lysosomal membrane permeabilization by targeted magnetic nanoparticles in alternating magnetic fields[END_REF]. Using this strategy, we showed that minute amounts of iron oxide MNPs targeting gastrin reoeptors (CCK2R), overexpressed in most endocrine tumors , caused the death of cancer cells upon AMF exposure, making this appro ach an attractive therapeutic option [START_REF] Sanchez | Targeting a G-proteincoupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death[END_REF][START_REF] Reubi | Targeting CCK receptors in human cancers[END_REF]. Strikingly, in this study, as in others, no perceptible temperature rise in the cell medium occurred during AMF exposure [ 4 6). Thus, MILH differs from standard magnetic hyperthermia whereby tumor eradication is achieved with large doses of MNPs which cause a temperature elevation of the whole tumor [START_REF] Silva | Application of hyperthermia induced by superparamagnetic iron oxide nanoparticles in glioma treatment[END_REF]. However, it cannot be excluded that MILH also contributes to cell death during standard magnetic hypa-thermi a experiments since small amounts of MNP injected intra tumourously could be intemalized by cancer cells. In MILH, it is expected that MNPs, even oflow heating power (S pecifi c Absorption Rate), generate a local temperature rise causing LMP (4 6,10). However, this ap pealin g hypothesis has never overnight and incubated with Gastrin MNPs (16 μg Fe/ml) for 24 h at 37 °C in medium buffered with 10 mM HEPES buffer pH 7.4 containing 0.5% FBS and 100 IU/ml penicillin streptomycin to allow Gastrin MNPs internalization and accumulation in lysosomes. Incubation medium was withdrawn and cells were rinsed twice with incubation medium and then incubated with 5 mM NAC, 1 mM DFO, 1 nM BafA1, 20 μM ART, 10 μM CA 074 Me, 2.5 μM Caspase 1 inhibitor or 10 μM Caspase 3 in hibitor for 1 h or 120 μM FeCl 3 for 4 h and exposed to AMF (275 kHz, 40 mT) for 2 h using a commercial magnetic inductor (Fives Celes, Lautenbach, France) as previously described [START_REF] Sanchez | Targeting a G-proteincoupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death[END_REF]. The temperature of the Cellview dish was maintained at 37.0 ± 0.2 °C and controlled using a thermal probe (Reflex, Neoptix, Canada) placed in the incuba tion medium of the cells. At the end of the experiments, cells were placed in a humidified atmosphere at 5% CO 2 and 37 °C. Different biological effects were analyzed following AMF exposure and sum marized in Supplementary Table 1.

Determination of cell death

The effects of AMF treatment were investigated as follows: first, cell survival was determined 24 h post AMF treatment using a MTT viability assay. Secondly, dead cells labeled with AnnexinV iFluor488 and pro pidium iodide (excitation: 488 and 540 nm respectively, AAT Bioquest) were counted 4 h after the end of AMF exposure as described before [START_REF] Sanchez | Targeting a G-proteincoupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death[END_REF]. To analyze cell death in INR1G9 CCK2R overexpressing GFP CathB or Casp1 fusion protein, INR1G9 CCK2R cells were seeded at a density of 25× 10 3 cells/cm 2 cells onto 35 mm dishes and transfected with 3 μg of pCathB eGFP, pCathB C29A eGFP (given by Klaudia Brix, Jacobs Uni versity Bremen, Germany) or pMSCV2.2 IRES GFP Caspase 1 C284A (given by Dr. Petr Broz, University of Basel, Switzerland) using Lipo fectamine 2000 reagent (Life technologies). 4 h post transfection, cells were seeded onto 4 compartment Cellview dishes (Greiner Bio One) at a density of 25× 10 3 cells/cm 2 , grown overnight and incubated with Gastrin MNPs (16 μg Fe/ml) for 24 h at 37 °C in medium buffered with 10 mM HEPES buffer pH 7.4 containing 0.5% FBS and 100 IU/ml pe nicillin streptomycin. Cells were rinsed twice with incubation medium, exposed to AMF for 2 h, labeled with AnnexinV iFluor555 (excitation: 540 nm, AAT Bioquest) 4 h after the end of AMF exposure and counted. Counting of labeled cells was carried out by analyzing confocal mi croscopy images (LSM510 confocal microscope, Zeiss) representing populations of 2000 3000 cells/experiment using ImageJ software.

Analysis of ROS production

INR1G9 CCK2R cells were seeded 24 h before the experiments onto 4 compartment Cellview dishes (Greiner Bio One) at a density of 25 to 50× 10 3 cells/cm 2 and incubated with Gastrin MNPs (16 μg Fe/ml) for 24 h. Cells were rinsed twice with incubation medium and incubated with 5 mM NAC, 1 mM DFO, 1 nM BafA1, 20 μM ART for 1 h or 120 μM FeCl3 for 4 h. For total ROS production analysis, cells were exposed to AMF for 2 h, rinsed with incubation medium, incubated with CellROX Green reagent (Molecular probes, Excitation wavelegnth: 488 nm), immediately after AMF exposure, in incubation medium according to manufacturer's instructions and quantification of ROS production was performed by analyzing the intensity of CellROX Green reagent labeling of confocal microscopy images (LSM780 confocal microscope, Zeiss) using Image J software. 2000 3000 cells/experiments were analyzed in at least 4 independent experiments. For initial ROS production analysis, cells having or not internalized Gastrin MNPs were incubated in the presence or absence of 1 mM DFO, 1 nM BafA1, 20 μM ART or 120 μM FeCl3 and then with CellROX Green reagent (Molecular probes, Excitation: 488 nm) in incubation medium according to manufacturer's instructions, exposed to AMF for 30 min, rinsed with incubation medium and ROS production was analyzed using confocal microscopy (LSM780 confocal microscope, Zeiss) immediately after AMF exposure. Initial ROS production was quantified by fluorescence intensity ratio of been directly proven experimentally. Moreover, the precise mechan isms leading to cell death by MILH remains largely unknown. Insight into these mechanisms is essential to optimize this approach in a therapeutic perspective.

To date, molecular mechanisms of lysosomal cell death of epithelial cells were mainly elucidated using lysosomotropic agents or death li gands which do not exclusively target lysosomes of cancer cells [11 13]. Lysosomal cell death through apoptosis was demonstrated to involve the release of lysosomal hydrolases into the cytosol, especially Cathepsin D and Cathepsin B (CathB) which digest apoptosis regulated proteins, leading to mitochondrial outer membrane permeabilization (MOMP) and apoptotic caspases activation [START_REF] Repnik | Lysosomal membrane permeabilization in cell death: concepts and challenges[END_REF][START_REF] Cirman | Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins[END_REF].

The aim of this study was to elucidate the cellular and molecular mechanisms involved in cancer cell death induced by MILH. Here, we report that, under an AMF, lysosome accumulated MNPs increase the temperature very locally and enhance reactive oxygen species (ROS) production through the Fenton reaction within lysosomes. This causes lipid peroxidation of lysosome membrane, LMP and subsequent cell death by a non conventional mechanism which is dependent on Caspase 1 and CathB but independent of apoptotic Caspase 3. These major advances in the understanding of the cell death mechanism oc curring during MILH will facilitate optimization of the strategy in a therapeutic perspective.

Material and methods

Nanoparticles functionalization

The synthesis method and characterization of the magnetic nano platform used in the present article have been previously described [START_REF] Sanchez | Targeting a G-proteincoupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death[END_REF]. The nanoplatform (Gastrin MNP) is composed of commercial iron oxide magnetic nanoparticles (MNPs) coated with PEG Amine (Gecco Dots, Sweden) and decorated with 100 molecules of a synthetic replicate of gastrin (Covalab) and 20 molecules of the fluorescent label NHS DY647 PEG1 (Dyomics GmbH, Jena, Germany). The size of the mag netic core determined by transmission electron microscopy was 8.7 ± 1.6 nm. The specific absorption rate of these MNPs is 13 W/g Fe at 275 kHz and 40 mT. N acetyl cystein (NAC), Desferroxamine (DFO), Bafilomycine A (BafA1), Artesunate (ART) and FeCl 3 were purchased from Sigma Aldrich. CA 074 Me and Caspase 3 inhibitors were from ApexBio. Caspase 1 inhibitor was from Santa Cruz Biotechnologies.

Cell lines

The glucagon producing Hamster tumoral cell line INR1G9 stably expressing CCK2R (INR1G9 CCK2R) obtained as previously described [START_REF] Leung-Theung-Long | Essential interaction of Egr-1 at an islet-specific response element for basal and gastrin-dependent glucagon gene transactivation in pancreatic alpha-cells[END_REF] and the Thp1 human macrophage/monocyte cell line were cul tured in RPMI1640 medium containing 10% fetal bovine serum (FBS) and 100 IU/ml penicillin/streptomycin (Life technologies). The HEK293 cells (Flp In system, Invitrogen) stably expressing the CCK2R, named HEK CCK2R, and the clone B13 of AR4 2J cells (kindly provided by Pr Timo Otonkoski, University of Helsinki, Finland, with the per mission of Dr. Itaru Kojima, Gunma University, Maebashi, Japan) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 100 IU/ml penicillin/streptomycin (Life technologies). The AGS gastric adenocarcinoma cell line was permanently transfected with CCK2R (AGS CCK2R) driven by the EF1 α promoter under puromycin selection and grown in DMEM/F 12 medium supplemented with 10% FBS and 100 IU/ml penicillin/streptomycin [START_REF] Watson | Transcriptional activation of the rat vesicular monoamine transporter 2 promoter in gastric epithelial cells: regulation by gastrin[END_REF]. The cells were grown in a humidified atmosphere at 95% air and 5% CO 2 at 37 °C.

Cell treatment by alternating magnetic field

Cell treatment by alternating magnetic field (AMF) was performed as follows. Cells were seeded into 4 compartment Cellview dishes (Greiner Bio One) at a density of 25 to 50 × 10 3 cells/cm 2 , grown CellROX Green reagent to Gastrin MNP labeling of confocal microscopy images (LSM780 confocal microscope, Zeiss) using Image J software. 20 30 cells/experiment were analyzed from at least 4 independent experiments.

Local heat quantification using molecular thermometers

INR1G9 CCK2R cells were seeded at a density of 25 × 10 3 cells/cm 2 onto 35 mm dishes and transfected with 0.1 μg of YFP Lamp1 (Addgene, plasmid #1816) and 2.9 μg of pcDNA3 using Lipofectamine 2000 reagent (Life technologies). 24 h post transfection, cells were in cubated with Gastrin MNP DY549 (16 μg Fe/ml) for 24 h, included in 0.5% agarose gel. For these experiments, the AMF was produced by a miniaturized home made electromagnet placed in a CELLView dish and covered with RPMI containing 0.5% FBS, as previously described [START_REF] Connord | Real-time analysis of magnetic hyperthermia experiments on living cells under a confocal microscope[END_REF]. This permits to follow in real time the evolution of the fluorescence of the MNPs under the application of AMF and thus to have an access to the local temperature. For these experiments, different positions were marked inside and outside the gap. Cells were exposed to the AMF (53 mT, 300 kHz). Before and during AMF application, fluorescence intensities of DY549 (excitation: 540 nm) and YFP Lamp1 (excitation: 510 nm) were measured from confocal microscopy images LSM780 confocal microscope, Zeiss) using ImageJ software. 20 30 cells/ex periment were analyzed from 4 independent experiments.

CathB leakage from lysosome

INR1G9 CCK2R cells were seeded at a density of 25 × 10 3 cells/cm 2 onto 35 mm dishes and transfected with 0.1 μg of RFP Lamp1 (Addgene, plasmid#1816), pCathB eGFP (given by Klaudia Brix, Jacobs University Bremen, Germany) and 2.9 μg of pcDNA3 using Lipofectamine 2000 reagent (Life technologies). 4 h post transfection, cells were seeded onto 4 compartment Cellview dishes (Greiner Bio One) at a density of 25 × 10 3 cells/cm 2 , grown overnight and in cubated with Gastrin MNPs (16 μg Fe/ml) for 24 h, rinsed twice with incubation medium and exposed to AMF for 2 h. CathB leakage from lysosome was analyzed by measuring the colocalization between the GFP CathB and RFP Lamp1 proteins and the fluorescence intensity ratio of GFP CathB to RFP Lamp1 labelings (excitation: 488 and 540 nm re spectively) of confocal microscopy images (LSM780 confocal micro scope, Zeiss, ImageJ software) immediately after AMF exposure. 20 30 cells/experiment were analyzed from 4 independent experiments.

For real time analysis of CathB leakage, INR1G9 CCK2R cells were seeded at a density of 25 × 10 3 cells/cm 2 onto 35 mm dishes and transfected with 0.1 μg of pCathB eGFP (given by Klaudia Brix, Jacobs University Bremen, Germany), 0.1 μg RFP Lamp1 (Addgene, plasmid #1817) and 2.8 μg of pcDNA3 using Lipofectamine 2000 reagent (Life technologies). 24 h post transfection, cells were incubated with Gastrin MNPs (16 μg Fe/ml) for 24 h. INR1G9 CCK2R cells expressing or not GFP CathB and having or not internalized Gastrin MNP were included in 0.5% agarose gel, poured into the electromagnet placed in a CELLView dish and covered with RPMI containing 0.5% FBS, as pre viously described [START_REF] Connord | Real-time analysis of magnetic hyperthermia experiments on living cells under a confocal microscope[END_REF]. Different positions were marked inside and outside the gap. Cells were exposed to the AMF (53 mT, 300 kHz) for 60 min. Before and during AMF application, CathB leakage from lyso some was analyzed by measuring the colocalization between the GFP CathB and RFP Lamp1 proteins labeling from confocal microscopy images of the marked positions by measuring the % of green pixels (GFP CathB) colocalized with red pixels (RFP Lamp1) (ImageJ soft ware). 20 30 cells/experiment were analyzed from 4 independent ex periments.

Analysis of Caspase 1 and 3 activation

Cells were seeded 24 h before the experiments onto 4 compartments Cellview dishes (Greiner Bio One) at a density of 25 to 50 × 10 3 cells/ cm 2 and incubated with Gastrin MNPs (16 μg Fe/ml) for 24 h and ex posed to AMF for 2 h. Immediately, 3 or 17 h after AMF exposure, cells were incubated with Fluorochrome Labeled Inhibitors of Caspase 1 (FLICA, FAM YVAD FMK, excitation: 488 nm) or Caspase 3 (FLICA, FAM DEVD FMK, excitation: 488 nm) reagent (Immunochemistry technologies) for 1 h according to manufacturer's instructions. As a positive control for Caspase 3 activation, INR1G9 CCK2R cells were incubated with 1 μM staurosporine for 4 h. In some experiments, cells were pretreated with 2.5 μM Caspase 1 inhibitor, 10 μM Caspase 3 in hibitor or 10 μM CA 074 Me CathB inhibitor for 1 h. Inhibitors were washed from the cells before adding FLICA. Next, cells were washed and analyzed by confocal micrsocopy (LSM510 confocal microscope, Zeiss). After Caspase 1 staining, cells were labeled with AnnexinV iFluor555 according to manufacturer's instruction (AAT Bioquest). Counting of labeled cells was carried out by analyzing confocal mi croscopy images representing populations of 2000 3000 cells/experi ment using ImageJ software.

Statistical analysis

Results are expressed as the mean ± SEM of at least three in dependent experiments. Statistical analysis was performed using ANOVA test. Differences were considered significant when p < 0.05. and pharmacological agents were carried out. Firstly, analysis of con focal microscopy images indicated that fluorescent ROS sensor co lo calized with Gastrin MNPs in lysosomes after 30 min of AMF exposure (Fig. 1c). Quantification of lysosomal ROS images showed that MILH enhanced ROS level by 2.0 ± 0.2 fold in the vicinity of Gastrin MNPs within lysosomes (% of relative fluorescence Gastrin MNP/ROS: 48.5 ± 3.9% vs 24.8 ± 1.1%). Moreover, addition of FeCl 3 aug mented MILH induced lysosomal ROS production by 1.3 ± 0.1 fold (% of relative fluorescence Gastrin MNP/ROS: 60.7 ± 6.1% vs 48.5 ± 3.9%), wheareas DFO inhibited it (37.8 ± 5.8%). Variation of lysosomal pH also modified lysosmal ROS production. Indeed, while ART treatment increased MILH induced lysosomal ROS production by 1.7 ± 0.1 fold (% of relative fluorescence Gastrin MNP/ROS: 80.9 ± 8.5% vs 48.5 ± 3.9%), BafA1 inhibited it (24.7 ± 4.2%). Together, these results demonstrate that MILH initially generates ROS through the Fenton reaction within lysosomes where Gastrin MNPs are accumulated.

However, as the ROS production can occur in other subcellular sites, we also determined whether MILH also generates ROS by activating, for instance, the mitochondrial respiratory chain complex or NADPH oxi dases which are transmembrane proteins mostly present at the plasma membrane, in endosomes and in endoplasmic reticulum [START_REF] Dikalov | Cross talk between mitochondria and NADPH oxidases, Free Radic[END_REF]. We found that rotenone, an inhibitor of the mitochondrial respiratory chain complex, did not modify cellular ROS generation by MILH, whereas diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, de creased it (5.9 ± 1.8 and 3.8 ± 1.3 fold over basal value in the presence of rotenone or DPI, respectively versus 5.7 ± 1.5 fold) (Fig. S2). Hence, MILH initially triggers ROS production through the Fenton reaction within lysosomes and, likely secondary, through NADPH oxi dases. Of note, no significant variation in ROS level was observed be tween the different conditions in the absence of AMF exposure (Fig. S1a,S2).

We further investigated the mechanism whereby MILH enhanced the Fenton reaction in lysosomes. Firstly, we examined the possibility that Fe ions, participating in the Fenton reaction, could originate from MNPs dissolution in lysosomes during AMF exposure. Indeed, it has been reported that iron oxide MNPs, in the absence of AMF application, are degraded through a lysosomal acidic and enzymatic lysis in the endo lysosome compartment [START_REF] Mazuel | Massive intracellular biodegradation of iron oxide nanoparticles evidenced magnetically at single-endosome and tissue levels[END_REF][START_REF] Mazuel | Magneto-thermal metrics can mirror the long-term intracellular fate of magneto-plasmonic nanohybrids and reveal the remarkable shielding effect of gold[END_REF]. On the other hand, another study performed at neutral pH, attributed ROS production to the catalytic reaction at the surface of MNPs rather than to being caused by dissolved iron from MNPs [START_REF] Wydra | The role of ROS generation from magnetic nanoparticles in an alternating magnetic field on cytotoxicity[END_REF]. Using particle electron paramagnetic resonance (pEPR) to determine the quantity of iron contained in the cells under the form of MNPs, we found that internalized Gastrin MNPs were not degraded following AMF application (0.94 ± 0.06 vs 0.92 ± 0.03 pg magnetic Fe/cell) (Fig. 1d). However, we cannot exclude that iron could have been released from Gastrin MNPs during their 24 h traf ficking in the endo lysosome compartment prior to magnetic field ap plication. Consequently, these results indicate that MILH induced ROS production was the result of enhanced Fenton reaction catalysis using cellular lysosomal iron or possibly iron from MNPs, dissolved most likely before AMF application.

Secondly, we evaluated the temperature rise during MILH experi ments. So far, our experimental data indicate that the death of cancer cells in MILH occurs without any perceptible rise in the global tem perature of the medium. The hypothesis that local heating occurs at proximity of MNPs has been supported by several experimental reports [START_REF] Riedinger | Subnanometer local temperature probing and remotely controlled drug release based on azo-functionalized iron oxide nanoparticles[END_REF], but direct data accounting for such a phenomenon in MILH were still lacking. In particular, no intracellular or intra lysosomal tem perature measurement under magnetic hyperthermia experiments has been reported so far in the literature. We therefore conducted such experiments, using fluorescent molecular thermometers and the pre viously described miniaturized high frequency electromagnet which enables confocal fluorescence imaging of cells during MILH experi ments [START_REF] Connord | Real-time analysis of magnetic hyperthermia experiments on living cells under a confocal microscope[END_REF]. Molecular thermometers were composed of the fluorescent moieties DY549 attached to MNPs via PolyEthyleneGlycol of 7 nm 2.10. Lipid peroxidation, mitochondrial membrane potential assay, DNA fragmentation, Il 1β secretion, Western blotting, analysis of lysosomal acidity See Supplementary information and Supplementary Table 1.

Results

Magnetic Intra Lysosomal Hyperthermia (MILH) enhances ROS production through the Fenton reaction within lysosomes

Over expression of hormone receptors is a hallmark of neuroendo crine tumors which thus represent a perfect model for the experi mentation of tumor cell targeting with ligand grafted MNPs. We pre viously showed that Gastrin grafted iron oxide MNPs (termed Gastrin MNP) are internalized specifically by the pancreatic endocrine tumoral cells INR1G9 overexpressing the CCK2R (INR1G9 CCK2R cells) through a CCK2R dependent physiological process, and are then trafficked to lysosomes where they accumulated (Fig. 1a). Of note, Gastrin MNPs exist under nano objects containing nanoparticles and serum adsorbed proteins whose distribution slightly increased from a size around 37 to 50 nm after 24 h of incubation in cell culture medium (data not shown). Upon AMF exposure, Gastrin MNPs killed 32.3 ± 2.7% of INR1G9 CCK2R cells by a phenomenon that we termed "Magnetic Intra Lyso somal Hyperthermia" (MILH) [START_REF] Sanchez | Targeting a G-proteincoupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death[END_REF] (Fig. 1a). 4 h after MILH treatment, the rate of cell damage reached 19.9 ± 1.5% (i.e. 3.5 ± 0.3 fold basal) as indicated by AnnexinV and propidium iodide (AnnV/PI) la belings (Fig. 1a). Both AnnV and PI labelings, which constitute early features of MILH cell death, were thus used throughout our study to appreciate cell death.

So far, our current knowledge on the mechanism of cancer cell death caused by MILH treatment is still elusive, although we and others identified cellular ROS production and lysosome leakage as biological events accompanying cell death [START_REF] Sanchez | Targeting a G-proteincoupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death[END_REF][START_REF] Domenech | Lysosomal membrane permeabilization by targeted magnetic nanoparticles in alternating magnetic fields[END_REF][START_REF] Connord | Real-time analysis of magnetic hyperthermia experiments on living cells under a confocal microscope[END_REF]. To gain insight into cell death mechanisms, we hypothesized that AMF application to cells containing MNPs would generate a local temperature elevation and, inasmuch as MNPs are concentrated into lysosomes, heating of lysosome content would trigger a death signaling cascade initiated by the enhancement of ROS production within lysosomes. Indeed, in addition to being a major digestive compartment, lysosomes are well known major sites of ROS production through the Fenton reaction (Fe 2+ +H 2 O 2 → Fe 3+ +OH +OH % ) which catalyzes the transformation of hydrogen peroxide to hydroxyl radicals. This reaction is dependent on low mo lecular weight iron derived from degraded iron containing proteins, lysosomal lumen acidity and temperature [18 20]. It is also known that high levels of ROS cause lysosome membrane permeabilization (LMP) whereby lysosomal cathepsins, as well as other hydrolytic enzymes, are released from the lysosomal lumen to the cytosol and trigger cell death (Fig. 1a b) [START_REF] Boya | Lysosomal membrane permeabilization in cell death[END_REF]. In INR1G9 CCK2R cells, MILH increased ROS levels by 5.7 ± 1.5 fold over the basal value (Fig. 1b,S1a). Such effect is po tentiated by the addition of FeCl 3 which increased ROS levels to 7.5 ± 0.7 fold. In contrast, intralysosomal chelation of iron by Des ferrioxamine (DFO) [START_REF] Cable | Cellular uptake and release of two contrasting iron chelators[END_REF] prevented MILH increased cellular ROS pro duction which returned nearly to the basal value (2.0 ± 0.5 fold over basal). Also, the inhibition of the vacuolar H + ATPase pump by Bafi lomicyne A (BafA1) treatment which caused intralysosomal pH rise (Fig. S1b) decreased MILH induced cellular ROS level to 2.7 ± 0.4 fold over basal value (Fig. 1b,S1a). Conversely, treatment of cells by Artesunate (ART), an activator of vacuolar H + ATPase pump de creasing intralysosomal pH (Fig. S1b) [START_REF] Yang | Artesunate induces cell death in human cancer cells via enhancing lysosomal function and lysosomal degradation of ferritin[END_REF], increased MILH enhanced cellular ROS production from 5.7 ± 1.5 to 8.3 ± 0.3 fold over basal value (Fig. 1b,S1a).

We previously showed that ROS production was significantly de tectable after 30 min of AMF exposure [START_REF] Connord | Real-time analysis of magnetic hyperthermia experiments on living cells under a confocal microscope[END_REF]. To precisely identify subcellular site(s) of ROS which are initially produced in MILH condi tion, a series of experiments combining confocal microscopy imaging length or the Yellow Fluorescent Protein (YFP) linked to Lysosome Associated Membrane Protein 1 (Lamp1) [START_REF] Sanchez | Targeting a G-proteincoupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death[END_REF]. Intensity of fluorescence emission of the two probes was previously shown to depend on tem perature [START_REF] Huang | Remote control of ion channels and neurons through magnetic-field heating of nanoparticles[END_REF]. This relationship was confirmed by the calibration curve showing linearity of fluorescence intensity decrease as a function of temperature increase (Fig. S3). Quantitative fluorescence imaging was then carried out on cells exposed to AMF for a limited duration (˂10 min) and incubated with ROS scavenger NAC in order to preserve ly sosome integrity and avoid possible chemical quenching of the fluor escent molecular thermometers by ROS. As shown on Fig. 1e, fluores cence intensity of DY549 grafted to MNPs declined with the time of AMF exposure. According to the calibration curve (Fig. S3), the per centage of decrease of fluorescence intensity observed after ~7 min of treatment corresponds to a temperature increase of 14.1 ± 1.4 °C, indicating that the temperature actually reached 51 °C at 7 nm distance from the MNP core. However, fluorescence intensity of YFP Lamp1 did not significantly change during MILH experiments (Fig. 1e). These re sults strongly support that AMF exposure to MNPs that have been ac cumulated in lysosomes causes a significant temperature increase at the immediate vicinity of the MNPs since it is not detected at the outer surface of lysosomes.

MILH enhanced ROS production within lysosomes causes lysosomal membrane permeabilization and cell death

We further assessed the relationship between ROS generation within lysosomes and cell damage. ROS can destabilize and permeabilize ly sosome membranes through lipid and protein peroxidation [START_REF] Repnik | Lysosomal membrane permeabilization in cell death: concepts and challenges[END_REF]. We performed experiments combining confocal microscopy imaging and pharmacological agents to analyze whether MILH induces the perox idation of lysosomal lipids (Fig. 2a). Analysis of confocal microscopy images indicated that MILH enhanced the level of lipid peroxidation which was detectable from 30 min of AMF application, and co localized with Gastrin MNPs (% of relative fluorescence: 74.4 ± 2.7% vs 41.5 ± 5.6%). Moreover, intralysosomal chelation of iron by DFO or BafA1 treatment decreased MILH induced lysosomal lipid peroxidation (54.4 ± 3.1% and 48.7 ± 15.1%). Together, these results demon strate that MILH induces lysosomal lipid peroxidation concomitantly with lysosomal ROS production.

Since MILH stimulates lysosomal ROS production and lipid perox ydation, it appeared likely that LMP subsequently occurs. Previously, we documented that, in MILH experiments, lysosomal integrity was affected as suggested by the decrease of lysosome labeling by LysoTracker. However, the criterium of LysoTracker labeling is insufficient to characterize LMP since it may account for leaking of low molecular weight components and not lysosomal enzymes which are key actors in lysosome functions. Herein, we determined whether ROS production in lysosomes truly triggered LMP. The decrease of CathB content in lysosomes (labeled with RFP Lamp1) was chosen to re present lysosomal enzyme leakage (Fig. 2b). In absence of AMF appli cation, INR1G9 CCK2R cells having internalized Gastrin MNPs pre sented a high level of GFP CathB/RFP Lamp1 colocalization which was reduced by the application of AMF (Pearson coefficient: 0.54 ± 0.03 vs 0.69 ± 0.02 in presence vs absence of AMF). The effect of MILH on GFP CathB/RFP Lamp1 colocalization was cancelled in the presence of the NAC scavenger (Pearson coefficient: 0.74 ± 0.03, Fig. 2b,S4). Moreover, quantification of fluorescence intensity showed that MILH decreased lysosomal CathB levels by 19.2 ± 3.7% (% of relative fluorescence: 30.7 ± 3.9% vs 50.5 ± 3.8%), whereas addition of NAC prevented it (50.3 ± 5.8%). These results demonstrate that lysosomal leakage was entirely related to MILH enhanced ROS production.

We then determined whether the leakage of CathB from lysosomes constituted an early event in the signaling cascade leading to cell death. During MILH experiments performed using the miniaturized electro magnet [START_REF] Connord | Real-time analysis of magnetic hyperthermia experiments on living cells under a confocal microscope[END_REF], we evaluated in real time the decrease of the colocaliza tion of GFP CathB with RFP Lamp1, a protein resident of lysosome membrane (Fig. 2c). Before or after AMF exposure, INR1G9 CCK2R cells having internalized Gastrin MNPs presented a high level of GFP CathB/RFP Lamp1 colocalization (Pearson coefficient > 0.70). How ever, the lysosomal CathB level decreased significantly by 25.4 ± 4.7% and 43.9 ± 6.3% after 30 and 60 min of MILH treat ment respectively, demonstrating that leakage of lysosome content is an early event occurring concomitantly with the generation of lysosomal ROS. Of note, RFP Lamp1 and GFP CathB fluorescence intensities, other LMP markers [START_REF] Xu | Protective mechanisms of CA074-me (other than cathepsin-B inhibition) against programmed necrosis induced by global cerebral ischemia/reperfusion injury in rats[END_REF], decreased respectively by 20 ± 2.4% and 16.5 ± 6.6% from 30 min of AMF exposure (Fig. S7), strongly sug gesting that the changes in RFP Lamp1 and GFP CathB fluorescence represent respectively lysosomal rupture and CathB leakage from ly sosome into the cytosol and that both events occur early in the signaling cascade leading to cell death.

Finally, we wished to establish the relationship between ROS gen eration within lysosomes and cell death MILH experiments. We found that the addition of FeCl 3 or ART, which enhance ROS production in the cell medium, augmented cell death induced by MILH (4.0 ± 0.3 and 4.1 ± 0.4 fold over basal value respectively vs 2.6 ± 0.2 fold in presence of Gastrin MNP alone, Fig. 2d,S5). In contrast, NAC, DFO and BafA1 treatments which trapped ROS or inhibited their formation, prevented MILH induced cell death (0.8 ± 0.1, 1.4 ± 0.1 and

GFP-CathB-WT

+ + + +
1.3 ± 0.1 fold over basal value respectively) (Fig. 2d, S4 S5). More over, MILH induced cell death was not inhibited by rotenone or DPI (Fig. S6), indicating that neither the mitochondria respiratory chain complex nor NADPH oxidases were involved in cell death, further supporting the idea that ROS generated by NADPH oxidases are sec ondary to lysosomal stress caused by MILH treatment. All together, these results demonstrate that lysosomal ROS production, which is enhanced by MILH through the Fenton reaction, is at the origin of cancer cell death.

Cancer cell death induced by MILH is dependent on CathB activity

In a previous study, we showed that MILH induced cell death de pends on lysosomal cysteine cathepsins which include cathepsins B, C, H, K, L, S and X [START_REF] Sanchez | Targeting a G-proteincoupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death[END_REF]. Herein, we performed experiments to more pre cisely identify the enzyme(s) involved. A likely candidate is CathB which is involved in lysosomal cell death, abundant in lysosomes, overexpressed in cancers and maintains its activity at neutral pH [30 32]. MILH experiments were performed in the presence of the se lective inhibitor of CathB CA 074 Me [START_REF] Buttle | CA074 methyl ester: a proinhibitor for intracellular cathepsin B[END_REF] or by overexpressing the enzymatically inactive mutant GFP CathB C29A. As shown in Fig. 3a b, both the inhibitor CA 074 Me and the inactive mutant GFP CathB C29A prevented MILH induced cell death (1.2 ± 0.1 and 1.5 ± 0.1 fold over basal value vs 2.5 ± 0.1 fold). In contrast, overexpression of wild type CathB (GFP CathB WT) augmented the efficiency of MILH to era dicate cancer cells (4.1 ± 0.2 vs 3.0 ± 0.3 fold over basal, Fig. 3c) reaching 34.8% of dead cells comparatively to 17.3% of dead cells in the absence of CathB WT overexpression. These results indicate that cell death induced by MILH is dependent on CathB activity.

MILH induces cell death through a Caspase 1 dependent but Caspase 3 independent mechanism

So far, in most circumstances, partial LMP induce cell death through an apoptotic pathway, by triggering a cascade of regulated molecular events leading to activation of the effector apoptotic enzyme Caspase 3 [START_REF] Repnik | Lysosomal membrane permeabilization in cell death: concepts and challenges[END_REF][START_REF] Cirman | Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins[END_REF]. We therefore investigated whether MILH also induces cell death through an apoptotic pathway. Surprisingly, the Caspase 3 inhibitor did not inhibit MILH induced cell death (2.7 ± 0.2% vs 2.8 ± 0.2 fold over basal) whereas it prevented cell death induced by staurosporine used as a reference agent to stimulate apoptosis (Fig. 4a). Moreover, Caspase 3 activation was not detected neither by confocal microscopy using a fluorescent substrate (Fig. 4b), nor by western blot as shown by the absence of Pro Caspase 3 cleavage (Fig. S8). We also showed that MILH did not modify mitochondrial outer membrane permeabilization (MOMP), which critically relies on apoptotic cell death, as demon strated with staurosporine treatment (Fig. 4c,S9), 4 h or 24 h after AMF exposure [34]. Hence, cell death induced by MILH does not involve an apoptotic pathway.

In some non epithelial cells, especially in macrophages exposed to infectious agents, the release of lysosomal hydrolases such as CathB induced after LMP was shown to activate a non apoptotic cell death pathway involving Caspase 1 activation [START_REF] Galluzzi | Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012[END_REF]. We therefore verified Caspase 1 expression in INR1G9 CCK2R cells (Fig. S10) and determined whether Caspase 1 was involved in MILH induced tumor cell death.

Incubation of INR1G9 CCK2R cells with Caspase 1 inhibitor prevented cell death induced by MILH (1.6 ± 0.2 vs 2.8 ± 0.2 fold over basal, Fig. 4d). Likewise, overexpression of Caspase 1 C284A inactive mutant inhibited cell death (1.5 ± 0.2 vs 3.0 ± 0.2 fold over basal, Fig. 4e). It was also noticed that overexpression of Caspase 1 C284A inactive mutant lowered basal rate of cell death (amount of dead cells: 1.2 ± 0.4% vs 6.5 ± 0.9%). Moreover, MILH increased Caspase 1 activation (3.5 ± 0.4, 9.0 ± 1.3 and 12.5 ± 2.4 fold over basal 1 h, 4 h and 18 h after AMF exposure) and this activation was inhibited by CathB inhibitor CA 074 Me (Fig. 4f,S11). It is also important to note that 84.5 ± 2.3% of cells presenting an upregulated Caspase 1 activity were labeled with AnnexinV (Fig. 4g). Taken together, these results demonstrate that cell death induced by MILH does not involve an apoptotic pathway, but requires Caspase 1 and Cath B activities.

An important function of Caspase 1 consists to process the precursor of the inflammatory cytokines, interleukine 1β (Il1β) and interleukine 18 (Il 18) into their active forms [START_REF] Fantuzzi | Interleukin-18 and interleukin-1 beta: two cytokine substrates for ICE (caspase-1)[END_REF]. Furthermore, activation of Caspase 1 in cells is a hallmark of pyroptosis, which is a form of regulated cell death, usually defined by several additional biochemical features such as independence of apoptotic caspases, Ann/PI positive labeling, DNA fragmentation [START_REF] Miao | Caspase-1-induced pyroptotic cell death[END_REF]. The current study demonstrates that MILH induced cell death is dependent on Caspase 1 activation and featured by Ann/PI labeling. Owing to functions of Caspase 1 in im mune cells, we first investigated whether MILH induced Caspase 1 ac tivation was associated with Il1β secretion (Fig. 5a). Our results show that, in INR1G9 CCK2R cells which were LPS primed or not, MILH did not increase Il1β secretion relative to control cells. In contrast, in LPS primed Thp1 macrophages, which were used as reference cells for Il1β processing and secretion, levels of Il1β secretion were increased by 27.7 ± 3.2 fold over basal value. The levels of Il1β secretion by INR1G9 CCK2R cells were very low (< 2 pg/mg of cell proteins) as was that of Pro Il1β expression, comparatively to macrophages cells (Fig. 5b). Hence, MILH induces Caspase 1 dependent cell death without activating the Caspase 1 pro inflammatory response in the endocrine tumoral INR1G9 CCK2R cells. However, we could not exclude that pro inflammatory response could be activated by MILH in cancer cells ex pressing higher level of Pro Il1β.

Then, we characterized DNA fragmentation caused by MILH using TUNEL assay which consists in the incorporation of fluorescent dUTP (deoxyuridine triphosphate) into free 3′ hydroxyl termini generated by genomic DNA fragmentation (Fig. 5c). Results indicate that, after 6 h of AMF exposure, DNA fragmentation increased by 3.4 ± 0.5 fold re lative to control cells (22.9 ± 3.8% vs 8.5 ± 1.4% of positive cells). Of note, DNA fragmentation induced by MILH was less abundant than that caused by staurosporine or by 42 °C water bath (40.1 ± 4.9% or 52.6 ± 14.0% of positive cells, respectively). Secondly, we char acterized the type of DNA fragmentation caused by MILH (Fig. 5d). In contrast to staurosporine treatment which provoked regular inter nu cleosomic DNA fragmentation characterized by DNA ladder [START_REF] Miao | Caspase-1-induced pyroptotic cell death[END_REF], MILH did not induce the formation of the characteristic laddering pattern associated with apoptosis. In conclusion, our results show that MILH induced a non apoptotic Caspase 1 dependent cell death related to pyroptosis which occurred without Il1β secretion and which was characterized by the formation of TUNEL positive cells without ex hibiting a laddering pattern [START_REF] Miao | Caspase-1-induced pyroptotic cell death[END_REF][START_REF] Watson | Salmonella enterica serovars typhimurium and Dublin can lyse macrophages by a mechanism distinct from apoptosis[END_REF]. .,. ... cells were killed using 16 μg/ml or 1 μg Fe/ml of Gastrin MNP re spectively (Fig. S13a b). Ann/PI labeling confirmed that MILH induced cell death was detectable 4 h post AMF (Fig. 6b) and dead cells were more often labeled with PI in AGS CCK2R and AR4 2 J cells than in INR1G9 CCK2R cells. MILH also triggered cell death in the non tumoral embryonic kidney cell overexpressing the CCK2R (HEK CCK2R: 2.0 ± 0.2 pmol/10 6 cells) previously used to characterize uptake and cellular trafficking of Gastrin MNPs (Fig. S13c) [START_REF] Sanchez | Targeting a G-proteincoupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death[END_REF][START_REF] Magnan | Regulation of membrane cholecystokinin-2 receptor by agonists enables classification of partial agonists as biased agonists[END_REF].

Determination of the uptake of Gastrin MNPs by the different cells indicates that receptor mediated uptake of MNPs by AGS CCK2R, AR4 2J and INR1G9 CCK2R cells was 0.83 ± 0.23, 0.60 ± 0.13 and 3.38 ± 0.74 pg of Fe/cell which corresponded to 0.52 ± 0.22, 0.14 ± 0.02 and 1.07 ± 0.28 pg of magnetic Fe/cell, respectively, as measured by pEPR, after 24 h of intracellular accumulation (Fig. 6c). Of note, in HEK CCK2R cells, Gastrin MNPs uptake was evaluated to be 4.01 ± 0.40 and 1.48 ± 0.22 pg of total and magnetic Fe/cell, re spectively (Fig. S13d).

We finally assessed that the signaling pathway involved in MILH induced cell death in AGS CCK2R, AR4 2J and HEK CCK2R cells was similar to that identified in INR1G9 CCK2R. Firstly, no significant Caspase 3 activation could be detected in the 3 cell lines, similarly to INR1G9 CCK2R cells (Fig. S13e,S14). Secondly, MILH increased Caspase 1 activation, 4 h after AMF exposure, by 3.5 ± 0.1, 3.6 ± 0.2 and 6.2 ± 0.6 fold in AGS CCK2R, AR4 2J and HEK CCK2R respec tively, similarly to INR1G9 CCK2R cells (Fig. 6d,S13f).

These above results demonstrate that MILH is effective in inducing the death of cancer cells from three different types of cancer (pancreatic endocrine, pancreatic exocrine and gastric) even if the targeted re ceptor is expressed at low levels and if minute amounts of Gastrin MNPs are internalized. Moreover, in the three cancers, as well as in HEK cells, MILH induced cell death occurs through activation of Caspase 1 and without activation of Caspase 3, indicating that MILH triggers a non apoptotic cell death pathway following lysosome damage which is de pendent on Caspase 1 activation.

Discussion

Following the publication of the proof of concept of anti cancer nanotherapy using ligand grafted MNPs specifically internalized via cell surface receptor, this study gained insight into the mechanisms at the origin of cell death induced by MILH (Fig. 6e). Using Gastrin MNPs which specifically target lysosomes of tumoral cells expressing the CCK2R as a killing agent, we demonstrate that tumor cell death induced by MILH occurs through a Caspase 1 dependent mechanism, but not an apoptotic signaling pathway, since Caspase 3 was not activated. This mechanism of MILH induced cell death was demonstrated in pancreatic endocrine tumor cells as well as in gastric and pancreatic exocrine tumor cells expressing the CCK2R.

MILH first upregulates the catalysis of ROS production through the Fenton type reaction within lysosomes which subsequently causes lipid peroxidation, followed by LMP and the leakage of lysosomal content into the cytosol. Among the lysosomal enzymes, CathB plays a critical role in cell death and activates Caspase 1. Thus, MILH induced tumor cell death can be related to pyroptosis mainly described in macro phages, but rarely in epithelial cells [START_REF] Galluzzi | Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012[END_REF][START_REF] Tseng | NLRP3 inflammasome activation in retinal pigment epithelial cells by lysosomal destabilization: implications for age-related macular degeneration[END_REF][START_REF] Derangere | Liver X receptor beta activation induces pyroptosis of human and murine colon cancer cells[END_REF]. Indeed, we showed that dead cells present AnnV and/or PI positive labelings and irregular DNA fragmentation (TUNEL positive staining without laddering pattern) as previously observed in pyroptotic macrophages [START_REF] Miao | Caspase-1-induced pyroptotic cell death[END_REF][START_REF] Watson | Salmonella enterica serovars typhimurium and Dublin can lyse macrophages by a mechanism distinct from apoptosis[END_REF][START_REF] Brennan | Salmonella induces macrophage death by caspase-1dependent necrosis[END_REF]. However, whilst Caspase 1 dependent cell death by pyroptosis in macrophages is usually associated with pro inflammatory responses resulting from Caspase 1 cleavage of Il 1β and 18, MILH did not increase Il 1β clea vage. These last results are in line with recent studies showing that Caspase 1 can induce pyroptotic cell death independently of Il1 β processing [ 44 46]. On the other hand, Pro Il1β is expressed at low levels in our tumor cell model. Thus, we cannot not exclude that pro inflammatory response would be activated by MILH in cancer cells expressing higher level of Pro IL 1β.

Our current knowledge about Caspase 1 indicates that Caspase 1 is synthesized as a cytosolic, inactive, monomeric zymogen (pro Caspase 1) that is thought to be activated by dimerization and autoproteolytic processing, resulting in generation of large and small subunits (called p20 and p10) of the catalytically active enzyme [START_REF] Martinon | The inflammasomes: guardians of the body[END_REF][START_REF] Thornberry | A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes[END_REF]. This activation step is preceded by recruitment of Pro Caspase 1 into inflammasomes, which are multiprotein signaling complexes. Furthermore, a direct processing of Caspase 1 by CathB [START_REF] Vancompernolle | Atractyloside-induced release of cathepsin B, a protease with caspase-processing activity[END_REF][START_REF] Hentze | Critical role for cathepsin B in mediating caspase-1-dependent interleukin-18 maturation and caspase-1-independent necrosis triggered by the microbial toxin nigericin[END_REF] or an indirect mechanism of activation through CathB induced activation of inflammasome com plexes [41, 50 52] were reported. In the current study of MILH induced cell death, CathB activation is critical for Caspase 1 activation and tumor cell death indicating that one of these two mechanisms may function, but this remains to be determined more precisely.

Conclusion

The present study provides essential new data on the mechanism whereby targeted MNPs induce the death of tumor cells when exposed to a high frequency magnetic field. The central role of the Fenton re action that produces ROS within lysosomes, together with that of CathB and Caspase 1, represent the key elements which are the basis for the optimization of Magnetic Intra Lysosomal Hyperthermia or MILH strategies to kill cancer cells and their future developments as an anti tumoral option.

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jconrel.2017.11.050. Fig. 6. MILH induces the death and activates Caspase-1 in different cancer cells. INR1G9-CCK2R, AR4-2J and AGS-CCK2R cells were incubated or not with Gastrin-MNPs and exposed or not to AMF. a) Cell survival was determined by MTT assay 24 h after the end of AMF exposure. Results are expressed as the % of inhibition of cell survival over control cells (in absence of Gastrin-MNP and AMF) for each cell line. b) Dead cells were counted 4 h after the end of AMF exposure by confocal microscopy analysis of cells labeled with AnnV/PI. Results are expressed as fold change of death rate over control cells (in absence of Gastrin-MNP and AMF) for each cell line. The % of dead cells is indicated above the histogram. 2000-3000 cells/experiments were analyzed and results are the mean ± SEM of at least 4 separate experiments. c) Gastrin-MNPs uptake was measured by colorimetric assay (total Fe) or by pEPR (Fe contained in superparamagnetic MNPs). d) Caspase-1 activation was analyzed by confocal microscopy of cells labeled with FAM-FLICA-Casp1, 4 h after AMF exposure. 2000-3000 cells/experiments were analyzed. Results are expressed in % of total cells labeled with fluorescent Caspase substrate and are the mean ± SEM of at least 4 separate experiments. Significant difference was analyzed comparatively to the control condition corresponding to cells devoid of Gastrin-MNP in absence of AMF exposure (a-d). e) Schematic representation of cell death mechanism induced by MILH. Application of AMF to Gastrin-MNPs specifically delivered to lysosomes of cancer cells increases the temperature at the nanoparticle periphery which enhances ROS production through the lysosomal Fenton reaction. Subsequently, ROS cause lipid peroxidation of lysosome membrane, LMP and the leakage of lysosomal enzymes into the cytosol, including CathB. Then, cytosolic CathB activates Caspase-1, but not the apoptotic Caspase-3, to induce the death of cancer cells.

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Fig. 1 .

 1 Fig.1. Magnetic Intra-Lysosomal Hyperthermia (MILH) induces ROS generation. a) Schematic representation of MILH approach. Gastrin-MNPs are specifically internalized through a CCK2R-dependent physiological process, and are then trafficked to lysosomes where they accumulated. Upon AMF exposure, Gastrin-MNPs cause LMP and the leakage of lysosomal enzymes into the cytosol including Cathepsins which trigger cell death. Subsequently, 19.9 ± 1.5% or 32.3 ± 2.7% of INR1G9-CCK2R cells were labeled by AnnV and PI or killed 4 h and 24 h after AMF exposure. b) Top panel: hypothesis of initiating event triggered by MILH. AMF application to cells containing MNPs would generate a local temperature elevation and enhance ROS production within lysosomes. Lysosomes are well-known major sites of ROS production through the Fenton reaction which catalyzes the transformation of hydrogen peroxide to hydroxyl radicals. Bottom panel: INR1G9-CCK2R cells having or not internalized Gastrin-MNPs were incubated in the presence or absence of 1 mM DFO, 120 μM FeCl 3 , 1 nM BafA1 or 20 μM ART, exposed or not to AMF and incubated with CellROX reagent. Quantification of ROS production was performed by analyzing the fluorescence intensity from confocal microscopy images and expressed as fold change of fluorescence intensity over control cells (in absence of Gastrin-MNP and AMF). Significant difference compared to the control condition corresponding to cells devoid of Gastrin-MNP in absence of AMF exposure was indicated above histogram bar. Statistical significances between other conditions are also indicated by lines/brackets. 2000-3000 cells/experiments were analyzed and results are the mean ± SEM of 4 separate experiments. c) INR1G9-CCK2R cells having or not internalized Gastrin-MNPs were incubated in the presence or absence of 1 mM DFO, 120 μM FeCl 3 , 1 nM BafA1 or 20 μM ART and incubated with CellROX reagent during the 30-min of AMF exposure. Top panel: confocal microscopy images illustrating ROS production, Gastrin-MNP labelings and line scan of fluorescence intensity. Bottom panel: Quantification of ROS production colocalizing with Gastrin-MNP was performed by analyzing the % of relative fluorescence intensities of ROS production over Gastrin-MNP from confocal microscopy images. Significant difference compared to the control condition corresponding to cells containing Gastrin-MNP in absence of AMF exposure was indicated above histogram bar. Statistical significances between other conditions are also indicated. 30-50 lysosomes of 20-30 cells/experiments were analyzed and results are the mean ± SEM of 4 separate experiments. d) INR1G9-CCK2R cells were incubated with Gastrin-MNPs and exposed or not to AMF. Fe contained in superparamagnetic MNPs was measured by particle electron paramagnetic resonance. e) INR1G9-CCK2R cells transiently expressing YFP-Lamp1 were incubated with Gastrin-MNP-DY549. AMF was applied using a miniaturized electromagnet. Temperature at MNPs surface and lysosome membrane was analyzed respectively using the molecular thermometers DY549 (red line) and YFP-Lamp1 (green line) by measuring the decrease of their fluorescence intensity. Significant difference was analyzed comparatively to t = 0 for each fluorophores. 20-30 cells/experiments were analyzed and results are the mean ± SEM of 5 separate experiments. As a control, no significant changes of the MNP surface (red dotted line) or lysosome membrane (green dotted line) temperatures were observed in cells not exposed to AMF. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2 .

 2 Fig. 2. MILH-induced ROS generation causes lysosome lipid peroxydation, lysosome membrane permeabilization and cell death. a) INR1G9-CCK2R cells having or not internalized Gastrin-MNPs were incubated in the presence or absence of DFO or BafA1 and incubated with Image-IT® lipid peroxidation reagent during the 30-min of AMF exposure. Left panel: confocal microscopy images illustrating lipid peroxidation in vesicles containing Gastrin-MNP labelings and line scans of fluorescence corresponding to peroxidized lipids colocalizing with Gastrin-MNPs. Right panel: Quantification was performed by measuring the % of fluorescence intensity of lipid peroxidation over that of Gastrin-MNP from confocal microscopy images. Significant difference compared to the control condition corresponding to cells containing Gastrin-MNPs in absence of AMF exposure was indicated above histogram bar. Statistical significances between other conditions are also indicated. 30-50 lysosomes of 20-30 cells/experiments were analyzed and results are the mean ± SEM of 3 separate experiments. b) INR1G9-CCK2R transiently expressing GFP-CathB and RFP-Lamp1 were incubated with Gastrin-MNPs and exposed to AMF in the presence or not of NAC. Left panel: LMP was evaluated by analyzing the colocalization between GFP-CathB and RFP-Lamp1 on confocal microscopy images using Pearson's coefficient. Significant difference compared to the control condition corresponding to cells devoid of Gastrin-MNP in absence of AMF exposure was indicated above histogram bar. Statistical significances between other conditions are also indicated. Middle panel: confocal microscopy images illustrating GFP-CathB in lysosome labeled by RFP-lamp1 and line scans of fluorescence corresponding to GFP-CathB colocalizing with RFP-Lamp1. Right panel: LMP was quantified by measuring the % of fluorescence intensity of GFP-CathB over that of RFP-Lamp1 from confocal microscopy images. Significant difference compared to the control condition corresponding to cells devoid of Gastrin-MNP and exposed to AMF was indicated above histogram bar. Statistical significances between other conditions are also indicated. 30-50 lysosomes of 20-30 cells/experiments were analyzed and results are the mean ± SEM of 4 separate experiments. c) INR1G9-CCK2R transiently expressing GFP-CathB and RFP-Lamp1 were incubated with Gastrin-MNPs and exposed or not to AMF. Different positions were marked inside (+ AMF) and outside ( AMF) the gap of the electromagnet allowing to visualize the effects of AMF under a confocal microscope. Top panel: Confocal microscopy images illustrating the lysosome integrity in cells before and 60-min after AMF exposure (53 mT, 300 kHz). Bottom panel: Before and during AMF application, LMP was analyzed by measuring the % of green pixels (GFP-CathB) colocalized with red pixels (RFP-Lamp1) (ImageJ software). Results are expressed as % of CathB/Lamp1 colocalization over control cells (in absence of Gastrin-MNP and AMF). Significant difference was analyzed comparatively to t = 0. 20-30 cells/experiments were analyzed and results are the mean ± SEM of 5 separate experiments. d) INR1G9-CCK2R cells were incubated with Gastrin-MNPs, exposed to AMF in presence or not of different agents. Dead cells were counted 4 h after AMF exposure by confocal microscopy analysis of cells labeled with AnnV/PI. Results are expressed as fold change of death rate over control cells (in absence of Gastrin-MNP and AMF). The % of dead cells are indicated above the histogram. Significant difference compared to the control condition corresponding to cells devoid of Gastrin-MNP in absence of AMF exposure was indicated above histogram bar. Statistical significances between other conditions are also indicated. 2000-3000 cells/experiments were analyzed and results are the mean ± SEM of 4 separate experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3 .

 3 Fig.3. Cell death induced by MILH is dependent on CathB activity. a) INR1G9-CCK2R cells having or not internalized Gastrin-MNPs were incubated with or without 10 μM CA-074-Me and exposed or not to AMF. Dead cells were counted 4 h after the end of AMF exposure by confocal microscopy analysis of cells labeled with AnnV/PI. Results are expressed as fold change of death rate over control cells (in absence of Gastrin-MNP and AMF). The % of dead cells are indicated above the histograms. b-c) INR1G9-CCK2R transiently expressing GFP-CathB-C29A inactive mutant (b) or GFP-CathB wild-type enzyme (c) were incubated with Gastrin-MNPs and exposed or not to AMF. Dead cells were counted 4 h after the end of AMF exposure by confocal microscopy analysis and expressed as the % of cells labeled with AnnV (red) among transfected (green cells) and non-transfected cells. Results are expressed as fold change of death rate over control cells (in absence of Gastrin-MNP and AMF). The % of dead cells are indicated above the histograms. Significant difference compared to the control condition corresponding to cells devoid of Gastrin-MNP in absence of AMF exposure was indicated above histogram bar. Statistical significances between other conditions are also indicated. 2000-3000 cells/experiments were analyzed and results are the mean ± SEM of at least 4 separate experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4 . 3 . 5 .0

 435 Fig. 4. Cell death lnduced by MILH 1s medlated tbrough a non..apoptotlc Caspase-1 slgnallng pa thway whlch 1s dependent on CathB actlvlty. INRlG9-ccK2R oells havlng or not lntemallzed Gastrin-MNPs were exposed or not to AMF ln the presence or ln absence ofCaspase-3 or Caspase-1 lnhibitor. As positive and negatlve controls of apoptosls, INR1G9-0CK2R cens were stlmulated wlth 1 µM stauroeporine ln the absence or ln the presence of Caspase-3 lnhlbitor. a) Dead cells were counted 4 h after the end of AMF exposure by confocal mlcroscopy analysls of cells labeled wlth AnnV /Pl. Results are expressed as fold change of death rate over control cells (ln absence of Gastrin-MNP and AMF). The% of dead cells 1s lndlcated above the histogram b) 4 h after AMF exposure or staurosporine treatment, Caspase-3 activation was analyzed by confocal mlcroscopy of cells labeled wlth FAM-PLI CA-Casp3. Results are expressed as the% of total cells labeled wlth fluorescent Caspase substrate c) 4 h after AMF exposure or stauroepcrine treatment, cells were lncubated wlth JC-10 reagent and were analyzed by confocal mlcroscopy. Quantification of MOMP was performed by anal)'2ing the JC-10 green/red fluorescence ratio from confocal mlcroscopy Images. d) Dead cens were counted 4 h after the end of AMF exposure by con focal mlcroscopy analysls cf cells labeled with Ann V/Pl. Results are expressed as fold change of death rate over control cells (ln the absence of Gastrin-MNP and AMF). The% of dead oells 1s lndlcated above the histogram e) INR1G9-CCK2R translentiy expresslng GFP-Casp l-C284A enzymatlcally Inactive mutant were lncubated wlth Gastrin-MNPs and exposed or not to AMF. Dead cells were counted 4 h after AMF exposure by confocal mlcroscopy ana lysls of cells labeled wlth Ann V. Results are expressed as fold change of death rate over control cells (ln absence of Gastrin-MNP and AMF). The% of dead cells 1s lndlcated above the hlstogram. 1) 4 h after AMP exposure, Caspase-1 activation was analy?Jed by confocal mlcroscopy of cells labeled wlth FAM-PLI CA-Caspl. Results are expressed ln% of total cells labeled wlth fluorescent Caspase substrate. g) 4 h after AMF application cells were labeled wlth FAM-FUCA-Caspl, then wlth AnnV (red) and observed by confocal mlcroscopy. Signiflcantdifference compared to the control condition correspondlng to cells devoid ci Gastrin-MNP ln absence of AMF exposure was lndlcated above histogram bar. Statlstical signiflcances between other conditions are also lndlcated. 2000-3000 cells/experiments were analyzed and results are the mean ± SEM of at least 4 separate experiments. (For lnterpret ation of the references to entour ln this figure legend, the reader 1s referred to the web version of this article.)

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Acknowledgements

PC and VG performed and analyzed studies of ROS production, lipid peroxidation, molecular thermometry, lysosome membrane permeabi lization, Caspases activation and cell death. PJ and BP performed and analyzed DNA fragmentation and Il 1β secretion studies. JC conceived the miniaturized electromagnet. NH and MG carried out magnetic field exposure using miniaturized electromagnet or magnetic inductor. VG, DF and JC wrote the manuscript.

We thank Cellular Imaging Facility Rangueil and Flow Cytometry platform of I2MC/INSERM for the excellent technical support. This research was partly funded by Ligue Nationale Contre le Cancer and None.

Conflicts of interest


Saptarshi De

Shiva Prasad

Rajiv Onkar Dusane

Lionel Presmanes

Yohann Thimont

Philippe Tailhades

Valérie Baco-Carles

Corine Bonningue

Sumangala Thondiyanoor Pisharam

Antoine Barnabé
email: barnabe@chimie.ups-tlse.fr

Narayanan Venkataramani De

N Venkataramani

Ethanol and Hydrogen Gas-Sensing Properties of CuO-CuFe 2 O 4 Nanostructured Thin Films

Keywords: hydrogen, nanocrystalline CuO-CuFe 2 O 4, thin film

Nanocrystalline CuO-CuFe 2 O 4 composite thin films were developed from CuFeO 2 ceramic target using a radiofrequency sputtering method followed by a thermal oxidation process. This fabrication process helps to develop porous sensing layers which are highly desirable for solid-state resistive gas sensors. Their sensing properties toward ethanol and hydrogen gas in dry air were examined at the operating temperatures ranging from 250 °C to 500 °C. The electrical transients during adsorption and desorption of the test gases were fitted with the Langmuir single site gas adsorption model. A composite thin film with a total thickness of 25 nm showed highest response (79%) toward hydrogen (500 ppm) at the operating temperature of 400 °C. The shortest response time (τ res ) was found to be ∼60 and ∼90 s for hydrogen and ethanol, respectively. The dependence of the response of the sensor on gas concentration (10-500 ppm) was also studied.

Index Terms-Ethanol, gas sensor

the gases listed above, CuO can also be interesting for CO 2 detection [START_REF] Samarasekara | Sputtered copper oxide (CuO) thin films for gas sensor devices[END_REF]. On the other hand, copper based spinel oxides such as copper ferrite (CuFe 2 O 4 ) having n-type semiconductor properties was also reported to show response toward H 2 [START_REF] Reddy | Preparation and characterization of ferrites as gas sensor materials[END_REF], LPG [START_REF] Singh | Synthesis of nanorods and mixed shaped copper ferrite and their applications as liquefied petroleum gas sensor[END_REF] and ethanol [START_REF] Tao | Preparation and gassensing properties of CuFe 2 O 4 at reduced temperature[END_REF]. In our previous work, maximum response (R/R) of 86% was obtained by CuFe 2 O 4 nano powder toward 500 ppm of ethanol [START_REF] Sumangala | Structural, magnetic and gas sensing properties of nanosized copper ferrite powder synthesized by sol gel combustion technique[END_REF], and this pure copper ferrite also showed a good response of 10% toward CO 2 [START_REF] Sumangala | Study on the effect of cuprite content on the electrical and CO 2 sensing properties of cuprite-copper ferrite nanopowder composites[END_REF].

Semiconductor nano composites with p-n junction were reported as a subject of interest for gas sensing regarding operating temperature (O.T.) and response. In particular, many authors have studied the combination of p-type CuO with various n-type oxides for CO 2 detection [START_REF] Ishihara | Application of mixed oxide capacitor to the selective carbon dioxide sensor: I. Measurement of carbon dioxide sensing characteristics[END_REF]- [START_REF] Zhang | Effect of particle size and dopant on properties of SnO 2 -based gas sensors[END_REF]. In the recent past, CuO/CuFe 2 O 4 composite thin films [START_REF] Chapelle | Improved semiconducting CuO/CuFe 2 O 4 nanostructured thin films for CO 2 gas sensing[END_REF] and powders [START_REF] Sumangala | Study on the effect of cuprite content on the electrical and CO 2 sensing properties of cuprite-copper ferrite nanopowder composites[END_REF] having spinel phase were also reported as CO 2 gas sensing material.

In this work, nanocrystalline CuO-CuFe 2 O 4 semiconductor thin films were developed from CuFeO 2 ceramic target using a radio-frequency (RF) sputtering method followed by a thermal oxidation process. This fabrication process may help to develop porous sensing layers which are highly desirable for solid state resistive gas sensors. These films were used as the sensitive material for reducing gases like hydrogen and ethanol. The effect of the operating temperature on the response, response time and recovery time of the active layer were studied to evaluate the merit of performance of the material. The effect of gas flow rate on the response time and recovery time of the active layer were also studied. To demonstrate its potential sensing application, the variation of response with different gas concentration has been shown. Here, the minimum operating temperature was chosen as 250 °C to avoid the effect of moisture on sensor samples during practical gas sensing application. We also tried to generate the values of activation energy for the gas (H 2 ) adsorption and intrinsic reaction on CuO thin film surface using Langmuir gas adsorption model, which may be useful in future to compare with other sensing material and gases for analysing the gas sensing properties.

II. PREPARATION OF THE GAS SENSITIVE LAYERS

Cu-Cu x Fe 3-x O 4 thin films were first deposited on fused silica substrate at room temperature with Alcatel A450 RF sputtering unit using a pure delafossite (CuFeO 2 ) ceramic target. The details of the deposition procedure were described Barnabé et al. [28]. Process parameters for the room temperature deposited samples are given in Table I. Two films having thicknesses 25 nm and 300 nm were deposited by varying the deposition time. Thickness of the deposited films was measured using a Dektak 3030ST profilometer and cross-sectional scanning electron microscopy (SEM) using JEOL JSM 6700F field emission SEM. Our previous studies (i.e. grazing incidence X-ray diffraction (GI-XRD), Raman spectroscopy and electron probe micro analysis (EPMA)) on the same samples have already revealed that the as-deposited films consisted of copper nano particles which were embedded in an oxide matrix which was made of cuprous oxide and mixed valence defect ferrite (Cu 2 O, Cu x Fe 3-x O 4 ) [START_REF] Barnabé | Copper and iron based thin film nanocomposites prepared by radio frequency sputtering. Part I: Elaboration and characterization of metal/oxide thin film nanocomposites using controlled in situ reduction process[END_REF], [START_REF] Chapelle | Copper and iron based thin film nanocomposites prepared by radio-frequency sputtering. Part II: Elaboration and characterization of oxide/oxide thin film nanocomposites using controlled ex-situ oxidation process[END_REF] (Equation 1a). In order to obtain the stable CuO/CuFe 2 O 4 nano composite, the as-deposited films were ex-situ annealed at 550 °C in air for 12 h (Equation 1b). The tenorite phase CuO then originated from the oxidation of the metallic copper in association with that of Cu 2 O. One can note that the reaction scheme could be more complex if we consider the formation of CuFeO 2 intermediate phase [START_REF] Chapelle | Copper and iron based thin film nanocomposites prepared by radio-frequency sputtering. Part II: Elaboration and characterization of oxide/oxide thin film nanocomposites using controlled ex-situ oxidation process[END_REF]. The annealing treatment of the as-deposited samples starting from delafossite target can be represented by the following simplified reaction scheme:

1) reduction during deposition step

CuFeO 2 → x Cu + (1 -x)/2 Cu 2 O + (9 -x)/24 Fe 24/(9-x) O 4 + x/3 O 2 (Target) ( as-deposited film) (1a)
2) oxidation during post deposition annealing

x Cu + (1 -x)/2 Cu 2 O + (9 -x)/24 Fe 24/(9-x) O 4 + (4x + 3)/12 O 2 → x/2 CuO + x/2 CuFe 2 O 4 (as-deposited film) (annealed film) (1b)
The SEM image in figure 1 shows that, as a result of annealing, the parent films were self-organized in a two layered stack with top to bottom layer thickness ratio of 1:2. These films were characterized by GI-XRD technique and Glow-discharge optical emission spectroscopy (GD-OES) profile [START_REF] Chapelle | Improved semiconducting CuO/CuFe 2 O 4 nanostructured thin films for CO 2 gas sensing[END_REF] and X-ray photo electron spectroscopy (XPS) [START_REF] Chapelle | Copper and iron based thin film nanocomposites prepared by radio-frequency sputtering. Part II: Elaboration and characterization of oxide/oxide thin film nanocomposites using controlled ex-situ oxidation process[END_REF] which confirmed that the top layer was tenorite CuO and that of the bottom layer was spinel CuFe 2 O 4 .

Interestingly, a 30% increase in the total thickness of the as-deposited thin film was observed after annealing which was possibly due to the porosity developed during postdeposition annealing [START_REF] Chapelle | Copper and iron based thin film nanocomposites prepared by radio-frequency sputtering. Part II: Elaboration and characterization of oxide/oxide thin film nanocomposites using controlled ex-situ oxidation process[END_REF]. This porosity in the two layered stack might be caused by the metallic copper diffusion during the oxidation process of the as-deposited samples. For thin film semiconductor metal oxide based gas sensors, the porosity of the sensing layer is an important parameter [START_REF] Seo | Gas sensing characteristics and porosity control of nanostructured films composed of TiO 2 nanotubes[END_REF] as the gas diffusion through the porosity can cause changes in electrical properties of the films, making the gas detection easier.

III. GAS SENSING MEASUREMENTS

Gas sensing experiments were carried out in a closed chamber with controlled operating temperature from 250 °C to 500 °C using a PID controller. The ambient gas environment was controlled by a continuous flow of the calibrated test gases or air using mass flow controller. For the hydrogen sensing, two gas cylinders were used-one with just zero air (moisture < 0.01%) and another with same zero air containing 500 ppm of hydrogen. The sensor samples were stabilized at each operating temperatures for at least 12 hours in zero air, prior to the gas sensing experiment. Resistancetransients of the sensing layer were measured in two probes mode using Keithley 2635B source meter. Similarly, for the ethanol sensing experiments, two gas cylinders were used, one with zero air and another with the same zero air containing 500 ppm of ethanol. The response (R s ) of the sensor samples is defined as the relative difference of the film resistance between air and test gas atmosphere (R gas -R air )/ R air ×100%, where R gas and R air are saturated resistance of the sensor in test gas atmosphere and in air respectively. The concentration of the test gases (C gas in chamber ) in the gas chamber was varied by diluting with zero air, and it can be calculated using the following relation:

C gas in chamber = [C test gas x(dV test /dt)]/[(dV test /dt) + (dV zero air /dt)] (2)
Where C test gas is the concentration of the test gas in gas cylinder and dV test /dt is the volumetric flow rate of test gas, similarly dV zeroair /dt is the volumetric flow rate of zero air.

IV. RESULTS AND DISCUSSION

Figure 2 shows the resistance-transients during the insertion of hydrogen (500 ppm) and recovery in air of the 25 nm thin film sensor at the operating temperature of 400 °C with 100 cc/min gas-flow rate. The sensing material showed good repeatability as the initial baseline was regained upon exposure to dry air. The increase in the electrical resistance of the sensors upon exposure to a reducing gas such as H 2 indicates that the obtained films have p-type semiconducting behaviour. It could be possible that only CuO is involved in sensing as it is the top layer.

The following reaction mechanism for the sensing of reducing gas by a p-type semiconductor can be summarized from several research reports [START_REF] Wang | Surface accumulation conduction controlled sensing characteristic of p-type CuO nanorods induced by oxygen adsorption[END_REF]. In a first step, at the operating temperature, oxygen is physisorbed on the sensor surface followed by electron transfer from the p-type semiconducting oxide CuO to the adsorbed oxygen, thus forming chemical bond between the adsorbed oxygen and the semiconducting oxide. Thus, the electrical resistance of the p-type semiconductors reduces during stabilization of the sensor material [see figure (3.a)].

These reactions are described in equations ( 3) and ( 4) respectively

1 2 O 2 + [sensor surface ] ↔ O ad-surface (physisorption) (3) O ad-surface + e -↔ O - ad (chemisorption) (4)
When the sensor is exposed to reducing gas ambient, the reducing gas is physically adsorbed on the active layer surface and reacts with the adsorbed oxygen according to the reaction ( 5) & ( 6) and the product (RO) goes out [eq. 7] [see figure (3.b)]. Thus, the resistance of the p-type sensor

Out of these reactions, the physisorption of oxygen as well as that of the reducing gas [Eq. ( 3) and ( 5)] are fast. On the other hand the reaction between the adsorbed gas and oxygen [Eq. ( 6)] is a slow process and therefore, the last one is the rate determining step for the response kinetics. This is easily corroborated from the reported data on surface reaction of adsorbed oxygen [START_REF] Ahn | Separation of adsorption and surface reaction rates: Dynamic studies in a catalytic slurry reactor[END_REF] and hydrogen [START_REF] Arrua | Effect of catalyst poisoning on adsorption and surface reaction rates in liquid-phase hydrogenation[END_REF]. According to Ahn et al. [START_REF] Ahn | Separation of adsorption and surface reaction rates: Dynamic studies in a catalytic slurry reactor[END_REF], the ratio of surface reaction rate constant to adsorption rate constant at adsorption equilibrium for oxidation of sulphur dioxide is 0.5, and Arrua et al. [START_REF] Arrua | Effect of catalyst poisoning on adsorption and surface reaction rates in liquid-phase hydrogenation[END_REF] reported the same ratio for hydrogenation using Pd/Al 2 O 3 catalyst in the range of 0.26-0.29. Assuming Langmuir single site gas adsorption model for the thin film sensors [START_REF] Mukherjee | Analyses of response and recovery kinetics of zinc ferrite as hydrogen gas sensor[END_REF], the response and the recovery transients were fitted well with the following two equations (eq. 8, 9) respectively (shown in coloured lines in fig. 2). The values of coefficient of determination (R 2 ) in this fitting for all response or recovery curves were in between 0.985-0.999.

R(t) response = R air + R 1 [1 -exp (-t/τ res )] (8) R(t) recovery = R air + R 1 [exp (-t/τ rec )] (9)
Where τ res and τ rec are the 'response time' and 'recovery time' respectively. And R 1 is a proportionality constant of the exponential term whose value is equal to the difference of the film resistance between air and test gas atmosphere (R gas -R air ).

The variation of response and recovery time with gas flow rate was observed and tabulated in Table II. Decrease in response and recovery time with gas flow rate indicates a mass transfer controlled reaction kinetics on this thin film surface. Therefore, the flow rate was kept fixed at 100 cc/min for the rest of the experiments performed.

Hydrogen sensing by a 25 nm thin film sensor was carried out at different operating temperatures and the variation of response time and recovery time are given in the table III. Response time seemed to be saturated above the operating temperature of 350 °C and the saturated value was found to be around 60 seconds. At the high operating temperatures, the reaction rate of eq. ( 6) became faster and the reaction might be limited by the test conditions, i.e., gas flow in the gas chamber. Recovery time decreased monotonically with the operating temperature until 500 °C. The bell shaped response curve with the operating temperature as shown in the figure 4 is a result of the competitive behaviour of eqs. ( 5) and [START_REF] Zhu | Facile fabrication and enhanced sensing properties of hierarchically porous CuO architectures[END_REF]. Similar bell shaped response curve had been reported by Ahlers et al. [START_REF] Ahlers | A rate equation approach to the gas sensitivity of thin film metal oxide materials[END_REF] and Biswas and Pramanik [START_REF] Biswas | Studies on the gas sensing behaviour of nanosized CuNb 2 O 6 towards ammonia, hydrogen and liquefied petroleum gas[END_REF]. According to them, response varies with operating temperature on the basis of two energy systems. E ads is dependent on the strength of the test gas binding onto the sensing material surface. On the other hand, E a is defined as the energy barrier required to be overcome by the adsorbed gas molecules for diffusion along the surface, resulting in catalysis induced surface combustion process. Initially, under clean air conditions, active sites on the surface of a sensor material had been covered by adsorbed oxygen. Then, relative occupancy of the test gas on the pre-adsorbed oxygen depends on partial pressure and operating temperature of the test gas. In the figure 4, simulated curves of Langmuir relative surface coverage (L), reaction rate (K) and the modeled response (R m ) are shown. Here, L = Pgas Pgas + Po [START_REF] Zhou | Study on sensing mechanism of CuO-SnO 2 gas sensors[END_REF] where P gas is partial pressure of test gas (H 2 ) at sensing layer and P o = k B T Vo exp ( -Eads k B T ), where v o is the quantum volume of the test gas species, given by v o = ( 2π h2 M gas M o k B T )

1.5
, where M gas is the relative atomic mass of the test gas (i.e. 2 for H 2 ); M o is the atomic mass unit (1.67 × 10 -27 kg); h is the reduced plank constant and k B , T are Boltzmann constant and absolute temperature respectively. The reaction rate of adsorbed test gas with chemisorbed oxygen ion is

K = A exp ( -Ea k B T ) ( 11
)
where A is a proportionality constant. The modeled response was obtained from the combination of Langmuir relative surface coverage and the reaction rate [START_REF] Ahlers | A rate equation approach to the gas sensitivity of thin film metal oxide materials[END_REF], and it could be given by

R m = Pgas Pgas+ k B T Vo exp( -Eads k B T ) A exp -Ea k B T (12)
In the case of tin dioxide (SnO 2 ) thin film sensors, the values of E ads varied from 130 to 145 kJ/mol (1.3-1.45 eV) and E a varied from 53 to 57 kJ/mol (0.53-0.57 eV) for different ethane concentrations [START_REF] Ahlers | A rate equation approach to the gas sensitivity of thin film metal oxide materials[END_REF]. Here, the values E ads and E a were obtained (by the fitting of experimental values with eq. 12) as 43 kJ/mol (0.45 eV) and 21 kJ/mol (0.22 eV) respectively. This sensor sample showed maximum response of 79% at the operating temperature of 400 °C toward 500 ppm of H 2 . A similar response of 70% was reported for H 2 but at a higher concentration (2500 ppm) with thicker copper oxidecopper ferrite sensor system [START_REF] Chapelle | Structural and gas-sensing properties of CuO-Cu x Fe 3-x O 4 nanostructured thin films[END_REF]. Hoa et al. [START_REF] Hoa | Synthesis of p-type semiconducting cupric oxide thin films and their application to hydrogen detection[END_REF] reported 40% response toward 10,000 ppm of H 2 at an operating temperature of 250 °C for CuO thin film, whereas at the same operating temperature, this CuO/CuFe 2 O 4 thin film exhibits 45% response only at 500 ppm of H 2 .

Similarly, ethanol sensing of the 25 nm thin film sensor was carried out at different operating temperatures and the variation of response time and recovery time are given in the table IV. Response time seemed to be saturated above the operating temperature of 450 °C and the value of that was found to be around 90 seconds. It was observed that the recovery time decreased to the range of 350 to 400 °C and after that it had increased. This increase at 500 °C is not well understood at present. Figure 5 shows the variation of response with operating temperature. In case of ethanol, the maximum response of this thin film sensor might be observed above 500 °C. As per literature, the best operating temperature to get maximum response for ethanol was reported to be higher than that of hydrogen [START_REF] Tonezzer | Dualselective hydrogen and ethanol sensor for steam reforming systems[END_REF]. The operating temperature was confined below 500 °C due to the (a) stability of the phase and microstructure in sensing layers and (b) instrumental limitation. Hence we may not be able to capture a similar behaviour as found in H 2 . Here, due to lack of the bell shape in the response curve, it could not be fitted with the chosen model (eq.12).

The gas sensing experiment was carried out with 300 nm thick film and the results are tabulated in table V. Thicker CuO-CuFe 2 O 4 thin film showed p-type response toward reducing gases, i.e., hydrogen and ethanol. So, the test gases were mostly interacting (adsorption/desorption) with the copper oxide layer located on the top of film.

Depending on the operating temperature, oxygen molecules adsorbed on semiconductor surface are in various ionic states, i.e. O - 2 , O -or O 2- [START_REF] Barsan | Fundamental and practical aspects in the design of nanoscaled SnO 2 gas sensors: A status report[END_REF]. So, adsorbed hydrogen (H 2ads ) may react with adsorbed oxygen (O ion ads ) as in the following equations.

2H 2 ads + O 2 - ads → 2H 2 O + e - (13)
or,

H 2 ads + O - ads → H 2 O + e - (14)
or,

H 2 ads + O 2- ads → H 2 O + 2e - (15)
Similarly, carbon dioxide and water are the final decomposition products of ethanol combustion in air. Acetaldehyde or acetic acid may also form as intermediate products during the oxidization of ethanol. Hence depending on the types of adsorbed oxygen and by-products of ethanol, various charge balance equations of ethanol decomposition are possible and given below.

2CH 3 CH 2 OH ads + O 2 -ads → 2CH 3 CHO + 2H 2 O + e - CH 3 CH 2 OH ads + O - ads → CH 3 CHO + H 2 O + e - CH 3 CH 2 OH ads + O 2- ads → CH 3 CHO + H 2 O + 2e - CH 3 CH 2 OH ads + O 2 -ads → CH 3 COOH + H 2 O + 1e - CH 3 CH 2 OH ads + 2O - ads → CH 3 COOH + H 2 O + 2e - CH 3 CH 2 OH ads + 2O 2- ads → CH 3 COOH + H 2 O + 4e - CH 3 CH 2 OH ads + 3O 2 -ads → 2CO 2 + 3H 2 O + 3e - CH 3 CH 2 OH ads + 6O - ads → 2CO 2 + 3H 2 O + 6e - CH 3 CH 2 OH ads + 6O 2- ads → 2CO 2 + 3H 2 O + 12e - ⎫ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎬ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎭ (16)
From equations ( 13), ( 14) and ( 15), for all metal oxide sensors a general rate equation of electron density can be written as

dn dt = K gas (T)[O ion ads ] a [R] b (17)
where, n is the electron density or electron concentration in the charge accumulation layer under the test gas (e.g. H 2 ) atmosphere, b is a charge parameter which might have value in the range of 0.5 to 2 for hydrogen and 0.08 to 2 for ethanol respectively. Similarly, a is a charge parameter which might have value in the range of 0.5 to 1 for oxygen ions. K gas (T) is the reaction rate constant or reaction rate coefficient described as

K gas (T) = A exp(-E a /k B T) (18)
where E a is the activation energy of reaction, k B is the Boltzmann constant, T is absolute temperature and A is proportionality constant. Integrating Eq. ( 17) leads to the solution as

n = K gas (T)[O ion ads ] a [R] b t + n o ( 19
)
where n o is the saturated electron concentration of sensor at an operating temperature in the air atmosphere. In the saturated ethanol, i.e., at equilibrium under ethanol and air atmosphere, carrier concentration n and n o could be considered as a constant with time.

n = K gas (T)[O ion ads ] a [R] b τ + n o ( 20
)
Where τ is a time constant. At a constant operating temperature the resistivity of a semiconductor is defined as ρ = α / n. Where α is a proportionality constant with '+' sign for n-type and '-' sign for p-type semiconductor, and can be substituted in equation [START_REF] Tao | Preparation and gassensing properties of CuFe 2 O 4 at reduced temperature[END_REF] as

1 Rg = (K gas (T)[O ion ads ] a [R] b τ )/α + 1 Ra (21)
Assuming the concentration of adsorbed test gas ([R] b ) on the sensor surface is linearly proportional to the gas concentration in gas chamber (C b g ), at constant operating temperature the sensor response relation can be obtained in a compact form R s = MC b g [START_REF] Sumangala | Study on the effect of cuprite content on the electrical and CO 2 sensing properties of cuprite-copper ferrite nanopowder composites[END_REF] where R s is response of the sensor and it could be defined as (R gas -R air )/R air and M is (K gas (T) [O ion ads ] a τ )R air / α, a constant at constant operating temperature. Figure 6 shows the variation of response of the 25 nm thin film sensor with gas concentration at the operating temperature of 400 °C. Gas sensing response is following the power law equation (eq. 22) for both the gases in the range of 10 ppm to 500 ppm. Response toward ethanol is slightly higher than that of hydrogen for similar concentration, i.e., this sensor is more sensitive toward ethanol than hydrogen. The obtained value of b is 0.65 for ethanol and 0.88 for hydrogen. This value of b toward ethanol is quite similar with the reported values, 0.677 and 0.54 for ZnO nano rods and nano structured sensing materials respectively [START_REF] Chen | Reduced-temperature ethanol sensing characteristics of flower-like ZnO nanorods synthesized by a sonochemical method[END_REF], [START_REF] Wongrat | Comparative study of ethanol sensor based on gold nanoparticles: ZnO nanostructure and gold: ZnO nanostructure[END_REF]. For hydrogen, the value of b was reported as 0.53 for ZnO thin films [START_REF] Rajan | An investigation on electrical and hydrogen sensing characteristics of RF sputtered ZnO thin-film with palladium Schottky contacts[END_REF]. The value of b of these sensors was not as close to 0.5. Such deviation might occur due to the fact that the surface depletion or accumulation layer has some effect on the oxygen adsorption species at metal oxide surface when the grain diameter is close to double of that layer thickness (2L d ) [START_REF] Rajan | An investigation on electrical and hydrogen sensing characteristics of RF sputtered ZnO thin-film with palladium Schottky contacts[END_REF]. At the operating temperature of 400 °C, both O -and O 2-ion species formation are possible on metal oxide surface [START_REF] Barsan | Fundamental and practical aspects in the design of nanoscaled SnO 2 gas sensors: A status report[END_REF]; quantitative comparison of these ions at this operating temperature is not available in literature. So, the value of b power law exponent for hydrogen can be in the range of 0.5 to 1 at the operating temperature of 400 °C. The value 0.88 for hydrogen in our case gives an indication of higher concentration of O -ion than O 2-on sensor surface at the operating temperature of 400 °C. Similarly, for ethanol the value can be in the range of 0.08 to 1 at the operating temperature of 400 °C, but the value 0.65 indicates the reduction of ethanol through the formation of acetaldehyde on the sensor surface.

The ethanol and H 2 sensing properties of various CuO nano structures in the literature are summarized in Table VI. Few of them reported higher response in comparison to the current work but at the cost of very high gas concentration [START_REF] Wang | Surface accumulation conduction controlled sensing characteristic of p-type CuO nanorods induced by oxygen adsorption[END_REF], [START_REF] Hoa | Synthesis of porous CuO nanowires and its application to hydrogen detection[END_REF], [START_REF] Aygün | Hydrogen sensitivity of doped CuO/ZnO heterocontact sensors[END_REF]. And short response time was observed in this present study among the values reported recently in the literature of CuO sensors.

V. CONCLUSION

The self-organized CuO-CuFe 2 O 4 thin films showed p-type semiconductor behaviour with increase in electrical resistance upon exposure to hydrogen or ethanol gas. Good fitting of response or recovery curve with single site gas adsorption model indicates that the reaction had occurred only on the surface of thin films. The developing process of this porous microstructure of top CuO layer is interesting as this kind of sensors have shown improved sensing properties toward reducing gases (e.g. ethanol and hydrogen) compared to the CuO thin film sensors fabricated by other techniques already reported. The best sensing performance was observed for the 25 nm thin film at an operating temperature of 400 °C with a response of 79% toward 500 ppm of H 2 and the response and recovery times obtained at this temperature are ∼60 s and ∼220 s, respectively. This 25 nm thin film sample also exhibited 128% response toward 500 ppm of ethanol with 90 seconds response time at the operating temperature of 400 °C. Also, we have demonstrated the variation of response of the sensors for a wide range of test-gas concentration. Due to these promising results, we believe that an optimised fabrication of this composite material could be a cheap potential gas sensing candidate only for local target oriented applications where the presence of other interfering gases is in negligible amount (e.g. in breathalyzer, water electrolysis plant). For example, the typical composition of exhaled air is 5.0-6.3% water vapour, 74.4% nitrogen, 13.6-16.0% oxygen, 4.0-5.3% carbon dioxide, <0.1% microbes or volatile organic compounds [START_REF] Guyton | Physical principles of gas exchange; diffusion of oxygen and carbon dioxide through the respiratory membrane[END_REF], [START_REF] Fenske | Human breath emissions of VOCs[END_REF]. Presence of humidity plays a significant role on sensing performance of metal oxide semiconductors at low operating temperatures (<250 °C) by the formation of HO -at the surface of the semiconductor. Wang et al. [START_REF] Wang | A highly sensitive gas sensor based on CuO nanoparticles synthetized via a sol-gel method[END_REF] reported decrease in response of CuO sensor toward reducing gas (e.g. ethanol) with increase in relative humidity at the operating temperature of 220 °C. Morimoto et al. [START_REF] Morimoto | Desorbability of chemisorbed water on metal oxide surfaces. I. Desorption temperature of chemisorbed water on hematite, rutile and zinc oxide[END_REF] showed that the most of the water content desorbed from metal oxides (e.g TiO 2 , ZnO, α-Fe 2 O 3 ) surface at the operating temperature of 400 °C. So, the effect of moisture on this sensor performance can be negligible as it will be operated at 450 °C. In our earlier studies, it was observed that the response of the same sample toward CO 2 was very slow with response time of ∼9.5 hour and maximum response of ∼50% was observed at 250 °C which reduced below 10% at higher operating temperature (400 °C) [START_REF] Chapelle | CO 2 sensing properties of semiconducting copper oxide and spinel ferrite nanocomposite thin film[END_REF]. So, fabrication of a practical breath-alcohol tester can be possible for its fast response time (1.5 min) and recovery time (7.5 min) with high response toward ethanol at 450 °C. Similarly, it can be used as cheap alarm for hydrogen-leakage, e.g., at water electrolysis plant (where the presence of alcohol or volatile organic compound is negligible or rare). A schematic illustration of the proposed sensing device is given in the figure 7. This proposed sensor should be operated at normal air atmosphere (∼20% O 2 ), where oxygen will not vary to that much extent in this environment. Though, in our future work, we plan to investigate the effect of oxygen concentration at ambient atmosphere on the sensing performance of this CuO-CuFe 2 O 4 composite sensing material.

Fig. 1 .

 1 Fig. 1. FE-SEM micrographs (a) plain view and (b) cross section view of the sample annealed at 550 °C for 12 hours in air.

Fig. 2 .

 2 Fig. 2. Resistance-transients (response and recovery) of the 25 nm thin film sensor at the operating temperature of 400 °C with 100 cc/min flow rate, fittings are shown in coloured lines.

Fig. 3 .

 3 Fig. 3. Schematic of the proposed sensing mechanism-(a) during stabilization of the sensor material (oxygen adsorption); (b) during sensing of the test gas (e.g. hydrogen).

Fig. 4 .

 4 Fig. 4.Experimental and simulated response vs. operating temperature of 25 nm composite thin film (test gas: 500 ppm of H 2 with 100 cc/min flow rate).

Fig. 5 .

 5 Fig. 5. Response vs. operating temperature of 25 nm thin film sensing material (test gas: 500 ppm of ethanol with 100 cc/min flow rate).

Fig. 6 .

 6 Fig. 6. Response vs. test gas concentration of 25 nm composite thin film at the operating temperature of 400 °C; gas flow rate: 100 cc/min.

Fig. 7 .

 7 Fig. 7. A schematic illustration of the proposed sensing device using CuO-CuFe 2 O 4 composite thin film.

TABLE I DEPOSITION

 I PARAMETERS FOR THE SPUTTERING by

TABLE II VARIATION

 II IN RESPONSE TIME AND RECOVERY TIME WITH GAS-FLOW RATE AT A FIXED OPERATING TEMPERATURE (500 °C); 25 nm COMPOSITE THIN FILM increases. RO ad + e - (6) RO ad ↔ RO gas ↑ + [sensor surface ]

R + [sensor] ↔ R ad (physisorption) (5) R ad + O - ad ↔

TABLE III VARIATION

 III IN RESPONSE TIME AND RECOVERY TIME OF 25 nm THIN FILM SENSOR WITH THE OPERATING TEMPERATURE (TEST GAS: 500 ppm OF H 2 WITH 100 cc/min FLOW RATE)

TABLE IV CHANGE

 IV IN RESPONSE TIME AND RECOVERY TIME OF 25 nm THIN FILM SENSING MATERIAL WITH THE OPERATING TEMPERATURE (TEST GAS: 500 ppm OF ETHANOL WITH 100 cc/min FLOW RATE)

TABLE V RESPONSE

 V TIME, RECOVERY TIME AND RESPONSE VS. FILM-THICKNESS

TABLE VI COMPARISON

 VI OF OUR EXPERIMENTAL DATA WITH AVAILABLE LITERATURE VALUES OF CuO BASED SENSING MATERIALS

ACKNOWLEDGMENT

The authors thank IRCC facility, IIT Bombay for the broadband dielectric spectrometer (BDS) facility.

This work was supported in part by the MonaSens Project DST under Grant 14IFCPAR001and in part by ANR under Grant 13-IS08-0002-01. The associate editor coordinating the review of this paper and approving it for publication was Dr. Camilla