Pierpaolo Pellicori

João Pedro Ferreira

Beatrice Mariottoni

Hans-Peter Brunner

La Rocca

Fozia Z Ahmed

Verdonschot Job

Tim Collier

Joe J Cuthbert

Johannes Petutschnigg

Blerim Mujaj

Nicolas Girerd

Arantxa González

Andrew L Clark

Franco Cosmi

Jan A Staessen

Stephane Heymans

Roberto Latini

Patrick Rossignol

Faiez Zannad

John Gf

Effects of spironolactone on serum markers of fibrosis in people at high risk of developing heart failure: rationale, design and baseline characteristics of a proof-of-concept, randomised, precision-medicine, prevention trial. The « Heart OMics in AGing » (HOMAGE) trial

Keywords: Peptides, Ablative Solutions, Corvidia, Relypsa

Background: Asymptomatic patients with coronary artery disease (CAD), hypertension and/or type-2 diabetes (T2DM) are at greater risk of developing heart failure (HF). Fibrosis, leading to myocardial and vascular dysfunction, might be an important pathway of progression.

Aims:

To investigate the effects of spironolactone on serum markers of collagen metabolism and on cardiovascular structure and function in people at risk of developing HF and potential interactions with a marker of fibrogenic activity, galectin-3.

Methods

: A prospective, randomised, open-label, blinded end-point (PROBE) trial comparing spironolactone (up to 50mg/day) and standard care over 9 months in people with clinical risk factors for developing HF, including hypertension, CAD and T2DM, and elevated plasma concentrations of NT-pro-BNP (125 to 1,000 ng/L) or BNP (35 to 280 ng/L). Exclusion criteria included left ventricular ejection fraction (LVEF)<45%, atrial fibrillation, severe renal dysfunction or treatment with loop diuretics. The primary endpoint was the interaction between change in serum concentrations of procollagen type III N-terminal propeptide (PIIINP) and treatment with spironolactone according to median plasma concentrations of galectin-3 at baseline.

Results:

For the 527 participants enrolled, median (inter-quartile range) age was 73 (69-79) years, 135 (26%) were women, 412 (78%) had hypertension, 377 (72%) CAD and 212 (40%) T2DM. At baseline, medians (IQR) were for LVEF 63 (58-67) %, for left atrial volume index 31 (26-37)mL/m 2 , for plasma ng/L, for serum PIIINP 3.9 (3.1-5.0) ng/mL and for galectin-3 16.1 (13.5-19.7) ng/mL.

Conclusions:

The HOMAGE trial will provide insights on the effect of spironolactone on pathways that might drive progression to HF.

Introduction

Despite advances in care, the prognosis of heart failure is generally poor once it is clinically obvious [START_REF] Rossignol | Heart failure drug treatment[END_REF]. Changes to lifestyle and treating modifiable risk factors, such as hypertension, hyperlipidaemia and diabetes, may have delayed the onset of heart failure but have not reduced the life-time risk. Only a few trials have specifically investigated interventions to delay or prevent the onset of heart failure [START_REF] Cleland | Prevention or Procrastination for Heart Failure?: Why We Need a Universal Definition of Heart Failure[END_REF]. Population screening, using blood tests or cardiac imaging, for early identification or prevention of heart failure has not yet been widely adopted, but there is a growing body of evidence, especially for natriuretic peptides, that such an approach might be useful [START_REF] Ledwidge | Natriuretic peptide-based screening and collaborative care for heart failure: the STOP-HF randomized trial[END_REF][START_REF] Huelsmann | PONTIAC (NT-proBNP selected prevention of cardiac events in a population of diabetic patients without a history of cardiac disease): a prospective randomized controlled trial[END_REF]. Pharmacological approaches to prevention might be most efficient when targeted at those both at greater risk and most likely to respond to a specific intervention. Markers of activity of pathways driving disease progression might provide such mechanistic biotargets, thereby providing a precision-or personalised-medicine approach.

Natriuretic peptides are released by the heart in response to cardiovascular stress. About 10% of adults have increased plasma concentrations of natriuretic peptides [START_REF] Linssen | N-terminal pro-B-type natriuretic peptide is an independent predictor of cardiovascular morbidity and mortality in the general population[END_REF], the proportion increasing with age and the development cardiovascular disease [START_REF] Scirica | SAVOR-TIMI 53 Steering Committee and Investigators. Heart failure, saxagliptin, and diabetes mellitus: observations from the SAVOR-TIMI 53 randomized trial[END_REF][START_REF] Mcgrady | N-terminal B-type natriuretic peptide and the association with left ventricular diastolic function in a population at high risk of incident heart failure: results of the SCReening Evaluation of the Evolution of New-Heart Failure Study (SCREEN-heart failure)[END_REF][START_REF] Bibbins-Domingo | Nterminal fragment ofthe prohormone brain-type natriuretic peptide (NT proBNP), cardiovascular events,and mortality in patients with stable coronary heart disease[END_REF][START_REF] Mureddu | Prevalence of preclinical and clinical heart failure in the elderly. A population-based study in Central Italy[END_REF]. Elevated plasma concentrations are associated with an increase in cardiovascular morbidity and mortality, including heart failure, whether or not the individual has clinically overt disease. Accordingly, low plasma concentrations of natriuretic peptides can be used to avoid unnecessary investigation and high concentrations used to target those in need of further investigation and treatment to delay or prevent the onset of clinically overt heart failure [START_REF] Huelsmann | PONTIAC (NT-proBNP selected prevention of cardiac events in a population of diabetic patients without a history of cardiac disease): a prospective randomized controlled trial[END_REF].

Fibrosis of the myocardium, vasculature and kidney and possibly other organs might be a key pathway of progression from asymptomatic cardiac dysfunction to heart failure [START_REF] Heymans | Searching for new mechanisms of myocardial fibrosis with diagnostic and/or therapeutic potential[END_REF] and therefore a therapeutic biotarget. Accordingly, markers of collagen synthesis and degradation might identify patients more likely to respond to an intervention targeting fibrosis.

Rationale for the trial

Serum concentrations of procollagen type I C-terminal propeptide (PICP) and procollagen type III N-terminal propeptide (PIIINP), indicating increased synthesis of type-1 and type-3 collagen, are increased in HF, regardless of LVEF, and are associated with a worse prognosis [START_REF] Querejeta | Increased collagen type I synthesis in patients with heart failure of hypertensive origin: relation to myocardial fibrosis[END_REF][START_REF] Krum | Relation of peripheral collagen markers to death and hospitalization in patients with heart failure and preserved ejection fraction: results of the I-PRESERVE collagen substudy[END_REF][START_REF] Klappacher | Galectin-3, a marker of cardiac fibrosis, predicts incident heart failure in the community[END_REF].

Serum PICP correlates directly with plasma concentrations of NT-proBNP and left atrial volume suggesting it is influenced by the degree of congestion (14). Increased serum concentrations of PIIINP in people without established CV disease are also associated with a higher risk of developing heart failure and of death (15,16). An increase in Type-I collagen predominates in the failing heart, contributing to increased myocardial stiffness (17). Serum concentrations of collagen type I C-terminal telopeptide (CITP), indicating increased degradation of Type-1 collagen, are also increased in heart failure [START_REF] Klappacher | Galectin-3, a marker of cardiac fibrosis, predicts incident heart failure in the community[END_REF] and associated with an adverse prognosis.

Raised plasma concentrations of galectin-3, a marker of inflammation involved in aldosteronemediated fibrogenesis (18,19), also predict incident heart failure in the general population (20).

Altogether, these findings suggest that there is both increased synthesis and degradation of collagen in heart failure and that high collagen turnover is associated with an adverse outcome.

Serum markers of collagen metabolism are also associated with an increase in myocardial extracellular matrix and therefore, presumably, collagen mass [START_REF] Querejeta | Increased collagen type I synthesis in patients with heart failure of hypertensive origin: relation to myocardial fibrosis[END_REF][START_REF] Klappacher | Galectin-3, a marker of cardiac fibrosis, predicts incident heart failure in the community[END_REF][START_REF] De Boer | Towards better definition, quantification and treatment of fibrosis in heart failure. A scientific roadmap by the Committee of Translational Research of the Heart Failure Association (HFA) of the European Society of Cardiology[END_REF]. It is likely that the pro-fibrotic state is systemic, with most collagen turnover occurring in organs such as bonemarrow and liver, but with the consequences expressed selectively by organs subject to dynamic change, such as the heart and blood vessels. Accordingly, serum markers reflecting collagen metabolism might be used to select patients more likely to benefit from anti-fibrotic interventions for the prevention of heart failure and might also be used as surrogate markers of a therapeutic effect.

Aldosterone plays an important role in regulating renal sodium and potassium exchange, plasma volume and systemic blood pressure. Experimental evidence also suggests that MRA have favourable effects on collagen metabolism [START_REF] Fuller | Mechanisms of mineralocorticoid action[END_REF]. Randomised trials show that MRA are effective anti-hypertensive agents and reduce morbidity and mortality for patients with heart failure and a reduced (HFrEF) and, perhaps, mid-range (HFmrEF) or even preserved left ventricular ejection fraction (LVEF) [START_REF] Pitt | The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators[END_REF][START_REF] Zannad | Eplerenone in patients with systolic heart failure and mild symptoms[END_REF][START_REF] Solomon | Influence of ejection fraction on outcomes and efficacy of spironolactone in patients with heart failure with preserved ejection fraction[END_REF]. These benefits of MRA may be mediated through diuresis, potassium retention, autonomic effects and reduction in pre-and after-load. However, clinical trials in patients with either HFrEF or HFpEF also suggest that administration of an MRA often exerts changes in serum concentrations of markers of collagen turnover in a pattern favouring reduced synthesis (26). This may reflect both direct, systemic effects of MRA on collagen metabolism and the effects of MRA on pre-and after-load that may specifically prevent, retard or reverse myocardial, vascular and renal fibrosis.

Effect of Spironolactone on Serum Markers of Collagen Metabolism in Clinical Trials

(Table 1)

The Randomized Aldactone Evaluation Study (RALES), a placebo-controlled trial of spironolactone in HFrEF, measured serum concentrations of collagen metabolism in a subgroup of 261 patients. Higher serum concentrations of PIIINP were associated with greater morbidity and mortality and, compared to placebo, treatment with spironolactone reduced serum PIIINP.

Amongst patients with a serum PIIINP below median, spironolactone did not reduce death or the composite of hospitalisation and death but exerted a substantial effect on both endpoints amongst those with greater than median serum PIIINP (27). However, there were too few patients in to provide robust evidence of an interaction. Similar patterns were observed for PINP and PICP.

Subsequently, other trials have variably confirmed (28, 29) or refuted (30) these results. Women and those with lower serum potassium concentrations, which might be associated with higher plasma concentrations of aldosterone, had a greater reduction in PIIINP following treatment with eplerenone in one trial (31). Smaller trials of HFpEF have also shown conflicting results (14,32,[START_REF] Deswal | Results of the Randomized Aldosterone Antagonism in Heart Failure with Preserved Ejection Fraction trial (RAAM-PEF)[END_REF]. Trials of patients with obesity and/ or hypertension have more consistently shown reductions in both PIIINP and PICP, as well as improvement in Doppler indices of diastolic LV dysfunction. [START_REF] Kosmala | Fibrosis and cardiac function in obesity: a randomised controlled trial of aldosterone blockade[END_REF][START_REF] Kosmala | A randomized study of the beneficial effects of aldosterone antagonism on LV function, structure, and fibrosis markers in metabolic syndrome[END_REF][START_REF] Ferreira | Potential spironolactone effects on collagen metabolism biomarkers in patients with uncontrolled blood pressure[END_REF]. Overall, the trials suggest that MRA lead to more consistent reductions in serum PIIINP than PICP, markers of synthesis, with little effect on CITP, a marker of collagen

Design of the trial

HOMAGE is a multicentre, proof of concept, prospective, randomised, open-label, blinded endpoint (PROBE) trial in patients at high risk of developing heart failure, comparing spironolactone titrated to a target dose of 50mg/day compared to no additional treatment. The primary endpoint is the interaction between changes in serum concentrations of PIIINP from baseline to nine months and baseline plasma concentrations of galectin-3 in participants assigned to spironolactone or not. The trial was conducted in ten centres in six European Countries:

France (Nancy, Corbeil-Essonnes and Toulouse), Italy (Cortona), United Kingdom (Kingston upon Hull, Manchester and Glasgow), The Netherlands (Maastricht), Germany (Berlin) and Ireland (Dublin). The protocol was approved by the institutional review board or the independent ethics committee for each institution before trial initiation. All patients provided written informed consent before participating in the trial.

Inclusion and Exclusion Criteria (Table 2)

People aged older than 65 years (later amended to >60 years) with established coronary artery disease or at least two criteria indicating CV disease (T2DM, hypertension, microalbuminuria, or an abnormal ECG) were invited to participate. Patients with a pre-existing diagnosis of heart failure or an LVEF <45% or currently treated with a loop diuretic were excluded. Patients with atrial fibrillation or severe renal dysfunction were excluded, as these conditions may cause increased plasma concentrations of natriuretic peptides without other evidence of CV disease.

MRA are contraindicated in severe renal dysfunction or hyperkalaemia (>5.0mmol/L) and therefore such patients were excluded.

At screening, after giving informed consent in writing, a blood test was taken and plasma concentrations of NT-proBNP or BNP measured in local-site laboratories. Plasma concentrations of NT-pro-BNP had to be in the 'window' between 125 and 1,000 ng/L or BNP between 35 and 280 ng/ml. The lower boundary for plasma natriuretic peptide concentration was based on ESC guidelines for the exclusion of heart failure in the presence of symptoms and signs suggesting such a diagnosis [START_REF] Ponikowski | Authors/Task Force Members; Document Reviewers.2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European HF Investigators and Committees. Angiotensin-Neprilysin Inhibition in Heart Failure with Preserved Ejection Fraction[END_REF]. The upper boundary of the 'window' was introduced in order to exclude patients who had a high probability of serious, undiagnosed cardiac or renal disease who required further investigation and treatment rather than participation in this trial.

Although people with an LVEF <45% or with moderate or severe valve disease were excluded, those with left ventricular hypertrophy or diastolic left ventricular dysfunction were not.

Accordingly, patients enrolled in HOMAGE could have cardiac dysfunction on imaging and increased plasma concentrations of natriuretic peptides, thereby fulfilling two of the three diagnostic criteria for heart failure according to ESC guidelines, and for stage B heart failure in the ACC/AHA guidelines [START_REF] Ponikowski | Authors/Task Force Members; Document Reviewers.2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European HF Investigators and Committees. Angiotensin-Neprilysin Inhibition in Heart Failure with Preserved Ejection Fraction[END_REF]38).

Randomization and Blinding

Patients were randomised in a 1:1 ratio to spironolactone or control using random permuted blocks stratified by centre. Randomisation lists for each centre were created by the trial coordinating centre in Leuven, using the statistical software SAS 9.4. Randomisation was carried out via a web-based management system. All persons evaluating key tests, the clinical endpoints committee, and central laboratories were kept blind to treatment allocation.

Trial Assessments (Figure 1)

Patients were screened in primary and secondary care. All patients provided informed consent in writing before undergoing any research related procedures. The first patient was randomised in February 2016. The plan was to randomise 800 patients by the end of 2017 and follow all patients for nine months, completing the trial by a grant-deadline of November 2018. Due to slow recruitment rate, the trial steering committee extended the enrolment period until June 2018 and reduced the minimum period of follow-up to 3 months. Eventually, 877 patients were screened, of whom 527 were randomised. (Figure 2)

Major visits: baseline, one month and end of trial.

Three major visits are planned, at baseline (randomisation), one month and nine months (modified to a minimum of 3 months for patients enrolled after January 2018). The one-month visit was designed to investigate short-term effects of spironolactone that might be attributed to direct effects on fluid and electrolyte balance and the later visit to investigate longer-term effects on cardiovascular structure and function.

At these visits, patients completed quality of life (EQ5D) and symptom questionnaires (appendix) and blood for local and central laboratory analysis is collected, and an ECG and echocardiogram done. Echocardiograms were analysed, blind to allocation, in a core lab located in Nancy (France). Participants also did incremental shuttle walk-tests (ISWT), using a 10m course (the 'shuttle) marked by two cones. The walking speed is determined by bleeps played from a compact disc: after every minute, walking speed is increased. There are up to 12 levels of speed and, potentially, 102 shuttles. Changes of 50-70 metres (5-7 shuttles) are considered clinically meaningful (39). Blood pressure was measured at the end of ISWT. During and after the ISWT, in a subgroup of participants, heart and respiratory rate were measured with a chestworn sensor (Equivital Inc, New York, USA) or heart sounds measured via acoustic cardiography (Audicor, Inovise Medical, Inc., Portland, USA).

Minor Follow-up visits: 7 days, three and six months of follow-up.

The protocol required at least three additional visits, on Day 7 and at 3 and 6 months, when serum potassium and creatinine is measured in order to adjust the dose of spironolactone (Table 3) and to ascertain adverse events.

Assays

Assays were done in a central laboratory. Plasma concentrations of galectin-3 were measured using an enzyme-linked immunosorbent assay (BG Medicine). Serum PIIINP and CITP were measured using a radio-immunoassay (Orion Diagnostica) and serum PICP using an enzyme immune-assay (Quidel Corp). Plasma NT-proBNP was measured using the Roche Elecsys assay (Roche Diagnostics GmbH, Mannheim, Germany).

Trial End-points

Primary Endpoint

The hypothesis was that the effects of spironolactone on serum concentrations of collagen markers would be greater in patients with increased plasma concentrations of galectin-3, a marker of aldosterone-mediated, fibrotic activity. The primary endpoint was the interaction between baseline plasma concentration of galectin-3 and the effect of randomised treatment (MRA or control) on changes, from baseline to nine months, in serum concentrations of PIIINP.

Secondary endpoints

Secondary endpoints will be assessed both at one and nine months and will include other biomarkers of extracellular matrix turnover, PICP (synthesis) and CITP (degradation). We will also assess the effects of spironolactone on weight, serum electrolytes, blood pressure, renal function, symptoms, quality of life, exercise capacity and cardiac function, assessed by echocardiography, and plasma concentrations of NT-proBNP and galectin-3. In addition, new onset atrial fibrillation, heart failure and other serious adverse events will be assessed.

Sample Size Calculation

The trial sample size was determined based on the ability to detect a difference of 0.79μg/l in the change in PIIINP amongst participants assigned to spironolactone or control with a baseline plasma galectin-3 above or below the median value using a two-sided significance level of 5% and 90% power and a standard deviation estimated at 1.73μg/l [START_REF] Kosmala | A randomized study of the beneficial effects of aldosterone antagonism on LV function, structure, and fibrosis markers in metabolic syndrome[END_REF]. The original calculation required 800 participants, with >700 having evaluable serum PIIINP data but, as only 527 patients were randomised, the trial has less power than originally conceived. Nonetheless, a trial of 500 patients provides 80% power to detect an interaction of 0.87μg/l in PIIINP with a twosided significance level of 5% (or 90% power to detect an interaction of 1.0μg/l in PIIINP).

Thus, the trial has sufficient power to provide estimates of the likely effect-size and interaction effects between 0.87 and 1.0μg/l. Further statistical power may be gained through the method of analysis in the final analysis plan, in particular by treating galectin-3 as a continuous variable and using methods for measurements from several time-points. No interim efficacy analyses were planned or done.

Statistical Analysis

Statistical analyses will be carried out using Stata ® version 15.1. Baseline characteristics are presented as median and inter-quartile range for continuous variables and frequencies and percentages for categorical. The primary efficacy analyses will be carried out on the Full Analysis Set, defined as all randomized patients who received at least one dose of the trial medication. Analysis will be conducted according to the intention to treat principle.

Analysis of the primary end-point will be carried out using analysis of covariance (ANCOVA).

A linear regression model will be fitted, including a binary variable to indicate the treatment group, a binary variable to indicate galectin-3 above or below the median and baseline PIIINP.

An interaction term will be included to evaluate the impact of spironolactone in those with galectin-3 above median. Residual analysis will be used to examine the fit of the model to the assumptions of linear regression and data may be transformed to meet the assumptions of linear regression. Sensitivity analyses will be carried out using multiple imputation with chained equations to impute missing baseline and/or final visit values. As this is a proof of concept trial, additional exploratory analyses may be carried out for the primary outcome, if appropriate, as pre-specified in a statistical analysis plan. There are no planned adjustments for multiple comparisons due to the exploratory nature of this trial.

Baseline Characteristics

The median (IQR) age of participants in the HOMAGE trial was 73 (69-79) years and a high proportion were men (74%), perhaps reflecting the use of CAD as a screening strategy (72%).

Most participants (78%) had treated hypertension, but the median systolic blood pressure at entry was still 140mmHg and 25% had a systolic blood pressure exceeding 154mmHg. Most patients had stage II chronic kidney disease (eGFR 60-89 ml/min/1.73m 2 ) and 40% had T2DM (Table 4) Median LVEF was normal at 63% (IQR 58-67%) and median plasma concentrations of NT-proBNP were 214 (IQR: 137-356) ng/L (Table 5). Left atrial volume and the ratio of E/E' were increased and left ventricular mass was at the upper limit of normal. On a symptom questionnaire (appendix), the median score in response to the question "do you get breathless on moderate exertion (eg:-walking quickly, climbing 2-3 flights of stairs)" the median score was 5 (2 -6) indicating that most participants found this amount of exertion troublesome. Baseline serum concentrations of collagen markers were similar to those observed in previous trials of hypertension and heart failure.

Potential impact

As life expectancy increases, so will the prevalence of heart failure (40). Preventing or delaying the onset of heart failure could prolong active life and potentially reduce healthcare costs. Some treatments for hypertension can delay or prevent the development of heart failure in older people, but not all anti-hypertensive agents are similarly beneficial (41-43). Spironolactone has been used to treat patients since 1959. Its side effects are well-known, reversible and, at the doses used in this trial, usually not severe. It is a highly effective treatment for resistant hypertension (44), and is known to reduce morbidity and mortality for HFrEF. MRA also appear to have favourable effects on myocardial fibrosis, which might be a key pathway for the development of heart failure in general, and perhaps specifically HFpEF.

Targeting specific mechanistic pathways involved in the progression to heart failure may increase the efficiency, safety and cost-effectiveness of pharmacological interventions for the prevention of heart failure. Patients with a biomarker profile suggesting increased fibrotic activity might receive greater benefit from an agent directed at fibrosis, such as an MRA.

Targeting treatment at those who have more to gain could also avoid treating those with a less favourable benefit/risk profile.

The HOMAGE trial enrolled people with few symptoms to suggest heart failure but with moderately elevated plasma concentrations of natriuretic peptides and a high prevalence of atrial dilation and left ventricular hypertrophy suggesting diastolic dysfunction. Indeed, patients enrolled in HOMAGE had higher plasma concentrations of NT-proBNP and greater left atrial dilatation than those enrolled in the ALDO-DHF (45) (Table 5), a trial of spironolactone in patients with symptomatic HFpEF. The patients in HOMAGE could be considered to have a "pre-HFpEF" syndrome. However, participants did report, on average, troublesome breathlessness on moderate exertion and therefore others might consider the patients already to have HFpEF. However, plasma concentrations of NT-proBNP were somewhat lower than in large clinical outcome trials of HFpEF, such as PEP-CHF (46), PARAGON-HF (47) or TOPCAT (48) (Table 5), which generally required patients to have symptoms of congestion treated with diuretics, prior hospitalisations for heart failure, echocardiographic evidence of LV disease or left atrial dilation in addition to high plasma concentrations of natriuretic peptides.

Trials of HFpEF have failed to show conclusive benefit, especially when those with HFmrEF are excluded. It is unclear whether women and men with HFpEF respond differently to treatments.

In PARAGON-HF women may have responded better (47). In TOPCAT, the overall effect was similar for men and women, but this masked a greater benefit for men with an LVEF <50% but a higher risk if LVEF was >65%, whereas woman obtained little or no benefit throughout the range of measured LVEF [START_REF] Solomon | Influence of ejection fraction on outcomes and efficacy of spironolactone in patients with heart failure with preserved ejection fraction[END_REF]. Patients with advanced disease may be less likely to respond to interventions. In a post-hoc analysis of the I-PRESERVE trial [START_REF] Anand | Prognostic value of baseline plasma amino-terminal pro-brain natriuretic peptide and its interactions with irbesartan treatment effects in patients with heart failure and preserved ejection fraction: findings from the I-PRESERVE trial[END_REF], for patients with HFpEF who had NT-proBNP below the median value (339ng/L), irbesartan reduced mortality by 25% and heart failure hospitalisation or death by 40% (both statistically significant) but had no benefit for patients with higher NT-proBNP values who were at greater risk. This suggests that whilst patients with less severe diastolic dysfunction may be at lower risk that are also more amenable to treatment. Further credence to this hypothesis comes from the HYVET trial, which showed that a combination of indapamide and perindopril administered to elderly patients with poorly controlled hypertension had a striking reduction in heart failure events and mortality (41). Many of these patients must have had left ventricular hypertrophy, dilated left atria, raised natriuretic peptides and, had they been assessed carefully, symptoms and signs of heart failure; in other words HFpEF. These considerations suggest that an effective method for the management of HFpEF might be early detection and treatment to delay or prevent progression to the more severe manifestations of this condition. Those with advanced disease may be recalcitrant to therapy.

Conclusions

HOMAGE is a proof-of-concept trial that should be a catalyst for further research on biomarkers that might also be biotargets that indicate activation of specific, modifiable pathological pathways. This research approach may help deliver precision-medicine for personalised care.

Further trials are required to ensure that theory translates into practice. serum of patients with idiopathic or ischemic dilated cardiomyopathy and impact on diagnosis and prognosis. Am J Cardiol 1995;75:913-8. ). ~Indicates significant changes during follow-up between the two arms (p<0.05). Δ: change from baseline, or between arms; cells with significant differences are also shaded for ease of reading; ** not indexed; # these values are extremely high, and higher than what has been reported in other studies, which is likely to reflect differences in kits and methods used to measure biomarker circulating levels.

Inclusion criteria

Exclusion criteria Corrected eGFR< 30ml/min/1.73m2, using the MDRD four-variable equation 6. Serum potassium >5.0 mmol/L and serum sodium <125 mmol/L 7. Treatment with an MRA or a loop diuretic (furosemide, bumetanide, ethacrynic acid or torasemide) in the previous three months 8. Potassium supplements or potassium-sparing diuretic at time of enrolment. 9. Atrial fibrillation within one month prior to inclusion 10. History of hypersensitivity to spironolactone or to any of its excipients 11.Patients who require treatment with prohibited medications according to the summary of product characteristics with the exception of ACE inhibitors or angiotensin receptor blockersalthough not their combination 12. Patients unable to give written informed consent 13. Participation in another interventional trial in the preceding month 14. Ability to walk is, in the investigators opinion, clearly limited by joint disease or other locomotor problems or lung diseases rather than by cardiorespiratory fitness

  degradation. The above observations provide the rationale for the « Heart OMics in AGing » (HOMAGE) trial, investigating whether spironolactone favourably alters markers of extra-cellular matrix remodelling for patients at increased risk of developing heart failure. The trial (clinicaltrials.gov: NCT02556450, EudraCT-No.: 2015-000413-48) was funded by the European Commission under the Health Cooperation Work Programme of the 7 th Framework Programme, under the Grant Agreement n. 305507.

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  Stienen S, Rossignol P, Barros A, Girerd N, Pitt B, Zannad F, Ferreira JP. Determinants of anti-fibrotic response to mineralocorticoid receptor antagonist therapy: insights from the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS) and Early Eplerenone Treatment in Patients with Acute ST-elevation 43) Parving HH, Brenner BM, McMurray JJ, de Zeeuw D, Haffner SM, Solomon SD, Chaturvedi N, Persson F, Desai AS, Nicolaides M, Richard A, Xiang Z, Brunel P, Pfeffer MA; ALTITUDE Investigators. Cardiorenal end points in a trial of aliskiren for type 2 diabetes. N Engl J Med. 2012;367:2204-13.

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  Williams B, MacDonald TM, Morant S, Webb DJ, Sever P, McInnes G, Ford I, Cruickshank JK, Caulfield MJ, Salsbury J, Mackenzie I, Padmanabhan S, Brown MJ; British Hypertension Society's PATHWAY Studies Group. Spironolactone versus placebo, bisoprolol, and doxazosin to determine the optimal treatment for drug-resistant hypertension (PATHWAY-2): a randomised, double-blind, crossover trial. Lancet. 2015;386(10008):2059-2068. 45) Edelmann F, Wachter R, Schmidt AG, Kraigher-Krainer E, Colantonio C, Kamke W, Duvinage A, Stahrenberg R, Durstewitz K, Löffler M, Düngen HD, Tschöpe C, Herrmann-Lingen C, Halle M, Hasenfuss G, Gelbrich G, Pieske B; Aldo-DHF Investigators.Effect of spironolactone on diastolic function and exercise capacity in patients with heart failure with preserved ejection fraction: the Aldo-DHF randomized controlled trial. JAMA. 2013;309(8):781-91. 46) Cleland JG, Taylor J, Freemantle N, Goode KM, Rigby AS, Tendera M. Relationship between plasma concentrations of N-terminal pro brain natriuretic peptide and the characteristics and outcome of patients with a clinical diagnosis of diastolic heart failure: a report from the PEP-CHF study. Eur J Heart Fail. 2012;14(5):487-94.

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  Solomon SD, McMurray JJV, Anand IS, Ge J, Lam CSP, Maggioni AP, Martinez F, Packer M, Pfeffer MA, Pieske B, Redfield MM, Rouleau JL, van Veldhuisen DJ, Zannad F, Zile MR, Desai AS, Claggett B, Jhund PS, Boytsov SA, Comin-Colet J, Cleland J, Düngen HD, Goncalvesova E, Katova T, Kerr Saraiva JF, Lelonek M, Merkely B, Senni M, Shah SJ, Zhou J, Rizkala AR, Gong J, Shi VC, Lefkowitz MP; PARAGON-evidence:results from the CHARM Echocardiographic Substudy-CHARMES. J Am Coll Cardiol. 2007;49(6):687-94. 53) Zile MR, Jhund PS, Baicu CF, Claggett BL, Pieske B, Voors AA, Prescott MF, Shi V, Lefkowitz M, McMurray JJ, Solomon SD; Prospective Comparison of ARNI With ARB on Management of Heart Failure With Preserved Ejection Fraction (PARAMOUNT) Investigators.Plasma Biomarkers Reflecting Profibrotic Processes in Heart Failure With a Preserved Ejection Fraction: Data From the Prospective Comparison of ARNI With ARB on Management of Heart Failure With Preserved Ejection Fraction Study. Circ Heart Fail. 2016;9(1). pii: e002551. 54) Solomon SD, Zile M, Pieske B, Voors A, Shah A, Kraigher-Krainer E, Shi V, Bransford T, Takeuchi M, Gong J, Lefkowitz M, Packer M, McMurray JJ; Prospective comparison of ARNI with ARB on Management Of heart failUre with preserved ejectioN fracTion (PARAMOUNT) Investigators.The angiotensin receptor neprilysin inhibitor LCZ696 in heart failure with preserved ejection fraction: a phase 2 double-blind randomised controlled trial. Lancet. 2012;380(9851):1387-95. 55) Zile MR, O'Meara E, Claggett B, Prescott MF, Solomon SD, Swedberg K, Packer M, McMurray JJV, Shi V, Lefkowitz M, Rouleau J.Effects of Sacubitril/Valsartan on Biomarkers of Extracellular Matrix Regulation in Patients With HFrEF. J Am Coll Cardiol. 2019;73(7):795-806. 56) de Boer RA, van Veldhuisen DJ, Gansevoort RT, Muller Kobold AC, van Gilst WH, Hillege HL, Bakker SJ, van der Harst P.The fibrosis marker galectin-3 and outcome in the general population. J Intern Med. 2012 ;272(1):55-64 Homage Design Paper R1_Final 27

  Abbreviations used: Procollagen type I C-terminal propeptide -PICP; Procollagen type III N-terminal propeptide -PIIINP; Collagen type I C-terminal telopeptide -CITP; Nterminal propeptide of collagen I -PINP. Left atrium -LA; Left atrial volume indexed -LAVI; Not reported -NR; Global longitudinal strain -GLS.

Figure 1 Figure 2

 12 Figure 1

Table 1 .

 1 RCTs of mineralo-corticoid receptor antagonists (MRA) investigating effects on serum collagen-derived markers, in patients with Heart Failure and Reduced (HFrEF; top panel) or Preserved (HFpEF; mid panel) Left Ventricular Ejection Fraction, and in those at high risk of developing heart failure (lower panel). Data for two trials of sacubitril/valsartan are also shown. Data are reported as median (Interquartile range) or mean (Standard deviation), when available. *Indicates significant changes during follow-up within same arm (p<0.05

	Trial		RALES 27		AREA-in-CHF 30	EPHESUS	28	PARADIGM-HF 55
	ation		HFrEF		HFrEF			AMI complicated by HFrEF	HFrEF
	Treatment		Spironolactone vs. Placebo	Canrenone vs. Placebo	Eplerenone vs. Placebo	Sac./Val. vs. Enalapril (E)
	N=		261			382			476		2067
	Follow-up		6 months		6 months (12m for echo)	Up to 9 months	8 months
			Controls		MRA	Controls		MRA	Controls		MRA	Enalapril	Sac/Val
	PIIINP -µg/L -pre	4.9 (2.5)	4.9 (2.7)	5.4 (4.0-7.4)		5.3 (3.9-6.9)	4.2 (1.5)		4.2 (1.7)	4.6 (3.6-5.9)
	PIIINP -µg/L -post	5.0 (2.8)	4.0 (1.9)	Δ:+5.0%		Δ:+3.7%	4.5 (6M)		4.0 (6M)~	-1.7%	-4.5%
			Δ: 0.11	Δ:-0.87*~				4.3 (9M)		3.7 (9M)~	Sac/Val vs E: -3% (p=0.086)
	PINP -µg/L -pre	51 (57)		41 (23)	NR		NR	35 (17)		33 (14)	36 (27-48)
	PINP -µg/L -post	57 (76)		36 (18)	NR		NR	44 (6M)		38 (6M)~	+0.6%	-6.2%
			Δ:6		Δ:-5*~				44 (9M)		36 (9M)~	Sac/Val vs E: -6% (p<0.001)
	CITP -µg/L -pre	NR		NR	NR		NR	6.7 (4.0)		6.4 (3.4)	NR	NR
	CITP -µg/L -post	NR		NR	NR		NR	5.3 (6M)		5.0 (6M)	NR	NR
									5.3 (6M)		4.6 (9M)	NR	NR
	PICP -µg/L -pre	129 (47)	139 (117)	NR		NR	NR		NR	NR	NR
	PICP -µg/L -post	142 (79)	131 (113)	NR		NR	NR		NR	NR	NR
			Δ:13		Δ:-8
	LAD -cm -pre	NR		NR	4.1 (0.6)		4.2 (0.8)	NR		NR	NR	NR
	LAD -cm -post	NR		NR	4.1 (0.7)		4.1 (0.8)~	NR		NR	NR	NR
	LAVI -ml/m	2	NR		NR	NR		NR	NR		NR	NR	NR
	Doppler		NR		NR	NR		NR	NR		NR	NR	NR
	Trial		PARAMOUNT	53,54	NCT00505336	32	RAAM-PEF 33	ALDO-DHF	14,45
	Population		HFpEF		HFpEF			HFpEF		HFpEF
	Treatment		Valsartan vs Sac/Val	Eplerenone vs. Placebo	Eplerenone vs. Placebo	Spironolactone vs. Placebo
	N=		301			44			44		381
	Follow-up		36 weeks		Up to 12 months (M)	Up to 26 weeks (W)	1 year
			Valsartan		Sac/Val	Controls		MRA	Controls		MRA	Controls	MRA
	PIIINP -µg/L -pre	5.6 (4.3-6.8)	5.5 (4.4-7.2)	5.0 (2.0)		6.2 (3.3)	6.3 (5.2-8.9)		6.0 (4.3-10.1)	NR	NR
	PIIINP -µg/L -post	5.4 (4.1-7.0)	5.3 (4.2-7.2)	4.7 (3.3) (6M)	5.1 (2.1) (6M)*	6.2 (5.0-9.6) (W14)	4.7 (3.8-8.9) (W14)*~	NR	NR
						7.3 (4.0) (12M) *~	5.7 (3.1) (12M)	5.9 (4.3-8.7) (W26)		5.8 (4.2-11.5) (W26)	NR	NR
	PINP -µg/L -pre	NR		NR	40 (17)		42 (21)	41 (29-60)		39 (26-66)	NR	NR
	PINP -µg/L -post	NR		NR	31 (16)* (6M)	32 (17)* (6M)	45 (35-61)(W14)*		31 (25-56) (W14)~	NR	NR
			NR		NR	48 (20)* (12M)	43 (14) (12M)	43 (35-61) (W26)		30 (22-52)(W26)*~	NR	NR

  Biological risk: NT-pro-BNP values between 125 and 1,000 ng/L or BNP values between 35 and 280 pg/ml 1. Recent wound healing/inflammation: • Surgical procedure, coronary, cerebral or peripheral vascular events or infection in the prior 3 months • Cancer (life limiting or less than 2 years in remission) • Autoimmune disease • Hepatic Disease 2. Pre-existing diagnosis of clinical HF 3. Moderate/severe LV systolic ventricular dysfunction, i.e. LVEF <45% 4. Moderate or severe valve disease (investigators opinion) 5.

	1.Ability to provide written informed consent prior to any study procedure
	2. Age >65years (modified to >60 years during the trial)
	3. Clinical risk factors for developing heart failure, either:
	A. Coronary artery disease (h/o myocardial infarction, angioplasty or
	coronary artery bypass)
	Or
	B. At least two of the following:
	• Diabetes Mellitus requiring Hypoglycaemic Pharmacotherapy
	• Receiving pharmacological treatment for Hypertension
	• Microalbuminuria
	• Abnormal ECG (left ventricular hypertrophy, QRS >120msec,
	abnormal Q-waves)
	4.

Table 2 .

 2 Main inclusion and exclusion criteria.

Table 3 .

 3 Dose adjustments after initiation of spironolactone treatment. Abbreviations used: EOD: Every Other Day; OD: Once daily. After withholding spironolactone due to high serum potassium, it can be re-started at a dose of 25 mg EOD when potassium levels have fallen below 4.5 mmol/L. Spironolactone must be discontinued if serum creatinine ≥220 μmol/L (or eGFR ≤30 ml/min/1.73m 2 ). Treatment may be restarted at the investigator's discretion once serum creatinine is <220 μmol/L and eGFR >30 ml/min/1.73m 2 .

Table 4 .

 4 Baseline characteristics of participants who were randomised or not in the HOMAGE trial. Abbreviations used: CAD -Coronary artery Disease; PCI -Percutaneous Coronary Intervention; CABG -Coronary Artery Bypass Grafting; TIA -Transient Ischaemic Attack; COPD -Chronic Obstructive Pulmonary Disease; ACE-I -Angiotensinconverting enzyme inhibitors; ARB -Angiotensin receptor blockers ; CCB -Calcium channel blockers; MRA-mineralocorticoid receptor antagonist; eGFR -Estimated glomerular filtration rate; BMI-Body Mass Index; NYHA -New York Heart Association; LV -Left Ventricular; EQ5D -EuroQol-5D. * Includes: Left Ventricular Hypertrophy, QRS >120msec, pathological Q-waves and standard deviation of patients reported to have normal or mild diastolic dysfunction and predominantly in sinus rhythm; # of which 26% were mineralocorticoid receptor antagonists; ~ required recent heart failure hospitalisation (within 9 months from screening) or elevated NT-proBNP (>300 ng/L if sinus rhythm, > 900 ng/L if atrial fibrillation) as inclusion criteria; ^ required recent heart failure

Acknowledgments: Authors are immensely grateful to Sylvia Van Haren, Heiko Breek and Stéphanie Grosjean for their valuable support throughout the duration of this trial. The research leading to these results has received funding from the European Union Commission's Seventh Framework programme under grant agreement N° 305507 (HOMAGE). We acknowledge the support to SH from the Netherlands Cardiovascular Research Initiative, an initiative with support of the Dutch Heart Foundation, CVON2016-Early HFPEF, 2015-10, CVON She-PREDICTS, grant 2017-21. We acknowledge the support to SH of the ERA-Net-CVD project MacroERA, 01KL1706. JF, NG, PR and FZ are supported by a public grant overseen by the French National Research Agency (ANR) as part of the second "Investissements d'Avenir" program FIGHT-HF (reference: ANR-15-RHU-0004) and by the French PIA project "Lorraine Université d'Excellence", reference ANR-15-IDEX-04-LUE, and by the Contrat de plan Etat-lorraine and FEDER lorraine.

02556450; European Commission 7th Framework Programme; Grant Agreement n. 305507).

Conflict of interest : PP, BM, FC, FZA, SH, JAS, BM, JJC, AG, PBLR, PJ did not report any conflict of interest. PR reports personal fees (consulting) for Idorsia and G3P, honoraria from AstraZeneca, Bayer, CVRx, Fresenius, Grunenthal, Novartis, NovoNordisk, Servier, Stealth Society of Cardiology (ESC). Developed with the special contribution of the Heart Failure Association (HFA) of the ESC. Eur J Heart Fail. 2016;18:891-975.


Marco Cirillo

Tangi Aubert

Raquel Gomes

Rik Van Deun

Philippe Emplit

Amelie Biermann

Holger Lange

Christian Thomsen

Edouard Brainis

Zeger Hens

Amelie Bier- Mann

'Flash' Synthesis of CdSe/CdS Core-Shell Quantum Dots

Keywords: semiconductor, nanocrystals, giant core/shell, Raman, non-blinking

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Quantum dots (QDs) show unique optical properties that make them a widely studied material for applications in several fields such as photonics, optoelectronics and biotechnology. [1][2][3][4] However, due to their nanometer size, the optical properties of QDs are strongly dependent on the properties of their surface. For instance, surface defects and poor surface passivation with organic ligands can lead to enhanced trap state emission or non-radiative recombination and consequently to a low photoluminescence quantum yield (PLQY) and photobleaching. 5 Moreover, an undesired characteristic of many semiconductor QDs is the on-off blinking of their emission. This blinking behavior has been extensively studied and is often explained by the recombination of electron-hole pairs through Auger processes. 6,7 To overcome these issues, the design of heterostructures such as core-shell QDs has been widely investigated. Indeed, capping QDs with a larger bandgap material results in an improved surface passivation and a better confinement of the excited carriers, thus decreasing trap emission and increasing PLQY. Core-shell structures have also proven to be very promising for non-blinking QDs. 8- 13 In particular, for CdSe/CdS core-shell QDs both the presence of large CdS shells [10][11][12] and alloyed interfacial layers 13 have been reported as efficient ways to decrease Auger recombination, and consequently reduce or even suppress blinking.

Several procedures have been developed for the synthesis of CdSe/CdS core-shell QDs, including continuous or layer-bylayer shell growth. 11,14,15 The most widely spread method is probably the successive ion layer adsorption and reaction process (SILAR), a layer-by-layer approach that allows for a precise tuning of the shell thickness and that has been successfully used for the synthesis of so-called 'giant' CdSe/CdS QDs featuring more than 10 CdS layers. 10,12,16 Many efforts have been devoted in the past years toward a better understanding of the synthetic parameters of this SILAR procedure in order to achieve optimal morphological and optical properties. 12,16,17 However, this method remains time consuming and complex as different temperatures, annealing times, or precursor concentrations are required depending on the deposited element (Cd or S), the core size, or the number of CdS layers. 12,16,17 Although 'giant' CdSe/CdS QDs were successfully synthesized with annealing times of 20-30 minutes per CdS layers, 16 Ghosh et al. recently reported that optimal optical properties could only be achieved with annealing times of 3.5-4 hours per CdS layers, 12 which corresponds to more than two days of continuous synthesis to grow 15 CdS layers. In addition, this method uses relatively low temperatures (below 250 °C). This can lead to residual strain and defects, two elements known to deteriorate PLQY [18][19][20] and promote blinking of the QDs. 21 Here we report a new, fast and efficient method for the synthesis of CdSe/CdS dot-in-dot QDs. This method, denoted as 'flash' synthesis, consists of a seeded growth at relatively high temperature (330 °C). This approach is adapted from a procedure reported in the literature for the synthesis of anisotropic heterostructures such as CdSe/CdS dot-in-rods 22 and tetrapods, 23,24 but which was also extended to the synthesis of (ZnSe/CdS)/CdS heterostructured nanorods. 25 In the present work, the method was modified to promote the isotropic growth of CdS shells on CdSe seeds through the use of a carboxylic acid instead of phosphonic acids. With this method, CdS shells with controlled thicknesses of up to 6.7 nm were grown in only 3 minutes while keeping state-of-the-art optical properties. The structure and morphology of the synthesized QDs were characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM). Raman spectroscopy was used to address the possible alloy formation at the core-shell interface. Finally, the optical properties of the CdSe/CdS QDs were characterized by means of UV-Vis absorption, steady-state and time resolved photoluminescence spectroscopies, highlighting the high PLQY and the low blinking rates of these QDs.

EXPERIMENTAL SECTION

Synthesis of the wurtzite CdSe QDs. CdSe QDs with a wurtzite crystal structure were synthesized according to a procedure described in the literature. 22 Briefly, 0.06 g of cadmium oxide (CdO, 99.998% Puratronic, Alfa Aesar), 3 g of tri-octylphosphine oxide (TOPO, 98%, Alfa-Aesar) and 0.280 g of octadecylphosphonic acid (ODPA, 98%, PCI) were mixed in a 25 mL three neck flask. The mixture was heated to 150°C under a vacuum for one hour. The solution was then put under a nitrogen atmosphere and heated to 345°C. As soon as the solution became colorless, indicating the dissolution of CdO, 1.8 mL of tri-octylphosiphine (TOP, 97%, Strem) were injected. After the temperature had recovered, a solution containing 0.058 g of selenium (99.999%, Sigma-Aldrich) dissolved in 0.360 g of TOP was injected. The reaction time was varied in order to tune the size of the QDs. Then, the reaction was quenched by a sudden drop of the temperature using a water bath and allowed to cool down to room temperature. The nanocrystals were purified by addition of isopropanol and methanol, centrifugation at 3000 rpm for 10 minutes and redispersion in toluene. The purification was repeated three times. The size of the particles was determined from the position of the first excitonic absorption peak using the sizing curve of Mulvaney et al.. 26 The concentration of the QD dispersion was determined based on the absorbance at 350 nm. 27 Flash synthesis of the CdSe/CdS dot-in-dot QDs. The synthesis of the CdSe/CdS dot-in-dots was performed by adapting the method developed by Carbone et al. for the synthesis of CdSe/CdS dot-in-rods, 22 except that a carboxylic acid (oleic acid) was used instead of phosphonic acids. CdO and oleic acid (OA, 90%, Sigma-Aldrich) were mixed with 3 g of TOPO in a three neck flask. The amount of CdO was varied between 0.5 and 3.47 mmol in order to tune the thickness of the CdS shell as detailed in the result section. The OA was introduced with an excess of 4 mmol considering that two equivalents of OA are needed to dissolve one equivalent of CdO. The reaction mixture was heated to 120°C while flushing with nitrogen for one hour. The temperature was then increased to 330°C. As soon as the solution became colorless, 1.8 mL of TOP were injected. After the temperature had recovered, 1.8 mL of a solution containing 87 nmol of CdSe core QDs and sulfur in TOP were injected. The sulfur was always kept in a 3 to 4-fold excess as compared to cadmium. After 3 minutes, the reaction was quenched by a sudden drop of the temperature using a water bath, followed by the injection of 10 mL of toluene. The nanocrystals were purified by the addition of isopropanol and methanol, centrifugation at 3000 rpm for 10 minutes and redispersion in toluene. The purification was repeated three times. The sizes of the coreshell structures were determined by TEM analysis and the numbers of CdS monolayers (MLs) grown were estimated by considering half of the wurtzite CdS c lattice parameter (0.3375 nm) as the thickness of one CdS monolayer.

Material characterization. Bright field transmission electron microscopy (TEM) images were taken using a Cs corrected JEOL 2200 FS microscope. The crystal structure of the QDs was determined by X-ray diffraction (XRD), using a Thermo scientific ARL X'TRA diffractometer. XRD samples were prepared by dropcasting a dispersion of QDs in an 80:20 hexane:heptane mixture on a glass slide. Absorption spectra were taken using a Perking Elmer Lambda 950 spectrometer. Photoluminescence measurements were done on an Edinburgh Instruments FLSP920 UV-vis-NIR spectrofluorimeter. For steady-state measurements, a 450W xenon lamp was used as the excitation source. The time resolved measurements were performed using a picosecond pulsed LED (pulse width 872 ps) as the excitation source. The signals were collected with a Hamamatsu R928P PMT detector, which has a response curve between 200 and 900 nm. All steady-state emission spectra were recorded for an excitation wavelength of 365 nm and were corrected for the detector sensitivity. The time resolved measurements were performed for an excitation wavelength of 331 nm with a bandwidth of 9.4 nm, which are the characteristics of the pulsed LED, and by collecting the signal at the maximum of emission. Photoluminescence quantum yields (PLQY) of the samples were determined for an excitation wavelength of 365 nm and by comparison with Coumarin 2, which has a known PLQY of 92% in ethanol. [START_REF] Montalti | Handbook of Photochemistry[END_REF] Blinking experiments were performed with a microluminescence setup. For these experiments, the QDs were deposited at very low concentration on a Suprasil® 2B quartz slide by spin coating. A single QD was excited using a pulsed laser at λ = 445 nm with a repetition rate of 2.5 MHz, and the emission of the QD was detected using a PerkinElmer photon counting diode (model SPCM-AQRH). Raman spectroscopy was used to study the internal structure of the core-shell QDs. Raman spectra were recorded in backscattering geometry using a Dilor XY800 triple monochromator Raman spectrometer with an N2 cooled charge-coupled-device detector. Laser excitation was provided by the 488 nm line of an Ar + -laser (Coherent). Each spectrum was calibrated with respect to neon and argon lines. This set-up provides an accuracy of the Raman shift of ± 0.4 cm -1 . The samples were prepared by dropcasting the QD solutions on Si wafers. After solvent evaporation, the wafers were transferred to a commercial cryostat (Oxford Instruments). All Raman measurements were performed at liquid helium temperature.

RESULTS AND DISCUSSION

Influence of oleic acid. During preliminary experiments and in a first attempt to synthesize CdSe/CdS dot-in-dots, we used the same amount of carboxylic acid (oleic acid) as of phosphonic acids (octadecylphosphonic acid and hexadecylphosphonic acid) reported in the method of Carbone et al. for the synthesis of CdSe/CdS dot-in-rods. 22 This means no excess of OA and just the two equivalents necessary to dissolve the amount of CdO. The TEM image of the result (Figure 1a) immediately revealed that this simple modification of the synthesis protocol led to the formation of spherical QDs. As a matter of fact, compared to phosphonic acids, oleic acid binds more loosely [START_REF] Gomes | [END_REF] and not selectively to the different facets of wurtzite CdSe nanocrystals. 30 Thus, the use of oleic acid results in an isotropic growth of the CdS shell instead of the preferential growth along the wurtzite c-axis observed with phosphonic acids. 22 XRD analyses evidenced that the CdSe/CdS dot-in-dots have a wurtzite structure (Figure 2a), indicating an epitaxial growth of the CdS shell on the wurtzite CdSe cores. The emission spectrum of this first sample (Figure 2b) featured, in addition to the main emission peak centered at 632 nm, another peak at 490 nm (inset Figure 2b). As the emission of the injected 4 nm sized CdSe cores should appear at around 600 nm, we attribute this peak to CdS nanocrystals, homogeneously nucleated during the reaction. Thus, even if the use of a carboxylic acid successfully promoted the isotropic growth of a CdS shell, the synthesis of CdSe/CdS dotin-dots still needed optimization. To prevent the nucleation of separate CdS QDs, the synthesis was repeated using an excess of 4 mmol of OA. The TEM image (Figure 1b) shows that the resulting QDs have a larger size than when synthesized without OA excess (16 and 8 nm respectively for 4 nm cores in both cases). In this case, all the cadmium was consumed for the formation of the shell. Due to the thicker CdS shell, the emission spectrum of the CdSe/CdS QDs synthesized with an excess of OA shifted to longer wavelengths (Figure 2b). More importantly, the emission spectrum of this sample does not show any additional peak, confirming that there is no homogeneous nucleation of separate CdS QDs. Thus, an excess of OA was necessary to prevent the formation of individual CdS QDs. This result is in line with the idea that an excess of acid increases the solubility of the growth species, 31 which will favor shell growth over homogeneous nucleation. Additional preliminary experiments showed that the concentration of sulfur did not play any important role unless it was introduced in a more than 6-fold excess compared to cadmium. In this case, the syntheses resulted in highly polydisperse particles. Thus, only the effect of Cd concentration was investigated and is reported in the following.

Tuning the thickness of the CdS shell. To investigate the effect of cadmium concentration, CdSe/CdS QDs were synthesized starting from 2.5 nm and 3.5 nm sized wurtzite CdSe cores, and using different amounts of CdO as summarized in Table 1. Figure 3 shows the TEM images and size distributions of samples QD1, QD2 and QD3 (see Supporting Information Figure S1 for samples QD4 to QD6). In Figure 4a, the average diameters of the CdSe/CdS QDs, determined from TEM images, are plotted against the amount of cadmium used. It appears that the size of the QDs, i.e. the thickness of the CdS shell, increases with the amount of cadmium involved in the synthesis. As illustrated in figure 3d the dispersion of the size distribution increases with the thickness of the CdS shell.. More precisely, the volume of the CdS shell reported on Figure 4b increases linearly with the amount of cadmium. The experimental sizes/volumes were always found to be in good agreement with the theoretical ones, indicating that all the cadmium introduced in the synthesis has reacted. Thus, owing to this full chemical yield of the shell formation reaction, the thickness of the CdS shell can be efficiently controlled by modifying the initial amount of CdO. In addition, the process is considerably faster than what is reported in the literature up to date. For example, a 6.7 nm thick shell (samples QD3 and QD6) corresponding to ca. 20 MLs of CdS was grown in no more than 3 minutes, whereas it could have taken up to several days to obtain the same result using the SILAR method. Raman analysis of the CdSe/CdS QDs. Besides enhanced passivation, core-shell structures have also proven to be of interest for reducing the Auger recombination rate, which plays a role in blinking processes and multi-exciton recombi-nation. 13 This is thought to be linked to a graded interface resulting from an alloying of the core-shell interfacial region. 32 We recently demonstrated the potential of Raman spectroscopy to investigate the structure of this interface. 33 Thus, we applied Raman spectroscopy on the 'flash' CdSe/CdS heteronanocrystals. Samples with thicker CdS shells show mostly a bulk-like CdS vibration and the CdSe-related features are relatively weak, making a detailed discussion difficult (see Supporting Information, Figure S2). Figure 5a shows the Raman spectrum of the two exemplary samples with moderately thick shells, QD1 and QD4. The intensities were normalized to the CdS-related peak around 300 cm -1 . The spectra feature CdS-and CdSe-related Raman modes and (mixed) overtones. On one hand, the CdS-related peaks are not significantly different between the two samples. This also applies to the first CdS-overtone around 600 cm -1 . The region in between fundamental and overtone shows CdSe-CdS combined modes. The proximity of the fundamental CdS-related peak to the CdS longitudinal optical phonon (LO) bulk frequency (305 cm -1 ) [START_REF]Cadmium sulfide (CdS) phonon wavenumbers, mean square displacements[END_REF] indicates a homogeneous shell with possible strain due to the lattice mismatch being mostly relaxed through the outer layers. On the other hand, the CdSe-related peaks are subject to stronger changes between the different samples. Figure 5b displays the Raman spectra limited to the frequency range of the CdSe-related vibrations. The spectra were normalized to the most prominent peak around 212 cm -1 . This peak is asymmetric and can be only well described by the sum of three Lorentzian functions. The background was accounted for with a linear function and a single Lorentzian function for the CdS shoulder at higher wavenumbers. The three underlying components for each sample are included in the figure. The peaks are significantly shifted in frequency, relative to bulk and between the samples. The most prominent feature around 216 cm -1 can be attributed to the CdSe LO. 33 Its shift relative to the bulk LO frequency (212 cm -1 ) 35 can be understood by a significant amount of compressive strain being present in the lattice. As the amount of core material is comparably small, the strain caused by the lattice mismatch between CdSe and CdS cannot relax and average out as it is the case for the CdS shell. Then, the strainand related frequency shiftis different between the samples due to difference in core size and shell thickness. The shoulder below 200 cm -1 can be attributed to surface optical phonon (SO) which exist in finite sized crystals. 36,37 The third feature, the shoulder around 209 cm -1 was previously attributed to an intrinsic vibration of a Cd(Se,S) alloy, stemming from a significant amount of alloyed material at the interface between the CdSe core and the CdS shell. 33 The intensity of this mode relative to the respective CdSe LO intensity is different between the samples, probably due to a comparable amount of alloyed material relative to a different amount of core material. This is in line with the different core diameters, and thus different surface/volume ratios, between the two samples. A comparison with the spectra discussed in the previous work about SILAR particles 33 suggests a similar interface composition, though the thickness of the interfacial layer cannot be estimated based on the available data. Thus, despite the fact that the reaction takes place in only 3 minutes, these Raman analyses show that the atoms at the core-shell interface still have enough time to rearrange and form alloyed layers, just as in other synthesis methods. Optical properties of the CdSe/CdS QDs. The optical properties, i.e. wavelength position of the emission peak, full width at half maximum (FWHM), lifetimes (τ1 and τ2) and PLQY of all the samples are reported in Table 1. The exact peak wavelength of the first excitonic absorption peak could hardly be determined, especially in the case of large CdS shell thicknesses. Indeed, as illustrated in Figure 6a and its inset for samples QD1 to QD3 (see Supporting Information Figure S3 for samples QD4 to QD6), the absorption spectra of the QDs are largely dominated by the absorption of the CdS, which represents most of the volume fraction of the QDs. For illustration, the emission spectra of samples QD1 to QD3 are shown in Figure 6b (see Supporting Information Figure S3 for samples QD4 to QD6). As expected, the emission spectra of the CdSe/CdS QDs shift towards longer wavelengths when the thickness of the CdS shell increases. This is due to the delocalization of the electron from the core into the shell following the small conduction band offset between CdSe and CdS. Figure 6b also shows that the emission peak becomes broader when the thickness of the CdS increases. Indeed, the FWHM values, which are reported in Table 1, reflect the concomitant increase of the size distribution already observed in Figure 3d. Nevertheless,even for the largest QDs the FWHM was always below 70 nm , which is suitable for many optical applications. From point of view of the photoluminescence lifetime, the decays reported in Figure 6c for samples QD1 to QD3 (see Supporting Information, Figure S3 for samples QD4 to QD6) were fitted with a double exponential. A short lifetime (τ1) and a longer one (τ2) were obtained for each samples and these values are reported in Table 1. The short lifetime can fairly be attributed to non-radiative processes involving the surface of the nanocrystals, and the longer lifetime corresponds most likely to the excitonic recombination. As expected, the growth of thicker CdS shells results in an increase of both lifetimes, which is due to a decrease of the overlap of the exciton wave functions. As for the red shift of the emission spectra, due to a smaller offset between the conduction bands of the CdSe and the CdS shell, the electrons are delocalized in the entire volume of the QD whereas the holes remain mainly confined in the core. 11 We did not observed any plateau value of the lifetime as reported by Ghosh et al., even if some of our shell volumes were larger than the 750 nm 3 threshold value these authors reported. 12 In core-shell QDs, with the highest PLQY values typically being observed for 2-3 monolayers, the subsequent PLQY decrease with additional shell layers is often explained by higher strain and defect densities induced by lattice mismatch or incoherent growth of thicker shells. [18][19][20] The CdSe/CdS QDs reported here showed PLQYs of 40-50% in the case of shells corresponding to about 9 CdS MLs (samples QD1 and QD4), 25-30% in the case of shells corresponding to about 15 CdS MLs (samples QD2 and QD5) and still 10-15% for QDs with up to almost 20 CdS MLs (samples QD3 and QD6). At similar shell thicknesses, these PLQY values are comparable or even higher than those obtained with a fully optimized SILAR procedure. 12 This is probably a benefit of the high synthesis temperature that allows a continuous coherent growth of the CdS shell, resulting in nanocrystals with high crystallinity. S3 for samples QD4 to QD6). The emission spectra were recorded for an excitation wavelength of 365 nm. The photoluminescence decays were recorded for an excitation wavelength of 331 nm.

In addition to the sharp and bright emission, CdSe/CdS QDs prepared with this 'flash' synthesis can show very low blinking behavior. In Figure 7 single QD measurements are reported for sample QD5. To ensure that the measured emission originated from a single photon emitter, anti-bunching experiments were performed (see Supporting Information, Figure S4). In Figure 7, the black curves represent the intensity of the luminescence, i.e., the number of detected photons per timebin of 10 ms, as a function of time, and the red curves show the corresponding statistical distribution of the number of counts. In Figure 7a, the fluence of each excitation pulse was set to produce an average number of νav = 0.15 excitons per pulse. During the 400 s of the record not a single off-period has been seen. In addition, the statistical distribution of the number of counts is single-peaked and has a mean value of 107 ± 21.2 counts. The fact that the distribution is broader than a Poisson distribution indicates that, despite no off-period is seen, the PLQY randomly changes in time by about 10% of its average value. These fluctuation periods are clearly seen on the black curves (Figure 7). Schlegel et al. already reported that fluctuating dynamic quenching processes could occur even at a single QD level depending on its local gaseous environment. 38 Indeed, photoluminescence measurements of QDs in different controlled atmospheres already showed that both water or oxygen molecules can have independent and synergistic effects on their optical properties. 39 Figure 7b shows the luminescence of the same QD, but using a stronger excitation (νav = 0.56). The QD again emits without intermittency during 400 s except for a short time interval of a few seconds around t = 242 s. In Figure 7b, the displayed time interval is reduced for a better visualization of the off-period. The statistics of the number of counts per 10-ms time-bin now shows a doublepeaked structure. The main peak (394 ± 86 counts) corresponds to the on-state while the small one (47 ± 10 counts) corresponds to the off-state. Thus, it appears that, despite the high excitation level, nearly no blinking occurred. A further increase of the excitation power tended to increase the duration of the off-periods which where nevertheless limited. It appears that the Auger processes responsible for blinking are strongly, if not completely, suppressed in the CdSe/CdS 'flash' QDs. This can be explained by the very thick CdS shell and the delocalization of the electron in this very large volume. [10][11][12] Chen et al. already reported a low blinking behavior of CdSe/CdS QDs with similar shell thickness. 10 However, we could achieve a similar result with a 3 minutes synthesis instead of several days using the SILAR approach. Furthermore, the observed alloyed interface between the CdSe core and CdS is of importance. García-Santamaría et al. recently demonstrated the importance of these alloyed interfaces on the reduction of Auger recombination through a "smoothing" of the confinement potential. 13 A smoothing of this potential was also reported by Wang et al. as an important parameter for the absence of blinking in their CdZnSe alloyed QDs coated with ZnSe shells. 9 More recently, Bawendi et al. reported nonblinking CdSe/CdS QDs with relatively thin CdS shells, 40 claiming that this was the result of the high synthesis temperature (310 °C) and the slow CdS formation which produces highly crystalline shells. The 'flash' CdSe/CdS QDs combine most of these characteristics, i.e. thick CdS shells, alloyed interfaces and high crystallinity thanks to high synthesis temperature, which explains the low blinking observed.

CONCLUSION

In conclusion, a new fast and efficient method for the synthesis of CdSe/CdS core-shell QDs was successfully developed. In this method, referred to as 'flash' synthesis, the use of a carboxylic acid (OA) instead of phosphonic acids led to the isotropic growth of CdS shells on wurtzite CdSe cores. It was found that an excess of OA compared to cadmium was necessary to prevent the secondary nucleation of individual CdS QDs. The thickness of the CdS shell could then be easily tuned by changing the amount of cadmium involved in the synthesis. This reaction is very fast, with full chemical yield, allowing the growth of very thick shell (up to 6.7 nm in the present study) in no more than 3 minutes. The synthesized CdSe/CdS QDs showed narrow emissions with enhanced PLQY even for very thick CdS shells. Finally, Raman spectroscopy analyses evidenced an alloying of the interface between the CdSe core and the CdS shell, which together with the thick CdS shell and probable high crystallinity, resulted in an important reduction of the Auger recombination and therefore of the blinking behavior as evidenced by single QD measurements for long periods. These QDs are therefore very promising for many applications where optical properties need to remain stable, such as lasing or biolabeling.

ASSOCIATED CONTENT

Supporting Information. Additional TEM images, Raman spectra, UV-vis absorption spectra, photoluminescence spectra, photoluminescence decays and anti-bunching experiment. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 1 .

 1 Figure 1. TEM images of CdSe/CdS QDs synthesized without OA excess (a) and with OA excess (b).

Figure 2 .

 2 Figure 2. (a) Typical XRD diffractogram of CdSe/CdS QDs. (b) Emission spectra of CdSe/CdS QDs synthesized with and without OA excess (inset: magnification of the wavelength range 450 to 530 nm). The emission spectra were recorded for an excitation wavelength of 365 nm.

Figure 3 .

 3 Figure 3. TEM images of samples QD1 (a), QD2 (b) and QD3 (c). Size distribution of those three samples (d) (see Supporting Information Figure S1 for samples QD4 to QD6).

Figure 4 .

 4 Figure 4. Average sizes (a) and volumes (b) of the CdSe/CdS QDs depending on the amount of cadmium used and for different core sizes.

Figure 5 .

 5 Figure 5. (a) Raman spectra of sample QD1 and QD4. (b) Focus on the CdSe-related peaks.

Figure 6 .

 6 Figure 6. Normalized (a) absorption spectra, (b) emission spectra, and (c) photoluminescence decays of samples QD1, QD2 and QD3 (see Supporting InformationFigureS3for samples QD4 to QD6). The emission spectra were recorded for an excitation wavelength of 365 nm. The photoluminescence decays were recorded for an excitation wavelength of 331 nm.

Figure

  Figure 6. Normalized (a) absorption spectra, (b) emission spectra, and (c) photoluminescence decays of samples QD1, QD2 and QD3 (see Supporting InformationFigureS3for samples QD4 to QD6). The emission spectra were recorded for an excitation wavelength of 365 nm. The photoluminescence decays were recorded for an excitation wavelength of 331 nm.

Figure 7 .

 7 Figure 7. : Luminescence intensity (black line, left axis) of a single CdSe/CdS QD as a function of time (bottom axis) for (a) νav = 0.15 and (b) νav = 0.56. The intensity of the luminescence is measured by the number of counts recorded by a photon counter during 10-ms time intervals. The statistical distribution of the number of counts is shown in red (top axis). In (b) the time interval is reduced for better visualization of the off-state, but the counts statistics (red line) are still calculated based on a 400 s experiment.

  12

Table 1 . Synthesis details, structural parameters and spectroscopy data of CdSe/CdS QDs synthesized with different core sizes and different amounts of CdO. The amount of TOPO was always 3 g, the OA was introduced with an excess of 4 mmol and the sulfur was in 3 to 4-fold excess compared to cadmium in all the syntheses.

 1

	sample	core size (nm)	CdO (mmol)	size (nm)	number of CdS MLs	em. peak (nm)	FWHM (nm)	τ1 (ns)	τ2 (ns)	PLQY (%)
	QD1	2.5	0.50	8.9 ± 1.3	9.5	597	34	5	24	40
	QD2	2.5	1.33	13.0 ± 1.4	15.6	623	54	30	100	26
	QD3	2.5	2.67	15.8 ± 2.7	19.7	635	63	32	145	11
	QD4	3.5	0.71	9.5 ± 1.2	8.9	631	36	7	33	47
	QD5	3.5	1.73	13.4 ± 1.4	14.7	647	48	30	117	31
	QD6	3.5	3.47	16.8 ± 3.1	19.7	653	67	42	191	15

ACKNOWLEDGMENT

The authors acknowledge BelSPo (IAP 7.35, photonics@be), FWO-Vlaanderen (Project G.0760.12), German Research Foundation (Project LA 2901/1-1), Max Kade Foundation, and Hercules Foundation (project AUGE/09/024 "Advanced Luminescence Setup") for financial support.

AUTHOR INFORMATION Corresponding Author

*E-mail: zeger.hens@ugent.be.

NOTES

The authors declare no competing financial interest.