Redouane Bouchaala

Nicolas Anton

Halina Anton

Thierry Vandamme

Julien Vermot

Djabi Smail

Yves Mély

Andrey S Klymchenko
email: andrey.klymchenko@unistra.fr

Light-triggered release from dye-loaded fluorescent lipid nanocarriers in vitro and in vivo

Keywords: Photo-release, controlled release, lipid nanoemulsions, lipophilic dyes, fluorescence correlation spectroscopy, fluorescence microscopy. Total number of worlds: 6236, Total Number of Figures/Tables: 5. Graphical

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Highlights:

-Light-triggered release from dye-loaded nanoemulsions is shown -Being stable in vitro and in vivo, nanoemulsion droplets can release their content after illumination with visible (green) light -Light does not break nano-droplets, but stimulates transfer of their content -Photo-release of dyes from nanocarriers involves at least in part oxygen photochemistry

Introduction

Triggered release of active molecules from nanocarriers has been a subject of intensive research in the last decades [1][2][3][4][5][6]. Various triggers, such as pH, reductive potential, enzymes, temperature, ultrasound, magnetic field and light have been utilized. Light is particularly attractive in this respect, as it can be focused at a desired position of a specimen with high spatiotemporal resolution. The most common approaches for light controlled release utilize molecules that undergo lightcontrolled isomerization, photo-polymerization and photo-cleavage [7,8]. Thus, azobenzene cistrans isomerization can trigger release from liposomes [9] polymer vesicles and micelles [10,11] and inorganic porous nanomaterials [12,13]. Photo cleavage reactions in o-nitrobenzyl [14][15][16], coumarin [17], spiropyran [18,19], malachite green [20], cynnamic acid [21,22] derivatives, etc can produce photo-driven hydrophobicity and charge changes, polymer/lipid fragmentation and de-crosslinking. However, majority of examples of the light-triggered release from nanocarriers reported to date require UV light or two-photon excitation [17,23]. The other approach utilizes surface plasmon and photo-thermal effects, to trigger the cargo release. In this case, most commonly gold nanoparticles, nano-rods or nano-shells, are used in combination with polymer, lipid nanocarrier or thermo-sensitive covalent bonds [24][25][START_REF] Cheng | Small[END_REF][27][28]. The plasmon absorption in this nanostructures was successfully tuned, so that they could be activated with almost any desired excitation wavelength from UV up to near-infrared (NIR) region [29,30]. However, photothermal effects using gold nanostrcutures generally require relatively strong illumination powers [31], which can be harmful for the biological sample. Upconverting nanoparticles, which convert NIR light into visible and further produce photochemical effect should be also mentioned, but so far all examples of photo-release with upconversion nanoparticles are limited to inorganic systems [32,33]. In order to develop organic photo-responsive systems one should use dyes encapsulated inside a nanocarrier. In this case, dyes operate as photosensitizers generating highly reactive singlet oxygen and/or as converters of light energy into heat. As photosensitizers, organic dyes, due to their capacity to disrupt biological membranes, were used for photochemical delivery into the cells of molecules and nanoparticles [34][35][36][37]. The use of organic dyes as triggers of molecular release from a nanocarrier were mainly realized in lipid vesicles, where local photothermal or photochemical effects were proposed as driving mechanisms of photo-release [38][39][40][41][42]. The photothermal effects were also reported for NIR dyes loaded into nanocarriers in cancer therapy applications [43][44][45]. Lipid nanoemulsions are an important nanocarrier platform, as they can be readily prepared from FDA approved agents and their hydrophobic core can encapsulate drugs and contrast agents [46][47][START_REF] Mcclements | Emulsion Design to Improve the Delivery of Functional Lipophilic Components[END_REF][49][50]. Previously, the group of Texier suggested these lipid nanocarriers as imaging agents by loading their core with apolar cyanines [51,52]. We further suggested a particular probe design, using long alkyl chains and bulky hydrophobic counterions, that can increase the dye encapsulation together with preservation of the highly efficient fluorescence [53,54]. Moreover, recent FRETbased studies revealed their high stability in vitro and in vivo and capacity to enter tumors in the intact form [55,56]. In particular, we showed that the use of highly hydrophobic substituents of Nile Red enables to significantly increase dye loading, preserve good fluorescence efficiency and drastically decrease the dye leakage [53]. However, the question remained: can a strong illumination of a large quantity of dyes confined on the nanoscopic space destabilize lipid droplets? This point was not studied up to now owing to the difficulty to encapsulate a large amount of dyes in these nanoscale reservoirs. Taking recently proposed approaches [53,54], the dye concentration can be increased up to 5 wt% along with conserving their fluorescent properties. This is precisely the point that can open the door to photo-triggered properties. Although Nile Red photosensitizing properties were not described yet, we have shown earlier that large concentrations of Nile Redbased membrane probe (NR12S) can produce some photo-damage on cell membranes [57], which suggests that Nile Red as photo-active molecule to destabilize lipid nanocarriers.

In the present work, we describe an unexpected phenomenon, which is a light-triggered release of nano-droplet content, when encapsulating a hydrophobic Nile Red analogue. We show that the dye release can be observed both in cell culture and in vivo on zebrafish model. The light-induced dye release was validated with different acceptor media, such as blank nanocarriers and 10% serum and it can be inhibited in the presence of oxygen scavenger. Finally, the light-driven release of coencapsulated molecule (second encapsulated dye) along with the Nile Red derivative was validated, which proposes the route to light-controlled drug release from lipid nano-droplets. The idea of light-triggered release from nano-carrier is summarized in Fig. 1. The nano-emulsion droplets can be considered as reservoir with high local concentration of dyes. Our question was whether illumination of this large ensemble of dyes within the small volume of lipid nanocarrier could produce release of its content due to photophysical or photochemical phenomena. As a dye for encapsulation, we selected a derivative of Nile Red bearing long alkyl chains (NR668, Fig. 1). Although this dye is expected to be cell permeant (like parent Nile Red), its high hydrophobicity ensures encapsulation into nano-droplets with minimal leakage effects, which prevents its fast accumulation in the cells [53].

Results and Discussion

We first prepared nanocarriers by spontaneous emulsification of Solutol, Labrafac and NR668 dye in water, which gave 32 nm particles of low polydispersity index (<0.1) according to dynamic light scattering. The phenomenon of dye release under light illumination was first studied in cell culture. Solutol ® (Kolliphor ® ) HS 15 / Labrafac ® WL nanoemulsions loaded with hydrophobic Nile Red derivative NR668 was incubated with cells and studied by wide-field fluorescence microscopy. Our earlier works showed that no internalization was observed for the same system even after 2h of incubation with HeLa cells [53], which showed that these nanocarriers cannot enter the cells on this time scale. Therefore, this system is ideal for testing the effect of light, as no artifacts due to particle internalization or dye leakage into the cells was observed. After 30 min of incubation of the cells with the emulsion of nanocarriers containing 1 wt% of NR668, the cells remained poorly fluorescent, as the fluorescence of the medium was more intense than that of the cells. Moreover, after washing of the cells, no significant fluorescence was detected inside the cells (Fig. S1), which confirmed that NR668 nano-droplets did not spontaneously internalize within this time scale. Then, we illuminated the cells surrounded by NR668-loaded nanocarriers for 30 seconds at 535 nm (absorption maximum of NR668 in nano-droplets is 526 nm [53]) using the maximal lamp power (~14 W/cm 2 power density at the sample level). The light action resulted in nearly complete photobleaching of the area of observation. Indeed, it was observed that initially very bright fluorescence field became non-fluorescent after the illumination (Fig. 2). Then, with time, the fluorescence of the medium started to recover, but surprisingly the fluorescence of the cells increased with time. This phenomenon was observed already at 1 wt% of dye loading and took about 10 min to achieve a clear intracellular staining (Fig. 2 A,B), characterized by a significant difference between intracellular and extracellular fluorescence intensity (Fig. 2F). At 5 wt% dye loading (Fig. 2 C,D), the cell staining after illumination was much stronger, so that the difference between intracellular and extracellular fluorescence intensity was >3-fold larger compared to that for 1 wt% loading. After cell washing from the nanocarriers, we could find the initially illuminated regions, where the fluorescence inside the cells was many-fold larger than that for the nonilluminated regions (Fig. 2 G,H). These experiments clearly showed that nanocarriers, unable to enter the cells in the dark, can deliver the dye after illumination. It should be noted that when NR668 dye without lipid nanocarrier was added directly to the cells from its DMSO stock solution (final DMSO concentration was 1%), the intracellular fluorescence of this dye was observed (Fig. S1). Earlier works showed that its parent analogue Nile Red could also spontaneously internalize [58], which implies that plasma membrane is not really a barrier for these hydrophobic dyes. These results highlight the role of the nanocarrier, which encapsulates NR668 preventing its leakage into the cells, but ensuring its release after the illumination. Then, we investigated whether the light-induced release can be also realized in vivo on zebrafish. Our earlier work showed that NR668-loaded nanocarriers remained in the blood circulation for at least 30 min without dye leakage into the endothelial cells of the vessels and surrounding tissues [53]. Here, we injected nanocarriers containing 5 wt% of NR668 and performed microscopy imaging as a function of time without and with illumination (through 450-490 nm bandpass filter). Without illumination, the red fluorescence of nano-droplets was observed exclusively inside the blood vessels for at least 60 min (Fig 3A,B), indicating that the nanocarriers remain in the blood circulation without dye leakage into the tissues. By contrast, after the illumination, we could observe a progressive increase of the fluorescence in surrounding tissues between the blood vessels (Fig. 3C-F). The fluorescence intensity, recorded in the tissue areas showed continuous increase in time for the observation period of 60 min (Fig. 3G). Thus, the illumination triggered release of the encapsulated NR668 dye, which then diffused freely into the surrounding tissues. To understand better the observed phenomenon of release, we performed fluorescence correlation spectroscopy (FCS) of our nanocarriers in different model biological media. FCS is a powerful technique to characterize nanoparticles in situ, which can provide information about size, concentration and brightness of particles [53,[START_REF] Haustein | Fluorescence correlation spectroscopy: Novel variations of an established technique[END_REF]60]. According to our FCS data, the particle size, measured from the diffusion correlation time of the emissive species was 33 nm (Table S1). This value is remarkably close to the hydrodynamic diameter obtained for these nanocarriers by DLS (32 nm). Moreover, the particles concentration for this sample at 1:10000 dilution, evaluated from the number of emissive species per focal volume, was 1.7 nM. Basic calculation based on known total lipid concentration in the used nanoemulsion (1.110 -3 %) and the mass of a single 33-nm nano-droplet (1.910 -17 g, assuming density of the droplet of 1 g/ml), we could estimate that the concentration of nanocarriers should be around 1.0 nM. The correspondence of the theoretical and experimental values of the particle concentration and the match of the particle size obtained from DLS and FCS suggest that after this 10000-fold dilution the nanocarriers remained practically intact. In order to monitor the photo-release of the dye, we studied the number of emissive species, which, according to our earlier studies, is an important indicator of the dye release [53]. Indeed, if the dyes are released into acceptor medium (blank particles or serum), the number of emissive species should increase (Fig. 4A). For example, in control experiments without illumination, Nile Red encapsulated into the nanocarriers showed low number of emissive species in water and Opti-MEM, where poorly soluble Nile Red cannot really escape (Fig. 4B). On the other hand, in the presence of blank nanocarriers or fetal bovine serum (FBS), the number of emissive species increased drastically, showing that Nile Red leaked from nanocarriers and distributed in the recipient medium. By contrast, the number of emissive species for the NR668-loaded nanocarriers was low for all four studied media (Fig. 4C) and the size of emissive species remained stable (Table S1). These results confirmed our earlier data on high stability of NR668-loaded nanocarriers against leakage [53]. After illumination with the laser at 532 nm (power density 60 mW/cm 2 ) the number of emissive species grew drastically in the presence blank nanocarriers and FBS, but did not change in water and Opti-MEM. The number of emissive species in the media with blank nanocarriers or FBS grew gradually on increase in the time of illumination (Fig. 4D). These results suggest that the dye photo-release is light-dose dependent and it requires biological medium containing hydrophobic microenvironment. This was expected, because NR668 is highly apolar (logP = 9.22 [53]) and practically insoluble in water. Moreover, as the number and the size (Table S1) of emissive species in water did not change after illumination, we could conclude that illumination did not split the nanocarriers into multiple species. Therefore, the observed photorelease in the presence of the acceptor medium as well as in cell and zebrafish embryo is probably related to the light-triggered dye leakage from the nanocarriers into the surrounding hydrophobic microenvironment. The illumination also produced the drop in the total fluorescence intensity (Fig. 4E), indicating the photobleaching of the dye. The latter indicates that NR668 underwent photochemical transformation, which could explain the destabilization of the nanocarrier with further release of the dyes into the medium. Photobleaching of dyes is commonly associated with generation of singlet oxygen that further oxidize the dye or other components of the nanocarrier. Therefore, we repeated our FCS experiments in the presence of sodium sulfite. The latter is a reductive deoxygenating agent, which depletes oxygen through oxidation into a sulfate anion [61][62][63]. Remarkably, sodium sulfite drastically decreased the release of the dye under illumination in the presence of blank nanocarriers and serum, as it can be seen from only small change in the number of emissive species (Fig. 4C). Moreover, sodium sulfite did not show a significant influence on the fluorescence intensity of the samples before illumination (Table S1), indicating that it did not act as quencher through electron transfer or other mechanisms. Overall, these results suggest that the key mechanism that drives the dye release is linked to the photo-oxidation that could destabilize the nanocarriers. The important question is whether illumination of NR668 can trigger release of other molecules form the nanocarriers. To this end, we encapsulated into our lipid nano-droplets a second dye, F888, which was also reported previously to remain inside the droplets without dye leakage [53], similarly to NR668. Importantly, the maxima of absorption and emission of F888 in nano-droplets at 1 wt% loading (388 and 455 nm, respectively) are well separated from those of NR668 (526 and 592 nm) [53], which should allow selective excitation and detection of three two dyes by fluorescence microscopy. We incubated the dually labeled nanocarriers with cells for 15 min and then illuminated them for 30 s at 535 nm. Before illumination, the intracellular fluorescence was weak confirming no leakage of the encapsulated dyes in the presence of cells (Fig. 5 A,B). After illumination, the fluorescence recorded at the NR668 channel showed expected behavior, where the total fluorescence decreased significantly after illumination, and then increased inside the cells in the time period of 10 min (Fig. 5B). Importantly, the intracellular fluorescence recorded at the channel corresponding to F888 dye (shown in green) increased after illumination and continued increasing together with that of NR668 (Fig. 5A). Importantly, the background fluorescence in F888 channel did not change after illumination, because this illumination cannot excite F888 dye. In the control experiment, we incubated the cells with only F888-loaded nanocarriers. After 30 s illumination at 535 nm, we found no changes in fluorescence inside the cells (Fig. S2), so that NR668 was absolutely required to induce release of F888 dye. These results showed that illumination that excites selectively NR668 dye inside nano-droplets can trigger the release of the second encapsulated dye F888. Thus, a prototype of a photo-release system is developed, where illumination of nanocarriers could induce the release a molecule of interest, for instance in the drug delivery applications.

Finally, to clarify whether the whole nanocarrier is delivered or just its content, we incubated the cells with a mixture of nano-droplets containing separately NR668 and F888. In this case, if the whole nanocarriers enter after the illumination, then both NR668-and F888-loaded nanocarriers should be detected inside the cells. However, it was found that after illumination, only NR668 signal was observed inside the cells, whereas F888 signal remained low (Fig. 5C,D). This suggested that light illumination triggered release of the dye content from NR668-loaded nanocarrier into the cells, but could not really induce internalization of the whole nanocarrier. Moreover, the fact that F888 did not enter the cells after illumination also suggested that the photorelease process did not disrupt the cell plasma membranes. This feature is important in the development of photo-release systems with minimal photo-damaging effects. Together with the FCS data, these observations suggest that the light acts at the level of the nano-droplet, producing destabilization and further cargo release into the cells. In this respect the described phenomenon is close to the light-triggered release from lipid vesicles, which was explained by photo-thermal or photochemical mechanisms [38][39][40][41][42]. The described phenomenon is also relevant to photochemical internalization [34][35][36][37], however, in the latter case light acts mainly at the level of cell membranes.

Conclusions

In the present work, a phenomenon of light-induced release from lipid nano-droplets using visible light is described for the first time. It was realized using nanoemulsion droplets of 32 nm size encapsulating hydrophobic analogue of Nile Red NR668. These nanocarriers do not enter the cells after several hours of incubation at 37 C, however, after illumination for 30 seconds under the microscope at the wavelength of NR668 absorption (535 nm), fast accumulation of the released dyes inside the cells was observed. The light-triggered release was also validated in vivo on zebrafish, where nano-droplets showing stable emission in the blood circulation, stained surrounding tissues after illumination. Using fluorescence correlation spectroscopy, we showed that laser illumination at relatively low power (60 mW cm -2 ) produced dye release into 10% serum or blank lipid nanocarriers. As this process is inhibited by the deoxygenating agent sodium sulfite, we can conclude that the mechanism is linked to oxygen-dependent photochemistry, which destabilizes the nanocarrier. Nevertheless, we cannot exclude that photo-thermal effect could also take part, as proposed in some recent literature for NIR dyes. Finally, we showed that illumination of NR668 can induce release of another highly hydrophobic dye from the lipid nanocarriers, which was observed as accumulation of both dyes inside living cells. Based on these results, dye-loaded lipid nano-droplets emerge as a promising platform for light-triggered release of active molecules in biomedical applications. We expect that this concept could be readily extended to other dyes, which could induce under light stimulus the release drugs from nano-droplets.

Materials and methods

Materials

All chemicals and solvents were purchased from Sigma-Aldrich. Kolliphor ® HS15 non-ionic surfactant (mixture of polyethylene glycol 660 hydroxystearate and free polyethylene glycol 660 and) obtained from BASF (Ludwigshafen, Germany) was a kind gift from Laserson (Etampes, France), Labrafac ® WL (medium chain triglycerides) was obtained from Gattefossé (Saint-Priest, France). MilliQ-water (Millipore) was used in all experiments. Culture reagents were obtained from Sigma (St. Louis, USA), Lonza (Verviers, Belgium) and Gibco-Invitrogen (Grand Island, USA). Opti-MEM ® reduced serum medium was from Gibco. Dihexylamino-2-(2-ethyl-hexyloxy)benzo[a]phenoxazin-5-one (NR668) and 4'-Dioctylamino-3-octyloxyflavone (F888) were synthesized as described before [53].

Formulation and characterization of lipid nanoparticle

Lipid nanocarriers were prepared by spontaneous nano-emulsification. Briefly, NR668 or F888 was solubilised in Labrafac ® WL. Then, Kolliphor ® HS 15 was added and the mixture was homogenized under magnetic stirring at 90° C. Nano-droplets of 32 nm diameter were prepared by adding 230 mg of ultrapure water to a mixture of 35 mg of Labrafac ® WL and 65 mg of Kolliphor ® HS 15 under intense stirring.

The size distribution of the nanoemulsions was determined by dynamic light scattering on a Zetasizer ® Nano series DTS 1060 (Malvern Instruments S.A., Worcestershire, UK) and by FCS (home-built setup, see below). In the DLS measurements statistics by volume was used.

Cell culture

HeLa cells were cultured in Dulbecco's modified Eagle medium (D-MEM, high glucose, Gibcoinvitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS, Lonza), 1% antibiotic solution (penicillin-streptomycin, Gibco-invitrogen) in a humidified incubator with 5% CO2 at 37 °C. Cells plated on a 75 cm 2 flask at a density of 10 6 cells/flask were harvested at 80% confluence with trypsin-EDTA (Sigma) and seeded onto a chambered coverglass (IBiDi) at a density of 0.1⨯ 10 6 cells/IBiDi. After 24 h, cells in the IBiDi dishes were washed 2 times with PBS (phosphate buffer saline) (Lonza). Then, a solution of dye-loaded nano-droplets diluted at 1 : 1000 in Opti-MEM was added. Microscopy images were taken after 30-min incubation at 37 C, unless indicated.

Microscopy

Fluorescence images were taken on a Leica DMIRE2 inverted microscope equipped with a Leica DC350FX CCD camera and a thermostated chamber (Life Imaging Services, Basel Switzerland) for maintaining the temperature at 37 °C. Cy3 filter cube (excitation 535/50 nm, emission 610/75 nm) and DAPI filter cube (excitation 360/40nm, emission 470/40nm) were used for detection of NR668 and F888, respectively. A 63⨯ HCX PLAPO (1.32 NA) was used as an objective. In the photo-release studies, the illumination for 30 s was done using the same Cy3 filter cube at ~14 W/cm 2 power density of light.

Fluorescence correlation spectroscopy (FCS) and dye release

FCS measurements were performed on a two-photon platform including an Olympus IX70 inverted microscope. Two-photon excitation at 830 nm (10 mW laser output power) was provided by a mode-locked Tsunami Ti : sapphire laser pumped by a Millenia V solid state laser (Spectra Physics). The measurements were realized in an 96 well plate, using a 50 uL volume per well. The focal spot was set about 20 µm above the plate . The normalized autocorrelation function, G(t) was calculated online by an ALV-5000E correlator (ALV, Germany) from the fluorescence fluctuations, δF(t), by G(τ) = < δF(t) δF(t + τ) > / < F(t) > 2 where < F(t) > is the mean fluorescence signal, and τ is the lag time. Assuming that lipid nanoparticle diffuse freely in a Gaussian excitation volume, the correlation function, G(τ), calculated from the fluorescence fluctuations was fitted according to Thompson [START_REF] Thompson | Topics in Fluorescence Spectroscopy Techniques[END_REF]:

𝐺(𝜏) = 1 𝑁 (1 + 𝜏 𝜏 𝑑 ) -1 (1 + 1 𝑠 2 𝜏 𝜏 𝑑 ) -1 2 (1)
where τd is the diffusion (correlation) time, N is the mean number of fluorescent species within the two-photon excitation volume, and S is the ratio between the axial and lateral radii of the excitation volume. The excitation volume is about 0.34 fL and S is about 3 to 4. Typical data recording times were 5 min, using dye-loaded lipid nanoparticle diluted 1 : 10 000 from the originally prepared nanoparticle. Using 6-carboxytetramethylrhodamine (TMR from Sigma-Aldrich) in water as a reference, the hydrodynamic diameter, d, of nanocarriers (NCs) was calculated as: dNCs = τd(NCs) / τd(TMR ) ⨯ dTMR, where dTMR is a hydrodynamic diameter of TMR (1.0 nm). Concentration of NCs was calculated from the number of species by: CNCs = NNCs/NTMR⨯CTMR, using a TMR concentration of 50 nM. The data were obtained based on 20 recorded correlation curves; the recording time for each curve was 10 s for NCs and 30 s for TMR. For the dye release studies by FCS, dye-loaded nano-droplets were diluted 10 000 fold to four different media: water, Opti-mem, blank nanocarriers at 1000-fold dilution (10-fold excess with respect to dye-loaded droplets) and 10 % of FBS in water (serum). Each sample was placed into special 50 µl cuvette and the whole sample was illuminated with 532-nm laser at ~60 mW/cm 2 power density for different time (10 and 30 s, 10, 20 30 and 60 min) and we measure number of species and total count rate.

In vivo studies in zebrafish

Zebrafish were kept at 28 °C and bred under standard conditions. The transgenic line, Tg(flk1:eGFP) [START_REF] Jin | [END_REF], expressing eGFP specifically in the endothelial cells, was used in order to visualize the vasculature. For the angiography, the embryos, 3 days after fertilization, were anaesthetized in egg water containing 0.04% tricaine and 0.05% phenyl thiourea (Sigma-Aldrich) and immobilized in 0.8% low melting point agarose (Sigma). The injections were performed using a Nanoject microinjector (Drummond Scientific, Broomal, PA, USA). The glass capillary was filled with NR668-loaded nanoparticle (5 wt% in oil) of the nanoparticle solution in 5 mM HEPES and 2.3 nL were injected in the sinus venosus of the embryos. The nanoparticle was immediately distributed in all the vasculature. The injected embryos were placed on the microscope stage and imaged within 5 min after injection. Intravital confocal microscopy was performed on a Leica SP5 fixed stage direct microscope with a 25⨯ (NA 0.95) and 10⨯ (NA 0.3) water immersion objectives. 488 nm argon laser line was used to excite both eGFP and the NR668 dyes, and their emission was detected by two separate PMTs in the spectral range 500-530 nm and 620-650 nm, respectively. At these conditions, no cross-talk between the channels was observed. The illuminations for the light-driven release were performed with the in epi fluorescence mode using a 450-490 nm band pass filter. Confocal z-stacks and time lapses were recorded and treated by the ImageJ software (rsbweb.nih.gov/ij/). The embryos were imaged during one hour post injection and no toxicity due to the injection or to the illumination was observed. All experiments performed with zebrafish complied with the European directive 86/609/CEE and IGBMC guidelines validated by the regional committee of ethics.

Fig. 1 .

 1 Fig. 1. Concept: lipid nanocarrier and the effect of light on the encapsulated Nile Red derivative NR668.

Fig. 2 .

 2 Fig. 2. Release of dye molecules in vitro. Brightfield (A and C) and fluorescence (B and D) images of Hela cells in the presence of nanocarrier loaded with at 1% (A, B) or 5% (C, D) of NR668. The first images on the left correspond to samples before illumination, while all others are after illumination for 30 sec at 535 nm with 2 min delay for each consecutive image. All the experiments were done at 37C. (F): difference between intracellular and extracellular fluorescence intensity, Iint-Iext, vs time delay after illumination. The error bars represent the standard deviation of the mean from three independent illumination cycles (n = 3). (G-H) Fluorescence images after washing (from nanocarriers at 5 wt% NR668) showing non-illuminated and illuminated regions.

Fig. 3 .

 3 Fig. 3. The photo-induced release of the NR688 dye from the nanoemulsions in the vasculature of living zebrafish embryo. Fluorescence images of tail vasculature (green) containing NR668 nanoemulsions (red). (A,B) Control non-illuminated embryo, showing no leakage of the dye. (C-F) Embryos illuminated for 5 min through 450-490 nm bandpass filter, showing a red fluorescence in the tissues surrounding the vessels. Scale bar is 100 µm. (G) The increase in the fluorescence intensity averaged in the ROIs shown in panel (C) with time. The error bars represent the standard deviation of the mean from two different regions of the embryo (n = 2).

Fig. 4 .

 4 Fig. 4. Fluorescence correlation spectroscopy (FCS) studies of the light-induced dye release into model biological media. (A) Scheme explaining the effect of the dye release on the number of the emissive species. (B) Control experiment showing Nile Red dye release from nanocarriers into blank nanocarriers (NCs) and 10% serum (FBS), which was recorded as number of emissive species. (C) The effect of 30 min illumination by 532-nm laser (power density 60 mW/cm 2 ) on lipid nanocarriers loaded with NR668 in different media: water, Opti-MEM, blank nanocarriers and FBS. Presence of oxygen-depleting agent sodium sulfite was also tested. (D) Effect of laser illumination time on the number of emissive species and the total fluorescence intensity of NR668loaded lipid nanocarriers in different media. The error bars in (B-E) represent the standard deviation of the mean (n = 20 recorded correlation curves).

Fig. 5 .

 5 Fig. 5. Fluorescence imaging of HeLa cells incubated for 15 min at 37 °C with nanocarrier before and after illumination at 535 nm for 30 s. Images were taken every 2 min. (A and B) Nanocarriers encapsulating both dyes F888-NR668 (1 wt% each). (C and G) Mixture of nanocarriers encapsulating separately F888 (1 wt%) and NR668 (1 wt%) dyes (1:1, v:v). F888 channel: excitation 360/40nm and emission 470/40nm. NR668 channel: excitation 535/50 nm and emission 610/75 nm.

Acknowledgements

This work was supported by ERC Consolidator grant BrightSens 648528. R. Bouchaala was supported by Ministry of Higher Education and Scientific Research of Algeria. We thank Ievgen Shulov for re-synthesizing NR668, L. Richert, P. Didier and F. Przybilla for help with FCS, we thank R. Vauchelles from PIQ imaging platform for help with fluorescence imaging. We also acknowledge help from the IGBMC imaging and zebrafish facilities.


Sandra Bertezene
email: sandra.bertezene@lecnam.net

Caroline Bayart
email: caroline.bayart@univ-lyon1.fr

David Vallat
email: david.vallat@univ-lyon1.fr

Jacques Martin
email: martin@univ-tln.fr

Determining factors of a CSR strategy: The case of French socio-medical establishments

Purpose: The socio-medical sector, like many others, is developing CSR actions and strategies to answer new expectations of residents, families and stakeholders. This research aims at proposing a model to show determining factors of CSR strategies, investigating the relations between a number of variables appertaining to CSR and evaluate their strength and their impact on some aspects of the performance of socio-medical organizations.

Methodology: The model rests on 18 hypotheses and uses a set of structural equations to measure the relations between the different variables.

Findings: The hypotheses proposed are largely validated and the model shows the determination of a number of factors on a CSR strategy by organizations. However it appears that non-observed factors in this study also have an impact, which will be the subject of further research. Globally the study shows that CSR strategies in these socio-medical establishments are more of an 'emergent' type than a 'deliberately planned' one.

Practical implications: This study can help socio-medical establishments to devise a CSR strategy and CSR actions, as very often they do not know well where to start from, where to go and how to evaluate the results.

Originality/value: This study is part of a vast programme to evaluate the strategy of sociomedical establishments in terms of CSR and assist them in conceiving and implementing a CSR strategy.

1 This paper uses the results of a survey carried out between 2012 and 2017 with the objective of promoting a CSR strategy adapted to homes for the elderly and care-at-home services.

Introduction

The care of elderly persons is undergoing dramatic changes everywhere in the West due to a number of mutations such as the lengthening of lifegoing with a constant increase of dependency -, the introduction of innovative and costly technologies (tele-medecine, robotics, connected objects, etc), a shortage of financial resources, increasing requirements from financers and the public in terms of quality and well-being, new investors from the private sector leading to the equitization of various groups of socio-medical establishments.

Establishments operate in a world which is definitely complex putting them in danger or at least creating uncertainty. In this context CSR can be considered by deciders as an asset permitting to find some stability in a very competitive environment. CSR tends to give confidence to investors and customers as it involves the social, environmental, ethical (human rights) of customers (elderly persons and relatives) in the strategy and operations of the establishment. It also requires a close collaboration between all the stakeholders (elderly persons, families, employees, overseeing authorities, suppliers, banks, insurance companies, societies of families, etc.) (European Union, 2011).

In this context, the central issue is that of the identification of determining factors for a CSR strategy in socio-medical establishments. To work out the model of these determining factors of a CSR strategy, we will present first the theoretical framework and the model of the research, then the survey by questionnaires to collect the field data 1 , the results and finally their analysis and discussion.

A model for determining factors of a CSR strategy

We propose a model permitting to understand better the determining factors of a CSR strategy in socio-medical establishments. It is presented below using elements of the literature to build the measurement scales.

The social aspect of CSR

The social aspect of CSR can be broken down into three parts: the responsibility towards the personnel, the responsibility towards elderly persons and families, the responsibility toward the community.

The responsibility toward the personnel is not always expressed spontaneously by the management and takes on several dimensions. The works of [START_REF] Berger-Douce | Rentabilité et pratiques de RSE en milieu PME. Premiers résultats d'une étude française[END_REF] put forward the improvement of working conditions, social dialogue, equity in the policy of human resource management and the development of competencies. Other authors such as Maignan and Ferrell (2004), [START_REF] Boal | The cognitive structure of corporate social responsibility[END_REF] and David et al. (2007 also take into account health, safety and the well-being of employees (hours, professional and personal life, etc.). We have considered all these items, which looked relevant to us, while trying to avoid redundant questions.

The responsibility of the establishment toward users (elderly persons and families) is measured thanks to the scales worked out by Maignan and Ferrell (2004). The items refer to three dimensions: the information given to elderly persons on the services offered, the tools used to grasp their expectations better, the measures taken to improve the quality of services.

The "societal" part of CSR (i.e. the responsibility toward the community and society at large) is rather difficult to translate to the management, although the directors questioned during the qualitative phase of the research often spoke of the insertion of their establishments into the local social and economic environment. To approach the societal responsibility, we have used the scales established by [START_REF] Maignan | Consumers' perceptions of corporate social responsibilities: A crosscultural comparison[END_REF] and [START_REF] David | Investor activism, managerial responsiveness, and corporate social performance[END_REF]. The purpose is to know the actions carried out in relation with the territory (hirings, partnerships, events, etc.) as well as the participation in the well-being of the community and the solving of social problems. The image of the establishment, translated into the trust granted to it, the quality of the services offered and the reputation noted are another important aspect of the societal engagement [START_REF] Fombrun | The Reputation Quotient : a multistakeholder measure of corporate reputation[END_REF].

The economic aspect of CSR

It is difficult to find scales which have been tested in socio-medical establishments to measure the economic aspect of CSR, we have then adapted a measurement scale of [START_REF] Maignan | Corporate citizenship: Cultural antecedents and business benefits[END_REF]. The items measuring the economic dimension of CSR are strongly linked to the financial dimension: generation of profit, creation of value and implementation of a long-term financial strategy. The analysis of the results shows that these indicators are relevant to measure the economic aspect of CSR in the organizations concerned by our study. Consequently our scale focuses on the financial elements of the organization (profit, turnover, variable costs, level of debt, etc.).

For the financial performance of the establishment, we refer to the works of [START_REF] Samiee | The Influence of Global Marketing S tandardizationon Performance[END_REF]. Their scale permits to know if the main indicators of financial management have improved over the previous period. In order to take into account some particular features of socio-medical establishments, we have added a more specific indicator about the cost of overtime for "normal" operations for the replacement of absentees.

The environmental aspect of CSR

This environmental aspect is the one that comes to mind most easily among the managers interviewed.

Following the work by Maignan and Ferrell (2004), we have adopted a global vision of environmental responsibility to grasp the managers' strategy. We have questioned them about the objectives of ecological performance, the financing of programmes, the actions implemented in the organization and tools for the analysis of impacts and the awareness of the personnel.

The levers (positive motivation) and the brakes (negative motivation) for the implementation of a CSR strategy

For the levers or brakes perceived by the management, we have adapted the scales proposed by [START_REF] Berger-Douce | Rentabilité et pratiques de RSE en milieu PME. Premiers résultats d'une étude française[END_REF].

Implementing a CSR strategy offers several advantages (positive motivations) that we divide into three categories: answering the expectations of financers and/or deciders, improving the relationships with the stakeholders, defending a philosophy.

We questioned the management about the incentives from the overseeing authorities, the pressure of stakeholders and the alignment on the strategy of other establishments.

The brakes can also be classified into three categories: the lack of resources (time, funds, personnel, competencies, technical support of authorities), doubts about such a strategy, legitimacy of the action (not a priority, not adapted to the culture of the establishment).

The knowledge of CSR

A factor that strongly influences the implementation of a CSR strategy is the knowledge of supporting organisms or referentials, particularly in that they can reduce certain brakes linked to the lack of resources of organizations. According to the works of Berger-Douce (2008), we have used a scale with four items to measure this knowledge. We have gone a little further by questioning the management about their knowledge of regulations about CSR.

The perceived uncertainty of the environment

The perceived uncertainty of the environment is a concept frequently studied in management studies as it is linked to a number of practices. For example organizations operating in an uncertain environment need more non-financial indicators [START_REF] Gosselin | Influence de la stratégie sur 'adoption des mesures de performance en vigueur dans le système de comptabilité de gestion[END_REF]. In our questionnaire we have adapted the scale of Govindarajan (1984) which breaks down this concept into seven items: demand, competition, customers, technology, purchases, regulations, trade-union actions.

The use of technologies is not a determining factor in homes for the elderly or services at home as would be in industry, and demand is not a particular problem as would be in retail trade for example. Consequently we have ignored these items and retained a five-item scale.

The evaluation of the impact of a CSR strategy

We refer to [START_REF] Mousli | Le déplacement du contrôle de gestion vers la RSE : un « éco-contrôle » plus juste ou juste l'écho d'un contrôle ? Cas de 50 sociétés cotées françaises[END_REF] for the practices of reporting. The author evidences indicators to evaluate the structure of the budgetary system. In our study, we try to know if the budgetary system of the establishments is adapted to measure the societal performance resulting from CSR actions. First we need to know if the organizations evaluate the impact of CSR actions.

Then we qualify the nature of the indicators used (qualitative and/or quantitative and/or financial). Finally we estimate the frequency of analysis of these indicators. We have questioned the establishments on indicators of measurement of societal performance to avoid getting answers only centred on accounting aspects of CSR.

Due to the lack of existing scales for our study, we have tested original scales. We have used the ISO 26000 standard to identify and measure the CSR process. The standard recommends the formalization and publicizing of the orientations and characteristics of societal responsibility in the establishment (ISO, 2010). The impacts of the CSR strategy are measured thanks to a scale constructed from various authors. For example, A CSR strategy enables the organization to be pro-active and have a strategic behavior which is more effective and less costly (Caroll and Buchholtz, 2011). As a result a CSR strategy contributes to the survival of the organization. It also, more specifically, favours a reduction of costs, of personnel turnover, the acquisition of new knowledge and a good reputation [START_REF] Luetkenhorst | Corporate Social Responsibility and the Development Agenda -A Case for Actively Involving Small and Medium Enterprises[END_REF]. It improves the corporate image, the morale and motivation of employees, reduces the risk of rejection from users or customers, and may also fend off excessive and constraining regulations imposed by the State (Caroll and Shabana; 2010).

Knowledge management and change management

The scales to measure knowledge management and change management are constructed on the basis of the works by [START_REF] Levitt | Organizational Learning[END_REF], [START_REF] Nonaka | la traduction française s'intitule La connaissance créatrice[END_REF], [START_REF] Davenport | Working knowledge: how organizations manage what they know[END_REF], [START_REF] Argyris | Knowledge for Action: A Guide to Overcoming Barriers to Organizational Change[END_REF], [START_REF] Senge | The Fifth Discipline -The Art and Practice of The Learning Organization[END_REF]. We have focused on the ways in which different types of knowledge (explicit and tacit) circulate in the organization and are capitalized, on the link between building of knowledge and development of competencies (training) and more generally on management of competencies and individual (productivity, quality of service) and collective (absenteeism, accidents, turnover, quality of service) performance.

The construction of the model

We propose here a model permitting to understand better the determining factors of a CSR strategy in socio-medical establishments. This model takes into account the articulation between the management's motives, the knowledge management practices, the CSR actions and the impacts on the establishments. Certain more general factors, such as the perceived uncertainty or the practices of change management also need to be considered to seize the phenomenon studied better. This model reflects a number of hypotheses formulated following twelve interviews conducted with directors of homes for the elderly and at-home care services:

-hQ 1: The positive motives of the management (or levers) have a positive impact on the CSR environmental actions. -hQ 2: The positive motives of the management (or levers) have a positive impact on the CSR economic actions. -hQ 3: The positive motives of the management (or levers) have a positive impact on the CSR social actions. -hQ 4: CSR social actions develop the organization's responsibility towards employees.

-hQ 5: CSR social actions develop the organization's responsibility towards elderly persons. -hQ 6: CSR social actions develop the organization's responsibility towards the community.

Hypotheses 4, 5 and 6 are necessary to check the stakeholders who are most impacted by CSR actions (personnel and customersprimary stakeholders with a contractual relationship -, the community or society).

-hQ 7: The perceived uncertainty of the environment has a positive impact on the CSR economic actions. -hQ 8: The CSR environmental actions contribute to the formalization of a CSR strategy.

-hQ 9: The CSR economic actions contribute to the formalization of a CSR strategy.

-hQ 10: The CSR social actions contribute to the formalization of a CSR strategy.

Collection, analysis of data, operationality of variables and estimation of the modèl

The survey was carried out on the internet. We will first present the reasons for using this medium, then we will present the sample selected and the rate of return, and we will order the data and qualify the sample. We finish by explaining the operationality of the variables and the estimation of the model.

The web questionnaire

Leading a survey about CSR strategies by the establishments selected requires interrogating a large sample of directors with a rather heavy questionnaire. As directors are not easily available and establishments are geographically scattered, it is not easy to carry out face-to-face interviews and not effective to do it on the phone. Over the last years, the methods used have evolved by integrating new technologies. It was then decided to resort to a web questionnaire which permits to contact a big number of establishments. The use of this medium also reduces the cost per contact and saves time for the respondent compared to a paper questionnaire.

The questionnaire contains several dozens of questions and a number of filters to avoid redundancies. The Sphinx Online software was used to carry out the survey. It offers good ergonomics ensuring a better rate of return. It also permits to guarantee the stocking and security of the data. In order to indentify CSR practices and also motivations and obstacles for the managers in this field, the questionnaire is divided into several themes: actions put in place in the 'three pillars' of CSR (economic, social, environmental); the knowledge of the management in terms of CSR and their perception of the impact of the strategies implemented; the CSR process (stakeholders, method, reporting); the ways and means of knowledge management in the establishments and departments. The items have been tested with measurement scales validated in the literature in order get a reliable evaluation of behaviours. 5 point Likert scales have been used for the respondents to express the degree of agreement (1 = totally disagree, 5 = totally agree). The characteristics of the establishments have also been taken into account. Organizations will indeed be different depending on their age, location and qualifications of the directors. Such criteria are likely to influence the will of implementing a CSR strategy. Therefore a number of questions concern the characteristics of the organization (status, opening date, number of places, location, etc.) and the characteristics of the director (age, sex, qualifications, seniority).

A satisfactory rate of return

The questionnaire was administered by mail by the directors of the establishments at the end of February 2014. These persons were first contacted to announce the launching of the survey and explain its objectives. After processing the e-mail addresses available, 4638 addresses were valid for the survey. After three recalls every fortnight, 537 persons accessed the website to try and answer the questionnaire (a rate of 11.57 %). However all the persons who accessed the site did not answer all the questions because of lack of time or lack of will to answer more complex or 'strategic' questions. Finally 486 persons gave exploitable answers, i.e. a rather satisfactory rate of return of 10.47% considering the length and complexity of the survey.

The processing of the data and the qualification of the sample

The analysis of the results had to overcome a number of difficulties. First the data need to be ordered to be exploitable. After checking that all the questions had been satisfactorily answered, and thus making sure that there would not be a significant bias in the results of the statistical tests, the answers were codified. This task consists in giving a specific code to each possible answer. The coding was done when the questionnaire was worked out for closed-end questions but could only be done after the answers had been collected for the open-ended questions.

It is also necessary to qualify the sample on the basis of socio-demographic characteristics. As the characteristics of the respondents were not known a priori, we based ourselves on the characteristics of the organization: nature of the activity, status, location, age, number of persons taken care of, etc. Then features of the directors were taken into account such as sex, age, level of qualifications… Then the test of equality of the means or proportions was used and results where the level of significance is more than 5% were retained.

The structure of the model

The model contains five exogenous variables: the levers in favour of CSR, the brakes to the implementation of a CSR strategy, the uncertainty of the environment, the knowledge about CSR, the knowledge management devices in the organization. These variables never appear as dependent variables in the equations of the structural model, but contribute to explain other variables of the model. In other words, no arrows point to them.

The model also contains ten endogenous variables: the responsibility towards employees, the responsibility towards elderly persons and families, the responsibility towards the community, the social aspect of CSR, the environmental aspect of CSR, the economic aspect of CSR, the CSR process, the impacts of the CSR strategy, the financial performance of the organization, the practices of change management in the organization. An endogenous variable is dependent in at least one equation of the structural model, and is explained by some exogenous variables. In other words, at least one arrow points to it.

It is interesting to note that the latent variable (not directly observed but constructed from other variables called manifest variables) "social aspect of CSR" is itself made up of three latent variables: responsibility towards employees, responsibility towards elderly persons and families, and responsibility towards the community. It is then a second order variable. To define it we have used all the manifest variables (directly observable and about which the directors have been interrogated) of the latent variables of order 1 which depend on them. To be more specific, the latent variable "social aspect of CSR" is defined by the following items:

-Concerning the responsibility towards employees ("Would you say that your establishment takes into account the interests of employees", etc.) -Concerning the responsibility towards elderly persons and families ("Would you say that your establishment uses the satisfaction level of elderly persons to improve the quality of service", etc.) -Concerning the responsibility towards the community ("Would you say that your establishment has a good reputation", etc.)

The reliability of the model

According to [START_REF] Chin | The partial least squares approach to structural equation modeling[END_REF] a model, to be reliable, needs to have a number of observations (i.e. answers given by respondents) higher than: -Ten times the number of structural relations (represented by the arrows in the figure); -Ten times the number of indicators (manifest variables associated with a latent variable) of the most complex formative latent variable. This is the case in our model as we have 191 respondents, 17 structural relations and 12 indicators maximum for the most complex formative latent variable (here the "knowledge management" variable).

The measures of this model are presented as formative indicators [START_REF] Fornell | Two Structural Equation Models: LISREL and PLS Applied to Consumer Exit-Voice Theory[END_REF], which means that the measures have an impact on (or cause) a unique construct. The direction of causality goes from the indicators (or manifest variables) to the construct (or latent variables). The latter can be considered as a combination of indicators which contribute to "shape" it. That is the reason why we speak of formative variables.

According to [START_REF] Diamantopoulos | Formative versus reflective indicators in organizational measure development[END_REF], the selection of the biggest number of indicators is interesting to validate the formative measurement models, which explains the sometimes big number of manifest variables associated to a latent variable (12 for the variable "practice of knowledge management"). It is also important to check two conditions to evaluate the capacity of the items (or manifest variables) of a scale to measure the latent (or variable) construct [START_REF] Nunnaly | Psychometric theory[END_REF][START_REF] Drucker | Validité et fiabilité de la recherche[END_REF]:

-Check if all the items refer to common notions, that is if each item is coherent with all the other items of the scale. In the PLS method the homogeneity of scales is evaluated with several indicators: Cronbach Alpha and rhô of Jöreskog.

-Check that the items of the scale are sufficiently distinguished from items meant to measure other close latent variables. This is what we call a discriminating validity. In the PLS method, we analyze the cross-loadings matrix. The values of Cronbach alpha and rhô of Jöreskog are all higher than 0.7 and often 0.8. The homogeneity of scales is then sufficient.

Then, if the model has been correctly specified, the manifest variables must have a strong relation with the latent variable that they define. Consequently it is necessary to analyze the corss-loadings matrix, which corresponds to the results of an exploratory analysis of principal components, in order to check that the most important factorial weights of each manifest variable are really linked to the corresponding latent variable, which is the case in our study for all the variables considered. The supposed links between the manifest variables and the latent variables that they characterize, do exist.

We note that the indicators (or manifest variables) which define the latent variables "responsibility towards employees, responsibility towards elderly persons, responsibility towards the community" are also strongly linked to the variable "social aspect of the CSR", which is explained by the fact that second order variable is the combination of the three latent variables.

Finally, the analysis of the correlations between the latent variables of our model shows indeed that the correlations exist and that they are significant.

The Bootstrap method consists in replication the estimation of the model on a big number of samples constituted randomly from the data collected. Starting from a sample giving information on a population, a new sample is extracted randomly (in the sub-population reduced to this sample) and the operation is repeated a big number of times (250 samples in our study). We then analyze the new observations obtained to refine the estimations realized on the initial observations. After this Bootstrap procedure, it is necessary to check that the indicators chosen (or manifest variables) significantly contribute to the latent variable to which they are linked. By definition, the value of the critical ratio for each manifest variable must be more than 1.96 (for a risk of error of 5%) or 1.64 (for a risk error of 10%). We note that five manifest variables present non-significant contributions with reference to their construct (or latent variable) at the level of significance of 5%, et only three at the level of 10%. The five variables are the following: -LEV2: "You have formulated CSR objectives in your project because the law imposes it"

(critical ratio = 1.026) -UNCER1: "Do you think that the actions of competing homes for elderly or at-home services are easily predictable (critical ratio = 1.492) -UNCER2: "Do you think that the evolutions of the expectations and needs of elderly persons are easily predictable?" (critical ratio = 1.411 -BRAKE1: "You have formalized CSR objectives in your project but you lack time to put in place your CSR strategy" (critical ratio = 1.704) -BRAKE2: "You have formalized CSR objectives in your project but you lack financial means to put in place your CSR strategy" (critical ration = 1.758)

Quality of adjustment of the model

Then, it is necessary to evaluate more globally the quality of adjustment of the model to the data of the survey. The PLS method gives the Goodness of Fit (GoF) as indicator of adjustment. It is an approximation of the global variance given by the model. The closer to 1 it is, the more the model is confirmed by the data. In our study, the GoF is 0.411, and very near its Bootstrap estimation (0.417). Consequently we can conclude that: -41% of the variance of the data is explained by the model proposed. The value of an observation (answer of a manager) can be broken down into two parts, a part explained by the model and a residual part. The dispersion of all the observations is then made up of the variance explained by the model and the unexplained residual variance. -The model is stable since the values of the GoF before and after the Bootstrap (absolute Gof) are not very different. This means that if we carry out the analysis on other samples of respondents (250 randomly built), the quality of adjustment of the model varies very little.

-The manifest variables are strongly linked to the latent variables that they define (external GoF = 0.977). This confirms that the manifest variables used in the questionnaire permit to define well the latent variables at the heart of the analysis. -The links defined between the latent variables, illustrated with arrows in the model we present, are relevant (internal GoF = 0.767), which confirms the hypotheses.

Analysis and discussion of the structural model

Once the measurement model has been studied, the structural model needs to be analyzed to be able to validate the hypotheses of the study. This validation depends on the importance and the significance of the structural relations obtained. The coefficients of determination (R2) reflect the part of the variance of the endogenous variables explained by the model. With a value between 0 and 1, they measure the adequacy between the model and the data observed. The values of the structural coefficients correspond to the different influences of the explaining (or exogenous) variables on the variable to explain (or endogenous).

A sound model must present coefficients of determination higher than 0.1 [START_REF] Falk | A primer for soft modeling[END_REF]. [START_REF] Chin | The partial least squares approach to structural equation modeling[END_REF] considers that the structural coefficients must be at least equal to 0.2, and even 0.3, to be judged as significant. As shown in the following table, the structural relations of the model are globally significant. Only some explaining variables of the endogenous variable "CSR strategy" do not seem to respect these criteria (structural coefficients of the variables "knowledge of CSR" and "economic aspect of CSR" are below 0.2). As the values of the critical ratio are all higher than 1.96, we admit the significance of the whole of the structural relations of our model at the level of significance of 5%. These results show the reliability and validity of the variables used in the study.

The different structural relations of our model of analysis are represented in the following figure with the structural coefficients to give a systemic view of the interrelations. The structural coefficients translate the importance of the explaining variables on the variable to explain. The higher the coefficient, the more its weight is important in the variable to explain. Thus, for the CSR economic variable, the impact of the 'lever' variable (positive motivation of the director) is more important (0.363) than that of the 'uncertainty of the environment' variable (0.227) on the estimation of the CSR economic latent variable. In other words, even if these two variables influence the decision to implement actions of the economic aspect of CSR, the motivation of the director has more importance than the will to adapt to the uncertainty of the environment.

As mentioned earlier, the value of an observation (e.g. economic CSR) can be broken down into two parts: one part explained by the model (e.g. the values of the 'lever' and 'uncertainties' variables) and a residual part (explained by other variables that we do not know or do not study here). This is the most important limitation of the model.

The dispersion of the whole of the observations (i.e. the distribution of the values around the mean, which indicates the heterogeneity of the answers) is then made up of the variance explained by the regression and theunexplainedresidual variance. The R2 (for each endogenous variable) is defined as the part of the variance explained (by the exogenous variables) with reference to the total variance. Then, the higher the R2, the more we can estimate the 'economic CSR' variable (in our example) thanks to the 'lever (positive motivation) and 'uncertainties' variables. In this case the R2 is 22.6%, which is not very high. This can be notably explained by the fact that we work on individual answers, which are nonaggregated data, submitted to strong variations. It also means, once again, that there are other variables (not studied) which have an impact on the putting in place of actions relating to the economic aspect of the CSR.

This model calls for four essential comments: First, the model is relevant to evaluate the putting in place of a CSR strategy in the organization (R2 = 37.6%, meaning that nearly 40% of the factors impacting the CSR strategy are represented in the model), thus permitting to validate largely the 18 hypotheses derived from the literature and the interviews. The model also shows the link between the implementation of a CSR strategy and the improvement of the economic performance of an establishment. This result is encouraging to foster the implementation of a CSR strategy, even more so as a recent study carried out in the same sector of activity [START_REF] Bertezene | Changement et engagement dans une stratégie RSE : le cas des établissements sociaux et médico-sociaux français[END_REF] shows that the perspective of improving the performance in a wide sense is the first lever in favour of the involvement in a CSR strategy.

This study reveals that three levers strongly influence the management in their involvement in a CSR strategy: the possibility to reduce the wastage of resources, to improve the quality of the offer and to answer the expectations of the persons received in terms of CSR. In parallel, three factors have a multiplying effect on all the others and thus favour even more the involvement of management in a CSR strategy: liberate the necessary time to define and implement a CSR strategy [START_REF] Berger-Douce | Rentabilité et pratiques de RSE en milieu PME. Premiers résultats d'une étude française[END_REF]Carroll and Shabana, 2010), acquire the adequate technical competencies to monitor and develop such a strategy [START_REF] Davis | Can Business Afford to Ignore Social Responsibilities ?[END_REF] et having laws on CSR be imposed [START_REF] Bertezene | An overview of the main strategies and approaches to CSR[END_REF][START_REF] Bertezene | Social responsibility in the medical and social sector : the French example[END_REF]. The model presented here completes these results and validates the existence of a positive link between the levers considered by the literature (the positive motivation of the management, that is the perception by the management of what CSR can bring) and the CSR strategies implemented in the establishments. This relation is not direct, other constructs interfere in the process (brakes, actions effectively implemented, knowledge management tools, knowledge of devices favouring CSR).

The implementation of CSR is explained in a systemic way: the management is globally motivated to put in place CSR actions, above all if they perceive their environment as uncertain. Once these actions have been done, the management thinks of formalizing a CSR strategy for their organization. At that moment, different brakes appear (lack of means, competencies, time, etc.). But logically the directors who know the means of monitoring the CSR better face the difficulties better and, in the end, the formalization of the CSR strategy has positive impacts on the organization. The financial performance is improved and the management of change becomes a real asset for the organization.

Even if the implementation of the CSR is explained in a systemic way, it is important de notice that the existence of knowledge management tools and devices in the organization leads to actions relating to the social aspect of CSR. Consequently KM favours the adaptation to the environment [START_REF] Vallat | Knowledge Management, An asset for managing change ?[END_REF], the changing environment favours the implementation of CSR strategies, and CSR strategies favour the improvement of the financial performance.

Although the value of R² is rather moderate, the "financial performance" variable does have observable positive impacts on the motivation of employees, the relationships with partners (suppliers, authorities, etc.), the image of the establishment, the satisfaction of elderly persons and families. This rather moderate value of the R² means that some non-observed factors also contribute to explain the financial performance, outside the impact of the CSR strategy and the economic or financial actions implemented in the organization. According to a previous study [START_REF] Bertezene | Changement et engagement dans une stratégie RSE : le cas des établissements sociaux et médico-sociaux français[END_REF], the factors explaining the impact of CSR on the financial performance could be in interaction and be the following: obtaining a financial help for the implementation of CSR actions, the improvement of the quality of the services, the implementation of CSR actions to meet the expectations of the residents, so that CSR offers ways and means to help the monitoring of the residents.

Finally, this model shows that CSR strategies are akin the model of "emergent" strategies. According to [START_REF] Mintzberg | Of Strategies, Deliberate and Emergent[END_REF], the strategy is constructed thanks to a continuous flux of actions. A real conscious and planned strategy (deliberate strategy) is in fact a very rare model. Most frequently it is composed of actions selected depending on events with which the organization is confronted (emergent strategy). These actions can originate from the management of the organization of course but also from other actors in the organization. According to Mintzberg (1990), organizations often have an interest in choosing an emergent strategy founded on the judgment capacity and experience of the members of the organization. This type of strategy is more implicit than explicit and is generally observed in organizations operating in an unstable world. This situation corresponds well to the elements observed: Homes for the Elderly and At-home service providers operate in a complex environment and organizations embark on a CSR strategy on the basis of a philosophical outlook and intention, which is made of a mix of formalized planned actions and non or little formalized un-planned actions as the implementation unfolds.

Conclusion

The objective of this study was to verify the existence of CSR actions implemented as the result of a more emergent than planned CSR strategy. We chose the context of French medical and social establishments which are undergoing heavy mutations, to try and answer this question. The answers of the medical and social establishments to a questionnaire have permitted to test a model, in which many variables interact, taking into account the (positive and negative) motivations of the management, the knowledge management practices, the CSR actions implemented and the impacts noticed in the establishments. Synthetically, this model reveals a number of significant results: -There is a positive link between the motivation of the management and the CSR strategies implemented, but this relation is not direct; -The implementation of CSR is explained in a systemic way: the management is motivated to implement CSR actions, even more when they perceive the environment as uncertain.

Then they consider formalizing a CSR strategy but encounter a number of obstacles that are better overcome when they are better informed and trained;

-Having knowledge management tools and devices has a positive impact on the implementation of CSR actions, especially in its social aspect; -The CSR strategy has positive impacts particularly on the financial performance, the motivation of employees, the relationships with partners, the image of the establishment, the satisfaction of elderly persons and families. There are, however, other factors, which are not observed, contributing to explain the phenomenon. Therefore further research needs to be carried out to comfort this still debated positive link between CSR and performance. It would also be of interest to study other sectors of activity to see if the same, or a similar, relation can be evidenced.

  -hQ 11: The management's perceived brakes (negative motives) slow down the development of a CSR strategy. -hQ 12: The management's knowledge of the CSR tools incites them to implement a CSR strategy. -hQ 13: The tools of knowledge management have a positive impact on the CSR social actions. -hQ 14: The tools of knowledge management have a positive impact on change management practices. -hQ 15: The formalized CSR strategy has a positive impact on the functioning of the organization. -hQ 16: The CSR strategy's impact on the organization contributes to improve the financial performance of the organization. -hQ 17: The CSR economic actions contribute to improve the economic performance of the organization. -hQ 18: The formalized CSR strategy influences positively the change management practices of the organization. -The model is illustrated below. The arrows show causal relations (e.g. « uncertainty of the environment impacts/determines/acts on « economic actions of CSR »).

Figure 1 .

 1 Figure 1. The links between a CSR strategy, the management's motives, the CSR and KM practicesCSR ActionsEnvironmental aspect

Figure

  Figure 2. Estimation of the stuctural model

Table 1 .

 1 Structural equation

	Environmental aspect of
	CSR	R²	R²(Bootstrap)	Critical ratio (CR)
		0.117	0.137	2.702
		Value (Bootstrap)	Standard deviation	Critical ratio (CR)
	Structural coefficients		(Bootstrap)
	Levers	0.365	0.059	5.757
	Economic aspect of CSR	R²	R²(Bootstrap)	Critical ratio (CR)
		0.205	0.226	3.981
		Value (Bootstrap)	Standard deviation	Critical ratio (CR)
	Structural coefficients		(Bootstrap)
	Levers	0.363	0.057	6.273
	Uncertainty of env.	0.227	0.050	4.170
	Social aspect of CSR	R²	R²(Bootstrap)	Critical ratio (CR)
		0.457	0.481	7.426
		Value (Bootstrap)	Standard deviation	Critical ratio (CR)
	Structural coefficients		(Bootstrap)
	Levers	0.349	0.035	9.600
	Knowledge Manag.	0.486	0.046	10.449
	Resp. employees	R²	R²(Bootstrap)	Critical ratio (CR)
		0.689	0.695	10.435
		Valeur (Bootstrap)	Standard deviation	Critical ratio (CR)
	Structural coefficients		(Bootstrap)
	Social aspect of CSR	0.833	0.040	20.827
	Resp. elderly persons	R²	R²(Bootstrap)	Critical ratio (CR)
		0.650	0.651	10.659
		Value (Bootstrap)	Standard deviation	Critical ratio (CR)
	Structural coefficients		(Bootstrap)
	Social aspect of CSR	0.806	0.038	21.053
	Resp. community	R²	R²(Bootstrap)	Critical ratio (CR)
		0.735	0.734	12.719
		Valeur (Bootstrap)	Standard deviation	Critical ratio (CR)
	Structural coefficients		(Bootstrap)
	Social aspect of CSR	0.856	0.034	25.211
	CSR strategy	R²	R²(Bootstrap)	Critical ratio (CR)
		0.352	0.376	6.077
		Value (Bootstrap)	Standard deviation	Critical ratio (CR)
	Structural coefficients		(Bootstrap)
	Env. aspect of CSR	0.200	0.027	7.187