Ch Nais É Beno

Morjani Hamid

Drapier Jean-Claude
email: claude.drapier@icsn.cnrs-gif.fr

M Dian

Impact of endogenous nitric oxide on microglial cell energy metabolism and labile iron pool

Keywords: Aconitate Hydratase, antagonists & inhibitors, metabolism, Adenosine Triphosphate, Animals, Cell Line, Cell Respiration, drug effects, Cytochrome c Group, Electron Transport, physiology, Electron Transport Complex I, Electron Transport Complex II, Energy Metabolism, physiology, Enzyme Activation, Interferon-gamma, pharmacology, Intracellular Fluid, Iron, Iron-Regulatory Proteins, Iron-Sulfur Proteins, Lipopolysaccharides, Mice, Microglia, cytology, Multienzyme Complexes, NADH, NADPH Oxidoreductases, Nitric Oxide, Oxidoreductases, Protein Binding, RNA, RNA-Binding Proteins, Succinate Dehydrogenase iron regulatory proteins, labile iron pool, microglial cells, neurodegenerative diseases, nitric oxide, respiratory chain

Microglial activation is common in several neurodegenerative disorders. In the present study, we used the murine BV-2 microglial cell line stimulated with -interferon and lipopolysaccharide to gain new insights into the effects of endogenously produced NO on γ mitochondrial respiratory capacity, iron regulatory protein activity, and redox-active iron level. Using polarographic measurement of respiration of both intact and digitonin-permeabilized cells, and spectrophotometric determination of individual respiratory chain complex activity, we showed that in addition to the reversible inhibition of cytochrome-c oxidase, long-term endogenous NO production reduced complex I and complex II activities in an irreversible manner. As a consequence, the cellular ATP level was decreased in NO-producing cells, whereas ATPase activity was unaffected. We show that NO up-regulates RNA-binding of iron regulatory protein 1 in microglial cells, and strongly reduces the labile iron pool. Together these results point to a contribution of NO derived from inflammatory microglia to the misregulation of energy-producing reactions and iron metabolism, often associated with the pathogenesis of neurodegenerative disorders.

INTRODUCTION

Microglial activation, oxidative stress, impairment of mitochondrial energy metabolism, and intracellular iron accumulation are features common to several neurodegenerative disorders ( ; ; ;

). There is a Beal, 1998[START_REF] Hirsch | Glial cells and Parkinson s disease[END_REF][START_REF] Murphy | Production of nitric oxide by glial cells: regulation and potential roles in the CNS[END_REF][START_REF] Schapira | Mitochondrial involvement in Parkinson s disease, Huntington s disease, hereditary spastic paraplegia and Friedreich s ataxia[END_REF] consensus that these metabolic dysfunctions play an important part in the pathophysiology of these diseases, but their interplay is complex and difficult to define. Even though the major physiological roles of microglial cells are host defense and tissue repair by phagocytosis, when activated they can also generate high levels of inflammatory cytokines, nitric oxide (NO) and reactive oxygen species (ROS), which may exacerbate neuronal damage ( ; ;

). [START_REF] Gonzalez-Scarano | Microglia as mediators of inflammatory and degenerative diseases[END_REF][START_REF] Nakajima | Functional roles of microglia in the central nervous system[END_REF]Stoll and Jander, 1999 Therefore, in Alzheimer s disease (AD), microglia proliferate around the pathological lesions and are believed to be important in plaque ' formation and neuronal degeneration ( ;Lee ., 1999;Wa , 1996). Microglial activation and Gonzalez-Scarano and [START_REF] Gonzalez-Scarano | Microglia as mediators of inflammatory and degenerative diseases[END_REF] NO, a signaling molecule responsible for a large number of physiological functions, may be harmful when produced in excessive concentrations and its implication in many neurodegenerative diseases has been suggested ( ; Dawson and Dawson, 1998 Heales ., et al ; ; ;

). NO and/or peroxynitrite, a strong oxidant generated from the combination of 1999 [START_REF] Hirsch | Glial cells and Parkinson s disease[END_REF][START_REF] Murphy | Production of nitric oxide by glial cells: regulation and potential roles in the CNS[END_REF]Toreilles ., 1999 et al NO and O , inhibit(s) several mitochondrial enzymes, including complexes of the respiratory chain and the Krebs cycle enzyme 2 aconitase ( ; ;

). Moreover, in parallel to impairment of mitochondrial functions, a [START_REF] Drapier | Interplay between NO and (Fe-S) clusters: relevance to biological systems[END_REF][START_REF] Clementi | Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione[END_REF][START_REF] Brown | Nitric oxide and mitochondrial respiration[END_REF] marked dysregulation of iron metabolism is also a feature common to many neurodegenerative disorders including PD, AD and Friedreich' ). Strikingly, NO production is responsible for alteration of iron metabolism in many cell types, and recent ., 1997 Smith ., 1998 et al results including ours pointed to a drastic change in iron regulatory protein (IRP) activity induced by NO and peroxynitrite, especially in macrophages (reviewed in ). We consider here whether NO generated by microglial cells exposed to inflammatory Drapier ., 2000 et al stimuli alters these cells mitochondrial and iron metabolisms. We found that NO production is responsible for alteration of specific sites of ' the electron transport chain associated with reduced ATP. In addition, we show for the first time that RNA-binding of IRP-1 is up-regulated by NO in microglial cells and that their labile iron pool (LIP) is decreased in an NO-dependent manner.

Materials

Murine recombinant -interferon (IFN-, specific activity 2 10 units/mg) was from R & D Systems, Abingdon, UK. France).

Cell culture

The BV-2 murine microglial cell line established by was kindly supplied by Dr. C. Demerl -Pallardy (Institut Henri Blasi ., 1990 et al é Beaufour, Les Ulis, France). Cells were routinely cultured in DMEM containing 1 g/l glucose, sodium pyruvate, pyridoxine, glutamax, and gentamycin antibiotic, and complemented with 10 fetal calf serum (FCS). For NO synthase (NOS) induction, semi-confluent cells were % treated with 20 U/ml IFN-and 50 ng/ml LPS for 16 h. γ

Preparation of mitochondrial and cytosolic fractions

Cells were detached, washed once in phosphate buffered saline (PBS) and centrifuged at 400 for 10 min. Cells were resuspended × g in 0.25 M sucrose, 40 mM KCl, 2 mM EGTA, 20 mM TRIS, pH 7.2, and lysed with 0.007 digitonin for 5 min on ice. After % centrifugation at 1,800 for 10 min, cytosol-free pellets (3 mg protein/ml) were dried and frozen at 80 C and were referred to as the × g -°mitochondrial fraction. Collected supernatants were ultracentrifuged at 150,000

for 60 min and the resultant cytosolic extracts (0.5 mg × g protein/ml) were kept at 80 C. The lactate dehydrogenase (LDH) and cytochrome-c oxidase (COX) activities were measured -°spectrophotometrically as markers of the cytoplasmic and mitochondrial fractions, respectively. The LDH activity was measured by the decline of NADH oxidation at 340 nm by using a commercially available kit (LD-L10, Sigma Diagnostics). LDH activity in the mitochondrial fraction was below 5 of total LDH activity (cytoplasmic mitochondrial fraction) indicating that very little cytoplasmic % + material remained in the mitochondrial fraction. Conversely, the 95 of COX activity, which was measured as described below, was % found in the mitochondrial fraction.

Polarographic measurement of cellular respiration and electron transport chain (ETC) activities

Oxygen consumption was measured with a Clarke-type electrode in a Peltier-controlled chamber (Hansatech oxytherm, EUROSEP, Cergy-St-Christophe, France). Polarography was used to determine activity of specific segments of the ETC using digitonin-permeabilized cells (1 10 cells/ml) in 0.25 M sucrose, 10 mM MgCl , 2 mM KH PO , 1 mM EGTA, 7 BSA, 0.005 digitonin, 20 mM HEPES pH

× 7 2 2 4 % %
7.2, as previously described ( ). Appropriate substrates and inhibitors were added as indicated. Acceptor control [START_REF] Drapier | Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells[END_REF] ratio was ~3.

Spectrophotometric determination of ETC activities

The activity of each complex of the ETC was assayed spectrophotometrically in the mitochondrial fraction (30 g protein) as equipped with multikinetic analysis software (SAFAS, Monaco). Complex I, NADH-ubiquinone reductase, activity was followed through the decline of NADH oxidation at 340 nm in the presence of decylubiquinone as an electron acceptor. Complex II, succinate-ubiquinone reductase, activity was determined by the reduction of the electron donor 2,6-dichloroindophenol at 600 nm in the presence of decylubiquinone and rotenone. Complex II III, succinate-cytochrome-c reductase, activity was measured by the reduction of + oxidized cytochrome-c at 550 nm in the presence of succinate as substrate. Complex IV, COX, activity was measured by the oxidation of reduced cytochrome-c at 550 nm. Complex V, ATPase, activity was determined by coupling the reaction to a pyruvate kinase and LDH system, and followed by monitoring the decline of NADH oxidation at 340 nm.

Electrophoretic mobility shift assay (EMSA)

The IRP-IRE interaction was analyzed as described ( ), by incubating 2 g protein of cell lysates with a molar Drapier and Hibbs, 1996 μ excess of P-CTP-labeled ferritin IRE probe in 20 l of 10 mM HEPES (pH 7.6), 40 mM KCl, 3 mM MgCl , and 5 glycerol. In parallel 32 μ 2 % experiments, samples were treated with 2 2-mercaptoethanol before addition of the RNA probe to allow full expression of the IRE % binding activity. After a 20-min incubation at room temperature, 1 l of RNase T1 (1 unit/ l) and 2 l of heparin (50 mg/ml) were μ μ μ sequentially added for 10 min each. The IRP-IRE complexes were resolved on 6 non-denaturing polyacrylamide gels and were % quantified with the IMAGEQUANT software from Molecular Dynamics (Amersham-Pharmacia Biotech, Orsay, France).

Determination of the labile iron pool (LIP)

LIP was determined by spectrofluorimetry as previously described ( ) with slight modifications. Briefly, 2.10 Epsztejn ., 1997 et al 6 cells were resuspended in 2 ml of prewarmed 20 mM HEPES, pH 7.3, 150 mM NaCl, 1 BSA buffer, and were incubated with 50 nM % calcein-AM for 15 min at 37 C. After centrifugation and washing, cells were resuspended in the same buffer, and the calcein fluorescence °intensity was recorded as a function of time. A stable baseline signal was recorded for 60 sec before the addition of 100 M SIH, a highly μ permeant iron chelator, which caused a rise in fluorescence corresponding to the LIP. LIP concentration was calibrated and calculated as described (

). The excitation and emission wavelengths were 483 nm and 505 nm, respectively. Epsztejn ., 1997 et al

Cytochrome-c immunostaining

Cells were grown in four-well Lab-Tek chamber slides (Nalgen Nunc International, Napperville, IL). After drug treatment, cells were fixed and permeabilized by 50 v/v Permeafix (Ortho, Paris, France) in water for 1 h. Then, cells were washed with PBS buffer % containing 1 BSA and 5 FCS, and incubated for 1 h with anti-cytochrome-c antibody diluted 1:300 in the same buffer. Then, cells were % %

washed again three times and incubated with FITC-conjugated mouse Ig (Dako, Copenhagen, Denmark) diluted 1:50 in the above buffer.

Immunostaining was performed at room temperature.

NOS activity assay

NOS activity of BV-2 cytosolic extracts was determined by the conversion of H-L-arginine in H-L-citrulline. Briefly, protein reaction mixture was then mixed with AG50W-X8 resin (Bio-Rad, Ivry-sur-Seine, France) and the radioactivity of the synthesized L-citrulline was counted in the supernatant.

Lactate measurement

BV2 cells were cultured in DMEM 10 FCS for 16 hours. At this time, the culture medium was removed and the incubation

+ %
continued with fresh medium for 5 hours. The medium was then removed, centrifuged, and aliquots of culture medium were assayed for lactate by monitoring the oxidation of L-lactic acid to pyruvate by NAD in the presence of lactate dehydrogenase (Sigma). NADH formation was monitored at 340 nm.

Other assays

The accumulation of the NO-end product nitrite in the culture media was determined spectrophotometrically at 543 nm by using the Griess reagent with final concentrations of 0.5 sulfanilamide and 0.05 N-(1-naphthyl)ethylenediamine hydrochloride in 45 acetic % % % acid. The intracellular ATP was measured in cell lysates by chemiluminescence with the ATP-bioluminescence-assay-kit HS-II (Roche Diagnostics, Meylan, France) and following the supplier s instructions. Aconitase activity was measured in both cytoplasmic and ' mitochondrial fractions by following the disappearance of cis-aconitate at 240 nm at 37 C as described previously ( °Drapier and Hibbs, ). Units are nmoles of substrate consumed/min. The protein content of both the mitochondrial fraction and cytoplasmic extracts was 1996 determined at 595 nm by using the Bio-Rad protein assay with BSA as a standard.

Statistics

Data represent the mean the mean S.D. of at least three independent experiments. Statistical significance was tested with one-way ± ANOVA with post-hoc Student-Newman-Keuls comparison (p<0.01 or p<0.05).

RESULTS

Stimulation of BV-2 microglial cells induces NO-synthesis and cytochrome-c release

The combination of 20 U/ml IFN-plus 50 ng/ml LPS was chosen because it induced a time-dependent NO production in the BV-2 γ cell line without triggering toxicity. Nitrite accumulation in media was detectable as early as 4 h after stimulation, and the increase was subsequently linear for the next 20 h. Increasing IFN-and/or LPS concentration did not increase nitrite production, whereas lower γ concentrations were not fully efficient (data not shown). The NOS inhibitors L-NMMA (2 mM) and S-ethylisothiourea (SEIT, 100 M) μ reduced nitrite accumulation by an average of 82 and 90 , respectively (

). In addition, nitrite production was fully inhibited by % % Fig. 1A the NOS-2 specific inhibitors 1400W (100 M) and L-NIL (500 M). NOS activity was further assayed by the production of ( It is worthy of note that apoptosis signalling occurred during the course of the 18-h stimulation of BV-2 cells by IFN-and LPS as γ testified by cytochrome-c release. Indeed, whereas immunostaining of cytochrome-c was mainly punctual and perinuclear in control cells, the fluorescence was more diffuse in the whole cytoplasm of stimulated cells and, in some of them, fluorescence was overspread (Fig. 1C ). This phenomenon was NO-dependent since the presence of SEIT efficiently inhibited the cytochrome-c release (

).

Fig. 1C

NO-mediated inhibition of cellular respiration

The effect of NO production on the different segments of the ETC was studied in digitonin-permeabilized cells to which were added exogenous substrates and selective inhibitors of the different complexes which compose the chain. The oxygen consumption rate in state 3, which is dependent on either complex I (malate/glutamate as substrates) or complex II (succinate as substrate in the presence of rotenone), was substantially decreased in permeabilized cells which had been previously exposed to IFN-plus LPS (

). BV2 cells stimulated in γ Fig. 2 the presence of L-NMMA, an inhibitor of NOS, did not exhibit such a decrease.

After cell permeabilization and in the absence of exogenously added L-arginine and NOS cofactors, the NO flux was stopped during the measurement of oxygen consumption rate. Therefore, in agreement with the fact that inhibition of complex IV activity by NO is reversible, the oxygen consumption using TMPD plus ascorbate as substrates was not markedly inhibited under these conditions ( ). Fig. 2

Inhibition of complexes I and II isolated activities by endogenous NO

The measurement of the activity of complexes I III by O consumption relies on the assumption that the downstream segment -2 (complex IV) is not limiting. As the latter is sensitive to NO, it is worth measuring the activities of complexes I III individually.

-Accordingly, the precise site of the NO-dependent blockade in the respiratory chain was assessed by measuring the activity of each complex of the chain, by means of spectrophotometry using mitochondrial fractions prepared from control and stimulated BV-2 cells. The results depicted in show that NO production by BV2 cells was associated with an average 45 inhibition of complex I and 59 Fig. 3 % % inhibition of complex II. In addition, the activity of complexes II III was inhibited to the same extent (57 ) than that of complex II,

+ %
implying that complex III was left intact. Furthermore, in the mitochondrial fraction from cells previously stimulated in the presence of L-NMMA, the average activity of complexes I and II was 89 and 103 of the control, respectively ( ), indicating that the % % Fig. 3 inhibitions observed in stimulated cells were NO-dependent. The activity of complex IV was not inhibited in the mitochondrial fraction from stimulated cells, which can be explained, as mentioned above, by the absence of NO flux during the assay.

NO-mediated decrease of intracellular ATP and mitochondrial aconitase activity

Intracellular ATP was measured in either control or NO-producing BV-2 cells. ATP was decreased by an average of 67 in IFN-

% γ
plus LPS treated cells and maintained at control level when L-NMMA was present during stimulation ( ). Nevertheless, ATPase Fig. 4A activity was unchanged in the mitochondrial fraction from control and stimulated cells ( ), suggesting that energetic impairment Fig. 4B occurred upstream of complex V.

In addition, the activity of the Krebs cycle enzyme aconitase was strongly inhibited in the mitochondrial fraction from NO-producing cells (1.11 0.52 U/mg protein 42.36 7.33 U/mg in control mitochondrial fraction, n 3, p<0.01). By contrast, the glycolysis

± versus ± =
pathway was not affected since lactate levels were not significantly changed in IFN-plus LPS treated cells (1.37 0.08 mM 1.24 γ ± versus ± 0.18 mM in control cells medium, n 3, p>0.3).

=

Increased IRE-binding activity of IRP-1 in NO-producing BV-2 cells

The effect of NO on IRP-1 and IRP-2 was investigated in the cytosol from control and IFN-plus LPS --treated BV-2 cells by an

γ γ
EMSA. IRE-binding activity of IRP-1 was strongly increased in NO-producing BV-2 cells ( ), and, as a counterpart, cytosolic Fig. 5 aconitase activity of IRP-1 which was 100.85 37.85 U/mg protein in control cytosol, was totally inhibited. The results of also ± Fig. 5 show that IRP-2 RNA binding was not significantly modified.

Decreased LIP in NO-producing BV-2 cells

Whether NO production and IRP-1 modulation affect intracellular iron and particularly the LIP was then assessed. LIP was determined by the method described by . This procedure takes advantage of the quenching of the calcein fluorescence by iron ). In this report, we focused on the relationship between NO synthesis in microglial cells and two parameters 1997 Meda ., 1995 et al crucial for cell survival: mitochondrial metabolism and the level of redox-active iron. Stimulation of BV2 microglial cells by a combination of IFN-and LPS induced NOS activity, and a marked flux of NO was released from cells for several hours. Several γ metabolic dysregulations dealing with cellular iron-related functions and specific sites of mitochondrial energy production were observed.

Over the last twenty years, several approaches have been followed to assess the NO-dependent dysfunction of the mitochondrial respiration. Originally, cells were harvested and permeabilized before measuring the oxygen uptake by polarography. These experimental conditions allowed demonstration that complex I and complex II activities were significantly reduced soon after the beginning of physiological production of NO ( ; ;

). It was also noticed [START_REF] Granger | Sites of inhibition of mitochondrial electron transport in macrophages-injured neoplastic cells[END_REF][START_REF] Drapier | cytotoxic activated macrophages inhibit aconitase in tumor cells. Inhibition involves the iron-sulfur prosthetic group and is reversible[END_REF][START_REF] Drapier | Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells[END_REF] that electron flow was reversibly inhibited at the complex IV level ( ; ). Alternatively, each segment of the Brown, 1999 Heales ., 1999 et al electron transport chain can be individually assessed by spectrophotometry (

) to avoid possible caveats linked to Rustin ., 1994 et al measurement of oxygen consumption. Our present study combined these different approaches in the same experimental cell model. Our data underlined that persistent exposure of microglial cells to endogenous NO or derivatives led to reduction of complex I and complex II activities. Few reports have addressed the question of whether NO-dependent reduction of mitochondrial respiration is accompanied by inhibition of ATP synthesis. It has been recently reported that short exposure of brain cells to NO gas is responsible for reversible inhibition of ATP synthesis by mitochondria (

). Here, we provide evidence that long-term blockade of ETC Brookes ., 1999 et al dependent on enzymatically-produced NO, combined with a decrease in the Krebs cycle aconitase activity, was reflected by a loss of chemical energy. The pool of cellular energy (ATP) was strongly decreased, showing that the stimulated microglial cells did not compensate for the energy loss by glycolysis.

The pattern of metabolic dysfunctions we have described, . inhibition of respiration, loss of mitochondrial aconitase activity or ATP i.e depletion, is often associated with iron overload within neuronal cells. This proved true in the Parkinsonian substantia nigra (Gu ., et IRPs control, via binding to IRE or IRE-like motifs, the translation of proteins playing a relevant part in energy metabolism, namely the Krebs cycle aconitase, succinate dehydrogenase subunit b (at least in insect) and the 75 kDa subunit of complex I ( ), and Lin ., 2001 et al of proteins involved in iron uptake or storage, like ferritin, transferrin receptor, divalent metal transporter (DMT) -1 (see Cairo and for a review). In BV-2 microglial cells, IRP1 contributes a higher proportion of total IRP activity. We showed that IRP-1 Pietrangelo, 2000 (but not IRP-2) activity was markedly increased in NO-producing BV2 cells, thus implying that IRE-containing mRNAs were subject to reduced translation or higher stability. NO-dependent modulation of IRPs that has previously been described in macrophages, neurons and fibroblasts ( ; ; ;

) is reported here in Drapier ., 2000 et al Jaffrey ., 1994 et al [START_REF] Pantopoulos | Nitric oxide signaling to iron-regulatory protein: direct control of ferritin mRNA translation and transferrin receptor mRNA stability in transfected fibroblasts[END_REF]Hentze, 1995 Wardrop andRichardson, 2000 microglial cells for the first time. A potential consequence of NO-dependent IRP-1 activation in glial cells would be repression of ferritin which has an IRE in the 5 UTR of its mRNA, and increased stability of transferrin receptor mRNA which has 5 IREs in its 3 UTR. As a ′ ′ consequence, increased iron uptake coupled to lessened storage may contribute to the cell damage often seen in mitochondria-related neurological disorders ( ; ;

). Hirsch and Faucheux, 1998 R tig ., 1997 ö et al Smith ., 1998 et al It is worth stressing that IRP-1 is itself an aconitase which converts citrate into isocitrate in the cytosol. When activating the IRE-binding activity of IRP-1, NO inhibits its aconitase activity. The aconitase function of IRP-1 is still elusive but a recent genetic study in yeast pointed to a possible role of its aconitase activity in glutamate synthesis and in NADPH replenishment of the anti-oxidant systems glutathione and thioredoxin/thioredoxin reductase (

). It is likely that the aconitase form of IRP-1 contributes to Narahari ., 2000 et al NADPH production in partnership with the cytosolic NADP-dependent isocitrate dehydrogenase in resting ( not producing NO) cells. i.e.

We checked that NO synthesis in BV2 microglial cells also led to loss of mitochondrial aconitase. In mitochondria, isocitrate dehydrogenase allows regeneration of NADPH required for reduction of glutathione disulfide (

) and thioredoxin Vogel ., 1999 et al

reductase (

). Altogether, NO-dependent inhibition of cytosolic and mitochondrial aconitases, by limiting isocitrate Watabe ., 1999 et al supply, may contribute to modulation of the NADPH-dependent redox balance in both cell compartments.

A parameter relevant to oxidative stress and cellular iron homeostasis which often accompanies various neurological disorders is the fraction of intracellular iron that is not present in protein prosthetic groups or stored in ferritin. This pool of iron is still ill-defined but recent progress has been made in evaluating its level (

) and it is now referred to as the labile iron pool or LIP. In this Epsztejn

, 1997 et al.

study, we showed that LIP reduction was dependent on NO synthesis and was even more pronounced than that exhibited by deferroxamine-exposed cells. Such a 40 change of LIP level was as high as what has been previously reported in the literature in the case % of regulation by ferritin ( ; ). Interestingly, this NO-dependent LIP decrease was significantly [START_REF] Kakhlon | Repression of the heavy ferritin chain increases the labile iron pool of human K562 cells[END_REF][START_REF] Picard | Role of ferritin in the control of the labile iron pool in murine erythroleukemia cells[END_REF] different from control as early as 8 h after the beginning of the activation, . shortly after the beginning of significant NO production. i.e This result indicates that LIP decrease is not the consequence of ferritin and transferrin receptor modulation through the IRP/IRE system.

Rather, reduction of LIP may contribute to IRP-1 enhancement in cooperation with a direct effect of NO. Elucidation of the exact mechanism by which NO affects LIP requires further investigation but, as NO has strong affinity for FeII and yields EPR-detectable complexes with non heme iron in NO-producing or NO-exposed cells ( ; ; ), it [START_REF] Drapier | Interplay between NO and (Fe-S) clusters: relevance to biological systems[END_REF][START_REF] Kennedy | An EPR investigation of the products of the reaction of cytosolic and mitochondrial aconitases with nitric oxide[END_REF][START_REF] Lancaster Jr | Coinduction of nitric oxide synthesis and intracellular nonheme iron-nitrosyl complexes in murine cytokine-treated fibroblasts[END_REF] can be proposed that formation of such nitrosyl-iron complexes may represent a sink for LIP or favor its intracellular transit and/or " " metabolism. Alternatively, the significance of decreased LIP could be the reduction of the level of the ROS-production catalyst iron as a resistance mechanism in the NO/ROS-producing cells.

In conclusion, our work shows that NOS-2 expression upon inflammatory stimulation of microglial cells leads to severe reduction in ATP supply and to misregulation of iron metabolism. Irreversible . deleterious metabolic impairments resulting from NOS-2-dependent i.e (i.e., long-term) production of NO are essentially inhibition of mitochondrial aconitase, inhibition of complex I and II, and increase in both IRP-1 activity and LIP. It is striking that these dysfunctions are closely related to the energetic and iron-linked neurological disorders such as PD, AD or HD. The present results indicate that inappropriate NO production in brain during the course of an inflammatory process could exaggerate the initial defects associated with neurological disorders.

Ackowledgements:

This work was supported in part by a joint grant from ARERS and the French Ligue Nationale contre le Cancer, Comit de l Aisne, and by the

FIG. 2

Activity of ETC segments in permeabilized BV-2 cells. Oxygen consumption of digitonin-permeabilized cells was recorded by polarography as described in Materials and Methods in the presence of either complex I substrates (malate 5 mM/glutamate 5 mM), complex II substrate (succinate 5 mM) and rotenone (3 nM), or complex IV substrates (TMPD 0.2 mM/ascorbate 10 mM). Cells were previously stimulated, or not, for 16 h with IFN-(20 U/ml) plus LPS (50 ng/ml) in the presence or absence of L-NMMA (2 mM). Data are the mean S.D. of five

  °spectrophotometer

  g) was incubated for 30 min at 37 C with 0.4 Ci of L-(2,3-H)-arginine (specific activity 44.2 Ci/mmol, NEN, Les Ulis, 20 M L-arginine, 0.5 mM NADPH, 4 M tetrahydrobiopterin, 2 mM magnesium diacetate, 100 mM Tris-HCl, pH 7.5. The μ μ

  citrulline from ( H)-L-arginine in cytosol. NOS activity was induced by IFN-plus LPS treatment and was fully inhibited by the 3 γ presence of L-NMMA during the stimulation period ( ) Fig. 1B

3 ±

 3 Abbreviations usedAD : Alzheimer s disease ' Calcein-AM : calcein-acetoxy-methylester COX : cytochrome-c oxidase EMSA : electrophoretic mobility shift assay ETC : electron transport chain FA : Friedreich s ataxia ' FITC : fluoresceine-isothiocyanate IFNγ : -interferon γ IRE : iron responsive element IRP : iron regulatory protein LDH : lactate dehydrogenase LIP : labile iron pool L-NIL : N -(1-imminoethyl)-L-lysine 6 L-NMMA : N-monomethyl-L-arginine LPS : lipopolysaccharide NO : nitric oxide NOS : nitric oxide synthase PBS : phosphate-buffered saline PD : Parkinson s disease ' ROS : reactive oxygen species SEIT : S-ethylisothiourea SIH : salicylaldehyde-isocotinoyl-hydrazone UTR : untranslated region References: Araujo DM , Cotman CW . 1992 ; Beta-amyloid stimulates glial cells in vitro to produce growth factors that accumulate in senile plaques in Alzheimer s disease ' . Brain Res . 569 : 141 -145 Beal MF . 1998 ; Mitochondrial dysfunction in neurodegenerative diseases . Biochim Biophys Acta . 1366 : 211 -223 Blasi E , Barluzzi R , Bocchini V , Mazzolla R , Bistoni F . 1990 ; Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus . J Neuroimmunol . 27 : 229 -237 Boje KM , Arora PK . 1992 ; Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death . Brain Res . 587 : 250 -256 Bradley JL , Blake JC , Chamberlain S , Thomas PK , Cooper JM , Schapira AHV . 2000 ; Clinical, biochemical and molecular genetic correlations in Friedreich s ataxia ' . Hum Mol Genet . 9 : 275 -282

  FIG. 3Activity of individualized complexes of the respiratory chain in BV-2 mitochondrial fractions. Cells were stimulated, or not, for 16 h with IFN-(20 U/ml) plus LPS (50 ng/ml) in the presence or absence of L-NMMA (2 mM). Mitochondrial fractions were prepared as described in γ the Materials and Methods and used for the spectrophotometric determination of each complex activity. Data are the mean S.D. of ten ± independent experiments. Statistical significance was tested with one-way ANOVA with post-hoc Student-Newman-Keuls comparison; columns with different letters within each complex group differ from each other (p<0.01).

  The pSPT-fer Suisse de Recherches Experimentales sur le Cancer, Epalinges, Switzerland). The highly cell permeant iron chelator salicylaldehyde-isocotinoyl-hydrazone (SIH) was a generous gift from Dr. P. Ponka (McGill University, Montreal). Low endotoxin fetal calf serum and DMEM were from Life Technologies (Les Ulis, France). Calcein-acetoxymethylester (calcein-AM) from Molecular Probes was purchased from Interchim, (Montlu on, France). The NOS-2 specific inhibitors 1400W and L-NIL were from Cayman Chemicals

	γ	γ	× 7
		ç
	(Spi-Bio, Massy, France). Calcein,	Escherichia coli	lipopolysaccharide (LPS), and all other chemicals were from Sigma (L Isle d Abeau, ' '

plasmid containing the iron responsive element (IRE) of human ferritin H-chain was kindly provided by Dr. Lukas C. K hn (Institut ü

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Jeanne Casetta

Danae Gonzalez Ortiz

Céline Pochat-Bohatier

Mikhael Bechelany
email: mikhael.bechelany@umontpellier.fr

Philippe Miele

Atomic layer deposition of TiO2 on porous polysulfone hollow fibers membranes for water treatment

Keywords: Membrane, Hollow fibers, Atomic layer deposition, Titanium dioxide, Polysulfone

hydrophilicity, surface charge or porosity [START_REF] Goh | Recent trends in membranes and membrane processes for desalination[END_REF][START_REF] Yong | Polymeric membranes incorporated with metal / metal oxide nanoparticles : A comprehensive review[END_REF] . Several modification techniques are developed like blending, grafting or mixed-matrix. Govardhan et al. prepared PES/Polyetherimide HF membranes by blending method and tested the modules in a laboratory membrane unit for water reclamation. They obtained a pure water permeability at 1 bar twice higher than the initial PES HF membrane reaching also a 99 % Bovine Serum Albumin (BSA) rejection [START_REF] Cai | Zwitterionic polymers grafted poly(ether sulfone) hollow fiber membranes and their antifouling behaviors for osmotic power generation[END_REF] . Cai et al. synthesized zwitterionic polymers grafted PES HF membranes for osmotic power generation. They observed that modified membranes exhibited improved fouling resistance to protein and bacterial adhesion without alteration of their morphology. Such systems also achieved higher water flux in pressure retarded osmosis tests compared to initial membrane [START_REF] Cai | Zwitterionic polymers grafted poly(ether sulfone) hollow fiber membranes and their antifouling behaviors for osmotic power generation[END_REF] . Wan et al. prepared PVDF HF membranes with UIO-66 metal organic frameworks for arsenate removal in water. They transformed the initial membrane that could not reject arsenate because of the presence of too large pores, into a hydrophilic mixed matrix membrane able to eliminate arsenate by sorption on UIO-66 [START_REF] Wan | Arsenate removal by reactive mixed matrix PVDF hollow fiber membranes with UIO-66 metal organic frameworks[END_REF] . Another possibility for surface modification is the use of coating method, which consist in depositing a thin layer of material on the membranes' surface. Different techniques [START_REF] Fotovvati | On coating techniques for surface protection: A review[END_REF] can be used, such as sol-gel, physical vapor deposition (PVD), chemical vapor deposition (CVD) or atomic layer deposition (ALD). Dong et al. synthesized PVDF/Al2O3 hybrid membrane via sol-gel process. They obtained a pure water flux increased by 83.4 % between the initial and modified membranes while the BSA rejection was improved from 88.3 to 96.6 % [START_REF] Dong | Preparation of PVDF/Al2O3 hybrid membrane via the sol-gel process and characterization of the hybrid membrane[END_REF] . Kowalik-Klimczak et al. modified polyamide membranes with copper oxide using PVD techniques. They achieved a stable coating during filtration of demineralized water and the modified membranes showed a strongest bactericidal activity, which allowed eliminating 99% of microorganisms [START_REF] Kowalik-Klimczak | The polyamide membranes modified by copper oxide using PVD techniques[END_REF] . Nomura developed silica membranes by CVD method on a porous ceramic substrate with controlled pore sizes and hydrothermally stable for the steam reforming reaction to extract H2 [START_REF] Nomura | Preparation of Silica Membranes by CVD Method. Current Trends and Future Developments on (Bio-) Membranes: Silica Membranes: Preparation, Modelling, Application, and Commercialization[END_REF] . All of these methods are interesting and provide promising results but ALD stands out from the crowd thanks to its very high surface conformality. Where PVD concerns only the surface , sol-gel blocks the pores and CVD will not provide a uniform layer all along, the surface-controlled film growth of ALD allows the obtaining of highly uniform coatings over surfaces of three-dimensional (3D) parts, deep trenches, porous materials and particles [START_REF] Pakkala | Atomic Layer Deposition[END_REF] . ALD is in fact one of the most recent techniques, a unique technology developed over the last four decades [START_REF] Nikkola | Surface modi fi cation of thin fi lm composite polyamide membrane using atomic layer deposition method[END_REF] , which allows the deposition of inorganic coating layers such as oxides; for example iron oxide FeO [START_REF] Gao | Cooperating effects of conformal iron oxide (FeOx) ALD coating and postannealing on Li-Rich layered cathode materials[END_REF] , aluminium oxide Al2O3 [START_REF] Neudeck | Effect of Low-Temperature Al 2 O 3 ALD Coating on Ni-Rich Layered Oxide Composite Cathode on the Long-Term Cycling Performance of Lithium-Ion Batteries[END_REF] , lanthanum oxide La2O3 [START_REF] Barhate | Spectroscopic study of La2O3 thin films deposited by indigenously developed plasma-enhanced atomic layer deposition system[END_REF] , silica SiO2 [START_REF] Mione | Atmospheric-Pressure Plasma-Enhanced Spatial ALD of SiO2Studied by Gas-Phase Infrared and Optical Emission Spectroscopy[END_REF] or tin oxide SnO [START_REF] Kim | Phase-controlled SnO2 and SnO growth by atomic layer deposition using Bis(N-ethoxy-2,2-dimethyl propanamido)tin precursor[END_REF] ; with high uniformity and precise sub-nanometer thickness control. As an environmental-friendly, low-cost and highly hydrophilic oxide, titanium dioxide TiO2 can be advantageously used for modifying membrane surface properties [START_REF] Feng | Article Atomic layer deposition of TiO 2 on carbonnanotubes membrane for capacitive deionization removal of chromium[END_REF] . Hamid et al. incorporated TiO2 nanoparticles into PSF HF membranes and observed a diminution of water contact angle (WCA) from 75° for raw membranes to 45° for TiO2 modified membrane. They also proved great antifouling properties against humic acid with a twice higher flux and a better flux recovery from 90 % to 95 % [START_REF] Hamid | Morphological and separation performance study of polysulfone/titanium dioxide (PSF/TiO2) ultrafiltration membranes for humic acid removal[END_REF] . Nechifor et al. produced polysulfone-silica microfiber grafted with TiO2 nanoparticles composite membranes. They obtained a 94% retention of BSA and a water permeability increase of 45%, as long as a great anti-bacterial action for PSF/SiO2 + TiO2 composite membranes [START_REF] Nechifor | Non-resorbable nanocomposite membranes for guided bone regeneration based on polysulfone-quartz fiber grafted with nano-TiO2[END_REF] . Recent studies have shown that ALD is applicable to flexible and temperature-sensitive polymeric membranes [START_REF] Lee | Environmental Science membrane surface engineering methods for water treatment : a short review[END_REF][START_REF] Weber | Atomic Layer Deposition for Membranes : Basics[END_REF] , the deposition of an oxide thin layer could increase the membrane hydrophilicity, modify its pore size and reduce the membrane fouling despite sacrificing the water permeability. Actually, some reviews are focused on the sensitization of nominally unreactive membrane materials for subsequent ALD and the use of ALD oxides as a hydrophilization coating on membranes. Yang et al. reviewed ALD/ sequential infiltration synthesis (SIS) engineering strategies with realization of efficient water treatment. They laid out a pathway to establish an ALD/SIS-based universal functionalization platform for water treatment, including sensitization strategies. [START_REF] Yang | Water treatment based on atomically engineered materials: Atomic layer deposition and beyond[END_REF] Wu et al. summarized the most recent advances in molecular engineering to tailor the properties of interfaces in composite membranes. They explained how the performances of membranes can be enhanced by the way of several surface modification techniques. [START_REF] Wu | Recent progress in molecular engineering to tailor organic-inorganic interfaces in composite membranes[END_REF] Yang et al. discussed the application of ALD and related techniques in the design of novel membrane interfaces and explained how ALD can be used for instance to modify the surface chemistry and interfacial properties of membranes and tailor the pore sizes. [START_REF] Yang | Atomic layer deposition for membrane interface engineering[END_REF] Feng et al. proceeded ALD of ZnO on carbon nanotubes. For 40 cycles they observed the highest permeability value at 430 L.m -2 h -1 bar -1 , a WCA reduced by a third and a six time higher BSA rejection. However, for more than 40 cycles the permeability decreased and the WCA did not change [START_REF] Feng | Atomic layer deposition of metal oxides on carbon nanotube fabrics for robust, hydrophilic ultrafiltration membranes[END_REF] . Wang et al. deposited polyimide on the surface of nanoporous anodized alumina by ALD. They showed that 50 cycles lead to the remarkable upgrade of retention from nearly none for the neat alumina substrate to 82 % while the water permeability almost three time lower was still maintained as high as 800 L/hm 2 bar [START_REF] Wang | Atomic-layer-deposition-enabled thin-film composite membranes of polyimide supported on nanoporous anodized alumina[END_REF] . The interest in using ALD technique on polymers is growing for different type of applications such as in barrier materials, protective coatings and gas and liquid filters. The mechanisms and reactions occurring during ALD process on some polymers had been studied by Parsons et al. They focused particularly on the common Al2O3 reaction sequence using trimethyl aluminum (TMA) and water on different kind of polymers such as polypropylene (PP), polyamide or polyester. They explained that reaction during TMA/H2O deposition on polymer materials depends strongly on the starting polymer, the precursor as well as the detailed process conditions. With inert polymer like PP, the TMA diffuses into the near surface region of the polymer and will then react with water during the second half-cycle step to form nuclei beneath or at the polymer surface. On polymers where there is a large density of reactive groups, the ALD reaction can be limited to the top surface of the polymer. Carbonyl site is the most Lewis basic functionality in some polymers, so it is expected to coordinate with the TMA Lewis acid to form an aluminum-oxygen-alkyl unit and to disrupt the hydrogen bonding between neighboring chains, which could act to "open up" the polymer chain network, promoting further TMA diffusion into the subsurface region. Like TMA, TiCl4 is also a strong Lewis acid, so it may diffuse into the polymer and react with it through similar Lewis acid/base adducts [START_REF] Parsons | Mechanisms and reactions during atomic layer deposition on polymers[END_REF] . To the best of our knowledge, there are several articles that focused on the insertion of TiO2 onto PSF membranes [START_REF] Hamid | Morphological and separation performance study of polysulfone/titanium dioxide (PSF/TiO2) ultrafiltration membranes for humic acid removal[END_REF][START_REF] Kusworo | Effect of nano-TiO2 loading in polysulfone membranes on the removal of pollutant following natural-rubber wastewater treatment[END_REF][START_REF] Zhang | Novel polysulfone hybrid ultrafiltration membrane prepared with TiO2-g-HEMA and its antifouling characteristics[END_REF][START_REF] Yang | Preparation and properties of polysulfone/TiO2 composite ultrafiltration membranes[END_REF][START_REF] Bidsorkhi | Preparation and characterization of a novel highly hydrophilic and antifouling polysulfone/nanoporous TiO2 nanocomposite membrane[END_REF] or on the deposition of oxides on polymeric membranes [START_REF] Wang | PVDF membranes with simultaneously enhanced permeability and selectivity by breaking the tradeoff effect via atomic layer deposition of TiO 2[END_REF][START_REF] Jia | Atomic layer deposition of Al2O3 on porous polypropylene hollow fibers for enhanced membrane performances[END_REF] but there is no study focusing on TiO2 ALD on PSF HF membranes. This study focus on the surface modification of PSF HF membranes by TiO2 using ALD technique and its influence on the fiber properties compared to raw PSF HF membranes. The effect of ALD treatment on the membrane morphology was investigated by scanning electron microscopy (MEB) and in terms of hydrophilicity and pores size. The performances of the HF membranes for water purification application were characterized: water permeability, antifouling using Bovine Serum Albumin (BSA) protein and mechanical properties. The modification of polymeric membranes by ALD requires a preliminary step of conditioning to enable them to withstand vacuum under heat treatment. To the best of our knowledge, there has not yet reported studies in the literature clarifying the conditioning of polymeric membranes for TiO2 deposition by ALD on PSF HF membranes. So in a first step, we will present the glycerin conditioning of the membrane and then the effect of deposition of 10, 20, 50 and 100 cycles TiO2 on the membrane structure and properties.

Materials and methods

Materials

PSF HF membranes supplied by Polymem were used as substrates for TiO2 ALD. The membranes were received after chlorine washing and glycerin conditioning steps. Titanium (IV) chloride (TiCl4, 99.9%, CAS: 7550-45-0) and deionized water were served as metallic and oxygen source for the ALD process, respectively. Argon gas was bought from Linde and used as received. Phosphate buffered saline (PBS) (Roth) and bovine serum albumin (BSA) (Mw=67 kDa, ≥96%, Sigma-Aldrich) solution were employed to assess the fouling resistance of both raw and modified PSF HF membranes. Dextran (Mw from 6 to 2,000 kDa, Sigma-Aldrich) were used for membrane pore size characterization. Deionized water (Millipore Milli-Q) was used in all aqueous solutions.

TiO2 ALD on PSF HF membranes

The commercial PSF membranes were received conditioned in a water/glycerin mixture. This processing is usually applied in industry to preserve the porous structure along storage. The glycerin must then be removed when the membranes are in use. Membranes called P, P-0 and P-10 have been washed in successive water baths to remove glycerin before experiments. Membranes called P-g-0, Pg-10, P-g-20, P-g-50 and P-g-100 undergone ALD process as received. After deposition, they were washed in successive water baths to remove glycerin to determine their functional properties. The commercial PSF membranes were introduced for a 15 min acclimation in the home-built ALD reactor under vacuum (~10 -2 mbar) and preheated at 100°C. The deposition process was achieved using TiCl4 and H2O as precursors contained in stainless steel cylinders which lines were heated at 80°C to avoid condensation. One ALD cycle consisted of 50 ms pulse of TiCl4, followed by 10 s exposure and 60 s purge with Ar gas, then H2O valve was opened for 2 s, followed by an exposure of 10 s and purge for 60 s. Several cycles were applied 10, 20, 50 and 100 that correspond respectively to 1, 2, 5 and 10 nm thick deposit according to a previous study conducted in this ALD chamber and using these operating conditions [START_REF] Sayegh | N-doped TiO2 nanotubes synthesized by atomic layer deposition for acetaminophen degradation[END_REF] .

Table 1 presents the nomenclature of the PSF HF membranes that have undergone the different tests of this study.

Table 1:

Membranes nomenclature (All membranes, except from P and P-g, were introduced in ALD chamber for a 15 min vacuum acclimation at 100°C) Fiber's name Description P PSF industrial fiber reference washed to remove glycerin P-0 PSF industrial fiber washed and placed in the ALD chamber under vacuum and at 100°C during 25 min without deposition P-g PSF industrial fiber reference conditioned with a water/glycerin mixture P-g-0 PSF industrial fiber conditioned with glycerin placed in the ALD chamber under vacuum and at 100 °C during 25 min without deposition P-10 PSF industrial fiber placed in the ALD chamber under vacuum and at 100 °C during 25 min for TiO2 deposition (10 cycles corresponding to 1 nm thickness) P-g-10 PSF industrial fiber conditioned with glycerin placed in the ALD chamber under vacuum and at 100 °C during 25 min for TiO2 deposition (10 cycles corresponding to 1 nm thickness) P-g-20 PSF industrial fiber conditioned with glycerin placed in the ALD chamber under vacuum and at 100 °C during 1 h for TiO2 deposition (20 cycles corresponding to 2 nm thickness)

Characterizations

The morphology of the membrane, both cross-section and surface, was observed using a Hitachi S4800 scanning electron microscopy system (SEM) after previous nitrogen cold-cutting. Energy-dispersive Xray spectroscopy analysis (EDX) was performed with a Zeiss EVO HD15 microscope coupled with an Oxford X-MaxN EDX detector. To investigate the changes in chemical structure and to confirm the successful deposition of TiO2 between the raw and the modified PSF HF membranes, Fourier Transform Infrared (FTIR) spectra were acquired using FTIR spectrometer NEXUS instrument equipped with an attenuated total reflection (ATR) accessory in the frequency range of 600-4000 cm -1 . The ATR-FTIR spectra were recorded with a resolution of 4 cm -1 , and the signals were averaged from 64 scans. The mechanical properties of the raw and modified PSF HF membranes were characterized using a dynamic mechanical analysis device (Z005, 5 kN Proline, Zwick Roell). A 10N sensor and a tensile testing speed of 0.4 mm•min -1 were applied. The Young's modulus of the membranes was calculated directly from the device. Water contact angles (WCA) were measured using a tensiometer/goniometer ILMS GBX, equipped with an optic telecentric F55 double focal monochrome and a GBX software. For each sample, about 0.5 μL of ultrapure water was deposited on the membranes using a needle. Furthermore, X-ray Photoelectron Spectroscopy (XPS) was carried out with a Kratos AXIS NOVA spectrometer (Kratos Analytical Ltd., Manchester, UK) using a monochromatized Al Kα X-ray source (1486.6 eV) operating at a power of 150 W (10 mA, 15 kV). Survey and high-resolution spectra were acquired at 160 and 20 eV pass energies, respectively. Mean pore diameter of raw HF membrane had been kindly determined by Porometer using the liquid-liquid porometry on a POROLIQ 1000ML.

Filtration experiments

A U-tube shaped hollow fiber bundle which consisted of 10 wet fibers measuring each 40 cm long (equivalent to 90 ± 5 cm 2 effective membrane area) was prepared using a homemade polyvinyl chloride (PVC) housing module. Epoxy resin was used to seal the module. The pure water flux (Jw) was measured for each membrane by circulating pure water through the membrane system using an applied pressure range of 0.5-1.5 bar. Each point was obtained measuring the flow passing through the membrane during 60 s. The pure water flux, Jw (L.h -1 m -2 ), was calculated using the following formula:

𝐽𝑤 = 𝑄 𝐴 (𝐿 • ℎ -1 • 𝑚 -2 ) (1)
where Q (L•h -1 ) is the amount of water that passed through the membrane and A (m 2 ) is the membrane area. The permeability was determined from the slope of the linear variation of JW versus the applied pressure.

P-g-50 PSF industrial fiber conditioned with glycerin placed in the ALD chamber under vacuum and at 100 °C during 3 h for TiO2 deposition (50 cycles corresponding to 5 nm thickness) P-g-100 PSF industrial fiber conditioned with glycerin placed in the ALD chamber under vacuum and at 100 °C during 8 h for TiO2 deposition (100 cycles corresponding to 10 nm thickness)

Rejection experiments were conducted to estimate the pore size and their reduction after ALD using 1 g/L aqueous solutions of Dextran solutes with Mw going from 6 to 2,000 kDa. A U-tube shaped hollow fiber bundle was prepared like described above. Dextran contents in the feed (Cf) and in the permeate (Cp) solutions were determined by flow injection analysis using refractometer 2414 (Waters Corporation). The solute rejection (R) was calculated using the following formula:

𝑅 = 1 - 𝐶 𝑝 𝐶 𝑓 × 100 (%) (2)
The membrane was first conditioned by applying a pressure of 1.5 bar with distilled water for 30 minutes. External-internal solute filtration of the membrane was then carried out for 20 min at 0.5 bar.

The hydrodynamic radius values, rp in Å, of the various Dextran molecules used in these experiments were estimated from the following equation [START_REF] Wickramasinghe | Relating the pore size distribution of ultrafiltration membranes to dextran rejection[END_REF] :

𝑟 𝑝 = 0.488 𝑀𝑤 0.437 ( 3
)
where MW is the molecular weight of the Dextran.

Static protein adsorption was performed to estimate the anti-fouling properties of the TiO2-deposited PSF HF membranes and the raw PSF HF membrane was used as reference. An I-shaped hollow fiber bundle, consisting of 10 wet fibers measuring each 27 cm long (equivalent to an effective membrane area of 63.28 cm 2 ), was prepared using a polyvinyl chloride (PVC) module and an epoxy resin as sealing agent. The protein solution was prepared by dissolving 2 g of BSA in 2 L of PBS solution at pH 7.4. The membrane was first conditioned by applying a pressure of 1.5 bar with distilled water for 30 minutes. Initial external-internal water filtration of the membrane is then carried out for 20 min at 0.5 bar and the steady pure water flux was recorded (Jw0) using a balance and data acquisition software. The tank was then filled with the BSA solution and filtration was carried out at 0.5 bar for 2 hours and foulant flux was recorded (Jwf). The BSA was removed by mechanical agitation and then backwashing with water applying a pressure of 1.5 bar internally-externally for 5 min. Finally, a last external-internal filtration cycle with water for 20 min at 0.5 bar was performed and the steady flux was recorded (Jw1). The total fouling ratio (Rt), flux recovery ratio (FRR), the reversible fouling ratio (Rr) and the irreversible fouling ratio (Rir) were estimated using equations ( 2)-( 5), respectively [START_REF] Alkhouzaam | Novel polysulfone ultrafiltration membranes incorporating polydopamine functionalized graphene oxide with enhanced flux and fouling resistance[END_REF] :

𝑅 𝑡 = 𝐽 𝑤 0 -𝐽 𝑤 𝑓 𝐽 𝑤 0 𝑥100 (%) (4) 𝐹𝑅𝑅 = 𝐽 𝑤 1 𝐽 𝑤 0 𝑥100 (%) (5)
𝑅 𝑟 = 𝐽 𝑤 1 -𝐽 𝑤 𝑓 𝐽 𝑤 0 𝑥100 (%) (6)
𝑅 𝑖𝑟 = 𝐽 𝑤 0 -𝐽 𝑤 1 𝐽 𝑤 0 𝑥100 (%) (7)
The feed BSA solution, BSA retentate and permeate concentrations are calculated from Beer Lambert's Law using the constant k = 0.6439 L/g and an ultraviolet (UV) spectrometer on the absorbance at 280 nm (Uviline Connect Series 940). The rejection of protein was obtained from equation (2) where Cp and Cf are the protein concentrations of BSA in permeate and feed.

Results and discussion

Glycerin conditioning

The successful deposition of TiO2 by ALD on HF membranes is challenging because of the temperature and pressure sensitivity of the polymers. A first study was carried out to observe the influence of glycerin conditioning on the morphology and water permeability of membranes after the deposition of TiO2. It is necessary to know whether the glycerin brings a protective effect to the polymeric membranes that underwent ALD process. The internal and external diameters of the membranes as well as their lengths were measured before and after ALD treatment. It can be seen that the diameters and lengths are not affected by vacuum (~10 -2 mbar), temperature (100°C) and TiO2 deposition as the values before and after are nearly identical. SEM images of the PSF HF membranes cross sections are presented in Fig. 1. All fibers present a sponge-like asymmetric structure characteristic of HF membranes. In the case of P-10 (without glycerin), SEM images show that most of the pores have collapsed during the deposition, which some are pointed out by the circles, whereas for P-g-10 (sample with glycerin) it is not possible to distinguish any significant change comparing to the raw PSF HF membrane. We assume that glycerin originating from conditioning and settled in the pores of the fiber allows protecting the structure during the preheating (100 °C) and under vacuum acclimation (~10 -2 mbar). Without glycerin, the HF structure is damaged by temperature and vacuum. No other difference was noticeable between raw and TiO2 deposited membranes on SEM images. The mean pore diameter of the raw PSF HF membrane was determined by liquid-liquid porometry to be 25 nm. It was believed that ALD deposition may reduce the pore size [START_REF] Wang | PVDF membranes with simultaneously enhanced permeability and selectivity by breaking the tradeoff effect via atomic layer deposition of TiO 2[END_REF] which was not possible to quantify via this technique as they are too small. Pore size reduction will be evaluated in the last part of this paper using dextran rejection.

The necessity of glycerin conditioning was also evidenced by the results of permeability measurements presented in Table 2. On one hand, membranes without glycerin conditioning P-0 and P-10 display a very low permeability (5±1 and 4±1 L/h -1 m -2 bar -1 respectively) after ALD process. In accordance with SEM images, the deposition damaged the structure of the membrane leading to the collapse of the pores and thus the diminution of water permeability. On the other hand, the glycerin allowed P-g-10 to show a 26 % higher permeability around 170 L/hm 2 bar against 135 L/hm 2 bar for raw membrane P. The 27 L/hm 2 bar permeability of P-g-0 pointed out that glycerin is no longer able to prevent the pore collapsing during the whole vacuum time residence. In fact, as presented in Table 2, P-0 and P-g-0 membranes spent the most time in the ALD chamber under vacuum: 40 min against 15 min for P-10 and P-g-10. So during the fifteen first minutes, the glycerin managed to counteract the pore reduction under vacuum at 100°C, then it is fully evaporated and the membrane structure is altered for the twenty-five other minutes. This explains the drop in permeability for P-g-0. For P-g-10, after the 15 min vacuum acclimation at 100°C, the deposition started and vacuum was stopped. According to SEM pictures and water permeability measurements, the efficiency of glycerin conditioning has been clearly proved. Moreover, the acclimation period before ALD cycles process was a key parameter to allow a good TiO2 deposition. Several tests have been performed and it was observed that, even for HF with glycerin, an acclimation time superior to 15 minutes deteriorate the HF membrane. After more than 15 minutes in the ALD chamber under vacuum at 100°C, the HF was totally dried and the result is a water permeability close to zero. This proved that glycerin conditioning is efficient only coupled with an acclimation no longer than 15 minutes. Indeed, the boiling temperature of glycerin was reduced from 290°C at atmospheric pressure to 96°C at the pressure applied during deposition (10 -2 mbar) [START_REF] Lide | CRC HANDBOOK of THERMOPHYSICAL and THERMOCHEMICAL DATA[END_REF] . We can propose that all the glycerin available on the HF surface is evaporated and only a few amount bounded onto the pores maintain the internal morphology at the end of acclimation at 100°C. The evaporation of glycerin was proved in Fig. 2 (a) with FTIR analysis. Indeed, pure glycerin showed two signals between 2800 and 3000 cm -1 corresponding to C-H stretching and one broad and intense band around 3300 cm -1 corresponding to O-H stretching. Three fibers were analyzed, the PSF industrial fiber reference conditioned with glycerin P-g, the PSF industrial fiber conditioned with glycerin placed in the ALD chamber under vacuum and at 100°C during 10 cycles for TiO2 deposition P-g-10 and the PSF industrial fiber reference washed after conditioning. The band around 3300 cm -1 also corresponds to water but the three fibers have been placed in a 60°C oven overnight before analysis, which lead to water evaporation as shown by the P spectrum (washed membrane containing no glycerin). The absorption band at 3300 cm -1 is not visible anymore. P-g spectrum presents the same O-H pic than glycerin but less intense whereas P-g-10 did not show any pic in this area. Therefore, it can be concluded that 15 min acclimation in the ALD chamber at 100°C under vacuum eliminate all the glycerin by evaporation as the IR characteristic signals of glycerin have disappeared of the sample after deposition. When the TiO2 deposition started, TiCl4 reacted directly with PSf support. Based on these observations, glycerin conditioning and 15 min acclimation were applied for all the following ALD tests.

Influence of ALD cycles on membrane properties

Structural and physico-chemical characterizations

The formation of TiO2 layers on the PSF HF membranes was investigated by ATR-FTIR spectroscopy. In Fig. 2 (b), the characteristic IR peaks of PSF are visible in raw and modified membranes. Signals observed at 1105, 1488, 1506 and 1586 cm -1 correspond to the vibrations of PSF aromatic ring (C=C stretching). Bands at 1151 cm -1 and 1240 cm -1 were attributed to the -O-S-O-symmetric and aromatic ether bond (-C-O-C-), respectively. The peak at 1295 cm -1 was related to SO2 group stretching vibration [START_REF] Cabasso | Polysulfone Hollow Fibers[END_REF] . Additionally, absorbance peak at 1676 cm -1 could be attributed either to the C=O stretching vibration of NMP or PVP that might remain on the membrane surface. Any new peaks could be observed for the ALD modified membranes in the investigated frequency range. Thus we can assume that the amount of TiO2 is too low to be detected via this technique, as a consequence of a low number of ALD cycles. Actually, Jia et al. studied the deposition of Al2O3 on porous polypropylene HF by ALD technique, and they only noticed a new absorption peak after more than 100 deposition cycles [START_REF] Jia | Atomic layer deposition of Al2O3 on porous polypropylene hollow fibers for enhanced membrane performances[END_REF] .

Moreover, Wang et al. studied the deposition of TiO2 by ALD on PVDF membranes and they suggested that the pic intensity was similar under 100 deposition cycles and began to drop after, as the number of ALD cycles increased. The TiO2 coating layer may reduce the depth that the light beam can reach in the material, thus the detected IR signal of the PVDF membrane is weaker. [START_REF] Wang | PVDF membranes with simultaneously enhanced permeability and selectivity by breaking the tradeoff effect via atomic layer deposition of TiO 2[END_REF] The same trend is observed for our samples, the intensity of the peaks decreases when increasing the number of ALD cycles. Any evidence of the deposition of TiO2 on the PSF HF membranes using ALD technique was found by ATR-FTIR spectroscopy technique. In addition, Waldman et al. focused on the SIS of Al2O3 in PES membranes and were able to understand the molecular interactions during ALD process through FTIR experiments. They explored in situ FTIR of TMA in-diffusion and out-diffusion to elucidate the nature and kinetics of an attractive interaction between the TMA and the PES moieties. All their FTIR measurements allowed them to confirm that TMA associates with the sulfonyl and aryl ether groups of the PES polymer in a qualitatively similar way to the association with the carbonyl and ester groups of PMMA. [START_REF] Waldman | Sequential Infiltration Synthesis of Al2O3 in Polyethersulfone Membranes[END_REF] This study could help us understanding the molecular interactions that took place in our ALD process supposing that TiO2 may react with sulfonyl and ether groups of the PSF membrane. SEM analyses performed on the top surface of raw PSF HF membrane P, and modified PSF HF membranes are presented in Fig. 3. They showed that for raw PSF HF membrane P and the membrane that undergone 10 cycles ALD no change was observed on the surface, which supposed a thin and uniform coating. However, for the PSF HF membrane with 50 ALD cycles, P-g-50, a geometric and regular structure on surface was observed most likely corresponding to the TiO2 thicker layer that may have cracked. Similar micrographs were obtained for P-g-100. In order to quantify the amount of TiO2 deposited on membranes, EDX of all raw and ALD modified PSF HF membranes was performed. The measured atomic percentages of C, O and Ti are presented in Table 3. The detected atomic percentages of Ti were very low for P-g-10 and P-g-20 samples ( 0.1) due to small layer thicknesses of TiO2 (1 and 2 nm respectively), but the Ti atomic percentage increases for P-g-50 and P-g-100 following the increasing number of ALD cycles. These results are in good agreement with SEM observations which allow the observation of the deposit starting from 50 ALD cycles. Moreover, we can add from EDX cross-sectioned mapping in Fig. S1 and Fig. S2 that TiO2 was uniformly deposited onto the membrane network. To go further, XPS analyses were performed on raw and modified membranes. No difference was observed between raw and P-g-10 and P-g-20. We assumed that, according to the other analyses methods, for 10 and 20 ALD cycles the amount of TiO2 was too low and under the detection limit. P-g-50 and P-g-100 presented the same spectra. The survey of C 1s, O 1s and Ti 2p deconvoluted spectra of P and P-g-50 are presented in Fig. 4. The XPS survey spectra of both membranes (Fig. 4a) consist of two peaks at the binding energies of 284.85 and 532.05 eV assigned to C and O atoms respectively with an additional peak around 458.36 eV for P-g-50 assigned to Ti atom. The C signal in the highresolution spectra of the raw membrane P could be deconvoluted into three peaks (Fig. 4b): Carbon atoms attached to hydrogen/carbon, sulfone and ether groups exhibit C 1s core-level binding energies of 284.80, 286.20 and 287.90 eV respectively. The C 1s spectra of P-g-50 (Fig. 4d) was similar which supports that the PSf matrix was not damaged during deposition. However, the deconvoluted spectra of O 1 s included two peaks for P (Fig. 4c) at 532.70 and 531.75 eV corresponding to O-C and O=S bonds respectively while a third peak appeared for P-g-50 (Fig. 4e) at 529.86 eV corresponding to the O-Ti bond. This confirmed that TiO2 was well deposited and reacted with the oxygen atoms of the PSf matrix. For P-g-50, in the deconvoluted Ti 2p spectra (Fig. 4f), two peaks are positioned at 458.36 and 464.16 eV, corresponding to Ti 2p3/2 and Ti 2p1/2 states indicating that Ti is 4+ valence.

Membrane performances

Mechanical tests were performed on raw and modified membranes. Young modulus and the strength at break values are presented in Table 5. The results showed that ALD-modified membranes displayed a higher Young modulus around 147 MPa in average compared to raw membrane (132 MPa), which corresponds to a lower resistance to stress deformation. Any correlation between the increasing number of ALD cycles and the increase Young modulus has been noticed. Because the ALD modified membranes were more rigid, the strength applied to break them was lower than for raw PSF HF membrane decreasing from 2.10 ± 0.02 to in average 1.76 N. However, TiO2 could act as crosslinker between the polymer chains, improving consequently the rigidity of the network and therefore, the mechanical strength [START_REF] Galiano | Novel photocatalytic PVDF/Nano-TiO2 hollow fibers for Environmental remediation[END_REF] . This observation is in accordance with Diez-Pascual et al., who studied the effect of TiO2 nanoparticles insertion on the performance of polyphenylsulfone. They observed a gradually increasing Young's modulus upon raising nanoparticle loading, by up to 62 % at the highest concentration tested. They attributed this improvement to the effect of the rigid TiO2 with the interactions (H-bonding) it established with the membrane matrix. This results in a decrease in polymer chain motion and in promoting the adhesion between the two phases [START_REF] Díez-Pascual | Effect of TiO2 nanoparticles on the performance of polyphenylsulfone biomaterial for orthopaedic implants[END_REF] . In Table 4 are also presented the permeability values which matched with the SEM observation. In fact, a thin and uniform TiO2 layer on the membrane surface for P-g-10 and P-g-20 allowed a 26 to 50 % higher permeability value compared to raw membrane P. However, starting from 50 cycles when the coating was thicker a drastic drop in permeability is observed. From these results, 50 and 100 cycles could be discriminated, supposing that the layer is too thick to allow great performance. For the rest of the study, P-g-100 was forsaken and P-g-50 was still studied to compare with P-g-10 and P-g-20. SEM did not permit to measure the pore size reduction reported in other works [START_REF] Wang | PVDF membranes with simultaneously enhanced permeability and selectivity by breaking the tradeoff effect via atomic layer deposition of TiO 2[END_REF] . To complete, different sizes of dextran were filtered through raw and ALD modified PSF HF membranes (P-g-10 and P-g-20). The evolution of the rejection rate versus molecular weight of dextran molecules is presented in Fig. 5 (a). The correspondence between the molecular weight and the pore diameter of dextran molecules was calculated based on Equation (3) and is presented in Table 5. All dextran molecules were able to pass through the raw PSF HF membrane pores from a rejection rate near zero for the smallest molecule to 80% rejection for the biggest at 55.3 nm. It can be observed that even if the mean pore size of raw PSF HF membrane was measured at 25 nm, there is a large distribution that allows molecules up to 55.3 nm going through the membrane. On the other side, P-g-10 presented already 20% rejection rate for smallest dextran at 4.7 nm, 50% for 10 nm sized molecules and reached 90% for 30.2 nm dextran. P-g-20 follows the same trend. Other works from the literature gathered in Table 6 confirmed a noticeable reduction of pore size and pore size distribution after ALD process. Dextran rejection rates support SEM observations to evidence the deposition of TiO2 on all the PSF HF membrane surface and subsurface. 3 It is well known that surface hydrophilicity affects the membranes antifouling properties. Membranes' WCA were measured for P, P-g-10 and P-g-20 and the results are presented on Fig. 5 (b). Initially, the raw membrane had a WCA of around 90° and this value was gradually reduced down to 70° as the number of ALD cycles increased. These results are in good agreement with literature as reported in Table 5. Indeed, Zhang et al. prepared PSF hybrid ultrafiltration flat membrane with TiO2-g-HEMA and studied its antifouling characteristics. The best hydrophilicity improvement they obtained with a WCA of 72° was for 2% concentration [START_REF] Zhang | Novel polysulfone hybrid ultrafiltration membrane prepared with TiO2-g-HEMA and its antifouling characteristics[END_REF] . The filtration performances of raw and ALD modified PSF HF membranes are depicted in Fig. 6 (a). The raw membrane exhibited a pure water permeability of 135 L•h -1 m -2 bar -1 and a retention of BSA around 80%. However, after the ALD deposition of TiO2, the permeability increases slightly reaching the higher value for 20 ALD cycles, then it decreases drastically for P-g-50 which displayed a water permeability of 15 L•h -1 m -2 bar -1 . P-g-10 and P-g-20 not only showed an increased water permeability but also a higher BSA retention compared to raw PSF HF membrane. Wang et al. deposited TIO2 on PVDF membranes via ALD process at 80°C. They observed a decrease of permeability that they attributed to the pore size diminution which is probably the result of the ALD process. Indeed, in addition to the surface, the TiCl4 molecules can also penetrate into the subsurface of the membrane. Water vapors pulsed into the chamber reacted with the TiCl4 entrapped in the free volumes between the polymer chains producing layers of TiO2. When the number of ALD cycles increases, the TiO2 keeps growing inside the membrane pores until they are completely blocked [START_REF] Wang | PVDF membranes with simultaneously enhanced permeability and selectivity by breaking the tradeoff effect via atomic layer deposition of TiO 2[END_REF] . In our case, a pore size reduction was (a) also noticed from dextran filtration accompanied by an increase in hydrophilicity. These two combined parameters explain the membrane filtration performances. Hypothetically, during acclimation all the glycerin was slowly evaporated which prevented the pores from collapsing and maintained the internal morphology. Above 20 ALD cycles, in accordance to SEM images, the observed TiO2 layer for P-g-50 and P-g-100 could block the pores on the membranes' surface causing the drop in water permeability.

Antifouling performances of the ALD modified membranes were investigated using BSA as a fouling agent, for that the FRR, Rt, Rr and Rir of the membranes are calculated from equations (4) to (7). All the calculated values are represented in Fig. 6 (b). FRR is at 100% for P-g-10 and P-g-20 compared to 88% for raw PSF HF membrane. This means that due to TiO2 addition, ALD modified PSF HF membranes were recovering all the flux after BSA filtration. Indeed, all ALD modified PSF HF membranes exhibited lower Rt, furthermore interestingly the Rr decreased with the increase of the number of ALD cycles. A reducing Rr means that BSA adsorbed reversibly was removed after water backwashing, which is a good result as membranes lifetime is threatened by fouling and most of all irreversible fouling and chemical backwashing needed to clean them. Rir initially at 12% for raw membrane was at 0% for both P-g-10 and P-g-20 and around 19% for P-g-50. P-g-100 was tested but the result was not detectable. On the one hand, the ALD modified PSF HF membranes demonstrated lower BSA fouling but also a 0% Rir which means that all the BSA adsorbed can be removed with water backwashing. This shows that with TiO2 the PSF HF membranes are less sensitive to BSA fouling and would have a longer lifetime. They display higher fouling resistance against protein compared to the raw PSF HF membrane. These results are very interesting since the values are even better than that reported in other studies (Table 6). Yang et al. [START_REF] Yang | Preparation and properties of polysulfone/TiO2 composite ultrafiltration membranes[END_REF] and Zhang et al. [START_REF] Zhang | Novel polysulfone hybrid ultrafiltration membrane prepared with TiO2-g-HEMA and its antifouling characteristics[END_REF] inserted for instance TiO2 nanoparticles into flat PSF membranes and obtained respectively 97 and 95% BSA rejection. Jia et al. [START_REF] Jia | Atomic layer deposition of Al2O3 on porous polypropylene hollow fibers for enhanced membrane performances[END_REF] deposited Al2O3 on PP HF membranes and obtained a maximum of 75% BSA rejection. To go further, BSA hydrodynamic radius is 10 nm and for P-g-10 40 % of corresponding Dextran molecule (Mw = 4.10 4 Da) passed through against 0 % of BSA molecule. This proved that BSA is not only rejected because of its size but thanks to the higher hydrophilicity of the membrane due to TiO2 deposit. Table 6. Comparison of this study with results reported in literature

Conclusion

Atomic layer deposition of TiO2 on PSF HF membranes was studied. The protective effect of glycerin to prevent pore collapsing during ALD process when the polymeric membrane is exposed under vacuum at 100°C was clearly demonstrated. By this way, TiO2 deposit onto the surface and the porous substructure can be achieved to give better performances in terms of permeability and anti-fouling properties with BSA. Moreover, the acclimation duration in the ALD chamber must not be too long because of the drastic reduction of glycerin boiling temperature under 10 -2 mbar vacuum (98°C against 290°C under atmospheric pressure). The duration of the ALD process cannot be much more longer than the glycerin evaporation. Based on these conditions, different number of ALD cycles were tested from 10 to 100 corresponding to thicknesses between 1 and 10 nm. The presence of TiO2 was proved by EDX analysis. On SEM images, TiO2 layer was observed only upon 50 ALD cycles. Thanks to XPS analyses, the reaction between TiO2 and the oxygen groups of PSF membranes was confirmed. Dextran molecules filtration showed a clear pore size and pore size distribution reduction for ALD modified PSF HF membranes compared to raw PSF HF membranes. Hydrophilicity improvement was confirmed by WCA reduction from 90° for raw PSF HF membrane to 70° for 20 ALD cycles modified PSF HF membranes. These two parameters explain the enhanced water filtration and fouling properties results of ALD modified PSF HF membranes. Indeed, the ones with 10 and 20 ALD cycles showed the best results, with water permeability up to 26 % higher compared to raw HF membrane and an optimum 100% FRR and a 0% Rir. All ALD modified PSF HF membranes' mechanical properties were not altered by ALD process. The obtained results are a first step for a more in-depth study on TiO2 coating by ALD on PSF HF membranes. It would be interesting to open the test in real membrane measurement and for filtration of other contaminants. Finally, to the best of our knowledge, this work is a first report on TiO2 ALD on PSF HF membranes and may pave the way for further studies.

Figure 1 .

 1 Figure 1. SEM pictures of raw PSf HF (P), PSf HF with 10 ALD cycles (P-10) and PSf HF conditioned with glycerin and with 10 ALD cycles (P-g-10)

Figure 2 .

 2 Figure 2. (a) Comparison of ATR-FTIR spectrum of glycerin with those of PSF membrane with glycerin (P-g), washed PSF membrane (P) and PSF membrane with glycerin after 10 ALD cycles and (b) Raw and ALD modified PSF membranes ATR-FTIR spectra

Figure 3 .

 3 Figure 3. SEM Images of raw (P) and ALD modified membranes surface (10 and 50 cycles)

Figure 4 :

 4 Figure 4: Survey XPS spectra (a) and deconvoluted XPS spectra of P C 1s (b) and O 1s (c) and P-g-50 C 1s (d), O 1s (e) and Ti 2p (f)

Figure 5 .

 5 Figure 5. (a) Different sized dextran rejection rate and (b) WCA of raw and ALD modified membranes (the error referred to standard deviation of 3 samples)

Figure 6 .

 6 Figure 6. (a) Water permeability and BSA retention and (b) Fouling resistance parameters for raw and ALD modified membranes (the error referred to standard deviation of 3 samples)

Table 1 :

 1 Water permeability values of P, P-0, P-g-0, P-10 and P-g-10 membranes (the error referred to standard deviation of 3 samples)

	Fiber's name	Residence time	Residence time	Permeability (L/h -1 m -
		under vacuum	with TiO2 deposit	2 bar -1 )
		(min)	(min)
	P	0	0	135 ± 14
	P-0	40	0	5 ± 1
	P-g-0	40	0	27 ± 4
	P-10	15	25	4 ± 1
	P-g-10	15	25	170 ± 17

Table 2 .

 2 EDX data showing the composition of the raw and ALD modified membranes (the error was calculated based on a 10% EDX measurement uncertainty)

		Atomic percentages (%)
	Samples	C	O	Ti
	P	86.8 ± 8.6 9.9 ± 0.9	0
	P-g-10	86.3 ± 8.6 11.3 ± 1.1	≤ 0.1
	P-g-20	87.2 ± 8.7 9.9 ± 0.9	≤ 0.1
	P-g-50	76.8 ± 7.7 20.5 ± 2.1 0.8 ± 0.1
	P-g-100 74.7 ± 7.5 22.2 ± 2.2 1.2 ± 0.1

Table 4 .

 4 Tensile properties of raw and ALD modified HF membranes (the error referred to standard deviation of 3 samples)

		Young Modulus (MPa)	Strength at break (N)	Permeability
				(L/hm 2 bar)
	P	132 ± 5	2.10 ± 0.02	135 ± 14
	P-g-10	153 ± 9	1.72 ± 0.11	170 ± 17
	P-g-20	143 ± 6	1.84 ± 0.05	200 ± 20
	P-g-50	144 ± 4	1.76 ± 0.06	15 ± 2
	P-g-100	150 ± 6	1.71 ± 0.08	< 1

Table 5 .

 5 Correspondence between molecular weight and hydrodynamic radius values of dextran molecules

	Mw (Da)	6.10 3	4.10 4	10 5	2.10 5	5.10 5	2.10 6
	rp (nm)	4.7	10	14.9	20.2	30.2	55.

(BSA)

Acknowledgment.

This project is part of INNOMEM and receives funding from the European Union's Horizon 2020 Research and Innovation Program under Grant Agreement N° 862330. Authors would like to thanks the companies Polymem for providing the PSF HF membranes and Porometer for the determination of HF membranes mean pore diameter. The authors acknowledge D. Cot and B. Rebiere for SEM and EDX observations and V. Bonniol for her technical support for Dextran analysis. Authors would like to thank Aurélien Renard from the SMI LCPME facility (Université de Lorraine-CNRShttp://www.lcpme.cnr-nancy.fr) for XPS analysis.