B Bouvry

L Ramiandrisoa

C Cheymol

C Gallou

H Maskrot

C Destouches

L Ferry

C Gonnier

T Duvaut

N Horny

Optical pyrometry measurement on oxidized zirconium alloy nuclear material cladding

In order to improve the safety of nuclear power plant, loss-of-coolant accident experiments are implemented in research reactor. In this framework, we develop an optical pyrometry device to measure surface temperature (700-1200°C) of Zircaloy cladding without contact. The whole set-up of the simplified device (under air, without radiation) and the measurement procedure including data treatment based on bichromatic pyrometry are presented, as well as results for various temperature levels. Temperature retrieval based on the hypothesis of emissivity ratio equal to a constant, is scanned over a large wavelength range. A constant surface temperature is obtained on the spectral range of measurement, confirming the relevancy of emissivity hypothesis. Differences between this non-contact temperature measurement and a complementary thermocouple temperature measurement are also discussed.

Introduction

Current energy needs of industrial and developing countries, combined with a strong increase of global warming induced by greenhouse gases like CO2 or N2O, leads to develop carbon-free energies. In this perspective, nuclear power is an important option, which needs research in term of recycling or security process. One of the most accidental scenario considered in nuclear power plant design is the LOCA (Loss Of Coolant Accident) which implies a large break on the primary circuit and a violent pressure decrease (from about 150 bars to few bars) [START_REF] Chung | Fuel behavior under loss-of-coolant accident situations[END_REF]. This dewatering makes the fuel clads overheat (up to 1200°C). The oxidation by steam at high temperature weakens clads which can burst and release fission products into the nuclear reactor. To simulate the LOCA, the Light-water One Rod Equipment for Loca Experimental Investigations (LORELEI) test device will be implemented on the Jules Horowitz reactor of CEA. The aim is mainly to test Zircaloy-4 claddings behavior in hard environment where steam oxidation is enhanced by high temperatures (from 700 up to 1200°C) [START_REF] Baek | Steam oxidation of Zr-1.5Nb-0.4Sn-0.2Fe-0.1Cr and Zircaloy-4 at 900-1200°C[END_REF]. A traditional temperature measurement by thermocouples is not accepted because it would create a cold point on the clad that generates a modification of burst conditions. This leads to design a non-contact real time (1Hz) measurement suited to these extreme conditions (high temperature, nuclear radiations and steam environment) and with an uncertainty about ±10°C.

In a first step, an optical pyrometry device is then developed to measure the surface temperature of Zircaloy cladding without contact, under air, without steam nor radiation. The aim is to test the measurement capability of optical pyrometry and study the emissivity property of cladding without additional perturbation. Thermocouples will be used on this experimental device to give a temperature reference.

Experiment

Zircaloy cladding

In nuclear reactor, pellet of nuclear material is inserted in a Zircaloy-4 clad and integrated inside heart of nuclear reactor. The visual aspect of a new clad is metallic color. Shortly, a thin layer of black color oxide covers the clad, this layer grows up to around a hundred of microns at the end of the life time of the fuel in the reactor core. So, we use an experimental facility named EDGAR 2 located in CEA Saclay research center to oxidize clad in air/steam environment at high temperature. The clad used in this study was oxidized at a temperature of 1100°C for 100s. Scanning Electron Microscopy (SEM) picture and Energy Dispersive Spectroscopy (EDS) measurement, presented in Figure 2, show the oxide layer, with thickness of about 15.7 µm, on the Zircaloy surface. This thickness is confirmed by the decrease of oxygen rate (bottom line with red color on Figure 2b) and the increase of zirconium rate from 76 to about 95% (top line with blue coloer on Figure 2b) in the volume of oxide layer compared to the rates deeper in the clad. The EDS analysis reveals that the oxide is only composed by Zirconium and Oxygen elements, which induces that oxide has a chemical composition close to zirconia material (ZrO2). Nevertheless, the black color aspect of the oxide (cf. Figure 1) implies that a non-stoichiometric zirconium oxide was created on the Zircaloy surface.

Optical pyrometry set-up

This optical pyrometry method is based on the comparison between the radiance emitted by the sample and by a heat source with known temperature and emissivity (ε), generally a black body (ε ~1) used as reference. The reference thermal source is a cylindrical black body furnace (HGH RCN1200N1) working up to a temperature of 1200°C, with an aperture of 12.5 mm.

An objective (made of two converging lens), collecting thermal radiation at about 10 cm from heat source or surface sample on an analysis area with a diameter of about 2 mm, is located first in front of the heat source. This set of lens focuses thermal flux emitted by the center of the aperture, on the end face of an optical fiber. We use an optical fiber in the pyrometry set-up to carry the optical power to the detector as in the future set-up in the research reactor. On the LORELEI device the fiber will be a pure silica core (or slightly doped silica core) fiber in order to limit the attenuation due to radiations, the optimum spectral range will be then: 0.9-1.8 µm [START_REF] Cheymol | High Level Gamma and Neutron Irradiation of Silica Optical Fibers in CEA OSIRIS Nuclear Reactor[END_REF]. The infrared spectrometer used on the pyrometry device is an Ocean Optics NIRquest 512. The photodiode detector allows measurement in a spectral range from 0.9 µm to 1.7 µm with a resolution of about 1.5 nm.

Figure 3.a) presents a picture of the heating sample holder, developed for this study, fitting by the cladding sample. The holder has a cylindrical shape and allows heating up to about 1000°C. Figure 3.b) presents a thermography picture of the Zircaloy cladding equipped with a welded thermocouple. The analysis area for pyrometry measurement, as well the thermocouple location, can be observe on this figure. This preliminary observation made by a bolometric infrared camera (FLIR systems, SC655 LS), working on the spectral range 7-12 µm, allows us to assess the temperature homogeneity on the sample.

The thermocouple is placed 1 cm below the pyrometry measurement area. To improve the grip of the thermocouple on the surface, oxide is withdrawn by polishing and thermocouple wires are welded on the surface of zirconium alloy. In this paper, the surface temperature of Zircaloy cladding will be presented for two temperatures of sample holder: 805°C and 905°C. The first step of the measurement procedure, the calibration, consists in collecting the power from the black body. The temperature of this source is chosen so that it emits power close to those emitted by the sample to study. In our procedure, four temperatures are used for calibration. So, black body is heated from 710 to 740°C, with a step of 10°C, and from 820 to 850°C, also with a step of 10°C, for sample holder temperature of 805 and 905°C, respectively. The (optical) power from the black body is measured for each temperature by the infrared spectrometer (result in "number of counts"). For each wavelength, the result is plotted as a function of the radiance, deduced from Planck's law. The linear regression obtained with the 4 points is the transfer function. Each wavelength has its own transfer function.

In a second step of procedure, the measuring device (lens, optical fibre and spectrometer) is translated in front of the cladding. Distance between lens and cladding is identical to those used for measurement on black body furnace.

The integrating times of the NIRquest 512 array detector vary with the aimed sample temperature and must be the same for sample measurement and calibration. For this study, 102 ms and 52 ms are selected for sample holder temperature of 805°C and 905°C, respectively. The integrating time of the spectrometer is set so that it works as much as possible on the linear range of the detector. Power from surface cladding is measured by the infrared spectrometer and is converting into radiance spectrum thanks to the transfer function.

Temperature retrieved by bichromatic method

Several pyrometry techniques may be applied to retrieve the temperature [START_REF] Ramiandrisoa | Pyrometry Techniques for Temperature Monitoring in Simulated LOCA (Jules Horowitz Reactor)[END_REF]. We focus here on the bichromatic method based on the hypothesis of known emissivity ratio between two wavelengths. Using Wien approximation, valid on the spectral range of measurement, the temperature can be retrieved as shown in [START_REF] Chung | Fuel behavior under loss-of-coolant accident situations[END_REF].

1 𝑇 = 𝜆 1 * 𝜆 2 𝜆 1 -𝜆 2 * 1 𝐶 2 * ln ( 𝑓 -1 (𝑆 1 ) 𝑓 -1 (𝑆 2 ) * 𝜀 2 𝜀 1 * ( 𝜆 1 𝜆 2 ) 5 ) (1)
With 𝑓, the transfer function of spectrometer, calculated from black body calibration, and, 𝑓 -1 (𝑆 1 ) and 𝑓 -1 (𝑆 2 ), the radiance calculated from signal 𝑆 1 received at wavelength 𝜆 1 and signal 𝑆 2 received at wavelength 𝜆 2 , respectively The values ε1 and ε2 are the emissivity at λ1 at λ2, respectively.

Based on the hypothesis of the emissivity ratio 𝜀 2 𝜀 1 ⁄ = 1, temperature is retrieved for each couple (λ1, λ2) selected in the spectral range considered. Several interval values λ2-λ1 (50, 100, 150, 200 and 250 nm) are taken into account on calculation. A mean temperature value is calculated as well as the standard deviation.

Results and discussion

The experimental procedure, described in the previous part, is used to measure the power from black body, in order to calculate the transfer function. Figure 5 presents intensity spectra measured on black body, for different temperatures (710, 720, 730 and 740°C). We can observe on Figure 5 (left) a depletion around 1.38 µm. This depletion is due to absorption bands of water vapour, present in atmosphere [START_REF] Yamanouchi | Absoprtion properties of the near-infrared water vapor bands[END_REF][START_REF] Smith | Absorption by water vapour in the 1 to 2 µm region[END_REF]. As the vapour concentration may fluctuate between calibration and sample measurements, the spectral range 1.3-1.45µm is removed. Figure 5 (right) presents also transfer functions calculated for two wavelengths (1 µm and 1.3 µm). The rather constant value of the temperature, for each experiment and wavelength gap presented on Figure 6, allows us to calculate a mean temperature, as well as the associated standard deviation. Table 1 gathers the thermocouple temperature and the mean of scanned bichromatic temperatures. All gaps of wavelengths lead to similar mean bichromatic temperature values for the two temperatures: 805 and 905°C. As shown in Figure 6, there is a strong fluctuation of the bichromatic temperature for the interval of 50 nm, confirmed by the highest value of the standard deviation of about 5.7°C (cf. Table 1). These temperature fluctuations decrease with the increase of the wavelength interval, as expected with noncorrelated fluctuation of the radiance [START_REF] Ramiandrisoa | Pyrometry Techniques for Temperature Monitoring in Simulated LOCA (Jules Horowitz Reactor)[END_REF]. By the knowledge of temperature and radiance, one can deduce an emissivity value of cladding around 0.9, constant on the spectral range and close to the value present in literature [START_REF] Sani | Diletta Sciti, Optical properties of black and white ZrO2 for solar receiver applications[END_REF]. However, are these temperature and emissivity retrieve the true ones? Indeed, others profiles of ε(λ) can also lead to constant profile. For example, bichromatic temperatures, for a gap of 100 nm and calculated for an emissivity ratio 𝜀 2 𝜀 1 ⁄ = 1.005 and 𝜀 2 𝜀 1 ⁄ = 0.995, corresponding to exponential emissivity profiles, are presented in Figure 7. They also lead to a constant profile, with mean temperatures 837.3°C and 826.2°C, respectively. Uncertainty about the real surface temperature value, when the emissivity profile is unknown, is the reason why a reference measurement by thermocouple was expected. However, we can observe a temperature difference of about 18°C between pyrometry temperatures and thermocouple values for the two temperatures levels (cf. Table 1). This discrepancy is not entirely due to the error of optical pyrometry measurement. Indeed, a temperature measurement by contact can lead to a heat lost due to a temperature gradient inside thermocouple wires and can create a cold point on the cladding surface. Gradient issues prevent accurate measurement of the surface temperature by thermocouples, and make difficult the comparison of optical pyrometry result with a reference measurement.

Conclusions

A non-contact temperature measurement is preferred rather thermocouple for the LORELEI test device because it will not disturb the burst conditions of the cladding. In this context, a simplified optical pyrometry set-up has been developed to test the temperature surface measurement capability of optical pyrometry on Zircaloy-4 cladding and study emissivity property of cladding under air without radiation. The whole set-up and the experimental procedure have been presented as well as the retrieval of temperature based on an extended bichromatic method. The hypothesis of an emissivity ratio equal to 1 appears to be relevant for both temperatures of sample heater: 805°C and 905°C. But, other emissivity ratio, close to 1 and corresponding to a little variation of ε(λ) also lead to relevant temperatures values with this method. One perspective of this study is a comparison of bichromatic results with those calculated by others pyrometry methods like monochromatic or polychromatic technics. This last method should allow us determining the true temperature and emissivity profile by a study of "residues" after minimization process. A miniaturization of optical pyrometry set-up is also expected for an introduction into LORELEI device.

Figure 1

 1 exhibits both new Zircaloy clad and oxidized clad used on pyrometry device.

Figure 1 :

 1 Figure 1: left: new Zircaloy-4 clad; right: oxidized clad with thin layer of black color oxide.

Figure 2 :

 2 Figure 2: a) SEM picture of pre-oxide Zircaloy cladding and b) EDS measurement (from outside surface of oxide to the depth of clad). Black layer on the left part of Figure 2a) corresponds to the resin coating sample used for SEM measurement.

Figure 3 :

 3 Figure 3: a) Description of sample holder and b) thermography picture, obtained by an infrared camera, for a temperature level: 805°C, showing analysis zone for pyrometry measurement.

Figure 4 :

 4 Figure 4: Diagram of pyrometry device for surface temperature measurement of Zircaloy-4 cladding and picture of black body furnace used like reference.

Figure 5 :

 5 Figure 5: left: intensity spectra from black body for temperatures from 710 to 740°C; right: transfer functions calculated at 1µm and 1.3 µm.

Figure 6

 6 Figure6shows a scanned bichromatic temperature calculation, in the spectral range from 1.0 µm to 1.3 µm, for sample holder temperature (TSH) : 805°C and 905°C as well as the temperature measured by thermocouples (TTC), located on the Zircaloy cladding surface, for each temperature level. Each point is the result of a bichromatic calculation for a wavelength couple. For example, the last point of the curve for a gap of 50 nm, corresponds to a calculation with λ1=1.25 µm and λ2=1.3 µm.

Figure 6 :

 6 Figure 6: Scanned bichromatic temperature as function of the wavelength λ1, with calculation made for several gaps (from 50 to 250nm) and both λ1 and λ2 included in the wavelength range: 1-1.3 µm, for two sample heater temperatures (TSH):805°C and 905°C, with an emissivity ratio hypothesis 𝜀 2 𝜀 1 ⁄ = 1

Figure 7 :

 7 Figure 7: Scanned bichromatic temperatures as function of the wavelength λ1, calculation made for a gap of 100 nm for a sample heater temperature of 905°C, with an emissivity ratio of 1.005 (green) and 0.995 (blue).

Table 1

 1

	: Thermocouple temperature (TTC) and bichromatic temperatures (hypothesis: 𝜀 2 𝜀 1 ⁄ = 1) for
	wavelength intervals from 50 to 250 nm and a step of 50 nm, obtained for two heater set points (TSH):
			805 °C and 905°C, and the standard deviation.
	TSH (°C)	TTC (°C)	T50nm	Bichromatic temperatures (°C) T100nm T150nm T200nm	T250nm
	805	727 ± 1.6	745.2 ± 5.7	745.2 ± 3.2	745.2 ± 2.1	745 ± 2.1	744.4 ± 2.2
	905	813.9 ± 2.5	832 ± 5.6	831.7 ± 3.1	831.8 ± 2.3 831.7 ± 1.9	832 ± 2.1

Acknowledgment

The authors are indebted to Ms. Lezaud-Chaillioux and Vandenberghe, and Mr. Hellouin de menibus for providing cladding sample as well as Ms. Blanc and Mr. Varlet for the SEM and EDS characterization.


Patrick Blaise

Francois Boussard

Paul Ros

Pierre Leconte

Marat Margulis

Gilbert Martin

Karine Blandin

Experimental R and D innovation for Gen-II,III et IV neutronics studies in ZPRs : a path to the future ZEPHYR facility in Cadarache

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

Introduction

For several decades, the French Atomic and Alternative Energy Commission has been undertaking experimental programs aimed at validating the calculation tools used to design standard and advanced LWRs. Two critical facilities [START_REF] Cathalau | More than forty years of neutronics experiments in critical facilities of Cadarache center: from EOLE and MINERVE to the future ZEPHYR reactor[END_REF] located at Cadarache have been mainly used to mock-up actual situations arising in LWRs in the EOLE facility, and measuring integral absorption cross-section of specific materials in the MINERVE pool reactor.

For example, after having investigated HCLWR concepts through the ERASME experiments in EOLE in the 80's, many programs were launched for mocking-up PWR situations in France, and then in ALWRs -in collaboration with Japanese industrial partners -from 1990 to 2005. The MINERVE facility was used to investigate burn up credit in collaboration with UK or to measure absorption cross-sections of heavy nuclides in collaboration with USA, through the INERI collaboration. More recently, MINERVE was used to determine integral capture cross-sections of current GEN-II&III absorbers and nowadays for measuring neutron characteristics of structural materials involved in the EPR™ GEN-III PWR reactor. Today, the EOLE facility is dedicated to investigate neutron vessel fluency ageing depending on both core loading and internal structures of NPP from the French fleet, through the FLUOLE2 program. In the same time, MINERVE should be used to perform the first comparison tests a new in-core instrumentation system.

These two facilities are connected to an experimental room where several -spectrometry benches are used to perform axial radial pin-by-pin fission ate measurements. Additionally, the MADERE platform is also used for specific measurements involving foils and dosimeters. Figure 1 and Figure 2 reproduce a bird's eye view of EOLE and MINERVE respectively.

The ZEPHYR Facility

The experimental programs performed in EOLE and MINERVE were very fruitful for the LWR physical phenomena understanding, but today these facilities approach fifty years old and should be strongly refurbished in order to satisfy the safety criteria against earthquake and to meet the post-Fukushima requirements. Instead of renewing these facilities, CEA and its partners have begun to design a new facility in Cadarache, called ZEPHYR 2 , aimed at offering at least, the same level of services as EOLE and MINERVE for the LWR codes experimental validation.

main experimental characteristics

The experimental capabilities expected for ZEPHYR will not only include those of EOLE and MINERVE, but also several ambitious improvements in terms of operation margins, fuel loadings, heterogeneous configurations, as well as innovative experimental techniques and associated instrumentation. One main ZEPHYR challenge is to combine the basic functionalities of EOLE and MINERVE in a single facility, which implies preserving both EOLE's experimental flexibility to validate a wide range of reactor physics methods and calculation procedures, as well as MINERVE's capabilities for oscillation measurements in various spectra for improving nuclear data. Other additional features of ZEPHYR are the following:

-Possibility to perform coupled core configurations: 2 ZEPHYR for Zero power Experimental PHYsics Reactor. o central area under water and self-critical (EOLE-like configurations) oro central area under air (harder spectrum with possibility of MOX fuel rods and/or a smaller moderation ratio for fast spectrum configuration, with a sufficient size and a modular driver core, -Multiple control rod mechanisms, adding more flexibility in the programmes with regard to the safety constraints, -Homogeneous sample heating and cooling system to measure high and low temperature Doppler effect under air or water configuration, -Mobile automatic pilot with controlled feed-back from the oscillation function (oscillator or handling cask) and from the function of sample permutation under air and water configurations, -Thermal/fast reference flux device for detector inter-calibration (fission chambers).

-Intensive training tool after the ISIS reactor shutdown, -Qualification of computational tools and experimental devices for Jules Horowitz Reactor (JHR), -Computation of irradiated fuel sample worth, -Reception of GEN-II,III, GEN-IV and MTR irradiated fuel samples for oscillation, prediction of reactivity loss, study of power distribution and neutron/gamma fluxes, using PIE on activated fuels under air or water by -spectrometry measurements.

Besides the above improvements, other potential ZEPHYR functions could include:

-Possibility to insert experimental loops of JHR type, such as NaK loop, possibly heated, -Enhanced dosimetry capacities. -Enhanced operation capabilities, particularly dedicated to education and training, -Criticality/safety representative configurations, -…

Status of engineering design

The main civil engineering work design is under completion. The reference design includes only one concrete structure to duplicate both EOLE and MINERVE specificities, namely the "2 in 1" concept but with a significant limitation regarding the operational point of view. Thus, the initial "2 in 1" concept has been revisited and other designs, among which a "2 in 2" with enhanced capacities. An example of a design very close to the current EOLE/MINERVE is given on figure 3. The decision between the different concepts will be made at the end of the preliminary design phase, foreseen at the end of 2016.

For the EOLE part, the core layout has been enlarged in order to treat coupled cores, as well as larger LWR lattices for the study of eigenvalue separation. The number of possible safety rods has been extended from 4 to 6. The EOLE part will mainly be dedicated to industrial applications (code validation, validation of innovative designs,…).

For the MINERVE part, the pool reactor concept has been maintained. The structures are reproduced as it is, but with the possibility to study coupled fast/thermal lattices.

Figure 3. Axial cross-sectional view of the main building

The facility storage has been designed to accept current EOLE and MINERVE fuels, as well as (limited amount of) rods and plates from MASURCA, for fast/thermal coupled core studies.

Focus on some on-going R&D work

The two following paragraphs will detail on going R&D studies on awaited experimental improvements. For the sake of synthesis, we will only focus on two main R&D innovations: fast/thermal coupling for spectral emphasis, and core degradation recriticality studies in fast reactors. Those R&D works are lead through 2 PhD theses.

Nuclear Data improvement through optimized fast/thermal coupled lattices

For the last decades, the MINERVE ZPR located in Cadarache has produced a large range of experimental data, mainly for LWRs but also for the fast spectrum reactors through the PHENIX and SUPERPHENIX projects [START_REF] Martin-Deidier | Mesure intégrale de la capture des produits de fission dans les réacteurs à neutrons rapides[END_REF][START_REF] Lehmann | A fast critical reactor assembly with strong energy dependence of adjoint flux[END_REF]. In parallel with R&D studies about sodium fast reactors, in particular the ASTRID technological demonstrator, a new interest for fast-thermal coupled cores has risen up. Those configurations consist of getting fast-spectrum neutronics characteristics in a reduced central zone, also called the "measurement zone" or "experimental zone" while criticality is achieved thanks to a thermal driver zone. As this latter gathers the majority of fissions, such configurations allow an important reduction of fissile materials and a higher flexibility due to the thermal-spectrum kinetics parameters. The main issue is then to provide a proper fast spectrum in the center thanks to an adapted spectral conversion zone, surrounded by the thermal spectrum zone.

In order to understand the neutronics specificities of fast/thermal coupled cores, simplified calculations were performed using the fast-lattice deterministic ECCO/ERANOS system of codes. It enables an accurate calculation of the fast zone -the one of interest -whereas the thermal zone is also well computed thanks to a 1968-group cell calculation based on subgroups and probability table methods for self-shielding. Core calculations are performed in (R-Z) geometry using the BISTRO transport code in S 4 -P1 and 33 energy-groups

From Thermal to Fast Spectrum: Conversion and Transition Zones

The challenge to convert a thermal spectrum into a fast one requires the conception of an adaptation zone. The reference fast cell is not efficient enough to lead to the targeted spectrum within an approximate 35 cm radius zone, the current radius of the experimental zone in MINERVE. Many calculations have been performed in order to improve the spectral adaptation. It seems that two successive zones with different and specific compositions is a satisfactory compromise:

 A conversion zone has to suppress the main part of thermal and epi-thermal neutron flux coming from the thermal zone so as to get a fast spectrum. Fissile isotopes have been selected, in order to convert efficient slowed-down neutrons into fission neutrons on the other hand, as it appears to be more efficient because neutrons from the thermal zone strongly contribute to the criticality.

 A transition zone stands as a first step to the targeted fast spectrum. The conversion zone softens the fission neutrons coming from the conversion zone and cuts off the residual thermalized neutrons. Then, several sizes of materials with scattering properties (stainless steel, graphite, natural UO 2 ) were tested. Thanks to its "natural" and "specific" slowing-down power, 𝜉𝛴 𝑠 and 𝜉𝛴 𝑠 𝛴 𝑎 ⁄ , natural UO 2 has appeared to be the best candidate.. In ZPR, highly precise measurement techniques such as pile oscillations enable to achieve uncertainties in the range of 0.01 pcm in thermal lattices. Nonetheless, total and integral effects can only be measured. The spectral emphasis technique, relying on the modification of the adjoint-flux energydependence [3][4], gives a way to separate reactivity effects in integral experiments.

Principle of the Spectral Emphasis Technique

For a non fissile isotope, the total reactivity variation splits up into two parts: ∆𝜌 𝑡𝑜𝑡 = ∆𝜌 𝑠 + ∆𝜌 𝑎 . The perturbation theory, here developed in a G energy groups in the diffusion theory, leads to : The main difference between those expressions is the adjoint flux contribution to the reactivity effect.

∆𝜌 𝑎 = -∑ 𝛷 𝑟𝑒𝑓,
Indeed, whereas the ∆𝛴 𝑔 𝛷 𝑝𝑒𝑟𝑡,𝑔 term is multiplied by 𝛷 𝑟𝑒𝑓,𝑔 † for absorption, it is multiplied by (𝛷 𝑟𝑒𝑓,𝑔′ † -𝛷 𝑟𝑒𝑓,𝑔 †

) for scattering. Consequently, if the adjoint flux is energy-independent, ∆𝜌 𝑠 = 0 and therefore, ∆𝜌 𝑡𝑜𝑡 = ∆𝜌 𝑎 . Using another configuration, with a strong energy dependency of the adjoint flux, we would be able to separate the absorption and scattering reactivity effects. Lattice design is slightly different, as the effect to be emphasized is different.

Nevertheless, the main principle of conversion/transition zone remains identical. Figure 4 hereafter reproduces a steep adjoint (left) as well as a flat adjoint (right), obtained in optimized configurations. A complete description of the preliminary design calculation for both optimized transition zones and spectral emphasis unit cells can be found in [START_REF] Ros | Fast/thermal coupled cores in ZPR revisited: physical specificities and potentialities for ZEPHYR[END_REF].

Severe accident modeling

Philosophy

The objective of the current study is to develop a new approach for experiments design in the frame of severe accidents studies in LWRs and FRs, due to the unique capabilities of the ZEPHYR. The underlying idea is to build a full program of tests in the future ZEPHYR facility and to develop innovative approaches to manage the measurement of reactivity effects. In order to address the objective of this work, several studies must be performed in order to provide the best design of the test lattices dealing with the following items:

 Departure from nucleate boiling and local void increase without fuel degradation  A possibility to use MASURCA (MSK) fuel plate to locally model a fuel bundle degradation, in both LWR and FR situations. This is in order to simulate a small portion of molten fuel, which leads to a bundle blockage and vapor expansion.

Under those constrains the effect of a local reflooding will be studied and characterized. This would allow developing a methodology for slow and precise reflooding, with implementation to actual LWRs and FRs. A suggested model of the fuel lattice is presented in Figure 5. Furthermore, several feasibility studies will be performed relative to several situations:  Problem of debris stratificationthis situation represents a very interesting problem of double heterogeneity for deterministic codes, as a function of debris size.  Problem of cladding oxidation, and in particular the ZrO 2 oxide layer and its impact on the reactivity.  Problem of boron presence in water and its a-posteriori deposition above the debris bed.

On-going studies for SFRs

The main objective of this work is to evaluate two scenarios of SFR core degradation sequences, small and large core distortions. The sequence leading to core damage is initiated by total sodium dry out of several channels, which is then followed with small and large fuel compactions around the core mid plane. At the final stage the compacted fuel is relocated to the bottom part of the core, where it forms a molten pool representative configuration. The objective of the work is included in a more general framework of developing a new approach for experiments design in the frame of severe accidents studies in LWRs and FRs. The underlying idea is to build a full program of tests in a dedicated facility and to develop innovative approaches to manage the measurement of reactivity effects and local power distributions.

In SFR, Current investigations show that, under typical accident conditions, a sufficient amount of core material can be removed from the core region within the right time slot, and a neutronically active fuel pool comprising the entire core, with all its potential for recriticality energy, can be prevented. In this way, an additional middle line of defense could be introduced into the safety strategy against recriticality.

Both models show a potential of reducing the probability for recriticality events in FRs. However, they are still under ongoing investigation, both experimental and theoretical studies [START_REF] Yamano | First 3-D calculation of core disruptive accident in a large-scale sodium-cooled fast reactor[END_REF][7] [START_REF] Rimpault | Challenges associated with the mitigation of unprotected loss of flow in sodium-cooled fast reactors cores[END_REF]. Therefore, there is a possibility to utilize the ZEPHYR unique capacities to investigate those models as part of the experimental program. Current R&D studies are focused on fast/thermal characteristics to reproduced in an condensed zone, severe situation that were modeled in the SNEAK facility [START_REF] Henneges | Validation of neutronic codes for distorted core configurations with the SNEAK-12 critical assemblies[END_REF] [START_REF] Helm | Measurements and calculations of reactivity effects of material rearrangement in a plutonium-fuelled fast reactor rod lattice[END_REF]. The experiments in the SNEAK-12A were focused on reactivity differences between an undistorted core and a series of disturbed configurations that simulated the mentioned earlier disruptions.. The scenario consists of 4 stages:

1. Normal core operation. 2. Small fuel slump in. 3. Large fuel slump in. 4. Molten pool formation.

The next Figure 6 and Figure 7 reproduce the philosophy of SCA in ZEPHYR using its fast/thermal capacities, as well as the results obtained in SNEAK-like configuration using TRIPOLI-4 [START_REF] Brun | TRIPOLI-4, CEA, EDF and AREVA reference Monte Carlo code[END_REF] and Serpent-2 [START_REF] Leppanen | Development of a New Monte Carlo Reactor Physics Code[END_REF] Monte Carlo codes, together with the JEFF3.1.1 nuclear data library . Several challenges remain, as the way to take into account temperature effects in severe accident sequences. This particular item is under analysis using representativity and transposition studies, are will be treated with the ECCO/ERANOS sensitivity capacities. The complete study on SCA is reproduced in [START_REF] Margulis | Advanced small and large core distortion modeling in ZPR to assess core recriticality scenarios of SFR core degradation sequences[END_REF] and extended in [START_REF] Margulis | Monte Carlo Analysis of SNEAK12 core disruption in LMFBRs-the path for innovative sever accidents modeling in ZPRs[END_REF].

Conclusions

ZEPHYR, the new facility to be built by CEA in Cadarache by 2025 includes innovative design features to answer key-problems in core physics and neutronics in the next decades. It reproduces, in an enhanced way, both EOLE and MINERVE characteristics, and extends their flexibilities to new features, such as nuclear data for Gen-IV reactors, coupled neutronics/thermalhydraulics capacities, criticality programs , and severe accident modeling for LWR and SFR recriticality problems. In parallel, the facility will be equipped with new capacities for instrumentation R&D, and its calibration in adapted neutron spectra, from thermal to fast.

ZEPHYR is open for international collaboration, within the frame of specific agreements for accessing the facility, and will also propose to the national and international community dedicated operation timeframe for "education and training" of young scientists, as well as operation teams for partners.

Figure 1 : 2 :

 12 Figure 1 : The EOLE facility Figure 2 : The MINERVE pool-type reactor

Figure 4 .

 4 Figure 4. Strongly energy-dependent adjoint flux in the center of fast-thermal coupled or fast configurations (left) and Energy-independent adjoint flux (right)

Figure 5

 5 Figure 5 Suggested model for re-criticality studies for LWRs in ZEPHYR

Figure 6 Figure 7 .

 67 Figure 6 Suggested model for re-criticality studies for SFRs in ZEPHYR

  a. Step s1 -Sodium void b. Step 2 -Small slump-in c. Step 3 -Large slump-in d. Step 4 -Molten pool

: corresponding author : patrick.blaise@cea.fr


Jasmin Matuszak

Philipp Dörfler

Stefan Lyer

Harald Unterweger

Maya Juenet

Cédric Chauvierre

Amr Alaarg

Danielle Franke

Gunter Almer

Isabelle Texier

Josbert M Metselaar

Ruth Prassl

Christoph Alexiou

Harald Mangge

Didier Letourneur

PhD Iwona Cicha
email: iwona.cicha@uk-erlangen.de

Comparative analysis of nanosystems' effects on human endothelial and monocytic cell functions

Keywords: nanoparticles, atherothrombosis, endothelial-monocyte interactions, endothelial migration, monocytic cell chemotaxis

The objective of our work was to investigate the effects of different types of nanoparticles on endothelial (HUVEC) and monocytic cell functions. We prepared and tested 14 different nanosystems comprising liposomes, lipid nanoparticles, polymer and iron oxide nanoparticles. Some of the tested nanosystems contained targeting, therapeutic, or contrast agent(s). The effect of particles (0-400 µg/mL) on endothelial-monocytic cell interactions in response to TNF-α was investigated using an arterial bifurcation model and dynamic monocyte adhesion assay. Spontaneous HUVEC migration (0-100 µg/mL nanoparticles) and chemotaxis of monocytic cells towards MCP-1 in presence of particles (0-400 µg/mL) were determined using a barrier assay and a modified Boyden chamber assay, respectively.

Lipid nanoparticles dose-dependently reduced monocytic cell chemotaxis and adhesion to activated HUVECs. Liposomal nanoparticles had little effect on cell migration, but one formulation induced monocytic cell recruitment by HUVECs under non-uniform shear stress by about 50%. Fucoidan-coated polymer nanoparticles (25-50 µg/mL) inhibited HUVEC migration and monocytic cell chemotaxis, and had a suppressive effect on monocytic cell recruitment under non-uniform shear stress. No significant effects of iron oxide nanoparticles on monocytic cell recruitment were observed except lauric acid and human albumin-coated particles which increased endothelial-monocytic interactions by 60-70%. Some of the iron oxide nanoparticles inhibited HUVEC migration and monocytic cell chemotaxis. These nanoparticle-induced effects are of importance for vascular cell biology and function and must be considered before the potential clinical use of some of the analyzed nanosystems in cardiovascular applications.

Introduction

Nanotechnology is expected to improve the management and detection of atherothrombotic diseases [START_REF] Mangge | Laboratory medicine for molecular imaging of atherosclerosis[END_REF], which constitute one of the biggest global health problems (Lozano, et al., 2012).

By coating diagnostic nanosystems with plaque-specific ligands, significantly increased accumulation of these agents at the atherothrombotic sites could be achieved [START_REF] Hamzah | Specific penetration and accumulation of a homing peptide within atherosclerotic plaques of apolipoprotein E-deficient mice[END_REF], leading to improved detection and characterization of the disease. Furthermore, the direct targeting of drugdelivery nanosystems at the affected artery region could increase their therapeutic efficacy in parallel with reducing the systemic side effects. Vast numbers of bench investigations published in the recent produced very promising results [START_REF] Cicha | Cardiovascular therapy through nanotechnology -how far are we still from bedside?[END_REF][START_REF] Bietenbeck | Remote magnetic targeting of iron oxide nanoparticles for cardiovascular diagnosis and therapeutic drug delivery: where are we now?[END_REF][START_REF] Karimi | Nanotechnology in diagnosis and treatment of coronary artery disease[END_REF], but the clinical impact of nanoparticulate agents has been negligible in terms of diagnosis or therapy of cardiovascular diseases so far. The reasons for that are mainly the safety requirements, which are much higher for nanoparticulate cardiovascular drugs than for nanoparticulate cancer therapies. Consequently, the numbers of nanomedicines for cardiovascular applications entering the clinical trials are substantially smaller than in case of anti-cancer nanodrugs.

For the development of clinically safe nanoparticle-based systems for intravascular administration, systematic toxicological analyses of the candidate nanosystems are necessary. In our previous work, we performed standardized investigation of 10 nanoparticle types concerning their basic physicochemical parameters, long-term stability and the biocompatibility with endothelial cells [START_REF] Matuszak | Nanoparticles for intravascular applications: physicochemical characterization and cytotoxicity testing[END_REF].

The purpose of the present study was to provide new insights into the effects of different types of nanoparticles on human umbilical vein endothelial cell (HUVEC) and monocytic cell functions. Both endothelial and monocytic cell activation plays a major role in the development and progression of atherosclerosis. In endothelial cells, constantly exposed to the blood flow, the physiological functions greatly depend on shear stress-activated mechanisms. In fact, the susceptibility to atherosclerosis is governed by the specific patterns of shear stress. Whereas laminar flow protects endothelial cells from atherogenic stimuli, non-uniform shear stress induces endothelial activation and inflammatory cell recruitment [START_REF] Cicha | Shear stress preconditioning modulates endothelial susceptibility to circulating TNF-alpha and monocytic cell recruitment in a simplified model of arterial bifurcations[END_REF]. Increased monocyte chemotactic response and their migration into atherosclerotic lesions are the driving force in the disease progression. Hence, inhibition of leukocyte chemotaxis represents a target for an effective anti-atherosclerotic therapy. During all stages of atherosclerosis, endothelial injury contributes to atherothrombosis and increased accumulation of blood components in the vessel intima. The capacity of endothelial cells to proliferate and migrate is therefore crucial for endothelial regeneration and providing anti-thrombogenic barrier.

Thus far, very little is known about the influence of circulating nanoparticles on the endothelial responses to cytokines, monocyte chemotaxis, or endothelial-monocytic cell interactions. We have therefore investigated whether the presence of circulating nanosystems affects the TNF-α-induced monocytic cell recruitment by endothelial cells grown under non-uniform shear stress conditions, and whether nanoparticles affect spontaneous endothelial cell migration and monocytic cells chemotaxis towards monocyte chemoattractant protein-1 (MCP-1). We analyzed diverse nanosystems, comprising liposomes, lipid nanoparticles, polymer and iron oxide nanoparticles. Some of the tested nanosystems contained a P-selectin targeting agent (fucoidan), a therapeutic substance (e.g. pravastatin, prednisolone), or a contrast agent (e.g. gadolinium chelate, iron oxide). In order to predict in vivo responses, we evaluated the effects of these nanosystems on primary HUVECs and a monocytic cell line, utilizing functional assays under conditions that resemble the physiological state.

Materials & methods

Reagents

Soybean oil and Myrj™ s40 (PEGylated surfactant) were purchased from Croda, Chocques, France.

Lipoid S75 and dipalmitoylphosphatidylcholine (DPPC) were from Lipoid GmbH, Ludwigshafen, Germany. Suppocire™ NB was from Gatefosse, Saint-Priest, France. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Dextran T70 was from Roth (Karlsruhe, Germany), or from Amersham Pharmacia Biotech (Freiburg, Germany), and dextran T40 from PharmaCosmos (Holbaek, Denmark). Carboxymethyl-dextran sodium salt (CM-Dextran) was purchased from Sigma Aldrich (Saint Quentin Fallavier, France) and diethylaminoethyl-dextran 20 (DEAE-dextran) from TdB Consultancy (Uppsala, Sweden). Low molecular weight Fucoidan (3-8 kDa, Ascophyscient®) was from Algues et Mer (Ouessant, France). IBCA (isobutylcyanoacrylate, Glue 368) was from Orapi (Saint-Vulbas, France). Bovine serum albumin (BSA) and iron (II) chloride tetrahydrate, were from Merck, Darmstadt, Germany. Recombinant human serum albumin (HSA) was purchased from Novozymes Biopharma (Bagsvaerd, Denmark). Lauric acid, epichlorohydrin and dextran T40 (Mw, 40 kDa) were from Sigma Aldrich, Munich, Germany. Ceric (IV) ammonium nitrate and tri-sodium citrate dihydrate were purchased from Fluka (Saint Quentin Fallavier, France). NaOH, HCl (25%), NH3 (25%), and nitric acid (65%w/w) were from Roth. Iron (III) chloride hexahydrate was purchased from Sigma-Aldrich or from Roth. Gadolinium chelate B22286 (DOTA-N-N-dioctadecylamide Gd(III)-complex) was kindly provided by Bracco (Milan, Italy).

Pravastatin sodium was from AIkon Chemical (China/Brunschwig Chemie, The Netherlands) and prednisolone phosphate from BUFA (Uitgeest, The Netherlands). MCP-1 was purchased from Peprotech (Hamburg, Germany) and TNF-α from Miltenyi Biotec (Bergisch Gladbach, Germany). All compounds used were of pharmaceutical (Ph. Eur) or highly pure (⩾99%) grade and were used without any further purification.

Endothelial Cell Growth Medium with endothelial cell growth supplement was purchased from PromoCell (Heidelberg, Germany). RPMI 1640 medium, glutamine and fetal calf serum were from Biochrom AG, (Berlin, Germany), penicillin and streptomycin were from Gibco (Fisher Scientific, Schwerte, Germany) and Trypan blue from Biowest (Th.Geyer, Renningen, Germany). Alexa488phalloidin was from PromoKine (Heidelberg, Germany), nuclear stain DAPI from Molecular Probes (Darmstadt, Germany) and hematoxylin-eosin stain from Dako (Hamburg, Germany).

Nanoparticle synthesis

In total, 14 nanoparticle systems were investigated, including 5 types of liposomes (LP-NPs), 3 types of lipid nanoparticles (LD-NPs), 2 types of polymer nanoparticles (PM-NPs) and 4 types of iron oxide nanoparticles (IO-NPs). The detailed description of nanoparticle characterization methods was reported before [START_REF] Matuszak | Nanoparticles for intravascular applications: physicochemical characterization and cytotoxicity testing[END_REF].

Liposomes: Liposomes are composed of a lipid bilayer that encloses an interior aqueous space [START_REF] Puri | Lipid-based nanoparticles as pharmaceutical drug carriers: from concepts to clinic[END_REF]. The sterically stabilized PEGylated liposomes (LP-NP1), containing POPC, cholesterol and DSPE-PEG2000 at molar ratios of 3:2:0.15, were made using the lipid film hydration technique, followed by extrusion, as described previously [START_REF] Almer | Adiponectin-coated nanoparticles for enhanced imaging of atherosclerotic plaques[END_REF][START_REF] Almer | Interleukin-10: an anti-inflammatory marker to target atherosclerotic lesions via PEGylated liposomes[END_REF]. Gadoliniumloaded liposomes (Gd-LP-NP1) were prepared using the same method, with the following components: POPC:cholesterol:DSPE-PEG2000:B22286 at molar ratios of 3:2:0.15:0.78.

The LP-NP2 liposomes, containing DPPC, DSPE-PEG2000 and cholesterol (at 1.85: 0.15: 1 M ratio), were prepared using the lipid injection method, by mixing the ethanolic lipid solution with the aqueous phase under magnetic stirring at 60˚C. The resulting coarse dispersion was downsized by multiple extrusion steps through polycarbonate filter membranes with decreasing pore sizes of (200-100 nm).

Subsequently, ethanol and dissolved lipids were removed by dialysis against phosphate buffered saline (PBS). Pravastatin-loaded liposomes (Prava-LP-NP2) were alike synthesized by injection of solubilized lipids and pravastatin sodium (2 mg/mL) into the aqueous phase. In order to remove ethanol, free drug and free lipids, the sample was dialysed by tangential flow filtration. Prednisolone-loaded liposomes (Predni-LP-NP2) were made by lipid injection into the aqueous solution containing 100 mg/ml prednisolone in water for injection (van der Geest, et al., 2015). Unencapsulated prednisolone was removed by dialysis against saline (molecular weight cut-off of 10 kDa).

Lipidots TM : Lipid nanoparticles (LD-NPs) were prepared by the sonication method [START_REF] Gravier | Lipidots: competitive organic alternative to quantum dots for in vivo fluorescence imaging[END_REF]. Briefly, the lipid phase was prepared by mixing Suppocire™ NB, soybean oil and lipoid S75.

The aqueous phase, containing Myrj™ s40 (PEGylated surfactant) in PBS, was heated to 50°C to melt the surfactant and then mixed with the lipid phase, followed by sonication and dialysis against PBS. The batches of particles with specified diameter were obtained by altering the lipid and surfactants ratios.

Three different sizes (diameters) were formulated: 50 nm (LD-NP1), 80 nm (LD-NP2), and 120 nm (LD-NP3).

Polymer nanoparticles: PM-NPs were synthesized by a redox radical emulsion polymerization method (Chauvierre, et al., 2003). The core of the nanoparticles used in our studies (approximately 80% of the total mass) was made of poly(isobutylcyanoacrylate) (PIBCA), which was covalently cross-linked with polysaccharides of the coating, forming a hydrophilic shell. Two different coatings were used: (a) 90% CM-Dextran/10% Fucoidan (FC-PM-NP1); and (b) 80% DextranT70/10% DEAE-dextran/10% Fucoidan (FC-PM-NP2). Targeting ligand fucoidan, which is contained in the particle shell, is a mimic of sialyl Lewis X, the natural ligand of P-selectin [START_REF] Bachelet | Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets[END_REF][START_REF] Rouzet | Radiolabeled fucoidan as a pselectin targeting agent for in vivo imaging of platelet-rich thrombus and endothelial activation[END_REF].

Iron oxide nanoparticles (IO-NP): Superparamagnetic iron oxide nanoparticles and ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) consist of an iron oxide core, which is coated with organic materials such as fatty acids, polysaccharides, or polymers. Four kinds of IO-NPs were used in the present study.

Lauric acid/BSA-coated magnetite nanoparticles (IO-NP1) were synthesized by coprecipitation under argon atmosphere, subsequent in situ coating with lauric acid, and formation of an artificial albumin corona as previously described (Zaloga, et al., 2014). Subsequently, IO-NP1 were purified by centrifugal ultrafiltration (molecular weight cut-off 100 kDa). IO-NP2 were prepared according to the same protocol, whereby BSA was replaced with clinically-approved HSA formulation. IO-NP3 nanoparticles were synthesized by the coprecipitation method under nitrogen atmosphere, followed by coating with carboxydextran as described previously [START_REF] Matuszak | Nanoparticles for intravascular applications: physicochemical characterization and cytotoxicity testing[END_REF]. For preparation of dextran-coated USPIOs (IO-NP4), the synthesis method developed by Unterweger et al. was used (Unterweger, et al., 2014). Particles were synthesized by coprecipitation under argon atmosphere, in the presence of dextran T40. To stabilize the dextran coating, crosslinking was performed using epichlorohydrine [START_REF] Unterweger | Non-immunogenic dextrancoated superparamagnetic iron oxide nanoparticles: a biocompatible, size-tunable contrast agent for magnetic resonance imaging[END_REF].

All particles used in this study were stored at 4°C and, prior to their use in cell culture experiments, sterilized by filtration through a 0.22 μm filter, with the exception of PM-NPs, for which 0.45 µm filters were used. This was due to the fact that the average hydrodynamic diameter of FC-PM-NP2 is around 227 nm, i.e. the same size as the pores of the 0.22 µm filters, which inevitably results in rapid clogging.

To be in accordance with the further in vitro tests, we also used the 0.45 μm filter for FC-PM-NP1, despite the fact that their size allowed filtration through a 0.22 μm filter.

Physicochemical characterization

Z-averaged hydrodynamic diameter, polydispersity (PDI) and ζ-potential were determined in water with a Zetasizer Nano ZS (Malvern). The detailed description of further characterization methods relevant for the respective nanoparticle types was reported previously [START_REF] Matuszak | Nanoparticles for intravascular applications: physicochemical characterization and cytotoxicity testing[END_REF]. The composition and basic characteristics of all tested nanosystems used in the present study are summarized in Table 1.

Cell culture and viability assays

Human umbilical vein endothelial cells (HUVECs) were isolated from freshly collected umbilical cords (kindly provided by the Dept. of Gynaecology, Prof. Beckmann, University Hospital Erlangen) [START_REF] Cicha | Shear stress preconditioning modulates endothelial susceptibility to circulating TNF-alpha and monocytic cell recruitment in a simplified model of arterial bifurcations[END_REF]. The use of human material was approved by the local ethics committee at the University Hospital Erlangen (review number 237_12B from 19.09.2012). Cells were cultured in Endothelial Cell Growth Medium with endothelial cell growth supplement containing 5% fetal calf serum, 4 μL/mL heparin, 10 ng/mL epidermal growth factor and 1 μg/mL hydrocortisone, at humidified 5% CO2 atmosphere. HUVECs at passage 1-2 were used. Prior to the functional assays, all tested nanosystems underwent the analysis of biocompatibility with HUVECs using real-time cell analysis and live-cell microscopy in static conditions, as well as the evaluation of potential cytotoxicity under arterial flow conditions, according to established methods [START_REF] Matuszak | Nanoparticles for intravascular applications: physicochemical characterization and cytotoxicity testing[END_REF]. Real-time cell analysis was used to estimate cell number, attachment and viability based on the impedance measurements, but did not allow the detailed observations on cell morphology. Therefore, live-cell microscopy was used as a complementary method to monitor HUVEC phenotype and confluence over incubation time.

THP-1 monocytic cells were cultured in RPMI 1640 medium supplemented with 2 mmol/L glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and 10% fetal calf serum. Viability of cells was greater than 98% as estimated by Trypan blue exclusion.

HUVEC confluence under flow conditions

The bifurcating flow-through cell culture slides were obtained from Ibidi® (Munich, Germany).

Numerical flow simulation [START_REF] Cicha | Shear stress preconditioning modulates endothelial susceptibility to circulating TNF-alpha and monocytic cell recruitment in a simplified model of arterial bifurcations[END_REF] distinguished the region of laminar shear stress (10.2-10.8 dyne/cm 2 at a flow rate of 9.6 mL/min) throughout the straight main channel, and the region of non-uniform shear stress at the outer walls of bifurcation (shear stress range from ~6.3 dyne/cm 2 to ~0.5 dyne/cm 2 ). HUVECs at 7x10 5 /mL were seeded in the bifurcating slides and grown until confluence.

Using a programmed peristaltic pump (Ismatec, Wertheim, Germany), the cell monolayer inside the slide channel was perfused with medium (with or without nanoparticles) at arterial shear stress for 18 h.

For the perfusion with nanoparticles, 2 different concentrations were used (100 µg/mL and 400 µg/mL).

Concentrations for iron oxide nanoparticles were calculated as total iron (Fe) concentration. The concentrations for lipid nanoparticles, liposomes and polymeric nanoparticles were calculated as total dry mass weight per volume. After 18 h, slides were detached from the pump system, washed with PBS and fixed with 4% formalin for 10 min at RT. HUVECs were stained with Alexa488-phalloidin. Cell nuclei were counterstained with DAPI. Images were obtained using fluorescence microscope Zeiss Axio Observer.Z1 (Carl Zeiss AG, Oberkochen, Germany). The confluence was determined on x20 objective magnification images using ImageJ software.

Dynamic monocyte adhesion assay

HUVECs were exposed to flow in the presence of circulating nanoparticles for 18 h, as described above.

Afterwards, HUVECs were stimulated with 2.5 ng/mL TNF-α for 2h, followed by perfusion for 1 h with fresh endothelial cell medium containing THP-1 monocytic cells (7x10 5 cells/mL) at the same flow rate [START_REF] Cicha | Shear stress preconditioning modulates endothelial susceptibility to circulating TNF-alpha and monocytic cell recruitment in a simplified model of arterial bifurcations[END_REF]. Non-adherent cells were removed by stringent washing. Following the fixation with 4 % paraformaldehyde, adherent monocytic cells were visualized using hematoxylin-eosin stain and digitally counted in minimum 8 image fields (0.89 mm 2 ) at x100 magnification in non-uniform shear stress area. The mean number of nanoparticle-untreated adherent monocytic cells in non-uniform shear stress region was set as 100%.

Endothelial cell migration assay

HUVEC migration was assessed in a modified barrier assay using silicone cell culture inserts from Ibidi (Munich, Germany). HUVECs were seeded in 2 wells separated by a 500 µm insert barrier, at a concentration of 3x10 5 /mL. The cells were pre-treated with 0, 50 or 100 µg/mL nanoparticles overnight.

Afterwards, the inserts were carefully removed, creating a 500 µm-wide gap between the two cell monolayers. HUVECs were washed then and further incubated with medium containing the appropriate concentrations of nanoparticles for additional 24 h, during which the closing of the gap created by insert removal was monitored. The gap size between the 2 monolayers was recorded at the insert removal point (0 h), at 12 and 24 hours using an Incucyte FLR system. Cell-free areas at different time points were measured using ImageJ software. The effect of nanoparticles on cell migration was assessed by calculating the increase in the area occupied by cells at later time points compared with the cell-covered area at 0 h.

Chemotaxis assay

The effect of nanosystems on monocytic cell migration was assessed in a 96-well Chemo-Tx plate (NeuroProbe, Gaithersburg, MD, USA). THP-1 monocytic cells were incubated with 0, 12.5, 25, 50, 100, 200 or 400 µg/mL particles for 2 h at 37°C and under constant stirring. Briefly, the microplate wells were filled with 30 µL serum-free RPMI 1640. MCP-1 (50 ng/mL) was used as a positive control.

After placing the filter frame, the filter top sites with 5 µm-pores were filled with 25 µL of nanoparticletreated monocytic cells at a concentration of 1x10 6 cells/mL. Following incubation for 1 h at 37°C, migrated non-adherent cells from the lower wells were fixed and counted using flow cytometry. All samples were run in quadriplicate and averaged. The mean number of migrated cells in the positive control (MCP-1 stimulated) samples was set as 100%.

Statistical analyses

The comparison between untreated and nanoparticle-treated samples was done using Student's T-test, for samples with normal distribution (according to Shapiro-Wilk test). Signed Rank test, Mann-Whitney test, or Kruskal-Wallis test (ANOVA on ranks) was used for the samples with non-parametric distribution. The multiple comparisons were performed with Tukey test. The analysis was done using SigmaStat/SigmaPlot statistical software. Data were expressed as mean ± SEM, unless stated otherwise. P<0.05 was considered statistically significant.

Results

Lipid nanoparticles

Because lipid nanoparticles at 200 µg/mL and above induced alterations in endothelial morphology (see also Supplementary Fig. 9; [START_REF] Matuszak | Nanoparticles for intravascular applications: physicochemical characterization and cytotoxicity testing[END_REF]), reduced LD-NP concentrations have been applied in the present experiments (50 and 100 µg/mL LD-NPs instead of 100 and 400 µg/mL). Independent of their size, all LD-NPs caused a reduction in monocytic cell recruitment by activated HUVECs, with a strongest, dose-dependent effect observed in cells treated with LD-NP1 (50 nm) (Fig. 2C). In samples treated with LD-NP3 (120 nm), a similar reduction was observed. LD-NP2 (80 nm) had somewhat weaker inhibiting effect on monocytic cell recruitment as compared to untreated controls (Fig. 2C).

Polymer nanoparticles

Because of endothelial toxicity observed at 100 µg/mL of polymer particles under flow conditions in our previous studies (see also Supplementary Fig. 5 and Suppl. Fig. 10; [START_REF] Matuszak | Nanoparticles for intravascular applications: physicochemical characterization and cytotoxicity testing[END_REF]), reduced nanoparticle concentrations have been applied in the present experiments (25 and 50 µg/mL FC-PM-NPs instead of 100 and 400 µg/mL). FC-PM-NP1 caused a strong 40-45% reduction in the number of recruited monocytic cells, which was statistically significant at both 25 and 50 µg/mL (Fig. 2D). Similar effect, and about 40% reduction in monocytic cell recruitment was observed in HUVECs exposed to 25 and 50 µg/mL FC-PM-NP2 (Fig. 2D, Supplementary Fig. 8B).

Iron oxide nanoparticles

Among iron oxide nanoparticles, no major effect of the treatment with IO-NP1, IO-NP3 or IO-NP4 on monocytic cell recruitment under non-uniform shear stress was detectable. Solely at 100 µg/mL IO-NP3, a slight reduction in the numbers of recruited monocytic cells was observed, but it was not detectable at the higher particle concentration. In HUVECs treated with IO-NP2, a significant increase in the numbers of adherent monocytic cells under non-uniform shear stress was detected at 100 and 400 µg/mL (Fig. 2E).

Nanoparticle effects on endothelial cell migration

The effect of nanosystems on spontaneous endothelial cell migration was assessed utilizing a modified wound-closing assay.

Liposomes

No effect of HUVEC incubation with LP-NP1 or Gd-LP-NP1 was observable. The analyses of the blank LP-NP2, Prava-LP-NP2 and Predni-LP-NP2 also showed no major effect of these particles on the spontaneous endothelial cell migration at 12 or 24 h (not shown).

Lipid nanoparticles

Independent of size, none of LD-NPs affected endothelial cell migration at 24 h. In the HUVEC samples treated with the smallest lipidots (LD-NP1), a transient reduction in gap-closure speed was detected at 12 h post-insert removal, but it was no longer observable after 24 h of migration (Fig. 3A).

Polymer nanoparticles

Treatment of HUVECs with FC-PM-NP1 resulted in a slight inhibition of migration, whereby at 24 h after the barrier removal, 75% of the gap was closed both at 50 and 100 µg/mL, as compared with 100% in untreated samples (Fig. 3B). In samples treated with FC-PM-NP2, very strong inhibition of migration was observed: At 24 h, only 50% of the gap was closed at nanoparticle concentration of 50 µg/mL, and only 20% at 100 µg/mL. This corresponded to 80% reduction in migration speed as compared to untreated controls (Fig. 3B, Supplementary Fig. 11).

Iron oxide nanoparticles

In HUVECs treated with IO-NP1, no effect on endothelial cell migration was observed at 12 h. After 24 h, inhibition of migration by about 35% at both tested concentrations was detected, as compared to untreated control (Fig. 3C). In the HUVEC samples treated with 100 µg/mL IO-NP2, a transient, significant reduction in gap-closure speed was detected at 12 h post-insert removal, but it was no longer observable after 24 h of migration.

Treatment with IO-NP3 more strongly affected endothelial cell migration: At 100 µg/mL IO-NP3, only 20% of the original gap was closed after 12 h, compared to nearly 60% in control. After 24 h of migration, untreated control samples showed a complete closure of the gap, whereas only 40% of the gap was covered by cells treated with 100 µg/mL IO-NP3 (Fig. 3C, Supplementary Fig. 11). Dextrancoated USPIO (IO-NP4) had no effect on spontaneous endothelial migration (not shown).

Nanoparticle effects on THP-1 monocytic cell chemotaxis

Monocyte chemotaxis and migration into the vessel wall are the driving force in atherosclerosis. We have therefore investigated the effects of the nanosystems on chemotactic response of THP-1 monocytic cells to MCP-1.

Liposomes

Upon 2 h-incubation of monocytic cells with LP-NP1, a dose-dependent reduction in transmigrated cell numbers was observed, reaching about 50% of positive control at 200 µg/mL. In contrast, this effect was abolished upon treatment of monocytic cells with Gd-LP-NP1 (Fig. 4A).

A different effect was observed for LP-NP2, which in their basic form had no effect on monocytic cell chemotaxis. Pravastatin-loaded liposomes (Prava-LP-NP2) caused a strong dose-dependent reduction in chemotaxis, whereby at 100 µg/mL, the numbers of transmigrated monocytic cells were decreased to 50% of positive control (nanoparticle-untreated). Interestingly, this decrease was not detectable in monocytic cells treated with Predni-LP-NP2 (Fig. 4B).

Lipid nanoparticles

Independent of their size, all LD-NPs caused a dose-dependent decrease in monocytic cell chemotaxis. This reduction was strongest for LD-NP2 and LD-NP3, with about 70% reduction relative of nanoparticle-untreated positive control observed at 100 µg/mL (Fig. 4C).

Polymer nanoparticles

Both types of FC-PM-NPs resulted in a very strong dose-dependent inhibition of monocytic cell chemotaxis (Fig. 5A). At 200 µg/mL, the numbers of transmigrated monocytic cells were reduced down to negative control levels (without MCP-1).

Iron oxide nanoparticles

In THP-1 samples treated with different concentrations of IO-NP1, a biphasic effect on monocytic cell chemotaxis was observed. Between 12.5-100 µg/mL, a decrease by 30% in numbers of transmigrated monocytic cells was detected, whereas at 200 µg/mL the numbers similar to positive control levels and at 400 µg/mL 50% increase in migrated monocytic cells was noted (Fig. 5B). In samples treated with IO-NP2 and IO-NP3, a similar but less pronounced effect was observed. At 50 µg/mL of IO-NP2 and 25 µg/mL IO-NP3, the numbers of transmigrated monocytic cells were decreased by 20%. A slight increase in monocytic chemotaxis was detected at 200 µg/mL of IO-NP2, and the number of migrated monocytic cells dramatically increased at 400 µg/mL (Fig. 5B). Slight increase in monocytic cell chemotaxis was also observed at 400 µg/mL IO-NP3. IO-NP4 had no effect on monocytic cell chemotaxis (Fig. 5C). Within 2h of incubation, no effect of nanoparticle treatment on THP-1 monocytic cell viability, as determined by flow cytometry, was detected (not shown).

Discussion

The concept of nanomedicine encompasses a localized delivery of nanosystems to the diseased tissues and minimized systemic side effects. However, an extended circulation time of nano-sized particles compared to free drugs may result in enhanced particle interactions with vascular and blood cells, potentially contributing to cytotoxicity [START_REF] Kumar | Genotoxic and carcinogenic potential of engineered nanoparticles: an update[END_REF], or immunogenicity [START_REF] Zolnik | Nanoparticles and the immune system[END_REF].

In this context, studies in cell culture models under physiological-like conditions represent a useful tool to predict nanoparticle behaviour in vivo [START_REF] Desai | Challenges in development of nanoparticle-based therapeutics[END_REF], including their cellular uptake and the effects on cell functions. Here, we investigated 14 nanoparticle systems, comprising lipid nanoparticles (Lipidots TM ), liposomes, polymeric nanoparticles, and iron oxide nanoparticles with regard to their functional effects on atherosclerosis-relevant cells. Our previous studies indicated that nanoparticle sedimentation in static conditions may increase their effective concentrations in the vicinity of cell monolayer, contributing to an enhanced cytotoxicity over time [START_REF] Matuszak | Nanoparticles for intravascular applications: physicochemical characterization and cytotoxicity testing[END_REF]. Although in vivo, doses above 100 µg/mL are not expected to occur systemically, they may be encountered locally at the vascular regions of bolus administration. We have therefore tested nanoparticle effects in the dynamic assays up to a concentration of 400 µg/mL, and correspondingly reduced the maximal nanoparticle concentrations in the HUVEC migration assay, which is performed in static conditions over prolonged period of time. As some of the nanosystems are expected to remain in circulation for several hours or more, we selected long-term monitoring for the investigations involving HUVECs. Short-term exposure was solely used in THP-1 monocytic cells (2 h incubation) to investigate the effect of nanosystems on chemotaxis. It must be therefore noted that more pronounced effects are possible upon prolonged exposure of these cells to nanoparticles. The diverse tested nanosystems and their utility for cardiovascular applications in the light of the obtained results are briefly discussed below.

Lipid-based nanoparticles

Apart from the ease of preparation, the advantages of lipid nanoparticles and liposomes as imaging or drug-delivery platforms also include their reported low immunogenicity [START_REF] Huwyler | Tumor targeting using liposomal antineoplastic drugs[END_REF][START_REF] Muller | 20 years of lipid nanoparticles (SLN and NLC): present state of development and industrial applications[END_REF], which is expected to enable safe and repeated administration. Liposomes, which are composed of a lipid bilayer that encloses an interior aqueous space [START_REF] Puri | Lipid-based nanoparticles as pharmaceutical drug carriers: from concepts to clinic[END_REF], are commonly functionalized with polymerizable moieties to improve stability (e.g. PEGylated stealth liposomes).

Concerning the endothelial compatibility, all liposome types tested in this study showed superb properties, with little or no effect on HUVEC viability or morphology up to 400 µg/mL. None of the formulations affected the migratory capacities of HUVECs. However, we observed other functional effects on HUVECs and monocytic cells, which were dependent on the type of liposomal formulation and/or the type of the loaded compound. Treatment with either empty or Gd-loaded LP-NP1 reduced monocytic cell recruitment by HUVECs grown under non-uniform shear stress conditions. Interestingly, whereas LP-NP1 dose-dependently reduced monocytic cell chemotaxis towards MCP-1, this effect was abolished in cells treated with Gd chelate-loaded liposomal particles. Both linear and macrocyclic Gd chelates have been previously shown to induce a proinflammatory phenotype in human monocytes in vitro [START_REF] Wermuth | Induction of a type I interferon signature in normal human monocytes by gadolinium-based contrast agents: comparison of linear and macrocyclic agents[END_REF]. Our present findings may thus indicate that the presence of chelator influences the cellular interactions and functional effects of liposomes on monocytic cells.

In case of LP-NP2 synthesized by lipid phase injection, non-uniform shear stress and TNF-α-induced recruitment of monocytic cells by HUVECs was significantly increased. This unexpected proinflammatory effect of basic LP-NP2 liposomes was drastically reduced by the presence of either pravastatin or prednisolone. Both these drugs belong to the classes of pharmaceutics known for their anti-inflammatory properties and were previously reported to reduce monocytic cell adhesion to endothelium [START_REF] Pozo | Simvastatin inhibits the migration and adhesion of monocytic cells and disorganizes the cytoskeleton of activated endothelial cells[END_REF][START_REF] Gelati | Reduced adhesion of PBMNCs to endothelium in methylprednisolone-treated MS patients: preliminary results[END_REF][START_REF] Simoncini | Estrogens and glucocorticoids inhibit endothelial vascular cell adhesion molecule-1 expression by different transcriptional mechanisms[END_REF]. Regarding monocytic cell chemotaxis, no effect of LP-NP2 was observed, except in cells treated with Prava-LP-NP2, which induced a strong dose-dependent decrease in chemotaxis. These findings were consistent with the previously reported interference of another nanoparticle-bound statin (pitavastatin) with MCP-1/CCR2 signaling [START_REF] Katsuki | Nanoparticle-mediated delivery of pitavastatin inhibits atherosclerotic plaque destabilization/rupture in mice by regulating the recruitment of inflammatory monocytes[END_REF], underscoring the anti-inflammatory and anti-atherogenic potential of this class of drugs [START_REF] Wong | Statins suppress THP-1 cell migration and secretion of matrix metalloproteinase 9 by inhibiting geranylgeranylation[END_REF].

Lipid nanoparticles used in this study (Lipidots™) display increased affinity for myeloid cells such as macrophages, or denditric cells [START_REF] Courant | Tailoring nanostructured lipid carriers for the delivery of protein antigens: Physicochemical properties versus immunogenicity studies[END_REF], and a biodistribution pattern with high uptake in lipid-rich areas, such as adrenals, ovaries and testis, and liver, where they are metabolized [START_REF] Merian | Synthetic Lipid Nanoparticles Targeting Steroid Organs[END_REF]. Their size is easily adjustable and lipid composition is similar to those of low density lipoproteins. Although Lipidots™ do not present apolipoprotein on their surface, it is highly probable that apolipoproteins efficiently bind to the LD-NP surface once in blood, as previously reported for similar lipid nanoparticles (Goppert, et al., 2005, Goppert, et al., 2005). Because of these features, LD-NPs are considered highly interesting nanosystems for atherosclerotic plaque targeting. Similar to liposomes, lipid nanoparticles used in this study had an overall good biocompatibility with HUVECs, whereby some effect on endothelial morphology and viability were previously observed at and above 200 µg/mL [START_REF] Matuszak | Nanoparticles for intravascular applications: physicochemical characterization and cytotoxicity testing[END_REF]. Independent of their size, LD-NPs had little effect on spontaneous HUVEC migration, but strongly and dose-dependently prevented monocytic cell chemotaxis. Since no effect of LD-NPs on monocytic cell viability during 2 h incubation was detectable, the effect was likely mediated at a CCR2 receptor level. All LD-NPs tested in the present study also strongly reduced endothelial recruitment of monocytic cell adhesion. Overall, these results indicate that empty LD-NPs possess certain anti-inflammatory potential, which could potentially be enhanced by appropriate drug payload.

Polymer nanoparticles

Synthetic and natural polymers have been previously used in the design of nanostructures for the treatment and diagnosis of cardiovascular diseases [START_REF] Mura | Nanotheranostics for personalized medicine[END_REF]. Their advantage is the presence of tunable surface properties that enable easy grafting of functional groups. In this work, redox radical emulsion polymerization of isobutylcyanoacrylate monomers was used to produce nanoparticles with a hydrophobic PIBCA core covered with a brush-like shell composed of dextran and fucoidan (Chauvierre, et al., 2003). PIBCA polymer has been chosen for its biodegradability and biocompatibility [START_REF] Muller | Invitro Model for the Degradation of Alkylcyanoacrylate Nanoparticles[END_REF]. For colloidal stability and biocompatibility [START_REF] Berry | Functionalisation of magnetic nanoparticles for applications in biomedicine[END_REF], dextrans, stable glucose polymers that contain functional groups for derivatization [START_REF] Sun | Engineering dextran-based scaffolds for drug delivery and tissue repair[END_REF], were used. Both types of PM-NPs were functionalized with 10% fucoidan, a negatively charged polysaccharide that contains high percentages of L-fucose and sulphate ester groups. Fucoidan, a mimic of sialyl Lewis X, the natural ligand of P-selectin expressed by activated endothelial cells and platelets [START_REF] Bachelet | Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets[END_REF][START_REF] Rouzet | Radiolabeled fucoidan as a pselectin targeting agent for in vivo imaging of platelet-rich thrombus and endothelial activation[END_REF], is of great interest for the treatment and diagnosis of atherothrombosis, e.g to detect intraluminal thrombi or activated endothelium in atherosclerotic lesions [START_REF] Porter | Cardiovascular imaging of remote myocardial ischemia: detecting a molecular trace of evidence left behind[END_REF][START_REF] Libby | The vascular biology of atherosclerosis and imaging targets[END_REF].

Concerning the functional effects of FC-PM-NPs observed in our study, both particle types strongly prevented monocytic cell recruitment by HUVECs exposed to non-uniform shear stress and TNF-α, independent of their dose within this setup. Moreover, a strong and dose-dependent reduction of monocytic cell chemotaxis towards MCP-1 was observed. These effects are likely to result from competitive receptor binding of fucoidan to selectins on HUVECs and their ligands on monocytic cells, which prevents subsequent ligand-receptor interactions [START_REF] Polley | Cd62 and Endothelial-Cell Leukocyte Adhesion Molecule-1 (Elam-1) Recognize the Same Carbohydrate Ligand, Sialyl-Lewis-X[END_REF][START_REF] Heinzelmann | Modulation of lipopolysaccharide-induced monocyte activation by heparin-binding protein and fucoidan[END_REF][START_REF] Zandberg | Metabolic inhibition of sialyl-Lewis X biosynthesis by 5-thiofucose remodels the cell surface and impairs selectin-mediated cell adhesion[END_REF], Cumashi, et al., 2007), thus contributing to anti-inflammatory effects. At the tested concentrations, no effects of FC-PM-NPs on cell viability were observed.

Concerning endothelial migration, strong to very strong dose-dependent inhibition in spontaneous gap closing was detected in samples treated with polymer nanoparticles. This effect was more pronounced in HUVECs treated with FC-PM-NP2, which at 100 µg/mL nearly prevented the migration of HUVECs.

At the same time, the viability of cells was not affected, indicating that the mechanism of this phenomenon was likely related to the particle interference with actin cytoskeleton regulation, which is necessary for cell motility. The strong differences observed between the two types of FC-PM-NPs may be related to their different sizes and surface charges: Zeta potential values (Table 1) indicate that the surface charge was greatly influenced by the charge of the shell polysaccharides [11], whereby CMdextran and fucoidan contained in FC-PM-NP1 shell are negatively charged. These polysaccharide chains are also shorter, which results is smaller particle size. On the contrary, DEAE-dextran is positively charged and dextran is neutral, resulting in a slightly positive charge of FC-PM-NP2, which together with their larger size may contribute to an increased internalization by HUVECs and enhancement of the elicited effects.

Iron oxide nanoparticles

SPION and USPIO have strong contrast-enhancing properties in magnetic resonance imaging (MRI), enabling plaque detection and characterization [START_REF] Trivedi | Identifying inflamed carotid plaques using in vivo USPIOenhanced MR imaging to label plaque macrophages[END_REF], Howarth, et al., 2009, Tang, et al., 2009[START_REF] Sadat | Sequential Imaging of Asymptomatic Carotid Atheroma Using Ultrasmall Superparamagnetic Iron Oxide-enhanced Magnetic Resonance Imaging: A Feasibility Study[END_REF], as well as cell-tracking in vivo [START_REF] Richards | In vivo mononuclear cell tracking using superparamagnetic particles of iron oxide: feasibility and safety in humans[END_REF]. Furthermore, the magnetic properties of these particles allow their remote targeting by means of external magnetic field [START_REF] Janko | Magnetic drug targeting reduces the chemotherapeutic burden on circulating leukocytes[END_REF][START_REF] Lyer | Visualisation of tumour regression after local chemotherapy with magnetic nanoparticles -a pilot study[END_REF], Tietze, et al., 2013), as well as their application for hyperthermiatherapy. IO-NPs have overall good biocompatibility when stabilized with coating, and the formulations used here were well tolerated by ECs as shown in our former [START_REF] Matuszak | Nanoparticles for intravascular applications: physicochemical characterization and cytotoxicity testing[END_REF] and present studies.

Compared to all other IO-based nanosystems tested, dextran-coated USPIO (IO-NP4) were most bioinert and did not elicit any functional effects on EC migration, monocytic cell chemotaxis or endothelial-monocytic interactions under flow conditions. The reason for this is likely related to the very low internalization of IO-NPs coated with crosslinked dextran by non-phagocytic cells, as previously shown for larger particles of this type [START_REF] Unterweger | Non-immunogenic dextrancoated superparamagnetic iron oxide nanoparticles: a biocompatible, size-tunable contrast agent for magnetic resonance imaging[END_REF].

Among the tested IO-NPs with fatty acid-coating, LA/ BSA-coated IO-NP1 did not significantly affect monocytic cell recruitment by activated ECs. In contrast, upon prolonged incubation, they induced an inhibitory effect on HUVEC migration. Moreover, a biphasic effect was observed in monocytic cell samples treated with IO-NP1, whereby between 12.5 and 100 µg/mL, chemotaxis was significantly reduced independent of the dose and returned to control levels at 200 µg/mL, followed by further nonsignificant increase above this concentration. In contrast to IO-NP1, IO-NP2 particles coated with LA and HSA induced a massive enhancement of the pro-inflammatory response in HUVECs. The reason for this strong effect is unknown. Physiological concentrations of BSA have been previously shown to inhibit TNF-α induced VCAM-1 expression in HUVECs [START_REF] Zhang | Albumin selectively inhibits TNF alpha-induced expression of vascular cell adhesion molecule-1 in human aortic endothelial cells[END_REF], whereas glycated BSA had opposite effect [START_REF] Desfaits | Gliclazide reduces the induction of human monocyte adhesion to endothelial cells by glycated albumin[END_REF]. It is therefore unclear what pro-inflammatory mechanism is induced or enhanced by HSA-coated particles in HUVECs exposed to atherogenic shear stress. At the same time, IO-NP2 did not affect endothelial migration and had no significant effect on monocytic cell chemotaxis up to 200 µg Fe/mL. Solely at the highest tested concentration (400 µg Fe/mL), a significant induction of chemotaxis was detected. It must be noted, however, that the concentrations of iron oxide nanoparticles are traditionally normalized to the total iron content, so the concentrations used here correspond to a much higher total dry mass weight e.g. about 10-fold higher in case of dextran-coated IO-NP4.

Concerning IO-NP3, no major effects of these particles either on endothelial-monocytic interactions, or monocytic cell chemotaxis were observed. Decreased numbers of adherent monocytic cells were detected in the samples treated with 100 µg/mL IO-NP3, but not at 400 µg/mL. At this highest tested concentration, a slight, but not significant, increase in monocytic cell chemotaxis was also observable.

However, IO-NP3 had a strong negative effect on endothelial migration, significantly and dosedependently increasing the time of gap closure. As previously reported by Soenen et al., high intracellular concentrations of certain iron oxide-based nanoparticles induce strong polarization of endothelial cells and affect actin cytoskeleton and microtubule network reorganization [START_REF] Soenen | High Intracellular Iron Oxide Nanoparticle Concentrations Affect Cellular Cytoskeleton and Focal Adhesion Kinase-Mediated Signaling[END_REF]. This effect was also observed in our studies, in particular in IO-NP1 and IO-NP3-treated HUVECs, which showed reduced cell spreading and strongly limited migratory capacity.

Taken together, our studies showed different nanoparticle type-and concentration-dependent effects and effects of the nanoparticle-loaded drug on endothelial and monocytic cell functions. In the performed assays, PEG-ylated liposomes and dextran-coated USPIO demonstrated the least toxicity among the tested nanosystems, indicating their potential for use in therapy and /or diagnosis of CVD.

Generally, the majority of the responses observed in unloaded nanoparticles can be considered mainly as nanoparticle type-dependent effects, but even among the particles of the same type (e.g. LD-NPs, PM-NPs or IO-NPs), the degree of exerted effect can vary, indicating that both nanoparticle toxicity and their non-cytotoxic biological effects depend on many characteristics apart from composition of the core. As an example, among the tested lipid nanoparticles, the best tolerated formulation was the one with largest diameter, thus the smallest surfactant to core ratio. It is known that certain physicochemical characteristics of nanoparticles can serve as predictors of increased toxicity (e.g. positive charge facilitating cellular uptake, aggregation tendency) and should be carefully avoided when designing a nanosystem for potential clinical application. Additionally, nanoparticle coating appears to play a major role in the cellular response to nanosystems. This may be due to the type of coating (composition), but also due to insufficient coating density or stability that can result in the exposure of the unmodified surface to cells. Therefore, each modification introduced in "basic" particle composition, size and coating should be treated with caution and ideally tested anew for potential toxicity and cellular effects.

Conclusions

Our studies advance the nanosafety by providing new insights into the nanoparticle effects on human endothelial and monocytic cells. Although the majority of the tested nanosystems had favourable biocompatibility profiles for potential cardiovascular imaging and drug-targeting, their specific effects on endothelial and monocytic cell functions should be taken into account when considering in vivo application. In many cases, the conjugated drug or compound was clearly responsible for the characteristic cellular effects of particles. Currently, testing of the most promising candidates for procoagulant activity and complement activation is ongoing, but a substantial amount of in vivo studies is necessary before the nanosystems with proven in vitro safety and efficacy can be translated into clinical trials. Despite multiple regulatory constraints, the future progress in diagnosis and treatment of cardiovascular disorders is expected to benefit strongly from the development of novel nanotechnologybased strategies. In case of multicore particles (IO-NPs), the Z-Avg value of hydrodynamic diameter refers to the whole multicore clusters, which however do not form aggregates. Abbreviations: DPPC, dipalmitoylphosphatidylcholine; DSPE-PEG2000, 1,2-distearoyl-phosphatidyl-ethanolamine-methyl-polyethyleneglycol conjugate-2000; POPC, 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

Figure 1 .

 1 Figure 1. The effects of tested nanosystems on HUVEC viability, monitrored by real-time cell analysis

Figure 2 .

 2 Figure 2. Effects of circulating nanoparticles on monocytic cell recruitment by HUVECs. HUVECs in

Figure 3 .

 3 Figure 3. Effect of nanoparticles on spontaneous HUVEC migration. HUVECs were pre-treated with 0,

Figure 4 .

 4 Figure 4. Effects of lipid-based nanoparticles on monocytic cell chemotaxis. THP-1 chemotaxis towards

Figure 5 .

 5 Figure 5. Effects of polymer-based and iron oxide-based nanoparticles on monocytic cell chemotaxis.

Table 1 .

 1 Composition and basic physico-chemical characteristics of the tested nanosystems.

	Type	Abbreviated	Nanoparticle composition	Z-avg d	ζ (mV)	PDI
		name		(nm)
		LP-NP1	Lipid composition: POPC, cholesterol DSPE-PEG2000 Method: Dry film rehydration	138.6	-16.3	0.104
		Gd-LP-NP1	Composition: POPC, cholesterol, DSPE-PEG2000, gadolinium-chelate (B22286) Method: Dry film rehydration	122.3	n.d.	0.040
	Liposome	LP-NP2	Lipid composition: DPPC, DSPE-PEG2000, cholesterol Method: Lipid injection in the aqueous phase	108.8	-9.0	0.034
		Prava-LP-NP2	LP-NP2 loaded with pravastatin sodium	108.0	-16.1	0.080
		Predni-LP-NP2 LP-NP2 loaded with prednisolone phosphate	110.0	-2.7	0.070
		LD-NP1	Core: Suppocire™ NB, soybean oil and lipoid S75; Shell: Myrj s40 Method: Sonication	53.3	-7.0	0.156
	Lipid NP	LD-NP2	Composition as described for LD-NP1 Method: Sonication	82.8	-9.0	0.191
		LD-NP3	Composition as described for LD-NP1 Method: Sonication	120.1	-8.8	0.151
			Core: Poly-isobutylcyanoacrylate (PIBCA); Shell: 90% carboxymethyl-dextran/ 10%
		FC-PM-NP1	Fucoidan	145.1	-51.0	0.072
	Polymer NP		Method: Redox radical emulsion polymerization Core: PIBCA; Shell: 80% dextran T70 /10% diethylaminoethyl-dextran 20/
		FC-PM-NP2	10% Fucoidan	226.9	3.3	0.194
			Method: Redox radical emulsion polymerization
		IO-NP1	Core: Iron oxide; Shell: Lauric acid/bovine serum albumin Method: Coprecipitation	78.7	-37.3	0.145
	Iron oxide NP	IO-NP2 IO-NP3	Core: Iron oxide; Shell: Lauric acid/human serum albumin Method: Coprecipitation Core: Iron oxide; Shell: Carboxydextran Method: Coprecipitation	68.3 79.6	-16.9 13.7	0.143 0.173
		IO-NP4	Core: Iron oxide; Shell: Dextran T40 Method: Coprecipitation	30.0	-6.3	0.102

Acknowledgements

The authors thank Prof. Beckmann (Dept. of Gynaecology, University Hospital Erlangen, Germany) for providing umbilical cords. We also thank Heike Kloos and Doris Flick (Laboratory of Molecular Cardiology, University Hospital Erlangen, Germany) for help with HUVEC isolation. Technical help of Julia Band is gratefully acknowledged.

Funding

This work was supported by the EU ("NanoAthero" project FP7-NMP-2012-LARGE-6-309820), the DFG (CI 162/2-1), and received the financial support from ANR-13-LAB1-0005-01 "FucoChem".

Nanoparticle characterisation and long-term effects on endothelial viability

The detailed description of physicochemical properties of respective nanoparticles was previously reported elsewhere [START_REF] Matuszak | Nanoparticles for intravascular applications: physicochemical characterization and cytotoxicity testing[END_REF][START_REF] Almer | Adiponectin-coated nanoparticles for enhanced imaging of atherosclerotic plaques[END_REF][START_REF] Almer | Interleukin-10: an anti-inflammatory marker to target atherosclerotic lesions via PEGylated liposomes[END_REF][START_REF] Gravier | Lipidots: competitive organic alternative to quantum dots for in vivo fluorescence imaging[END_REF], Zaloga, et al., 2014, Unterweger, et al., 2014). Prior to the functional assays, all tested nanosystems underwent the analysis of biocompatibility with HUVECs. Figure 1 shows the heat-map summarizing the effects of diverse nanosystems on HUVEC viability, measured by real-time cell analysis and livecell microscopy, and on their confluence under flow conditions. The corresponding graphs showing the effect of each type of nanoparticles on HUVEC viability at 24 h and example images are presented in Supplementary Fig. 1234567.

Nanoparticle effects on monocytic cell recruitment by HUVECs

Lining the lumen of the entire vascular tree, endothelial cells are constantly exposed to the blood flow, the patterns of which determine their responses to stimuli. Whereas laminar flow protects endothelial cells from harmful stimuli, non-uniform shear stress induces endothelial activation [START_REF] Cicha | Shear stress preconditioning modulates endothelial susceptibility to circulating TNF-alpha and monocytic cell recruitment in a simplified model of arterial bifurcations[END_REF], and contributes to atherosclerotic plaque development. To investigate the effect of nanosystems on the endothelial-monocytic cell interactions in response to inflammatory stimulus and non-uniform shear stress, HUVECs were perfused with medium containing 100 or 400 µg/mL of nanoparticles for 18 h. Subsequently, the cells were treated with TNF-α for 2 h, followed by the dynamic adhesion assay.

Liposomes

The experiments with LP-NP1 and Gd-LP-NPs showed a significant reduction in monocytic cell adhesion to activated endothelium both at 100 and 400 µg/mL (Fig. 2A, Supplementary Fig. 8A). In contrast, the treatment with LP-NP2 resulted in a dose-dependent increase in the monocytic cell recruitment under non-uniform shear stress. This effect was statistically significant at both 100 and 400 µg/mL, reaching about 30% increase at 400 µg/mL (Fig. 2 B). In samples treated with 100 µg/mL of pravastatin-loaded liposomes (Prava-LP-NP2), a 20% decrease in monocytic cell recruitment occurred, which was not observed in samples treated with 400 µg/mL. Compared to untreated samples, Predni-LP-NP2 had no effect on monocytic cell recruitment. LETI/DTBS is part of the Arcane Labex program, funded by the French National Research Agency (ARCANE project n° ANR-12-LABX-003).

Disclosure statement

The authors have no conflict of interests to disclose.

Supplementary data

Matuszak et al.: Comparative analysis of nanosystems' effects on human endothelial and monocytic cell functions.

Supplemetary Fig. 1 Suppl. Figure 1 Note that the growth of cells monitored with real-time cell analyzed was very slow, even in control (untreated cells) during the first 24h, which did not allow a clear estimation of particle effect. Longerterm analysis and complementary methods (live cell imaging) was necessary to confirm that these particles did not exert any toxic effect on HUVECs up to 400 µg/mL. Supplementary Fig. 4 Suppl. Figure 4 HUVECs were seeded in 2 wells separated by a 500 µm insert barrier and pre-treated with 50 µg/mL of FC-PM-NP2 or IO-NP3 overnight. Afterwards, the inserts were carefully removed, and HUVECs were washed and further incubated with medium containing the appropriate concentrations of nanoparticles for additional 24 h, during which the closing of the created gap was monitored. The gap size between the 2 monolayers was recorded at the insert removal point (0 h), at 12 and 24 h. Representative images obtained with live-cell imaging system are shown.