Carlos A Aguilar Salinas

M John Chapman
email: john.chapman@upmc.fr

BSc (Hons John Chapman

Remnant lipoproteins: Are they equal to or more atherogenic than LDL

Keywords: Apo, apolipoprotein ASCVD, atherosclerotic cardiovascular disease VLDL, very low-density lipoproteins IDL, intermediate-density lipoproteins HDL, high-density lipoproteins LDL, low-density lipoproteins CE, cholesteryl ester CETP, cholesteryl ester Remnant cholesterol, LDL cholesterol, dysbetalipoproteinemia, apolipoprotein B, apolipoprotein E, apolipoprotein CIII ; matrix proteoglycans

Purpose of review

To critically appraise new insights into the biology of remnant lipoproteins and their putative role in the pathophysiology of atherosclerotic cardiovascular disease, and to compare the atherogenicity of remnant particles with that of LDL.

Recent findings

New in vivo stable isotope tracer studies of the kinetics of apoB48-and apoB100-containing lipoproteins in postprandial conditions have revealed that apoB48-containing VLDL accumulated markedly in hypertriglyceridemic subjects. These intestinally-derived particles were cleared slowly, and represented up to 25% of circulating VLDL; as part of the remnant particle population, they may increase cardiovascular risk. Importantly, the PCSK9 inhibitor, evolocumab, was shown to reduce remnant levels (-29%) during the postprandial period in diabetic subjects on statin therapy, an effect which may be additive to that of LDL cholesterol reduction in conferring cardiovascular benefit. In recent Mendelian randomization studies, the effect of lowering triglyceride-rich lipoproteins or LDL cholesterol translated to similar clinical benefit per unit of apoB. Finally, in randomized trials involving statin-treated patients with ASCVD, remnant cholesterol levels were associated with coronary atheroma progression independently of LDL cholesterol.

Summary

Overall, data from observational studies in large cohorts, Mendelian randomization studies, metaregression analyses and post-hoc analyses of randomized trials are consistent with the contention that remnants are highly atherogenic particles and contribute to the atherosclerotic burden in an equivalent manner to that of LDL.

Introduction :

Recent findings have furthered our understanding of the complex biology of circulating triglyceride-rich lipoproteins (TGRL) and their remnants, and of their relevance to the pathophysiology of atherosclerotic cardiovascular disease (ASCVD). Indeed, we now recognize that in both the fasting and post-prandial periods, remnant particles are produced by intravascular remodelling of both intestinally-derived chylomicrons and very low-density lipoproteins (VLDL) of hepatic origin. In plasma, such particles are distributed widely across the density intervals of both VLDL (d<1.006 g/mL; including chylomicrons defined as Sf >400) and intermediate density lipoproteins (IDL; d<1.006-1.019 g/mL) [1,2••]. Equally present as components of these highly heterogeneous, density-defined, lipoprotein fractions are newlysecreted chylomicrons and VLDL. Importantly, new evidence in human subjects shows that such VLDL may be of both intestinal as well as hepatic origin, the former containing apoB48 rather than apoB100. As a consequence, both intestinal chylomicrons and their remnants are identified by their content of a single copy of apolipoprotein (apo) B48, while particles isolated in the d<1.006 g/mL density fraction may contain either apoB48 or apoB100 according to their tissue origin [1,2••].

As a function of triglyceride pool size, and of the metabolic, genetic and nutritional background in a given individual, part of the hepatic apoB100-containing remnant pool is converted to cholesterol-rich LDL by further remodeling by lipases and cholesteryl ester transfer protein (CETP); the remainder is taken up by hepatocytes via multiple mechanisms [START_REF] Havel | Triglyceride-rich lipoprotein remnants[END_REF][START_REF] Björnson | Apolipoprotein B48 metabolism in chylomicrons and very low-density lipoproteins and its role in triglyceride transport in normo-and hypertriglyceridemic human subjects[END_REF][START_REF] Boren | Low-density lipoproteins cause atherosclerotic cardiovascular disease: pathophysiological, genetic, and therapeutic insights: a consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Mahley | Atherogenic remnant lipoproteins: role for proteoglycans in trapping, transferring, and internalizing[END_REF]. The relative contents of apoE and apoCIII in TGRL particles are critical not only in the formation of VLDL remnants, but also in their rates of hepatic clearance relative to their propensity to form LDL [3•,5,6•].

A substantial body of evidence now attests to the indisputable causal role of LDL in the pathophysiology of ASCVD, and is based upon the fulfillment of eight key criteria of causality (Table 1) [START_REF] Ference | Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF]. A critical question now arises: are remnants of triglyceride-rich lipoproteins, the direct precursors of LDL, of similar, lesser or greater atherogenic potential than LDL particles themselves ? In this review, we present a comprehensive evaluation of recent evidence from pathophysiological, epidemiologic, genetic, Mendelian randomisation, and intervention studies that links remnant particles with atherosclerosis and cardiovascular events. Such evidence has prompted the proposal that TGRL and their remnants represent targets for the therapeutic lowering of circulating concentrations of these particles, with subsequent reduction in cardiovascular risk [3•,8,9].

Atherobiology of remnants :

The physicochemical characteristics of remnant particles are intimately linked to their atherogenic potential. Firstly, the particle size of small chylomicron and VLDL remnants ( ≈250-700 Å) is consistent with their entry by transcytosis into the arterial intima at sites of predilection of atherosclerotic lesion formation, where they may be retained by electrostatic interaction between their principal apolipoproteins (apoB and apoE) and matrix proteoglycans [START_REF] Havel | Triglyceride-rich lipoprotein remnants[END_REF][START_REF] Boren | Low-density lipoproteins cause atherosclerotic cardiovascular disease: pathophysiological, genetic, and therapeutic insights: a consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Mahley | Atherogenic remnant lipoproteins: role for proteoglycans in trapping, transferring, and internalizing[END_REF][START_REF] Havel | Remnant lipoproteins as therapeutic targets[END_REF][START_REF] Dallinga-Thie | Triglyceride-rich lipoproteins and Remnants: Targets for Therapy?[END_REF][START_REF] Boren | The central role of arterial retention of cholesterol-rich apolipoprotein Bcontaining lipoproteins in the pathogenesis of atherosclerosis : a triumph of simplicity[END_REF]. Importantly, and unlike LDL, remnants efflux slowly in relation to their rates of entry [START_REF] Schwartz | Lipolysis of triglyceride-rich lipoproteins, vascular inflammation and atherosclerosis[END_REF]. Furthermore, the enrichment of remnants in apoCIII amplifies their retention to matrix proteoglycan biglycan by an unidentified mechanism [START_REF] Krauss | ApoC-III content of apoB-containing lipoproteins is associated with binding to the vascular proteoglycan biglycan[END_REF].

Significantly, the intravascular cholesteryl ester transfer protein (CETP)-mediated enrichment of remnants in cholesteryl ester (CE) confers them with elevated cholesterol content, thereby favouring arterial cholesterol accumulation. Indeed, it is estimated that a typical remnant in the IDL range of size and molecular weight containing some 30% by weight of cholesterol (after correction for the CE fatty acid) may contain up to a maximum of a fourfold greater absolute number of cholesterol molecules (up to ≈ 8600 per particle) as compared to an LDL particle (2000-2700) [3••,13]. Secondly, in addition to containing a single copy of either apoB48 or B100, the proteome of remnant particles is typically enriched in apoE relative to other TGRL, but equally features apoCIII, apoCII, apoCI, apoAV and angiopoietin-like protein (ANGPTL) -3, together with traces of apoAI and apoAII [START_REF] Havel | Triglyceride-rich lipoprotein remnants[END_REF][START_REF] Mahley | Atherogenic remnant lipoproteins: role for proteoglycans in trapping, transferring, and internalizing[END_REF][START_REF] Havel | Remnant lipoproteins as therapeutic targets[END_REF][START_REF] Dallinga-Thie | Triglyceride-rich lipoproteins and Remnants: Targets for Therapy?[END_REF][START_REF] Nordestgaard | Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology[END_REF]. ApoE is a major determinant not only of the binding and uptake of remnants by both receptor-dependent and receptor-independent mechanisms in hepatocytes, but importantly, the receptor-mediated endocytosis of native remnants by surface receptors (LDL receptor, LDL receptor-related protein 1) of subendothelial monocyte-derived macrophages [START_REF] Havel | Triglyceride-rich lipoprotein remnants[END_REF][START_REF] Mahley | Atherogenic remnant lipoproteins: role for proteoglycans in trapping, transferring, and internalizing[END_REF][START_REF] Dallinga-Thie | Triglyceride-rich lipoproteins and Remnants: Targets for Therapy?[END_REF][START_REF] Nordestgaard | Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology[END_REF]. Remnant lipoprotein uptake by intimal macrophages is thus a key driver of macrophage foam cell formation, the hallmark cell of the atherosclerotic plaque. Such remnant-mediated cholesterol accumulation favours macrophage M1 polarisation in the intimal microenvironment, with an enhanced inflammatory response [START_REF] Pourcet | Alternative macrophages in atherosclerosis: not always protective![END_REF]. Moreover, proinflammatory macrophages secrete a spectrum of factors which promote lesion development and ultimately destabilisation; these include matrix-degrading proteases, prooxidant enzymes, lipases, growth factors, proinflammatory cytokines, eicosanoids, proteoglycans and procoagulant factors [16]. Triglyceride-rich lipoproteins and their remnants may equally enhance local vascular inflammation in the subendothelial space by virtue of the release of free fatty acids and other hydrolytic lipid derivatives, such as proinflammatory lysolipids, upon lipolysis induced by lipoprotein lipase (LPL), phospholipases and sphingomyelinases [START_REF] Schwartz | Lipolysis of triglyceride-rich lipoproteins, vascular inflammation and atherosclerosis[END_REF].

Overall, the contribution of lipoproteins to the atherogenic burden depends on several factors, including plasma abundance and residence time, as well as endothelial entry rates via transcytosis, the degree of retention in arterial tissue and the propensity to elicit both foam cell formation and a maladaptive response. The remarkably greater abundance and long half-life of LDL particles (≈250 mg/dL; up to 3 days) relative to that of remnants argue strongly for LDL particles as the main target for prevention of ASCVD. However, recent estimates suggest that up to 30% of the cholesterol load in apoB-containing lipoproteins is transported in remnant particles under nonfasting conditions [START_REF] Balling | A third of nonfasting plasma cholesterol is in remnant lipoproteins: Lipoprotein subclass profiling in 9293 individuals[END_REF]. In this context, in vivo kinetic studies of remnant particle metabolism, performed under metabolic conditions during which the arterial wall is most exposed to these particles, ie. the post-prandial period, are of immediate relevance. Indeed, TGRL and remnant levels are typically present at elevated levels over periods as long as 8 hours or more depending on the lipolytic capacity of the individual. Thus, using a new stable isotope-based tracer approach and an integrated non-steady state multicompartmental model, Björnson and colleagues showed that intestinally-derived, apoB48-containing chylomicrons and VLDL remain in plasma for a markedly longer period of time than previously observed (several hours in the VLDL range instead of a few minutes) [2••]. Such particles may be remodelled to remnants, particularly in hypertriglyceridemic subjects. Consequently, apoB48containing lipoproteins potentially contribute significantly to the remnant-related atherogenic burden.

Considered together, the above pathophysiological and metabolic evidence highlights the multiple, interactive and mutually complementary mechanisms which underlie the atherogenicity of both apoB48-and apoB100-containing remnants.

Epidemiologic studies of remnant cholesterol:

Extensive observational evidence has linked plasma accumulation of both LDL and remnants with the incidence of cardiovascular events [START_REF] Lewington | Blood cholesterol and vascular mortality by age, sex, and blood pressure: a meta-analysis of individual data from 61 prospective studies with 55,000 vascular deaths[END_REF][START_REF] Varbo | Extreme nonfasting remnant cholesterol vs extreme LDL cholesterol as contributors to cardiovascular disease and all-cause mortality in 90000 individuals from the general population[END_REF][START_REF] Varbo | Remnant cholesterol as a cause of ischemic heart disease: evidence, definition, measurement, atherogenicity, high risk patients, and present and future treatment[END_REF][START_REF] Varbo | Remnant lipoproteins[END_REF][START_REF] Varbo | Elevated remnant cholesterol causes both low-grade inflammation and ischemic heart disease, whereas elevated low-density lipoprotein cholesterol causes ischemic heart disease without inflammation[END_REF]. The association between LDL cholesterol levels and the incidence of cardiovascular events has been the subject of multiple studies with many thousands of participants [START_REF] Ference | Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF]. Comparable evidence for remnants is strong but less substantial.

The Copenhagen General Population Study and the Copenhagen City Heart Study, in which nearly 90,000 subjects have participated, provides seminal information however [START_REF] Nordestgaard | Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology[END_REF][START_REF] Varbo | Extreme nonfasting remnant cholesterol vs extreme LDL cholesterol as contributors to cardiovascular disease and all-cause mortality in 90000 individuals from the general population[END_REF][START_REF] Varbo | Remnant cholesterol as a cause of ischemic heart disease: evidence, definition, measurement, atherogenicity, high risk patients, and present and future treatment[END_REF][START_REF] Varbo | Remnant lipoproteins[END_REF][START_REF] Varbo | Elevated remnant cholesterol causes both low-grade inflammation and ischemic heart disease, whereas elevated low-density lipoprotein cholesterol causes ischemic heart disease without inflammation[END_REF].

Confirmatory reports have been published in Chinese populations [START_REF] Xiang | Comparison of remnant cholesterol levels estimated by calculated and measured LDL-C levels in Chinese patients with coronary heart disease[END_REF] and in selected groups as exemplified by type 2 diabetes [START_REF] Nguyen | Remnant lipoproteinemia predicts cardiovascular events in patients with type 2 diabetes and chronic kidney disease[END_REF]. Nonetheless, the strength of the evidence is limited by methodological issues, as the concentration of remnant particles was estimated indirectly using levels of either plasma triglycerides or calculated remnant cholesterol (as total cholesterol -LDLcholesterol -HDL-cholesterol). Plasma triglyceride is not an ideal marker, as it may be determined in part by the presence of other particles that are not strongly associated with atherosclerosis (ie. chylomicrons and large VLDLs); a similar comment applies to determination of remnant cholesterol levels when measured in a corresponding manner [START_REF] Nordestgaard | Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology[END_REF][START_REF] Varbo | Extreme nonfasting remnant cholesterol vs extreme LDL cholesterol as contributors to cardiovascular disease and all-cause mortality in 90000 individuals from the general population[END_REF][START_REF] Varbo | Remnant cholesterol as a cause of ischemic heart disease: evidence, definition, measurement, atherogenicity, high risk patients, and present and future treatment[END_REF][START_REF] Varbo | Remnant lipoproteins[END_REF][START_REF] Varbo | Elevated remnant cholesterol causes both low-grade inflammation and ischemic heart disease, whereas elevated low-density lipoprotein cholesterol causes ischemic heart disease without inflammation[END_REF][START_REF] Xiang | Comparison of remnant cholesterol levels estimated by calculated and measured LDL-C levels in Chinese patients with coronary heart disease[END_REF][START_REF] Nguyen | Remnant lipoproteinemia predicts cardiovascular events in patients with type 2 diabetes and chronic kidney disease[END_REF]. In addition, the contribution of remnants could be under-represented in many studies, as determinations have frequently been performed under fasting conditions. Despite such potential limitations, those subjects in the highest versus the lowest percentile of non-fasting remnant cholesterol concentration in the prospective Copenhagen cohorts displayed a 2.4 fold increased risk for ischemic heart disease [START_REF] Varbo | Extreme nonfasting remnant cholesterol vs extreme LDL cholesterol as contributors to cardiovascular disease and all-cause mortality in 90000 individuals from the general population[END_REF]. When an equivalent comparison was made for LDLcholesterol, the corresponding hazard ratio for risk was 2.3. The hazard ratio for myocardial infarction attained a maximum of 3.4 for remnant cholesterol and 4.7 for LDL-cholesterol.

Remarkably, remnant cholesterol concentration was superior in predicting all-cause mortality relative to LDL cholesterol [START_REF] Varbo | Extreme nonfasting remnant cholesterol vs extreme LDL cholesterol as contributors to cardiovascular disease and all-cause mortality in 90000 individuals from the general population[END_REF].

Evidence from Mendelian randomization studies:

Sets of genetic variants that are implicated in the determination of LDL cholesterol levels are strongly associated with cardiovascular events, thereby constituting robust evidence that these particles exert a causal role in atherosclerosis [START_REF] Ference | Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF]. In contrast, the case for remnants is more complex as the genetic variants that influence their circulating concentrations exert pleiotropic effects, having additional targets and impact on lipid and lipoprotein metabolism [START_REF] Nordestgaard | Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology[END_REF][START_REF] Varbo | Elevated remnant cholesterol causes both low-grade inflammation and ischemic heart disease, whereas elevated low-density lipoprotein cholesterol causes ischemic heart disease without inflammation[END_REF][START_REF] Varbo | Remnant cholesterol as a causal risk factor for ischemic heart disease[END_REF]. However, a recent analysis based on the UK biobank (n=654,783 participants) compared the association between coronary heart disease (n=91,129 cases) and sets of genetic variants that themselves are associated with lower levels of either LDL (via the LDL receptor) or triglycerides (by modulating lipoprotein lipase (LPL) activity) [26 •• ]. As LPL action represents a critical step in the progressive transformation of chylomicrons or VLDL to remnants, this approach has the potential to compare the contribution of LDL relative to that of remnants with respect to cardiovascular risk. Thus, variants associated with lower triglyceride levels should equally have an effect on apoB concentrations in order to be associated with CHD, even if this effect is associated with a small increment in LDL cholesterol concentrations.

Remarkably, both LPL and LDLR scores were associated with similar reduction in risk of CHD per 10mg/dL lower level of apoB [26••]. This finding indicates that the effect of lowering TGRL on the one hand, and LDL cholesterol on the other, translates into clinical benefit which is proportional to the absolute reduction in their content of apoB. Such evidence again suggests that a particle per particle equivalence may exist when comparing the atherogenic potential of TGRL and remnant particles with that of LDL.

Evidence from genetic disorders of lipid metabolism:

Monogenic conditions, as exemplified by elevated circulating concentrations of LDL in familial hypercholesterolemia (FH), and of lipoprotein remnant particles in dysbetalipoproteinemia, are natural experiments in which the respective atherogenic roles of these lipoproteins can be prospectively assessed. These disorders are characterised by selective clearance defects giving rise to LDL or remnant accumulation, the latter primarily in the IDL density interval [START_REF] Santos | International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel. Defining severe familial hypercholesterolaemia and the implications for clinical management: a consensus statement from the International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel[END_REF][START_REF] Marais | Dysbetalipoproteinemia: an extreme disorder of remnant metabolism[END_REF]. In FH, the genetic defect is located mainly in the LDL receptor, and to a lesser degree in the apoB and PCSK9 genes [START_REF] Santos | International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel. Defining severe familial hypercholesterolaemia and the implications for clinical management: a consensus statement from the International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel[END_REF].

By contrast, the most common cause of dysbetalipoproteinemia is homozygosity for the receptor binding-defective form of apoE (apo E2/E2 genotype), which is the main ligand for the family of LDL receptors that remove remnants from plasma [START_REF] Marais | Dysbetalipoproteinemia: an extreme disorder of remnant metabolism[END_REF]. ApoE2/E2 status requires the coexistence of a secondary cause of hyperlipidemia for phenotypic expression with severe accumulation of remnants.

Although both conditions are associated with a markedly increased risk for premature ASCVD, there are major differences in the clinical expression between these two entities. The age of onset of hyperlipidemia is greater in dysbetalipoproteinemia; as a consequence, dysbetalipoproteinemia cases may present normal lipid concentrations for many years. In contrast, hypercholesterolemia is present at birth in FH. In addition, the clinical phenotypes tend to be distinct; thus, palmar crease xanthomata are seen in dysbetalipoproteinemia, while this cutaneous manifestation is rare in FH. Both disorders feature premature coronary atherosclerosis; peripheral artery disease is as common as coronary atherosclerosis however in dysbetalipoproteinemia [START_REF] Koopal | Vascular risk factors, vascular disease, lipids and lipid targets in patients with familial dysbetalipoproteinemia:a European cross-sectional study[END_REF]. These observations suggest that the impact of the plasma accumulation of LDL particles on the arterial wall is not the same as that for remnants, and may differ according to sites in the arterial tree. Regrettably, the lack of registries of patients with dysbetalipoproteinemia precludes comparison of the magnitude of cardiovascular risk with that in FH.

Finally, in some cases of FH, concurrent accumulation of both LDL and remnants occurs, with more severe atherosclerotic lesions resulting [START_REF] Tada | Remnant-like particles and coronary artery disease in familial hypercholesterolemia[END_REF]. Furthermore, remnant cholesterol concentration was an independent predictor of the presence of cardiovascular disease in FH patients (n=282) in this study [START_REF] Tada | Remnant-like particles and coronary artery disease in familial hypercholesterolemia[END_REF].

Evidence from randomized controlled studies:

LDL-centered therapies, notably statins, ezetimibe and PCK9 antibodies, have consistently proven to be effective in reducing the incidence of cardiovascular events; some 95% of the beneficial effects of such therapies can be accounted for by the magnitude of the absolute LDL cholesterol reduction [START_REF] Havel | Triglyceride-rich lipoprotein remnants[END_REF].

Interestingly however, Taskinen and colleagues have recently provided evidence that the PCSK9 inhibitor, evolocumab, significantly reduces remnant cholesterol levels (-29%) during the postprandial response in diabetic subjects on background statin therapy, a finding consistent with the higher absolute cardiovascular risk reduction observed in patients with diabetes (2.7%) versus patients without diabetes (1.6%) in the Fourier trial [31•, 32].

Ideally, clinical intervention trials in dyslipidemic patients with lipid lowering agents targeted specifically to remnant particles would permit a more direct comparison of their atherogenic potential relative to that of LDL. The design of such trials would prove singularly challenging however, particularly given the intricate metabolic relationships of remnants with their upstream TGRL precursors and downstream LDL products, and the need to neutralise the component of cardiovascular risk due to LDL and possibly Lp(a). Moreover, the link between lipolysis of TGRL and the role of the sequestration of surface lipolytic fragments to maintenance of the HDL pool should not be ignored.

Nonetheless, remnant lipoproteins (measured as remnant-cholesterol) were clearly identified as a component of the residual risk in ASCVD patients with on-statin LDL cholesterol at the guidelinerecommended goal of <70mg/dL, consistent with the proposal that therapeutic targeting of remnants is desirable [START_REF] Fujihara | Remnant Lipoproteins Are Residual Risk Factor for Future Cardiovascular Events in Patients With Stable Coronary Artery Disease and On-Statin Low-Density Lipoprotein Cholesterol Levels <70 mg/dL[END_REF].

Fibrates, niacin and omega-3 fatty acids are currently indicated for the treatment of hypertriglyceridemic states; statins, ezetimibe and PCSK9 inhibitors exert modest effects (typically <15%) which depend largely on triglyceride levels at baseline [6•,14,33]. However, fibrates, niacin and omega-3 fatty acid preparations influence concentrations of all major lipoprotein classes by direct or indirect mechanisms, have minor effects on LDL cholesterol, and therefore cannot be considered simply as remnant-lowering agents. In particular, the effect of fibrates and niacin in the prevention of cardiovascular events has been inconsistent, particularly when evaluated in combination with statin therapy [6•,14,33]. These drugs lower apoB concentrations to a minor degree (≤20%) [16,[START_REF] Nordestgaard | Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology[END_REF][START_REF] Laufs | Clinical review on triglycerides[END_REF]; consideration of issues including study design, the enrolment of patients who were moderately hypertriglyceridemic at most, together with methodological issues, may explain the negative or neutral results of prevention trials based on fibrate and niacin therapies [START_REF] Nordestgaard | Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology[END_REF][START_REF] Laufs | Clinical review on triglycerides[END_REF].

A recent meta-regression analysis of trial level data has addressed the generic question as to whether reduction in triglyceride levels translates into cardiovascular benefit across three lipid-lowering therapeutic classes, and included fibrates, niacin and omega-3 fatty acids; data from an earlier metaregression of statin trials was equally integrated into the analysis [START_REF] Marston | Association Between Triglyceride Lowering and Reduction of Cardiovascular Risk Across Multiple Lipid-Lowering Therapeutic Classes: A Systematic Review and Meta-Regression Analysis of Randomized Controlled Trials[END_REF]. Although triglyceride lowering translated overall to a lower risk of vascular events, even after adjustment for changes in LDL cholesterol, these findings should be regarded with caution, as driven by the positive results obtained with high dose eicosapentanoic acid (EPA)(icosapent ethyl; 4g/day) in high risk patients on background statin therapy in the REDUCE-IT trial [35••]. Indeed, although the cardiovascular benefit (a 25% reduction in the primary composite cardiovascular endpoint in the intervention group relative to placebo) in the REDUCE-IT trial occurred across a wide range of baseline triglyceride levels, the mechanism(s) which underlies such benefit remains speculative. Nonetheless, we cannot exclude the possibility that hypertriglyceridemic participants with either ASCVD or diabetes and other risk factors in REDUCE-IT, and who would be anticipated to exhibit an amplified postprandial response with elevated remnant levels, may have gained major benefit from attenuation of remnant atherogenicity consistent with a plasma triglyceride reduction on treatment of 20% versus placebo.

The relationship of remnant cholesterol levels to the progression of atherosclerotic disease is of special interest. In this context, Elshazly and colleagues estimated the contribution of remnant cholesterol to changes in % coronary atheroma volume (determined by intravascular ultrasound) and 2-year major cardiovascular events in 10 clinical trials (n=5754) involving both statin and non-statin therapies [36•]. Remnant cholesterol was determined as non-HDL cholesterol -LDL cholesterol. In multivariable analyses, on-treatment remnant cholesterol was significantly correlated with change in plaque volume, even after adjusting for LDL cholesterol and other risk factors. A positive correlation was equally found between the highest on-treatment remnant cholesterol quartile and the incidence of cardiovascular events as compared to the lowest. Overall, the correlations were stronger for ontreatment remnant cholesterol than for on-treatment LDL cholesterol. Thus, these reports support the contention that remnant cholesterol constitutes a complimentary target to LDL cholesterol for the prevention of cardiovascular events.

Conclusions

Table 1 summarizes and compares the evidence for key criteria supporting a causal role for remnants on the one hand, and LDL on the other, in the pathophysiology of ASCVD. Indeed, both LDL and remnants fulfill eight criteria consistent with the assumption of causality (Table 1). However, the relative contribution of LDL relative to that of remnants to the atherogenic process may differ substantially as a function of the nutritional, metabolic, genetic and pharmacological profile in any given individual [START_REF] Aguilar-Salinas | Genetic and environmental determinants of the susceptibility of Amerindian derived populations for having hypertriglyceridemia[END_REF]. Nonetheless, we submit that particle for particle, the weight of the evidence to date is consistent with a markedly similar atherogenic potential for both an LDL particle and a cholesterol-, apoE-rich remnant. Remnant particles may therefore be considered as a target for therapy, and especially in very high and high risk statin-treated patients with established ASCVD in which guideline recommended LDL cholesterol goals have been achieved, but in whom residual atherosclerotic cardiovascular disease risk subsists [8,9,38•].

It is in this light that the ongoing development of innovative agents which will have potentially major impact on remnant levels is especially encouraging; notable among them are pemafibrate, a new generation fibrate currently under evaluation in a cardiovascular outcome trial (PROMINENT) in dyslipidemic diabetic subjects, as well as agents targeted to upregulate TGRL lipolysis-and potentially hepatic remnant clearance -by virtue of the inhibition of apoCIII or ANGPTL3 [START_REF] Florentin | Recent developments in pharmacotherapy for hypertriglyceridemia: what's the current state of the art?[END_REF][START_REF] Tsimikas | RNA-targeted therapeutics for lipid disorders[END_REF].

Finally, a review of the multiple facets of the atherogenicity of remnant particles leads inevitably to the identification of some of the key, unsolved questions of both clinical and scientific relevance, which are summarised in Table 2. Such questions can be anticipated to stimulate further researchand ultimately to advance our understanding of the complex biology of remnant particles.

KEY POINTS:

1) In vivo turnover data in non-fasting hypertriglyceridemic subjects indicate that the intestine produces apoB48-containing VLDL in addition to chylomicrons, and that such VLDL give rise to remnants with prolonged plasma residence.

2) The evidence base for both LDL and remnant particles fulfils eight key criteria of causality for ASCVD.

3) On a particle for particle basis, the atherogenic potential of a cholesterol-rich remnant is similar to that of an LDL particle.

4 )••

 4 Remnant cholesterol constitutes a complimentary target to that of LDL cholesterol in the prevention of ASCVD. 5) Recent findings (epidemiological, meta-analyses and post hoc analyses of randomized trials) confirm the atherogenic risk associated with plasma remnant accumulation.Legend to Figure 1:A comparison of the key biological features of LDL as compared to remnant particles in relation to their atherogenicity. The figure highlights the key aspects of the evidence supporting the atherogenicity of both classes of particles, and emphasises the lack of experimental evidence (principally placebo-controlled, randomised intervention trials) for remnants.LDL Elevated particle numbers in plasma• Up to 2700 cholesterol molecules / particle • Half-life = up to 3 days • Bind matrix proteoglycans via apoB100 Lower particle numbers in plasma as compared to LDL • Up to 8600 cholesterol molecules/IDL particle • Half-life = < 24 hours • Bind matrix proteoglycans and biglycan via apoE, apoCIII and apoB; low intimal efflux rates

Acknowledgements

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Conflicts of interest

There are no conflicts of interest for the preparation of this review.

Criterion LDL Remnants Plausibility

Strong experimental evidence [START_REF] Boren | Low-density lipoproteins cause atherosclerotic cardiovascular disease: pathophysiological, genetic, and therapeutic insights: a consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Mahley | Atherogenic remnant lipoproteins: role for proteoglycans in trapping, transferring, and internalizing[END_REF][START_REF] Ference | Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Boren | The central role of arterial retention of cholesterol-rich apolipoprotein Bcontaining lipoproteins in the pathogenesis of atherosclerosis : a triumph of simplicity[END_REF]16] No animal model is representative of human remnant metabolism [START_REF] Havel | Triglyceride-rich lipoprotein remnants[END_REF][START_REF] Björnson | Apolipoprotein B48 metabolism in chylomicrons and very low-density lipoproteins and its role in triglyceride transport in normo-and hypertriglyceridemic human subjects[END_REF]. Remnant components are found in human atherosclerotic plaques [START_REF] Mahley | Atherogenic remnant lipoproteins: role for proteoglycans in trapping, transferring, and internalizing[END_REF][START_REF] Boren | The central role of arterial retention of cholesterol-rich apolipoprotein Bcontaining lipoproteins in the pathogenesis of atherosclerosis : a triumph of simplicity[END_REF][START_REF] Krauss | ApoC-III content of apoB-containing lipoproteins is associated with binding to the vascular proteoglycan biglycan[END_REF]] Strength Monogenic and polygenic lifelong elevations lead to higher lifetime risk of ASCVD [3,27,] Monogenic and polygenic lifelong elevations lead to higher lifetime risk of ASCVD [START_REF] Nordestgaard | Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology[END_REF][START_REF] Varbo | Elevated remnant cholesterol causes both low-grade inflammation and ischemic heart disease, whereas elevated low-density lipoprotein cholesterol causes ischemic heart disease without inflammation[END_REF]26,[START_REF] Marais | Dysbetalipoproteinemia: an extreme disorder of remnant metabolism[END_REF]] Biological gradient A dose-dependent association between the absolute magnitude of exposure to LDL and risk of ASCVD [START_REF] Boren | Low-density lipoproteins cause atherosclerotic cardiovascular disease: pathophysiological, genetic, and therapeutic insights: a consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Ference | Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Boren | The central role of arterial retention of cholesterol-rich apolipoprotein Bcontaining lipoproteins in the pathogenesis of atherosclerosis : a triumph of simplicity[END_REF][START_REF] Santos | International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel. Defining severe familial hypercholesterolaemia and the implications for clinical management: a consensus statement from the International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel[END_REF] A dose-dependent association between the absolute magnitude of exposure to remnant cholesterol and risk of ASCVD [START_REF] Chapman | Further resolution of the low density lipoprotein spectrum in normal human plasma: physicochemical characteristics of discrete subspecies separated by density gradient ultracentrifugation[END_REF][START_REF] Nordestgaard | Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology[END_REF][START_REF] Varbo | Extreme nonfasting remnant cholesterol vs extreme LDL cholesterol as contributors to cardiovascular disease and all-cause mortality in 90000 individuals from the general population[END_REF][START_REF] Varbo | Elevated remnant cholesterol causes both low-grade inflammation and ischemic heart disease, whereas elevated low-density lipoprotein cholesterol causes ischemic heart disease without inflammation[END_REF]26,[START_REF] Tada | Remnant-like particles and coronary artery disease in familial hypercholesterolemia[END_REF][START_REF] Marston | Association Between Triglyceride Lowering and Reduction of Cardiovascular Risk Across Multiple Lipid-Lowering Therapeutic Classes: A Systematic Review and Meta-Regression Analysis of Randomized Controlled Trials[END_REF][START_REF] Bhatt | REDUCE-IT Investigators. Cardiovascular risk reduction with icosapent ethyl for hypertriglyceridemia[END_REF] Temporal sequence Exposure to elevated LDL precedes the onset of ASCVD [START_REF] Boren | Low-density lipoproteins cause atherosclerotic cardiovascular disease: pathophysiological, genetic, and therapeutic insights: a consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Ference | Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Santos | International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel. Defining severe familial hypercholesterolaemia and the implications for clinical management: a consensus statement from the International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel[END_REF] Exposure to elevated remnant cholesterol precedes the onset of ASCVD [START_REF] Nordestgaard | Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology[END_REF][START_REF] Varbo | Remnant cholesterol as a cause of ischemic heart disease: evidence, definition, measurement, atherogenicity, high risk patients, and present and future treatment[END_REF][START_REF] Varbo | Elevated remnant cholesterol causes both low-grade inflammation and ischemic heart disease, whereas elevated low-density lipoprotein cholesterol causes ischemic heart disease without inflammation[END_REF]26,[START_REF] Marais | Dysbetalipoproteinemia: an extreme disorder of remnant metabolism[END_REF][START_REF] Aguilar-Salinas | Genetic and environmental determinants of the susceptibility of Amerindian derived populations for having hypertriglyceridemia[END_REF] Specificity LDL is associated with ASCVD independent of other risk factors [START_REF] Boren | Low-density lipoproteins cause atherosclerotic cardiovascular disease: pathophysiological, genetic, and therapeutic insights: a consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Ference | Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF] Remnant cholesterol is associated with ASCVD independent of other risk factors [START_REF] Nordestgaard | Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology[END_REF][START_REF] Varbo | Remnant cholesterol as a cause of ischemic heart disease: evidence, definition, measurement, atherogenicity, high risk patients, and present and future treatment[END_REF][START_REF] Varbo | Elevated remnant cholesterol causes both low-grade inflammation and ischemic heart disease, whereas elevated low-density lipoprotein cholesterol causes ischemic heart disease without inflammation[END_REF]26,[START_REF] Marais | Dysbetalipoproteinemia: an extreme disorder of remnant metabolism[END_REF]

Consistency

Multiple sources of evidence demonstrate a dose-dependent, log-linear association between the absolute magnitude of exposure to LDL and risk of ASCVD [START_REF] Boren | Low-density lipoproteins cause atherosclerotic cardiovascular disease: pathophysiological, genetic, and therapeutic insights: a consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Ference | Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Santos | International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel. Defining severe familial hypercholesterolaemia and the implications for clinical management: a consensus statement from the International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel[END_REF] Observational and post-hoc analyses of intervention trials show a dose-dependent association between the absolute magnitude of exposure to remnants and risk of ASCVD [START_REF] Nordestgaard | Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology[END_REF][START_REF] Varbo | Elevated remnant cholesterol causes both low-grade inflammation and ischemic heart disease, whereas elevated low-density lipoprotein cholesterol causes ischemic heart disease without inflammation[END_REF]26,[START_REF] Marais | Dysbetalipoproteinemia: an extreme disorder of remnant metabolism[END_REF]

Coherence

Multiple sources of evidence replicate the association LDL-ASCVD [START_REF] Boren | Low-density lipoproteins cause atherosclerotic cardiovascular disease: pathophysiological, genetic, and therapeutic insights: a consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Ference | Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Boren | The central role of arterial retention of cholesterol-rich apolipoprotein Bcontaining lipoproteins in the pathogenesis of atherosclerosis : a triumph of simplicity[END_REF]16,[START_REF] Santos | International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel. Defining severe familial hypercholesterolaemia and the implications for clinical management: a consensus statement from the International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel[END_REF] Several sources of evidence replicate the association of remnants with premature ASCVD [START_REF] Nordestgaard | Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology[END_REF][START_REF] Varbo | Remnant cholesterol as a cause of ischemic heart disease: evidence, definition, measurement, atherogenicity, high risk patients, and present and future treatment[END_REF][START_REF] Varbo | Elevated remnant cholesterol causes both low-grade inflammation and ischemic heart disease, whereas elevated low-density lipoprotein cholesterol causes ischemic heart disease without inflammation[END_REF]26,[START_REF] Marais | Dysbetalipoproteinemia: an extreme disorder of remnant metabolism[END_REF]] Reduction in risk with intervention Multiple randomized trials evaluating LDL lowering therapies demonstrate that reducing LDL cholesterol reduces the risk of ASCVD events [START_REF] Boren | Low-density lipoproteins cause atherosclerotic cardiovascular disease: pathophysiological, genetic, and therapeutic insights: a consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Ference | Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Santos | International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel. Defining severe familial hypercholesterolaemia and the implications for clinical management: a consensus statement from the International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel[END_REF] Post-hoc analyses of randomized trials and meta-regression analyses suggest that lowering remnant cholesterol reduces the risk of ASCVD events; additional evidence is needed. [START_REF] Havel | Remnant lipoproteins as therapeutic targets[END_REF][START_REF] Nordestgaard | Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology[END_REF][START_REF] Bhatt | REDUCE-IT Investigators. Cardiovascular risk reduction with icosapent ethyl for hypertriglyceridemia[END_REF] Modified from reference [START_REF] Ference | Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel[END_REF].


Mushtaq Ali
email: mushtaqali.zada@gmail.com

Muhammad Saad

Mamatimin Abbas

Arshad S Bhatti

Luca Ottaviano

Marianna Ambrico

Andrea Di Cicco

Roberto Gunnella

Andrea Di Cicco

Formation of TiO2 nanostructures modified Eumelanin films with enhanced properties for biopolymer implementations

Keywords: hybrid nanostructures, absorption features, spectroscopy, optoelectronics

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

The recent advancements in the research of pigments like eumelanin (termed as melanin) are preluding to applications of these biopolymers in the fields of bioelectronics, sensors, and (opto)electronic devices [START_REF] D'ischia | Chemical and structural diversity in eumelanins: Unexplored bio-optoelectronic materials[END_REF][START_REF] Meredith | The physical and chemical properties of eumelanin[END_REF][START_REF] Ambrico | From commercial tyrosine polymers to a tailored polydopamine platform: Concepts, issues and challenges en route to melanin-based bioelectronics[END_REF][START_REF] Bonavolontà | Anomalous evolution of broadband optical absorption reveals dynamic solid state reorganization during eumelanin build-up in thin films[END_REF]. Among the main physicochemical properties of melanin are the well-known strong UV-Vis optical absorption, relatively high ionic/electronic conductivity, photoconductivity (PC), exceptional biocompatibility, antioxidant and free-radical scavenging activities [START_REF] Ambrico | From commercial tyrosine polymers to a tailored polydopamine platform: Concepts, issues and challenges en route to melanin-based bioelectronics[END_REF][START_REF] Wünsche | Eumelanin thin films: Solutionprocessing, growth, and charge transport properties[END_REF][START_REF] Abbas | Control of structural, electronic, and optical properties of eumelanin films by electrospray deposition[END_REF]. They are at the basis of the diverse functions of the natural melanin in the human body as a photoprotective agent and its involvement in some diseases like melanoma skin cancer, Parkinson's and Alzheimer neurodegenerative disease. However for study purpose and applications point of view, synthetic melanin guarantees a more convenient and reliable model instead of natural melanin due to inherent heterogeneity and difficulty in separation of the latter one [START_REF] Ambrico | From commercial tyrosine polymers to a tailored polydopamine platform: Concepts, issues and challenges en route to melanin-based bioelectronics[END_REF][START_REF] Wünsche | Eumelanin thin films: Solutionprocessing, growth, and charge transport properties[END_REF]. Monomeric units of melanin structure are sketched in Fig. 1: (a) 5-6-dihydroxyindole (DHI or HQ), (b) 5,6-dihydroxyindole-2-carboxylic acid (DHICA), (c) indolequinone (IQ), and (d) semichinone (SQ), further details about melanin structure can be found elsewhere [START_REF] Ambrico | From commercial tyrosine polymers to a tailored polydopamine platform: Concepts, issues and challenges en route to melanin-based bioelectronics[END_REF][START_REF] Abbas | Control of structural, electronic, and optical properties of eumelanin films by electrospray deposition[END_REF][START_REF] Meredith | Towards structure-property-function relationships for eumelanin[END_REF].

Previous studies started from characteristics of pellet type melanin samples [START_REF] Ambrico | From commercial tyrosine polymers to a tailored polydopamine platform: Concepts, issues and challenges en route to melanin-based bioelectronics[END_REF], and subsequently developed in various directions, allowed the study of the secondary structure and the dependence of functional properties on the molecular organization and film quality [START_REF] Bonavolontà | Anomalous evolution of broadband optical absorption reveals dynamic solid state reorganization during eumelanin build-up in thin films[END_REF][START_REF] Meredith | Towards structure-property-function relationships for eumelanin[END_REF][START_REF] Bothma | Device-quality electrically conducting melanin thin films[END_REF]. Comprehensive results were obtained in terms of film smoothness, continuity and overall quality, followed by controlled structural, electronic and optoelectronic properties that have connected the fundamental features of the melanin organization, its feasible functionalizations [START_REF] Wünsche | Eumelanin thin films: Solutionprocessing, growth, and charge transport properties[END_REF][START_REF] Abbas | Control of structural, electronic, and optical properties of eumelanin films by electrospray deposition[END_REF][START_REF] Abbas | Structural, electrical, electronic and optical properties of melanin films[END_REF] and practical applications [START_REF] Ambrico | From commercial tyrosine polymers to a tailored polydopamine platform: Concepts, issues and challenges en route to melanin-based bioelectronics[END_REF][START_REF] Ambrico | Hysteresis-type current-voltage characteristics in Au/eumelanin/ITO/glass structure: Towards melanin based memory devices[END_REF][START_REF] Cicco | A water-soluble eumelanin polymer with typical polyelectrolyte behaviour by triethyleneglycol N-functionalization[END_REF][START_REF] Ambrico | Memory-like behavior as a feature of electrical signal transmission in melanin-like bio-polymers[END_REF][START_REF] Ambrico | Melanin layer on silicon: An attractive structure for a possible exploitation in bio-polymer based metal-insulator-silicon devices[END_REF]].

An intriguing aspect of this research is involving the interactions of melanin with other organic or inorganic nanoparticles in hybrid/nanocomposite materials. Most common drawbacks are complete insolubility in organic solvents, the lack of appropriate deposition techniques that prevent the development of homogeneous and reproducible blended thin films, and the lack of a solid conceptual frame to understand the structure-property-function relationships upon the interaction of nanoparticles with synthetic melanin, hindering the full implementation of the bio(electronic) devices. Titanium dioxide (TiO 2 ) nanoparticles are low cost, non-toxic, stable oxide semiconductor with distinctive physical and chemical properties, applied in some photovoltaic devices and several photocatalysts applications. The combination of metal/metal oxide nanoparticles like TiO 2 with organic structured materials is very attractive for the electronic, morphological properties modifications [START_REF] Fakharuddin | Vertical TiO 2 Nanorods as a Medium for Stable and High-Efficiency Perovskite Solar Modules[END_REF][START_REF] Boucle | Hybrid solar cells from a blend of poly (3-hexylthiophene) and ligand-capped TiO2 nanorods[END_REF][START_REF] Garusinghe | Water Resistant Cellulose-Titanium Dioxide Composites for Photocatalysis[END_REF][START_REF] Nam | A New Mussel-Inspired Polydopamine Sensitizer for Dye-Sensitized Solar Cells: Controlled Synthesis and Charge Transfer[END_REF] and towards practical applications. To date, the formation of hybrid functional materials based on TiO 2 and monomer precursors, like DHICA were established using different synthesis methods i.e., DHICA polymerization through coated TiO 2 nanoparticles and solution mixed DHICA/TiO 2 hybrid nanostructures [START_REF] Vitiello | Antimicrobial activity of eumelanin-based hybrids: The role of TiO2 in modulating the structure and biological performance[END_REF][START_REF] Vitiello | 5,6-Dihydroxyindole-2-carboxylic Acid-TiO 2 Charge Transfer Complexes in the Radical Polymerization of Melanogenic Precursor(s)[END_REF], but complete picture of such hybrid materials in the form of thin films, ideal to investigate nanohybrid materials is still lacking.

Hence, melanin-TiO 2 nanostructure is an open issue for understanding the mechanism of hybrid formation, as their properties are strongly dependent on the particle size and aggregation mechanisms that vary while passing from solution to the solid film.

Here, we report the fabrication of melanin blended TiO 2 nanoparticle thin films by solution processed electrospray deposition (ESD) technique. The aim to develop thin films of these kind of hybrid nanostructures was multifold: the foremost, is to enhance the photo absorption for extended range of wavelength by exploiting the broadband absorption of melanin and TiO 2 nanoparticles. Also important is the response of melanin coupled to strong electron acceptor/donor materials. Last but not least, the structure-function relation and mutual dependence of these components must be critically investigated. By the way, properties of melanin (particularly the transport) are complicated by the presence of two kinds of water, the weakly and strongly bounded to the structure [START_REF] Ambrico | From commercial tyrosine polymers to a tailored polydopamine platform: Concepts, issues and challenges en route to melanin-based bioelectronics[END_REF][START_REF] Abbas | Control of structural, electronic, and optical properties of eumelanin films by electrospray deposition[END_REF][START_REF] Abbas | Structural, electrical, electronic and optical properties of melanin films[END_REF]. Adding the TiO 2 which has been widely used for water splitting, purification or treatment applications will bring experimental insights for the charge transport mechanisms and its response towards charge carrier recombination activities. In addition, the motivation of this work was to explore in details the characteristics of melanin and melanin: TiO 2 nanoparticle active layers, when heated at a high temperature as required for many device preparations (T˃ 120°C). To investigate all these issues, we analyzed morphological, structural, electrical and optical properties of the active layer melanin and blend (melanin: TiO 2 ) thin films for as prepared and treated samples, grown by ESD deposition technique looking forward to organic/inorganic photovoltaics and other (bio)electronic device applications.

Experimental details

Synthetic melanin powder, prepared by oxidation of tyrosine with hydrogen peroxide (M8631 synthetic melanin) and TiO 2 nanoparticles (about 25 nm) were purchased from Sigma-Aldrich.

Melanin is notoriously insoluble pigment, but dimethyl sulfoxide (DMSO) is the most effective solvent for its use. We dissolved melanin in Dimethyl Sulfoxide Methanol Solution (DMSO: CH 3 OH; 1:20) with the concentration of 0.2 mg/ml. Similarly, for TiO 2 solution we used the same concentration of 0.2 mg/ml but different solvent (dichlorobenzene). After the preparation of the solution, the melanin and melanin: TiO 2 (with 1:1 ratio) were deposited by electrospray technique 4 on indium-tin-oxide (ITO) and corning glass substrates. To realize the formation of hybrid nanostructures through enhanced interfaces area the melanin and TiO2 proportion were kept the same, without going into the details of ratio optimization, which is essential for the purpose of efficient electronic devices in the future and is beyond the scope of this paper.

The schematic of ESD developed system [START_REF] Ali | Variability of physical characteristics of electro-sprayed poly ( 3-hexylthiophene ) thin films[END_REF][START_REF] Ali | Realization of solution processed multi-layer bulk heterojunction organic solar cells by electro-spray deposition[END_REF] during thin films deposition is presented in the Fig. 2. A solution container was pressurized with the N 2 gas and the flow rate was controlled by measuring the time required to move a solution of 20 µL through 1 cm gap marked at various position on Teflon tube. The flow rate (1.3 µL/s) and applied voltage (2 kV) were kept constant during the deposition [START_REF] Abbas | Control of structural, electronic, and optical properties of eumelanin films by electrospray deposition[END_REF]. ESD melanin and blend films of the average thickness of 200 nm on glass and ITO substrates were characterized with various microscopic and spectroscopic techniques. The morphological studies of these melanin and blend films were performed by Scanning Electron Microscopy (SEM-SU-1500 Hitachi) operating at 2-5 kV and Atomic Force Microscopy (AFM-Agilent's Pico Plus) working in tapping mode with cantilever frequency 160 kHz. The AFM images were analyzed quantitatively by Gwyddion Software [START_REF] Nečas | Gwyddion: an open-source software for SPM data analysis[END_REF]. Optical absorption spectra of melanin and blend films were measured in the ultraviolet, visible and near infrared regions. Absorption measurements were carried out by measuring the absorption light intensity obtained by spectrophotometer (Perkin, Elmer Lambda 950 UV/Vis/NIR).

Photoluminescence (PL) study was carried out by using Dongwoo Optron W500i with He-Cd laser having wavelength 325 nm (3.8 eV) for excitation purpose.

The identification and investigation of functional group vibrations of melanin and blend (melanin: TiO 2 ) were performed using NICOLET 6700 Fourier Transform infrared spectroscopy (FTIR). Argon ion laser with laser lines, of wavelength 514 nm as excitation source was used in dark ambient at room temperature for Raman spectra (Renishaw 1000). Finally, the electrical characterization was taken under the dark and illumination conditions. The current-voltage (J -V) curves were obtained by using a Keithley 4200 semiconductor analyzer in dark and under illumination of an AM1.5 (100 mW/cm 2 ) calibrated with IL1400BL radiometer. Gold stripes were evaporated with a channel length of 50μm onto glass substrates after film deposition to get planar geometry for J -V measurements in both forward and backward voltage sweep directions.

Results and discussion

Film morphology

The morphology of synthetic melanin film is quite different and of better quality than that of natural melanin, due to the successive improvements in the methods of fabrication and the choice of solvents that affect the structure, and thin film quality [START_REF] Bothma | Device-quality electrically conducting melanin thin films[END_REF][START_REF] Lorite | Synthetic melanin films: Assembling mechanisms, scaling behavior, and structural properties[END_REF][START_REF] Ligonzo | Electrical and optical properties of natural and synthetic melanin biopolymer[END_REF]. In the present work, we report the thermal heat treatment effect on the morphology of synthetic melanin film and of its blend with TiO 2 nanoparticles. Previously [START_REF] Abbas | Control of structural, electronic, and optical properties of eumelanin films by electrospray deposition[END_REF], we found that the pure melanin thin films deposited at 3 kV applied voltage by ESD technique showed 2D regions with separated boundaries between flat zones formed on sample surface due to strain and to the process of drying. Therefore, here in this work we effectively lowered the applied voltage to 2 kV, for all the samples. SEM analysis show that the thin films fabricated at 2 kV by controlled electrospray deposition process [START_REF] Abbas | Control of structural, electronic, and optical properties of eumelanin films by electrospray deposition[END_REF][START_REF] Ali | Variability of physical characteristics of electro-sprayed poly ( 3-hexylthiophene ) thin films[END_REF] are quite homogenous, with complete flat coverage of the substrate with planar regular structures without any cracks or boundaries as shown in Fig. 3(a-d). SEM images for the annealed (at 120° C for 5 minutes) samples deposited on glass were presented in Fig. 3(a andb) for melanin and melanin: TiO 2 blended films respectively, while same annealed samples deposited on ITO substrate are in Fig. 3(c andd). Annealing is particularly important in the case of the hybrid films, to reach an acceptable homogeneity. The pristine hybrid film on ITO substrate (not shown here) indicated small clusters of TiO 2 nanoparticles coated and /or wrapped by melanin polymorphic organized layers. After annealing, the homogeneity of the blend film enhanced significantly and moderate distribution of TiO 2 nanoparticles (Fig. 3(b andd)) on the whole substrate was observed. Thermal annealing certainly affects the uniformity of thin films deposited on all kinds of substrates especially the conducting surfaces. In discussing samples deposited on glass substrates (upper panel) vs. samples on ITO substrates (lower panel) in Fig. 3, the latter ones probably exhibited more wetting character in the blend film, preferentially due to the interaction between melanin and titania nanoparticles or interfaces with the substrate. These observations are favorable to conditions of molecular orbital hybridization and functionalization to be employed in real devices. AFM images of melanin and blend (melanin: TiO 2 ) deposited on glass (upper panel) and ITO substrate (lower panel), after heat treatment are reported in Fig. 4, respectively. While comparing annealed melanin thin films grown on ITO with those on glass substrates, the first one shows clear improvement in the surface morphology, in terms of film roughness, homogeneity and oligomers structures planarity, as shown in Fig. 4(a andc). AFM images of blended titania films annealed at 120°C are reported in Fig. 4(b andd). Thermal annealing might affect polymers film thickness, by a more organized molecular spacing and in addition, helping supramolecular aggregation with increased uniformity and film quality. It is evidenced from the images that the signatures of titania nanoparticles are very clear, especially on ITO substrates, and showed almost no difference in root-mean-square roughness (before annealing 1.02 nmafter annealing 1.05 nm) associated with different regions of the blend film after annealing. In case of melanin films in the left panels of Fig. 4, the morphology disclosed overall uniform lengthened platelet [START_REF] Abbas | Control of structural, electronic, and optical properties of eumelanin films by electrospray deposition[END_REF][START_REF] Bothma | Device-quality electrically conducting melanin thin films[END_REF]. Compared to the latter, slightly corrugated surface of melanin: TiO 2 samples (right panel of Fig. 4) was observed when blended with polymers by solution process, obviously due to presence of titania particles agglomeration (although reduced with annealing) [START_REF] Boucle | Hybrid solar cells from a blend of poly (3-hexylthiophene) and ligand-capped TiO2 nanorods[END_REF]. As a final observation, the films grown on conducting surfaces (Fig. 4d) with better adhesion are highly organized in terms of substructures, supported by the optical analysis in the coming section.

Optical absorption

The normalized absorption spectra of thin films melanin and its blend with TiO 2 as prepared on glass and ITO substrates were determined in the region (300-950 nm) and shown in the Fig. 5(a andb), respectively. Before annealing the absorption spectra of melanin, particularly on glass substrates (Fig. 5(a), solid curve), showed the typical wide band character consisting of overall featureless smooth absorption without any apparent edge. However, an increase in absorption spectra in the visible wavelength range (380-750 nm) observed for pure melanin films, after annealing (Fig. 5(a andb), solid and dot curves) is suggesting more compacts films (especially on conducting substrate, probably due to adhesion properties of melanin [START_REF] Mosurkal | Organic photosensitizers with catechol groups for dye-sensitized photovoltaics[END_REF][START_REF] Nighswander-Rempel | Quantitative Fluorescence Excitation Spectra of Synthetic Eumelanin[END_REF]), and improved self-assembly relating to monomeric components bonding. The most intriguing results were obtained after embedding TiO 2 nanoparticles in the melanin matrix particularly on conducting surfaces. The blended (melanin: TiO2) dash and dash dot curves in Fig. 5(a andb) show enhanced absorption spectra compared to melanin, due to the photocatalytic activity of TiO2 nanoparticles and better mutual adhesion during the ESD process [START_REF] Ali | Realization of solution processed multi-layer bulk heterojunction organic solar cells by electro-spray deposition[END_REF][START_REF] Ali | Film forming properties of electrosprayed organic heterojunctions[END_REF]. Moreover, the normalized absorption spectra of melanin blending TiO 2 nanoparticles on ITO substrates showed (Fig. 5b) completely twisted behavior composed of new features different from the melanin characteristic broadband monotonic absorption [START_REF] Ambrico | From commercial tyrosine polymers to a tailored polydopamine platform: Concepts, issues and challenges en route to melanin-based bioelectronics[END_REF][START_REF] Wünsche | Eumelanin thin films: Solutionprocessing, growth, and charge transport properties[END_REF]. These spectra with even broader and dominant absorption (350-800 nm) are similar to the prominent absorption of conventional low band gap conducting polymers in the range of 400-750 nm due to π-π * bonding [START_REF] Boucle | Hybrid solar cells from a blend of poly (3-hexylthiophene) and ligand-capped TiO2 nanorods[END_REF][START_REF] Ali | Variability of physical characteristics of electro-sprayed poly ( 3-hexylthiophene ) thin films[END_REF]. The peak observed near UV region at about 350 -400 nm is due to the fundamental absorption of the Ti-O bond at that wavelength as shown in the inset of Fig. 5(a). Moreover, the wet nature of ESD thin films on ITO substrates reflect the slow growth rate for the dynamics of molecular organization/oligomerization, as compared to the fast growth rate on non-wet insulating glass substrates. These effects on conducting substrates were also accompanied by slight differences in morphology observed in SEM and AFM analysis. In addition, the photocatalytic properties of TiO 2 nanoparticles also played a significant role in the DHICA/DHI polymerization [START_REF] Vitiello | 5,6-Dihydroxyindole-2-carboxylic Acid-TiO 2 Charge Transfer Complexes in the Radical Polymerization of Melanogenic Precursor(s)[END_REF] that alters the absorption spectra of melanin in a different fashion from that of amorphous semiconductor [START_REF] Ambrico | From commercial tyrosine polymers to a tailored polydopamine platform: Concepts, issues and challenges en route to melanin-based bioelectronics[END_REF]. Although melanin layer absorption features on conducting surface in the UV-Vis-NIR range are in agreement with the previous experimental observations [START_REF] Bonavolontà | Anomalous evolution of broadband optical absorption reveals dynamic solid state reorganization during eumelanin build-up in thin films[END_REF] and theoretical simulations [START_REF] Tuna | Onset of the electronic absorption spectra of isolated and π-stacked oligomers of 5, 6dihydroxyindole: An ab initio study of the building blocks of eumelanin[END_REF][START_REF] Stark | Effect of stacking and redox state on optical absorption spectra of melanins --comparison of theoretical and experimental results[END_REF], and attributed to an increase in the degree of πstacking and to the oxidation state of melanin, we emphasize here that the presently observed physical properties are the consequence of a controlled film fabrication by efficient ESD system able to blend melanin with TiO 2 nanoparticles which leads to a photocatalytic oxidative action on conducting surface. These examined absorption features are enhanced under thermal annealing with an increase of two orders of magnitude of absorption coefficient around 600 nm (Fig. 5b) in comparison to pure melanin suggesting the augmented adhesion between the two constituents. In fact, melanin similar to polydopamine [START_REF] Nam | A New Mussel-Inspired Polydopamine Sensitizer for Dye-Sensitized Solar Cells: Controlled Synthesis and Charge Transfer[END_REF][START_REF] Mosurkal | Organic photosensitizers with catechol groups for dye-sensitized photovoltaics[END_REF] shows significantly strong adhesion to TiO 2 nanoparticles in the hybrid structure due to catechol groups.

We recently applied the Tauc model [START_REF] Bonavolontà | Anomalous evolution of broadband optical absorption reveals dynamic solid state reorganization during eumelanin build-up in thin films[END_REF][START_REF] Ligonzo | Electrical and optical properties of natural and synthetic melanin biopolymer[END_REF][START_REF] Sangaletti | Polymerization effects and localized electronic states in condensed-phase eumelanin[END_REF][START_REF] Kim | Electrochemical growth of synthetic melanin thin films by constant potential methods[END_REF] for the calculation of energy gap value from the optical band edge as the absorption curve is not showing any of the exponential behavior of more disordered natural melanin. Equation of the form (αhν) 1/2 ∝ (hν -E g ) according to the Tauc model for indirect band gap semiconductor was used, where α is the absorption coefficient (obtained from the absorption curves in Fig. 5), hν is the incident photon energy on the sample, and E g gives the optical gap value by plotting the term (αhν) 1/2 as a function of energy. The result of extrapolated onset on the horizontal axis of the absorption curves for a set of all films in this study was in the range of 1.25-1.55 eV. The optical gap value 1.4 eV for melanin film deposited on ITO substrates was obtained using the Tauc plot as shown in the inset of Fig. 5(b). The estimated energy band gap values for melanin are slightly dependent on the choice of substrates, annealing and on the interaction of TiO 2 nanoparticles as photocatalytic that induced stacking ordering in the polymeric chains. These values are comparable to the melanin E g values observed in the range from 1.0 to 1.4 eV [START_REF] Bonavolontà | Anomalous evolution of broadband optical absorption reveals dynamic solid state reorganization during eumelanin build-up in thin films[END_REF][START_REF] Abbas | Control of structural, electronic, and optical properties of eumelanin films by electrospray deposition[END_REF][START_REF] Ligonzo | Electrical and optical properties of natural and synthetic melanin biopolymer[END_REF][START_REF] Sangaletti | Polymerization effects and localized electronic states in condensed-phase eumelanin[END_REF][START_REF] Eom | Melanin: A naturally existing multifunctional material[END_REF][START_REF] Capozzi | Raman and optical spectroscopy of eumelanin films[END_REF][START_REF] Sangaletti | Electronic excitations in synthetic eumelanin aggregates probed by soft X-ray spectroscopies[END_REF]. We must be aware of the effect of Mie scattering that may be present at the longer wavelengths in the tail part of the spectra [START_REF] Ambrico | From commercial tyrosine polymers to a tailored polydopamine platform: Concepts, issues and challenges en route to melanin-based bioelectronics[END_REF][START_REF] Bonavolontà | Anomalous evolution of broadband optical absorption reveals dynamic solid state reorganization during eumelanin build-up in thin films[END_REF][START_REF] Abbas | Control of structural, electronic, and optical properties of eumelanin films by electrospray deposition[END_REF][START_REF] Abbas | Structural, electrical, electronic and optical properties of melanin films[END_REF] affecting the energy band edge.

Photoluminescence spectroscopy

To examine further the optical properties of melanin and blend films of melanin: TiO 2 a PL study was carried out at room temperature in order to understand the interaction between the two components. As a comparison, Fig. 6(a andb) show the PL spectra of melanin and blend thin films excited with the excitation wavelength of 325 nm. It is found that different behavior of the PL spectra in the range of 300-950 nm before annealing supports the significant substrate effects obtained previously from morphological and absorption analysis. The emission spectrum for 325 nm (3.8 eV) excitation reached the peaks maximum at around 550 nm obtained from melanin on glass substrates and then decreased monotonically with increasing emission wavelength [START_REF] Eom | Melanin: A naturally existing multifunctional material[END_REF][START_REF] Perna | Fluorescence properties of natural eumelanin biopolymer[END_REF], while the emission spectrum for melanin: TiO 2 blend on glass substrates (Fig. 6a) was reduced due to incorporation of the TiO 2 nanoparticles in the melanin matrix.

Similar emission spectra related to the DHICA anions were obtained with maximum peak intensity at wavelength of 425 nm [START_REF] Vitiello | 5,6-Dihydroxyindole-2-carboxylic Acid-TiO 2 Charge Transfer Complexes in the Radical Polymerization of Melanogenic Precursor(s)[END_REF] compared to 550 nm in this case. Furthermore, it has been shown in the literature that melanin PL quantum yield is small, proved by the probe beam attenuation method and origin of some unexpected observations [START_REF] Meredith | Towards structure-property-function relationships for eumelanin[END_REF][START_REF] Nighswander-Rempel | Quantitative Fluorescence Excitation Spectra of Synthetic Eumelanin[END_REF][START_REF] Nofsinger | Ultrafast Nonradiative Relaxation Dynamics of Eumelanin[END_REF]. We supposed that the reason of unforeseen wide PL (FWHM ~ 1eV) behavior of pure melanin and blend films, on insulating substrates could be that the exciton emission originating from a hole-electron recombination, was most probably due to trapped states on the surface [START_REF] Boucle | Hybrid solar cells from a blend of poly (3-hexylthiophene) and ligand-capped TiO2 nanorods[END_REF], resulting from the combination of disorder of melanin monomers, defects (oxygen vacancies/structural defects due to agglomeration) and impurities including two kinds of water bonded in structure of melanin. Such impurities and defects could be eliminated by thermal, physical or chemical processes. In the case of thin films of organic species such as melanin and its blend with TiO 2 anatase phase over the ITO conductive (Fig. 6b) layer, electron and hole scavengers and the charge recombination mechanism in the visible and up to near infrared region are dominant [START_REF] Nighswander-Rempel | Quantitative Fluorescence Excitation Spectra of Synthetic Eumelanin[END_REF][START_REF] Paulin | Structural and optical properties of soluble melanin analogues with enhanced photoluminescence quantum efficiency[END_REF][START_REF] Zhu | The photoluminescence mechanism in carbon dots (graphene quantum dots, carbon nanodots, and polymer dots): current state and future perspective[END_REF].

The PL spectra after annealing (Fig. 6b) for a set of samples were shifted to an energy gap values near IR region (700-900 nm) compared to that on glass substrates before annealing in the wavelength range 350-550 nm. Such observations are suggesting that the recombination of a hole with an electron due to surface trapped states/ disordering was reduced after annealing. The intense peaks in overall spectra for melanin and titanium dioxide were observed at the high wavelength near IR band gap region (Fig. 6b) for melanin, suggesting larger π-stacking oligomerization related to red-shifts in the absorption spectra and then emission. The intense excitation peaks in annealed PL emission spectra of the thin films started at offset of 1.3-1.45 eV and reach their maximum at 1.55 eV. These observations are in agreement with the estimated band gap values obtained from absorption analysis. The PL peaks (probably affected by spectrometer background) in the range of 1.5-1.65 eV may correspond to different chromophores [START_REF] Ambrico | From commercial tyrosine polymers to a tailored polydopamine platform: Concepts, issues and challenges en route to melanin-based bioelectronics[END_REF][START_REF] Meredith | Towards structure-property-function relationships for eumelanin[END_REF] of melanin DHI and DHICA, the latter one at lower energy compared to DHI that has dissimilar reaction times and extinction coefficients at different wavelengths [START_REF] Micillo | Eumelanin broadband absorption develops from aggregation-modulated chromophore interactions under structural and redox control[END_REF].

In the Fig. 6(b), these observations further elucidate the trend of PL peaks intensity that quenched significantly in a systematic way from the pristine melanin/ITO film (curve-a) by annealing the same sample (curve-b) and then by adding TiO 2 nanoparticles as an electron acceptor with respect to the donor melanin material (curve-e). It reveals the strong interface formation between melanin and TiO 2 nanoparticles as a nanohybrid structures fostering robust devices applications. On the contrary, on insulating surface almost the same PL peaks intensity for thin films of melanin/glass (curve-c) and the blend/glass (curve-d) are observed, suggesting the weak interface formation between melanin and TiO 2 nanoparticles, that results in the TiO 2 nanoparticles PL response at around the wavelength values (450-550 nm).

To put into relation the absorption and PL spectra, we observed an increase in the absorbance with dominants absorption feature for melanin: TiO 2 samples and unusual emission spectra from PL spectroscopy. The comparison of annealed melanin: TiO 2 /ITO absorbance ( in Fig. 5(b), dash-dot curve) and PL spectrum shown in Fig. 6 (curve-e) revealed our observations further.

Here, we must point out that efforts are required to quench completely the PL spectra for the melanin-TiO 2 hybrid nanostructures peculiarly presented here in the near IR band gap region.

For such a complex nanohybrid system, this could be done in the future by balancing the charge carrier mobilities while changing the ratio of melanin: TiO 2 (1:1) nanoparticles that will eventually reduce charge recombination processes.

Raman and Fourier transform infrared spectroscopy

Raman spectra were collected from melanin and its blend with TiO 2 (Melanin: TiO 2 ) films which were deposited on glass and ITO substrates through ESD technique. All ESD films investigated through visual inspection with microscope and by Raman spectroscopic analysis were quite homogenous, which permitted their study by a spatial resolution on the surface of the samples around 1 µm [START_REF] Abbas | Control of structural, electronic, and optical properties of eumelanin films by electrospray deposition[END_REF]. The Raman spectra of melanin are similar to the amorphous carbon graphitic structure having two dominant peaks at about 1600 cm -1 and 1300 cm -1 , which are related to the vibrational modes. These vibrational modes involving different atoms (carbon, oxygen, nitrogen and hydrogen) are organized according to different functional groups, bonded to indolic structure that gives the formation of basic monomeric units of melanin such as DHI, DHICA, SQ and IQ.

The Raman spectra of thin films taken at room temperature consists of Raman active vibrational modes involving these different functional groups like, hydroxyl groups OH, carbonyl groups C=O, carboxylic acid COOH and NH groups, as reported in Fig. 7. In blended samples a slight shift was observed in main G (~ 1600 cm -1 ) and D band (~ 1300 cm -1 ) of carbon structures due to incorporation of TiO 2 nanoparticles of anatase phase. Moreover, the G and D band on ITO substrates were more intense than on glass. The Raman spectrum was fitted using the Gaussian function indicating the melanin (900-1800 cm -1 ) and anatase TiO 2 (400-900 cm -1 ) vibrational modes. The enclosed fitted area of blended Raman spectrum is reported in the left inset of Fig. 7(a) that indicates the presence of TiO 2 nanoparticles modes in the range of 400-900 cm -1 . The spectrum exhibit Raman fingerprints for anatase phase with important modes B 1g (419 cm -1 ), A 1g (517 cm -1 ) and E g (639 cm -1 ) in agreement with previous results [START_REF] Farahani | An Investigation into W or Nb or ZnFe2O4 Doped Titania Nanocomposites Deposited from Blended Powder Targets for UV/Visible Photocatalysis[END_REF].

In the recent literature, nine and eleven components fitted Raman spectrum has been already observed which were correlated with different vibrational modes of melanin [START_REF] Abbas | Control of structural, electronic, and optical properties of eumelanin films by electrospray deposition[END_REF][START_REF] Perna | Vibrational Characterization of Synthetic Eumelanin by Means of Raman and Surface Enhanced Raman Scattering[END_REF]. In Fig. 7(a), The peaks centered at about 1700 and 1775 cm -1 related to the C=O stretching vibrational modes in carboxylic acid or in quinone or ketone group. However, at 1775 cm -1 and above weak peaks are observed corresponding to the stretching of carbonyl functional group vibrations in carboxylic acid [START_REF] Rossella | TiO2 thin films for spintronics application: a Raman study[END_REF]. The pyrrole di-and tri-carboxylic groups are also investigated during the subsequent oxidization of DHI and DHICA, which described the structure of melanin [START_REF] Centeno | Surface enhanced Raman scattering (SERS) and FTIR characterization of the sepia melanin pigment used in works of art[END_REF].

These Gaussian peaks value corresponding to the various vibrational modes for melanin and TiO 2 anatase peaks in the melanin: TiO 2 sample obtained from the fitting of the experimental Raman spectrum are listed in Table 1.

On the other hand, Raman spectra of the melanin on glass and ITO substrates before and after annealing are given in Fig. 7(b). It is important to note that the Raman spectrum of the melanin on glass reflect the structure like graphitic amorphous carbon, with a band D at 1300 cm -1 and a strong band G at 1600 cm -1 [START_REF] Abbas | Control of structural, electronic, and optical properties of eumelanin films by electrospray deposition[END_REF][START_REF] Perna | Vibrational Characterization of Synthetic Eumelanin by Means of Raman and Surface Enhanced Raman Scattering[END_REF]. A small shift was observed in the G band position of melanin spectra deposited on ITO compared to glass substrates, and after annealing compared to as prepared melanin film on ITO. This regular small shift in the position of G strong band after annealing of melanin Raman spectra is similar to aromatic character changing from amorphous carbon graphitic structure to nanocrystalline diamond and/or nanocrystalline carbon graphitic structure. Furthermore, the shift in the G position is related to the optical Tauc gap [START_REF] Ferrari | Interpretation of Raman spectra of disordered and amorphous carbon[END_REF],

supporting our results obtained from absorption and PL analysis. in references [START_REF] Okuda | Mid-infrared absorption spectrum of 5, 6-dihydroxyindole-2-carboxylic acid[END_REF]. A wide band centered at ~3000 cm -1 in both spectra was observed, which is corresponding to O-H stretching due to the presence of water in the melanin structure. IR absorption peaks in the range of 1460-1730 cm -1 are assigned to the bonding vibrations of C═C, C═O and to the stretching of COOH double bonds. Moreover, in the blend thin film (melanin:

TiO 2 ) the broad peaks observed at 3450-2400, 1609 and 720 cm -1 were ascribed to the presence of OH, C═O and Ti-O in the catalyst, and is suggesting the oxidative polymerization in the formation of hybrid nanostructure [START_REF] Erdem | XPS and FTIR Surface Characterization of TiO 2 Particles Used in Polymer Encapsulation[END_REF].

Electrical characterization

The charge transport characteristics of ESD synthetic melanin blended with TiO 2 nanoparticles films were measured under nitrogen-gas condition to avoid any surrounding effects. In Fig. 9 the Current-Voltage (J -V) curves were obtained by using a Keithley 4200 semiconductor analyzer in dark and under illumination of an AM1.5 (100 mW/cm 2 ) calibrated with IL1400BL radiometer. Gold stripes were evaporated with a channel length of 50μm onto 7059 Corning glass substrates after film deposition to get planar geometry for J -V measurements in both forward and backward voltage sweep directions. In principle, more detailed analysis and improved performance could be obtained in the future by sandwiching the blended layer between two electrodes for an ideal devices modeling. At room temperature, the hysteretic behavior of melanin: TiO 2 films as shown in Fig. 9, contracted noticeably compared with hysteretic behavior that was reported in the previous work of ESD melanin films [START_REF] Abbas | Control of structural, electronic, and optical properties of eumelanin films by electrospray deposition[END_REF], due to the presence of shallow trap states and to the existence of two types of water weakly and strongly bounded in melanin pores structures [START_REF] Abbas | Structural, electrical, electronic and optical properties of melanin films[END_REF][START_REF] Ambrico | Memory-like behavior as a feature of electrical signal transmission in melanin-like bio-polymers[END_REF].

Previously, it was described that the electrical transport across the melanin active layers could strongly be affected by hydration state [START_REF] Ambrico | From commercial tyrosine polymers to a tailored polydopamine platform: Concepts, issues and challenges en route to melanin-based bioelectronics[END_REF][START_REF] Wünsche | Eumelanin thin films: Solutionprocessing, growth, and charge transport properties[END_REF][START_REF] Abbas | Structural, electrical, electronic and optical properties of melanin films[END_REF][START_REF] Ambrico | Memory-like behavior as a feature of electrical signal transmission in melanin-like bio-polymers[END_REF] Such effects in the J-V curve for hybrid nanostructures layer were compensated by the presence of TiO 2 as an electron acceptor in the melanin matrix by filling the pores structures, while in case of ESD melanin films these trapped charges were released due to phonon-assisted hopping with increase in temperature. Moreover, the photocatalytic and water splitting characteristics of TiO 2 played crucial role to alter the hydration state for melanin charge transport mechanism in a reorganized structure. To assess the photo response of such active layer for organic/inorganic solar cells, the J-V curves for melanin: TiO 2 films under illumination were obtained. We found a strong photo response in the photoactive layer of melanin: TiO 2 thin film. The resistance of ESD melanin: TiO 2 film is very low at room temperature compared with ESD melanin films [START_REF] Abbas | Structural, electrical, electronic and optical properties of melanin films[END_REF]. It is worth to note that TiO 2 nanoparticles as an electron acceptor semiconducting materials probably enhanced the charge transport towards electrodes by the reduced charge recombination effect in the active layer when added with melanin. These results are in agreement with optical, morphological and structural analysis, and revealed that hybrid melanin: TiO 2 nanostructures are active semiconducting agents that can be employed as potential biocompatible nanomaterials in organic/inorganic bio/ (opto)electronic devices technology.

Conclusions

In this work, we described the formation of hybrid nanostructures of melanin with TiO 2 nanoparticles by ESD technique. Morphological analysis of the ESD thin films revealed quite homogeneous and uniform flat surfaces covering the complete substrate area. After subsequent thermal annealing, the oligomeric aggregates characteristic of melanin, as well as the hybrid nanostructures interfaces area are increased due to the mild distribution of titania particles in the melanin matrix and the strong adhesion between them that strongly influenced the physicochemical properties. The broadband absorption of the melanin: TiO 2 hybrid films also increased as a result of the interaction of TiO 2, nanoparticles especially on the conducting substrates. In fact, after heat treatments the absorption features in the optical absorption and PL characteristics of melanin: TiO 2 layers on ITO substrates were enhanced. These facts preannounced the reorganized growth mechanism of self-assembled supramolecular structures with increasing degree of oligomerization and π-stacking of oligomers. In our opinion this result opens the way to a possible tuning of the band gap values and increase in the External Quantum Efficiency (EQE) in the infrared range of wavelengths. However, further quenching of the PL broad emission peaks is still an open issue for experimental researchers to balance the charge carrier mobility of optoelectronic devices with higher efficiency. These experimental results including: efficient charge transport mechanism, rationalized feature of absorption and quenching of PL emission, robust formation of melanin: TiO 2 hybrid nanostructures (elucidated by Raman and IR spectroscopies), represent a significant step toward future implementations of optoelectronic and bio-electronic/sensor devices using this important bio-polymer. .

Fig. 1 .

 1 Fig. 1. Monomeric units for melanin structure: (a) DHI or HQ, (b) DHICA, (c) IQ, (d) SQ. Red (oxygen atoms), light blue (nitrogen atoms), white (hydrogen atoms), and black (carbon atoms).

Fig. 2 .

 2 Fig. 2. Schematic of the ESD system used for the film growth on respective substrates.

Fig. 3 .

 3 Fig. 3. SEM images comparison: (a) melanin film on glass substrate; (b) blend (melanin: TiO 2 ) on glass substrate; (c) melanin film on ITO substrate; (d) blend (melanin: TiO 2 ) on ITO substrate. Furthermore, all SEM images were from samples annealed at 120° C.

Fig. 4 .

 4 Fig. 4. AFM images in tapping mode of melanin thin film deposited on glass substrate (a), and melanin: TiO 2 blend on glass substrate (b) -(upper panel). AFM image of melanin thin film deposited on ITO substrate (c), and blend (melanin: TiO 2 ) deposited on ITO substrate (d) -(lower panel). Here, we presented the samples annealed at 120° C.

Fig. 5 .

 5 Fig. 5. Normalized absorption spectra of melanin and its blend (melanin: TiO 2 ) films on glass and on ITO substrates are shown in Fig. (a) and (b), respectively. The absorption spectra of TiO 2 nanoparticles deposited on ITO substrate by ESD deposition is reported in the inset of (a) and energy band edges of melanin film on ITO is described in the inset of (b), respectively.

Fig. 6 .

 6 Fig. 6. (a) PL spectra of melanin and blend samples deposited on glass substrates before annealing. (b) PL spectra of melanin and hybrid melanin: TiO 2 samples deposited on insulating and conducting substrates before and after annealing at 120° C for 5 minutes. All PL spectra were recorded with He-Cd laser having excitation wavelength 325 nm (3.8 eV).

Fig. 7 .

 7 Fig. 7. (a) The normalized Raman spectra of blend (melanin: TiO 2 ) deposited on ITO and glass substrates, the inset showed the fitting plot of the experimental Raman spectrum along with chemical structure for both melanin (right) and TiO 2 (left), respectively. (b) Raman Spectra for melanin on ITO and glass substrates, before and after annealing of 120 ºC.

2 the

 2 Raman spectra of blended films confirmed that titania nanoparticles are not perturbing the spectral response of melanin functional group. The best-fitting curve of melanin experimental Raman spectrum composed of ten components is shown in the right inset (zoomed from solid curve) of Fig.7(a). The peaks around 800 to 1000 cm -1 in the melanin structure are due to the deformation of modes of vibration in the pyrrole and indole structure. The broad peaks about 1400 cm -1 overlap different vibrational modes namely C=C, C=N ring stretching in pyrrole structure. The lower intensity bands around 900 -1200 cm -1 are due to the vibration modes of CH, OH and NH with in plane or out of plane deformation. The broad band feature at 1550 and 1622 cm -1 is the contribution to the Raman bands of pyrrole and indole ring stretching vibrations.

Fig. 8

 8 Fig. 8 depicts further the identification and investigation of functional group vibrations of melanin and melanin: TiO 2 hybrid nanostructures with a more comprehensive spatial range by using NICOLET 6700 Fourier Transform infrared spectroscopy (FTIR). The IR spectra indicated clearly that the large absorption due to functional group of melanin and hybrid melanin-TiO 2 nanostructures were characterized with different bonds in their respective regions as depicted on top of Fig. 8. The peak values assigned to different modes of vibrations at 1150-1371 due to C-C, C-O, and at 1460 cm -1 due to C-N vibrations are similar to the reported values

Fig. 8 .

 8 Fig. 8. IR absorption spectra of melanin thin films (lower-solid curve) and melanin: TiO 2 thin films (upper-dot curve) deposited by controlled ESD methods.

Fig. 9 .

 9 Fig.9. J-V curve of a planar blend (melanin: TiO 2 ) structure both in forward and backward voltage sweep directions, measured by two Au electrodes separated by 50 μm. In the inset a planar geometry for J-V measurements on glass is shown.

Table 1 .

 1 Raman Peaks related with modes in melanin blended TiO 2 nanoparticles complex system.

	Raman Peaks (cm-1)	Assignments
		Melanin Peaks in blend (melanin: TiO 2 )
	994 ± 2	O-H out of plane deformation
	1078 ± 2	C-H deformation modes in or Out of plane
	1218 ± 2	N-H in plane deformation and stretching C-C ring breathing
	1290 ± 2	C-O stretching and OH deformation in COOH
	1379 ± 2	Stretching (C-N)/indole ring vibration
	1478 ± 2	C=C and C=N in plane vibration in pyrrole
	1546 ± 2	Pyrrole ring stretching vibration/ C=C aromatic
	1592 ± 2	Indole ring vibration / stretching antisymmetric (COO-)
	652 ± 2	C=C aromatic ring vibration
	1737 ± 2	Stretching C=O in DHICA, Quinone or Ketone
		TiO 2 Peaks in Blend
	639 ± 2	E g mode for anatase TiO 2

Acknowledgments

Mushtaq Ali acknowledges the financial support of Higher Education Islamabad, under the Pakistan Program for Collaborative Research (PPCR-HEC) and the Department of Physics University of Camerino (MC), Italy. We are thankful to Assoc. Prof. N. Abbas for providing absorption spectroscopy facilities, Dr. Zia-ur-Rehman for FTIR and Dr. S. K. Shah for I-V technical assistance.

Author Information

*E-mail:mushtaqali.zada@gmail.com (M Ali)

Declaration of interests

x The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

X The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: