Emilie Faure

Eric Bertrand

Amélie Gasté

Elise Plaindoux

Valérie Deplano
email: valerie.deplano@univ-amu.fr

PhD Stéphane Zaffran
email: stephane.zaffran@univ-amu.fr

Valérie

Side-dependent effect in the response of valve endothelial cells to bidirectional shear stress

Keywords: hemodynamic, aortic valve, valvular endothelial cells, wall shear stress

Endothelial cells covering the aortic and ventricular sides of the aortic valve leaflets are exposed to different stresses, in particular wall shear stress (WSS). Biomechanical stimuli actively regulate valve tissue structure and induce remodeling events leading to valve dysfunction.

Endothelial to mesenchymal transformation (EndMT), for example, has been associated with aortic valve disease. The biomechanical response of cells at different sides of the leaflets has not been clearly characterized. To analyze the mechanical response of valve endothelial cells (VECs) we developed a unique fluid activation device that applies physiologically relevant pulsatile WSS. We characterized the morphology and function of adult porcine aortic VECs derived from the opposite sides of aortic valve leaflets following exposure to different pulsatile WSS. We found that elongation and orientation of cells in response to pulsatile WSS depends on their side of origin. Quantification of gene expression confirms phenotypic differences between aortic and ventricular VECs. Aortic VECs exposed to pulsatile WSS similar to that in vivo at the tip of aortic side of the valve leaflet upregulated pro-EndMT (ACTA2, Snail, TGFb1) and inflammation (ICAM-1, VCAM-1) genes, whereas expression of endothelial markers like PECAM-1 was decreased. Conversely, ventricular-VECs showed strong increase of PECAM-1 expression and no activation of pro-EndMT marker. Finally, we found that stress-induced genes are upregulated in both cell types, at higher levels in ventricular compared to aortic VECs.

Application of physiological shear stress levels using a fluid activation device therefore reveals functional differences in VECs derived from opposite sides of the aortic valve leaflets.

Introduction

The aortic valve maintains unidirectional blood flow between the left ventricle and the ascending aorta. Aortic valve leaflets are composed of three highly organized layers of extracellular matrix (ECM) populated by valve interstitial cells and covered by a monolayer of valve endothelial cells (VECs). The distinct layers of ECM within the leaflet are mainly composed of collagen fibers at the aortic side (fibrosa), elastin at the ventricular side (ventricularis) and proteoglycan and glycosaminoglycan (spongiosa) in between [START_REF] Hinton | Heart valve structure and function in development and disease[END_REF]. The composition of ECM components contributes to the structural and mechanical proprieties of the valve. Collagen provides tensile strength to the valve leaflet during opening and transfer the load to the aortic wall when the valve is closed [START_REF] Schoen | Mechanisms of function and disease of natural and replacement heart valves[END_REF]. Elastin facilitates the fast opening of the valve leaflet. The spongiosa absorbs shocks during cardiac cycle and also facilitates the relative internal rearrangements to protect the valve leaflet from damage [START_REF] Duan | Three-dimensional printed trileaflet valve conduits using biological hydrogels and human valve interstitial cells[END_REF]. VECs surrounding the valve leaflets are critical for circulatory function and blood-tissue interaction and can respond to changing stimuli by altering their normal functions [START_REF] Rabkin-Aikawa | Heart valve regeneration[END_REF]. Experimental studies have reported that aortic valve leaflets experience a wide range of wall shear stress (WSS) depending on the side (aortic or ventricular) and region (the base, belly and tip regions) at which they are located [START_REF] Butcher | Valvular endothelial cells and the mechanoregulation of valvular pathology[END_REF]. The three sub-regions of the aortic valve leaflet have been anatomically characterized by [START_REF] Cao | Computational comparison of regional stress and deformation characteristics in tricuspid and bicuspid aortic valve leaflets[END_REF] as a region aligned along the circumferential direction, and spanning on third of the total length of the leaflet (Figure S1). While the ventricular side experiences a strong pulsatile unidirectional WSS, the aortic side experience a much lower recirculating WSS.

Therefore, a WSS is a mechanical stimulus that greatly differs on either sides of the valve leaflets, and plays an important role in the development of side-dependent valve disease [START_REF] Arjunon | Aortic valve: mechanical environment and mechanobiology[END_REF][START_REF] Holliday | Discovery of shear-and side-specific mRNAs and miRNAs in human aortic valvular endothelial cells[END_REF]. Indeed, abnormal blood velocity and pressure can apply environmental changes, which will have consequences at the macro-to microscales promoting maladaptive tissue remodeling. Such mechanical stimuli can activate VECs, which transduce this mechanical signal to a biochemical signal that leads to changes in gene expression and/or protein secretion, particularly of ECM components in the valve [START_REF] Balachandran | Hemodynamics and mechanobiology of aortic valve inflammation and calcification[END_REF][START_REF] Steed | Hemodynamics driven cardiac valve morphogenesis[END_REF]. At the macroscale, hemodynamic forces induce WSS on the side of the valve leaflets as well as deformation of the valve tissue under cyclic stretch. WSS modulates the function of valve cells and impacts the physiology and pathologies of these cells [START_REF] Butcher | Transcriptional profiles of valvular and vascular endothelial cells reveal phenotypic differences: influence of shear stress[END_REF]. Recent 3D fluid structure interaction valve numerical modeling has investigated the macroscopic scale temporal and regional WSS characteristics on aortic valve leaflets under physiological flow [START_REF] Cao | Three-dimensional macro-scale assessment of regional and temporal wall shear stress characteristics on aortic valve leaflets[END_REF][START_REF] Cao | Computational comparison of regional stress and deformation characteristics in tricuspid and bicuspid aortic valve leaflets[END_REF]. These studies confirmed side-specific differences in WSS pulsatility and magnitude between the base, belly and tip regions of the leaflets. High WSS, essentially unidirectional and pulsatile, with values comprised between 46 and 70 dyne/cm 2 near the base and the belly and reaching 93 dyne/cm 2 at the tip of the leaflet were found on the ventricular side, but were one order of magnitude lower and bidirectional on the aortic side.

Identifying how VECs respond to mechanical forces is critical to better understand aortic valve disease. The side-specificity of valve disease such as calcification might be attributed to the inherent differences in the VECs lining the aortic and ventricular sides of valve leaflets [START_REF] Simmons | Spatial heterogeneity of endothelial phenotypes correlates with side-specific vulnerability to calcification in normal porcine aortic valves[END_REF].

Interestingly, ~10% of VECs in human valve have been shown to undergo endothelial to mesenchymal transformation (EndMT) during development decreasing to ~1% in adult valves, suggesting heterogeneity in the phenotype of these cells [START_REF] Paruchuri | Human pulmonary valve progenitor cells exhibit endothelial/mesenchymal plasticity in response to vascular endothelial growth factor-A and transforming growth factor-beta2[END_REF]. To further investigate sidedependent differences in VECs we designed and used an original fluid activation device to apply controlled in vivo like WSS on the surface of the cells. The flow profile within the bioreactor was characterized both computationally and experimentally. Exposure of different pulsatile shear stress magnitudes to VECs confirmed a side-dependent effect. Porcine aortic VECs isolated from the two sides of the leaflets elongated and aligned differently according to the pulsatile WSS applied. Moreover, we observed a side-dependent gene expression pattern that reflected a differential response of these cells to biomechanical forces. Our results showed that aortic-VECs undergo EndMT associated with a decrease of endothelial gene expression and an increase of inflammation genes, whereas expression of PECAM-1 was highly increased in ventricular-VECs. Therefore, by using an original fluid activation device we highlight the side-specific phenotype of VECs lining on the two sides of the aortic valve leaflets. These data help to better understand how heterogeneity of VECs contributes to their function on different sides of the aortic valve leaflets under physiological and pathophysiological conditions.

Materials and Methods

Bioreactor design, development and functioning

A schema of the bioreactor composed of an original homemade fluid activation device linked to a specific 2D flow chamber is shown in Figure 1. An upstream glass reservoir bottle with screw cap pressure compensation (0.2 and 0.45µm filters) contains the culture medium (Figure 1A). Its volume depends on the duration to which the valve cells are submitted to WSS. Two stainless steel syringes (kdScientific) of 4.85mm inside diameter and 168.7mm overall barrel length were used (Figure 1B). One of the two syringes, filled with medium before the beginning of the experiment, injects medium in the 2D flow chamber in response to a computer generated signal. This syringe is emptied, whereas the other one, mechanically enslaved to it in an opposite manner, fills up. When the medium volume contained in one syringe tends to zero, a 3-way solenoid operated pinch valve (Bio-Chem, 100PD3MP12-02S) is automatically actuated. Meanwhile the second syringe, which is full, injects the medium into the flow chamber. C-flex tubing (3.2mm OD, 1.6mm ID, St Gobain) was used for the pinch valve to avoid potential contaminations. A downstream glass reservoir bottle was connected to the flow chamber outlet to collect the medium. Special attention was paid to avoid air bubbles within the syringes, tubing, and flow chamber.

A computer-controlled trio motion coordinator was used to drive a stepper motor (HS200-2231, Danaher motion) linked to a rolled ball screw (W1001MA-3PY-C3Z2, NSK). A gear ratio system was also added to improve displacement accuracy. This set up, using preloaded rolled ball screws with 0mm of axial play and a stepper motor with a small rotor inertia (J=340 g cm 2 ) enabled fast accelerations and decelerations with high accuracy.

The choice of the technical specificities used to move both syringes as well as the linking system between the engine and the syringes are the major key points that allow this original fluid activation set-up to reproduce the WSS to which the valve cells are exposed in vivo in a perfectly controlled manner (see below for the validation of the activation device). This fluid activation device is actually more adapted than commercial systems to mimic the rapid changes in flow direction. It is able to master pulsatility, accelerations, decelerations and fast variations of flowrate generating pathophysiological flow waveforms. This demonstrated its superiority in comparison to previous approaches [START_REF] Butcher | Porcine aortic valve interstitial cells in three-dimensional culture: comparison of phenotype with aortic smooth muscle cells[END_REF][START_REF] Butcher | Unique morphology and focal adhesion development of valvular endothelial cells in static and fluid flow environments[END_REF][START_REF] Mahler | Effects of shear stress pattern and magnitude on mesenchymal transformation and invasion of aortic valve endothelial cells[END_REF] that used 2D flow chamber, plastic syringes and a peristaltic pump that generated steady or oscillatory flows, which do not achieve hemodynamic conditions on valvular leaflets. Finally, although Mohammed et al. [START_REF] Mohammed | Studying the Response of Aortic Endothelial Cells under Pulsatile Flow Using a Compact Microfluidic System[END_REF] have used an interesting piezoelectric pumping system for studying aortic endothelial cells under pulsatile flow, their system cannot drive the values of flow rate we need in our system.

In order to impose controlled WSS on a surface we needed to master the flow behavior in a flow chamber. It is now well known that a 2D parallel plate flow chamber, with specific geometrical dimensions, permits such a control. The designed flow chamber was composed of three parts (Figure 1A). The upper and lower covers, 10mm thick, were machined using plexiglass. In between, the flow chamber itself, l=21mm and L=90mm, was machined using a 250µm thickness Lexan 8010 film sheet; inlet and outlet diameters were of 4mm. The fourteen stainless steel screws used for assembling the three parts ensured that the flow chamber was leak-tight. Tygon™ tubing (3.2mm OD, 1.6mm ID, St Gobain) and connectors in polypropylene (Ark Plas Products™) were used to associate the flow chamber to the activation device components.

As this flow chamber is 2D, it was important to include a 3D scaffold to culture the VECs, which did not change the shear stress at the flow chamber walls but allowed for a 3D cell culture environment. To do so, elliptical holes, with major and minor axis dimensions of 26mm X 9.8mm respectively and 2mm of depth, were drilled within the lower cover of the flow chamber (Figure 1A). They were then filled with a 3D collagen gel. 3D collagen gels at a concentration of 1.5 mg/mL collagen type I were made by addition of Dulbecco's Modified Eagle's Medium (DMEM, Thermo Fischer Scientific), 10% fetal bovine serum (FBS, Thermo Fischer Scientific), 0.1M NaOH and rat tail collagen (10 mg/mL concentration, BD Biosciences). An aliquot of the collagen solution was pipetted into each well and allowed to gel for at least 1hr at 37°C in 5%CO2. VECs were seeded on the surface of the 3D collagen gels. It is worth noting that the 3D collagen gel is not a model of the leaflet tissue, but provides a 3D cell culture environment.

The dynamic viscosity of the working medium fluid was measured using a rheometer HaakeMars III, fluid µ=0.945e -3 Pa.s. The entire set up was placed in an incubator and kept at 37°C and 5% CO2 during all the experiments.

Porcine Aortic Valve Endothelial Cells Isolation and Culture

Porcine aortic valve endothelial cells (VECs) were isolated from the aortic or the ventricular side of the aortic valve leaflets as previously described [START_REF] Butcher | Unique morphology and focal adhesion development of valvular endothelial cells in static and fluid flow environments[END_REF]. Porcine hearts were kindly donated by the "abattoir Saint-Saturnin-lès-Apt". Briefly, aortic valve leaflets were isolated and incubated in collagenase type II/DMEM (Life Technologies) for 3x7 min. After incubation cells were gently scraped to isolated VECs from the two sides of the leaflets. After isolation aortic or ventricular VECs were seeded and cultured on flask coated with 50ul/mL rat collagen type I (10 mg/mL concentration, BD Biosciences). VECs were cultured at 37°C and 5% CO2 in Endothelial Cell Growth Medium (Promocell) supplemented with manufacturer's adjuvants and 5% FBS. Endothelial cell phenotype was confirmed by qPCR. Thus, the low expression levels of ACTA2 validated the lack of contamination with other cell types such as interstitial cells in the culture. The cobblestone-like morphology of valvular endothelial cells was confirmed at passage 3, and cells were used at passages 3 to 6 [START_REF] Butcher | Unique morphology and focal adhesion development of valvular endothelial cells in static and fluid flow environments[END_REF].

Immunohistochemistry

Cells on collagen gels were fixed in 4% buffered paraformaldehyde during 10 min at room temperature. Fixed cells were washed for 15 min three times with PBS and permeabilized with 0.2% Triton-X 100 (Sigma) in PBS for 10 min. Cells were washed another three times with PBS before a saturation with 3% BSA and 10% Fetal Bovine Serum (Life Technologies). Cells were incubated overnight with Phalloidin-iFluor 488 Reagent (F-actin; abcam) and DAPI (4',6diamidino-2-phenylindole) was used at 300 nM as a counterstain to distinguish cell nuclei. Cells were photographed with a confocal LSM800 (Zeiss).

Morphology Analysis

10 representative confocal images were taken on static and exposed cells on 3D collagen I gels that were fixed and stained with Phalloidin-iFluor 488 Reagent. The images were analyzed using Image J2 software [START_REF] Rueden | ImageJ2: ImageJ for the next generation of scientific image data[END_REF]. Images from gels exposed to WSS were aligned so that the image horizontal corresponded to the direction of flow. Using Morpholib plugins on Image J2 software, cell area and cell perimeter were analyzed. Orientation angle between the horizontal (flow direction) and the majority of F-actin filaments was also determined. Based on cell area (A) and cell perimeter (P), Elongation index (Elong I) and Circularity index (shape index -SI) of cells (static or exposed to the flow) were determined (SI = 4 π A / P^2 and Elong I = P^2 / 4 π A). Cell alignment was assessed using angle orientation data. Frequency of cells upon angle orientation between 0-90°C were analyzed using Matlab software. Angle 0° were used as reference to cell parallel with the flow and 90° as reference to cell perpendicular with the flow.

Angle orientation data from static conditions or cells exposed to fibrosa WSS (base, belly and tip) were represented in polar histogram obtained using Matlab software.

Real time quantitative PCR (qPCR)

Cells were first removed from the collagen gels. Cells were lysed on Trizol (Life Technologies) and RNAs were extracted using RNeasy mini Kit (Qiagen). Reverse transcriptions were performed by using first strand cDNA synthesis kit (Agilent) per manufacturer's instructions.

LightCycler 480 SYBR Green I Master mix (Roche) was used for quantitative real-time qRT-PCR analysis with a LightCycler 480 (Roche) following the manufacturer's instructions.

Each experiment was performed with n³3. Samples were normalized to TBP as an endogenous housekeeping gene. mRNA expression levels for each gene were calculated using the comparative cycle threshold (ΔΔCT) method. Expression levels in the static condition were set to 1. Primers used are listed in Table 1.

Statistical Analysis

All experiments were performed with n³3. Data is expressed as mean ±SEM. Statistical significance was determined using Student's t test to compare variances. For non-parametric data, the Mann-Whitney test was used to calculate significance between the medians.

Interactions between conditions were initially tested with ANOVA statistics (http://biostatgv.sentiweb.fr/). This test was used when it was necessary to decide whether several independent groups defined by the k modalities of the study factor were from the same population. p value < 0.05 was considered significant for this study.

Results

During the cardiac cycle, wall shear stress (WSS) is considered as one of the most important hemodynamic parameters for valve endothelial cells (VECs). Previous transcriptional analysis of VECs isolated from aortic valve leaflets have identified side-specific differences in VECs expression suggesting heterogeneity in the VEC phenotype [START_REF] Simmons | Spatial heterogeneity of endothelial phenotypes correlates with side-specific vulnerability to calcification in normal porcine aortic valves[END_REF][START_REF] Hulin | Maturation of heart valve cell populations during postnatal remodeling[END_REF]. In order to assess the sidespecificity of VECs we designed and developed an original fluid activation device able to apply a physiological controlled pulsatile WSS on the surface of VECs isolated from alternate sides of the leaflets. The temporal evolution of the WSS radial component obtained by Cao et al, 2016 (Figure 2A-C) at the base, belly and tip regions of the aortic side of the leaflets have been modeled using this original fluid activation device (see Figure S1) [START_REF] Cao | Three-dimensional macro-scale assessment of regional and temporal wall shear stress characteristics on aortic valve leaflets[END_REF].

To analyze WSS amplitudes and waveforms, it was important to determine their amplitude WSS variation (DWSS) as well as their maximum, minimum and mean (WSS 333333 ) values at base, belly and tip regions (Table 2).

Table 2 shows large amplitude WSS variations, more particularly at belly and tip regions. Both D, [START_REF] Ku | Pulsatile flow and atherosclerosis in the human carotid bifurcation. Positive correlation between plaque location and low oscillating shear stress[END_REF], was also calculated. |WSS| is the norm of WSS and T, the period of cardiac cycle. This index, which is a hemodynamic factor related to oscillation flow, can vary from 0 to 0.5. When considering a WSS component, OSI will be equal to 0 if WSS is consistent and 0.5 if highly oscillatory. In the present study, we found OSIbase=0.38, OSIbelly=0.4 and OSItip=0.45. With such a high temporal oscillation of WSS during the cardiac cycle, it is obvious that VECs are exposed to strongly oscillating WSS signals far from static and steady flow conditions. In this latter case, we can also note that it would not be appropriate to subject VECs to WSS mean values that are low compared to maxima or minima values encountered.

Validation of the flow device

The flow rates were generated by controlling the displacements of the syringes, (Ds), using a To validate our original fluid activation device, it was therefore important to record the syringeimposed displacements to assess their spatio-temporal accuracy. Three different WSS signals computed at base, belly and tip of the aortic side were extracted from results reported in Cao et al., 2016 (Figure 2A-C). Flow rates corresponding to these signals were then determined using equation ( 1) and associated displacements of the syringe calculated using equation (2). Figure 2 shows the comparison between syringe-imposed displacements to generate radial WSS signals related to the belly, base and tip of the aortic side and the measured ones using Solartron Metrology displacement sensors (Figure 2G-I). The results highlight that the fluid activation device is able to precisely reproduce the complex and fast displacement changes, which, in turn, induce the WSS changes occurring during the cardiac cycle.

Response of endothelial cells to mechanical environment

We investigated the orientation and shape of aortic and ventricular VECs (aortic-VECs and vent-VECs) following static culture or 1hr exposure to different pulsatile WSS as predicted in the belly, base and tip regions of the aortic side [START_REF] Cao | Three-dimensional macro-scale assessment of regional and temporal wall shear stress characteristics on aortic valve leaflets[END_REF]. Under static conditions, we found no difference between aortic-and vent-VECs since the two cell types were randomly oriented (Figure 3). Following exposure to pulsatile WSS predicted in the tip region of aortic side of the leaflets, aortic-VECs did not display clear orientation. Cells were randomly oriented, as more than 60% had the angle of orientation ranged from 30° and 60° (Figure 3A,B). Elongation and shape index showed that aortic-VECs were mainly rounded (Figure S2). However, ventricular-VECs exposed to a similar pulsatile WSS adopted an azimuthal orientation (Figure 3C,D). We found that 60% of vent-VECs were orientated with the angle of orientation ranged from 45° and 90°. Cell shape index analysis revealed that at equal magnitude of pulsatile WSS, vent-VECs have a more elongated shape than aortic-VECs (Figure S2). Together these results show that VECs isolated from the opposite sides of aortic valve leaflets have different phenotypes.

Side-dependent differential gene expression

To further investigate the phenotype of the two cell types we analyzed the expression of different markers after an exposure of 1hr to different pulsatile WSS (related to the belly, base and tip regions of the aortic side). For the aortic-VECs we quantified changes in pro-EndMT (ACTA2, Snail, TGFb1) genes following exposure to pulsatile WSS (Figure 4A). Compared to static control, exposure to pulsatile WSS predicted in the tip region at the aortic side significantly upregulated pro-EndMT gene expression (ACTA2 4.3±0.3 fold increase over static control, p<0.05). Interestingly, the upregulation of ACTA2 expression was proportional to the magnitude of the pulsatile WSS applied (Figure 4B). However, ventricular-VECs exposed to similar pulsatile WSS did not show comparable activation of ACTA2 compared to aortic-VECs (Figure 4B). Similarly, we did not observe significant fold change for other pro-EndMT markers (Figure S3). We further analyzed the expression of the endothelial gene PECAM-1 in the two cell types. Expression of PECAM-1 was increased in aortic-VECs exposed to pulsatile WSS predicted in the base region of the aortic side of the leaflets (3.2±0.7 fold increase over static control, p<0.05), whereas it was downregulated in cells exposed to pulsatile WSS predicted in the belly and tip regions (Figure 5A). Conversely, PECAM-1 expression was strongly upregulated in ventricular-VECs after exposure to the three pulsatile WSS applied (Figure 5B). For instance, expression of PECAM-1 was highly increased in vent-VECs exposed to pulsatile WSS predicted in the tip region (60±10 fold increase over static control, p<0.05).

As activation of inflammatory mediators is induced by abnormal shear stress [START_REF] Sucosky | Altered shear stress stimulates upregulation of endothelial VCAM-1 and ICAM-1 in a BMP-4-and TGF-beta1dependent pathway[END_REF], we analyzed the expression of 2 inflammatory markers, VCAM-1 and ICAM-1. Expression of these genes was upregulated in both cell types with some differences (Figure 6). Expression of VCAM-1 and ICAM-1 was increased in aortic-VECs, with the highest activation observed in cells exposed to pulsatile WSS with the greatest differential (Figure 6A; 49.5±3.7 fold increase after exposure as predicted in tip region over static control, p<0.05). Upregulation of NFkB expression in aortic-VECs confirmed the activation of inflammation in this cell type with (Figure S4). Although expression of VCAM-1 and ICAM-1 were both increased in vent-VECs (Figure 6), activation of VCAM-1 was significantly greater in this cell type compared to its upregulation in aortic-VECs (Figure 6B; 265±17 fold increase after exposure as predicted in tip region over static control, p<0.05). Thus, these results suggest that the pulsatile fibrosa WSS activates expression of inflammatory markers in the vent-VECs. To analyze the activation of stress-related genes in both cell types we quantified expression of NRF-2 and EGR-1, which are known to respond to shear stress in endothelial cells [START_REF] Havis | EGR1 Transcription Factor is a Multifaceted Regulator of Matrix Production in Tendons and Other Connective Tissues[END_REF][START_REF] Hosoya | Differential responses of the Nrf2-Keap1 system to laminar and oscillatory shear stresses in endothelial cells[END_REF]. Expression of NRF-2 and EGR-1 was upregulated in the two cell types, but was greater in vent-VECs compared to aortic-VECs (Figure 7A,B). Together these findings indicate that aortic-and vent-VECs behave differently to pulsatile WSS predicted in the different regions of the aortic side of aortic valve leaflets.

Discussion

Although the aortic valve is covered by a monolayer of endothelial cells on its two sides (aortic and ventricular) the response of valve endothelial cells (VECs) to shear stress remains poorly understood. Here we used a fluid activation device to show that VECs respond differently to similar WSS depending on their side of origin. Shear stress is known to play a leading role in endothelial cell migration and vessel remodeling. Shear stress has also been shown to result in directed migration of endothelial cells against the direction of flow [START_REF] Simmers | Arterial shear stress regulates endothelial celldirected migration, polarity, and morphology in confluent monolayers[END_REF]. Our findings showed differences in the alignment of aortic-vs vent-VECs to the direction of the flow. We found that the orientation angle of 60% of vent-VECs was ranged between 45° to 90° when the magnitude of the pulsatile WSS was highest (corresponding to WSS at the tip of the leaflet). Conversely, at a similar pulsatile WSS magnitude less aortic-VECs were oriented with this angle suggesting these two cell types align differently in response to flow. The alignment behaviors that we observed in these two cell types may thus reflect a specific response to high WSS. The exact mechanism by which VECs sense shear stress remains unclear. Several endothelial cell flow sensors have been proposed for endothelial cells including integrin complexes, ion channels, caveolae and complex including PECAM-1, VE-cadherin and VEGFR2 [START_REF] Chatterjee | Mechanotransduction in the endothelium: role of membrane proteins and reactive oxygen species in sensing, transduction, and transmission of the signal with altered blood flow[END_REF]. Our observations suggest that the expression of PECAM-1 was highly upregulated in vent-VECs compared to aortic-VECs confirming that cell-cell contacts may be implicated in flow sensing [START_REF] Conway | Fluid shear stress on endothelial cells modulates mechanical tension across VE-cadherin and PECAM-1[END_REF]. Our data are consistent with previous study that suggests distinct phenotype between VECs and vascular endothelial cells in their response to blood flow [START_REF] Butcher | Unique morphology and focal adhesion development of valvular endothelial cells in static and fluid flow environments[END_REF]. Analysis at the transcriptional and protein level has confirmed a transcriptional difference between VECs and aortic endothelial cells [START_REF] Butcher | Transcriptional profiles of valvular and vascular endothelial cells reveal phenotypic differences: influence of shear stress[END_REF]. These data suggest that valvular endothelium acts as a distinct organ system, which may explain the difference find between valvular and vascular pathology.

Several studies have also shown EndMT on the aortic side of adult valves, which is exposed to bidirectional shear stress and is often the site of inflammatory and calcific degeneration [START_REF] Mohler | Mechanisms of aortic valve calcification[END_REF][START_REF] Mohler | Bone formation and inflammation in cardiac valves[END_REF]. On the contrary, EndMT is absent at the ventricular side of the valve [START_REF] Mahler | Inflammatory cytokines promote mesenchymal transformation in embryonic and adult valve endothelial cells[END_REF]. At this side, the VECs are exposed to high unidirectional shear stress. Consistent with these observations, our data show that bidirectional WSS signal induces EndMT in aortic-VECs but not ventricular-VECs.

We showed that inflammatory markers (VCAM-1 and ICAM-1) are upregulated in both populations of VECs in response to physiological bidirectional WSS. The difference of VCAM-1 upregulation between the aortic-versus vent-VECs is consistent with a previous study showing differential expression of VCAM-1 on opposite sides of normal aortic valves [START_REF] Simmons | Spatial heterogeneity of endothelial phenotypes correlates with side-specific vulnerability to calcification in normal porcine aortic valves[END_REF]. The upregulation of VCAM-1 and ICAM-1 has already been demonstrated in response to TNF-a, suggested a role for the hemodynamic environment in valve inflammatory process [START_REF] Aikawa | Human semilunar cardiac valve remodeling by activated cells from fetus to adult: implications for postnatal adaptation, pathology, and tissue engineering[END_REF].

However, our results differ from a previous study, which demonstrated that exposure of the aortic, but not ventricular, surface of the aortic valve leaflet to bidirectional shear stress increases expression of inflammatory markers [START_REF] Sucosky | Altered shear stress stimulates upregulation of endothelial VCAM-1 and ICAM-1 in a BMP-4-and TGF-beta1dependent pathway[END_REF]. The WSS signal used in this previous study, although bidirectional, was not physiological. More particularly the accelerating and decelerating slopes as well as the frequency of flow direction changes were lower than those physiologically observed. Another difference is that we used isolated VECs and not a leaflet. Therefore, cells within the endothelium in intact leaflets, may be less sensitive to shear stress than isolated cells. Indeed, it is known that the trilaminar structure of the leaflet protects the valve, and so the VECs, from its environment (hemodynamic and biomechanics). Although VECs were cultured directly on the collagen gel we observed that exposure to particular conditions induced EndMT and probably invasion in the matrix, even though the collagen gel has different stiffness than the ECM forming the leaflet tissue. Assessment of the effect of the difference in stiffness has demonstrated that the phenotype of VECs responds to changes of the 3 D culture environments [START_REF] Duan | Active tissue stiffness modulation controls valve interstitial cell phenotype and osteogenic potential in 3D culture[END_REF][START_REF] Yip | Calcification by valve interstitial cells is regulated by the stiffness of the extracellular matrix[END_REF]. Thus, it would be important to work on a 3D cell culture environment that reproduces the physiological condition.

Our results confirm a previous study showing that VECs on aortic and ventricular surfaces have differential gene expression profiles and, therefore, distinct phenotypes [START_REF] Simmons | Spatial heterogeneity of endothelial phenotypes correlates with side-specific vulnerability to calcification in normal porcine aortic valves[END_REF]. The observation of morphological and functional differences between VECs isolated from the two sides of aortic valve leaflets exposed to identical conditions suggests that the side-specific phenotypic differences are in part intrinsic. Recent transcriptomic analysis of postnatal heart valves at single cell resolution demonstrated that the endothelial cell population is separated into three distinct subpopulations that display specific spatial locations in the valve leaflets [START_REF] Hulin | Maturation of heart valve cell populations during postnatal remodeling[END_REF]. The VECs subset, which is characterized by classic endothelial markers, can be found on both sides of porcine aortic valve leaflets. However, a particular subset of VECs is present in regions of high mechanical stress, where valve leaflets are exposed to high shear [START_REF] Hulin | Maturation of heart valve cell populations during postnatal remodeling[END_REF]. Interestingly, a recent study demonstrated restricted expression of Wnt9b in endothelial layer of zebrafish heart valves in response to fluid forces [START_REF] Goddard | Hemodynamic Forces Sculpt Developing Heart Valves through a KLF2-WNT9B Paracrine Signaling Axis[END_REF]. Altogether these findings suggest a contribution of biomechanical forces to maintain VEC diversity in different region of the valves.

Conclusions

In the current study, we designed and developed a unique fluid activation device that applies physiologically relevant pulsatile WSS (related to the belly, base and tip regions of the leaflet).

We identified a clear phenotypic difference between VECs isolated from the opposite sides of aortic valve leaflets. Our model replicates the physiological radial component of WSS predicted in different regions of the aortic side of the aortic valve leaflet. We observed that aortic-VECs undergo EndMT when bidirectional WSS, characterized by high OSI value, is applied, whereas vent-VECs are less sensitive to this WSS feature. We found that VECs isolated from the two sides of aortic valve leaflets induced inflammatory process, which probably respond to the activation of a stress program. Our findings provide an improved understanding of phenotypic Expression levels in the static condition were set to 1 and all conditions were compared to this reference. fold increased over static control (p<0.05) in vent-VECs. All experiments were performed in triplicate (n= 3 for each conditions) and expressed as mean ±SEM (*p<0.05 versus control (static condition) according to ANOVA followed by Mann-Whitney test). Expression levels in the static condition were set to 1 and all conditions were compared to this reference.

  Assuming fully developed, laminar and incompressible flow, there exists an analytical relation between the imposed flow rate Q within the chamber and the WSS induced on its walls: 𝑄 = $%%&' ( )* where l, h and µ are the width, the thickness of the flow chamber and the fluid dynamic viscosity respectively. This relation remains true when assuming unsteady flow given the value of the frequency parameter. Consequently, 𝑄(𝑡) = $%%(.)&' ( )* (equation 1) and the wall shear stress on endothelial cells will be 𝑊𝑆𝑆(𝑡) = )* &' ( 𝑄(𝑡).

  positive and negative WSS values are high compared to the mean values and the radial WSS component temporal evolutions (Figure2A-C) highlight a significant alternation between these positive and negative values. To more accurately capture this bidirectional oscillatory waveform, oscillatory shear index,

  computer. A direct relation exists between Ds and the fluid volume, 𝜗 F , injected, by the syringe: 𝜗 F = 𝑆 HI 𝐷 H .with Ssy being the syringe section. Considering ti the time of fluid injection Q(t) is derived by 𝑄(𝑡)

Figure 1 :

 1 Figure 1: Schematic illustration of the fluid activation device. (A) Scheme illustration of the

Figure 2 :

 2 Figure 2: A unique fluid device system producing a pulsatile WSS. Comparison between

Figure 3 :

 3 Figure 3: Valve endothelial cell alignment following exposure of wall shear stress. (A-D)

Figure 4 :

 4 Figure 4: Valve endothelial cell phenotype following exposure to wall shear stress. (A)

Figure 5 :

 5 Figure 5: PECAM-1 expression following exposure of wall shear stress. (A,B) PECAM-1

Figure 6 :

 6 Figure 6: Expression of inflammatory markers after exposing valve endothelial cells to

Figure 7 :

 7 Figure 7: Expression of stress-related genes after exposing valve endothelial cells to wall

differences between VEC populations in their environment and suggest that vent-VECs are less subject to valve disease.

Acknowledgments

We thank Dr. Gaëlle Odelin for her help in collecting pig hearts. We are grateful to Dr. Robert Kelly for critically reading our manuscript. This work was supported by the "Association Française contre les Myopathies" (TRIM-RD Project), and the "Institut National de la Santé et de la Recherche Médicale" to S.Z. E.F received a postdoctoral fellowship from the "Association Française contre les Myopathies" and the "Fondation Lefoulon Delalande".

Author Contributions: conceptualization, E.F., V.D. and S.Z.; validation, E.F. and E.B.; formal analysis, E.F., E.P., A.G.; investigation, S.Z.; writing-original draft preparation, V.D. and S.Z.; writing-review and editing, V.D. and S.Z.; supervision, S.Z.; funding acquisition, S.Z.

Conflicts of Interest:

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. and expressed as mean ±SEM. Expression levels in the static condition were set to 1 and all conditions were compared to this reference.

Supplementary figure legends


Jesus G Ovejero

Irene Morales

Patricia De La Presa

Nicolas Mille

Julian Carrey

Miguel A Garcia

Antonio Hernando

Pilar Herrasti

Hybrid nanoparticles for magnetic and plasmonic hyperthermia

The present manuscript studies the use of hybrid magneto-plasmonic nanoparticles (HMPNP) based on iron oxide nanoparticles and Au nanorods as colloidal nanoheaters. The individual synthesis of the magnetic and plasmonic components allowed to optimize their feature for heating performance separately, before they were hybridized. Besides, detailed characterizations and finite element simulations were carried out to explaine the interaction effects observed between the phases of the HMPNP. The study also analyzed the heating power of these nanostructures when were excited with infrared light and AC magnetic fields, and compared it with the heating power of their plasmonic and magnetic component. In the latter case, the AC magnetization curves revealed that the magnetic dipolar interactions increase the amount of heat released by the hybrid nanostructures.

Introduction

Inorganic nanoparticles (NPs) with active properties for heat dissipation, such as magnetic and plasmonic NPs, open the possibility of inducing a contactless and local heating. The heat released by these NPs can be employed in a plethora of technological applications such as cancer treatment 1 , catalysis 2 or water purification 3 . One of the most interesting applications in the biomedical field is the so-called hyperthermia therapy. This technique treats local tumors by inducing a thermal shock in cancer cells that activates apoptotic death mechanisms 4,5 . The most popular modalities of hyperthermia treatments mediated by NPs are magnetic hyperthermia 6 and plasmonic photothermia 7 .

In magnetic hyperthermia, the heat is produced by the hysteresis losses generated by magnetic NPs when are exposed to high frequency alternating magnetic fields. The magnetic nanoagents employed in this approach must present a superparamagnetic behavior at low frequency fields, high saturation magnetization and high magnetic susceptibility 6,8 . Iron oxide nanoparticles (IONPs) are the most widely employed material for this purpose due to their good magnetic features and their high biocompatibility with human cells 9 .

A novel strategy proposed to enhance the heating power in magnetic hyperthermia consists on controlling the effect of dipolar interactions between the magnetic NPs 10,11 . It has been postulated that, in a linear arrangement of IONPs, the dipolar interactions modify the effective anisotropy field (HK) of the aggregates and can increase the hysteresis losses of the system [10][11][12] . However, such increment can only be achieved if the maximum field applied (HMAX) is larger than the HK of the system 12,13 . Thus, the enhancement of heating performance via dipolar interactions demands a careful analysis of the ratio between the HMAX and the HK generated in each specific system 10 .

On the other hand, the plasmonic photothermia take advantage of the heat generated by metallic nanoparticles when are irradiated with laser light. The electron oscillation induced by the electric field of the laser, known as localized surface plasmon, derives in the generation of phonons in the atomic lattice that are eventually dissipated to the media as heat 14 . Au NPs, and most specially Au nanorods (AuNRs) 15 , present excellent plasmonic properties for tailoring their optical spectra in the visible and infrared (IR) range maximizing their light absorption 16 and heat generation in deep tissues 17 .

Recently, it has been observed that, using the appropriate NPs, these two approaches can be combined 18 . The large amount of heat dissipated by such magneto-plasmonic approach can help to enhance the effectivity of the in vivo treatment reducing the amount of nanoparticles and radiation dose required for the therapy 19 . Besides, the combination of magnetic and plasmonic components brings new biomedical possibilities such as theragnostic approaches 20,21 and magnetically enhance treatments 22 . However, there is still a lack of biocompatible materials that combine suitable magnetic and plasmonic properties to be use for this purpose.

Hybrid magneto-plasmonic nanoparticles (HMPNP) have been developed to address the issue combining magnetic and metallic phases in a single nanostructure. However, in this kind of nanostructures, the interactions between the phases can compromise the magnetic and plasmonic responses. An interesting strategy to address this issue consist in introducing a third biocompatible material (polymers, inorganic materials, etc.) that acts as spacer 23 and avoids common interfaces between them. Silica (SiO2) has outstand over other alternatives due to its high thermal, mechanical and chemical stability 24,25 In this article, we explore the possibility of using recently developed HMPNPs in which IONPs and AuNRs are coupled with a silica shell 26 , as magnetic and plasmonic nanoheaters. The HMPNP response at high frequency magnetic fields and IR laser excitation were studied in detail to analyze their heating power as well as the effect of the interaction between the different phases on the hybrid nanostructures.

Experimental

Reactants

Ethanol, hexadecyltrimethylammonium bromide (CTAB), chloroauric acid (HAuCl4), sodium borohydride (NaBH4), silver nitrate (AgNO3), sodium hydroxide (NaOH), ascorbic acid, poly(ethylene glycol) thiol (PEG-SH) and tetraethyl orthosilicate (TEOS) were purchased from Sigma-Aldrich (www.sigmaaldrich.com). Ultrapure water, tetrabutylammonium bromide (TBAB) and ammonia (NH3) were purchased from Panreac (www.panreac.es). Fe slides were purchased from GoodFellow (www.goodfellow.com). Citric acid was purchased from Merk (www.merck.es).

Synthesis of AuNR

AuNRs were synthesized using the seed-mediated growth method of El-Sayed et al 27 . Firstly, seeds were prepared using a solution of CTAB (5 mL, 0.1 M) and HAuCl4(0.25 mL, 10 mM) gently mixed. 0.3 mL of 10 mM ice cooled NaBH4 were added drop by drop to the solution and the mixture was allowed sitting for 1 hour in a thermostatic bath at 30 °C.

For AuNRs growth, 250 mL of CTAB solution 0.1 M were mixed with 12.45 mL of HAuCl4 (10 mM) and 2 mL of AgNO3 (10 mM). Au ions were reduced by adding ascorbic acid (1.2 mL, 0.1 M). AuNR with different aspect ratio (AR) were prepared by adding different amount of Au seeds solution to the growth solution: 30 µL (AR=3.15), 60 µL (AR=3.38), 120 µL (AR=3.86) and 240 µL (AR=4.38). The reaction mixture was sat overnight at 30 °C. The suspension of AuNRs was cleaned with water by triple centrifugation of 10000 rpm for 30 min.

AuNRs were concentrated to 0.8 mM (OD400nm=2). 25 mg of SH-PEG (Mw=6000) were dissolved in 2.5 mL of water and introduced in an ultrasound (US) bath for 25 min to dissolve them. Then, 8.75 mL of AuNR and 2.32 mL of SH-PEG solution were mixed under gentle stirring for 6 h. After the pegylation, the samples were centrifugated at 10000 rpm for 30 min and the medium was progressively substituted by pure ethanol.

Synthesis of IONP

The electrochemical synthesis of IONPs was carried out oxidizing a metallic electrode in a conductive solution of tetrabutylammonium bromide 40 mM applying a current density of 45 mA cm -2 for 30 minutes. Two flat Fe plates of 2 cm 2 (electrode) and 8 cm 2 (counterelectrode) were faced with a 1 cm gap between them. The solution was mixed at 400 rpm using a magnetic stirrer. The reaction temperature was fixed to 5ºC using a thermostatic glass. After three magnetic decantation and cleaning, the IONPs were immediately coated with citric acid by adding 25 mL of 0.1 M citric acid to the NPs under stirring. After adjusting the pH to 5 using diluted NaOH, the dispersion was heated up to 80 °C for 2 h. Ferrite NPs were cleaned again by triple magnetic decantation.

Synthesis of hybrid AuNR-IONP

Hybrid (AuNR/IONP)@SiO2 NPs were prepared using a modification of Stöber method 26,28 . Optical density of AuNRs was adjusted to 1 mM (OD400nm=2.4) by adding pure ethanol and IONPs were suspended in water at a concentration of 0.7 gFe L -1 . In a 12.5 mL plastic tube, 2.04 mL of IONP dispersion were added to 7 mL of AuNR in order to establish a solvent ratio of ethanol/water= 3.5. The mixture was shaken and sonicated for 5 min. The pH was risen to ~8.5 adding 0.04 mL of NH3 (28%). Then, 2.04 mL of TEOS were added and the dispersion was shaken and sonicated for other 5 min. Thereafter, the mixture was placed in an ultrasound bath for 2 hours maintaining the temperature bellow 50 °C. The HMPNP produced were cleaned by triple centrifugation of 30 min at 3000 rpm. Supernatant was discarded and the HMPNPs were stored in pure ethanol. For hyperthermia experiments the medium was substituted by water following a similar centrifugation process.

Characterization

Size and geometry of AuNRs, IONPs and HMPNPs were studied using a transmission electron microscopy (TEM) JEM-2100F (JEOL Ltd.). The average size of each collection of NPs was estimated measuring more than 150 NPs and fitting the data with a log-normal distribution. Colloidal features such as hydrodynamic diameter (DH) and polydispersity index (PDI) were obtained by means of Dynamic Light Scattering (DLS) a Zetasizer Nano (Malvern) using the values of scattered light intensity (intensity mode). The X-ray difraction patterns were acquired using a PANalytical X'Pert Pro Multi-Purpose Diffractometer with a CuKa (λ = 0.15418 nm). Raman experiments were carried out in a confocal Raman microscope (Witec ALPHA 300RA) equiped with a Nd:YAG laser (532 nm) in p-polarization. Fe and Au concentration of IONP and HMPNP colloids were estimated by inductively coupled plasma optical emission spectroscopy (ICP-OES), digesting an aliquot of the sample in aqua regia and diluting to a known Mili-Q® water volume (18.2 MΩ•cm @ 25 °C).

The optical extinction spectra were recorded using a UV-Vis-IR spectrometer Perkin Elmer Precisely Lambda 35 with a quartz cuvette of 1 cm path length. Quasistatic magnetic response of IONPs and HMPNPs were measured using a MPMS Quantum Design SQUID magnetometer. For these measurements, two sample preparations were studied: i) powder samples were measured by means of gelatin capsules with the residue of lyophilized colloids compacted with cotton; ii) colloidal samples were measured by using homemade paraffin sealed containers of 50 µL. The magnetization value of the colloids was estimated using the Fe concentration obtained by ICP-OES.

Magnetization curves were obtained applying a maximum magnetic field of 3 T. The susceptibility dependence with temperature (ZFC-FC curves) was obtained by applying a magnetic field of 10 mT. To record thermoremanence curves (TMR), the sample was cooled down with an applied field of 10 mT that was switched off at low temperatures. Then, the remanent magnetization was recorded in the absence of field while increasing the temperature. High frequency magnetization curves of colloid samples (0.5mL, 2.8 gL -1 ) were Please do not adjust margins Please do not adjust margins measured using a home-made magnetometer, described in details in ref. 29 . The set-up reaches a maximum field of 65 mT at 56 kHz.

Theoretical calculations

Mie-Gans theory was employed to study the optical properties of the HMPNPs 30 . Equations 1-3 describe the extinction cross section of a metallic prolate spheroid (a=b≠c) with a permittivity ε= ε1 +iε2, surrounded by a dielectric medium with a real permittivity εm:

Where ω is the frequency of the incident light. The presence of magnetite nanoparticles in the silica shell was included in the equations by assuming an effective medium, in which εeff is the weighted average of the silica permittivity and magnetite permittivity, as shown in Equation 4:

𝛜 𝐞𝐟𝐟 (𝛚) = 𝚽 • 𝛜 𝐅𝐞𝟑𝐎𝟒 (𝛚) + (𝟏 -𝚽) • 𝛜 𝐒𝐢𝐎𝟐 (𝛚) ( 4
)
Where 𝚽 is the amount of magnetite in the volume of the silica shell.

The near field enhancement of these nanostructures was simulated using the commercial software package COMSOL Multiphysics that employs finite element method (FEM) for the analysis of the plasmonic response 31 . The extinction spectrum was obtained from the electric field (𝑬 ⃗ ⃗ ) and electric vector (𝑫 ⃗⃗ ) using Equations 5:

𝝈 𝒂𝒃𝒔 = 𝟐 𝒄 𝟎 𝜺 𝟎 𝒏|𝑬 𝒊𝒏𝒄 | 𝟐 ∫ 𝑹𝒆𝒂𝒍(𝝈 𝑬 ⃗ ⃗ • 𝑬 ⃗ ⃗ * -𝒊𝝎𝑬 ⃗ ⃗ • 𝑫 ⃗⃗ * )𝒅𝑽 𝑽 (5)
Reflected and scattered waves were cancelled using a spherical perfect matching layer technique. The geometry of the HMPNP was reproduced as a Au proloid with the dimensions obtained in TEM pictures, surounded by 6 spheres of Fe3O4 inserted in a concentric proloid of silica.

Calorimetric measurements

The heating power of the NPs was evaluated registering the increment of temperature when irradiated with high frequency magnetic fields and laser. Equations 6 was used to evaluate the Specific Absorption Rate (SAR) of the samples.

𝑺𝑨𝑹 = 𝑪 𝑽 [𝑿] 𝚫𝑻 𝚫𝒕 Equation 6
Where CV is the specific heat of water (4.186 kJ L -1 K -1 ) and [X] is the concentration of the sample in g L -1 . In the case of magnetic hyperthermia, fields between 6-65 mT and a frequency of 100 kHz were applied to a total sample volume of 0.5 mL. The increase of the temperature as a function of time was measured with two fiber optic thermometers placed at slightly different heights inside the colloid, acquiring the mean value of both temperature measurements.

In laser irradiation experiments (photothermia), the samples were measured with a non-adiabatic crystal capillary of 0.8 mm using a volume of 50 𝛍L of colloidal NPs. This volume was irradiated using a 785 nm laser (LDM785, Thorlabs) of 20 mW focused to cover the whole diameter of the capillary. The fiber optic thermometer was inserted directly in the dispersion and carefully placed out of the laser spot.

Results and discussion

Synthesis of HMPNP

The TEM images of the IONPs and AuNRs employed in the synthesis of HMPNP are presented in TEM images of Figure 1a and1c, respectively. The electrochemical synthesis of IONPs produces quasispherical ferrite NPs with an average size of 12.0 nm and a size dispersion of 0.5 (Figure S1). Figure 1b shows that these IONPs present magnetization curves with a high saturation magnetization value (MS= 79 Am 2 kg -1 ) and small coercivity (HC) at room temperature if they are measured in a quasistatic regime (dc-field), what indicates a superparamagnetic behavior. X-ray diffraction patterns and Raman spectrum (Figure S4) indicate that the magnetic phase of IONP is mainly magnetite (Fe3O4) although a small fraction of maghemite (-Fe2O3) could be present as well.

On the other hand, the synthesis of AuNRs by the method of El-sayed produces a monodisperse population of NPs in which transversal and longitudinal sizes can be tailored adjusting the amount of seed employed in the synthesis (SI: Figure S2). Figure 1d shows the extinction spectrum of the samples prepared adjusting the aspect ratio (AR) to 3.15, 3.38, 3.86 and 4.38. Changing the AR of the AuNRs, it was possible to modify the frequency of the longitudinal plasmon resonance. The IR absorption peak (associated to longitudinal plasmon) shifts to higher wavelengths as long as the AR of the AuNRs grows, what opens the possibility of fitting the position of the peak to the wavelength of the excitation laser.

HMPNP presented in Figure 1e were prepared functionalizing the IONPs and AuNRs with PEG and CA molecules respectively. Previous studies 26,32 showed that these two molecules bind each-other during the growth of silica coating and produces hybrid nanostructures with a core@satellites arrangement. In these nanostructures, the silica shell acts as a matrix that holds the magnetic and plasmonic component in a robust and chemically stable structure 23 . Figure 1g shows that silica shell grows between IONPs and the AuNR avoiding common interfaces of these two phases. Consequently, the interplanar distances registered in their closer surfaces results similar to their corresponding bulk materials in (111) and (220) crystalline directions. The X-ray diffraction patterns of HMPNPs confirmed that the interatomic distances of the individual components were preserved after the hybridization (SI: Figure S4). In spite of this, the short-distance interactions between the different phases of the hybrid nanostructure can modify the magnetic and plasmonic properties of the components. This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins

Magnetic properties of IONP and HMPNP

Figure 2 shows the magnetization curves of IONPs and HMPNPs prepared as powders and colloids. It can be observed that the MS of HMPNPs is drastically reduced to 17 A m 2 kg -1 as a consequence of the non-ferromagnetic phases present in the sample (Au and SiO2). The insets of these figures show the first and second quadrant of normalized curves (dashed lines have been included as eye-guide).

Comparing curves of powder preparation (black curves) of both systems, it is possible to observe that the HMPNP curves presents larger 𝝌 and higher HC.

The direct interaction between Fe3O4 and Au phases is avoided by the silica interphase, which acts as a insulator spacer blocking the charge transference between them. Thus, in the study of the magnetic properties, the most relevant difference between both systems is the distance among magnetic NPs. In powder preparation, the IONPs are in close proximity to one another, while in HMPNPs, the silica coating increases the distance between IONPs and the shape of the AuNR favors their linear arrangement around the Au nucleus 26 . These two conditions modify substantially the effect of dipolar interactions in each case.

Using Monte-Carlo simulations, Serantes et al. observed that dipolar interactions can increase or reduce the effective HK of a collection of magnetic NPs depending on their relative disposition 10 . In a linear arrangement, the effective HK of the system is increased and their magnetization curve presents higher 𝝌 and HC. On the contrary, for more compact arrangements (cubic, hexagonal, etc.), the dipolar interactions produce the opposite effect on the magnetic response.

In the case of the HMPNPs, the presence of a Au nucleus prevents adjacent positions between IONPs and favors the linear arrangements of the IONP (Figure S3), what explains the higher HC and 𝝌 observed for hysteresis loops of HMPNP measured as powder.

On the other hand, both systems present magnetization curves with larger 𝝌 and smaller HC in liquid phase (red curves) respect to powder preparations (black curves). This effect can be attributed to the possibility of the particles to rotate freely in the liquid and align their easy magnetization axes with the field, increasing the magnetic susceptibility of the system.

In order to analyze further this effect, the zero field cooled (ZFC), field cooled (FC) and thermoremanence (TMR) curves were obtained for liquid phase preparation of both systems, as presented in When the dispersing media melts, the NPs are unblocked and the torque generated by the magnetic field can make them rotate, which increases the magnetization of the system. However, the HMPNPs comprise several IONPs with different magnetization easy axis and thus the torque generated by the magnetic field is not uniform in the whole nanostructure. In this kind of superparamagnetic systems, the effective torque induced by the field is divided by √𝑵 33 , where N is the number of NPs contained in the agglomerates (~6 in this case, details in SI). Besides, the DLS measurements indicated that hydrodynamic diameter is significantly higher for HMPNPs (DH =266 nm, PDI=0.20) than IONPs (DH= 57 nm, PDI=0.17), which also hampers their Brownian rotation in a viscous media and hinders the magnetization process. This effects explain the higher susceptibility observed in the hysteresis loops of IONP in liquid phase.

Plasmonic properties of HMPNP

Figure 4a shows the optical extinction spectra measured for AuNR, silica coated AuNR (AuNR@SiO2) and HMPNP samples. As it can be observed in AuNR@SiO2, the longitudinal plasmon resonance shifts to higher wavelengths and becomes wider when the AuNRs are surrounded by dielectric media of higher permittivity such as silica (𝜺 𝒓 ~𝟐. 𝟏). The shifting and widening of the peak become more significant in the case of HMPNP. Figure 4b compares the experimental spectrum of HMPNPs with the one predicted by Mie-Gans theory when assuming a 9% of magnetite in the surrounding media. It can be observed that although the position of both peaks matches, the width at half maximum registered for the longitudinal peak results much larger than the theoretical prediction (Table 1).

FEM simulations were carried out to analyze the discrepancies between experimental data and Mie-Gans spectrum. Figure 4d displays the geometry designed for the simulation of HMPNP plasmonic response. It is composed by a single Au proloid (a=51.9 nm, b=c=13.7 nm) surrounded by a silica shell 15 nm thick in which 6 Fe3O4 spheres of = 12 nm were randomly inserted (see details and other geometries in SI). The experimental permittivity values of Au 34 , Fe3O4 [START_REF] Querry | Contract. Rep[END_REF] and SiO2 36 were implemented in the COMSOL Multiphysics simulations ad oc. Please do not adjust margins

Please do not adjust margins

As shown in Figure 4c, the spectra obtained in AuNR, AuNR@SiO2 and HMPNP simulations, reproduce the position of the longitudinal plasmon resonance peak (Table 1). However, the single nanoparticle simulations do not take into account the size dispersion of the samples, which generates much narrower absorption peaks. Despite that, the widening obtained for the longitudinal peak of an HMPNP respect to an uncoated AuNR resulted to be in good agreement with the one observed in experimental data (~1.9).

The patterns of electric field enhancement presented in Figures 4e-f for the transversal and longitudinal plasmon resonances of HMPNP explain this effect. In contrast to AuNR and AuNR@SiO2 simulations (SI:Figure S6), the enhanced field around HMPNP does not follows to the radiation pattern of a dipolar antenna. The insets in these figures highlight the confinement of the electric field generated between IONP and AuNR surfaces. In appropriate conditions, such confinements increase the intensity of the plasmonic resonance and thus the amount of heat dissipated [START_REF] Ahijado-Guzmán | [END_REF] . However, the disordered arrangement of the IONPs around the AuNRs produced by this synthesis method generates a heterogeneous polarization of the surrounding media that damps the IR extinction peak and explain the extra widening observed in the spectrum of HMPNP.

Magnetic hyperthermia in IONP and HMPNP Figure 5 compares the magnetic response of IONPs and HMPNPs at high frequency. The high-frequency hysteresis curves in Figure 5a show that, at low HMAX, the IONPs curves present an elliptical shape similar to the one predicted by linear response models 38 . According to these models, the magnetization of the sample varies linearly with the applied field and the delay between them determines curve parameters such as HC or remanent magnetization. In this kind of cycles, the hysteresis area grows quadratically with HMAX. When µ0HMAX is over 29 mT, the magnetization starts to saturate and the curves do not present an elliptic shape anymore. This kind of curves needs to be explained using different approaches such as Stoner-Wohlfarth models 39 .

The growth of the cycle area (A) with HMAX 2 , presented in Figure 5d, shows also two regimes. When µ0HMAX < 31 mT, the area of the cycle grows linearly respect to HMAX 2 with a slope of 1.72 J g -1 T -2 , while at higher magnetic fields, the slope is reduced to 0.22 J g -1 T -2 . There is a linear relation between the area of the cycles and the power dissipated at a specific frequency. The corresponding SAR values were included in the right axis of the graph. They were calculated multiplying the area of the loops by the frequency of the excitation field (𝝂) (Equation 7).

𝑺𝑨𝑹 = 𝑨 • 𝝂 Equation 7

In Figure 5b, the hysteresis curves of HMPNPs present a different dependence with HMAX. At low fields, the curves are closed and thus magnetization reversal takes place without hysteresis. Only for µ0HMAX>23 mT it was possible to observe a significant area inside the curve. The growth of the area with HMAX 2 in Figure 5e shows two regimes as well, with slopes of 1.34 J g -1 T -2 and 0.37 J g -1 T -2 for low and high fields respectively. In this case, the transition field between them is 42 mT. Please do not adjust margins Please do not adjust margins Such difference in transition fields can be related to the different effective HK induced by dipolar interactions in IONPs and HMPNPs. According to Stoner-Wohlfarth theory, for a thermally blocked system of magnetic NPs with magnetization easy axis randomly oriented, the effective HK of the system is 2.08 times the HC of the curves 39 . In this way, HK values were calculated from the HC value of the loop measured at large magnetic field, obtaining 29 mT and 38 mT for IONP and HMPNP, respectively. In both cases, the transition between both regimes take place when HMAX is close to 1.1 times the effective HK of the system. According to numerical simulations of magnetic NP chains, the optimum HMAX value permitting to maximize the area of the curves with respect to the field energy is approximately 1.1 times HK 10 . These results indicate that the theoretical optimum value for HMAX corresponds to the field at which the transition between the two growth regimes occurs.

On the other hand, comparing the maximum value of SAR in Figure 5d (SAR = 115 W g -1 ) and Figure 5e (SAR = 134 W g -1 ), it is possible to observe that HMPNPs are able to dissipate a larger amount of heat when the field is strong enough. Figure 5c presents together the hysteresis loops of IONP and HMPNP at the highest field (65 mT). It shows that, at this field, the HMPNPs generate loops with a higher HC, which implies a larger area and thus larger SAR values. The HC enhancement in HMPNPs agrees with the quasistatic curves of powder samples (Figure 2b).

Calorimetric measurements in Figure 5f are in good agreement with the conclusions extracted from high frequency loops. At low fields (µ0HMAX ≤ 40 mT), the SAR of both samples grows quadratically with the field. The IONPs presents higher values of SAR in this range. However, when HMAX is increased, the growth of SAR decays and HMPNPs overcomes IONPs. The enhancement of SAR values for IONPs in linear assemblies has been previously reported 11 and has also been associated with the effect of dipolar interactions.

Photothermia experiments

Figure 6a shows the extinction spectrum of the AuNR, AuNR@SiO2 and HMPNP samples prepared for photothermal assays. The dash blue line displays the extinction spectra of IONPs employed in this study. The samples were prepared with AuNRs of different AR so that the maximum of the IR absorption peak matched with the wavelength of the excitation laser (SI: Figure S7). Besides, the number of NP was adjusted in each colloid to obtain similar optical extinction coefficient at 785 nm. In photothermal experiments, all the samples were irradiated with the maximum laser power accepted in human tissue (0.33 W cm -2 ).

The SAR values obtained for different colloids are presented in Figure 6b. In order to be comparable, SAR values were obtained substituting in Equation 7 the Au concentration of the colloids. Only in the case of IONPs the increment of temperature was divided by Fe3O4 concentration of the sample. It can be observed that although IONPs are not metallic and thus have not plasmonic response, they generate a certain amount of heat when are irradiated with an IR laser. To the best of our knowledge, the origin of this effect has not been fully explained yet [START_REF] Sadat | This journal is[END_REF]41 , since at the wavelength employed, the absorption of Fe3O4 is minimum (Figure 6a). However, the heat produced by laser irradiation has been observed to be effective to enhance the hyperthermia performance of IONPs 19 .

Comparing the SAR values of AuNR, AuNR@SiO2 and HMPNP, it can be concluded that the presence of IONPs in the HMPNPs improves the photothermal performance of hybrid nanostructures. However, to distinguish the contribution of the different phases, an additional colloid of IONPs and AuNRs, with Au and Fe concentrations similar to HMPNP sample, was included in the study.

The turquoise column shows that this colloid presents the highest SAR value. The differences between this colloid and HMPNP should be associated to the interaction between AuNRs and IONPs observed in Figure 4. In this case the creation of inhomogeneous concentrations of electric field between the surfaces of both phases reduce the heating power of these kind of nanostructures. Further development of self-assembling techniques would be required to produce a cooperative interaction between plasmon resonance of AuNRs core and IONPs around them.

Conclusions

In this work Au nanorods and iron oxide nanoparticles were combined in a hybrid nanostructure by controlling the sol-gel silanization process. The resultant nanostructures present suitable magnetic and plasmonic properties for nanoheating purposes in biomedical applications. It has been observed that the hybrid samples display a larger coercive field, which induces larger heating at high magnetic fields than the IONPs. Besides, the heat generated by IONPs and AuNRs in photothermal heating can be combined using HMPNPs. However, it was observed that the plasmonic interaction between the phases reduces the amount of heat released respect to a mixed colloid. Figure 6. Extinction spectrum of IONP (blue), AuNR (purple), AuNR@SiO2 (red) and HMPNP (green) samples used in photothermal assays. SAR values obtained for colloids of IONP, AuNR@CTAB, AuNR@SiO2, HMPNP and a mixture colloid of AuNR@SiO2 and IONP (turquoise).

Figures 3 .

 3 It can be observed that in ZFC and FC curves the magnetization grows drastically at the melting temperature of water (273 K), and in TMR curves drop to nearly zero values. It can also be observed that in the case of HMPNPs, the curves change more progressively with temperature.

Figure 1 .

 1 Figure 1. TEM images of: (a) iron oxide nanoparticles (IONP), (c) Au nanorods (AuNR) and (e, f) hybrid magneto-plasmonic nanoparticles (HMPNP). (g) HRTEM picture of HMPNP shows the atomic planes of IONP (upper inset) and AuNR (lower inset) in proximal sufaces. (b) Magnetization curve of IONPs at 300K measured in powder. (d) Normalized absorption spectra of Au nanorods with different AR: AR=3.15 (red), AR=3.38 (yellow), AR=3.86 (green) y AR=4.38 (blue).

Figure 2 .

 2 Figure 2. Magnetic hysteresis curves at 300 K of (a) IONP and (b) HMPNP. The insets present the normalized curves in which dash lines have been included as eye-guides. Both samples were measured as a powder (black) and as a colloidal suspension (red).

Figure 4 .

 4 Figure 4. Plasmonic response of AuNR, AuNR@SiO2 and HMPNP: (a) Experimental optical extinction spectra, (b) Comparative plot of experimental (continuous) and Mie-Gans theory (dash) extinction spectra of HMPNP, (c) Theoretical extinction spectra obtained by finite element method simulations (FEM). (d) Geometry used in FEM studies to simulate the plasmonic response of HMPNP. Near field enhancement generated at (e) transversal and (f) longitudinal plasmon resonances in the X-Z plane of HMPNP.

Figure 3 .

 3 Figure 3. Zero Field Cooled (black squares), Field Cooled (red dots) and thermoremanence (green triangles) curves for (a) IONP and (b) HMPNP in liquid phase. Vertical line indicates the fusion temperature of water (273K).

Figure 5 .

 5 Figure 5. High frequency magnetic curves of (a) IONP and (b) HMPNP at different maximum fields (HMAX). (c) Comparative graph of high frequency magnetic curves at µ0HMAX = 65 mT. Dependence of the area with HMAX 2 in magnetic curves of (d) IONP and (e) HMPNP. Dash and dot lines indicate the linear fittings of the data at low and high magnetic fields, respectively. The right axis indicates the corresponding SAR values. (f) Calorimetric measurements of SAR at different HMAX for IONP and HMPNP. Dash line shows the quadratic fitting of the data at low fields.

Table 1 .

 1 IR

		EXP	FWMHEXP	MG	FWMHMG	FEM	FWMHFEM
		(nm)	(nm)	(nm)	(nm)	(nm)	(nm)
	AuNR	765	138	765	129	760	44
	AuNR@SiO2 785	165	782	151	790	50
	HMPNP	857	261	860	195	861	86
		maximum wavelengths () and full width at half maximum
	(FWHM) according to experimental data (EXP), Mie-Gans theory (MG)
	and finite element method simulations (FEM).

Published on 24 August 2018. Downloaded by Freie Universitaet Berlin on 8/24/2018 4:31:16 PM. View Article Online DOI: 10.1039/C8CP02513D

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

Acknowledgements

The authors would like to thank the Dr. Ueslen Silva and Prof. Daniel Jaque for their help with the initial test of these systems. This work was supported by the Spanish Ministry of Science and Innovation: MAT2015-67557-C2-2-P and MAT2015-67557-C1-2-P. Financial support from COST Action TD1402 RADIOMAG for a STSM at LPCNO, Tolouse, is also acknowledged.

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