M.D Nicolas Hoertel

M.D Hélène Cipel

M.D Carlos Blanco

M.D Maria A Oquendo

M.D Pierre Ellul

M.D Edourd Leaune

M.D Frédéric Limosin

M.D Hugo Peyre

M.D Jean-François Costemale-Lacoste
email: costemale-lacostedr.jfclacoste@gmail.com

Cerebrospinal fluid levels of monoamines among suicide attempters: a systematic review and random-effects meta-analysis Running title: CSF monoamines among suicide attempters

Keywords: suicide attempt, serotonin, dopamine, noradrenalin, biomarker, cerebrospinal fluid, violent, meta-analysis

Background: It remains unclear whether the dopaminergic and noradrenergic systems may be implied in suicide attempt risk. In addition, although the serotonergic system has been extensively studied, no formal meta-analysis has been performed to examine its association with suicide attempt.

Methods: Using PRISMA methodology, we performed a systematic literature review and random-effects meta-analyses of the differences in cerebrospinal fluid (CSF) levels of 5-HIAA, HVA and MHPG between suicide attempters and individuals who never attempted suicide.

Results:

We identified 30 studies including 937 suicide attempters and 1128 non-attempters; 29 of them measured CSF levels of 5-HIAA, 22 measured CSF levels of HVA and 14 measured CSF levels of MHPG. CSF levels of 5-HIAA and HVA were significantly lower in suicide attempters than in non-attempters [SMD= -0.43 (95% CI: -0.71 to -0.15; p<0.01) and SMD= -0.45 (95% CI: -0.72 to -0.19; p<0.01), respectively]. We did not find a significant association between CSF MHPG levels and suicide attempt.

Limitations: Our analyses relied on a limited number of studies of good quality and most studies included small sample sizes.

Conclusion:

Both serotonin and dopamine systems may play a role in suicide attempt risk.

Our findings suggest that a silo approach to biomarkers should be phased out in favor of the study of multiple systems in parallel and in the same populations to progress in the 2 identification of the biological components independently associated with suicide risk, with the goal of identifying new treatment targets and improving suicide risk prediction.

Introduction

Worldwide, about 800,000 people take their own life every year ("WHO | Preventing suicide," 2017). To date, suicide risk assessment is based solely on clinical evaluations, which are hampered by the elevated number of medical, psychological and social predictors, their frequent co-occurrence, and their limited predictive power [START_REF] Geoffroy | Sleep complaints are associated with increased suicide risk independently of psychiatric disorders: results from a national 3-year prospective study[END_REF][START_REF] Ghasemi | Measurement Scales of Suicidal Ideation and Attitudes: A Systematic Review Article[END_REF]Kovacs, Beck, & Weissman, 1975;Hamilton, 1960;Hoertel et al., 2015[START_REF] Hoertel | A Comprehensive Model of Predictors of Suicide Attempt in Depressed Individuals and Effect of Treatment-Seeking Behavior: Results From a National 3-Year Prospective Study[END_REF]Maris, 2002;[START_REF] Mcmahon | Childhood maltreatment and impulsivity as predictors of interpersonal violence, self-injury and suicide attempts: A national study[END_REF]Oquendo, Currier, & Mann, 2006;[START_REF] Oquendo | Life Events: A Complex Role In The Timing Of Suicidal Behavior Among Depressed Patients[END_REF][START_REF] Pascal De Raykeer | Effects of Psychiatric Disorders on Suicide Attempt: Similarities and Differences Between Older and Younger Adults in a National Cohort Study[END_REF][START_REF] Peyre | Contributing Factors and Mental Health Outcomes of First Suicide Attempt During Childhood and Adolescence: Results From a Nationally Representative Study[END_REF]Posner et al., 2011;Sheer et al., 2020). The lack of robust clinical predictors of suicide risk has led researchers to search for biomarkers that may help improve suicide risk prediction and suggest mechanism-based specific interventions.

The stress-diathesis model posits that suicidal behavior is triggered by a stressful life event or a psychiatric episode in an individual with a predisposing diathesis [START_REF] Van Heeringen | The neurobiology of suicide[END_REF]. Several lines of evidence implicate dysregulation in stress response systems, especially the hypothalamic-pituitary-adrenal (HPA) axis, and abnormalities in the serotonergic system, as a diathesis for suicide risk (Oquendo, Sullivan, et al., 2014). However, the HPA axis is linked to other biological systems, such as the serotonergic, opioid, and glutamate systems, inflammatory pathways, lipid status, and neuroplasticity or neurogenesis system (Oquendo, Sullivan, et al., 2014), suggesting that a battery of biomarkers, rather than a single one, will likely be needed to identify the components underlying this diathesis.

The earliest promising cerebrospinal fluid (CSF) monoamine biomarkers of suicidality were the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), the dopamine metabolite homovanillic acid (HVA), and the norepinephrine metabolite 3-methoxy-4hydroxyphenylglycol (MHPG) [START_REF] Asberg | 5-HIAA in the cerebrospinal fluid. A biochemical suicide predictor?[END_REF][START_REF] Engström | Reduced cerebrospinal HVA concentrations and HVA/5-HIAA ratios in suicide attempters. Monoamine metabolites in 120 suicide attempters and 47 controls[END_REF][START_REF] Gardner | CSF metabolites in borderline personality disorder compared with normal controls[END_REF][START_REF] Linnoila | Low cerebrospinal fluid 5-hydroxyindoleacetic acid concentration differentiates impulsive from nonimpulsive violent behavior[END_REF]. A literature review published in 1995 [START_REF] Lester | The concentration of neurotransmitter metabolites in the cerebrospinal fluid of suicidal individuals: a meta-analysis[END_REF] indicated that most studies found significantly decreased levels of CSF levels of 5-HIAA among suicide attempters compared to nonattempters, whereas most studies did not find significantly decreased CSF levels of HVA or MHPG among suicide attempters compared to non-attempters. However, that review examined each study individually and did not use meta-analytic methods to obtain pooled estimates of the associations between suicide attempt and CSF levels of these monoamines. Furthermore, multiple studies have been conducted since 1995, allowing for increased power to examine those associations. Another literature review [START_REF] Mann | Can biological tests assist prediction of suicide in mood disorders?[END_REF] indicated that CSF levels of 5-HIAA may be useful to predict suicide in mood disorders, although with limited positive predictive value. However, no study to date has established quantitatively synthesized results on this association. Thus, there is a need to re-examine whether CSF levels of 5-HIAA, HVA and MHPG are significantly associated with suicide attempt and to estimate the magnitude of those associations. Such knowledge is essential to advancing our understanding of the neurobiological mechanisms underlying suicidal behaviors. Specifically, the dopaminergic system and noradrenergic system have received relatively limited attention in the past decade as potential contributors to the predisposing diathesis for suicide risk (Oquendo, Sullivan, et al., 2014).

However, dopamine, acting through multiple receptors, may play a substantial role in suicidal behavior as it exerts a stimulatory role on the activation of the HPA axis in response to a severe stressor and is involved in the maintenance of post-stress activation of the HPA axis [START_REF] Belda | Dopamine D1 and D2 dopamine receptors regulate immobilization stress-induced activation of the hypothalamus-pituitary-adrenal axis[END_REF]. Some findings also suggest lower norepinephrine function in suicide risk (Oquendo, Sullivan, et al., 2014), which, if confirmed, could indicate either a direct contribution to suicidal behavior or reflect norepinephrine depletion due to downregulation in excessive response to stress (Oquendo, Sullivan, et al., 2014). Finally, although the serotonergic system has been extensively studied in suicide, we did not find any report of formal meta-analysis of the associations between suicide attempt and CSF levels of 5-HIAA.

Therefore, examining if CSF levels of serotonin, dopamine and norepinephrine metabolites are significantly decreased in suicide attempters constitutes an important step to help identify relevant potential biomarkers of suicide risk and guide future research aiming at clarifying the role of each and their interplay, with the ultimate goal of identifying new treatment targets and improving suicide risk prediction [START_REF] Fischer | An Update on the Role of Serotonin and its Interplay with Dopamine for Reward[END_REF][START_REF] Niederkofler | Functional Interplay between Dopaminergic and Serotonergic Neuronal Systems during Development and Adulthood[END_REF][START_REF] Seo | Role of Serotonin and Dopamine System Interactions in the Neurobiology of Impulsive Aggression and its Comorbidity with other Clinical Disorders[END_REF].

In this report, we performed a systematic literature review and the first random-effects meta-analysis of the associations of CSF levels of serotonin, dopamine and norepinephrine metabolites (i.e., 5-HIAA, HVA and MHPG) with suicide attempt. Because prior research (Asberg & Träskman, 1981;[START_REF] Lester | The concentration of neurotransmitter metabolites in the cerebrospinal fluid of suicidal individuals: a meta-analysis[END_REF] has suggested that abnormalities in neurotransmitters levels in individuals who attempted suicide might be more characteristic of those who use violent methods (i.e., suicide attempt by hanging, drowning, gunshot wound, gas poisoning, or multiple deep cuts) rather than less violent ones (i.e., oral drug overdoses or single wrist cuts), we also examined CSF levels of these metabolites among suicide attempters who used violent methods.

1.0.

Material and methods

Protocol and registration

This study is a systematic literature review of trials and meta-analyses performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [START_REF] Shamseer | Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation[END_REF]. The study protocol has been registered in the international prospective register of systematic reviews (PROSPERO, CRD42019125208).

Outcomes

The primary outcome was the difference in CSF levels of 5-HIAA, HVA and MHPG between suicide attempters and controls (i.e., individuals who had no history of attempted suicide). The secondary outcome was the difference in CSF levels of these metabolites between individuals who made a violent suicide attempt versus those who had no history of attempted suicide.

Search criteria

We searched for all studies that examined CSF levels of 5-HIAA, HVA and MHPG in suicide attempters. To rate studies' quality (good, fair and poor), values were attributed to each grade of the Newcastle-Ottawa Scale by awarding stars in each domain following the guidelines of the Newcastle-Ottawa Scale. We used the following cut-offs: association of 3 or 4 stars in selection, 1 or 2 stars in comparability, and 2 or 3 stars in outcomes was referred to as "good" quality score; association of 2 stars in selection, 1 or 2 stars in comparability, and 2 or 3 stars in outcomes was referred as to "fair" quality score; finally, 0 or 1 star in selection, or 0 star in comparability, or 0 or 1 star in outcomes reflected a "poor" quality score. We used those cutoffs as done in prior research (Sharmin et al., 2017a(Sharmin et al., , 2017b;;[START_REF] Vivekanantham | The association between headache and low back pain: a systematic review[END_REF].

Statistical analysis

We used random-effects meta-analysis models, which assume that the observed estimates of CSF levels of 5-HIAA, HVA and MHPG can vary across studies because of real differences in each study as well as sampling variability [START_REF] Riley | Interpretation of random effects meta-analyses[END_REF]. Measures of associations are presented as standardized mean difference (SMD), also known as Cohen's d (Cohen, 2013). Heterogeneity was assessed using the I 2 statistic and Tau 2 . The I 2 score represents the percentage of variation across studies due to heterogeneity, and ranges from 0 to 100 %; higher values indicate greater heterogeneity across studies [START_REF] Higgins | Measuring inconsistency in meta-analyses[END_REF].

Tau 2 estimates represent the between-study variance in a random-effects meta-analysis. A

Tau² value greater than 1 suggests the presence of substantial heterogeneity, whereas a value close to 0 indicates homogeneity, meaning that all studies show the same effect (Borenstein, Hedges, & Rothstein, 2007;[START_REF] Deeks | Analysing Data and Undertaking Meta-Analyses[END_REF][START_REF] Suijkerbuijk | Interventions for obtaining and maintaining employment in adults with severe mental illness, a network meta-analysis[END_REF].

Publication bias was assessed using a funnel plot.

If I² was above 25%, a meta-regression analysis was performed to explain statistical heterogeneity in terms of study-level variables. Random-effects meta-regression analyses quantified the association of certain sociodemographic characteristics [age, sex (i.e. male-tofemale ratio in cases / male-to-female ratio in controls), the region of the World where the study has been performed, which has been dichotomized into Europe and North America as only 1 study was conducted elsewhere], and diagnosis (i.e., mood disorders versus schizophrenia, mood disorders versus other psychiatric diagnoses (i.e. other than mood or schizophrenia), and schizophrenia versus other psychiatric diagnoses) of studies' participants with study-level effects. Studies were weighted by the inverse of the sum of the within-and between-study variance. Analyses were performed on complete cases.

We also performed post-hoc power analyses to determine whether potential nonsignificant associations might have resulted from limited statistical power [START_REF] Valentine | How Many Studies Do You Need?: A Primer on Statistical Power for Meta-Analysis[END_REF]. To examine the robustness of our results, we repeated those analyses while including only studies of good quality according to the Newcastle-Ottawa Scale. Because psychotropic medications are known to influence CSF concentrations of monoamine metabolites (Scheepers, Wied, Westenberg, & Kahn, 2001; Sheline, Bardgett, & Csernansky, 1997), additional sensitivity analyses were done while excluding studies in which lumbar puncture was performed within two weeks of the administration of any psychotropic medication and those that did not specify the date of last use of psychotropic medications. These analyses were performed using the function "metareg" of R package "meta" [START_REF] Schwartze | meta: An R Package for Meta-Analysis[END_REF].

Statistical analyses were performed using the R package "meta" [START_REF] Schwartze | meta: An R Package for Meta-Analysis[END_REF] and statistical significance was evaluated using a two-sided design with α set at 0.05.

Results

2.1.

Characteristics of studies included in the meta-analysis

Thirty case control studies were included in the present analysis ( 1b).

Violent suicide attempts

Ten studies (n=595) compared CSF 5-HIAA levels between 126 violent suicide attempters and 469 controls (mean age=38.4 years (SD=6.4), sex ratio=0.8) (eFigure 3a). Of these 10 studies, 5 found that violent suicide attempt was significantly associated with lower CSF 5-HIAA levels and 5 reported a non-significant association. Results from the randomeffects meta-analysis showed that CSF 5-HIAA levels were significantly lower in violent suicide attempters than in controls [SMD= -0.90 (95% CI: -1.47; -0.32), p=0.0024, 2b).

Violent suicide attempts

Seven studies (n=416) compared CSF HVA levels between 108 violent suicide attempters and 308 controls (mean age=36.6 (SD=6.9), sex ratio=0.7) (eFigure 6a). Of these studies, none reported a significant association between CSF HVA levels and violent suicide attempts. Results from the random-effects meta-analysis showed a negative trend towards significance between CSF HVA levels and violent suicide attempt [SMD = -0.16 (95% CI: -0.39 to 0.07), p=0.1746, Tau 2 =0.00, I 2 =0.00]. Sensitivity analysis restricted to the 6 studies of good or fair quality (n=309, mean age=36.6 years (SD=6.9), sex ratio=0.7) yielded similar results [SMD= -0.23 (95% CI: -0.48 to 0.03), p=0.0866, Tau 2 =0.00, I 2 =0.00] (eFigure 6b).

Sensitivity analysis restricted to the 15 studies comparing CSF HVA levels between 336 nonviolent suicide attempters and 553 controls (mean age=43.1 years (SD=9.1), sex ratio=0.71)

showed that CSF HVA levels were significantly lower in non-violent suicide attempters than in controls [SMD= -0.67 (95% CI: -1.06; -0.27), p=0.0009, Tau 2 =0.47, I 2 =0.83] (eFigure 7).

Finally, additional meta-regression analysis indicated that CSF levels of HVA did not significantly differ between individuals who did a violent versus a non-violent suicide attempt (SMD=0.5156; 95%CI: -0.0855; 1.1166, p-value=0.0927).

MHPG

Full sample

Twelve studies reported a non-significant association, 2 found that suicide attempt was significantly associated with lower CSF MHPG levels and 1 found an association in the opposite direction. Results from the random-effects meta-analysis of these studies did not show an association between CSF MHPG levels and suicide attempt [SMD= -0.33 (95% CI: -0.77 to 0.11), p=0.1407, Tau 2 =0.61, I 2 =0.89] (Figure 3a). whereas we did not find a significant association between CSF MHPG levels and suicide attempt among participants with mood disorders [SMD= -0.28 (95% CI: -0.68 to 0.12), p=0.1698] or other psychiatric diagnoses [SMD= -0.33 (95% CI: -1.04 to 0.39), p=0.3725].

Sensitivity analysis restricted to the 11 studies of good or fair quality (n=753, mean age=37.5 years (SD=6.4), sex ratio=0.8) yielded similar results [SMD= -0.31 (95% CI: -0.86; 0.24), p= 0.2621, Tau 2 =0.77, I 2 =0.91] (Figure 3b).

Violent suicide attempts

Three studies (n=160, sex ratio=1.1, mean age=39.5 years (SD=0.6)) compared CSF MHPG levels between 41 violent suicide attempters and 119 controls (eFigure 8). None of these studies reported a significant association between CSF MHPG levels and violent suicide attempts. Results from the random-effects meta-analysis of these studies showed a nonsignificant association between CSF MHPG levels and violent suicide attempts [SMD= -0.20 (95% CI: -0.79 to 0.38), p-value=0.4955, Tau 2 =0.15, I 2 =0.58] (eFigure 8). Additional metaregression analysis indicated that CSF levels of MHPG did not significantly differ between individuals who did a violent versus a non-violent suicide attempt (SMD= 0.3337; 95%CI: -0.6840; 1.3513, p-value=0.5205).

Publication bias

Funnel plots of studies comparing CSF levels of 5-HIAA, HVA and MHPG between suicide attempters and controls highlighted relatively symmetrical plots, with few studies with elevated standard errors for 5-HIAA and HVA studies, and none for MHPG studies, suggesting a low likelihood of publication estimates bias (Figure 4). In addition, sensitivity analyses excluding study outliers did not modify the significance of our results.

Post-hoc power analyses

Statistical power for detecting a small effect size of SMD=0.2 was >80% for randomeffects meta-analysis models of 5-HIAA studies (i.e., 86.1%) and HVA studies (i.e., 83.0%).

However, it was lower for MHPG studies (i.e., 60.2%), for which our meta-analysis had 80%

power to detect a SMD of 0.3. For meta-analysis models performed in individuals who made violent suicide attempts, statistical power for detecting a small effect size of SMD=0.2 was limited, at 27.4% for 5-HIAA studies, 23.2% for HVA studies and 13.6% for MHPG studies.

Discussion

In the first meta-analysis to our knowledge of the relationship between CSF levels of serotonin, dopamine and norepinephrine metabolites and suicide attempt, we found that suicide attempt was significantly associated with diminished CSF levels of 5-HIAA and HVA. These results suggest that lower serotonin and dopamine levels in the central nervous system may be implicated in suicide attempts. The magnitude of these associations was small to medium, with effect sizes of 0.43 for CSF 5-HIAA and 0.45 for CSF HVA.

While the finding of lower CSF levels of 5-HIAA among suicide attempters converges with prior research (Asberg & Träskman, 1981;[START_REF] Lester | The concentration of neurotransmitter metabolites in the cerebrospinal fluid of suicidal individuals: a meta-analysis[END_REF], we also found a significant association between diminished CSF levels of HVA and suicide attempt, contrasting with a previous literature review [START_REF] Lester | The concentration of neurotransmitter metabolites in the cerebrospinal fluid of suicidal individuals: a meta-analysis[END_REF]. The use of meta-analytic methods, which combine estimates from multiple studies to increase statistical power, permitted detection of an effect that had been previously concealed by the examination of individual studies. Associations of diminished CSF levels of 5-HIAA and HVA with violent and non-violent suicide attempt were all significant, except for that between CSF HVA levels and violent suicide attempt, which showed only a trend towards significance, possibly because of limited statistical power.

These results suggest that differences in CSF levels of 5-HIAA and HVA may not be specific to violent suicide attempts but rather associated with suicide attempt regardless of the method used. Finally, in accord with a prior literature review [START_REF] Lester | The concentration of neurotransmitter metabolites in the cerebrospinal fluid of suicidal individuals: a meta-analysis[END_REF], but contrasting with some findings suggesting lower norepinephrine function in suicide risk (Oquendo, Sullivan, et al., 2014), we did not find a significant association between CSF MHPG levels and suicide attempt. Because our meta-analysis had 80% power to detect a SMD of at least 0.3 for CSF MHPG levels, our study suggests that any association between CSF MHPG levels and suicide attempt, if it exists, is likely to be small.

The finding of a possible implication of serotonin in suicide attempts is consistent with prior research. Prior genetic studies, although not all [START_REF] Brezo | The genetics of suicide: a critical review of molecular studies[END_REF][START_REF] Lutz | Neuropathology of suicide: recent findings and future directions[END_REF], have found that serotonin transporter genetic polymorphisms [START_REF] Clayden | The association of attempted suicide with genetic variants in the SLC6A4 and TPH genes depends on the definition of suicidal behavior: a systematic review and meta-analysis[END_REF][START_REF] Daray | Serotonin transporter gene polymorphism as a predictor of short-term risk of suicide reattempts[END_REF][START_REF] De Medeiros Alves | Genetic Polymorphisms Might Predict Suicide Attempts in Mental Disorder Patients: A Systematic Review And Meta-Analysis[END_REF][START_REF] Lee | Possible Association between Serotonin Transporter Gene Polymorphism and Suicide Behavior in Major Depressive Disorder[END_REF][START_REF] Li | Meta-analysis supports association between serotonin transporter (5-HTT) and suicidal behavior[END_REF], tryptophan hydroxylase (TPH) genetic polymorphism [START_REF] Bellivier | Association between the TPH gene A218C polymorphism and suicidal behavior: a meta-analysis[END_REF][START_REF] Clayden | The association of attempted suicide with genetic variants in the SLC6A4 and TPH genes depends on the definition of suicidal behavior: a systematic review and meta-analysis[END_REF][START_REF] Pawlak | Suicide behavior as a quantitative trait and its genetic background[END_REF] and serotonin receptor genetic polymorphisms [START_REF] Pawlak | Suicide behavior as a quantitative trait and its genetic background[END_REF] may influence suicide risk. Moreover, prior studies suggest that individuals with a lifetime history of suicide attempt who have low CSF 5-HIAA levels may be at greater risk for reattempting or completing suicide [START_REF] Nordström | CSF 5-HIAA predicts suicide risk after attempted suicide[END_REF][START_REF] Roy | Cerebrospinal fluid monoamine metabolites and suicidal behavior in depressed patients. A 5-year follow-up study[END_REF][START_REF] Träskman | Monoamine metabolites in CSF and suicidal behavior[END_REF]. Prior research also supports the potential implication of impaired serotoninergic system in violent behaviors [START_REF] Fanelli | The influence of the serotonin transporter gene 5-HTTLPR polymorphism on suicidal behaviors: a meta-analysis[END_REF][START_REF] Gerra | Association between lowactivity serotonin transporter genotype and heroin dependence: behavioral and personality correlates[END_REF][START_REF] Hallikainen | Association between low activity serotonin transporter promoter genotype and early onset alcoholism with habitual impulsive violent behavior[END_REF] and the lower plasma 5-HIAA and platelet 5-HT concentrations in suicide attempters compared to healthy controls [START_REF] Spreux-Varoquaux | Differential abnormalities in plasma 5-HIAA and platelet serotonin concentrations in violent suicide attempters: relationships with impulsivity and depression[END_REF]. Finally, our results are also in line with studies indicating that use of SSRIs may be associated with a reduced risk of suicide in adults with major depressive disorder [START_REF] Barbui | Selective serotonin reuptake inhibitors and risk of suicide: a systematic review of observational studies[END_REF].

Importantly, we found that the dopaminergic system may play a substantial role in suicide attempt, in line with several findings. A prior study suggested that patients with major depression who had attempted suicide may have a greater reduction in urinary metabolites of dopamine than depressed non-attempter individuals or healthy controls [START_REF] Roy | Marked reduction in indexes of dopamine metabolism among patients with depression who attempt suicide[END_REF]. [START_REF] Fitzgerald | Dysregulation of Striatal Dopamine Receptor Binding in Suicide[END_REF] also found in a post-mortem analysis of brain tissue of 17 individuals who died by suicide a loss of correlation between D1 and D2 receptor binding, suggesting a dysregulation of dopamine receptors. [START_REF] Bowden | Reduced dopamine turnover in the basal ganglia of depressed suicides[END_REF] previously showed a reduction of dihydroxyphenylacetic acid, a dopamine metabolite, in the caudate, putamen and nucleus accumbens in a postmortem study of depressed individuals who died by suicide.

Finally, our results are also consistent with studies indicating that use of ketamine, which may increase dopamine levels in the cortex, the striatum and the nucleus accumbens [START_REF] Kokkinou | The effects of ketamine on dopaminergic function: meta-analysis and review of the implications for neuropsychiatric disorders[END_REF] through a downstream effect from its glutamatergic effects, may lead to short-term reductions in suicidal ideation [START_REF] Murrough | Ketamine for rapid reduction of suicidal ideation: a randomized controlled trial[END_REF][START_REF] Wilkinson | The Effect of a Single Dose of Intravenous Ketamine on Suicidal Ideation: A Systematic Review and Individual Participant Data Meta-Analysis[END_REF].

Our study has important implications. First, our results suggest that both the serotoninergic pathway and the dopaminergic pathway may be implicated in suicide attempts.

Specifically, dopamine has received relatively little attention as a potential contributor to suicide risk, especially recently (Oquendo, Sullivan, et al., 2014). However, dopamine may play a substantial role in suicidal behavior due to its stimulatory role on the activation of the HPA axis in response to a severe stressor and involvement in the maintenance of post-stress activation of the HPA axis [START_REF] Belda | Dopamine D1 and D2 dopamine receptors regulate immobilization stress-induced activation of the hypothalamus-pituitary-adrenal axis[END_REF], which evinces multilevel dysfunction associated with increased suicide risk (Oquendo, Sullivan, et al., 2014). Furthermore, serotonin and dopamine interact with each other [START_REF] Daw | Opponent interactions between serotonin and dopamine[END_REF][START_REF] Di Giovanni | Role of serotonin in central dopamine dysfunction[END_REF], 2008;[START_REF] Esposito | Serotonin-dopamine interaction as a focus of novel antidepressant drugs[END_REF][START_REF] Olvera-Cortés | Serotonin/dopamine interaction in learning[END_REF] and may be both involved in the individual's ability to cope with suicidal thoughts [START_REF] Ryding | The role of dopamine and serotonin in suicidal behaviour and aggression[END_REF]. Serotonin-dopamine interaction may act as a moderator of decision making [START_REF] Rogers | The roles of dopamine and serotonin in decision making: evidence from pharmacological experiments in humans[END_REF], commonly observed to be impaired during suicidal crises [START_REF] Szanto | Decision-making competence and attempted suicide[END_REF]. Interactions between serotonin and dopamine systems in the prefrontal cortex may also underlie the link between impulsive aggression and suicidal behavior [START_REF] Seo | Role of Serotonin and Dopamine System Interactions in the Neurobiology of Impulsive Aggression and its Comorbidity with other Clinical Disorders[END_REF]. Whether serotonergic and dopaminergic abnormalities observed in individuals who attempted suicide or have died by suicide are independent of each other or stress response abnormalities remains a crucial unanswered question. Taken together, these findings suggest that a silo approach to biomarkers should be phased out in favor of the study of multiple systems in parallel and in the same populations to progress in the identification of the components independently associated with suicide risk, with the goal of identifying new treatment targets and improving suicide risk prediction.

Finally, our results suggest that differences in CSF levels of metabolites of serotonin and dopamine may not be specific to violent suicide attempts, but rather associated with suicide attempt regardless of the method used, suggesting that these biomarkers might be useful in predicting all types of suicide attempt. However, since there are likely different biological pathways to suicide attempt, future work on the neurobiology of suicide risk would benefit in using finer-grained phenotyping of suicidal behavior, based for example on patterns of suicidal thinking and stress responsivity [START_REF] Bernanke | Toward fine-grained phenotyping of suicidal behavior: the role of suicidal subtypes[END_REF], to examine whether each potential biomarker candidate is specific to one or different suicidal behavior phenotypes.

Our study has several limitations. First, most studies included in our meta-analysis relied on small sample sizes (i.e., less than 30 participants). However, our meta-analysis models examining the relationship between suicide attempt and CSF levels of 5-HIAA, HVA and MHPG were appropriately powered (i.e. ≥80%) to detect a small effect size of SMD=0.3.

Second, an important issue is whether the biochemical abnormalities observed in our study are independent of psychiatric diagnosis. Because only a minority of studies included participants with the same psychiatric diagnosis, our study was insufficiently powered to perform analyses stratified by diagnosis (e.g. only one study measured CSF HVA levels among suicide attempters with schizophrenia). Although we did not find any significant moderating effect of the diagnosis of participants on the association between suicide attempt and CSF levels of 5-HIAA and HVA, future studies are needed to examine this issue. Third, our analyses relied on a limited number of studies of good quality. However, our significant results held in sensitivity analyses restricted to good or fair quality. Fourth, in all studies except one [START_REF] Chatzittofis | CSF 5-HIAA, cortisol and DHEAS levels in suicide attempters[END_REF], individuals were prescribed psychotropic medications, which are known to influence CSF concentrations of monoamine metabolites (Scheepers et al., 2001;[START_REF] Sheline | Correlated reductions in cerebrospinal fluid 5-HIAA and MHPG concentrations after treatment with selective serotonin reuptake inhibitors[END_REF], and date of last use of psychotropic medications varied substantially across studies. However, the exclusion of studies in which lumbar puncture was performed within two weeks of the administration of any psychotropic medication and those that did not specify when participants used for the last time psychotropic medications, did not alter the significance of our results. Fifth, measures of association do not necessarily imply causal association (Le [START_REF] Strat | Correlation is no causation: gymnasium proliferation and the risk of obesity[END_REF]. Finally, we cannot rule out that some significant results, such as the moderating effects of participants' characteristics, might result from type I error inflation due to multiple testing.

Research implications

Whether serotonergic and dopaminergic abnormalities in individuals who attempted suicide are independent of each other or stress response abnormalities is a crucial unanswered question. Our findings suggest that a silo approach to biomarkers should be phased out in favor of the study of multiple systems in parallel and in the same populations to progress in the identification of the components independently associated with suicide risk, with the goal of identifying new treatment targets and improving suicide risk prediction.

Figure 1 .

 1 Figure 1. Forest plot comparing CSF 5-HIAA levels between suicide attempters and non-attempters using a random-effects meta-analysis model.

  suicide attempt type based on the suicide method used [categorized as violent (i.e., suicide attempt by hanging, drowning, gunshot wound, gas poisoning, or multiple deep cuts), non-violent (i.e., oral drug overdoses or single wrist cuts) and nonspecified], scores on suicide scales, and CSF levels of 5-HIAA, HVA and MHPG.

	[Suicide attempt]. Studies cited in the reference list of each eligible systematic review were Using a standardized form, we systematically recorded from each eligible article the
	also examined and included in this review if they fulfilled eligibility criteria. following data: publication date, authors' names, study quality according to the Newcastle-
	Ottawa Scale (Williams, Gierisch, McDuffie, Strauss, & Nagi, 2011), sample size,
	1.4. participants' age and sex and their main psychiatric diagnosis, medication taken before the Eligibility criteria
	We restricted our search to case control and cohort studies published between January lumbar puncture,
	1980 and December 2019, including both women and men without any age limitation. Studies
	that did not include a control group or did not measure, among attempters, CSF levels of
	monoamines after the suicide attempt were excluded. Although studies in which lumbar
	puncture was performed within two weeks of the administration of any psychotropic
	medication or that did not specify the date of last use of psychotropic medications were
	included in the main analyses, sensitivity analyses excluding them were also performed to
	examine the robustness of the findings. If full text articles were not available or if there were
	missing values of CSF levels of metabolites, authors were systematically contacted by
	electronic mail.
	1.5.	Data collection process
	Two authors (JFC and HC) conducted the search. They first examined all titles and
	abstracts independently. Then, they independently extracted the data using a standard form as
	described below. Disagreements in study eligibility or data extraction were resolved by
	consensus. The flowchart is given in eFigure 1. Of the 106 articles meeting eligibility criteria,
	The search was conducted on March 2019 using PUBMED, WEB OF 74 records were excluded because the studies did not include any measure of CSF levels of
	SCIENCE, EMBASE and SCOPUS. Potential gray literature was identified searching in monoamines after the suicide attempt (n=61) or a control group (n=13), and 30 articles
	OPENGREY database. We used the following key words: [Suicide attempt] AND [CSF] matched eligibility criteria and were included in qualitative and quantitative synthesis.
	AND ([5-HIAA] OR [HVA] OR [MHPG]). To ensure a complete search, we also used the
	following key words: [Suicidal] AND [CSF] AND ([5-HIAA] OR [HVA] OR [MHPG]) NOT 1.6. Data collected

Table 1

 1

	), involving

Of these 30 studies, 10 studies (33.3%) included participants with mood disorders (9 studies comprised individuals with unipolar depression and 1 study included individuals with bipolar depression), 2 studies (6.7%) focused on individuals with schizophrenia, 4 studies (13.3%) comprised participants with other psychiatric diagnoses (e.g. personality disorders), and 14 (46.7%) included mixed populations (i.e. individuals with different diagnoses).

Of 29 studies, 12 found that suicide attempt was significantly associated with lower CSF 5-HIAA levels, 2 indicated a significant association in the opposite direction, and 15 reported a non-significant association. (Figure

1a

). This heterogeneity could not be explained


Julie Oniszczuk

Imane El Joukhar

Fayida Ali Said

Sabrina Belbekhouche
email: belbekhouche@icmpe.cnrs.fr

Glucose-and H2O2-dual responsives drug delivery particle based on the boronic acid chemistry

Keywords: particle, glucose-responsive, polysaccharides, layer-by-layer, phenylboronic acid chemistry, hydrogen peroxide-responsive

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Stimuli-responsive particles have still attracted scientific interest as for diagnosis, sensing, or drug delivery. [START_REF] Deshmukh | Stimuli-sensitive layer-by-layer (LbL) self-assembly systems: targeting and biosensory applications[END_REF][START_REF] Sukhorukov | Intelligent micro-and nanocapsules[END_REF] This undeniable success can be rationalized by the possibility to tune the resulting property by changing the environnmental condition as pH or ionic strenght. This allows a control and a specific release of the drug.

Several methods have been successfuly developed to produce particles. One may cite pH induced micellization of a grafted polymer, [START_REF] Dou | pH-dependent self-assembly: micellization and micelle-hollow-sphere transition of cellulose-based copolymers[END_REF] emulsion polymerization, crosslinking of micelles, self-assembly and layer-by-layer (LbL) deposition of polyelectrolytes onto a sacrificial template. [START_REF] Gittins | Tailoring the polyelectrolyte coating of metal nanoparticles[END_REF][START_REF] Gittins | Multilayered polymer nanocapsules derived from gold nanoparticle templates[END_REF] Herein, we focus on the LbL process [START_REF] Borges | Molecular interactions driving the layer-by-layer assembly of multilayers[END_REF] as it is proved to be efficient for the chemical modification of a plethora of substrates. [START_REF] Shimomura | Bottom-up strategy of materials fabrication: a new trend in nanotechnology of soft materials[END_REF][START_REF] Szarpak | Designing hyaluronic acid-based layer-by-layer capsules as a carrier for intracellular drug delivery[END_REF] This approach allows developing particle with well-tuned property. The overall strategy combines particles templating and self-assembly methods. More precisely, it lies on the alternative deposition of charged polyelectrolyte to form a multilayer film on particles used as templates. The polyelectrolyte excess could easily be removed by a simple washing step before the deposition of the next layer. This strategy enables to easily and efficiently control the size and the film thickness of the as-obtained material. Herein, we focus on calcium carbonate microparticle cores because it presents several advantages as biocompatibility, low cost, mild decomposition and its intrinsic porosity which can be beneficial for providing high drugs loading. [START_REF] Volodkin | Protein encapsulation via porous CaCO3 microparticles templating[END_REF][START_REF] Volodkin | Matrix polyelectrolyte microcapsules: new system for macromolecule encapsulation[END_REF][START_REF] Sukhorukov | Porous calcium carbonate microparticles as templates for encapsulation of bioactive compounds[END_REF][START_REF] Stein | Real-time assessment of spatial and temporal coupled catalysis within polyelectrolyte microcapsules containing coimmobilized glucose oxidase and peroxidase[END_REF][START_REF] Petrov | Protein-calcium carbonate coprecipitation: a tool for protein encapsulation[END_REF] Moreover, the CaCO3 micro-sized particles are easily obtained, for instance by mixing calcium chloride and sodium carbonate. [START_REF] Zhao | Assembly of multilayer microcapsules on CaCO3 particles from biocompatible polysaccharides[END_REF] For promising applications in the drug-delivery field, the most fundamental requirement is the use of biocompatible and nontoxic polymer. In this aims, polysaccharides reveal to be very competitive candidates. Their hydroxyl-or amine-rich chemical structure enables to bring new interesting functionality as stimuli-responsiveness. They present also the interest of being pH-dependent charged which is interesting for LbL assembly. We can cite the alternative deposition of polysaccharides to form multilayers walls on microparticles, as dextran sulfate/protamine. [START_REF] Balabushevich | Loading the multilayer dextran sulfate/protamine microsized capsules with peroxidase[END_REF] Bringing stimuli-responsiveness to drug system may open new route to trigger the drug release. Numerous studies reported on responsive particles to a broad range of external stimuli such as pH, ionic strength or even temperature. [START_REF] Geest | Ultrasound-triggered release from multilayered capsules[END_REF][START_REF] Sukhishvili | Responsive polymer films and capsules via layer-by-layer assembly[END_REF][START_REF] Geest | Release mechanisms for polyelectrolyte capsules[END_REF] Although glucose has a crucial biological function in living systems, only few groups have being working on this chemical. [START_REF] Geest | Glucose-responsive polyelectrolyte capsules[END_REF][START_REF] Belbekhouche | Glucose-sensitive polyelectrolyte microcapsules based on (alginate/chitosan) pair[END_REF] For instance, cancer cells contain a very high glucose concentration in comparison to normal cells [START_REF] Aykin-Burns | Increased levels of superoxide and H2O2 mediate the differential susceptibility of cancer cells versus normal cells to glucose deprivation[END_REF] or we may simply think about diabetes treatment. The design of glucoseresponsive systems is then still a challenging and interesting task. [START_REF] Wu | Organization of glucose-responsive systems and their properties[END_REF] The phenylboronic acids chemistry could be successfully implied for developing glucose-responsive system. [START_REF] Kazunori | Sensitive glucose-induced change of the lower critical solution temperature of poly[N,N-(dimethylacrylamide)-co-3-(acrylamido)-phenylboronic acid] in physiological saline[END_REF] Indeed, this functional group is recognized to form covalent bonds with polydiols as polysaccharides. [START_REF] Geest | Glucose-responsive polyelectrolyte capsules[END_REF] The complexation occurs mainly in a pH range above the pKa of the boronic moieties (pKa~9). [START_REF] Geest | Glucose-responsive polyelectrolyte capsules[END_REF][START_REF] Wu | Organization of glucose-responsive systems and their properties[END_REF][START_REF] Brooks | Synthesis and applications of boronic acid-containing polymers: from materials to medicine[END_REF] Levy et al. have prepared glucose-sensitive capsules, stabilized by the formation of ester bonds between mannan and phenylboronic acid moieties grafted onto a polyacid. [START_REF] Levy | Polymer microcapsules with carbohydratesensitive properties[END_REF] Glucose-sensitive capsules have been produced by De Geest et al. by the layer-by-layer assembly of a copolymer of dimethylaminoethylacrylate and acrylamidophenylboronic acid and poly(styrene sulfonate). They demonstrated glucoseinduced change in the electrostatic interactions between the polyanion and the phenylboronic acid-containing polycation. [START_REF] Geest | Glucose-responsive polyelectrolyte capsules[END_REF] Belbekhouche et al. reported assemblies based on alginate derivative containing phenylboronic acid side group and chitosan [START_REF] Belbekhouche | Glucose-sensitive polyelectrolyte microcapsules based on (alginate/chitosan) pair[END_REF] or poly(vinylpyrrolidone). [START_REF] Belbekhouche | Glucose-sensitive capsules based on hydrogen-bonded (polyvinylpyrrolidone / phenylboronic -modified alginate) system[END_REF] Furthermore, it is important to note that phenylboronic acid chemistry may also be used for developing hydrogen peroxide responsive system. This could be interesting to develop reactive oxygen species detection systems. [START_REF] Van De Bittner | In vivo imaging of hydrogen peroxide production in a murine tumor model with a chemoselective bioluminescent reporter[END_REF][START_REF] Srikun | Organelle-targetable fluorescent probes for imaging hydrogen peroxide in living cells via SNAP-Tag protein labeling[END_REF][START_REF] Lee | In vivo imaging of hydrogen peroxide with chemiluminescent nanoparticles[END_REF][START_REF] Chang | A selective, cell-permeable optical probe for hydrogen peroxide in living cells[END_REF][START_REF] Yu | Physiologically relevant oxidative degradation of oligo(proline) cross-linked polymeric scaffolds[END_REF] Such systems are implied in several physiological processes such as apoptosis or proliferation Excess ROS production is often correlated to diseases such as tumors and inflammatory pathologies. [START_REF] Song | Oxidation-responsive poly(amino ester)s containing arylboronic ester and self-immolative motif: synthesis and degradation study[END_REF][START_REF] Song | Oxidation-accelerated hydrolysis of the ortho ester-containing acid-labile polymers[END_REF] Cellular components can be damaged out of the balance due to ROS, e.g. hydrogen peroxide, superoxide or even hydroxide radicals. [START_REF] Geronikaki | Antioxidants and inflammatory disease: synthetic and natural antioxidants with anti-inflammatory activity[END_REF] Thus, some ROS can be considered as a key to cell signalling [START_REF] Finkel | Oxidant signals and oxidative stress[END_REF] for instance.

In this paper, the boronic acid chemistry is used to develop a new smart responsive material presenting a dual response to glucose and hydrogen peroxide. Our work consists in the building-up and the characterization of multilayer films on flat substrate based on either ((diethylaminoethyl-dextran hydrochloride/alginate derivative) polyelectrolytes pair for hydrogen peroxide-response or (polylysine/alginate derivative) polyelectrolytes pair for glucose-response. This strategy is then extended to the fabrication of stimuli-responsive microparticles which can be loaded with a probe and further be triggered released according to glucose or hydrogen peroxide.

Experimental parts.

Chemicals:

These chemicals were purchased diethylaminoethyl-dextran hydrochloride (Sigma-aldrich), polylysine (Sigma-aldrich), alginate with mannuronic / guluronic ratio 1.4 (Cargill (Baupte, France, Table 1), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Sigma-aldrich, > 98.5%), N-hydroxysuccinimide (NHS, Sigma-aldrich, > 98%), mercaptosulfonate (Sigma-aldrich, > 89.5%), calcium chloride (CaCl2, rectapur, 99%), sodium nitrate (rectapur, 99%), 4-aminophenylboronic acid hydrochloride (Alfa Aesar, 97 %), acetic acid (Alfa Aesar,+99%), sodium acetate (Alfa Aesar, 99%) and ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich, 98%). [36]).

Introduction of phenylboronic group on polyanionic polysaccharide

Phenylboronic-modified alginate derivative was obtained by adding an amine-containing phenylboronic groups onto the carboxylic acid groups of the alginic acid via the formation of amide linkage through the EDC/NHS coupling reagent. The used EDC/NHS ratio is the optimized condition for this alginate modification. More precisely, 500 mg of alginate was dissolved in 100 mL of aqueous solution (potassium phosphate buffer (0.1 M, pH 7.2))

overnight. Further, a mixture of 10 mg EDC and 10 mg NHS was added on the solution of alginate and stirred for 45 min. A solution of 4-aminophenylboronic acid (100 mg in 1 mL of methanol) was then dropwise added to the solution of alginate (mixing 1 day). A dialysis step was implied to purify the product (6 kDa cut off). The product was obtained via freeze-drying (yield 90%).

[20]

Monitoring of the multilayer buildup and stimuli-response with QCM measurement

After cleaning (rinsing with water and ethanol, treatment in a UV/ozone chamber for 20 min and additional flushing with ethanol), the gold coated QCM Sensor Crystal was pretreated with sulfonated thiol (5 g.L -1 in water). 1 mg.mL -1 of polyelectrolytes was injected into the cell with a flow rate of 500 μL.min -1 . The following polyelectrolytes pair were used ((diethylaminoethyl-dextran hydrochloride/alginate derivative) and (polylysine/alginate derivative).

After the multilayer built-up, the glucose-or hydrogen hydroxide-response was investigated by flushing the resulting film by solution containing this chemical at different concentration.

Fabrication of glucose-and H2O2-responsive[ particles.

CaCO3 microparticles were fabricated according to Belbekhouche et al. [START_REF] Belbekhouche | Glucose-sensitive capsules based on hydrogen-bonded (polyvinylpyrrolidone / phenylboronic -modified alginate) system[END_REF] Multilayer buildup was done via the LbL technique from 1 mg.mL -1 of each polyelectrolytes (namely diethylaminoethyl-dextran hydrochlorid, polylysine and alginate derivative). After each adsorption process, the excess of polyelectrolyte was removed via two washing steps. A centrifugation process (5 min, 10 000 rpm) was implied after each adsorption process. The particles were washed with water two times during 5 min.

Probe-loaded hybrid microcapsules.

10 mg.mL -1 of a suspension of microparticles were added into the solution containing a probe (0.5 mg rhodamine b in 5 mL of deionized water). After 12 h, the loaded-CaCO3 microparticles were centrifuged and washed to remove free rhodamine derivative. The LbL process was done as explained in section 2.4. The probe loading and the entrapment efficiency were calculated by using equations 1 and 2.

probe loading = X 100%

(1)

entrapment efficiency = * X 100% (2)
The release behavior was investigated by introducing in a dialysis bag the probe-loaded microcapsules and then immersing it in glucose or hydrogen peroxide solution. The probe concentration was determined by absorbance measurements at 550 nm.

Methods

Fourier transform infrared spectra were recorded on a Bruker Tensor 27 instrument equipped with a Digi TectDLaTGS detector.

Results and discussion

As shown in figures 1 and 2 A, our overall strategy consists in the layer-by-layer assembly of charged biopolymer, namely diethylaminoethyl-dextran hydrochloride or polylysine (polycation) and an alginate derivate (polyanion). For providing glucose-and hydrogen peroxyde sensitivity, phenylboronic acid moiety is incorporated inside the alginate. Subjected to these stimuli (figure 2B), the as-obtained multilayer is supposed to totally be removed from the substrate. Then, this strategy is transposed to spherical substrate leading to stimuliresponsive particle. hydrochloride. [START_REF] Kitano | Glucose-responsive complex formation between poly (vinyl alcohol) and poly (N-vinyl-2-pyrrolidone) with pendent phenylboronic acid moieties[END_REF][START_REF] Kitano | A novel drug delivery system utilizing a glucose responsive polymer complex between poly (vinyl alcohol) and poly (N-vinyl-2-pyrrolidone) with a phenylboronic acid moiety[END_REF] After a purification process (dialysis and freeze drying), a white chemical is obtained and labeled alg-bor (Figure 3A). The 1 H NMR spectra show the presence of both chemical shift of the protons of the alginate chain (from 3 to 4 ppm) and phenyl groups (from 6.7 to 7.2 ppm) (Figure 3A). From this technique, the presence of the aromatic group is qualitatively checked and allows estimating a degree of substitution which is found to be around 20%. [START_REF] Belbekhouche | Glucose-sensitive polyelectrolyte microcapsules based on (alginate/chitosan) pair[END_REF] From FTIR spectra (Figure 3B), we show the presence of two peaks at 867 cm -1 and 945 cm -1 which are assigned respectively to the guluronate and mannuronate residues of alginate and alg-bor samples and a band around 3300 cm -1 attributed to the -OH A)

Figure 3. A) 1 H NMR spectrum (D2O) of (A) alginate and (B) phenylboronic acid modified alginate; B) FTIR spectra of alginate and phenylboronic-modified alginate derivative.

Multilayers self-assembly on flat gold surfaces

The Lbl process is performed in an aqueous solution between obtain alginate derivative (presence of carboxylate groups) and diethylaminoethyl-dextran hydrochloride or polylysine (presence of ammonium group) as oppositely charged polyelectrolytes. Electrostatic attractions are then implied as the driving force during the multilayer assembly. First, the gold substrate is pretreated with a sulfonated thiol derivative leading to high density of negative surface charges. This enables to adsorb the polycation as the first layer, and then this is followed by the adsorption of the alginate derivative (polyanion). This step is repeated to form the multilayer build-up.

To demonstrate the efficiency of this method, the LbL assembly is investigated by wettability and then in real time by QCM experiments on a flat surface.

Static contact angle measurement are performed on the flat gold pretreated substrate before and after the multilayer build-up with (alg-bor/diethylaminoethyl-dextran hydrochloride)5 or (alg-bor/polylysine)5 to show qualitatively the elaboration of the multilayer film onto the substrate. The contact angle values decreased from 70°±2° to 20°±3° upon the coating independently of the polyelectrolytes pair. This result could be correlated to the hydrophilic nature of these polysaccharides (Figure 4). Interestingly, the flushing of this modified substrate with a solution of glucose or hydrogen peroxide results in a contact angle values above 70°±2° (i.e. removal of the multilayer from the surface). Note that flushing this Then, the LbL assembly was monitored by QCM experiments to (i) demonstrate the stepwise adsorption of polycation/ phenylboronic acid-containing alginate polyelectrolytes pair, (ii) investigate the adsorption kinetics and (iii) evidence the impact of glucose or hydrogen peroxide on the resulting multilayer film. In aqueous solution, the implied build-up procedure ensures both the presence of oppositely charged carboxylate and ammonium groups and, that boronic acid function remained uncharged during the layer-by-layer buildup (pKa of phenylboric acid 8.9). [START_REF] Westmark | Selective monosaccharide transport through lipid bilayers using boronic acid carriers[END_REF] Upon addition of the polyelectrolytes, a decrease of the frequency signal is seen, showing that mass was being added onto the surface (Figure 5). Seven layers of the polyelectrolytes were then successfully deposited onto the quartz substrates. After adsorption, the final frequency shift is around -150 Hz and -200 Hz for (phenylboronic acid modified alginate/ polylysine)n and (b) (phenylboronic acid modified alginate/ diethylaminoethyl-dextran hydrochloride) n respectively.

Figure 5. Evolution of the frequency shift values vs time during the successive polyelectrolytes addition, (a) (phenylboronic acid modified alginate/ polylysine)n and (b) (phenylboronic acid modified alginate/ diethylaminoethyl-dextran hydrochloride) n (standard deviation was always lower than 5%).

To demonstrate the glucose and/or hydrogen peroxide sensitivity, various concentrations of these chemicals were flushed on the resulting multilayer film (Figure 6). As discussed before, the final equilibrium frequency shift values after the LbL build-up of the dextran derivative/alg-bor films were -200 Hz. These values can then be used as references to show either the glucose or hydrogen peroxide response of the films. Indeed, the total destabilization of the films should involve a variation of the equilibrium frequency shift of about +200 Hz.

The same hypothesis could be used if implying (phenylboronic acid modified alginate/ polylysine)n as polyelectrolytes pair.

As observed from figure 6A and 6B, the frequency shift parameter increases upon exposing the film to both glucose and H2O2 to finally reach a stable value. The critical concentration is found to be less than 100 µM for H2O2 and around 10 -2 M for glucose, meaning near biologically relevant chemical concentrations. Our results are in accordance with the results obtained by (i) Levy et al. who studied glucose-sensitive system based on mannan and phenylboronic acid moieties grafted onto a polyacid, [START_REF] Levy | Polymer microcapsules with carbohydratesensitive properties[END_REF] (ii) De Geest et al. who fabricated glucose-responsive layer-by-layer assembly of a copolymer of dimethylaminoethylacrylate and acrylamidophenylboronic acid and poly(styrene sulfonate) and (iii) Sato et al. who designed hydrogen peroxide responsive multilayer film based on phenylboronic acid-bearing poly (allylamine) and poly (vinyl alcohol). [START_REF] Sato | H2O2-induced decomposition of layerby-layer films consisting of phenylboronic acid-bearing poly (allylamine) and poly (vinyl alcohol)[END_REF] The film decomposition upon the addition of these chemicals is then successfully demonstrated. In contrast, in a buffer solution meaning without H2O2 nor glucose, the frequency shifts does not change indicating that the multilayer film remained stable on the substrate. These results clearly pointed out that these films are sensitive under oxidizing condition and glucose condition (explained through the suggested mechanism in Figure 2B).

To our point of view, the as-observed mass behavior change is mainly due to the incorporation of glucose within the multilayer films through complexation with the phenyl boronic moieties. This assumption is confirmed by the fact that no significant frequency shift was seen upon flushing glucose solutions with high concentration (up to 0.1 M) over multilayer films composed by diethylaminoethyl-dextran hydrochloride and native alginate.

A)

B)

Figure 6. Effect of increasing concentrations of (A) glucose and B) hydrogen peroxide (NB:

no variation of the frequency without glucose or hydrogen peroxide)

Figure 7 shows how phenylboronic acid-containing alginate interacts with the diol of the glucose. [START_REF] Springsteen | A detailed examination of boronic acid-diol complexation[END_REF] The result strongly suggests that there is formation of boronate ester bonds during the contact with the flushing with glucose which leads to (i) the complexation caused by the electrostatic interactions in the anionic glucose/borate complex and (ii) repulsion between the carboxylate groups of the alginate and the glucose/borate complex. The combination of these two phenomena seems to result in repulsion phenomena between the multilayers and then to the multilayer removal (Figure 8). The hydrogen peroxide response could be explained by the scheme presented in Figure 2B.

Multilayers self-assembly onto spherical surfaces

(diethylaminoethyl-dextran hydrochloride / phenylboronic acid-based alginate) and

(polylysine / phenylboronic acid-based alginate) polyelectrolytes pairs have been evidenced to form multilayer film and to present glucose-or hydrogen peroxide responsive behavior.

Further, these conditions will be implied for the fabrication of smart particles (Figure 9). CaCO3 particles have been used for the fabrication of responsive microparticles due to their intrinsic porosity which may enable the entrapment of small molecules. They can easily synthesize in a reproducible and size-controlled way. Furthermore, they are non-toxic. As shown in Figure 9, the CaCO3 core particle was coated with the studied polyelectrolyte bilayers. The conditions of the multilayer deposition were identical to those implied during the coating of the quartz surfaces but no precoating step with the thiol derivative was required. Indeed, pristine CaCO3 particles present negative surface charges allowing the direct deposition of positively charged polyelectrolytes as pointed by the measured zeta-potential value of -5 mV for the uncoated calcium carbonate material (Figure 10A). This parameter was then measured after each adsorption step to evidence the successive deposition of each polyelectrolyte on the particles surface. [START_REF] Belbekhouche | Latex nanoparticles surface modified via the layer-by-layer technique for two drugs loading[END_REF] A variation of the zeta-potential value is observed i.e. between +40 and -20 mV upon successive adsorption of polycation and alg-bor, respectively. As seen from SEM picture, the as-obtained microparticles present well-defined spherical shape and micrometer size (Figure 10B). The release rate of the entrapped probe within the interior cavity of the LbL coated microparticles may be highly impacted by the permeability of the material. For polyelectrolytes LbL assemblies, the inter-polyelectrolyte interactions can be tuned by various stimuli as pH or ionic strength. One may cite as example, particles based on alginate/polylysine or DNA/spermidine which were evidenced to provide a cargo release due to change in salt concentration. [START_REF] Schüler | Decomposable hollow biopolymer-based capsules[END_REF] In the present work, the release behavior of the coated microparticles loaded with the rhodamine b derivative inside its cavity is supposed to be triggered by both glucose and hydrogen concentration. Initially, the rhodamine b was loaded within the porous structure of the CaCO3 material prior to coat it with the multilayer film. The entrapment efficiencies efficiency of the material are is up to 60% and the probe loading is above 4 % (determined via equations 1 and 2). Without chemical stimulus the coated microparticles stay stable meaning that the remained loaded with the probe and those even after more than two weeks. However, when the coated microparticles are suspended in glucose-or hydrogen peroxide containing solution a release of the rhodamine derivative is evidence. It deserves noting that the threshold concentrations of each of this chemical has been estimated during the QCM experiment. These concentrations were used for monitoring the rhodamine b release. A fast probe release of 85% is observed after 24 h up to this critical concentration. The probe is progressively released below this critical concentration. This result is illustrated in Figure 11.

Conclusion

Novel multilayer assemblies based on alginate derivative (chemically modified to introduce phenylboronic acid group) have been implied for the design of both glucose-and hydrogen peroxide responsive system. Herein, the implemented strategy is based on the alternative adsorption of the polycation and the phenylboronic acid-containing polyanion on the surface of CaCO3 microparticles. After each deposition, the reversal of the surface charge was shown by zeta potential measurements, which is in accordance with the successful multilayer assemblies. Spherical shape was evidenced by SEM measurements. The glucose-and hydrogen peroxide sensitive character was related to these chemicals concentration in the surrounding buffer and implied due to the phenylboronic acid incorporation inside the polymer chain. The present work mainly focuses on the chemical modification and the physico-chemical aspect of the systems, a biological study will further be done. Indeed, the novel smart microparticle may further be employed for specific biological applications. One may cite diabetes disease (glucose-sensing device) or to detect oxidative stress (hydrogen peroxide-sensing system).

Figure 1 .Figure 2 .

 12 Figure 1. Biopolyelectrolytes used for the elaboration of glucose-and/or hydrogen peroxide responsive substrate namely phenylboronic acid modified alginate as polyanion and diethylaminoethyl-dextran hydrochloride or polylysine as polycation.

  groups. The C-O stretching vibration of alcoholic hydroxyl group and C-O-C stretching vibration remain unchanged, i.e. at 1143 cm -1 and 1023 cm -1 respectively. Moreover, a new peak attributed to the C-N stretching vibration (at 1080 cm -1 ) appeared after the chemical modification. At 1595 cm -1 and 1412 cm -1 peaks obtained are correlated to N-H deformation and C-N stretching vibrations. The presence of the carbonyl group from the amide is observed at around 1740 cm -1 .

  the buffer does not lead to a change in the contact angle value (i.e. remains near 20°±3°).

Figure 4 .

 4 Figure 4. Wettability measurement on bare gold substrate (70°±2°) and after the multilayer film build-up (20°±3°).

Figure 7 .

 7 Figure 7. Equilibrium between phenylphenylboronic acids-containing alginate and diolsbased glucose in aqueous solution.

Figure 8 .

 8 Figure 8. Proposed mechanism of the glucose-induced disassembly of (phenylboronic acid modified alginate/ polylysine)n

Figure 9 .

 9 Figure 9. Schematic illustration of the elaboration of the stimuli-responsive microcapsules based on phenylboronic acid containing polyanion.

Figure 10 .

 10 Figure 10. A) Zeta potential variation according to the adsorption step during the film build up on the CaCO3 microparticles (PC = polycation and MP = microparticles). B) SEM images of modified microspheres constituted by (polycation/alg-bor)n polyelectrolytes pair.

Figure 11 :

 11 Figure 11: Release of rhodamine b from ((phenylboronic acid modified alginate/ polylysine)n particles in presence of glucose solution at • 7.5 10 -3 M and ○ 10 -2 M.

Table 1 :

 1 Characterization of the alginate derivative.

	Samples	(g.•mol -1 )	(g.•mol -1 )	DS*
	alginate	2.7.10 5 (± 1.3 %) 3.8.10 5 (± 0.4 %)	-
	alginate-g-boronic	2.5.10 5 (± 1.3 %) 6.2.10 5 (± 0.4 %) 0.20 ± 0.04
	* grafting ratio of phenylboronic acid groups (estimated from 1 H NMR from Pettignano et al.

Acknowledgements

The authors wish to sincerely thank the UPEC and CNRS for financial support.