Nicolas Marie

Corinne Canestrelli

Florence Noble
email: florence.noble@parisdescartes.fr

Role of pharmacokinetic and pharmacodynamic parameters in neuroadaptations induced by drugs of abuse, with a focus on opioids and psychostimulants

Role of pharmacokinetic and pharmacodynamic parameters in neuroadaptations induced by drugs of abuse, with a focus on opioids and psychostimulants

Nicolas Marie, Corinne Canestrelli, Florence

Introduction :

It is now well admitted that addiction is a brain disease [START_REF] Leshner | Addiction is a brain disease, and it matters[END_REF] and that neuroadaptations are a core neurobiological feature of this pathology. The neuroadaptations are complex and may occur not only into the mesocorticolimbic dopamine system, a key neurotransmitter system involved in addictive behaviors, but also in other numerous systems (review in [START_REF] Koob | Neurobiology of addiction: a neurocircuitry analysis[END_REF]. Addiction is characterized by a compulsive behavior, a continued abuse of drugs despite negative consequences, craving when drugs are not more available and high risk of relapse. Moreover addicts may also be drug tolerant, physically and psychologically dependent. These characteristics involve persistent changes in the brain's structure and function (neuroadaptations). However, not everyone who uses drugs becomes addicted. Thus, depending on the drugs used, between 10-30% of users will develop an addictive behavior [START_REF] Flórez-Salamanca | Probability and predictors of transition from abuse to dependence on alcohol, cannabis, and cocaine: results from the National Epidemiologic Survey on Alcohol and Related Conditions[END_REF]. No single factor can predict whether a person will become addicted to drugs. But a combination of factors including psycho-biological factors (e.g., genetic factors, personality traits, co-morbidities), environmental factors (e.g., family, peer influence or sociocultural context), and neuroadaptations induce by drugs themselves influence risk for addiction. Several parameters are directly involved in the pharmacological effects induced by a drug, and consequently in the neuroadaptations that may occur following repeated administrations: i) the bioavailability of the drug itself (importance of the route of administration); ii) the transport across the blood brain barrier (BBB); iii) the rate of delivery to the brain, and iv) the efficacy of the intracellular responses, depending on the interactions (e.g., affinity, partial or full agonist) between the ligand and its target (Figure 1). This review will summarize the behavioral and neurochemical neuroadaptations observed following repeated administration of drugs of abuse with different routes, speeds and patterns of administration, with a focus on opioid ligands, but also with results obtained following repeated administration of cocaine or other psychostimulants.

In human, drug use is a voluntary act. In rodents, distinct neuroadaptations have been reported in studies comparing active (self-administration) versus passive (experimenter-administered, or yoked animals) drug administrations, suggesting that this parameter is also crucial regarding neuroadaptations induced by drugs of abuse, and development of drug addiction [START_REF] Jacobs | Gene transcripts selectively down-regulated in the shell of the nucleus accumbens long after heroin self-administration are up-regulated in the core independent of response contingency[END_REF][START_REF] Jacobs | Neuroadaptive effects of active versus passive drug administration in addiction research[END_REF][START_REF] Jacobs | Long-term gene expression in the nucleus accumbens following heroin administration is subregion-specific and depends on the nature of drug administration[END_REF][START_REF] Fernàndez-Castillo | Active and passive MDMA ('ecstasy') intake induces differential transcriptional changes in the mouse brain[END_REF]. Because selfadministration paradigm in rodents involves active drug administration, it mimics some aspects of human addiction behavior with a crucial role of cognitive processes (e.g., reinforcement learning, impulsivity, attention). This model is very helpfull to try to decipher the cellular and molecular mechanisms involved in drug addiction, even if the doses and the frequencies of administration are not controlled, as each animal will have its own behavior, and each animal will decide when pressing a lever or poking its nose into a hole to get drug. However, the main goal of this review is to demonstrate the importance of pharmacodynamic and pharmacokinetic parameters in neuroadapations, and thus only studies reporting results with experimenteradministered drugs of abuse have been considered. From these studies, the routes of administration, the patterns of administration, the pharmacological efficacy of ligands can be directly compared.

Importance of the route of administration:

The route of administration will define the plasma concentration that can be reached, and thus the amount of compound able to cross the BBB. It is well known that the rewarding effects are dependent on the peak effect. Thus, imaging studies in human, and preclinical studies in animals have demonstrated that the speed with which a drug enters and leaves the brain are important factors in determining its reinforcing effects. Thus when the Cmax and Tmax are reached few minutes after absorption, they are related to the intensity of high (subjective experience of euphoria) that subjects experience. This could be referred to the peak effect that predicts abuse liability, i.e. when the concentration of the drug in the brain increases very rapidly, higher is the risk of dependence (see [START_REF] Volkow | Addiction: decreased reward sensitivity and increased expectation sensitivity conspire to overwhelm the brain's control circuit[END_REF][START_REF] Allain | How fast and how often: The pharmacokinetics of drug use are decisive in addiction[END_REF].

Studies investigating the pharmacokinetic properties of morphine have demonstrated that the relative onset of drug effects and the time necessary to reach the peak concentration in the brain are increased from intravenous to intramuscular/subcutaneous, and to oral routes [START_REF] Upton | Pharmacokinetic optimisation of opioid treatment in acute pain therapy[END_REF]. After intravenous administration, morphine brain concentration is the result of passage from the blood across the BBB to the brain. After nasal administration, the level of morphine into the brain could be the result of both distribution across the BBB and transfer via direct olfactory pathways. Indeed, it has been demonstrated in rats that morphine is directly and very rapidly transferred from the nose, via olfactory pathways, to the olfactory bulbs and the brain hemispheres [START_REF] Westin | Direct nose-tobrain transfer of morphine after nasal administration to rats[END_REF]. In a study comparing the effects of intranasal and intravenous heroin self-administration in heroin-dependent patients, it was demonstrated that the reinforcing effects of heroin are similar by the two routes of administrations, but that intranasal heroin is less potent than intravenous heroin when the subjective effects (e.g., "I feel a good drug effect", "I feel high") are reported [START_REF] Comer | Comparison of intravenous and intranasal heroin self-administration by morphine-maintained humans[END_REF].

The psychostimulant methylphenidate is used for the treatment of attentiondeficit/hyperactivity disorder and narcolepsy but it also has a history of being misused as a 'smart drug'. Methylphenidate is a blocker of dopamine and noradrenaline transporters [START_REF] Gatley | Affinities of methylphenidate derivatives for dopamine, norepinephrine and serotonin transporters[END_REF]. Thus, this drug is able to increase the synaptic dopamine concentration. The effects induced by methylphenidate have been compared following either intravenous or oral administration. In the striatum the higher concentration of the psychostimulant was observed 10 minutes after intravenous administration (Cmax value), and 90 minutes after oral injection [START_REF] Volkow | Variables that affect the clinical use and abuse of methylphenidate in the treatment of ADHD[END_REF]. Both routes of administration are able to increase dopamine levels in the striatum with the same magnitude, but the effect is faster following intravenous injection compared to oral administration. The difference in the delivery rate of methylphenidate may explain why the oral route did not induce a « high », whereas for intravenous injection the magnitude of the dopamine increase was associated with the intensity of the « high » [START_REF] Volkow | Variables that affect the clinical use and abuse of methylphenidate in the treatment of ADHD[END_REF]. This is in good agreement with the "peak effect" theory, which identifies reinforcers with addictive potential as those that have rapid onset and short time to peak effect [START_REF] Hatsukami | Crack cocaine and cocaine hydrochloride. Are the differences myth or reality?[END_REF][START_REF] De Wit | Subjective and behavioral effects of diazepam depend on its rate of onset[END_REF][START_REF] Abreu | Effect of intravenous injection speed on responses to cocaine and hydromorphone in humans[END_REF][START_REF] Allain | How fast and how often: The pharmacokinetics of drug use are decisive in addiction[END_REF] 2. Importance of the rate of drug delivery to the brain Besides the route of administration that plays an important role in bioavailability of the drug, the speed of administration and the passage across the BBB are also very important factors.

They will determine the quantity of drugs reaching the brain which will have a strong impact on the neuroadaptations observed.

The importance of the speed of administration is well illustrated in a paper published by Comer et al. [START_REF] Comer | Relationship between rate of infusion and reinforcing strength of oxycodone in humans[END_REF]. They clearly demonstrated a relationship between rate of infusion and reinforcing strength of the opioid ligand, oxycodone in humans. Thus, while the same dose of oxycodone was administered intravenously over 2, 15, 30, 60, or 90 minutes in different groups, it is only when oxycodone was delivered in 2 or 15 minutes that participants reported reinforcing effects. This is in good agreement with the notion of peak effect, as already evoked above.

The importance of speed of administration was also investigated in a preclinical study using cocaine. The same dose of cocaine was intravenously administered in rats in either 5, 25 or 100 seconds. In these different conditions, the authors investigated the regulation of c-Fos expression and behavioral sensitization [START_REF] Samaha | The rate of cocaine administration alters gene regulation and behavioral plasticity: implications for addiction[END_REF]. Whatever the time of administration, cocaine is able to induce an increase in c-Fos immunoreactivity particularly in the striatum, but with a much stronger effect when cocaine is injected in 5 seconds. Then this increase diminishes as the duration of injection lengthens.

The authors have also explored the behavioral consequences of the speed of cocaine administration using the locomotor sensitization model, a behavior usually observed during repeated administration of drugs of abuse. On day 1, administrations of cocaine increased the locomotor activity in the same magnitude whatever the infusion rate. On day 2, a significant behavioral sensitization was observed after intravenous administration of cocaine in 5 seconds, while the same dose of cocaine administered in 25 or 100 seconds did not induce this behavioral adaptation [START_REF] Samaha | The rate of cocaine administration alters gene regulation and behavioral plasticity: implications for addiction[END_REF]. Using a different strategy, we highlighted the influence of delivery rate on neuroadaptations with cocaethylene, a psychoactive metabolite produced from a concomitant cocaine and ethanol consumption. Indeed, when cocaethylene was dissolved in an emulsion that is supposed to slow its brain delivery, instead of saline, behavioral sensitization was less robust [START_REF] Noble | Interest for delivery of cocaethylene in a sustained release emulsion vs saline evaluated on behavioral sensitization in naive and cocaine-sensitized mice[END_REF]. Thus it clearly appears that the rate at which cocaine or its metabolites are delivered influences the development of locomotor sensitization, an expression of neuroadaptations occurring in the brain upon repeated drug administration.

The second important factor, which will define the concentration of the drug at the site of action in the brain, is the permeability of the BBB. This layer protects the brain and is composed of endothelial cells joined together with tight junctions. These cells express transporters that may either prevent intracellular accumulation into the brain of circulating compounds and drugs (efflux pumps), or facilitate their entrance (influx pumps) (review in Theodorakis et al., 2017). Among the efflux pumps, P-glycoprotein (Pgp ; MDR1 ; ABCB1) significantly contributes to BBB functions, both preventing the influx of agent from the blood into the brain and facilitating the efflux of compounds from the brain into the blood. Using Pgp knockout mice, Xie et al. [START_REF] Xie | The role of P-glycoprotein in blood-brain barrier transport of morphine: transcortical microdialysis studies in mdr1a (-/-) and mdr1a (+/+) mice[END_REF] nicely demonstrated that this efflux pump participates in regulating the amount of morphine transport across the BBB. Thus, while the brain to plasma morphine concentration ratio was 0.5 for wild-type animals, it was of 1.1 in knockout mice. This has an impact on the pharmacological responses induced by morphine that involved supraspinal structures. In Pgp knockout mice, a leftward shift of the morphine dose-response curve was observed in the tail flick test as compared to wild-type animals [START_REF] King | Transport of opioids from the brain to the periphery by P-glycoprotein: peripheral actions of central drugs[END_REF]. All these results suggest that Pgp is able to decrease the permeability of BBB to morphine.

In the same way, using a selective inhibitor of Pgp, PSC833, we have also observed a leftward shift of the dose-response curve of morphine in the hot plate test in mice. Interestingly, no difference was observed in presence or absence of Pgp inhibitor on the analgesic responses obtained with heroin strongly suggesting that while morphine is a Pgp substrate, heroin is not [START_REF] Seleman | Impact of P-glycoprotein at the blood-brain barrier on the uptake of heroin and its main metabolites: behavioral effects and consequences on the transcriptional responses and reinforcing properties[END_REF]. Inhibiting Pgp has consequences not only on the analgesic effects of morphine, but also on regulation of gene expression. We have investigated the regulation of different genes including Rnd3, which is known to regulate actin cytoskeleton, and is an early common effector of several drugs of abuse [START_REF] Marie-Claire | Rnd family genes are differentially regulated by 3,4-methylenedioxymethamphetamine and cocaine acute treatment in mice brain[END_REF][START_REF] Seleman | Impact of P-glycoprotein at the blood-brain barrier on the uptake of heroin and its main metabolites: behavioral effects and consequences on the transcriptional responses and reinforcing properties[END_REF]. At a dose of morphine unable to regulate Rnd3 expression, co-administration of the Pgp inhibitor PSC833 + morphine, revealed an effect, similar with the one obtained when heroin was administered alone or in association with PSC833 [START_REF] Seleman | Impact of P-glycoprotein at the blood-brain barrier on the uptake of heroin and its main metabolites: behavioral effects and consequences on the transcriptional responses and reinforcing properties[END_REF]. Finally, regarding the rewarding effects of morphine, it clearly appears in the conditioning place preference (CPP) that co-administration of the selective Pgp inhibitor with morphine, potentiated the rewarding effects of this opioid agonist [START_REF] Seleman | Impact of P-glycoprotein at the blood-brain barrier on the uptake of heroin and its main metabolites: behavioral effects and consequences on the transcriptional responses and reinforcing properties[END_REF].

Very interestingly chronic morphine treatment in rats was able to increase the levels of Pgp in different brain structures, including the cortex and the hippocampus, without modification of the integrity of the BBB [START_REF] Yousif | Effect of chronic exposure to morphine on the rat blood-brain barrier: focus on the Pglycoprotein[END_REF]. This increase could be interpreted as a protective reaction to avoid excessive stimulation by morphine, by preventing entry of large amount of the opioid ligand into the brain.

In human positron-emission tomography (PET) was used to investigate Pgp function in the BBB of patients with depression, schizophrenia or progressive neurodegeneration [START_REF] De Klerk | Regional increase in P-glycoprotein function in the blood-brain barrier of patients with chronic schizophrenia: a PET study with [(11)C]verapamil as a probe for Pglycoprotein function[END_REF][START_REF] De Klerk | Locally increased P-glycoprotein function in major depression: a PET study with [11C]verapamil as a probe for P-glycoprotein function in the blood-brain barrier[END_REF][START_REF] Bartels | Blood-brain barrier P-glycoprotein function decreases in specific brain regions with aging: a possible role in progressive neurodegeneration[END_REF], but to the best of our knowledge never in addiction field. These investigations could be very interesting as Pgp may modulate the entrance into the brain of different opioid ligands, and thus may also explain variability in the responses to opioid maintenance treatment [START_REF] Linnet | A review on the impact of P-glycoprotein on the penetration of drugs into the brain. Focus on psychotropic drugs[END_REF].

Importance of interactions with receptors

Receptors bind drugs (also referred to as ligand), with a relatively high degree of specificity, and after binding of the ligand, initiate a signalling cascade. Membrane receptors, when activated, usually result in activation of secondary enzymes or ion channels via the heterotrimeric G proteins. The pharmacological responses will depend on two main factors: affinity and efficacy.

The affinity refers to the ability of a given drug to bind to its receptor by direct chemical interactions with the receptor binding site. It is an intrinsic property of the drug. The efficacy can be defined as the ability of a drug, once bound to its receptor, to activate it and thus initiate cellular signalling pathways that lead to pharmacological responses. A relative efficacy may be defined, as the relative maximum response from the drug. Drugs that produce less than the maximum activation of a given receptor are referred to as partial agonists (in opposite to full agonists, that lead to the maxima responses) (review in Strange, 2008).

Opioid receptors are members of the G protein-coupled receptor (GPCR) superfamily, and several receptors have been cloned : mu opioid receptor (MOR), kappa opioid receptor (KOR), delta opioid receptor (DOR), and ORL1/nociceptin receptor [START_REF] Dhawan | International Union of Pharmacology. XII. Classification of opioid receptors[END_REF]. At the cellular level, opioid receptors are mainly coupled to Gi/o and Gq proteins and their activations lead to inhibition of adenylyl cyclase activity and voltage-gated Ca2+ channels, increase in mitogenactivated protein kinase (MAPK) phosphorylation and in the activity of inwardly rectifying K+ channels and phospholipase C beta. In general, studies show that MOR-or DOR-selective agonists can induce both analgesia and reward [START_REF] Abdallah | The Delta Opioid Receptor in Pain Control[END_REF][START_REF] Klenowski | The role of δ-opioid receptors in learning and memory underlying the development of addiction[END_REF][START_REF] Matthes | Loss of morphineinduced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioidreceptor gene[END_REF]. The use of transgenic mice has provided greater insight into the potential roles of individual opioid receptor subtypes. These studies indicate that the analgesic effects and the rewarding properties of opioids such as morphine are primarily mediated by the activation of MOR [START_REF] Charbogne | 15 years of genetic approaches in vivo for addiction research: Opioid receptor and peptide gene knockout in mouse models of drug abuse[END_REF]. However, DOR has been shown to have a role in the regulation of emotional responses associated with opioid use [START_REF] Peppin | Delta opioid agonists: a concise update on potential therapeutic applications[END_REF]. Thus, to demonstrate the importance of the binding parameters in the neuroadaptations that may be induced by chronic treatments with opioid agonists, we will review the differences that can be observed with 3 different opioid agonists : morphine, methadone, and buprenorphine, and we will give results essentially on MOR.

These 3 MOR agonists are largely used in humans, as methadone and buprenorphine are used in the treatment of opioid addiction. It is also important to compare the long-term effects of morphine, methadone and buprenorphine as they show differences in their binding parameters.

Buprenorphine has a unique profile, significantly different from morphine, or methadone. It has a high affinity for MOR but low intrinsic activity, with partial agonist properties.

To complexify the picture, most of the compounds administered in animals are subjected to biotransformations that generate active and inactive metabolites. This is well illustrated with heroin which displays a low affinity towards MOR [START_REF] Inturrisi | Evidence from opiate binding studies that heroin acts through its metabolites[END_REF]) and whose action is mediated by its main metabolites produced sequentially : 6-monoacetylmorphine and morphine (review in [START_REF] Rook | Pharmacokinetics and pharmacokinetic variability of heroin and its metabolites: review of the literature[END_REF]. Heroin is thereby considered as a prodrug. Morphine is in turn transformed into morphine-3-glucuronide (review in De [START_REF] De Gregori | Morphine metabolism, transport and brain disposition[END_REF], which has a low affinity and activity on MOR [START_REF] Frölich | Distinct pharmacological properties of morphine metabolites at G(i)-protein and β-arrestin signaling pathways activated by the human μ-opioid receptor[END_REF][START_REF] Roeckel | Morphine-induced hyperalgesia involves mu opioid receptors and the metabolite morphine-3-glucuronide[END_REF]. In humans, morphine is also transformed in morphine-6-glucuronide (review in De [START_REF] De Gregori | Morphine metabolism, transport and brain disposition[END_REF], an active metabolite [START_REF] Handal | Pharmacokinetic differences of morphine and morphine-glucuronides are reflected in locomotor activity[END_REF]. 6-monoacetylmorphine has an affinity for MOR comparable to morphine [START_REF] Inturrisi | Evidence from opiate binding studies that heroin acts through its metabolites[END_REF] and behaves as a partial agonist (with an higher efficacy than morphine) [START_REF] Selley | mu Opioid receptor-mediated G-protein activation by heroin metabolites: evidence for greater efficacy of 6monoacetylmorphine compared with morphine[END_REF]. Methadone is also metabolized, but its main metabolite, 2ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, has a poor affinity for MOR (Ki > 1 µM) [START_REF] Lötsch | Modulation of the central nervous effects of levomethadone by genetic polymorphisms potentially affecting its metabolism, distribution, and drug action[END_REF]. Buprenorphine is mainly metabolized in norbuprenorphine, and both ligands have a similar affinity for MOR, DOR and KOR. Norbuprenorphine behaves as a partial agonist toward MOR (but with an higher efficacy than buprenorphine), DOR and KOR [START_REF] Huang | Comparison of pharmacological activities of buprenorphine and norbuprenorphine: norbuprenorphine is a potent opioid agonist[END_REF]. However, its contribution to buprenorphine effects in vivo is very modest as its passage to brain is limited by the BBB [START_REF] Ohtani | Comparative analysis of buprenorphine-and norbuprenorphine-induced analgesic effects based on pharmacokinetic-pharmacodynamic modeling[END_REF]. Buprenorphine and norbuprenorphine are subjected to glucuronidation that generates buprenorphine-3glucuronide, buprenorphine-6-glucuronide and norbuprenorphine-3-glucuronide, norbuprenorphine-6-glucuronide, respectively [START_REF] Bruce | Pharmacokinetic interactions between buprenorphine and antiretroviral medications[END_REF]. Despite the fact that these metabolites display high affinity towards opioid receptors, they have modest pharmacological effects [START_REF] Brown | Buprenorphine metabolites, buprenorphine-3-glucuronide and norbuprenorphine-3-glucuronide, are biologically active[END_REF]. In summary, except when the opioid is a prodrug such as heroin, all these metabolites will play a modest role in pharmacological effects and it is more likely the variability in the metabolism that will influence the responses by changing the availability of the active compounds.

Binding parameters and signalling

Whereas morphine and methadone bind to MOR, with high affinities (in the nanomolar range), buprenorphine has an higher affinity (around 0.1 nM) and more importantly, it also binds DOR and KOR contrary to morphine and methadone [START_REF] Lutfy | Buprenorphine: a unique drug with complex pharmacology[END_REF]). On KOR, buprenorphine is usually described as an antagonist that mediates its anti-depressant effects [START_REF] Falcon | Antidepressant-like Effects of Buprenorphine are Mediated by Kappa Opioid Receptors[END_REF], but a recent study also suggests a role of MOR in these effects [START_REF] Robinson | A role for the mu opioid receptor in the antidepressant effects of buprenorphine[END_REF]. Buprenorphine is also unique as once bound to the receptor, it slowly dissociates [START_REF] Rance | Animal and molecular pharmacology of mixed agonist-antagonist analgesic drugs[END_REF]. We investigated this characteristic ex vivo by measuring the ability of [ 3 H]-D-Ala2, N-MePhe4, Gly-ol5-enkephalin (DAMGO), a selective MOR ligand to displace buprenorphine on rat brain membranes. We found that in membranes pre-incubated with buprenorphine, [ 3 H]-DAMGO was not able to completely displace buprenorphine. The same results were obtained with [ 3 H]-naltrindole (a DOR ligand) and [ 3 H]-CI977 (a KOR ligand) but to a lesser extent [START_REF] Mégarbane | Buprenorphine is protective against the depressive effects of norbuprenorphine on ventilation[END_REF]. This slow dissociation of buprenorphine from the MOR, resulting in a long residence time of the ligand on the receptor, could explain why the intensity of withdrawal signs is lower after chronic buprenorphine treatment as compared to morphine or methadone (Figure 2) (N. Marie and F. Noble, unpublished results). This might have consequences on the ability of buprenorphine to promote behavioral sensitization (see below 4.1).

Although, morphine, methadone, buprenorphine are MOR agonists, they have different efficacies to couple receptor to G protein and to mobilize intracellular pathways. Indeed, in most of the studies measuring second messengers, morphine and methadone are usually being considered as full agonist with methadone > morphine, whereas buprenorphine is depicted as a partial agonist [START_REF] Saidak | Differential activation of Gproteins by mu-opioid receptor agonists[END_REF].

Transcriptional responses

Using morphine, methadone and buprenorphine we investigated changes in gene expression over time (30 minutes, 1h and 4h after drug administration) in three cerebral brain structures, the thalamus involved in analgesic responses observed with opioid ligands [START_REF] Dong | Morphine applied to the thalamic nucleus submedius produces a naloxone reversible antinociceptive effect in the rat[END_REF][START_REF] Yen | Distribution of thalamic nociceptive neurons activated from the tail of the rat[END_REF][START_REF] Saadé | Augmentation of nociceptive reflexes and chronic deafferentation pain by chemical lesions of either dopaminergic terminals or midbrain dopaminergic neurons[END_REF], and the ventral (nucleus accumbens) and dorsal striatum, both implicated in the transition from recreational drug use to compulsive consumption of drugs of abuse [START_REF] Lesscher | Compulsive drug use and its neural substrates[END_REF]. The dose of morphine (10 mg/kg s.c.) used was based on previous studies in the same rat strain showing gene regulations and conditioned place preference [START_REF] Garcia | Alterations in immediate-early gene proteins in the rat forebrain induced by acute morphine injection[END_REF][START_REF] Benturquia | Effects of nitrous oxide on dopamine release in the rat nucleus accumbens and expectation of reward[END_REF][START_REF] Marie-Claire | Fos but not Cart (cocaine and amphetamine regulated transcript) is overexpressed by several drugs of abuse: a comparative study using real-time quantitative polymerase chain reaction in rat brain[END_REF][START_REF] Gutstein | Pattern of c-fos mRNA induction in rat brain by acute morphine[END_REF]. This dose corresponded to 3 x ED50 value of morphine determined in the tail-flick assay [START_REF] Belkaï | Comparison of the transcriptional responses induced by acute morphine, methadone and buprenorphine[END_REF]. The corresponding buprenorphine (0.2 mg/kg) and methadone (3.7 mg/kg) doses are within range commonly used in studies investigating their rewarding properties in the conditioned place preference [START_REF] Rowlett | Dissociation of buprenorphine-induced locomotor sensitization and conditioned place preference in rats[END_REF][START_REF] Steinpreis | Methadone produces conditioned place preference in the rat[END_REF][START_REF] Tzschentke | Reassessment of buprenorphine in conditioned place preference: temporal and pharmacological considerations[END_REF].

Regulations of some immediate early genes (Fos, Egr1, Arc and Homer1) (Figure 3) as nonspecific markers of neural activation, and of six opioid system genes (encoding peptides and receptors) (Figure 4 and figure 5) were investigated [START_REF] Belkaï | Comparison of the transcriptional responses induced by acute morphine, methadone and buprenorphine[END_REF] ; E Belkaï, C Marie-Claire, F. Noble, unpublished results). The overview of the results shows that the regulations are opioid ligand-dependent, time-dependent, and brain structure-dependent.

Regarding Fos mRNA expression, 30 minutes after acute administration of buprenorphine, a decrease was observed in the nucleus accumbens, while an increase was quantified in the thalamus 1 hour after treatment compared to saline-treated rats. With morphine and methadone, a delayed regulation of Fos mRNA was observed, with an increase 4h after administration of the opioid ligands in the nucleus accumbens, dorsal striatum and thalamus (Figure 3). For the other immediate early genes investigated, differences in the regulation of Egr1 induced by morphine, methadone and buprenorphine were also observed depending on the brain structure and the withdrawal period after acute administration. In the thalamus, only methadone was able to increase the Egr1 gene expression 1h after injection, while 4h after, the three opioid ligands increased expression of this immediate early gene. Moreover, 4 hours after administration morphine and methadone induced an increase of Egr1 in the dorsal striatum, while a decrease was observed with buprenorphine in the nucleus accumbens. Arc was also regulated by acute administration of the opioid agonists, but here again these regulations are ligand-, structure-and time-dependent. A reduction in Homer1 expression was only observed in the dorsal striatum, 4 hours after buprenorphine administration. Currently the functional consequences of these complex regulations are not known, but they could be involved in neuroadaptations induced by opioids, as for instance Homer1 as been shown to tune synaptic plasticity in excitatory neurons by regulating the synaptic distribution of GluA2-containing AMPA receptors [START_REF] Rozov | Homer1 gene products orchestrate Ca(2+)permeable AMPA receptor distribution and LTP expression[END_REF].

Acute injection of the three opioid ligands modulated the expression of the genes encoding endogenous opioid peptides (Pdyn, Penk, POMC) (Figure 4) and receptors (Oprm1, Oprd1 and Oprk1) (Figure 5) with different time courses and intensities in the three brain structures studied. The expressions of the genes studied were modulated preferentially in the thalamus and nucleus accumbens at the earliest time point, and in the dorsal striatum at the latest time point studied. Interestingly, methadone and buprenorphine did not produce any common gene expression regulations in the three tested brain structures at any time studied. Moreover the regulations observed suggest that the transcriptional effects of methadone resemble those of morphine more closely than those of buprenorphine in these structures [START_REF] Belkaï | Comparison of the transcriptional responses induced by acute morphine, methadone and buprenorphine[END_REF] probably because buprenorphine is not a MOR selective agonist.

Importance of administration patterns

Behavioral and cellular neuroadaptations induced by morphine, methadone and buprenorphine

Very few studies have investigated the influence of the drug administration pattern on both behavioral and neurochemical levels. So, we conducted a series of experiments to explore this factor using two patterns of treatment: one daily repeated injection (ODRI) (one daily injection for 5 or 7 days) or multiple daily repeated injection MDRI (MDRI) (three daily injection for 5 or 7 days) with morphine, methadone or buprenorphine. After the ODRI or MDRI treatment, animals were challenged after different periods of withdrawal either with the same opioid used for the repeated treatment (homologous sensitization) or a different opioid (heterologous sensitization). The three opioids were injected intraperitoneally and except for morphine, equiactive doses of methadone and buprenorphine were used to design the treatment regimen and animals received the same amount of drugs in ODRI and MDRI treatments.

We found that ODRI treatments promoted a more robust behavioral sensitization for all the three opioid agonists as compared to the MDRI patterns. The most important differences were reported with buprenorphine [START_REF] Allouche | Different patterns of administration modulate propensity of methadone and buprenorphine to promote locomotor sensitization in mice[END_REF][START_REF] Marec | Chronic and intermittent morphine treatment differently regulates opioid and dopamine systems: a role in locomotor sensitization[END_REF]. Indeed, no behanvioral sensitization was observed with the MDRI treatment that may be due to a lack of withdrawal periods with this pattern of treatment (see above), a state well known to play a key role in the acquisition of sensitization [START_REF] Rothwell | Episodic withdrawal promotes psychomotor sensitization to morphine[END_REF]. However, a study conducted by

Trujillo and co-worked demonstrated that a continuous morphine or fentanyl administration using either osmotic pump or pellets produced locomotor sensitization [START_REF] Trujillo | Continuous administration of opioids produces locomotor sensitization[END_REF].

Thus, one might argue that the lack of behavioral sensitization with the MDRI buprenorphine treatment is rather due to the partial agonist property of the compound as compared to morphine or fentanyl. Interestingly, the MDRI morphine treatment promoted a transient behavioral sensitization but with a delay (after 14 days of withdrawal), while after one day of withdrawal a reduction of locomotor activity was observed in mice repeatedly treated with morphine. This could be qualified as a tolerance to locomotor effect induced by morphine (Le [START_REF] Marec | Chronic and intermittent morphine treatment differently regulates opioid and dopamine systems: a role in locomotor sensitization[END_REF]. This tolerance was probably attributed to the administration pattern, indeed when the same dose of morphine was administered in a ODRI regimen, no tolerance was observed (T Le Marec, F Noble, N Marie, unpublished results).

In order to investigate if some neurochemical modifications could correlate with these behavioral modifications, we measured D1 and D2 dopamine receptor densities in striatum following ODRI and MDRI treatments using radioligand binding assays on brain slices. With morphine, we found that D1 receptors were increased when behavioral sensitization was observed whereas D2 receptors were diminished in the same time. In parallel, when tolerance to locomotor effects of morphine was observed on WD (withdrawal day) 1 after the MDRI treatment, an increase in D2 receptors with a decrease in D1 receptors were measured (Le [START_REF] Marec | Chronic and intermittent morphine treatment differently regulates opioid and dopamine systems: a role in locomotor sensitization[END_REF]. These data are in accordance with a role for D1 receptors in acute effects of morphine in locomotion [START_REF] Serrano | Effects of DA D1 and D2 antagonists on the sensitisation to the motor effects of morphine in mice[END_REF]. Concerning D2 receptors, our data might be in apparent contradiction with literature where D2 receptor antagonists were found to block morphine sensitization [START_REF] Serrano | Effects of DA D1 and D2 antagonists on the sensitisation to the motor effects of morphine in mice[END_REF]. D2 receptors are expressed as two isoforms, D2L mainly described as a post-synaptic receptor and D2S described as a pre-synaptic receptor responsible for the negative feedback on dopamine release [START_REF] De Mei | Getting specialized: presynaptic and postsynaptic dopamine D2 receptors[END_REF]. One might hypothesize that we detected a decrease of D2S that would reduce the brake on DA release thus facilitating locomotor activity. We also found that MOR was down-regulated in the ventral tegmental area after chronic MDRI morphine treatment. This would reduce inhibitory action of opiates on GABA interneurons, leading to a decrease on dopamine neurons activity, contributing to a lower locomotor activity (tolerance) (Figure 6).

With the opioid substitution treatments, we found that both ODRI and MDRI treatments induced regulation of D1 and D2 receptors in the striatum with the ODRI treatment promoting a higher number and long-lasting modifications. For instance, ODRI buprenorphine or methadone regimen promoted an increase of D1 receptors and a decrease of D2 receptors at WD35, respectively [START_REF] Allouche | Striatal dopamine D1 and D2 receptors are differentially regulated following buprenorphine or methadone treatment[END_REF]. Whereas correlations between striatal expression of D1 and D2 receptor and sensitization were found with morphine, no evidences for such links were demonstrated with methadone and buprenorphine. One day after withdrawal, an increase in D1 receptors concomitant to a decrease in D2 receptors were measured following buprenorphine and methadone treatment (except for the ODRI methadone treatment) but no behavioral sensitization was observed suggesting a decoupling between dopamine receptor expression and sensitization.

Overall, our data demonstrated that short term (5 or 7 days) repeated treatments with morphine and more importantly with methadone and buprenorphine were able to promote long-lasting neurochemical and behavioral changes in a dynamic way. It can be speculated that these changes also occur in patients, who are very often treated for many years. Our results also strongly suggest that more than the dose, the administration pattern is crucial to influence these changes. This is emphasized by the pioneer works of Vanderschuren and colleagues where they observed a higher behavioral sensitization in rats after a morphine ODRI regimen as compared to the MDRI treatment despite the fact that the rats received a greater amount of morphine in the MDRI regimen [START_REF] Vanderschuren | Morphine-induced long-term sensitization to the locomotor effects of morphine and amphetamine depends on the temporal pattern of the pretreatment regimen[END_REF].

Behavioral and cellular neuroadaptations induced by cocaine

Cocaine is a widely abused drug in the world, and addiction to this drug is a major public health problem since there is a lack of specific medication. While the acute effect of this psychostimulant is well characterized (i.e., increases of monoamine levels in the synaptic space by inhibiting their reuptake into presynaptic terminals), the neuroadaptations following a repeated intake remain highly complex. Analysis of literature clearly shows many discrepancies in the results that may be due to distinct experimental procedures. As described above, different patterns of opioid treatment induced distinct behavioral and neurochemical consequences with different time-course (Le [START_REF] Marec | Chronic and intermittent morphine treatment differently regulates opioid and dopamine systems: a role in locomotor sensitization[END_REF][START_REF] Allouche | Striatal dopamine D1 and D2 receptors are differentially regulated following buprenorphine or methadone treatment[END_REF], thus it could also be speculated that the patterns of cocaine injections play a key role in the development of neuroadaptations observed. To investigate this hypothesis a recent series of experiments have been performed with two different patterns of cocaine treatments. Animals were treated by an ODRI (one administration per day) or a MDRI (three administrations per day) pattern.

As expected, an acute cocaine challenge, one day after the last injection of the chronic treatment (WD1) induced a behavioral sensitization. But more interestingly, the expression of cocaineinduced behavioral sensitization was related to the profile of administration. The MDRI treatment led to sensitization of locomotor effects of cocaine, whereas the ODRI treatment did not [START_REF] Puig | Short-and long-lasting behavioral and neurochemical adaptations: relationship with patterns of cocaine administration and expectation of drug effects in rats[END_REF]. These results are in good agreement with the literature, showing that the duration and intensity of sensitization are dependent on the administration patterns [START_REF] Kalivas | Time course of extracellular dopamine and behavioral sensitization to cocaine. II. Dopamine perikarya[END_REF][START_REF] Davidson | Behavioral sensitization is greater after repeated versus single chronic cocaine dosing regimens[END_REF]King et al., 1994b). Interestingly, the locomotor sensitization observed in the MDRI group was associated to a dopamine release sensitization in the nucleus accumbens after a cocaine challenge [START_REF] Puig | Short-and long-lasting behavioral and neurochemical adaptations: relationship with patterns of cocaine administration and expectation of drug effects in rats[END_REF]. As previous studies showed that D1 and D2 dopamine receptors play a role in the development and expression of behavioral sensitization [START_REF] Li | Pharmacological reversal of behavioral and cellular indices of cocaine sensitization in the rat[END_REF][START_REF] Mccreary | Cocaine-induced behaviour: dopamine D1 receptor antagonism by SCH 23390 prevents expression of conditioned sensitisation following repeated administration of cocaine[END_REF][START_REF] Nelson | A comparison of striatal-dependent behaviors in wild-type and hemizygous Drd1a and Drd2 BAC transgenic mice[END_REF][START_REF] Sim | Role of dopamine D2 receptors in plasticity of stress-induced addictive behaviours[END_REF][START_REF] Tobón | Preadolescent drd1-EGFP mice exhibit cocaine-induced behavioral sensitization[END_REF][START_REF] Thompson | Altered ratio of D1 and D2 dopamine receptors in mouse striatum is associated with behavioral sensitization to cocaine[END_REF], it was also interesting to evaluate the consequences of the ODRI and MDRI cocaine administration patterns on dopamine receptor regulations, in different brain structures forming the two major dopaminergic pathways in the brain: the ventral tegmental area and the nucleus accumbens for the mesolimbic pathway, and the substantia sigra and the caudate putamen for the nigrostriatal pathway. These two pathways play a key role in the rewarding effects [START_REF] Hyman | Addiction to cocaine and amphetamine[END_REF] and the locomotor adaptations [START_REF] Kalivas | Cellular mechanisms of behavioral sensitization to drugs of abuse[END_REF] following repeated administrations of cocaine. On WD1, using autoradiography approaches we observed modifications of D1 receptors after the MDRI chronic cocaine treatment pattern in the substantia nigra (increase) and in the caudate putamen and nucleus accumbens (decrease), while no modifications were observed after the ODRI pattern, suggesting that multiple daily injections are needed to induce early D1 receptor modifications [START_REF] Puig | Influence of cocaine administration patterns on dopamine receptor regulation[END_REF]. Regarding regulation of D2 receptors, densities were modified by both cocaine administration patterns, and interestingly they were opposite depending on the administration patterns, in the four brain structures studied [START_REF] Puig | Influence of cocaine administration patterns on dopamine receptor regulation[END_REF]. Regulations of dopamine receptors are long lasting, as on WD14 densities of D1 and D2 receptors were still different in cocaine treated animals as compared to control rats. Surprisingly the modifications observed were different from those observed on WD1 for both cocaine administration patterns, leading to the down-regulations of D1 and D2 receptors in most of the brain structures studied [START_REF] Puig | Influence of cocaine administration patterns on dopamine receptor regulation[END_REF]. Interestingly, these changes in dopamine receptor densities are certainly indirect via modulation of synaptic dopamine concentrations, as cocaine is not a dopamine ligand, but inhibits dopamine reuptake via blockade of the transporter located on the presynaptic neurons.

All together, these results show that the behavioral and neurochemical adaptations induced by chronic cocaine treatments are depending on the cocaine administration patterns, the brain structures considered, and the withdrawal periods as described in other studies (King et al., 1994a;[START_REF] Izenwasser | Tolerance and sensitization to the locomotor-activating effects of cocaine are mediated via independent mechanisms[END_REF][START_REF] Calipari | Temporal pattern of cocaine intake determines tolerance vs sensitization of cocaine effects at the dopamine transporter[END_REF][START_REF] Calipari | Intermittent cocaine selfadministration produces sensitization of stimulant effects at the dopamine transporter[END_REF][START_REF] Zhou | Persistent increases in rat hypothalamic POMC gene expression following chronic withdrawal from chronic "binge" pattern escalating-dose, but not steady-dose, cocaine[END_REF]. As previously described with opioid ligands, the changes observed are dynamic as they develop in a time-dependent manner.

Conclusions

The neuroplasticity, both at behavioral and neurochemical levels, observed following chronic opioid or cocaine treatments (even for short period) are dynamic and long lasting, and are dependent on numerous factors. The objectives of this review were to illustrate that the complexity of the neuroadaptations and the divergent results reported in the literature following chronic treatments with drugs of abuse are specific to the agonists used, the patterns of administration, and the withdrawal periods. The behavioral and neurochemical neuroadaptations are different on early, intermediate and protracted abstinence. They develop in a dynamic way, and they are also dependent on several factors, including pharmacodynamic factors (e.g., binding parameters of different ligands on the same target/receptor), and pharmacokinetic factors (e.g., speed and route of administration, transport across the BBB, patterns of administration). This review was focused on the results obtained with experimenteradministered drugs of abuse, the only method to control the exact doses and frequencies of administration. Other data in the literature also report different neuroadaptations depending on the temporal pattern of drug administration in a model of self-administration in rats, with short-, long-and intermittent-access to the drugs [START_REF] Calipari | Temporal pattern of cocaine intake determines tolerance vs sensitization of cocaine effects at the dopamine transporter[END_REF][START_REF] Calipari | Intermittent cocaine selfadministration produces sensitization of stimulant effects at the dopamine transporter[END_REF][START_REF] Allain | How fast and how often: The pharmacokinetics of drug use are decisive in addiction[END_REF], highlighting the importance of pharmacokinetics in the molecular and cellular mechanisms critical for addiction.

As the same factors of variability are also encountered in drug users, they can certainly largely contribute to the heterogeneity of patients and explain the difficulties encountered by clinicians in the therapeutic management. The clinical challenge is to stratified the patients with biological and/or behavioral markers, to propose a personnalized treatment adapted to specific neuroadaptation developed, certainly the only way to reduce relapse, and to aid in recovery from addiction. This is a huge challenge, because the full medical history of drug-dependent patients is rarely available. One way to improve this aspect would be to systematically include questionnaires about their drug consumption (e.g., quantities, frequencies) in the interviews. Sprague-Dawley rats were treated with saline, morphine, methadone or buprenorphine and killed 30 min, 1h and 4h after drug injections. The brains were rapidly removed and the nucleus accumbens, dorsal striatum and thalamus were dissected. Real-time quantitative PCR were performed. (n = 9-12 per group) (modified from [START_REF] Belkaï | Comparison of the transcriptional responses induced by acute morphine, methadone and buprenorphine[END_REF]. Legends: Oprm1, mu opioid receptor; Oprd1, delta opioid receptor; Oprk1, kappa opioid receptor.

Figure 4 :

 4 Figure 4: Time-course of changes in mRNA encoding endogenous opioid peptides.

Figure 5 :

 5 Figure 5: Time-course of changes in mRNA encoding opioid receptors.

Figure 6 :

 6 Figure 6: Mechanisms leading to sensitization or tolerance following different patterns of

Legends of figures:

Sprague-Dawley rats were treated with saline, morphine, methadone or buprenorphine and killed 30 min, 1h and 4h after the drug injections. The brains were rapidly removed and the nucleus accumbens, dorsal striatum and thalamus were dissected. Real-time quantitative PCR were performed. (n = 9-12 per group) (modified from [START_REF] Belkaï | Comparison of the transcriptional responses induced by acute morphine, methadone and buprenorphine[END_REF].


Rihab Benabbas

Noelia Sanchez-Ballester

Bernard Bataille

Laurent Leclercq

Tahmer Sharkawi

Ian Soulairol

Structure-Properties Relationship in the Evaluation of Alginic Acid Functionality for Tableting

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Structure-Properties Relationship in the Evaluation of Alginic Acid Functionality for Tableting Rihab Benabbas,1 Noelia M. Sanchez-Ballester,1 Bernard Bataille,1 Laurent Leclercq,2 Tahmer Sharkawi,1 and Ian Soulairol1,3,4

Abstract. The aim of this study is to investigate the relationship between the structural, molecular, and particulate properties of alginic acid and its functional characteristics in direct compression (tabletability, compressibility, elasticity, deformation mechanism, and disintegration ability). Therefore, accurate characterization of two different batches of alginic acid was executed (X-ray powder diffraction, Fourier-transform infrared spectroscopy, thermogravimetric analysis, scanning electronic microscopy, 1H nuclear magnetic resonance, size exclusion chromatographymulti angle light scattering, viscosimetry, carboxylic acid titration, powder flowability, true density, laser granulometry). Results showed that molecular weight seems to affect tablet properties and that the alginic acid with the lowest molecular weight provides the hardest tablets with the lowest elastic recovery. Furthermore, these results show the potential interest of exploiting alginic acid as filler excipient in tablet formulation. Finally, disintegration properties of tested materials were found to be close to that of commercial superdisintegrants (Glycolys® and Kollidon Cl®) but not correlated to their swelling force. It can be concluded, for the first time, that the determination of alginic acid molecular weight seems key for applications in direct compression and in particular for obtaining tablets with reproducible strength.

KEY WORDS: alginic acid; physicochemical characterization; direct compression; swelling force; disintegration.


Laurent Leclercq

Charly Renard

Antonio Stocco

Herve Cottet
email: herve.cottet@umontpellier.fr

Hervé Cottet

Superhydrophobic capillary coatings: Elaboration, characterization and application to electrophoretic separations

Keywords: Superhydrophobic coating, wettability, adsorption, capillary electrophoresis

Introduction

Superhydrophobic surfaces (SHS) are generally generated from the combination of chemical hydrophobicity with micro and nano-structuration that makes the surface entirely water repellent. An air layer is formed between the surface and water solution poured on it. The most well-known examples can be found in Nature with lotus leaves, butterfly wings, petals of rose, or legs of gerris [START_REF] Barthlott | Purity of the sacred lotus, or escape from contamination in biological surfaces[END_REF][START_REF] Nine | Facile Adhesion-Tuning of Superhydrophobic Surfaces between "Lotus" and "Petal" Effect and Their Influence on Icing and Deicing Properties[END_REF][START_REF] Zhang | Recent developments in superhydrophobic surfaces with unique structural and functional properties[END_REF]. Direct applications of SHS include self-cleaning, antifreeze, anticorrosion, and anti-friction surfaces, together with droplet handling [START_REF] Barthlott | Purity of the sacred lotus, or escape from contamination in biological surfaces[END_REF][START_REF] Nine | Facile Adhesion-Tuning of Superhydrophobic Surfaces between "Lotus" and "Petal" Effect and Their Influence on Icing and Deicing Properties[END_REF][START_REF] Zhang | Recent developments in superhydrophobic surfaces with unique structural and functional properties[END_REF]. Other applications exist in pharmaceutical and biomedical fields, such as the study of cellular communication or the reduction of bacterial adhesion on biomedical implants [START_REF] Falde | Biomaterials Superhydrophobic materials for biomedical applications[END_REF].

HS can be characterized by the measurement of the static contact angle (CA), which is the macroscopic angle between the solid-liquid interface and the liquid-air interface when a water droplet is deposited on a solid surface. The higher the CA value, the more spherical the droplet is, and so, the more repellent to water the surface is. A surface is hydrophilic when CA < 90°, while it is considered as hydrophobic when 90° < CA < 120°, and superhydrophobic when CA > 120° [START_REF] Law | Definitions for hydrophilicity, hydrophobicity, and superhydrophobicity: Getting the basics right[END_REF].

SHS can be divided into two categories with antagonistic effects. The lotus effect prevents water molecules from adhering to the surface, while the petal effect corresponds to a sticking (but non-wetting) water drop on the surface. The difference between these two categories is generally related to pattern periodicity and surface roughness [START_REF] Nine | Facile Adhesion-Tuning of Superhydrophobic Surfaces between "Lotus" and "Petal" Effect and Their Influence on Icing and Deicing Properties[END_REF]. Apart from these two effects, surface wetting on SHS can be characterized by the so-called Cassie (nonwetting) [START_REF] Cassie | Wettability of porous surfaces[END_REF] and Wenzel (wetting) states (Figure 1). In the Cassie state, air bubbles are present in the anfractuosities of the surface, while the liquid fill most of the space in the Wenzel state [START_REF] Lafuma | Superhydrophobic states[END_REF][START_REF] Quéré | Modulation du mouillage par des microtextures[END_REF]. Some SHS can lead to slippage (Cassie state with a lotus effect), others not (Cassie state with a petal effect). In this context, Wenzel state corresponds to simple hydrophobic wetting. Due to the presence of air, water friction on SHS is reduced and generally characterized by the slip length that is defined as the length corresponding to the intercept along the axis orthogonal to the interface when a tangent line is drawn along the velocity profile at the interface [START_REF] Jung | Biomimetic structures for fluid drag reduction in laminar and turbulent flows[END_REF][START_REF] Maali | Measurement of slip length on superhydrophobic surfaces[END_REF][START_REF] Avier | M moire sur les Lois du Mouvement des Fluides[END_REF].

SHS can be prepared in several ways that are generally based on silica nanoparticles, carbon nanotubes, SU-8 polymeric photoresist, polydimethylsiloxane (PDMS) or lithography [START_REF] Yoon | Hierarchical micro/nano structures for super-hydrophobic surfaces and super-lyophobic surface against liquid metal[END_REF]. For instance, microchannels (30 µm deep x 300 µm wide x 8 cm long) coated by octadecyltrichlorosilane (OTS), give a CA of 120° and 0.92 µm slip length [START_REF] Tretheway | A generating mechanism for apparent fluid slip in hydrophobic microchannels[END_REF]. Other examples can be found in the literature. Lotus-leaf layered columns (7 to 19 mm diameter, 145 mm height) were obtained by lotus leaf print on flat flexible cellophane paper (100 µm thick) and then filled with steel balls of 4.6 mm diameter, giving a CA higher than 150° and 47-117 µm slip length [START_REF] Saha | Slip-enhanced flow through thin packed column with superhydrophobic wall[END_REF]. Cylindrical microchannels (150 µm diameter) were prepared with negative print of lotus leaf surface in polystyrene, giving a CA of 160° [START_REF] Das | Fabrication of Cylindrical Superhydrophobic Microchannels by Replicating Lotus Leaf Structures on Internal Walls[END_REF]. Microchannels were coated with carbon nanotubes, giving a CA of 165° and 20 µm slip length [START_REF] Joseph | Slippage of water past superhydrophobic carbon nanotube forests in microchannels[END_REF]. Superhydrophobic cells consisting of two surface-modified glass plates with nanostructured silica wall (by deposition of soot, tetraethoxysilane and alkyl trichlorosilane layers) were used with two platinium electrodes, giving a CA of 169°, less than 1 µm roughness, up to 30 nm slip length under 0 to 50 V/cm electrical field, but no increase of electroosmotic flow [START_REF] Papadopoulos | Electrokinetics on superhydrophobic surfaces[END_REF].

Capillary electrophoresis (CE) is a separation technique especially well-suited for the analysis of (bio)macromolecules such as peptides, proteins and polysaccharides. In the ideal case, when separation efficiency is controlled by molecular diffusion, the theoretical plate number in CE is inversely proportional to the molecular diffusion coefficient [START_REF] Kasicka | Recent developments in capillary and microchip electroseparations of peptides[END_REF][START_REF] Stepanova | Recent applications of capillary electromigration methods to separation and analysis of proteins[END_REF][START_REF] Dawod | Recent advances in protein analysis by capillary and microchip electrophoresis[END_REF][START_REF] Mantovani | Recent advances in capillary electrophoresis separation of monosaccharides, oligosaccharides, and polysaccharides[END_REF].

Therefore, the larger the solute is, the lower its molecular diffusion coefficient, and the higher the separation efficiency. However, other dispersive phenomena can occur, such as solute adsorption onto the capillary wall, which tend to decrease the separation efficiency [START_REF] Kenndler | Theory of Capillary Zone Electrophoresis[END_REF]. In order to minimize protein adsorption, capillaries can be physically or chemically coated with a neutral [START_REF] Mesbah | A neutral polyacrylate copolymer coating for surface modification of thiol-ene microchannels for improved performance of protein separation by microchip electrophoresis[END_REF] or charged polymer [START_REF] Towns | Synthesis and evaluation of epoxy polymer coatings for the analysis of proteins by capillary zone electrophoresis[END_REF] or even using polyelectrolyte multilayers [25][26]. Njoya et al. proposed the use of silica colloidal crystals (with ordered arrangements of silica spheres coated with polyacrylamide) for protein electrophoresis leading to plate height of only 0.15 µm [START_REF] Njoya | Silica Colloidal Crystals as Emerging Materials for High-Throughput Protein Electrophoresis[END_REF]. An interesting alternative to avoid solute adsorption onto the capillary wall would be to limit the wettability of the water-based electrolyte onto the capillary surface. To our knowledge, the idea of using SHS in CE has not been yet investigated. When hydrophobic surfaces are used in CE or miniaturized chip electrophoresis [START_REF] Papadopoulos | Electrokinetics on superhydrophobic surfaces[END_REF][START_REF] Duffy | Rapid prototyping of microfluidic systems in poly(dimethylsiloxane)[END_REF][START_REF] Bocquet | Flow boundary conditions from nano-to micro-scales[END_REF][START_REF] Wang | Covalent modified hydrophilic polymer brushes onto poly(dimethylsiloxane) microchannel surface for electrophoresis separation of amino acids[END_REF][START_REF] Bouzigues | Nanofluidics in the debye layer at hydrophilic and hydrophobic surfaces[END_REF], researchers generally try to reduce the analyte adsorption by modifying/coating the capillary/channel wall and making them more hydrophilic. For instance in ref [START_REF] Wang | Covalent modified hydrophilic polymer brushes onto poly(dimethylsiloxane) microchannel surface for electrophoresis separation of amino acids[END_REF], electrophoretic separations of amino acids were compared in PDMS microchannels without and with an in situ polyethylene glycol covalent modification using air-plasma treatment and silanization [START_REF] Wang | Covalent modified hydrophilic polymer brushes onto poly(dimethylsiloxane) microchannel surface for electrophoresis separation of amino acids[END_REF]. Coated PDMS microchannels gave much better separation efficiency (up to 20 times) due to higher hydrophilicity leading to lower CA (64° instead of 106° for non-coated PDMS). In this work, we propose a paradigm change by making the capillary wall so hydrophobic that the capillary surface should become unwettable by the background electrolyte, and by the solute zone. By this way, the interactions between the solutes and the capillary wall should be reduced.

Finally, it is worth noting that slip flow occurring in chromatography using colloidal crystals made of 470 nm silica spheres that are chemically modified with hydrocarbon considerably enhance the separation efficiency [START_REF] Wei | Slip flow in colloidal crystals for ultraefficient chromatography[END_REF].

Glaco TM is a commercial isopropyl alcohol suspension containing hydrophobically modified silica nanoparticles used as a spray to make car windshields and wing mirrors superhydrophobic to improve visibility during rainy conditions. Glaco TM coating has been used and reported in literature based on a simple coating protocol consisting in the contact of the surface to be treated with the Glaco TM solution, followed by heating at 200°C for 30 min [START_REF] Lv | Departure of condensation droplets on superhydrophobic surfaces[END_REF]. This process was generally repeated 3-4 times [START_REF] Lv | Departure of condensation droplets on superhydrophobic surfaces[END_REF]. Atomic Force Microscopy (AFM) studies of the Glaco TM treated surface showed that particle size was about 30 to 50 nm, with 50 to 200 nm roughness, a CA of minimum 155° and a sliding angle less than 5° [START_REF] Lv | Departure of condensation droplets on superhydrophobic surfaces[END_REF][START_REF] Vakarelski | Stabilization of Leidenfrost vapour layer by textured superhydrophobic surfaces[END_REF].

Superhydrophobicity was observed on Glaco TM treated microscope glass blade (with receding angle of 150° and advancing angle of 164°) [START_REF] Li | Monostable Superantiwettability[END_REF].

This work aims at elaborating, characterizing and using superhydrophobic coatings in fused silica capillaries using Glaco TM deposition for electrophoretic separations.

Experimental section

Chemicals and samples

Glaco Mirror Coat Zero™ was purchased from Soft99 (Osaka, Japan). It is made of silica nanoparticles (hydrophobically modified with alkyl functions) suspension in isopropyl alcohol (see Material safety data sheet [START_REF] Yeast | SOFT99 corporation[END_REF]). The nanoparticle concentration in the Glaco™ solution is about 4 g/L, as quantified by weighting the dry residue after evaporation of the solvent. Ultrapure grade water (18 MΩ.cm) was produced on a Milli-Q equipment (Merck Millipore, USA). Sodium hydroxide (NaOH) and N,N-dimethylformamide (DMF, 99% m/m) were purchased from Sigma-Aldrich (St Quentin Fallavier, France). 4-(2-hydroxyethyl)-1piperazine ethanesulfonic acid (HEPES) from VWR (Fontenay-sous-bois, France), tris(hydroxymethyl)aminomethane (Tris) from Thermo Fischer (Karlsruhe, Germany) and sodium phosphate, monobasic 99% m/m from Aldrich Chemical Company (Milwaukee, USA) were used as received to prepare the different background electrolytes in CE.

Creatinine, alanine dimer (Ala-Ala), alanine trimer (Ala-Ala-Ala), alanine tetramer (Ala-Ala-Ala-Ala), Lys-His-Gly and Bursin tripeptides were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Brilliant Blue FCF (food coloring, E133) was used as a water colorant purchased from Vahiné (Avignon, France) and used as is.

Glaco™ coating

Glass blades (76 x 26 mm, Thermo Scientific, Germany) were coated with Glaco TM by pouring Glaco TM solution on them using a 5 mL plastic pipette. Isopropyl alcohol was then evaporated and the coating was stabilized by placing the glass blades in an oven for 15 minutes at 250°C. The same process was repeated 3 times to obtain maximum covering of the glass surface by the Glaco TM nanoparticles.

Fused silica capillaries (PolyMicro Technologies, Photonlines, Saint-Germain-en-Layes, France) of 360 µm external diameter, various internal diameters (180, 100 and 50 µm), and 40 cm total length (30 cm to the detector window) were coated with Glaco TM by flushing at a given pressure and for a given duration that depended on the internal diameter (see Table 1). Excess of Glaco TM solution was removed by flushing with air at a well-defined pressure / duration depending on the capillary internal diameter (I.D.) (see Table 1). Coating was then thermally stabilized by placing the capillaries in a GC-2010 Plus oven (Shimadzu, Marne-la-Vallée, France) for 15 min at 250°C without any gas flow. The same process was repeated 4 times by alternating the flushing direction in the capillary (alternatively from the starting or the ending ends of the capillary) to obtain maximum covering of the capillary walls by the Glaco TM nanoparticles. Capillaries were finally let overnight under ambient air at room temperature before use.

Surface characterization methods

Contact Angle (CA)

Contact angle between droplets of 20 mM HEPES + 10 mM NaOH (pH 7.46) solution and Glaco TM treated glass blades was measured using PAT tensiometer (Sinterface, Berlin, Germany). Dropsnake plugin on ImageJ software was used for processing the picture of the droplet. Advancing contact angle was measured by growing gradually (with 1 µL steps) the droplet volume from 20 µL to 30 µL, while receiding angle was measured by decreasing gradually the droplet volume from 30 µL to 20 µL.

2.3.2Ellipsometry

Multiple angles of incidence ellipsometry (Multiskop Optrel, Sinzing, Germany) was used to measure the thickness d Glaco and refraction index n Glaco of the Glaco TM coating on a glass blade and the thickness of the air layer on a Glaco TM coated glass blade submerged in deionized water. The incidence angle of the laser beam (533 nm) could be finely modified and the reflected polarized light was detected by a four quadrant photodiode (2×2 cm 2 ). Ellipsometry at the water-solid interface was measured by using light guides immersed in water in order to detect the reflection from the water-solid interface avoiding the reflection coming from the air-water interface [START_REF] Benjamins | New experimental setup to use ellipsometry to study liquid-liquid and liquid-solid interfaces[END_REF].

Atomic Force Measurement (AFM)

Tapping Mode Atomic Force Microscopy (TM-AFM) images were acquired with a Bruker Nanoman (Bruker SAS, Palaiseau, France) driven by Nanoscope 3A Quadrex electronics. The cantilever tips were Silicon Point Probe Plus TM NCSTR (Nanosensors, Neuchâtel, Switzerland) used at a constant force of 6.5 N/m, resonance frequency of 157 kHz. All the image treatments and the thickness measurements were performed using WSxM 4.0 Beta 8.2 and the resulting data were treated with the Fityk software to correct the background of the image.

Dynamic Light Scattering (DLS)

Glaco TM nanoparticles size was measured with a Litesizer™ 500 (Anton Paar, Graz, Austria) dynamic light scattering (DLS) equipment mounted with a BM10 module, driven by Kalliope 1.8.4 software. Quartz cells (10 mm x 10 mm x 45 mm) were used. Refraction index of isopropyl alcohol was 1.3749 at 25 °C. Transmittance was 98.3% at 90°. Glaco TM solution (4 g/L in nanoparticle) was used as received for the DLS measurement.

Scanning Electron Microscopy (SEM)

Scanning Electron Microscopy (SEM) was realized using a Hitachi S4800 machine (Hitachi High Technologies America, Inc., Schaumburg, Illinois, USA). Thickness, roughness and nanoparticle size were measured on Glaco TM coated glass blades and capillaries. Inner surface of capillaries were made accessible to SEM by burning the external polyimide protection layer of the capillaries and then by shattering it with a ceramic capillary cutter on a double faced tape support to obtain some shards turned face up. Acceleration voltage varied from 0.1 to 30 kV depending on the magnification and sample conductivity.

Optical Microscopy

USB optical microscope n° 44302 (Celestron, California, USA) was used to observe the capillary wall-air-electrolyte interface in Glaco TM coated capillaries at ×150 magnification.

Capillaries (40 cm total length) of various internal diameters (50, 100 and 180 µm) were observed after being partially filled with brilliant blue dyed water (1 g/L) and air bubble.

Dyed water and air were introduced with a syringe fixed on capillary by a LuerTight™ 360 µm outside diameter (O.D.) tubing to Luer connection (P-662 rapid connection, Idex Health and Science, California, USA).

C4D profiling of the Glaco TM coated capillary

The capacitively coupled contactless conductivity detector (C4D) was a TraceDec (Innovative Sensor Technologies GmbH, Innsbruck, Austria) used with the following settings: frequency 2xHIGH, voltage -12 Db, gain 50% and offset 0. Conductivity measurements were performed along the capillary under a 1 mL/min flow rate of 20 mM HEPES + 10 mM NaOH electrolyte, pH 7.46. The flow rate was ensured by a syringe-pump (KD Scientific, USA) connected to the capillary by a LuerTight™ connection (P-662 rapid connection, Idex Health and Science, California, USA). The C4D cell was moved along the capillary by steps of 3 cm, and the conductivity was measured at each point. Fused silica capillaries of different I.D. were tested as reference and compared to a 100 µm I.D. Glaco™ coated capillary.

Hydrodynamic behavior: Poiseuille law

The flow rate under given hydrodynamic pressure was measured using the CE equipment on Glaco TM coated capillaries and fused silica capillaries ( The flow rate Q (in m 3 /s) at a given pressure ΔP (in Pa) was calculated from the detection time t DMF (in s) of the DMF marker according to:

2 c DMF lR Q t   ( 1
)
where l is the length of the capillary to the UV detection window and R c is the capillary radius. Poiseuille law (Q vs ΔP) was systematically plotted. The slope a of the Q vs ΔP relationship depends on the apparent radius R e of the capillary, according to the following equation:

4 8 e R P Q L     (2)
where η is the viscosity of the solution (in Pa.s) and L is the total length of the capillary (in m). It is therefore possible to determine the apparent radius R e from the slope a using the following equation:

1/ 4 8 e La R          (3)
where t m is the migration time and δ is the half-height peak width determined using CEval 0.6 software available at the following address [https://echmet.natur.cuni.cz/software/ceval].

RESULTS AND DISCUSSION

Superhydrophobic glass blade

Glaco™ coated glass surfaces were first prepared on glass blades, based on simple contact of Glaco™ solution on the surface followed by a heating treatment at 250°C, both repeated three times (see the experimental protocol described in the experimental part). Figure 2 displays a picture of a ~20 µL water droplet deposited on the Glaco™ coated glass surfaces.

A static contact angle (CA) of 159° (± 6°, which is the standard deviation based on n= 4 repetitions on 3 different blades) was observed for advancing droplet (i.e. growing droplet)

and 145° (± 12°) for receding (i.e. decreasing droplet size), clearly demonstrating that Glaco TM coated glass blades present superhydrophobic behavior (defined as CA > 120°).

Figure SI1 shows a photography of an immersed glass blade and the air layer covering it. The silver-mirror sheen of the immersed coated glass blade is due to a thin air layer retained on the surface, and is the signature appearance of Cassie state [START_REF] Vakarelski | Stabilization of Leidenfrost vapour layer by textured superhydrophobic surfaces[END_REF].

To better characterize the surface, the thickness of the Glaco™ layer and of the air layer between the immerged Glaco™ coated glass surface and water were measured by ellipsometry (Figure 3). Uncoated glass blade in contact of water shows the absence of an air layer at the solid-liquid interface (Figure 3A). Figure 3A shows scans of the ellipsometric amplitude (tanΨ) and phase (Δ) as a function of the incident angle (φ) for a bare and a

Glaco™ coated glass surfaces exposed to air. Around the Brewster angle (φ Br =arctan(1/n Glass =56.8), tanΨ and Δ change sharply for the uncoated surface confirming the absence of an interfacial layer and the cleanness of the glass substrate. In the presence of a Glaco™ coating, tanΨ and Δ deviate strongly from the ones of the bare surface, which allows us to evaluate the average thickness and refractive of the coating: n Glaco =1.58±0.1 and d Glaco =270±5 nm (see inset of Figure 3), assuming a single layer interfacial profile between two macroscopic media: water and air. Ellipsometry was also performed for glass blades in water, as shown in Figure 3B. In order to fit these results, we introduce an additional interfacial layer of refractive index n Air and thickness d Air , fixing the refractive index of the glass substrate n Glass and the thickness of the Glaco TM coating (as obtained from Figure 3A).

From the fits, we obtain n Air =1.17-1.24 and thicknesses d Air = 124-208 nm. Being n Air < n Water = 1.333, we can interpret this result as the observation of gas bubbles trapped in water on the hydrophobic Glaco TM surface. The interfacial layer between water and the Glaco TM coating could be modelled as a mixed layer composed by gas bubbles immersed in water. The volume fraction of air f in the interfacial layer of thickness d Air could be evaluated accounting for the Wiener effective medium approximation [START_REF] Aspnes | Optical properties of thin films[END_REF]:

(5) which leads to f = 0.31-0.52 .

Figure 4 displays the surface topology of the Glaco™ onto the glass blade as observed by AFM (tapping mode). Excellent coverage of the surface was observed, with a thickness of about 200 nm (Figure 4A and4B). Particle size was about 30 nm (Figure 4C) and roughness was about 100 nm (Figure 4B). Granulometric distribution of the Glaco™ solution (in isopropyl alcohol) measured by DLS showed an intensity average hydrodynamic diameter of 135 nm (with a polydispersity index of 23%). Glaco™ suspension is made of stable raspberry shaped clusters of smaller nanoparticles (~30 nm diameter) as shown in Figure 4C. These results are in agreement with a self-similar fractal structure [START_REF] Cottin-Bizonne | Scaling laws for slippage on superhydrophobic fractal surfaces[END_REF]. Neither ultrasounds, nor dilution, were found to modify cluster size (monitored by DLS).

Superhydrophobic capillary coating

Glaco™ coated capillaries were then prepared according to an optimized protocol described in the experimental part. In brief, this protocol is based on the 4-times repetition of a three steps procedure based on: (i) flushing of the capillary with the Glaco™ suspension; (ii) controlled air flushing of the capillary leading to a Glaco™ film deposition onto the capillary surface and (iii) the thermal stabilization of the coating at 250°C in a GC oven.

Step (ii) was found to be crucial to control the quantity of Glaco™ deposited onto the capillary surface.

The thickness (ε) of the Glaco™ film remaining on the fused silica capillary when flushing with air strongly depends on the applied air pressure to remove the Glaco™ suspension, as

shown by equation ( 6) from the Landau, Levich and Derjaguin (LLD) theory (also known as the Bretherton law) [START_REF] Quéré | Fluid Coating on a Fiber[END_REF] :

2 / 3 1.34 c R Ca   ( 6
)
where Ca is the dimensionless capillary number defined by equation ( 7):

V Ca    (7)
γ is the surface tension of the coating solution (in N/m) and V is the linear velocity of the coating solution flushed by air pressure (in m/s). Combining equations ( 6), [START_REF] Lafuma | Superhydrophobic states[END_REF] with the Poiseuille law leads to: 2/ 3 7 / 3 1. [START_REF] Vakarelski | Stabilization of Leidenfrost vapour layer by textured superhydrophobic surfaces[END_REF] 8

c P R L        ( 8
)
where ΔP is the applied pressure at which the air replace the Glaco TM solution, and L is the capillary length. It is worth noting that equations ( 6) and ( 8) are only valid for Ca lower than 10 -2 . The pressure ΔP were adjusted so that the coating thickness was similar for all capillary I.D.. Taking γ = 0.0217 N/m at 20°C for isopropyl alcohol (solvent of Glaco TM suspension),

and the ΔP values set in Table 1 for each capillary I.D., leads to Glaco thickness ε = 1.1, 1.2 and 1.0 µm for capillary I.D. of 50, 100 and 180 µm, respectively. Ca numbers are 5.9 x 10 -3 , 2.3 x 10 -3 , 7.6 x 10 -4 respectively, demonstrating the validity of equation [START_REF] Quéré | Modulation du mouillage par des microtextures[END_REF]. Following this experimental protocol, very good coating coverage of the capillary inner wall surface was observed, as shown by scanning electron microscopy (SEM) in Figure 5, even if the surface coverage was found less effective in capillaries (Figure 5C to 5F) than in glass blade (Figure 5A).

Scanning contactless conductivity detector (sC4D) was next used to sense the capillary inner surface (as discussed in a review paper [START_REF] Gillespie | Using scanning contactless conductivity to optimise photografting procedures and capacity in the production of polymer ion-exchange monoliths[END_REF]) by measuring the conductivity at different positions along the capillary under constant flow rate with a 20 mM HEPES electrolyte (pH 7.46). The conductivity measurements confirm that the thickness of the glaco coating is negligible compared to the capillary I.D., as the conductivity was similar on Glaco TM coated and on a fused silica capillaries (see Figure SI2).

To demonstrate that the superhydrophobicity was also observed inside the capillaries (and not only on the coated glass blades), the meniscus of a blue colored water solution injected in Glaco™ coated capillaries was compared to the one observed in a fused silica capillary (Figure 6). As expected for a SHS, the meniscus was reversed in the case of Glaco™ coated capillaries for all internal diameters (50, 100 and 180 µm I.D.). It is worth noting that flushing the capillary with air by hand using a syringe and LuerTight™ connector in step (ii) of the coating protocol did not give reversed meniscus (only flat profile of the interface; not shown).

Similarly, using lower pressure in step (ii) that those given in Table 1, leads to thinner film deposition (< 1 µm) and weaker SH properties.

Hydrodynamic behavior

Before studying the hydrodynamic behavior (Poiseuille law), it was first observed that a minimal pressure of 0.3 psi was required to flush a freshly prepared 180 µm I.D. Glaco™ coated capillary, initially filled with air, with a HEPES / Na BGE. Below that limiting value, the BGE did not enter in the capillary. This is in good agreement with the minimum pressure predicted by the Young-Laplace law: Figure 7A displays the BGE fronts (0.5% v/v DMF was added in the BGE as a marker) obtained at different applied pressures in Glaco™ coated capillaries, initially filled with HEPES / Na BGE at 1 psi 2 min, and in fused silica capillary for comparison (all capillaries with 180 µm I.D.). The higher the applied pressure, the shorter the elution time. Flow rates were plotted against the applied pressure (Figure 7B). The slope is directly related to the apparent capillary radius, R e , that can be determined using equation [START_REF] Zhang | Recent developments in superhydrophobic surfaces with unique structural and functional properties[END_REF]. The blue trace in Figure 7B corresponds to the behavior of a fused silica capillary, leading to an effective radius of 94 µm which is very close to the expected value of 90 µm, assuming no-slip boundary conditions at the silica surface. As for the superhydrophobic 180 µm I.D. Glaco™ coated capillary, it was first observed a shift in the Poiseuille law with a pressure drop of about 0.3 psi (or 2100 Pa, yellow trace in Figure 7B), which is due to the presence of multiple air bubbles in the capillary. Using equation ( 9), we can estimate that a bubble separating two plugs of BGE in a 180 µm I.D. capillary generate a pressure drop ΔP bubble of ~100 Pa taking γ =72 N/m, R c = 90 µm,  r =145° and  a =160°:

(cos( ) cos( )

) ra P R      (10)
This observation means that the pressure drop would correspond to a train of approx. 20 bubbles in the whole capillary. Experimentally, using a USB optical microscope, 14 to 24 bubbles could be observed in the Glaco™ coated capillary, in good agreement with the expectations.

The Poiseuille law measurements was duplicated one week later on the same capillary, leading to the red trace with apparent slippage (slope higher than bare silica slope) corresponding to a slip length of 23.3 µm, calculated from the Poiseuille law corrected from the slip length factor [START_REF] Lauga | Apparent slip due to the motion of suspended particles in flow of electrolyte solutions[END_REF] leading to:

3 2 4 La R b R     (11)
with b the slip length and a the slope of the Poiseuille profile (i.e. Q/ΔP). The poor linearity of the Poiseuille law at low flow rate can be explained by unstable flows (i.e. the flow can be temporally stopped), leading to non-linear behavior.

Finally, the same experiments performed one week later, leads to the green trace, which is similar to the fused silica capillary. These observations are in agreement with a progressive coating degradation that led to the loss of the meniscus inversion upon time. The fact that the slipping behavior (higher slope in the Poiseuille law than for fused silica capillary) was observed at intermediate state (in week 2), could be related to the pattern and the density of the Glaco™ coating, which is affected by the progressive coating degradation. Indeed, it was shown recently that the effective slip length strongly depends on the meniscus angle  meniscus of the nanobubble trapped between the surface anfractuosities and the liquid (see e.g. Figure 3 in ref. [START_REF] Steinberger | High friction on a bubble mattress[END_REF]). In the Cassie regime, when bubbles fill the holes,  meniscus between -30• and 45° leads to positive slip length with a maximum slip at ~0°. For  meniscus > 45•, the slip lengths become negative, i.e. smaller than in the Wenzel case (without any gas phase trapped on the surface = no slip). Negative effective slip lengths can be explained by an immobilized layer of the liquid close to the wall. In this limit, the liquid-solid friction is higher than on a nonslipping surface (without holes) [START_REF] Steinberger | High friction on a bubble mattress[END_REF]. We can imagine that the freshly prepared Glaco™ SHS leads to high  meniscus (>45°) with no slipping surface. Then, the aging of the coating may favor the formation of less protruding gas bubbles and the slip condition at the SHS surface occurs (ideal metastable Cassie slipping state), before recovering a more classical no-slip condition (Wenzel state), leading to similar hydrodynamic behavior as observed on silica surface.

Similar results were obtained on 100 µm and 50 µm I.D. Glaco™ coated capillaries (see Figures SI3 andSI4), even if the intermediate slipping state was not observed; probably because it is metastable.

To get more insight on the reason of the coating degradation upon time, and to investigate if the degradation was caused by the mechanical erosion due to the capillary flushing between hydrodynamic experiments (1 psi ~70 mbar, 2 min), a freshly coated capillary was left in the HEPES / Na pH 7.5 BGE to see if the meniscus was lost with time in absence of capillary flushing. The meniscus starts to be altered after 4 to 7 days (see Figure SI5 for more details), while the SHS was conserved even after 18 days of contact with a Tris / Phosphate pH 2.5 BGE. This is why the pH 2.5 BGE was used to study the electrokinetic behavior.

Electrokinetic Behavior of SHS coated capillaries

Freshly prepared 100 µm I.D. Glaco™ coated capillaries were used to separate a peptide mixture composed of Bursin, Lys-His-Gly, creatinine, Ala-Ala, Ala-Ala-Ala and Ala-Ala-Ala-Ala in acidic conditions (Tris / Phosphate 6.5 mM / 14.1 mM at pH 2.5). Separation efficiencies were compared with those obtained on a fused silica capillary in the same conditions. Figure 8 shows the electrophoregrams and the corresponding current intensity obtained using the fused silica capillary (Figure 8A) and the Glaco™ coated capillary at different times (Figure 8B to 8E).

Separations could not be obtained on the freshly prepared Glaco™ coated capillaries (Figure 8B) with reversed meniscus (see insert in Figure 8B), due to unstable current intensity probably because of the presence of large air bubbles in the capillary. This phenomenon was observed despite the degassing of the BGE (using boiled water) and the application of a +3 psi pressure on both ends of the capillary to help to stabilize the current intensity. After 5 runs, the meniscus was less reversed, and stable separations could be obtained with +3 psi pressure (Figure 8C). Figure 9 gives the separations efficiencies (in plate number) obtained on a fused silica capillary (blue) and on three different Glaco™ coated capillaries. Much better separation efficiencies were obtained on Glaco™ coated capillaries (up to 60 000 plates) compared to fused silica capillary (less than 25 000 plates). These results were reproducible, as shown by the data reported on 3 different Glaco™ coated capillaries. These encouraging results demonstrate that a low wettability of the capillary surface can increase the separation efficiency (with an average gain by a factor 2 on the separation efficiency). However, the stabilization of the current intensity is difficult to obtain on freshly SHS due to air bubble formation.

Increasing the total number of runs leads to some coating degradation as demonstrated by the shape of the meniscus (see Figures 7C to 7E), which tends to become flat (Figure 7D), or not well defined (Figure 7E), probably because of the occurrence of coating heterogeneities. This degradation tends to increase the peak dispersion, and thus, to decrease the separation efficiencies. It is worth noting that a progressive wetting of the surface leading to the Wenzel state, instead of a coating degradation, was discarded since the SH state was not recovered after heating of the degradated capillary in a GC oven at 250°C for 15 min.

Coming back to freshly prepared SHS capillaries, the +3 psi stabilization pressure (both ends) used to try to stabilize the current intensity was arbitrarily chosen. Conceptually, it only had to be lower than P lim , the pressure above which wetting of the coating is forced by the compression of the air bubble. This limiting pressure value P lim was evaluated to be ~100 psi (7.1 bars) using equation ( 12):

lim P r   (12)
taking γ the superficial tension of the BGE (72 x 10 -3 N/m), and r the roughness of the capillary wall (100 nm, see AFM measurement). Stabilization pressures higher than 3 psi were tested (up to 80 psi), but none of those tests gave better results regarding current stabilization (or separation efficiency) compared to the application of 3 psi. The current intensity was not stabilized faster (there were still approx. 5 runs similar to Figure 8B before the capillary behavior was similar to figure 8C).

CONCLUSION

In this work, we report for the first time a simple coating protocol to obtain superhydrophobic coatings in fused silica capillaries with I.D. comprised between 50 µm and 180 µm. The pressure of the air flush to remove the Glaco TM coating suspension was found to be crucial to control the thickness, the homogeneity and the reproducibility of the coating layer. The coating layer was ~200-300 nm thick, with 100 nm roughness and ~100-200 nm air layer when dipped in water. SHS were obtained in the 50-180µm I.D. fused silica capillaries as demonstrated by the shape of the meniscus and the existence of a limiting pressure to introduce water in a capillary previously filled with air. The hydrodynamic behavior of the superhydrophobic capillaries revealed the presence of air bubbles in the capillary, as observed optically and by the pressure drop in the Poiseuille law. Initially, the superhydrophobic capillaries did not display any slipping behavior, but the hydrodynamic behavior changed with time with intermediate metastable slipping state (slipping length ~23 µm).

Electrophoretic separations were difficult to obtain in freshly prepared superhydrophobic capillaries due to the presence of air bubbles leading to unstable current intensity. After a few (typically 5) instable runs, electrophoretic separations of peptides display improved separation efficiencies (by a factor ~2) compared to fused silica capillaries, demonstrating the possibility to improve electrophoretic separations by using superhydrophobic coatings. This first proofof-concept should be further studied to find better coating long-term stability and to improve the current intensity stability. E133 at 1 g/L) and air. Glaco™ coated capillaries are prepared according to the protocol given in section 2.2 (see also Table 1).

  40 cm total length, 30 cm to detector) of three different I.D. (50, 100 and 180 µm), by applying different pressures with the 20 mM HEPES + 10 mM NaOH electrolyte, pH 7.46, containing 0.5% v/v DMF as UV marker. Between each run, the capillary was flushed by the same background electrolyte (BGE), without UV marker, at a given pressure depending of the capillary I.D. (3 psi 2 min for I.D. 100 µm, 10 psi 5 min for I.D. 50 µm, and 1 psi 2 min for I.D. 180 µm. 1 psi = 0.06 bar). For 50 µm (resp. 100 µm and 180 µm) I.D. capillaries, applied pressure range was 3 to 14 psi (resp. 1 to 3 psi and 0.2 to 1.2 psi). Detection of the marker was monitored by UV absorbance at 190 nm. Flow-to-pressure relationship was used to calculate the apparent capillary diameter, and to estimate the slip length.

2. 3 . 9 .

 39 Capillary electrophoresisCapillary electrophoresis experiments were carried out on a P/ACE™ MDQ Beckman-Coulter (Sciex, Villebon sur Yvette, France) piloted by 32 Karat software. Bare silica and Glaco TM coated capillaries of various internal diameter (I.D.), and of total capillary length 40 cm and effective capillary length 30 cm, were tested for the electrophoretic separation of peptides mixtures. Unless otherwise specified, all electrolytes were prepared with boiled ultrapure water to obtain gas-free solutions. Electrophoretic separations were performed in a Tris / Phosphate 6.5 mM / 14.1 mM electrolyte at pH 2.5 (10 mM ionic strength). Before each run the capillary was flushed at 3 psi for 2 min (I.D. 100 µm). Peptides mixture for analysis (0.1 g/L in water of Lys-His-Gly, Arg, creatinine, Ala, Ala-Ala and Ala-Ala-Ala) was injected into the capillary at 0.2 psi, 2 s (1.06 nL for a capillary of 50 µm I.D.).Separations were realized at 10 kV with a pressure of 3 psi at both capillary ends of the capillary to stabilize the electrical current. Between each run, the capillary was flushed by the BGE at a pressure depending of the capillary I.D. (3 psi 2 min for I.D. 100 µm, 10 psi 5 min for I.D. 50 µm, and 1 psi 2 min for I.D. 180 µm). The temperature of the capillary cartridge was set at 25°C. UV detection was performed at 214 nm. Separation efficiency was determined using the following equation:

  γ = 72 N/m, R c = 90 µm, leading to P min = 0.23 psi. This experimental observation is in agreement with the SHS of the Glaco™ coated capillary.

Figure 1 .

 1 Figure 1. Scheme of a droplet in the non-wetting Cassie state (A), with the presence of air

Figure 2 .

 2 Figure 2. Picture of a ~20 µL droplet (20 mM HEPES, pH 7.45) deposited on a Glaco™

Figure 3 .

 3 Figure 3. Ellipsometric scans of Glaco™ coated glass blade at the solid-air interface (A) and

Figure 4 .

 4 Figure 4. (A) Tapping mode AFM scan of a Glaco™ coated glass blade surface with a bare

Figure 5 .

 5 Figure 5. SEM pictures of Glaco™ coated surfaces. Glaco™ coated glass blade (A). Beveled

Figure 6 .

 6 Figure 6. Visualization of the meniscus in bare silica (A: 100 µm ID) and in Glaco™ coated

Figure 7 .

 7 Figure 7. DMF fronts obtained at different applied pressures on Glaco™ coated capillary (A):

Figure 8 .

 8 Figure 8. Electrophoregrams (blue traces) of a peptide mixture obtained on bare silica

Figure 9 .

 9 Figure 9. Separation efficiencies for each peptide obtained on bare silica capillary and on

ACKNOWLEDGMENTS

The authors thank Michel Ramonda (UM) for AFM, and Didier Cot (IEM, UM) for SEM measurements. José Bico (ESPCI), Anne-Laure Biance and Christophe Ybert (iLM, Université de Lyon) are gratefully acknowledged for fruitful discussions.

AUTHOR INFORMATION Corresponding Authors

*herve.cottet@umontpellier.fr