Mark S Bentley

Roland Schmied

Thurid Mannel

Klaus Torkar

Harald Jeszenszky

Jens Romstedt

Anny-Chantal Chantal Levasseur-Regourd

Iris Weber

Elmar K Jessberger

Pascale Ehrenfreund

Christian Koeberl

Ove Havnes

Aggregate dust particles at comet 67P/Churyumov-Gerasimenko

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validate the aggregate model of cometary dust, they are also an important input for understanding comets and their formation. The measured grain elongations are similar to the value inferred for interstellar dust and support the idea that such grains could represent a fraction of the building blocks of comets. In the subsequent growth phase, hierarchical agglomeration can be a dominant process [START_REF] Dominik | Physical Processes : Dust Coagulation and Fragmentation[END_REF] and would produce aggregates that stick more easily at higher masses and velocities than homogenous dust particles [START_REF] Johansen | Protostars Planets VI[END_REF] . The presence of hierarchical dust aggregates in the surface layers of the nucleus also provides a mechanisms for lowering tensile strength and aiding dust release [START_REF] Skorov | Dust release and tensile strength of the non-volatile layer of cometary nuclei[END_REF] .

MIDAS, the Micro-Imaging Dust Analysis System [START_REF] Bentley | MIDAS : Lessons learned from the first spaceborne atomic force microscope[END_REF][START_REF] Riedler | MIDAS -The micro-imaging dust analysis system for the Rosetta mission[END_REF] , is the first spaceborne atomic force microscope (AFM) and a unique instrument designed to measure the size, shape, texture and microstructure of cometary dust. Flying on the Rosetta spacecraft, it collects dust on sticky targets during passive exposures and images its 3D topography with an unprecedented nanometre to micrometre resolution [START_REF] Bentley | MIDAS : Lessons learned from the first spaceborne atomic force microscope[END_REF] .

Cometary dust was first collected in mid-November 2014. In this work we focus on particles collected from then until the end of February 2015. The collected particles cover a range of sizes from tens of micrometres down to a few 100 nanometres, and have various morphologies, from single grains to aggregate particles with different packing densities. Five examples are presented here.

Figure 1 shows topographic images (height fields) of three particles (A, B and C). Particles A and C will be referred to as compact, since their sub-units (hereafter grains) are tightly packed, and B appears to be a homogeneous grain. The next example (D) is also a compact particle scanned with a higher lateral resolution of 80 nm (Fig. 2) -a factor four better than the previous scan. The final particle (E), presented in Fig. 3, is best described as a loosely packed "fluffy" aggregate comprising many grains. Detailed collection times and geometries for all particles can be found in Extended Data Fig. 123.

Aided by the 3D nature of the data, individual grains can be identified, as shown in Fig. 1b, Fig. 2b and Fig. 3b. The properties of these particles and their grains are summarised in Tab. 1 for particles A-D, and in Fig. 3d for particle E. Since particle E extends beyond the edge of the scanned area only lower limits for its dimensions can be given. All further calculations and discussion refer only to this visible region.

Compact particles A and C are both approximately 5.6 µm in effective diameter (hereafter size -see Methods), and are built from grains in the size range 1.93 -1.22 +0.10 µm to 3.31 -1.23 +0.06 µm. The compact grain B is 2.76 -0.61 +0.07 µm in size, comparable to the dust grains of particles A and C. In fact, the topographic image suggests that this grain was originally part of C but detached on impact with the target. Particle D is 1.09 -0.25 +0.01 µm in size, again similar to the grains in A-C. However, the higher resolution reveals that this micrometre-sized particle is itself an aggregate of smaller units; seven grains can be resolved, with sizes ranging from 260 -120 +50 nm to 540 -250 +20 nm. The visible part of particle E has a maximum extent of 14 µm in X and 37 µm in the Y direction. Analysis of its component grains (Fig. 3d) shows sizes in the range from 0.58 -0.20 +0.15 to 2.57 -0.51 +004 µm with the grain heights ranging between 0.2 µm and 3 µm with 90% smaller than 1.7 µm. These measurements are the first evidence for a continuation of the aggregate nature of dust particles below the size range observed by COSIMA (10s-100s micrometres) [START_REF] Langevin | Typology of dust particles collected by the COSIMA mass spectrometer in the inner coma of 67P/Churyumov Gerasimenko[END_REF] .

Particle E also shows a morphology strongly reminiscent of stratospheric chondritic porous (CP) IDPs, long suspected of having a cometary origin. This link is consistent with observations by COSIMA for larger dust particles, which also measured similar compositions for dust at 67P and IDPs [START_REF] Langevin | Typology of dust particles collected by the COSIMA mass spectrometer in the inner coma of 67P/Churyumov Gerasimenko[END_REF][START_REF] Hilchenbach | Comet 67P/Churyumov-Gerasimenko: Close-Up on Dust Particle Fragments[END_REF] . One notable difference to IDPs is the extremely flat nature of particle E, which has a height an order of magnitude lower than its (minimal) lateral dimension. Indeed all of the particles presented here have flattened shapes to some degree (see Tab. 1). It is not yet clear if this is an intrinsic property of cometary dust or the result of a rearrangement of grains on impact. COSIMA has observed that sub-mm aggregate particles undergo rearrangement of their grains on impact, producing flattened shapes [START_REF] Langevin | Typology of dust particles collected by the COSIMA mass spectrometer in the inner coma of 67P/Churyumov Gerasimenko[END_REF] . Additionally, COSIMA collected small, apparently compact particles which are also flat, but the resolution is insufficient to determine if they are single grains, or indeed aggregates themselves. On the other hand, cluster-cluster aggregation with rotating grains can form elongated structures with very high aspect ratios [START_REF] Paszun | The influence of grain rotation on the structure of dust aggregates[END_REF] , and laboratory experiments have produced dust "flakes" [START_REF] Stephens | Laboratory Reflectance Measurements of Analogues to ' Dirty ' Ice Surfaces on Atmosphereless Solar System Bodies[END_REF] .

Investigation of the size distribution of CP IDPs and fine-grained material returned by the Stardust mission [START_REF] Rotundi | Combined micro-Raman, micro-infrared, and field emission scanning electron microscope analyses of comet 81P/Wild 2 particles collected by Stardust[END_REF][START_REF] Stodolna | Characterization of preserved primitive fine-grained material from the Jupiter family comet 81P/Wild 2 -A new link between comets and CP-IDPs[END_REF] showed that the majority of their component grains are smaller than 500 nm [START_REF] Stodolna | Characterization of preserved primitive fine-grained material from the Jupiter family comet 81P/Wild 2 -A new link between comets and CP-IDPs[END_REF][START_REF] Wozniakiewicz | Pre-Accretional Sorting of Grains in the Outer Solar Nebula[END_REF] . Fig. 3d shows that 90% of the grains in particle E are smaller than 2 µm, comparable to particle D. which itself is built from grains smaller than about 500 nm. This suggests that the grains of the fluffy aggregate particle E are also aggregates of sub-micrometre components similar to those in CP IDPs and points towards a hierarchical structure. Hierarchical growth (i.e. aggregates of smaller aggregates) has been proposed as a growth mechanism in the protoplanetary disc when fragmentation of larger particles provides a population of smaller aggregates available for agglomeration [START_REF] Dominik | Physical Processes : Dust Coagulation and Fragmentation[END_REF] . The sticking probability of such particles can be higher for a given mass and velocity and need to be accounted for in dust particle growth models [START_REF] Johansen | Protostars Planets VI[END_REF] . Hierarchical aggregates have also been invoked to produce a cometary dust layer with sufficiently low tensile strength to allow for dust release [START_REF] Skorov | Dust release and tensile strength of the non-volatile layer of cometary nuclei[END_REF] .

Since MIDAS provides, for the first time, real measurements of the grain shapes, it is possible to go further and evaluate which models support the observations. The elongation of the grains is found by calculating the ratio of the longest and shortest perpendicular axis. Further details are described in the Supplementary Information. For particle E the grain heights are almost all smaller than their in-plane diameters, suggesting that it comprises a single layer of grains, allowing accurate grain heights to be determined. The elongation is calculated for 114 grains (the 11 omitted grains show strong distortions due to tip convolution), giving an average elongation of 2.87 -0.44 +1.90 (i.e. the largest axis is 3 times longer than the smallest). The compact particles show similar values (Tab. 1).

Elongated grains are considered in several models of cometary dust. For example Greenberg and

Gutasfson [START_REF] Greenberg | A Comet Fragment Model for Zodiacal Light Particles[END_REF] suggested that comets aggregate from interstellar grains. They modelled the dust as cylinders with aspect ratios of 2-4 and found good agreement between light scattering experiments and observations.

Other authors have similarly found good agreement between simulations using aggregates of spheroidal particles and observational data [START_REF] Levasseur-Regourd | Physical properties of cometary and interplanetary dust[END_REF][START_REF] Lasue | Porous irregular aggregates of sub-micron sized grains to reproduce cometary dust light scattering observations[END_REF] . The elongated nature of interstellar dust can be inferred from linear polarisation of starlight due to partially aligned grains [START_REF] Mathis | Interstellar Dust and Extinction[END_REF] . The core-mantle structure proposed for interstellar and cometary dust [START_REF] Greenberg | From interstellar dust to comets: a unification of observational constraints[END_REF] cannot be confirmed by MIDAS data alone, but the elongation measurement supports the idea of a common precursor grain, or growth mechanism. A-D) and their component dust grains . d is the diameter of a circle with equivalent area, zmax is the maximum height above the substrate surface. For particle A grain 3, particle D grain 4, 6 and 7 the maximal elongation is found for the ratio of the two lateral dimensions that are attached with especially large uncertainties. Thus for particle A grain 3 no accurate elongation can be given and for the grains of particle D the elongation is attached with extensive uncertainties.

Methods

Data acquisition and calibration

Exposure durations and times were planned by estimating the dust flux using the predicted spacecraft position, pointing and a dust flux model for 67P derived from observational data [START_REF] Fulle | Comet 67P/Churyumov-Gerasimenko: the GIADA dust environment model of the Rosetta mission target[END_REF] . For a graphical visualization of the exposure geometries, see Extended Data Fig. 123.

MIDAS operates in a slightly different way than most terrestrial AFMs, by making a careful approach to the sample at each pixel position and then moving away by a so-called retraction distance before moving to the next pixel, resulting in long scan times and possible distortion [START_REF] Bentley | MIDAS : Lessons learned from the first spaceborne atomic force microscope[END_REF][START_REF] Riedler | MIDAS -The micro-imaging dust analysis system for the Rosetta mission[END_REF] . Distortion correction is performed using scans of on-board calibration targets, and polynomial background correction is used to remove height drifts.

This procedure was performed with the data used to produce Fig. 1 and3. The scan shown in Fig. 2 was much shorter and no significant distortion was observed hence only background subtraction was performed.

Particle and grain heights are measured relative to the substrate surface, which is very clear for Fig. 1 and Fig. 2, but the zero reference level had to be set manually for each grain in Fig. 3, since the steps would otherwise distort the measurements.

The lateral extent of both particles and grains is characterised by an effective size (d), which is the diameter of a circle with the same area as the projection of all pixels forming the unit; if not stated elsewise, all references to size refer to this effective value. The peak height (z max ) is the maximum elevation above the target for a given grain. Identification of particles and their sub-units is performed by visual inspection of the calibrated data and, when necessary, cross sections through the 3D data are used, see Extended Data Fig. 4.

For particle E (Fig. 3) a manual levelling of the surface was necessary due to the visible steps (imaging artefacts). Repeating this manual levelling process several times showed that the induced error was negligible. In addition, the height of a grain can only be measured precisely if the grain is directly on the surface and not on another grain. For particle E most of the grains seem to fulfil this requirement, as the mean heights of the grains are smaller than their mean diameters.

Error analysis

In principle, since AFM tips cannot be infinitely sharp, the size of every particle is overestimated due to the tip sample convolution (i.e. the recorded image reflects a combination of both tip and sample shapes). The convolution uncertainty is generously estimated here to give an upper limit. Since the particle diameter cannot be underestimated by this convolution, the uncertainty interval becomes asymmetric. Values for sizes quoted in the text, and the error bars in Fig. 3 (d) reflect this calculation.

The elongation of particles and grains is calculated by determining its equivalent ellipse (the ellipse with the same second order moments) and choosing the maximum ratio of the largest to smallest of (i) the height of the particle to the major axis, (ii) the height to the minor axis and (iii) the ratio of the major and minor axes.

The uncertainties in these ratios take into account the statistical uncertainty due to the manual masking of the particles and the systematic uncertainty due to the tip-sample convolution for the axis lengths. The ratio of the major to minor axis suffers from a large convolution uncertainty which, in some cases (typically particles with steep slopes), prevents a clear statement about their orientation. In these cases no elongation is given. The final uncertainty for the ratio is a worst case estimate which overestimates the uncertainty for non-isolated flat grains.

Extended Data legends

Extended Data Table 1: Scan parameters of the primary AFM topography scans discussed in the paper as Figures 123. The number of pixels and thus the pixel resolution at a given scan size was limited by the time available and chosen to maximize the resolution. The filename corresponds to that used in the Planetary Science Archive. shows the spherical particle, while the blue line depicts the topography as measured with the delta-shaped and pyramidal-shaped tip, respectively. The measurement of the volume of the spherical particle is exaggerated by 25% for the delta-shaped tip and by 50 % for the described cone-shaped tip. The height measurement is not affected by the tip sample convolution.

Extended

Figure 1 :

 1 Figure 1: AFM topographic images of particles A, B and C and their sub-units. (a) 20x50 µm overview

Figure 2 :

 2 Figure 2: AFM topographic images of particle D and its sub-units. (a) 5x5 µm overview image with a

Figure 3 :

 3 Figure 3: AFM topographic images of particle E showing its sub-units and their size distribution. (a)

Data Figure 1 :Extended Data Figure 3 :Extended Data Figure 4 :

 134 The geometry of the exposures where particle A, B and C were collected. All exposures are marked by green bars. The top panel shows the distance of Rosetta from the comet (red) and the off-nadir angle (blue). The lower panel shows the latitude and longitude in red and blue. The heliocentric distance during this exposure was 2.25 au. Extended Data Figure 2: The geometry of the exposures where particle D was collected. All exposures are marked by green bars. The top panel shows the distance of Rosetta from the comet (red) and the offnadir angle (blue). The lower panel shows the latitude and longitude in red and blue. The heliocentric distance during this exposure varied between 2.54 and 2.41 au. The geometry of the exposures where particle E was collected. All exposures are marked by green bars. The top panel shows the distance of Rosetta from the comet (red) and the offnadir angle (blue). The lower panel shows the latitude and longitude in red and blue, respectively. The heliocentric distance during this exposure varied between 2.85 and 2.52 au. Topographic cross-sections demonstrating the identification of sub-units. (a) Topographic image of particles A, B and C. Dashed blue, red and green lines show where the cross sections of particle A, B and C, respectively, were made. (b) Height profiles of the 3 cross sections shown in (a) demonstrating how sub-grains were identified, see blue and green arrows, and also revealing the slope of 60 -70° with the substrate surface. Extended Data Figure 5: Tip-sample convolution effects. Comparison of a spherical particle imaged with an ideal delta-shaped tip (a) and a cone -shaped tip with an opening angle of 30° (b), which is similar to that of the MIDAS tips 14 showing a simulated AFM image (where the colour scale indicates the height) and (c) an (d) showing the corresponding cross-section through the centre of the structure. The black dashed curve

Figure 1 Figure 2 Figure 3

 123 Figure 1

Figure 1 Figure 2 Figure 3

 123 Figure 1

Table 1 : Size, height and elongation data of the presented dust particles (

 1

	Tables
		type	d ± Δd [µm] zmax [µm]	elongation
	particle A	compact particle	𝟓. 𝟒𝟖 -𝟏.𝟏𝟎 +𝟎.𝟎𝟒	1.79	𝟑. 𝟑𝟐 -𝟎.𝟒𝟏 +𝟎.𝟏𝟒
	grain 1	dust grain	𝟑. 𝟑𝟏 -𝟏.𝟐𝟑 +𝟎.𝟎𝟔	1.79	𝟐. 𝟗𝟒 -𝟎.𝟒𝟑 +𝟎.𝟏𝟐
	grain 2	dust grain	𝟐. 𝟔𝟐 -𝟎.𝟖𝟕 +𝟎.𝟎𝟖	1.33	𝟑. 𝟎𝟒 -𝟎.𝟒𝟐 +𝟎.𝟏𝟓
	grain 3	dust grain	𝟏. 𝟗𝟑 -𝟏.𝟐𝟐 +𝟎.𝟏𝟎	1.57
	grain 4	dust grain	𝟐. 𝟔𝟐 -𝟏.𝟎𝟕 +𝟎.𝟎𝟖	1.55	𝟏. 𝟗𝟔 -𝟎.𝟒𝟎 +𝟎.𝟎𝟗
	particle B	dust grain	𝟐. 𝟕𝟔 -𝟎.𝟔𝟏 +𝟎.𝟎𝟕	1.02	𝟑. 𝟏𝟒 -𝟎.𝟒𝟐 +𝟎.𝟏𝟖
	particle C	compact particle	𝟓. 𝟕𝟗 -𝟎.𝟖𝟕 +𝟎.𝟎𝟒	1.39	𝟒. 𝟕𝟕 -𝟎.𝟓𝟎 +𝟎.𝟐𝟒
	grain 1	dust grain	𝟐. 𝟔𝟔 -𝟎.𝟗𝟐 +𝟎.𝟎𝟕	1.33	𝟐. 𝟐𝟔 -𝟎.𝟒𝟐 +𝟎.𝟏𝟏
	grain 2	dust grain	𝟐. 𝟓𝟕 -𝟎.𝟕𝟐 +𝟎.𝟎𝟖	1.14	𝟐. 𝟖𝟎 -𝟎.𝟒𝟏 +𝟎.𝟏𝟓
	grain 3	dust grain	𝟐. 𝟏𝟖 -𝟎.𝟖𝟑 +𝟎.𝟎𝟗	1.28	𝟐. 𝟐𝟑 -𝟎.𝟑𝟗 +𝟎.𝟏𝟐
	grain 4	dust grain	𝟐. 𝟒𝟐 -𝟎.𝟗𝟎 +𝟎.𝟎𝟖	1.39	𝟐. 𝟑𝟏 -𝟎.𝟑𝟗 +𝟎.𝟏𝟏
	grain 5	dust grain	𝟐. 𝟑𝟏 -𝟎.𝟖𝟕 +𝟎.𝟎𝟖	1.38	𝟐. 𝟑𝟐 -𝟎.𝟑𝟗 +𝟎.𝟏𝟏
	particle D	compact particle	𝟏. 𝟎𝟗 -𝟎.𝟐𝟓 +𝟎.𝟎𝟏	0.42	𝟑. 𝟑𝟔 -𝟎.𝟒𝟕 +𝟎.𝟐𝟑
	grain 1	dust grain	𝟎. 𝟐𝟔 -𝟎.𝟏𝟐 +𝟎.𝟎𝟓	0.17	𝟏. 𝟖𝟗 -𝟎.𝟑𝟔 +𝟎.𝟏𝟗
	grain 2	dust grain	𝟎. 𝟒𝟖 -𝟎.𝟏𝟔 +𝟎.𝟎𝟑	0.22	𝟐. 𝟓𝟐 -𝟎.𝟒𝟕 +𝟎.𝟐𝟎
	grain 3	dust grain	𝟎. 𝟒𝟏 -𝟎.𝟏𝟒 +𝟎.𝟎𝟑	0.31	𝟏. 𝟔𝟐 -𝟎.𝟐𝟕 +𝟎.𝟏𝟏
	grain 4	dust grain	𝟎. 𝟑𝟑 -𝟎.𝟏𝟑 +𝟎.𝟎𝟒	0.25	𝟏. 𝟕𝟒 -𝟎.𝟕𝟏 +𝟐.𝟓𝟏
	grain 5	dust grain	𝟎. 𝟒𝟔 -𝟎.𝟏𝟕 +𝟎.𝟎𝟑	0.37	𝟏. 𝟓𝟑 -𝟎.𝟐𝟖 +𝟎.𝟎𝟗
	grain 6	dust grain	𝟎. 𝟓𝟒 -𝟎.𝟐𝟓 +𝟎.𝟎𝟐	0.42	𝟐. 𝟎𝟎 -𝟎.𝟖𝟐 +𝟓.𝟎𝟕
	grain 7	dust grain	𝟎. 𝟐𝟔 -𝟎.𝟏𝟓 +𝟎.𝟎𝟓	0.32	𝟐. 𝟎𝟎 -𝟎.𝟗𝟕 +𝟖.𝟎𝟑

Table 1

 1

	target	14	12	12
	cantilever	9	9	7
	image resolution	256 x 256	256 x 256	192 x 192
	image size	80 x 80 µm²	20 x 20 µm²	40 x 40 µm²
	pixel resolution	312 nm	80 nm	210 nm
	z step size	0.7 nm	0.7 nm	0.7 nm
	retraction height	1095 nm	977 nm	734 nm
	duration	1 day, 05:05:33	08:14:15	11:16:30
	start time	2015-04-29T05:21:40Z	2015-03-13T08:44:38Z	2015-01-18T20:59:28Z
	filename	IMG_1509813_1512600_054_ZS	IMG_1507001_1508813_005_ZS	IMG_1501323_1504200_013_ZS
		Extended Data

Acknowledgements

Rosetta is an ESA mission with contributions from its member states and NASA. We also thank the Rosetta Science Ground Segment and Mission Operations Centre for their support in acquiring the presented data.

MIDAS became possible through generous support from funding agencies including European Space

Agency, PRODEX programme, the Austrian Space Agency, the Austrian Academy of Sciences and the German funding agency DARA (later DLR). ACLR acknowledges support from the French Space Agency, CNES. TM gratefully acknowledges the Steiermärkische Sparkasse and the Karl-Franzens Universität Graz for their financial support. PE acknowledges support from the NASA Astrobiology Institute. RS thanks Ferdinand Hofer and Harald Plank for their discussions and the Austrian Research Promotion Agency (FFG) for financial support. All data presented in this paper will be made available in the ESA Planetary Science Archive.

Code and data availability

Extended Data Tab. 1 summarises the key parameters for the AFM scans used to produce Fig. 123. The filenames listed refer to products available in the ESA Planetary Science Archive where all data used in this paper are freely available. The open source package Gwyddion 28 was used to perform calibration, grain identification and analysis throughout this paper.

Supplementary Information is linked to the online version of the paper at www.nature.com/nature

Author contributions

RS, TM and MSB planned the experiments on MIDAS, analysed and interpreted the data and wrote the manuscript. MSB developed the planning and data processing pipelines. RS and TM implemented the elongation calculations. RS performed the post-processing and calibration as well as the particle/grain measurement and is responsible for the graphical data presentation. TM considered the uncertainties for all data. ACLR provided information on cometary dust derived from polarimetric observations and its interpretation. HJ supported the experiments with software updates. All authors discussed the results and commented on the manuscript.

Author information

Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper.

Correspondence and requests for materials should be addressed to MSB (mark.bentley@oeaw.ac.at).


Microcell Substrate

Sebastien Jutteau
email: sebastien.jutteau@edf.fr

Myriam Paire

Laurent Lombez

Jean-François Guillemoles

Design and fabrication of a micro CPV system based on Cu(In,Ga)Se2 microcells array

Keywords: Photovoltaic, CPV, Concentrator, CIGS, Microcells, Microlenses Physics, Simulation, and Photonic

INTRODUCTION

The domain of the low and middle concentration is dominated by Si solar cells, adapting flat-plate technologies to a use under concentration or developing new approaches (MEPV concept developed by Sandia 1 ). We propose to use a low to middle range concentrating system on a well-known PV material yet rarely used in CPV applications, Cu(In,Ga)Se2. Recently, a record of 22.3% has been achieved, by Solar Frontier under AM1.5, making CIGS cells more efficient than polysilicon cell (record of 20.4% 2 ) . Moreover, because Cu(In,Ga)Se2 is a thin film material, it has the intrinsic advantage that Cu(In,Ga)Se2 solar cell arrays could be directly fabricated on the substrate by a top-down or a bottom-up approach, such as impression techniques. We studied ( [START_REF] Paire | Cu(In, Ga)Se2 microcells: High efficiency and low material consumption[END_REF][START_REF] Paire | Cu(In,Ga)Se2 mesa diodes for the study of edge recombination[END_REF][START_REF] Paire | Resistive and thermal scale effects for Cu(In, Ga)Se2 polycrystalline thin film microcells under concentration[END_REF] ) the miniaturization of the Cu(In,Ga)Se2 solar cell in order to develop efficient concentrator cells. The present work develops a prototype concentrating system adapted and integrated to these thin films microcells arrays.

A CU(IN,GA)SE2 CONCENTRATOR SYSTEM

Our work on Cu(In,Ga)Se2 microcells has covered different aspects. After a first theoretical study of miniaturization effect [START_REF] Paire | Toward microscale Cu(In,Ga)Se2 solar cells for efficient conversion and optimized material usage: Theoretical evaluation[END_REF] , we fabricated proof of concepts devices with dimensions from 5 to 500µm, made of coevaporated absorbers and a structured window layer to create the microcells. The test under concentrated illumination highlighted an increase of the conversion efficiency of 2% absolute for each decade of concentration (16% efficiency under 1x and 21.3% efficiency under 475x, a relative increase of the efficiency of 30%), if parasitic losses are kept low, and very limited temperature elevation under high flux [START_REF] Paire | Cu(In, Ga)Se2 microcells: High efficiency and low material consumption[END_REF] . We also tested Cu(In,Ga)Se2 microcells with absorbers grown by low cost techniques, such as electrodeposition [START_REF] Paire | Characterization of CIGS ED and coevaporated devices by means of concentrated illumination[END_REF] . This technique allows to demonstrate the proof of concept but fails to save material. Then, we fabricated localized microcells using etching techniques and we assessed that edge recombination is very limited in these devices, making them adequate and efficient microconcentrator cells [START_REF] Paire | Cu(In,Ga)Se2 mesa diodes for the study of edge recombination[END_REF] . These microcells are closer to an application as the absorber layer is not kept intact on the substrate, but patterned to create mesa diodes and save material. Finally, a technique of localized grow of Cu(In,Ga)Se2 is being developed, which will lead to real material saving.

Figure 2. Top left, efficiency as a function of short-circuit current density or concentration ratio for the best microcell of diameter 50µm. Measurements under 532nm laser light. The orange star corresponds to AM1.5G measurement [START_REF] Paire | Cu(In, Ga)Se2 microcells: High efficiency and low material consumption[END_REF] . Top right, efficiency as a function of concentration ratio for mesa diodes with various diameters [START_REF] Paire | Cu(In,Ga)Se2 mesa diodes for the study of edge recombination[END_REF] . Bottom left and bottom right, illustration of the generation 1 and 2 of the Cu(In,Ga)Se2 microcells

NUMERICAL STUDY OF A CONCENTRATING SYSTEM

In this work, we decided to design the system only with plano-spherical lenses. Spherical lenses are not the best system because of aberrations but they are easier to fabricate as an array, which leads to lower costs, especially for a large area production. The first system has been designed to achieve an increase of the absolute efficiency close to 4%. Based on Paire et al [START_REF] Paire | Cu(In, Ga)Se2 microcells: High efficiency and low material consumption[END_REF] , we have chosen an optical system with a geometrical ratio of 100. The geometrical ratio is defined as the ratio between the surface of the primary lens and the surface of the microcell. To design the system, we used a ray tracing software, Zemax OpticStudio.

To have a low cost sun tracking system, our optical system needs to have a large acceptance angle. So, based on a geometrical ratio of 100x, our objective is to obtain the maximum optical efficiency of the system, maintaining the Optical efficiency acceptance angle as high as possible. The influence of the number of optical elements and the geometrical parameters of the spherical lenses have been studied [START_REF] Jutteau | Micro Solar Concentrators: Design and fabrication for microcells arrays[END_REF][START_REF] Jutteau | Micro Solar Concentrators: Design and fabrication for microcells arrays[END_REF] . To design a very thin optical system, we have chosen to work with a high index material, the Polycarbonate (PC).

We achieved a final design of 1mm thickness, with two levels of optics and a theoretical concentration factor of 85x and an angular acceptance of +/-3°, without anti-reflective coating. With an anti-reflective coating, we can expect a concentration factor around 95x.

Figure 3. Map of the optical efficiency, depending the radius of curvature of the primary and secondary lenses, for an incident angle of 3°

ESTIMATION OF PERFORMANCES IN REAL CONDITIONS

Using a MATLAB code that we developed, we evaluated the performances of such a system in real conditions, at different locations. The code is based on four key parameters: meteorological data (from NREL NSRDB), number of axis of the tracking system, the optical parameters of the designed system and the electrical behavior of the solar cell.

To calculate the average increase of efficiency and the annual production of our system we chose a solar microcell working at 17% efficiency under AM1.5G. The optical system used is described in the numerical study, taking into account the angular dependency of the optical efficiency and an anti-reflective coating. The sun tracking system is a twoaxis tracker.

In sunny regions, the increase of the efficiency is important, which results in a system being more efficient than a standard flat panel on a 2-axis tracker. Moreover, it saves rare-earth material which supply will be difficult in the coming years [START_REF] Redlinger | Evaluating the availability of gallium, indium, and tellurium from recycled photovoltaic modules[END_REF][START_REF] Jean | Pathways for solar photovoltaics[END_REF][START_REF] Duan | Rethinking China's strategic mineral policy on indium: implication for the flat screens and photovoltaic industries: Rethinking China's strategic mineral policy on indium[END_REF] .

PROTOTYPING

Fabrication process

To fabricate our microlenses we use the thermal reflow process. First, cylinders with a controlled diameter and height are fabricated using photolithography. Then, these structures are melted in an oven and take a spherical shape by minimizing the surface energy. Adjusting the dimensions of the initial cylinders allows us to control the geometrical parameters of the microlenses, such as the radius of curvature.

To show that the photoresist itself can be used to fabricate the optics of our prototype, at a laboratory scale, and be able to concentrate the light under AM1.5, we realized a first coupling experiment. We coupled a 16µm diameter microcell with a 500µm lens. The numerical evaluation of this system allowed us to expect not more than 38x. We obtained a concentration factor of 33x under a non-CPV solar simulator, which confirm that the photoresist can be used as an optical material for our prototype [START_REF] Jutteau | Micro Solar Concentrators: Design and fabrication for microcells arrays[END_REF] .

Finally, we fabricated an array with 2500 microcells of 54µm diameter, all connected in parallel, for a total active area of 5,72x10 -2 cm².On top of these microcells we fabricated 2500 secondary microlenses. On the other hand we fabricated the 2500 primary microlenses on a 1mm glass substrate.

Figure 4: 54µm microcells through secondary lenses

Coupling 2500 microcells and microlenses arrays

To couple the two arrays, we used a mechanical set-up composed of two angles to adjust the parallelism of the two substrates, two axis and one angle to align the optical axis of the primary and secondary lenses, and one axis to adjust the height of the primary lenses. The entire set-up has been installed under an AM1.5G solar simulator. The best alignment of the two arrays has been adjusted by maximizing the output current. J-V curves have been measured for different configurations, results are presented in Figure 5 and Table 2. Combining the array of microcells and the two arrays of optics, we achieved a concentration ratio of 65x, for a theoretical value of 85x. The difference between theoretical and experimental value can be mainly explained as we use a non-CPV solar simulator and the precision of the alignment set-up.

Concerning the electrical behavior of the microcells, we observe an increase of the open circuit voltage of +165mV. The fill factor also increases from 50% to 64% when the secondary lenses are added, due to the lower contribution of the shunt resistance. Unfortunately, the quality of our contacts is not good enough to be able to extract the high current density generated when the primary lenses are aligned, the series resistance is to strong and so the fill factor drops to 52%. The initial low fill factor, due to the fabrication process, and the bad quality contacts are being improved.

CONCLUSION

We propose a new type of concentrated photovoltaic system based on thin film microcells. A numerical study has been performed to design a very simple micro optical system based on microlenses, adapted to these microcells.

An estimation of the annual production of such a system, depending on the location, has been realized and shows that this system can be efficient in sunny region.

The measurements of the prototype showed good results. Improving the measurement set-up and the alignment of the arrays, we expect to achieve the theoretical performances of the system.

This concentrating system is a promising CPV solution for low and middle concentration because of easy microcells array fabrication associated with a compact and lightweight concentrating system.

Figure 1 .

 1 Figure 1. Sketch of the thin film microconcentrator concept

Figure 5 :

 5 Figure 5: Left, J-V curves of the reference sample (green), reference sample and secondary optics (orange) and full system (blue). Right, picture of the first prototype under a solar simulator AM1.5G

Table 1 :

 1 Annual production of a micro-CPV system based on CIGS solar microcells

		Meteorological Data	CIGS microcells on 2-axis tracker	CIGS flat panel on 2-axis tracker
	Location	DNI (%)	DNI (kWh.m -2 )	Average absolute efficiency (%)	Production (kWh.m -2 )	Production (kWh.m -2 )
	Las Vegas	81	2726	+ 3	540	505
	San Francisco	73	2165	+ 2.3	427	415
	Seattle	60	1498	+ 0.7	294	302
	New-York	56	1415	+ 1.2	277	303

L.

Table 2 :

 2 Jsc, Voc, Fill Factor and concentration factor of the first prototype

		Jsc (mA.cm -2 ) Voc (mV) FF (%) C factor (exp) C factor (th.)
	Reference array 2500 microcells	30,6	580	50	1	1
	Array + secondary lenses	305	675	64	10	24
	Array + secondary + primary lenses	2011	745	52	65	85

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Mathilde Ménard

Florent Meyer
email: fmeyer@unistra.fr

Christine Affolter-Zbaraszczuk

Morgane Rabineau

Alexandre Adam

Paula Duenas Ramirez

Sylvie Bégin-Colin
email: sylvie.begin@ipcms.unistra.fr

Damien Mertz
email: damien.mertz@ipcms.unistra.fr

Design of hybrid protein-coated magnetic core-mesoporous silica shell nanocomposites for MRI and drug release assessed in a 3D tumor cell model

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

INTRODUCTION

The blossoming growth of nanotechnologies has brought rapid progress in the design of multifunctional nanoplatforms called "nanotheranostics" which combine diagnosis and targeted therapy. [START_REF] Janib | Imaging and drug delivery using theranostic nanoparticles[END_REF][START_REF] Lammers | Theranostic nanomedicine[END_REF][START_REF] Lee | Multifunctional nanoparticles for multimodal imaging and theragnosis[END_REF] Among functional nanoparticles (NPs) for theranostic applications, superparamagnetic iron oxide nanoparticles (IO NPs) are of particular interest. Such NPs, apart from there magnetic resonance imaging (MRI) properties are currently emerging as powerful nano-heating mediators for magnetic hyperthermia making them nanotheranostic platforms by themselves. [START_REF] Mahmoudi | Superparamagnetic iron oxide nanoparticles (SPIONs): development, surface modification and applications in chemotherapy[END_REF][START_REF] Kievit | Surface engineering of iron oxide nanoparticles for targeted cancer therapy[END_REF][START_REF] Blanco-Andujar | Design of iron oxide-based nanoparticles for MRI and magnetic hyperthermia[END_REF][START_REF] Walter | Mastering the shape and composition of dendronized iron oxide nanoparticles to tailor magnetic resonance imaging and hyperthermia[END_REF] Moreover, various organic or inorganic coatings [START_REF] Stanicki | Chapter 4 Iron-oxide Nanoparticle-based Contrast Agents[END_REF][START_REF] Knežević | Magnetic mesoporous silica-based core/shell nanoparticles for biomedical applications[END_REF] have been investigated to allow drug loading, furtivity and/or targeting ability to address treatment side effect issues. The design of magnetic nanoplatforms with drug delivery capability through local or remote stimuli is therefore of outmost importance because of high expectations raised by synergistic combined therapies. [START_REF] Hervault | Magnetic nanoparticle-based therapeutic agents for thermo-chemotherapy treatment of cancer[END_REF][START_REF] Oliveira | Magnetic field triggered drug release from polymersomes for cancer therapeutics[END_REF][START_REF] Mertz | Drug releasing nanoplatforms activated by alternating magnetic fields[END_REF][START_REF] Kralj | Design and fabrication of magnetically responsive nanocarriers for drug delivery[END_REF][START_REF] Hervault | Doxorubicin loaded dual pH-and thermo-responsive magnetic nanocarrier for combined magnetic hyperthermia and targeted controlled drug delivery applications[END_REF] Organic molecules or polymer nanocoatings at the surface of IO NPs can be used for the loading and release of drugs. The drug can be bound either via covalent or non-covalent ways to the NPs.

Non covalent binding of the drug is typically achieved through electrostatic or H-bonding established with organic anchors/polymers at the NP surface and the drug release from the NPs is usually obtained through pH-conformational changes of such nanocoatings. [START_REF] Nigam | Poly (ethylene glycol)modified PAMAM-Fe3O4-doxorubicin triads with the potential for improved therapeutic efficacy: generation-dependent increased drug loading and retention at neutral pH and increased release at acidic pH[END_REF][START_REF] Gaihre | Gelatin-coated magnetic iron oxide nanoparticles as carrier system: drug loading and in vitro drug release study[END_REF] The main limitations of such approaches are in the low drug loading capacity (DLC) associated with a nonnegligible drug leakage through the NPs coatings. Alternatively, covalent binding of the drugs to NPs that can be cleaved upon various local or external stimuli were investigated [START_REF] Lee | Multifunctional mesoporous silica nanocomposite nanoparticles for pH controlled drug release and dual modal imaging[END_REF][START_REF] N'guyen T T T | Functional Iron Oxide Magnetic Nanoparticles with Hyperthermia-Induced Drug Release Ability by Using a Combination of Orthogonal Click Reactions[END_REF] to afford smart responses and few leaking. However, drug payload per particle is usually there rather moderate when using such strategy and the drug/nanoparticle ratio is a key parameter in the toxicity evaluation. Inorganic coatings like mesoporous silica (MS) tend to overcome this issue as they allow, in comparison with organic coatings, higher DLC. MS NPs (without magnetic/plasmonic core) were reported reaching up to 30-48% DLC. [START_REF] Yuan | Preparation of pH-responsive mesoporous silica nanoparticles and their application in controlled drug delivery[END_REF][START_REF] Shen | Mesoporous silica nanoparticles loading doxorubicin reverse multidrug resistance: performance and mechanism[END_REF] However such NPs must be associated with an external organic/polymer coating acting as a gatekeeper to avoid spontaneous drug release.

Different strategies can be used depending on the stimulus (pH, redox, temperature) to trigger drug release. For example, Yuan et al. made use of poly(acrylic acid) polymer hydrogel grafted on MS to ensure the pH-responsive release of doxorubicin (Dox), a chemotherapeutic agent, triggered when pH was lowered from pH=7.4 to 5.6. [START_REF] Yuan | Preparation of pH-responsive mesoporous silica nanoparticles and their application in controlled drug delivery[END_REF] In another work, Duguet and coworkers used a thermo-sensitive lipidic-based gatekeeper to encapsulate Dox in IO@MS NPs and release it upon lipid phase melting above 40°C. [START_REF] Liu | Heat-triggered drug release systems based on mesoporous silica nanoparticles filled with a maghemite core and phase-change molecules as gatekeepers[END_REF] Among biological stimuli, drug release ensured through enzyme biodegradation/conversion action is of particular interest. In fact, in vivo, enzyme expression is specifically located either in the cell surrounding or inside the cell in subcellular compartments. Such strategy was usually investigated on drug releasing polymer films coated on planar surfaces. [START_REF] Andreasen | Biocatalytic polymer thin films: optimization of the multilayered architecture towards in situ synthesis of anti-proliferative drugs[END_REF][START_REF] Vogt | Layer-by-Layer Enzymatic Platform for Stretched-Induced Reactive Release[END_REF][START_REF] Barthes | Stretch-induced biodegradation of polyelectrolyte multilayer films for drug release[END_REF] However, to the best of our knowledge, the design of biodegradable gatekeeper shell releasing drugs initially embedded within porous nanomaterials upon protease action is not yet reported. Furthermore, among the diversity of polymer nanocoating approaches that may be used to encapsulate drugs, we developed the use of isobutyramide (IBAM) grafts and its derivatives as an innovative way to form non-covalent biofunctional and tight nanoassemblies. [START_REF] Mertz | Bromoisobutyramide as an Intermolecular Surface Binder for the Preparation of Free-standing Biopolymer Assemblies[END_REF][START_REF] Mertz | Protein capsules assembled via isobutyramide grafts: sequential growth, biofunctionalization, and cellular uptake[END_REF][START_REF] Mertz | Ultrathin, bioresponsive and drug-functionalized protein capsules[END_REF][START_REF] Mertz | Templated assembly of albumin-based nanoparticles for simultaneous gene silencing and magnetic resonance imaging[END_REF] Using such strategy, we successfully formulated self-supported protein-based hollow microcapsules and NPs. The formation of tight IBAM-HSA coatings over magnetic mesoporous silica may be thus of particular interest to design responsive coatings acting as drug gatekeepers using enzymatic degradation as stimulus to release the drugs.

Herein, we report the design of hybrid protein-coated magnetic core-mesoporous silica shell nanocomposites for MRI and drug release assessed. As described in Figure1, our strategy consists in building a thin mesoporous silica (MS) shell (pores of 3 nm) around superparamagnetic IO NPs, obtained by thermal decomposition. This synthesis leads to uniform and individual magnetic core covered by MS shell, called IO@MS NPs. Then, after surface modification with aminopropyltriethoxysilane (APTS), Dox is loaded by simple soaking into the porosities of the silica shell. HSA is then deposited on the silica shell surface, which is previously modified with IBAM binders. The so-obtained final system is called IO@MS-Dox-HSA. In this study, we report: i) the synthesis process and the deep characterization of the IO@MS-HSA NPs, ii) the evaluation of the contrast efficiency for MRI especially in T2-weighted MRI, iii) the investigation of the Dox loading procedure, iv) the biological studies with a 3D model called multicellular tumor spheroid (MCTS). Finally, HSA biodegradation and subsequent Dox release is assessed by incubation of the nanocomposites in a protease media and in vitro in a 3D cancer cell model. Synthesis of spherical IO NPs by thermal decomposition method. Uniform 10 nm sized Fe3O4 nanocrystals were synthesized using a previously reported method. [START_REF] Baaziz | Magnetic iron oxide nanoparticles: reproducible tuning of the size and nanosized-dependent composition, defects, and spin canting[END_REF] Briefly 1.38 g (2.22 mmol) of iron stearate was thermally decomposed in dioctylether (20 mL) in presence of 1.25 g (4.44 mmol) of oleic acid. First the mixture was heated to 130 °C under stirring for 1 h to dissolve the reactants and remove water residues. Then by applying a heating rate of 5 °C/min the solution reached the boiling temperature (ca 287 °C) and was kept under reflux for 2 h. After washing the final product by centrifugation with a mixture of chloroform/acetone (1/3) the IO NPs stabilized with oleic acid were dispersed in chloroform with a concentration of 2 mg Fe •mL - 1 measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES).

Coating of small pore MS around IO NPs. The MS coating method was adapted from previous reported methods. [START_REF] Kim | Multifunctional uniform nanoparticles composed of a magnetite nanocrystal core and a mesoporous silica shell for magnetic resonance and fluorescence imaging and for drug delivery[END_REF][START_REF] Ye | Uniform mesoporous silica coated iron oxide nanoparticles as a highly efficient, nontoxic MRI T2 contrast agent with tunable proton relaxivities Contrast Media[END_REF] The procedure described below is the typical procedure to synthesize IO@MS NPs of 30 nm in size and with a core of 10 nm.

First an aqueous solution of CTAB at 28 mM was prepared (500 mg in 50 mL of Milli-Q water).

After complete dissolution of CTAB, 3 mL of IO NPs dispersed at 2 mg Fe/mL were added under vigorous magnetic stirring for 30 min. At this step a brown turbid solution was obtained. Then the mixture was heated up to 65°C and maintained at this temperature for 10 min under stirring to evaporate the chloroform. The solution obtained was black and transparent. A solution of 150 mL of Milli-Q water and 60 µL of a 2M NaOH solution were added to the mixture. The so obtained solution was heated up to 70°C under vigorous stirring. 1.5 mL of TEOS and 2.55 mL of EtOAc were added and this final mixture was stirred for 3h at 70°C. The particles were then washed 3 times in ethanol by centrifugation.

Silanization of IO@MS: IO@MS-NH2. After CTAB washing via ammonium nitrate ion exchange, the IO@MS NPs were dispersed in 30 mL of EtOH and mixed overnight with 960 µL NH3 and 4.5 mL of APTES. Then the particles were washed by centrifugation 3 times in EtOH and 3 times in water. The resulting IO@MS-NH2 NPs were finally dispersed in 25 mL in Milli-Q water which correspond to a NPs concentration of 0.6 mg•mL -1 . Dox loading. Dox loading into IO@MS-NH2 NPs was performed by simple soaking of Dox with NPs in Milli-Q water. The soaking was achieved at a so called feed weight ratio (fwr) corresponding to the percentage of the ratio between the weight of Dox fed initially in the soaking media and the weight of NPs: Thus after 24 h of mixing at a certain fwr, the Dox loaded IO@MS-NH2 NPs were collected by centrifugation. The supernatants were measured by absorbance to calculate the amount of Dox loaded into the NPs. Furthermore 500 µL of each batch of IO@MS-NH2 NPs dispersed in Milli-Q water were dry-freezed and then weighted to determine the initial concentration of IO@MS-NH2 NPs. From these measurements, to evaluate the drug loading abilities of NPs, two essential entities were deduced: Drug Loading Content (DLC) and the Drug Loading Efficiency (DLE).

Fluorescent labeling of HSA. 531 µL of FITC in DMSO at 10 mg•mL -1 were mixed to 30 mL of HSA at 10 mg•mL -1 in 0.1 M sodium bicarbonate (NaHCO3) buffer (pH 8.5). The mixture was stirred overnight and then dialyzed in MilliQ water to remove free FITC. The final volume was adjusted to 60 mL to have a HSA FITC concentration of 5 mg•mL -1 .

Protocol of HSA coating. After the Dox loading, the HSA final coating was performed by using IBAM grafts. To functionalize the NPs surface with IBAM moieties, the sample was redispersed in 500 µL of DMF and 200 µL of Et3N directly after Dox loading. Then a mixture of 270 µL of IBC in 2,5 mL of DMF was quickly added to the sample. A precipitate was formed immediately. These mixtures were stirred for 1 h and then 10 mL of HSA at 2 mg•mL -1 in Milli-Q water was added.

After the addition of the HSA solution the precipitates were immediately dissolved. The solutions were stirred for additional 1h30 before being washed twice by centrifugation in water.

Finally the resulting IO@MS-Dox-HSA NPs were dispersed in 3 mL of Milli-Q water (freezedrying and subsequent weighing of the sample indicated a concentration of 0.4 mg•mL -1 ).

The exact same protocols were carried out again on the previous silanized IO@MS NPs to produce the corresponding final NPs with HSA coating but without the loaded Dox. Two samples were executed: once using naturel HSA and a second with FITC-labelled HSA.

Protease degradation study. 200 µL of Streptomyces griseus proteases dissolved respectively at 25, 50 and 100 µg•mL -1 in PBS, were mixed with 200 µL of the previous IO@MS-HSA FITC and IO@MS-Dox-HSA NPs. The mixture was incubated in water bath at 37°C and after a certain time was centrifuged (7000 g / 10 min) in order to measure the fluorescence signal in the supernatant. After the fluorescent spectroscopy measurement, the supernatant was replaced in the mixture vial, the NPs were re-dispersed and put back at 37°C. This measure was repeated regularly over 20 hours.

Biological study.

Cell. Huh7-Luc, human hepatocarcinoma (ATCC®) were cultivated in Dulbecco's modified Eagle's medium supplemented with 10 % of decomplemented fetal bovine serum, penicillin streptomycine and non essential amino-acids.

Huh7-Luc multicellular tumor spheroid (MCTS) formation. Huh7-Luc MCTS were prepared using the hanging drop method. Previously a 96 well plate (Corning™ Costar™ Flat Bottom Cell Culture Plates, Fisher scientific, Ref 10792552) was prepared to be non-adhesive. 100 μl of poly(2-hydroxyethyl methacrylate) (poly-HEMA; sigma P3932-10G) 0.5 % (W/V) in 100% ethanol solution was added to each well. The plate was then incubated in a dry oven at 38 °C until complete evaporation of the solution. A Huh7-Luc cell suspension of 5300 cell/mL was prepared. 15 μL drops were put down the inside face of the coverslip of the 96 well plate. Each drop was centered on a well. 100 μL of PBS were added to each well and the coverslip replace on the plate. Having made so each drop of cell suspension hangs from the coverslip above a well of the 96 well plate. Plate was incubated for 3 days at 37°C and 5% CO2. PBS was then discarded and replace by 200 L of complete medium. Plate was centrifuged at 500 G for 30 sec in order to harvest the Huh7-Luc MCTS. At this stage we can expect one spheroid, about 100 µm diameter size, per well.

Confocal laser scan microscopy. Huh7-Luc MCTS were incubated for 24 h with IO@MS-Dox-HSA and IO@MS-HSA FITC NPs. Half of the medium was then discarded from each well and replaced by PFA 8% in PBS. Huh7-Luc MCTS were then incubated for 1h at room temperature.

Once fixation completed Huh7-Luc MCTS were first rinced in three consecutive bath of PBS and mounted on a glass slide in Antifade Mounting Medium (Vector laboratories, H-1000, Burlingame, CA). Manipulation of Huh7-Luc MCTS was performed with a 400 µm nylon loop under microscope. A glass coverslip of 12 mm diameter was used and sealed with nail varnish. Huh7-Luc MCTS observation was performed with a Zeiss LSM 710 confocal microscope and with a Plan Apochromat 40X objective. The following conditions were defined: FITC excitation = 488 nm (argon laser), emission = 496-540 nm; Doxorubicin excitation = 488 nm, emission = 540-600 nm. Images were analyzed with the software ImageJ.

Assessment of the antitumoral effect on Huh7-luc MCTS. The experiments were carried out in octuplicate in 96-well plate using the spheroids as previously described. The IO@MS-Dox-HSA NPs or IO@MS-HSA NPs were diluted in complete cell culture medium. Nanoparticles (11 µL) were then immediately added to each spheroid by further dilution into the cell culture medium (100 µL). Final concentrations were 50, 100, 250, 300 and 500 µg•mL -1 . The spheroid growth was examined using a Nikon Eclipse TS100 microscope and a 10 X objective. Images were recorded at days 0, 2 and 6 with a Nikon Color CCD DS-Fi2 Camera and a DS-L3 camera controller. The area of each spheroid was measured using the NIH Image J software and reported in µm 2 . The MCTS growth was calculated relative to the initial area (A0; day 0).

CHARACTERIZATION Transmission Electron Microcopy (TEM) and Energy dispersive X-ray (EDX) analysis. NPs

were deposited on Formvar-coated copper grids. TEM images and EDX spectra were aquired with a JEOL 2100 TEM instrument operating at 200 kV coupled with energy dispersive X-ray (EDX) spectroscopy. The size distribution of NPs was measured on more than 200 NPs.

Dynamic Light Scattering (DLS).

The colloidal stability of NPs suspensions was assessed by measuring the particle size distribution using a nano-sizer MALVERN (nano ZS) zetasizer at a scattering angle of 173°.

Spectrofluorimetry.

A SAFAS (Monaco) spectrophotometer was used to measure the amount of Dox loaded within the IO@MS NPs and to follow the HSA FITC degradation under proteases action.. The Dox loading was estimated by analyzing the absorbance of the supernatant after soaking the free Dox with IO@MS for 24h and centrifugation. The HSA FITC degradation by proteases was followed by analyzing the fluorescence of the supernatant after centrifugation at different time points.

ICP-AES. IO@MS

N2 isotherm adsorption.

Nitrogen adsorption isotherms were obtained on a Micrometrics ASAP 2420 Accelerated Surface Area and Porosimetry System at 77K under continuous adsorption conditions. Brunauer-Emmett-Teller (BET) method was used to determine the surface area as well as the total internal pore volume at P/P0 = 0.97. By applying the Barrett-Joyner-Halenda (BJH) method, the surface area, the cumulative pore volume, and the mesoporous pore size distribution (PSD) are determined during the adsorption and desorption steps.

Fourier Transform Infra-Red Spectroscopy (FTIR). The composition of the IO@MS NPs was verified by Fourier Transform Infra-Red Spectroscopy (FTIR) (Perkin Elmer instrument FTIR Spectrum Two).

RESULTS AND DISCUSSION

Synthesis and characterization of magnetic core@mesoporous silica shell NPs

First, we established a suitable protocol, inspired from the literature, to synthesize spherical, monodispersed in size, IO@MS NPs. The IO NPs were synthesised by thermal composition as described previously [START_REF] Baaziz | Magnetic iron oxide nanoparticles: reproducible tuning of the size and nanosized-dependent composition, defects, and spin canting[END_REF]. The resulting IO NPs, presented in Figure 2.A-C were monodispersed and spherical with a size of 9 ± 1 nm. Then, a MS shell was build at the surface of the IO NPs by CTAB surfactant mediated sol-gel approach according to a procedure adapted from Hyeon et al. [START_REF] Kim | Multifunctional uniform nanoparticles composed of a magnetite nanocrystal core and a mesoporous silica shell for magnetic resonance and fluorescence imaging and for drug delivery[END_REF]. CTAB surfactant removal was achieved by nitrate ammonium washing and followed by IR spectroscopy (Figure S1). TEM (Figure 2 calculations, shows pore sizes about 2.3 nm. As these core-shell NPs are very small in size, a large signal was also obtained for porosities higher than 10 nm resulting from the interparticle voids. A specific surface area of ~120 m 2 /g was calculated. This value is a bit lower than the values found in the literature on similar systems, nevertheless, specific surface areas of 100 m 2 /g are suitable for the loading and release of drug molecules.

HSA coating on IO@MS NPs

In this section, with the aim to provide a biocompatible coating at the surface of the IO@MS, HSA was adsorbed at the surface of the IO@MS by using IBAM grafts as non-covalent binders. First, the surface of the IO@MS was modified with APTES. Then, the amine moieties reacted with isobutyrylchloride in DMF to form IBAM grafts. Finally, HSA was coated by simple adsorption in water. The TEM pictures, in Figure 4. A and B, of the protein coated NPs, IO@MS-HSA NPs,

show that the IO@MS structure was not affected by this further sequential synthesis steps.

Furthermore, we can distinguish that the porosities and the borders of the NPs appeared blurry, which could be ascribed to the HSA coating. In addition, the protein coating is also clearly visible between the NPs. TGA was performed to quantify the amount of the IBAM-HSA layer respectively to the inorganic core-shell. Figure 6. (black curve) shows the progressive calcination and evaporation of the organic layer from 25 to 600°C. Then the increase of mass observed is attributed to oxidation of the magnetic core. The first loss of mass of 5% was attributed to the water remaining in the system despite freeze-drying. Then IBAM-HSA layer was found to contribute to ca. 16.5% in mass which correspond to a coating of ca. 210 µg IBAM/HSA per mg of IO@MS core shell composite.

Evaluation of MRI contrast properties of IO@MS-HSA

Thanks to the superparamagnetic properties of the magnetite core, the IO@MS-HSA NPs may be used as T2 contrast agents. This property was assessed by proton NMR relaxometry at 37 °C and 1.41 T (60 MHz). These NPs were dispersed in PBS buffer in order to avoid pH variation between measurements. Longitudinal (T1) and transverse (T2) relaxation times of protons were The specific relaxivity values of IO@MS-HSA were of r1= 0.15 mM -1 s -1 and r2= 70 mM -1 s -1 . In a previous study on the same kind of IO NPs coated with TPEDTA siloxane (free silica) instead of MS-HSA shell, we obtained a r1 of 13.9 mM -1 s -1 and a r2 of 88.6 mM -1 s -1 . [START_REF] Wang | Optimizing the silanization of thermally-decomposed iron oxide nanoparticles for efficient aqueous phase transfer and MRI applications[END_REF] Our present study here on IO@MS-HSA NPs showed that the MS-HSA coating on the IO NPs did not modify importantly the r2 value as compared to IO@TPEDTA but strongly decreased the r1 leading to a higher r2/r1 ratio. This low r1 value could be explained by a limited water accessibility to the magnetic core due to the MS-HSA coating. Furthermore, the r2 value of IO@MS-HSA NPs was in the same range than r2 of commercially used IO, 120 mM -1 s -1 for Feridex, 186 mM -1 s -1 for

Resovist and 65 mM -1 s -1 for Combidex. [START_REF] Na | Inorganic nanoparticles for MRI contrast agents[END_REF] Thus these results place our system as potential candidate for T2 contrast agents.

Dox encapsulation in IO@MS -HSA NPs

Our IO@MS-HSA NPs are promising T2 contrast agent candidates. However, in order to make them good candidates as theranostics agents, their cargo capacities for drug delivery had to be tested. Hence, in this part, the IO@MS-HSA sample was loaded with Dox before the HSA coating step. To be sure to reach the maximum DLC possible, a high fwr of 170% was choosed. The DLC and DLE values measured by indirect method of the supernatant absorbance analysis at this fwr were respectively of 34w% and 20w% (see calibration curve in Figure S3). In the final formulation IO@MS-Dox-HSA, it was important to determine if some Dox was released during

A B

the subsequent synthesis steps (IBAM modification and HSA coating). Thus comparative TGA analyses between Dox-loaded IO@MS-Dox-HSA and non-loaded IO@MS-HSA NPs were also run (Figure 6). Free Dox and HSA TGA analyses (Figure S4) show similar calcination signals allowing predicting similar behaviors on the IO@MS composites. According to the TGA curves of IO@MS-HSA and the IO@MS-Dox-HSA NPs, 58 % of IO@MS-Dox-HSA sample corresponded to the inorganic IO@MS NPs whereas 5 % came from the remaining water, 16,5 % from the IBAM-HSA coating and 20 % from the Dox loading. Thus, this corresponds to a DLC of 34 % of Dox loaded into raw IO@MS NPs. This result is matching the DLC value obtained with the supernatant analysis after Dox impregnation suggesting that a negligeable amount of Dox was released from the NPs during the synthesis steps for the protein coating. The colloidal stability of IO@MS-Dox-HSA NPs was assessed in water. Figure 7.A presents the DLS measurement in volume of IO@MS and IO@MS-Dox-HSA NPs in water (Figure S5 shows the DLS measurements in intensity and number). These results showed a similar behavior for both raw IO@MS and final IO@MS-Dox-HSA NPs meaning that the modification of the IO@MS with Dox loading and IBAM-HSA coating did not change the colloidal stability of the system. No NPs aggregation can be observed. Hence, a major contribution with hydrodynamic sizes of ca. 58 nm and second minor contribution at ca. 200 nm were observed.

The Zeta-potential measurements, in

Dox release through degradation of the protein shell.

To test the bio-responsiveness of the protein nanocoating for controlled drug delivery, IO@MS-Dox-HSA NPs were incubated with protease solutions at various concentrations. The objective here was to investigate the Dox release after HSA shell enzymatic degradation.

For this purpose, a first sample without Dox but with FITC-labelled HSA (called IO@MS-HSA FITC ) and a second sample with Dox but with non-labelled HSA (called IO@MS-Dox-HSA) were prepared. The NPs were incubated at 37 °C in PBS at pH 7.4 with various proteases concentrations: 1000, 500 and 100 µg•mL -1 . At predetermined time intervals, the mixtures were centrifuged and the supernatants were analyzed by fluorescence spectroscopy with λexc= 488 nm and λem=520 nm for IO@MS-HSA FITC and with λexc= 488 nm and λem=595 nm for the Dox-loaded sample. Analysis of the spectra indicates that the spectral characteristics of Dox are very similar for free Dox and IO@MS-Dox. Indeed Dox presents two fluorescence emission maxima at ca. 570 nm and 599 nm. This indicates that Dox does not suffer any change of chemical structure after impregnation in the core-shell system. Regarding the spectral characteristics of IO@MS-Dox-HSA, some changes can be observed especially in the second band of emission which was reduced. This result suggests that a non-negligible part of the Dox molecules was degraded during the process IBAM-HSA. Thus, Dox molecules impregnated at the outer surface of the NPs are probably altered by the IBAM grafting procedure while the Dox molecules impregnated in depth in the pores are more likely not altered. At least, to elucidate the reactant responsible for the Dox alteration, we acquired spectrofluorometric spectra in the reaction media in DMF of Dox, Dox with Et3N and Dox with Et3N + isobutyrylchloride (IBC). Data in figure S8 show that while Dox spectra are very similar between Dox and Dox+Et3N, the absence of spectrum for Dox+Et3N+IBC demonstrated the clear degradation of Dox under these conditions. These data

A B A

show that the acidity generated by IBC is probably the main responsible agent for Dox degradation.

Finally, the preservation of both the red color of our suspension (Fig. S6) and the two emission peaks, strongly indicates that the Dox degradation is only partial when loaded into IO@MS NPs, while it is total for free Dox in solution. Hence the nano-confinement within the pores, like a "ship in a bottle", may ensure retention of Dox structure and its activity however it may also explain why the Dox hardly release upon protease action despite effective HSA shell degradation. Thus, bench conditions where only one biochemical stimulus is applied (protease at 1mg•mL -1 maximum) is not sophisticated enough to allow an effective Dox release. . With the aim to investigate if Dox release would occur in a more complex media endowed with various biochemical stimuli, we directly assessed, in the next paragraphs, our IO@MS-Dox-HSA NPs on 3D cellular models (spheroids).

Evaluation of IO@MS-HSA NPs diffusion and Dox release in Huh7-Luc MCTS model.

One of the major drawbacks concerning the use of NPs in medicine is their poor diffusion in tissue. Indeed, depending on their size but as well as the type of tissue, the diffusion can be rather limited. Even if two-dimensional (2D) cell monolayers have been conventionally used by the scientific community to evaluate drug efficacy, NPs cytotoxicity and their internalization, they poorly represent the complex tumor 3D microenvironment. Indeed, cells grown in 2D lack the extracellular matrix (ECM), as well as cell-cell contact and cell-matrix interactions. Thus, The developpement of 3D culture models have emerged as an additional in vitro step study to assist drug product development and tend to reduce the requirement for unnecessary animal studies [START_REF] Hamilton | Multicellular spheroids as an in vitro tumor model[END_REF][START_REF] Kunz-Schughart | Multicellular tumor spheroids: intermediates between monolayer culture and in vivo tumor[END_REF][START_REF] Lin | Recent advances in three-dimensional multicellular spheroid culture for biomedical research[END_REF][START_REF] Hirschhaeuser | Multicellular tumor spheroids: An underestimated tool is catching up again[END_REF][START_REF] Hutmacher | Biomaterials offer cancer research the third dimension[END_REF][START_REF] Mehta | Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy[END_REF][START_REF] Fitzgerald | Life in 3D is never flat: 3D models to optimise drug delivery[END_REF][START_REF] Asghar | Engineering cancer microenvironments for in vitro 3-D tumor models[END_REF]]. However, based on the so-called enhanced permeation and retention effect described for some tumors, NPs could help to deliver locally the chemotherapeutic agent and avoid side effects as it would only diffuse in the tumor. In our strategy we aim to deliver Dox in a controlled manner: by the protease degradation of the HSA coating. Dox is a small molecule able to diffuse freely through cell plasma membrane. Thanks to its inner fluorescence, it is easily detectable by microscopy. We thus tested the internalization of the NPs and their diffusion in a Huh7-Luc MCTS model and follow the subsequent diffusion of Dox loaded in the nanoparticles. To do so, Huh7-Luc MCTS were incubated 24h with IO@MS-HSA FITC and IO@MS-Dox-HSA samples. We then assessed the diffusion of Dox from the IO@MS-Dox-HSA NPs internalized in Huh7-Luc MCTS. First, Z-stack images were performed on spheroids incubated for 24 h at various concentrations of free Dox (0, 0.01 and 3 μg•mL -1 ) to evaluate Dox behavior in our model. As pictured on the images summarized in Figure S9, Dox accumulated after 24 h of incubation into the nuclei of the cells. Fluorescence intensity recorded, keeping the same setting parameters, are correlated with the various Dox concentrations tested. For 3 µg•mL -1 of Dox, the laser intensity used in the imaging parameters was too high and led to saturated images. Then, Dox localisation into MCTS was assessed after internalization of IO@MS-Dox-HSA NPs by Z-stack analyses with the same setting as previously. IO@MS-Dox-HSA NPS were studied at Fe concentrations of 0.8 and 0.4 µg•mL -1 (Figure 10). As seen in Figure 10, after 24h incubation Dox seems to be disseminated into the entire MCTS. It proves that once the IO@MS-Dox-HSA NPs are internalised in the border cells they underwent protease degradation. Surprisingly no Dox was found accumulated at the border with the same images we get with the FITC labeled particles. It could be interpreted as a complete release of the Dox in the incubation time.

Finally, we can state that even if the NPs were stayed only at the periphery of the spheroids, they could ensure the local release of Dox that can then diffuse into the entire 3D model. Such behavior could sign the lag between the particle internalization and Dox release as it is triggered by HSA digestion probably by proteases found in endo-lysosome. Such 3D culture system is a model of early tumorogenesis process. So these results help to envision how such particles could work on in-vivo models.

Assessment of the antitumoral effect on Huh7-luc MCTS.

To assess the cell cytotoxicity and the antitumoral effect of the core-shell nanoparticles, we used Huh7-Luc MCTS growth monitoring. Huh7-Luc MCTS were cultivated during 6 days in presence of IO@MS-Dox-HSA NPs or IO@MS-HSA NPs of various concentrations. At day 2 and 6 the Huh-7

Luc MCTS area was measured and compared to area of the control Huh7-Luc MCTS that have grown without particles. As shown in the figure below, for the samples up to 100 µg•mL -1 no cytotoxicity is recorded with a growth of the spheroids in presence of NPs comparable to the control. From 250 µg•mL -1 up to 500 µg•mL -1 , the NPs show some cytotoxicity that is correlated to the concentration. However, we can clearly note a difference in the Huh7-Luc growth between IO@MS-Dox-HSA NPs or IO@MS-HSA NPs. Indeed, at 250 and 300 µg•mL -1 , IO@MS-Dox-HSA NPs show more growth inhibition than IO@MS-HSA NPs (respectively 54 % vs 32 % inhibition for 250 µg•mL -1 and 75% vs 61 % inhibition for 300 µg•mL -1 at D6). This difference could be attributed to the release of Dox into the spheroids by the time. Nevertheless, this effect is abolished at the highest concentration of 500 µg•mL -1 showing a severe cell cytotoxic effect for both cases (86 % growth inhibition at D6). Altogether those results show that we do have an active release of Dox by the time once the NPs have been internalized in the Huh7-luc spheroids, but the inherent cytotoxicity impedes the use of a higher concentration that could have a more potent antitumor effect. Finally, in this biological study, we confirmed that in a cell media like a spheroid, the various and complex biochemical stimuli secreted by the cells were able to unstick the Dox confined and ensure its release in the spheroids as observed by confocal microscopy. Moreover, the cytotoxic study above indicates a cytotoxic effect of the Dox. These results are in agreement with study that we published recently, where we used the same chemical strategy to immobilize Dox in HSA capsules (DOX loading between APTS and IBAM in HSA wall) [START_REF] Ménard | Mesoporous silica templated-albumin nanoparticles with high doxorubicin payload for drug delivery assessed with a 3-D tumor cell model[END_REF]. The Dox-loaded HSA nanocapsules were formed after sacrificial dissolution of the silica template. Here we also observed the same behavior than in the present work: no Doxorubicin release was observed after HSA capsule incubation with protease while a strong cytotoxicity was observed on spheroids as compared to the non-loaded HSA capsules.

Overall, the results addressed in this work, suggest that in such core shell constructs, an important part of Dox keeps its structural properties, is able to diffuse into the spheroid, and, acts as cytotoxic agents by killing human cancer cells.

Conclusion

In this work, the design and full characterization of new hybrid spherical monodisperse IO@MS nanocomposites covered with IBAM-HSA coating, and denoted IO@MS-HSA NPs was achieved. These nanocomposites were analyzed by proton relaxometry, and the HSA coated IO@MS NPs were shown to display significantly high T2 contrast properties, with a transverse relaxivity r2 of 70 Mm.s -1 . Then, impregnation of Dox achieved on IO@MS NPs modified with APTS in water, led to a very high DLC value in the range of 35% which was also confirmed in the final IO@MS-Dox-HSA. Furthermore, regarding the drug release, we found out that, after Dox loading and capping by a tight HSA shell, an efficient HSA shell degradation was observed under enzymatic action. Dox release was objectivized in Huh7-Luc MCTS. Indeed, after only 24h incubation, IO@MS-Dox-HSA internalized into the border cell of the spheroid release their Dox that can freely diffuse into the tissue and inhibit the spheroid growth. This new hybrid protein coated magnetic core-MS shell could thus be envisionned as a new type of theranostic agents displaying in the same formulation abilities for antitumoral drug release and T2 contrast for MRI diagnosis. Supporting information. S1. FTIR spectra of IO@MS NPs before CTAB washing, and after nitrate ammonium washings, S2. SEM image of the IO@MS NPs, S3. TGA of free Dox and free HSA, S4. DLS measurement of IO@MS and IO@MS-Dox-HSA in A) intensity an B) number. S5.

Photograph of IO@MS@Dox-HSA NPs highlighting their colloidal stability, S6. CLSM images of free Dox penetration into spheroids

Figure 1 :

 1 Figure 1: Schematic illustration of the synthetic procedure for IO@MS-Dox-HSA NPs and the Dox release by HSA degradation via proteases action

  NPs samples were digested in closed vessels with trace metals grade nitric acid (TraceSELECT, Fluka) at 200 °C. The amount of Fe and Si in mineralized samples was measured with ICP-AES (Varian 720ES). Emission intensities were measured at 259.940 nm for Fe and 252.851 nm for Si and compared to emission intensities of standards (0.05, 0.2, 1, 5 mg•L -1 ) prepared from a certified standard (1000 mg•L -1 , CPI International)." Relaxivity measurements. T1 and T2 relaxation times measurements of IO@MS NPs were performed with a Bruker Minipsec 60 (Karlsruhe,Germany) working at a Larmor frequency 60MHz (1.41 T) at 37°C. The longitudinal and transverse relaxivity values r1 and r2 were obtained according to the general equation of relaxivity R=R0+r[CA] where R is the relaxation rate (1/T) in the presence of the contrast agent (CA), R0 the relaxation rate of the aquous medium in the absence of the CA and r the relaxivity value of the CA.

  .D and E) and SEM (Figure S2) images, confirmed the formation of individual IO@MS core-shell NPs composed of a IO core of 9 nm in diameter surrounded by a porous silica shell of about 12 nm thickness. The monodispersity in size was assessed by measuring the size of at least 500 NPs from the TEM images. The resulting TEM size distribution graph, Figure 2.F, shows a narrow size distribution of the IO@MS NPs of 31 ± 3 nm.

Figure 2 .

 2 Figure 2. A) low and B) high magnification TEM images of IO NPs of 9 nm made by thermal decomposition. D) low and E) high TEM images of core-shell IO@MS NPs of 31 nm. C) and F) histograms of the TEM size distribution measured of both NPs on more than 500 particles.

Figure 3 .

 3 Figure 3. A) FTIR spectra of IO@MS NPs, B) N2 adsorption-desorption isotherms and C) corresponding PSD curve for IO@MS NPs.

Figure 4 .

 4 Figure 4. A) Low and B) high magnified TEM images of IO@MS NPs of 31 ± 3 nm coated with IBAM-HSA (denoted IO@MS-HSA)

  Fe concentrations, [Fe]. The highest [Fe] was measured initially by ICP-AES, and the lower [Fe] were obtained by dilution. As expected, the resulting relaxivity curves 1/T1 and 1/T2 in function of [Fe], presented in Figure 5.A and B respectively, showed linear behaviors. The slopes of the linear fit of these curves gave the r1 and r2 values.

Figure 5 .

 5 Figure 5. T1 and T2 relaxometry curves of IO@MS-HSA of 30 nm in diameters (A and B respectively).

Figure 6 .

 6 Figure 6. TGA curves of IO@MS-HSA and IO@MS-Dox-HSA NPs

Figure 7 .

 7 B, show that IO@MS-NH2 NPs have an IEP around 7.7. Therefore, they were positively charged before the Dox loading, with a Zeta potential over +20 mV, in the pH range of Milli-Q water, pH 6-7. As the Dox hydrochloride salt is well positively charged in Milli-Q water we can conclude through this Zeta potential measurements that the interactions between the amino-modified silica surface and the Dox were not of electrostatic nature. After the Dox loading and HSA coating, the IEP of these particles decrease to 6. IO@MS-Dox-HSA NPs are then slightly negatively charged, with a Zeta potential between 0 and -20 mV, in the Milli-Q water pH range. This should have led to poor colloidal stability according to such low ZP values. However, according to the DLS results for this compound and the excellent colloidal stability observed (see photograph of a suspension in FigureS6), the NPs were found to have a great colloidal dispersion. This good colloidal stability can be more attributed to steric effects of the HSA layer than to repulsive forces with less contribution of electrostatic interactions.

Figure 7 .

 7 Figure 7. A) DLS measurement of IO@MS and IO@MS-Dox-HSA NPs and B) Zeta potentials of IO@MS-NH2 and IO@MS-Dox-HSA NPs.

Figure 8

 8 shows the profiles of HSA degradation (8.A) and Dox release (8.B) of the A B A samples. The increase of fluorescence intensity in IO@MS-HSA FITC in contact with proteases confirmed that HSA FITC was progressively degraded in the presence of proteases. Degradation rate is driven by enzyme concentration. No fluorescence was recorded in control sample, in the absence of protease. It confirms that the HSA shell is stable by the time course of the experiment and that the proteases are mandatory for the shell degradation. However, no significant Dox release was recorded in presence of protease.

Figure 8 .

 8 Figure 8. Kinetic profiles under various proteases concentration (() 1000 g/mL, () 500 g/mL () 500 g/mL, () 0 g/mL) at 37 °C in PBS at pH 7.4 of A) HSA FITC degradation from IO@MS-HSA FITC sample, B) Dox release from IO@MS-Dox-HSA sample.

First, internalization ofFigure 9 .Figure 9 .

 99 Figure 9. CLSM imaging of Huh7-Luc MCTS after 24h incubation with IO@MS-HSA FITC NPs at 0.4 and 0.8 µg Fe•mL -1 . Images show the entire spheroid analysis (Z-stack). The magnification used in CLSM is x 40.

Figure 10 .

 10 Figure 10. High resolution CLSM imaging of Huh-7 MCTS after 24h incubation with IO@MS-Dox-HSA NPs at 0.4 and 0.8 µg Fe•mL -1 . The magnification used in CLSM is x 40.

Figure 11 .

 11 Figure11. Evolution of % spheroid growth vs control of Huh7-luc MCTS at days 0, 2 and 6 carried out in octuplicate after incubation of IO@MS-Dox-HSA NPs or IO@MS-HSA NPs. NPs concentrations are 50 (A), 100 (B), 250 (C), 300(D) and 500 µg•mL -1 . (E). The MCTS growth was calculated relative to the initial area (A0; day 0).

Acknowledgements. C. Ulhaq and D. Ihiawakrim are acknowledged for assistance with TEM microscope. D. Bégin and K. Parkhomenko are acknowledged for helpful discussion. A. Boos and P. Ronot are acknowledged for ICP-AES experiments. D.M. thanks the University of Strasbourg for financial support from IDEX-Attractivité framework. S. B.C, D. M. and F.M. thank ARC (Association de recherche contre le Cancer), IHU (Institut Hospitalo-Universitaire) de Strasbourg for financial support under the TheraHCC framework and Alsace contre le Cancer for financial support under the Venacoco project. The Institut National du Cancer (INCa) is also acknowledged for the financial support (project PRTK14,THERAMAG 2014-225).


S Béchu

A Loubat

M Bouttemy

M Balestrieri

S Gaiaschi

T Hildebrandt

V Achard

M Frégnaux

D Aureau

J Vigneron

F Donsanti

M Jubault

P Chapon

D Lincot

J F Guillemoles

A Etcheberry

Surface reactivity of CIGS absorber on soda-lime and flexible substrates studied by XPS: a global approach of deoxidation, ageing and alkali elements distribution

Keywords: CIGS, XPS, ageing, surface deoxidation, flexible substrates

Surface reactivity of CIGS absorber on soda-lime and flexible substrates studied by XPS: a global approach of deoxidation, ageing and alkali elements distribution

I. INTRODUCTION

Photovoltaic cells based on CIGS (Cu(In,Ga)Se 2 ) absorbers are among the most efficient in photo-conversion. In the worldwide competitive context of photovoltaic market, the cells architectures are in constant evolution to still gain efficiency while decreasing the production cost. In addition, new developments are considered to address new emerging market orientation, such as flexible solar cells (for transport, energy productive buildings, high technology and intelligence…) widely dominated at the moment by the organic and nanomaterial sectors [START_REF] Kaltenbrunner | Ultrathin and lightweight organic solar cells with high flexibility[END_REF]. Among the thin films technology, CIGS has proven to be relevant and recent evolutions are oriented towards its adaptation on flexible substrates. In this particular case, unlike the classical soda lime substrates on which was developed the initial CIGS cell design, an internal source of alkali elements is missing. Past studies [START_REF] Kronik D. Cahen | Effects of Sodium on Polycrystalline Cu(In,Ga)Se 2 and Its Solar Cell Performance[END_REF]- [START_REF] Chirila | Potassium-induced surface modification of Cu(In,Ga)Se 2 thin films for high-efficiency solar cells[END_REF] have clearly demonstrated that alkali presence and localization in the CIGS network (mainly at grain boundaries) is a key parameter of efficiency increase. Therefore, different routes are considered to provide alkali in the absorber from an external source. This additional but essential step is usually performed from the front side of the absorber (post-deposition treatment). Consequently, the surface composition of CIGS is a fundamental point as it will condition the absorber/buffer layers and their interface properties in the final stack.

Indeed, whatever the substrate on which CIGS is deposited, it is well known that the performance of these solar cells is mainly governed by the bulk absorber properties. However, the interfaces with the front and back contact layers are also crucial for the performance improvement and have to be finely optimized. X-ray Photoelectron Spectroscopy (XPS) enables to obtain overall physico-chemical information by the determination of the composition and chemical environments at the surface, bulk and interfaces of CIGS layers. Specific spectroscopic signatures of deposition process and storage conditions can be highlighted and bring insight about the quality of the absorber considering different surface issues such as oxidation/deoxidation, presence of binary compounds and deeper aspects involving alkali elements distribution or absorbers ageing evolution…

This work is mainly focused on the surface reactivity. In the first part, the study of CIGS surface chemistry (classical architectures on soda lime substrates) after the reactor extraction, after further preparation by specific chemical engineering (HCl [START_REF] Loubat | Chemical engineering of Cu(In,Ga)Se2 surfaces: An absolute deoxidation studied by X-ray photoelectron spectroscopy and Auger electron spectroscopy signatures[END_REF], NH 3 -H 2 O, HCl-KCN [START_REF] Canava | Increasing solar cell efficiencies based on Cu(In,Ga)Se2 after a specific chemical and oxidant treatment[END_REF], [START_REF] Canava | XPS and electrical studies of buried interfaces in Cu(In,Ga)Se 2 solar cells[END_REF] and NH 3 -H 2 O-KCN) and the chemical evolution of those surfaces in time at ambient atmosphere, is presented. Indeed, this work aims to find an effective and easy to use universal treatment (here dipping) to adjust the surface composition, control the doping and contamination levels, eliminate oxides and binary compounds. Likewise, since surface composition of the CIGS differs from the bulk one, a specific preparation has to be performed before the buffer layers deposition, in order to obtain the best band alignment. To complete this study, the XPS monitoring of the surface evolution during 4 months is performed in order to determine the acceptable delay before implementing the next steps of the cell fabrication.

In the second part of this work, the study of CIGS deposited on flexible substrates with specific NaF and KF postdeposition treatments is presented. A focus is made on CIGS surface evolution arising from these treatments. For this purpose un-rinsed samples are first studied, to maintain a capping and protective layer and avoid unwanted alkali surface diffusion. Then, water rinsed samples are examined, just after elimination of the residual alkali charge and along time evolution, with a specific attention on the alkali elements behavior.

A cross analysis of the results on the flexible and soda-lime substrates is presented and completed by the evolution of alkali distribution in-depth.

II. EXPERIMENTAL RESULTS

A. Deoxidation treatment

A major issue of CIGS based solar cells concerns the control of the surface composition to ensure optimal interface properties. This surface composition can be finely adjusted using a chemical engineering procedure relying on dipping in selected solutions (HCl, NH 3 -H 2 O, HCl-KCN and NH 3 -H 2 O-KCN). Indeed, initially, the surface composition of an asgrown sample differs strongly from the one of the bulk and is representative of the absorber elaboration process. ) ratio, mainly governed by the Se content, discrepancies are also observable with EDS values but the consistency within the XPS set of values evidences a constant global stoichiometry. It is worth noting that EDS values are only indicative as the probed depth is not the same as the one of XPS (around 1.5 µm vs 10 nm), as well as the accuracy of quantitative data. Indeed, the fine analysis of the Se 3d photopeaks evidences the presence of superficial Se 0 (Figure 2) for both formulations employed lowering the CIG/Se. Finally, the additional use of KCN dipping is considered. This treatment has proven to efficiently eliminate elementary Se (Se 0 ) [START_REF] Bandyopadhyay | Effect of Surface Treatment on the Photoanodic Behaviour of Thin Film n-CdSe[END_REF]. By using either HCl-KCN or NH 3 -H 2 O-KCN treatments (HCl or NH 3 -H 2 O dipping following by a 5 minutes dipping in KCN solution at 1M), similar results are obtained with the thinning of the Se 3d peak (Figure 2), due to the disappearance of the Se 0 contribution at 56 eV. Consequently, the spin-orbit splitting of Se 3d peak is fully recovered. The use of KCN leads to CGI ratio lower than the one obtained for HCl, NH 3 -H 2 O and the overall EDS value. This confirms that the Cu content at the surface is directly modulated by the nature and formulation of the dipping solution.

B. Ageing at ambient atmosphere

Starting from those pristine surfaces, samples have been aged in air for 4 months to study their reoxidation kinetics. Whatever the treatment, oxidation is only observed after almost 2 days air storage as illustrated by the evolution of O 1s and Se 3d peaks (Fig. 3).

The oxide contribution to the O 1s signal (531.00 ± 0.05 eV) increases in time and the Se oxide contribution (59.00 ± 0.05 eV) is detected on the 4 days spectrum. During ageing, the surface progressively becomes again Cu-poor and the Na also migrates to the surface. In addition, Cu oxide becomes detectable only after 4 months. Longer time evolution experiments are ongoing to determine if this trend continues. Note that the CGI remains higher than the one measured on the as-grown surface after 4 months (0.32 instead of 0.29).

C. CIGS thin films on flexible substrates

For CIGS deposition on flexible substrates (such as polyimide), alkali are introduced from the top side by additional post-treatments, evaporated under Se flux. Consequently, chemical modifications are expected on CIGS surface, but also concerning alkali incorporation and migration during ageing. Cu(In,Ga)Se 2 layers have been deposited at IPVF on 800 nm DC sputtered molybdenum layer on polyimide foils (Upilex 50S). CIGS films have been grown with a multi-stage co evaporation process developed at low temperature (<400°C).

Both as deposited samples, which present the initial CIGS chemical surface, and freshly rinsed ones (extra-pure water), to reproduce the process stage before the buffer layer deposition, have been investigated. Specific spectroscopic signatures and alkali incorporations are observed, depending on the nature of the alkali. For rinsed CIGS sample, unusual surface evolutions are evidenced during air ageing, with the absence of oxidized Se contribution (Fig. 4 a)) even after 4 months. This behavior is strongly different to the one observed on conventional CIGS/soda-lime (section B) and concomitant with a surprising reemergence of the spin-orbit splitting. Regarding the alkali behavior, the increase with time of Na 1s photopeak (Fig. 4 b)) evidences its migration to the surface, similar to the soda-lime system. No reemergence of the counter-ion was observed in this particular case.

The un-rinsed sample presents similar characteristics, with an equivalent Na migration and also no evolution of the counter-ion over ageing time. Moreover, the Se 3d photopeak doesn't present any oxide traces, even after 6 months of ageing.

The exact depth distribution of alkali is investigated by XPS profiling over the first hundreds of nanometers (Ar + sputtering 2000 eV, zalar rotational mode) and completed by GD-OES qualitative profiling. Different distributions are pointed out for NaF and KF as the incorporation of the counter ion depends on its mobility. For a sample with two alkali post-treatments deposition (in a first time, NaF, followed by a KF deposition), it is shown that the counter ion of alkali element is localized deeper in the layer (Fig 5 .) while two kinetics are observed for the two different alkali elements. Indeed, the KF post-deposition treatment is performed after the NaF post-deposition treatment, which is in good agreement with the evolution of the XPS atomic percentages observed here.

III. CONCLUSION

Comparison of the surface reactivity of CIGS between sodalime and flexible substrates was underlined in this study. While deoxidation treatment is necessary to restore as pristine CIGS surface in the case of soda-lime substrates, a specific surface chemistry is shown for the CIGS elaborated on flexible substrates. In addition, a different surface reactivity is observed, as evidenced by the absence of Se oxide even after 4 months ageing. Concerning alkali, their depth distribution differs with the source (soda-lime, NaF or KF post-treatment) but ageing leads to a migration of alkali elements toward the surface in all cases. Ongoing experiments will provide the complementary information to fully determine the position of alkali elements in CIGS absorber layers and their mobility to optimize the elaboration process on flexible substrate.

Fig. 1 .

 1 Fig. 1. a) Ga 3d -In 4d and b) O 1s photopeaks acquired on CIGS surfaces (on soda-lime substrate) of an aged raw sample (black) and after treatment of this surface by a subsequent HCl dipping (red) or a NH 3 -H 2 O (blue) dipping.

Fig. 1 a

 1 Fig. 1 a) shows the Ga 3d -In 3d photopeaks monitored on CIGS (on soda-lime) absorbers after different surface preparations. This spectral window, rarely considered in XPS studies, presents the advantage of enabling a direct comparison of the In and Ga proportions, the photoelectrons collected having similar escape depths. This represents then an original and powerful tool to GGI (namely [Ga]/([Ga]+[In]) ratio) determination. Firstly, in the case of the as-grown CIGS surface, the Ga 3d -In 3d photopeak is characterized by a specific

Fig. 2 .

 2 Fig. 2. Se 3d photopeaks acquired on CIGS surfaces of an aged as grown sample (black) and after different chemical treatments of this surface (HCl, NH 3 -H 2 O, HCl-KCN and NH 3 -H 2 O-KCN).

Fig. 3 .

 3 Fig. 3. Time evolution of CIGS deoxidized surfaces: a) O 1s and b) Se 3d (after HCl+KCN treatment) photopeaks acquired just after treatment until 4 months air ageing. The as-grown spectra are shown for comparison.

Fig. 4 .

 4 Fig. 4. a) Se 3d and b) Na 1s photopeaks evolution over air ageing for a rinsed CIGS sample deposited on flexible substrate with NaF post-deposition treatment.

Fig. 5 .

 5 Fig. 5. XPS atomic percentage evolution over etching time for the different elements present in the CIGS sample.

TABLE I SUMMARY

 I OF THE XPS SPECIFIC RATIOS OF CIGS SURFACES FOR THE DIFFERENT CHEMICAL TREATMENTS STUDIED

	Sample	GGI	CGI	CIG/Se
	As-grown	0.20 ± 0.03	0.24 ± 0.03	0.62 ± 0.03
	HCl	0.32 ± 0.03	0.91 ± 0.03	0.49 ± 0.03
	NH3-H2O	0.32 ± 0.03	0.74 ± 0.03	0.47 ± 0.03
	HCl-KCN	0.31 ± 0.03	0.68 ± 0.03	0.63 ± 0.03
	NH3-H2O-KCN 0.31 ± 0.03	0.62 ± 0.03	0.63 ± 0.03
	EDS	0.28 ±0.05	0.86 ± 0.05	1.17 ± 0.04

ACKNOWLEDGEMENTS

This work was carried out in the framework of the projects I and B of IPVF (Institut Photovoltaïque d'Ile-de-France). This project has been supported by the French Government in the frame of the program of investment for the future (Programme d'Investissement d'Avenir -ANR-IEED-002-01).

Authors thank Wolfram Hempel from ZSW (Zentrum für Sonnenenergie-und Wasserstoff-Forschung, Germany) for CIGS samples on glass supply.