Fanny Mousseau
email: fanny.mousseau@univ-paris-diderot.fr

Jean-François Berret
email: jean-francois.berret@univ-paris-diderot.fr

Evdokia K Oikonomou
email: evdokia.oikonomou@univ-paris-diderot.fr

Design and applications of a fluorescent labeling technique for lipid and surfactant preformed vesicles

Keywords:

Amphiphilic molecules such as surfactants, lipids and block copolymers can be assembled into bilayers and form vesicles. Fluorescent membrane labeling methods require the use of dye molecules that can be inserted in the bilayers at different stages of the synthesis. To our knowledge there is no generalized method for labeling preformed vesicles. Herein we develop a versatile protocol that is suitable to both surfactant and lipid preformed vesicles and requires no separation or purification steps. Based on the lipophilic carbocyanine green dye PKH67, the methodology is assessed on zwitterionic phosphatidylcholine vesicles. To demonstrate its versatility, it is applied to dispersions of anionic or cationic vesicles, such as a drug administrated to premature infants with respiratory distress syndrome, or a vesicle formulation used as a fabric softener for homecare applications. By means of fluorescence microscopy we then visualize the interaction mechanisms of nanoparticles crossing live cell membranes and of surfactant adsorbing on cotton fabric. These results highlight the advantages of a membrane labeling technique that is simple and applicable to a large number of soft matter systems.

In this context, it is important to control the membrane labeling techniques. For surfactants, the monoand bilayer labeling takes advantage of fluorescently-tagged amphiphiles [START_REF] Barni | Dye surfactant interactions and their applications[END_REF][START_REF] Fallest | Fluorescent visualization of a spreading surfactant[END_REF][START_REF] Yin | A Double-Tailed Fluorescent Surfactant with a Hexavanadate Cluster as the Head Group[END_REF] or solvatochromic surfactant-dyes, [START_REF] Chen | Visualization of Surfactant Dynamics to and along Oil-Water Interfaces Using Solvatochromic Fluorescent Surfactants[END_REF] the latter having the property to change the emission wavelength depending on their environment. With surfactants however, it is often found that the dye insertion modifies the overall assembly properties, inducing a disruption of micellar or membrane structures. With lipids, the most common labeling methods are based on lipid film hydration [START_REF] Morales-Penningston | GUV preparation and imaging: Minimizing artifacts[END_REF][START_REF] Akashi | Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope[END_REF][START_REF] Alekseeva | Liposomal formulation of a methotrexate lipophilic prodrug: assessment in tumor cells and mouse T-cell leukemic lymphoma[END_REF][START_REF] Ishmukhametov | A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes[END_REF] or electroformation [START_REF] Morales-Penningston | GUV preparation and imaging: Minimizing artifacts[END_REF][START_REF] Montes | Giant unilamellar vesicles electroformed from native membranes and organic lipid mixtures under physiological conditions[END_REF] in which a small proportion of fluorescently modified lipids is added beforehand. This technique is suitable for the production of giant uni-lamellar vesicles and has shown excellent results. [START_REF] Morales-Penningston | GUV preparation and imaging: Minimizing artifacts[END_REF][START_REF] Akashi | Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope[END_REF][START_REF] Bagatolli | To see or not to see: Lateral organization of biological membranes and fluorescence microscopy[END_REF][START_REF] Alekseeva | Liposomal formulation of a methotrexate lipophilic prodrug: assessment in tumor cells and mouse T-cell leukemic lymphoma[END_REF] The previous methods are however not suitable for living cell membranes or for preformed vesicles. Here, we use the term "preformed vesicles" to describe surfactant or lipid compartments obtained using various chemical or biological processes and which structure must be preserved upon staining. Preformed vesicles are for instance present in cosmetics or homecare formulations. [START_REF] Kumar | Intact deposition of cationic vesicles on anionic cellulose fibers: Role of vesicle size, polydispersity, and substrate roughness studied via streaming potential measurements[END_REF][START_REF] Oikonomou | Fabric Softener-Cellulose Nanocrystal Interaction: A Model for Assessing Surfactant Deposition on Cotton[END_REF][START_REF] Seth | Origins of microstructural transformations in charged vesicle suspensions: The crowding hypothesis[END_REF] In biology they are found in lung fluids or they are the exosomes secreted by live cells associated to regular trafficking functions. [START_REF] Cairo | Fluorescent small-molecule probes of biochemistry at the plasma membrane[END_REF][START_REF] Morales-Kastresana | Labeling Extracellular Vesicles for Nanoscale Flow Cytometry[END_REF][START_REF] Van Der Vlist | Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry[END_REF] In the recent literature, we have not found any vesicle labeling protocol that is simple enough and suitable for a broad range of amphiphilic molecules, including surfactants and lipids. After evaluating several biology-based staining methods, we show here an easy and versatile solution to this problem.

In cellular biology, membrane labeling is commonly achieved using lipophilic carbocyanine dyes. 22- 23 These dyes intercalate spontaneously and non-covalently into the bilayers thanks to their aliphatic pending chains and rapidly stain the outer cellular membrane through lateral diffusion. [START_REF] Almendro-Vedia | Supramolecular zippers elicit interbilayer adhesion of membranes producing cell death[END_REF][START_REF] Apellaniz | Confocal microscopy of giant vesicles supports the absence of HIV-1 neutralizing 2F5 antibody reactivity to plasma membrane phospholipids[END_REF] With time, the dye molecules also spread to intracellular organelles as a result of lipid exchange. Well-known examples are the carbocyanine fluorescent dyes PKH (e.g. PKH26, PKH67) and DiO, DiI, DiD and DiR which cover the visible and infrared emission spectrum. [START_REF] Van Der Vlist | Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry[END_REF][START_REF] Horan | Stable cell membrane labelling[END_REF][START_REF] Nagyova | A comparative study of PKH67, DiI, and BrdU labeling techniques for tracing rat mesenchymal stem cells[END_REF][START_REF] Franzen | Characterization of Uptake and Internalization of Exosomes by Bladder Cancer Cells[END_REF][START_REF] Hood | Exosomes Released by Melanoma Cells Prepare Sentinel Lymph Nodes for Tumor Metastasis[END_REF] Direct labeling using lipophilic dyes can however lead to significant issues. To speed up the insertion of fluorescent molecules into the membrane, large concentrations of lipophilic molecules and the use of specific solvents are prescribed, leading to further complex separation protocols. [START_REF] Morales-Kastresana | Labeling Extracellular Vesicles for Nanoscale Flow Cytometry[END_REF][START_REF] Van Der Vlist | Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry[END_REF][START_REF] Franzen | Characterization of Uptake and Internalization of Exosomes by Bladder Cancer Cells[END_REF] For preformed vesicles, the difficulty is augmented because the vesicles and the non-solubilized dye aggregates have comparable sizes and densities. In such cases, more sophisticated separation techniques based on sucrose gradient or on chromatography are to be implemented. [START_REF] Van Der Vlist | Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry[END_REF] Herein we develop a versatile labeling membrane protocol that is suitable to both surfactant and lipid preformed vesicles and requires no separation or purification steps. The methodology is based on direct staining using the PKH67 liphophilic green dye (Scheme 1). The protocol design and optimization are first assessed using dipalmitoylphosphatidylcholine (DPPC) preformed vesicles. The DPPC vesicle features such as the size and the charge are thoroughly investigated and found in agreement before and after the labeling treatment. The protocol is then extended to two types of vesicle dispersions: i) an exogenous pulmonary surfactant drug administrated to premature infants with respiratory distress syndrome and ii) a vesicle formulation used as a conditioner in homecare applications. These vesicles differ by their surface charges, the former being anionic and the latter cationic. In the end, we show the benefits of such a staining protocol in two applications, namely the role of pulmonary surfactant in the particle internalization in live cells and the surfactant deposition on cotton fabric.

-Results and Discussion

-PKH67 behavior in polar solvents

Dynamic light scattering was used to study the PKH67 dye solubility in solvents such as ethanol, DIwater, PBS phosphate buffer and Diluent C ® . In ethanol, no aggregates nor micelles are observed with light scattering indicating a good solubility in this medium. Diluent C ® is the commercially available solvent for carbocyanine PKH dyes. It is used primarily to maintain the cell viability whilst maximizing the dye solubility in the culture medium. As Diluent C ® components may change the vesicle properties (it contains surfactants, proteins and complexing agents), its use requires post-labeling treatment such as purification and separation steps. Alternative solvents to Diluent C ® are water and phosphate buffer saline (PBS), this later being the reference buffer in many biological studies. Figs. 1a-c displays the intensity distributions of 5 µM PKH67 dispersions in DI-water, PBS and Diluent C ® respectively. The size distributions are obtained from the deconvolution of the scattered light autocorrelation function and reveal the existence of nanometer (for DI-water and Diluent C ® ) to micrometer (for PBS) sized aggregates. Obtained by fitting, the continuous lines show maxima at 400 nm, 1.40 µm and 430 nm. The results suggest that the dyes are partially soluble in either of the solvents, a result that is explained by the hydrophobicity of the fluorescent molecule (Scheme 1). From the aggregate sizes, it is inferred that the PKH67 solubility in DI-water is comparable to that in Diluent C ® and is better than in PBS. Since the membrane labeling efficiency is linked to this solubility, we conclude that DI-water can be an alternative solvent to Diluent C ® for staining vesicles.

-Incorporating PKH67 dyes into lipid vesicles

Systematic studies were performed by mixing DPPC vesicles at a concentration of 1 g L -1 in DI-water together with dye dispersions of increasing concentrations. We followed the scheme displayed in Fig. 2 for the sample preparation and increased the PKH67 molar concentration from 0.2 to 5 µM. These concentrations correspond to a dye-to-lipid ratio varying between 1:300 and 1:7000. It was found that at a ratio of 1:1400, the dyes did not alter the vesicular structure and the vesicles were fluorescent. The vesicle properties prior and after labeling were further examined using fluorimetry, light scattering and microscopy. 3a shows the fluorescence excitation (𝜆 "# = 490 𝑛𝑚) and emission (𝜆 "+ = 502 𝑛𝑚) spectra for a 50 nM PKH67 aqueous suspension and for a PKH67/DPPC dispersion prepared in DI-water as described above. In particular the dye-to-lipid ratio was set at 1:1400. The fluorescence signal observed for DPPC/PKH67 is 40 times higher than that of PKH67 alone at the same concentration. The low fluorescence level found for pure PKH67 dispersion is explained by the phenomenon of aggregationcaused quenching. [START_REF] Ma | Fluorescence Aggregation-Caused Quenching versus Aggregation-Induced Emission: A Visual Teaching Technology for Undergraduate Chemistry Students[END_REF][START_REF] Zhegalova | Minimization of selfquenching fluorescence on dyes conjugated to biomolecules with multiple labeling sites via asymmetrically charged NIR fluorophores: Bright NIR label[END_REF] Aggregation-caused quenching takes place when poorly soluble molecules associate into micelles and aggregates, resulting in concentration induced emission quenching. In contrast, the strong signal obtained in the presence of lipids suggests that the dyes have been incorporated in the membranes and are spatially dispersed. There, the distance between dye molecules is estimated at 30 nm, preventing the self-quenching to take place. In both cases the emission occurs at the same wavelength, showing that the presence of DPPC molecules does not alter the PKH67 photophysical properties.

Labeled and unlabeled vesicles were further studied using dynamic light scattering, zeta potential measurements and optical microscopy. Fig. 3b displays the size distribution obtained for a 1 g L -1 dispersion with and without PKH67. In the two experiments the distributions peak is around 0.8 -1 µm. In dilute solutions, the vesicles observed in 60× phase-contrast microscopy are identified as separated and immobile objects adsorbed at the glass surface (Figs. 3c). With fluorescence, the vesicles appear as bright spots co-localized with those observed in phase-contrast (Fig. 3d). An analysis of the merge image (Fig. 3e) leads to the result that more than 80% of the treated vesicles have been labeled and that their size is not modified by the staining. These findings confirm that the above protocol is efficient and does not change the overall vesicle geometry.

-Labeling biological lipid and industrial surfactant vesicles

The previous method was applied to vesicles of different origin and surface charge. Vesicles made from biological lipids coming from the pulmonary surfactant drug Curosurf ® or those made from the commercial double-chain surfactant TEQ were assessed. We recall that Curosurf ® is a lung fluid substitute used for the treatment of respiratory distress syndrome. [START_REF] Mousseau | Supported pulmonary surfactant bilayers on silica nanoparticles: formulation, stability and impact on lung epithelial cells[END_REF][START_REF] Waisman | Nanostructure of the aqueous form of lung surfactant of different species visualized by cryo-transmission electron microscopy[END_REF][START_REF] Curstedt | A Unique Story in Neonatal Research: The Development of a Porcine Surfactant[END_REF] Its composition comprises a wide variety of lipids (Scheme 2) which impart an average negative charge to the membrane (Table I). The TEQ surfactant in turn is a quaternary ammonium esterquat entering topical softener formulations. In Supplementary Information S1 and S2 cryo-TEM images confirm that both Curosurf ® biological lipids and TEQ esterquat surfactants associate locally into bilayers with a thickness of 4 -5 nm and that on a larger scale the bilayers form unilamellar and multivesicular vesicles. [START_REF] Mousseau | Supported pulmonary surfactant bilayers on silica nanoparticles: formulation, stability and impact on lung epithelial cells[END_REF] Figs. 4a and4b display the size distributions for PKH67 labeled and unlabeled Curosurf ® and TEQ dispersions respectively obtained from light scattering at 1 g L -1 . In this assay, the Curosurf ® vesicle size is controlled via extrusion through a 100 nm pore polycarbonate membrane. [START_REF] Mousseau | The role of surface charge in the interaction of nanoparticles with model pulmonary surfactants[END_REF] The data indicate that there is a good superimposition of the distributions for the two dispersions. Electrophoretic mobility experiments show that the zeta potential remains unchanged with and without labeling, the values being around -20 mV and +60 mV for Curosurf ® and TEQ respectively (Table I). In Figs. 4c-e and f-h, phase-contrast and fluorescent microscopy images of native non-extruded Curosurf ® and TEQ are presented. The figures reveal well-contrasted spherical objects of average size 1 µm. As for DPPC, the vesicles appear as bright spots co-localized with those observed in phase-contrast mode. 85 % of them are fluorescent and their size distribution remain unchanged. These findings confirm that the staining protocol developed for zwitterionic DPPC vesicles can be extended to anionic lipids from biological origin and to cationic surfactants coming from an industrial source. In the next sections, we take advantage of this labeling technique to investigate the interaction mechanisms of nanoparticles crossing live cell membranes and of surfactant adsorbing on cotton fabrics.

Vesicles

-Role of the pulmonary surfactant in the cellular uptake of nanoparticles

When inhaled, nanoparticles are able to reach the respiratory zone and enter in contact with the alveolar region. [START_REF] Bajaj | Advances and Challenges in Recapitulating Human Pulmonary Systems: At the Cusp of Biology and Materials[END_REF][START_REF] Puisney | Brake wear (nano)particle characterization and toxicity on airway epithelial cells in vitro[END_REF][START_REF] Geiser | Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells[END_REF] Various scenarios of nanoparticles passing from the alveolar spaces towards the blood circulation have been examined and in some instances translocation has been demonstrated. [START_REF] Geiser | Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells[END_REF][START_REF] Puisney | Mechanisms of Uptake and Translocation of Nanomaterials in the Lung[END_REF] Our research group has recently explored the behavior of 50 nm particles and of surfactant substitutes in controlled physico-chemical conditions. Three types of surfactant mimetics, including the exogenous substitute Curosurf ® were assessed together with aluminum oxide, silicon dioxide and latex nanoparticles. [START_REF] Mousseau | The role of surface charge in the interaction of nanoparticles with model pulmonary surfactants[END_REF] The result that emerged from this survey (particles were selected to display different shapes and surface charge densities [START_REF] Mousseau | Polyelectrolyte assisted charge titration spectrometry: Applications to latex and oxide nanoparticles[END_REF] ) was the observation of the spontaneous nanoparticle-vesicle aggregation induced by Coulomb attraction. The aggregation was strongly enhanced for oppositely charged species. Labeling pulmonary surfactant vesicles, as shown previously represents hence an opportunity to further study the aggregate structure. Fig. 5a displays a high magnification view of a silica/ Curosurf ® aggregate observed under phase-contrast (Fig. 5a1), green (Fig. 5a2) and red (Fig. 5a3) emission. In this assay, the particles are aminated and fluoresce in the orange-red at 590 nm due to rhodamine molecules present in the silica structure. The experimental conditions are a total concentration of 1 g L -1 , a volumetric lipid-to-nanoparticle ratio of 2 and a temperature of 37 °C. [START_REF] Mousseau | The role of surface charge in the interaction of nanoparticles with model pulmonary surfactants[END_REF] The merge image of Figs. 5a2 and 5a3 exhibits an excellent superimposition of the green and red channels over the entire object, indicating that the aggregates contain both fluorescent species (Fig. 5a4). These results, together with those of Supplementary Information S3 ascertain that the aggregates are made of vesicles and particles and that both are intertwined at the micron scale (Fig. 5b). In the alveolar spaces, inhaled nanoparticles enter first in contact with the lipid monolayer at the airliquid interface, and then with the hypophase. [START_REF] Lopez-Rodriguez | Structure-function relationships in pulmonary surfactant membranes: From biophysics to therapy[END_REF][START_REF] Muhlfeld | Interactions of nanoparticles with pulmonary structures and cellular responses[END_REF] This interaction leads to an aggregate formation comparable to the one discussed previously and is susceptible to modify the fate of the nanoparticles towards the alveolar cells located beneath the hypophase. In particular, the lipids are expected to mitigate the protein adsorption. [START_REF] Falahati | A health concern regarding the protein corona, aggregation and disaggregation[END_REF] Here, the double labeling permits to visualize the vesicle and particle localization following the cellular uptake. A549 lung epithelial carcinoma cells were incubated for 4 h with the nanoparticle-vesicle aggregates shown in Fig. 5a. Due to their size and density, the aggregates sedimented at the bottom of the Petri dish [START_REF] Puisney | Brake wear (nano)particle characterization and toxicity on airway epithelial cells in vitro[END_REF][START_REF] Deloid | Preparation, characterization, and in vitro dosimetry of dispersed, engineered nanomaterials[END_REF] and enter in contact with the cell layer. Fig. 5c exhibits confocal images of a cluster of five cells in bright field (Fig. 5c1) and in green and red fluorescence (Figs. 5c2 and 5c3 respectively). The nuclei are stained in blue with DAPI. Fig. 5d exhibits the merge image of the three fluorescent signals, together with close-up views of the plasma membrane and of the cytoplasm. These images demonstrate that in the presence of lipid membranes, the particles are internalized in the cells in a significant amount (see Supplementary Information S4 for additional data). In this respect, the vesicular membrane seems not to have a protective role towards cells, as it is the case with supported lipid bilayers. [START_REF] Mousseau | Supported pulmonary surfactant bilayers on silica nanoparticles: formulation, stability and impact on lung epithelial cells[END_REF] Yellow patches associated with nanoparticlevesicle co-localization are essentially visible at the outer cellular range, but not internally or in a lesser amount. This suggests that after passing through the outer lipid barrier a separation of the two components occurs and that the preformed aggregates break. Fig. 5e illustrates schematically this process.

To our knowledge, it is the first time that assays using membranes of biological origin reveal a separation mechanism for the organic and inorganic species in cells.

-Double-chain surfactant deposition on cotton fibers

Recently, we show that PKH67 labeled vesicles made from TEQ double-tailed surfactant could be also used to decipher the interaction mechanism with cellulose nanocrystals. [START_REF] Oikonomou | Fabric Softener-Cellulose Nanocrystal Interaction: A Model for Assessing Surfactant Deposition on Cotton[END_REF] Obtained from natural cellulose, [START_REF] Elazzouzi-Hafraoui | The Shape and Size Distribution of Crystalline Nanoparticles Prepared by Acid Hydrolysis of Native Cellulose[END_REF] cellulose nanocrystals are rod-shaped particles in the form of 200 nm laths. Being negatively charged, these anisotropic particles interact strongly with the TEQ cationic vesicles and form mixed aggregates comparable to those obtained with the silica nanoparticles and discussed in the previous section. Examples are given in Supplementary Information S5.

To evaluate the performances of a home care product such as a fabric softener, it is more convenient to use actual fabric materials for deposition assessment. Pertaining to the deposition on cotton, important issues are the amount of adsorbed surfactants and the morphology of the deposited layer, questions to which labeled vesicles could provide an answer. [START_REF] Oikonomou | Design of eco-friendly fabric softeners: Structure, rheology and interaction with cellulose nanocrystals[END_REF][START_REF] Gerhardt | Tribological investigation of a functional medical textile with lubricating drug-delivery finishing[END_REF][START_REF] Igarashi | Elucidation of Softening Mechanism in Rinse Cycle Fabric Softeners. Part 1: Effect of Hydrogen Bonding[END_REF][START_REF] Mishra | Esterquats: the novel class of cationic fabric softeners[END_REF] In a typical experiment, a 1×1 cm 2 piece of woven cotton fabric is treated with a dispersion containing TEQ fluorescent vesicles at 1 g L - 1 during 10 minutes and then rinse with DI-water. The PKH67 concentration was set at 6 µM for these assays, so slightly higher than for DPPC and Curosurf ® vesicles. Fig. 6a displays a phase-contrast microcopy view of a treated fabric submerged in water. There, the 200 µm sized yarns are recognizable and separated by large voids. Additional SEM data confirm the woven structure (Supplementary Information S6). In the emission mode (Fig. 6b), the fibers exhibit an intense fluorescence signal, illustrating that TEQ surfactants have been adsorbed on the cellulose substrate in a significant manner. The bright spots (arrows) are assigned to large TEQ vesicles coating the cellulose substrate. The vesicle adsorption is explained by Coulomb attraction between the cationic vesicles and the anionic fibers. Control experiments performed with unlabeled surfactant show that in the present conditions the fibers are not fluorescent and appear dark (Supplementary Information S7). An analysis of the distribution of the fluorescence signal along the fiber cross section shows an enhanced signal on the edges, the intensity showing an M-shape signal (Fig. 6c, 6d and inset in Fig. 6c). Such M-shape signals are characteristic from fluorescence emission arising from the fluorescent surfaces or interfaces. [START_REF] Akashi | Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope[END_REF][START_REF] Campillo | Volume transition in composite poly(NIPAM)-giant unilamellar vesicles[END_REF] Concerning the nature of the absorbed layer, it could come either from supported lipid bilayers or from a supported vesicle layer. In the first scenario, the fibers would be coated with a single or with a stack of surfactant bilayers, whereas in the second the vesicles remain intact and stick to the cellulose fibers. Figs. 6b and 6d actually suggest that both scenarios are here plausible. These two types of surfactant deposition are schematically presented in Fig. 6e. In conclusion, the present technique allows a direct visualization of surfactant adsorbed on cotton and should permit to assess the formulation deposition performance.

-Conclusion

In this work we report a fluorescent labeling technique based on the spontaneous insertion of lipophilic molecules into membranes made either from surfactants or lipids. The protocol is derived from methods developed for live cell imaging. Thanks to an accurate dosage of the dye molecules in solution, here the PKH67 lipophilic green fluorophore, issues related the removal of the excess dyes or to the use of a specific solvent are avoided. The PKH67 concentration is namely adjusted to that of the lipids in a way that the fluorescence signal remains high and that at the micron scale the vesicular structures are not altered by the dye insertion. Optimum ratios are in the range 1 dye molecule per 1400 lipids. The vesicular resilience upon dye addition is evaluated on DPPC vesicles using light scattering, zeta potential and fluorescent microscopy. With the above dye-to-lipid ratio, we insure moreover that phenomena such as aggregation-caused quenching do not occur and mitigate the fluorescence intensity. As anticipated, we also found that separation and centrifugation techniques usually necessary to remove the dye excess are here not required. To demonstrate its versatility, the protocol is applied to two other vesicular systems, a lipid drug administrated to premature infants with respiratory distress syndrome (Curosurf ® ) and a surfactant formulation used as fabric conditioner. In Curosurf ® the vesicles are negatively charged, whereas in the surfactant formulation they are positively charged. Interestingly, the amounts of dye added to optimize the fluorescence signals depend little on the system or on the electrostatic charge, around 1 µM for a lipid or surfactant solution at 1 g L -1 . To complete this study, we illustrate the benefits of this labeling method in evaluating the internalization of silica nanoparticles in cells after their interactions with Curosurf ® vesicles. With the fabric softener formulation, we also investigate the deposition of surfactant on cotton fibers. In both examples, we are able to visualize using fluorescence microscopy the fate of the vesicles after their interactions with live cells or with cotton. These results allow us to establish the interaction patterns specific to each system and to assign the relevant nanoscale interaction mechanisms thanks to fluorescence microscopy.

-Materials and Methods

-PKH67 fluorescent dye

Developed for live cells, PKH linkers are lipophilic molecules that incorporate spontaneously into the lipid bilayer. Their photophysical properties cover the visible and infrared emission spectrum and the molecules are described as non-toxic effects towards cells. [START_REF] Morales-Kastresana | Labeling Extracellular Vesicles for Nanoscale Flow Cytometry[END_REF][START_REF] Horan | Stable cell membrane labelling[END_REF][START_REF] Nagyova | A comparative study of PKH67, DiI, and BrdU labeling techniques for tracing rat mesenchymal stem cells[END_REF] The 1 mM PKH67 solution in ethanol was purchased from Sigma-Aldrich and used as received. The molecule is made of two fluorescent head groups and two aliphatic chains. Its chemical structure as provided by the supplier is shown in Scheme 1. PKH67 fluoresces in the green, its excitation and emission wavelengths being at 410 nm and 502 nm respectively. More information can be found in the website in Supplementary Information S5 and S7.

-Phospholipids and surfactants

Dipalmitoylphosphatidylcholine (DPPC, Scheme 2a) was purchased from Sigma-Aldrich. It is a phosphatidylcholine lipid with a gel-to-fluid phase transition temperature of 41 °C. [START_REF] Liu | Interfacing Zwitterionic Liposomes with Inorganic Nanomaterials: Surface Forces, Membrane Integrity, and Applications[END_REF] For vesicle preparation, DPPC was initially dissolved in methanol at 10 g L -1 . After a 30 min solvent evaporation at low pressure and 60 °C, a lipid film was formed on the glass surface. It was hydrated with the addition of deionized (DI) water at 60 °C and stirred at atmospheric pressure for another 30 minutes. DI-water was added to obtain a 1 g L -1 dispersion. In these conditions, DPPC was found to form uni-and multilamellar vesicles.

Curosurf ® , also called Poractant Alfa (Chiesi Pharmaceuticals, Parma, Italy) is an extract of whole mince of porcine lung tissue purified by column chromatography. [START_REF] Curstedt | A Unique Story in Neonatal Research: The Development of a Porcine Surfactant[END_REF][START_REF] Notter | Lung surfactant: Basic science and clinical applications[END_REF] It is indicated for the rescue treatment of respiratory distress syndrome in premature infants and is administered intratracheally at a dose of 200 mg per kilogram. Curosurf ® was kindly provided by Dr. Mostafa Mokhtari and his team from the neonatal service at Hospital Kremlin-Bicêtre, Val-de-Marne, France. Curosurf ® is produced as a 80 g L -1 phospholipid and protein suspension where the phospholipids are assembled in the form of multi-lamellar anionic vesicles. It contains, among others, phosphatidylcholine lipids, sphingomyelin, phosphatidylglycerol and the membrane proteins SP-B and SP-C. [START_REF] Waisman | Nanostructure of the aqueous form of lung surfactant of different species visualized by cryo-transmission electron microscopy[END_REF][START_REF] Mousseau | The role of surface charge in the interaction of nanoparticles with model pulmonary surfactants[END_REF][START_REF] Lopez-Rodriguez | Structure-function relationships in pulmonary surfactant membranes: From biophysics to therapy[END_REF][START_REF] Alonso | Keeping lung surfactant where it belongs: Protein regulation of two-dimensional viscosity[END_REF] Some of the lipids present in Curosurf ® are displayed in Scheme 2b (Supplementary Information Table S1). According to the manufacturer, the pH of Curosurf ® is adjusted with sodium bicarbonate at pH 6.2. [START_REF] Mousseau | Biophysicochemical Interaction of a Clinical Pulmonary Surfactant with Nanoalumina[END_REF] In the conditions used in this work, Curosurf ® is organized in anionic vesicles with a large size distribution (50 nm -2 µm), as shown by cryogenic transmission electron microscopy (cryo-TEM [START_REF] Dubochet | On the Development of Electron Cryo-Microscopy (Nobel Lecture)[END_REF] ). The gel-tofluid transition of Curosurf ® membranes was measured and found at 𝑇 6 = 29.5 °C (Supplementary Information S8).

The esterquat cationic surfactant ethanaminium, 2-hydroxyN,N-bis(2-hydroxyethyl)-N-methyl-esters with saturated and unsaturated C16-18 aliphatic chains (Scheme 2c), abbreviated TEQ in the following was provided by Solvay®. Its gel-to-fluid phase transition temperature is observed at 60 °C and the counterions associated with the quaternized amines are methyl sulfate anions. [START_REF] Oikonomou | Fabric Softener-Cellulose Nanocrystal Interaction: A Model for Assessing Surfactant Deposition on Cotton[END_REF] TEQ is a cationic surfactant widely used in the industry as the main component of hair and fabric conditioners. It forms unilamellar and multi-vesicular vesicles in water, as shown by cryo-TEM in Supplementary Information S2. 17 S1.

-Silica nanoparticles

The positively charged silica particles were synthetized using the Stöber synthesis. [START_REF] Mousseau | Supported pulmonary surfactant bilayers on silica nanoparticles: formulation, stability and impact on lung epithelial cells[END_REF][START_REF] Reinhardt | Quaternary Ammonium Groups Exposed at the Surface of Silica Nanoparticles Suitable for DNA Complexation in the Presence of Cationic Lipids[END_REF] Briefly, fluorescent silica seeds were prepared in three steps. Rhodamine derivative rhodamine red c2 maleimide (Aldrich) was first covalently bound to silica precursor (3-mercaptopropyl)-trimethoxysilane (MPS, Aldrich). The rhodamine-MPS compound was then mixed with a tetraethyl orthosilicate silica precursor (TEOS, Aldrich) and the Stöber synthesis was performed. With this approach the dyes were covalently bound to the silica matrix. In a third step, a non-fluorescent silica shell was grown with TEOS to increase the particle size and prevent leakage of the dyes out of the particles. Functionalization by amine groups was then performed, resulting in a positively charged coating. [START_REF] Reinhardt | Quaternary Ammonium Groups Exposed at the Surface of Silica Nanoparticles Suitable for DNA Complexation in the Presence of Cationic Lipids[END_REF] Aminated silica was synthesized at 40 g L -1 and diluted with DI-water at pH 5. The hydrodynamic and geometric diameters were determined as 𝐷 3 = 60 nm and 𝐷 7 = 41.2 nm. The fluorescence properties were characterized using a Cary Eclipse fluorimeter (Agilent), with leading excitation and emission peaks at 572 nm and 590 nm, respectively. A particle identity card displaying UV-Vis spectrometry, fluorimetry, TEM and light scattering data is provided in Supplementary Information S9.

-Cellulose fibers

1×1 cm 2 woven cotton fabrics were used to investigate the softener surfactant deposition onto fibers.

The fabrics are made of cotton yarns of diameters 300 ± 100 µm, each of them being constituted of approximately 20 cotton fibers of diameter 10 to 20 µm. A representative SEM image of the woven cotton fabric used in this work is provided in Supplementary information S6. All fabrics were first treated with DI-water for 10 min and dried at 35 °C under air circulation before use.

-Vesicle labeling

The 1 mM commercial PKH67 stock solution in ethanol was first diluted 10 times using pure ethanol. 2 µl of the diluted stock was then mixed rapidly with 100 µl of DI-water and vortexed for 10 s. It was then added to a 100 µl of a DPPC or Curosurf ® vesicle aqueous dispersion at the concentration of 2 g L -1 . The final dye concentration was then 1 µM. Rapid vortexing for ca. 10 s was followed to ensure the dye insertion in the lipid membranes. The dispersion was let to rest in the dark for 10 minutes. For labelling TEQ vesicles, the dye concentration used was 6 times larger than the previous one. In particular, 2 µl of the PKH67 stock solution were rapidly mixed with 100 µl of DI-water and vortexed for ca. 10 s. This solution was then added to 100 µl of a TEQ vesicle dispersion at the concentration of 2 g L -1 and the mixture was vortexed.

-Sample preparation

To evaluate the PKH67 solubility, solutions were prepared at 5 µM in different solvents, such as ethanol, PBS, Diluent C ® and water. Diluent C ® is a commercial product developed for cell labeling. It contains glycerol (50%), Triton X100 (0.15 wt. %), bovine serum albumin (200 mg L -1 ), dithiothreitol (1 mM), EDTA (0.1 mM), NaCl (250 mM) and Tris-HCL (10 mM). Pertaining to the vesicles, Curosurf ® , DPPC and TEQ dispersions were prepared at room temperature using DI-water and a concentration of 2 g L -1 . Before use, the dispersions were characterized by dynamic light scattering and ζ-potential measurements. In some cases, the dispersions were extruded through a polycarbonate membrane following a protocol described in Ref. [START_REF] Mousseau | The role of surface charge in the interaction of nanoparticles with model pulmonary surfactants[END_REF] to reduce the vesicle size dispersity. To study the interaction of silica nanoparticles with lipids, we use the method of continuous variation developed by Paul Job to determine the stoichiometry of binding (macro)molecular species in solutions. [START_REF] Job | Studies on the formation of complex minerals in solution and on their stability[END_REF] The method is here combined with static and dynamic light scattering, leading to Job scattering plots. [START_REF] Oikonomou | Fabric Softener-Cellulose Nanocrystal Interaction: A Model for Assessing Surfactant Deposition on Cotton[END_REF][START_REF] Mousseau | The role of surface charge in the interaction of nanoparticles with model pulmonary surfactants[END_REF][START_REF] Mousseau | Biophysicochemical Interaction of a Clinical Pulmonary Surfactant with Nanoalumina[END_REF][START_REF] Fresnais | Nanoparticle Aggregation Controlled by Desalting Kinetics[END_REF] In brief, stock solutions (1 mL) were prepared in the same conditions of pH and concentration (1 g L -1 ) and mixed at the volumetric ratios 𝑋 = 𝑉 :; 𝑉 <; = 2 ⁄ where 𝑉 :; and 𝑉 <; are the volumes of the surfactant and particle solutions. The particle and lipid concentrations (0.66 and 0.33 g L -1 , respectively) correspond to identical surface area concentrations in membrane and in silica. [START_REF] Mousseau | The role of surface charge in the interaction of nanoparticles with model pulmonary surfactants[END_REF]

-Deposition of labeled TEQ vesicles on cotton fabrics

10 µl of PKH67 were dispersed in 1500 µl of DI-water and mixed rapidly with vortex. This mixture was then added in 1500 µl of TEQ prepared at 1 g L -1 and mixed rapidly with the vortex. 0.03 g of cotton fabrics cut in small pieces were immersed for 10 minutes in this dispersion.

-Cell culture

Adenocarcinomic human alveolar epithelial cells A549 (ATCC reference CCL-185 TM ) were grown in T75-flasks in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Exponentially growing cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. When the cell confluence reached 80%, cell cultures were passaged using trypsin-EDTA. All products used in cell culture came from Gibco, Life Technologies.

-Light scattering

The scattering intensity 𝐼 ? and hydrodynamic diameter 𝐷 3 were measured using the Zetasizer NanoZS spectrometer (Malvern Instruments, Worcestershore, UK). A 4 mW He-Ne laser beam (𝜆 = 633 nm) is used to illuminate the sample dispersion, and the scattered intensity is collected at a scattering angle of 173°. The second-order autocorrelation function 𝑔 (B) (𝑡) is analyzed using the cumulant and CONTIN algorithms to determine the average diffusion coefficient 𝐷 E of the scatterers. The hydrodynamic diameter is then calculated according to the Stokes-Einstein relation, 𝐷 3 = 𝑘 G 𝑇/3𝜋𝜂𝐷 E , where 𝑘 G is the Boltzmann constant, 𝑇 the temperature and 𝜂 the solvent viscosity. Measurements were performed in triplicate at 25 °C after an equilibration time of 120 s.

-Zeta potential

Laser Doppler velocimetry (Zetasizer, Malvern Instruments, Worcestershore, UK) using the phase analysis light scattering mode and a detection at an angle of 16 degrees was performed to determine the electrophoretic mobility and zeta potential of the different studied dispersions. Measurements were performed in triplicate at 25 °C, after 120 s of thermal equilibration.

-Phase-contrast and fluorescence optical microscopy

Images were acquired on an IX73 inverted microscope (Olympus) equipped with a 60× objective. An Exi Blue camera (QImaging) and Metaview software (Universal Imaging Inc.) were used as the acquisition system. The illumination system "Illuminateur XCite Microscope" produced a white light, filtered for observing a green signal in fluorescence (excitation filter at 470 nm -bandwidth 40 nm and emission filter at 525 nm -bandwidth 50 nm). Thirty microliters of the vesicles dispersion (DPPC, Curosurf ® or TEQ) or nanoparticles / vesicles dispersions (Silica particles / Curosurf ® or nanocellulose / TEQ) were deposited on a glass plate and sealed into a Gene Frame dual adhesive system (Abgene/ Advanced Biotech). In case of DPPC and Curosurf ® vesicles, the glass slides were coated using a cationic polymer (PDADMAC) to improve their adhesion properties based on electrostatic interaction. Images were digitized and treated by the ImageJ software and plugins (http://rsbweb.nih.gov/ij/).

-Confocal microscopy

Images were acquired on an LSM 710 microscope (Zeiss) equipped with a 40× immersion objective and a temperature and carbon dioxide content controller. The A549 cells (ATTC) were first seeded on a glass slide in a 6 wells plate at 200 000 cells per well. After 48 hours of growth in complete white DMEM, the cells were rinsed with PBS and incubated 4 h at 37 °C with a silica-Curosurf ® dispersion.

The concentrations are 0.1 g L -1 for the particles and 0.015 g L -1 for Curosurf ® in white DMEM. Cells were then fixed with PFA and nuclei were stained in blue with DAPI. Finally, slides were sealed into a Gene Frame dual adhesive system (Abgene/Advanced Biotech). Images were digitized and treated by the ImageJ software and plugins (http://rsbweb.nih.gov/ij/).

-Absorbance and fluorescence measurements

A UV-visible spectrometer (SmartSpecPlus from BioRad) was used to measure the absorbance of PKH67 aqueous dispersions at 0.05 and 2 µM. The fluorescence properties of PKH67 at 0.05 and 2 µM and of DPPC/PKH67 at 0.05 g L -1 /0.05 µM were characterized using a Cary Eclipse fluorimeter (Agilent) with PMT at 860, 980 and 860 respectively.

Scheme 1 :

 1 Scheme 1: Chemical structure of PKH67 amphiphilic dye. The molecule is made of the fluorescent head groups Z1 and Z2 emitting in the green (502 nm) and of two aliphatic chains that insert into the bilayer (see Materials and Methods for details).

Figure 1 :

 1 Figure 1: Dynamic light scattering size distributions of 5 µM PKH67 dispersions using a) de-ionized water, b) PBS and c) Diluent C ® as a solvent.

Figure 2 :

 2 Figure 2: Schematic representation of the protocol used for labeling dipalmitoylphosphatidylcholine (DPPC) vesicles in DI-water.

Fig.

  Fig.3ashows the fluorescence excitation (𝜆 "# = 490 𝑛𝑚) and emission (𝜆 "+ = 502 𝑛𝑚) spectra for a 50 nM PKH67 aqueous suspension and for a PKH67/DPPC dispersion prepared in DI-water as described above. In particular the dye-to-lipid ratio was set at 1:1400. The fluorescence signal observed for DPPC/PKH67 is 40 times higher than that of PKH67 alone at the same concentration. The low fluorescence level found for pure PKH67 dispersion is explained by the phenomenon of aggregationcaused quenching.[START_REF] Ma | Fluorescence Aggregation-Caused Quenching versus Aggregation-Induced Emission: A Visual Teaching Technology for Undergraduate Chemistry Students[END_REF][START_REF] Zhegalova | Minimization of selfquenching fluorescence on dyes conjugated to biomolecules with multiple labeling sites via asymmetrically charged NIR fluorophores: Bright NIR label[END_REF] Aggregation-caused quenching takes place when poorly soluble molecules associate into micelles and aggregates, resulting in concentration induced emission quenching. In contrast, the strong signal obtained in the presence of lipids suggests that the dyes have been incorporated in the membranes and are spatially dispersed. There, the distance between dye molecules is estimated at 30 nm, preventing the self-quenching to take place. In both cases the emission occurs at the same wavelength, showing that the presence of DPPC molecules does not alter the PKH67 photophysical properties.

Figure 3 :

 3 Figure 3: a) Fluorescent excitation and emission spectra of a 50 nM PKH67 aqueous dispersion in presence and in absence of dipalmitoylphosphatidylcholine (DPPC) vesicles. The lipid concentration is 50 mg L -1 and the temperature 25 °C. The arrow indicates that with DPPC the fluorescence signal is enhanced by a factor

Figure 4 :

 4 Figure 4: a) Dynamic light scattering size distribution of 1 g L -1 extruded Curosurf ® vesicles with and without PKH67 dyes. The extrusion was made using a 100 nm pore polycarbonate membrane. b) Similar to a) for native TEQ vesicles. c-d) Phase-contrast and fluorescence optical microscopy showing non-extruded Curosurf ® vesicles. e) Merge of the patterns found in phase-contrast and fluorescence. f-h) Same as c-e) for TEQ quaternary ammonium esterquat surfactant.

Figure 5 :

 5 Figure 5: a) A silica nanoparticle/ Curosurf ® aggregate observed under phase-contrast (a1), green (a2) and red (a3) illumination. The silica fluoresce in the orange-red at 590 nm and the vesicles are labeled with a green fluorescent dye (PKH67) emitting at 502 nm. The merge image of the red and green signals is shown in a4. b) Schematic representation of a nanoparticle/ Curosurf ® aggregate derived from optical microscopy. c) Bright field (c1) and confocal microscopy (c2,c3) of A549 alveolar epithelial cells incubated with silica nanoparticle/Curosurf ® aggregates at concentrations 0.10/0.015 g L -1 . The nuclei are labeled in blue with DAPI. The white lines are the cell contours. d) Three-color merge images showing cells (d1), plasma membrane (d2) and cytoplasm (d3). The bars in d2 and d3 are 2 µm and 4 µm respectively. e) Schematic representation of a cell with adsorbed and internalized silica nanoparticles.

Figure 6 :

 6 Figure 6: Phase-contrast (a,c) and fluorescence (b,d) microscopy images of a woven cotton fabric treated with fluorescent vesicles. Magnification are 20× for a and b, and 40× for c and d. In b) fluorescent vesicles are indicated with yellow arrows. Inset in c): fluorescence profile along the line A depicted in d). The fluorescence maxima are ascribed to the fiber edges. e) Schematics of surfactant deposition on cotton fibers assuming either supported lipid bilayer or supported vesicle layer models.

Scheme 2 :

 2 Scheme 2: Chemical formulae of a) dipalmitoylphosphatidylcholine (DPPC), b) most common lipids present in Curosurf ® formulation and c) ethanaminium, 2-hydroxyN,N-bis(2-hydroxyethyl)-N-methyl-esters abbreviated TEQ. Curosurf ® contains zwitterionic (phosphatidylcholine (PC), sphingomyelin(SM), phosphatidylethanolamine (PE)) and anionic lipids (phosphatidylinositol (PI), phosphatidylglycerol (PG)). Its composition is available in Supplementary Information TableS1.

Table I :

 I Hydrodynamic diameter (𝐷 3 ) and zeta potential (𝜁) of the native and fluorescent DPPC, Curosurf ® and TEQ vesicles. The star (*) indicates that the vesicles are extruded with a 100 nm pore polycarbonate membrane.

		Native Fluorescent Native Fluorescent
	DPPC	1350	1080	0	0
	Pulmonary surfactant substitute Curosurf®*	130	135	-20	-18
	Double-tail esterquat surfactant TEQ	550	530	+ 60	+ 58

𝑫 𝑯 (nm) 𝜻 (mV)

Acknowledgement

We thank Armelle Baeza-Squiban, Victor Baldim, Mélody Merle, Mostafa Mokhtari, Jesus Perez-Gil, Chloé Puisney, Milad Radiom for fruitful discussions. We also acknowledge the support from Galder Cristobal and Jean-Christophe Castaing from Solvay during the research program on novel softener formulations. ANR (Agence Nationale de la Recherche) and CGI (Commissariat à l'Investissement d'Avenir) are gratefully acknowledged for their financial support of this work through Labex SEAM (Science and Engineering for Advanced Materials and devices) ANR 11 LABX 086, ANR 11 IDEX 05 02. We also thank Stéphane Mornet from the Institut de Chimie de la Matière Condensée de Bordeaux (Université Bordeaux 1) for the synthesis of the aminated fluorescent silica nanoparticles. We acknowledge the ImagoSeine facility (Xavier Baudin, Jacques Monod Institute, Paris, France), and the France BioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04, « Investments for the future »). This research was supported in part by the Agence Nationale de la Recherche under the contract ANR-13-BS08-0015 (PANORAMA), ANR-12-CHEX-0011 (PULMONANO), ANR-15-CE18-0024-01 (ICONS), ANR-17-CE09-0017 (Alveo-lusMimics) and by Solvay.

Conflict of interest

The authors declare no conflict of interest

Supplementary information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.XXX Cryo-TEM images of Curosurf® vesicles, CryoTEM images of TEQ vesicles and analysis, Fluorescence optical microscopy images of Silica -Curosurf® aggregates, Confocal image of silica nanoparticles in cells, Phase contrast and fluorescence microscopy of mixed vesicle-cellulose nanocrystal aggregates, Scanning Electron Microscopy (SEM) of cotton fabric, Control experiments for surfactant deposition on cotton fabric, Differential scanning calorimetry of Curosurf®, Silica particle identity card, Curosurf® composition in lipids and proteins.


C Cristobal

L Bourgaux

I Heux

Jean-Francois Boucenna
email: jean-francois.berret@univ-paris-diderot.fr

Berret

E K Oikonomou
email: evdokia.oikonomou@univ-paris-diderot.fr

N Christov

G Cristobal

C Bourgaux

L Heux

I Boucenna

J.-F Berret

Design of eco-friendly fabric softeners: structure, rheology and interaction with cellulose nanocrystals

Keywords: Cationic surfactant, vesicles, fabric softeners, guar polymers, cotton fibers, cellulose nanocrystals

Design of eco-friendly fabric softeners: Structure, rheology and interaction with cellulose nanocrystals

products are used since the 1960's to make cotton fabrics soft, fresh smelling and wrinkle free. For the last decade, efforts have been made to develop conditioners based on natural and less aquatoxic materials [START_REF] Rosalind | Respiratory toxicity of fabric softener emissions[END_REF]. The first step towards more eco-friendly formulations has been taken with the replacement of dialkyldimethylammonium surfactants by esterquats. Esterquats are quaternary ammonium compounds having two (C(16)-C( 18)) fatty acid chains with two weak ester linkages. The inclusion of ester linkages has significantly improved the biodegradation kinetics of these materials, lowering the environmental exposure [START_REF] Mishra | Ester quats: The novel class of cationic fabric softeners[END_REF]. The second step for the development of eco-friendly products is modern and concerns the reduction of the surfactant concentration used in house and personal care formulations. In this work we explore this possibility to lessen the surfactant content in fabric softeners while preserving the initial deposition and softening performances.

Although softeners were used for decades [START_REF] Murphy | Fabric softener technology: A review[END_REF], there is still a lack of consensus pertaining to the active deposition on cotton fibers as well as on the softening mechanism. It has been shown that the surfactants used in conditioners are self-assembled into micron size vesicles [START_REF] Basavaraj | Nanovesicle formation and microstructure in aqueous ditallowethylesterdimethylammonium chloride (DEEDMAC) solutions[END_REF][START_REF] Kumar | Intact deposition of cationic vesicles on anionic cellulose fibers: Role of vesicle size, polydispersity, and substrate roughness studied via streaming potential measurements[END_REF][START_REF] Oikonomou | Fabric softener-cellulose nanocrystal interaction: A model for assessing surfactant deposition on cotton[END_REF]. In general it is assumed that the softening effect is due to the formation of a lubricating surfactant layer on the fibers [START_REF] Chugg | Boundary lubrication and shear properties of thin solid films of dioctadecyl dimethyl ammonium chloride (TA 100)[END_REF][START_REF] Serreau | Adsorption and onset of lubrication by a double-chained cationic surfactant on silica surfaces[END_REF][START_REF] Gerhardt | Tribological investigation of a functional medical textile with lubricating drug-delivery finishing[END_REF]. Recent studies have correlated fabric frictional characteristics with smoothness and softness [START_REF] Liao | A simultaneous measurement method to characterize touch properties of textile materials[END_REF], while others have put forward that the friction between the human skin and fabrics has a more prominent role [START_REF] Vilhena | Friction of Human Skin against Different Fabrics for Medical Use[END_REF]. Following Igarashi et al. the softening effect would come from the reduction of H-bonding between the bound water molecules and the cotton fibers [START_REF] Igarashi | Elucidation of Softening Mechanism in Rinse Cycle Fabric Softeners. Part 1: Effect of Hydrogen Bonding[END_REF][START_REF] Igarashi | Elucidation of Softening Mechanism in Rinse-Cycle Fabric Softeners. Part 2: Uneven Adsorption-The Key Phenomenon to the Effect of Fabric Softeners[END_REF]. Crutzen has suggested that hydrophobic interactions derived from the long alkyl chains of cationic surfactants are the primary driving force for adsorption [START_REF] Crutzen | Study of the ditallowdimethylammonium chloride interaction with cellulose[END_REF], whereas Kumar et al. proposed that the deposition process is electrostatically driven and that intact vesicles adsorb on cotton or viscose fibers [START_REF] Kumar | Intact deposition of cationic vesicles on anionic cellulose fibers: Role of vesicle size, polydispersity, and substrate roughness studied via streaming potential measurements[END_REF][START_REF] Kumar | Electrokinetic investigation of deposition of cationic fabric softener vesicles on anionic porous cotton fabrics[END_REF]. The deposition of vesicular structures (including uni/multilamellar and mutivesicular vesicles) on solid substrates results in the formation of a socalled supported vesicular layer (SVLs) [START_REF] Serreau | Adsorption and onset of lubrication by a double-chained cationic surfactant on silica surfaces[END_REF][START_REF] Reimhult | Intact vesicle adsorption and supported biomembrane formation from vesicles in solution: Influence of surface chemistry, vesicle size, temperature, and osmotic pressure[END_REF]. Understanding the SVL formation is also important for conditioner and active delivery applications. In fabric softeners, surfactant vesicles act as vehicles for driving water insoluble molecules (e.g. fragrances) on cotton [START_REF] Saha | Hydrocolloids as thickening and gelling agents in food: a critical review[END_REF]. In a recent paper, we have shown that in contact with cellulose nanocrystals, vesicles maintain their shape, in agreement with the work by Kumar an coworkers [START_REF] Kumar | Intact deposition of cationic vesicles on anionic cellulose fibers: Role of vesicle size, polydispersity, and substrate roughness studied via streaming potential measurements[END_REF] and are strongly associated to the surfactant membrane via electrostatic forces [START_REF] Oikonomou | Fabric softener-cellulose nanocrystal interaction: A model for assessing surfactant deposition on cotton[END_REF]. To produce the next conditioner generation, it is essential to understand the intrinsic processes pertaining to the deposition and softening mechanism, as well as being able to control them through a solid physicochemistry approach.

Recently the Solvay application laboratories have filed a series of patents that describe the benefits of adding minute amounts of polysaccharides to actual formulations in order to improve their softening and fragrance delivery performances [START_REF] Zhang | Composition comprising a quaternary ammonium compound, a cationic polysaccharide and a nonionic polysaccharide, Patent number WO 2015/192971 Al[END_REF][START_REF] Zhang | Compositions Comprising Quat and Polysaccharides, Patent number WO 2017/101798 Al[END_REF]. Natural hydrocolloid polymers such as cationic guar gum (C-Guar) and a hydroxypropyl guar (HP-Guar) extracted from the seeds of cyamopsis tetragonalobus plant were considered as additives for this application. In these patents, it is reported that towels treated with different formulations, including additive-free benchmark were evaluated from independent panellists in double-blinded tests. The softness of the treated towels was rated in a scale of 1 to 5, wherein 1 represents the lowest softness and 5 represents the highest. According to this survey, esterquat formulations modified with the C-Guar and HP-Guar polymers received the highest ranking for the softening performance, 4.4 against 4.0 for the additive-free benchmark. At the same time the surfactant concentration could be reduced by about half, from 10.5 wt. % to 4 -6 wt. %.

In this work, we investigate the effects of cationic and hydroxypropyl guar polymers focusing on the structural and rheological properties of novel conditioner formulations. The dispersions are investigated using a combination of techniques including optical and cryo-transmission electron microscopy, small-angle X-ray scattering and rheology. It is found that at low concentration, the polymers do not alter the local structure of the cationic esterquat vesicles, whereas at higher concentrations the phase and rheological behaviors are modified. By adjusting the polymer concentration at around 1/10 that of the surfactant, it is possible to offset the decrease of the elastic property associated with the surfactant reduction. The deposition on model cotton fibers is simulated following the interaction of vesicles and polymers with cellulose nanocrystals [START_REF] Berret | Stoichiometry of electrostatic complexes determined by light scattering[END_REF][START_REF] Fresnais | Nanoparticle aggregation controlled by desalting kinetics[END_REF][START_REF] Job | Studies on the formation of complex minerals in solution and on their stability[END_REF][START_REF] Renny | Method of continuous variations: Applications of job plots to the study of molecular associations in organometallic chemistry[END_REF].

Cellulose nanocrystals are here used as a stand-in for cotton to facilitate the assessment of the interactions in bulk phases. We finally show that the physicochemical study of conditioners in the dilute and concentrated regimes, combined with the testing of their deposition and softening performances are useful to help guide the development of novel and environmentally friendly softeners.

II -Experimental Section

II.1 -Materials and sample preparation

Materials. The esterquat surfactant ethanaminium, 2-hydroxyN,N-bis(2-hydroxyethyl)-Nmethyl-esters with saturated and unsaturated C16-18 aliphatic chains, abbreviated TEQ in the following was provided by Solvay®. For TEQ, the gel-to-fluid transition related to the longrange order of the surfactant molecules in the membrane is found at 𝑇𝑇 ! = 60 °C [START_REF] Oikonomou | Fabric softener-cellulose nanocrystal interaction: A model for assessing surfactant deposition on cotton[END_REF]. The counterions associated to the quaternized amines are methyl sulfate anions. The polysaccharide polymers are a cationic guar gum (C-Guar, molecular weight 2 × 10 6 g mol -1 ) and a hydroxypropyl guar gum (HP-Guar, molecular weight 0.5×10 6 g mol -1 ), both synthesized by Solvay® (Scheme 1). Guar is a natural polymer extracted from the seeds of cyamopsis tetragonalobus plant. HP-Guar is widely used in oil and gas recovery, paints, cosmetics and biology [START_REF] Gangotri | Evaluation of guar gum derivatives as gelling agents for microbial culture media[END_REF] as a thickening and lubricant agent [START_REF] Asbell | Modulation of HLA-DR in dry eye patients following 30 days of treatment with a lubricant eyedrop solution[END_REF][START_REF] Jamshidian | Stretching properties of xanthan and hydroxypropyl guar in aqueous solutions and in cosmetic emulsions[END_REF]. Cationic guar is known for its conditioning properties, notably on hair [START_REF] Hössel | Conditioning polymers in today's shampoo formulations -efficacy, mechanism and test methods[END_REF][START_REF] Jordan | Evaluation of novel synthetic conditioning polymers for shampoos[END_REF][START_REF] Ungewiß | Quantitative determination of cationic modified polysaccharides on hair using LC-MS and LC-MS-MS[END_REF]. They were obtained by introducing positively charged trimethylamino(2-hydroxyl)propyl into the backbone.

Sheme I: Chemical structures of the cationic (a) and hydroxypropyl modified (b) guar polymers used in this work. These polymers are abbreviated as C-Guar and HP-Guar respectively.

Cellulose nanocrystals (CNC) was prepared according to earlier reports using catalytic and selective oxidation. 30 Briefly, cotton linters provided by Buckeye Cellulose Corporation were hydrolyzed according to the method described by Revol et al. treating the cellulosic substrate with 65% (w/v) sulfuric acid at 63 °C during 30 min [START_REF] Revol | Helicoidal self-ordering of cellulose microfibrils in aqueous suspension[END_REF]. The suspensions were washed by repeated centrifugations, dialyzed against distilled water until neutrality and sonicated for 4 min with a Branson B-12 sonifier equipped with a 3 mm microtip. The suspensions were then filtered through 8 µm and then 1 µm cellulose nitrate membranes (Whatman). At the end of the process, a 2 wt. % aqueous stock suspensions was obtained. The resulting nanoparticles are in the form of laths and have a length of 180 ± 30 nm, a width of 17 ± 4 nm and a height of 7 nm. These values were confirmed by Cryo-TEM (see Supplementary Fig. S1). Using light scattering, the CNC dispersion prepared at pH 4.5 and at a concentration of 0.1 wt. % displays a single relaxation mode in the autocorrelation function. 𝑔𝑔 ! (𝑡𝑡) is decreased rapidly above delay times around 10 3 µs, indicating that the nanofibers are not aggregated while the size studies revealed an intensity distribution centered around 120 nm (see Supplementary Fig. S1). Water was deionized with a Millipore Milli-Q Water system. All the products were used without further purification.

Formulation preparation. All concentrated aqueous formulations were prepared using MilliQ filtered water at 60 °C following the protocol described in Refs. [START_REF] Zhang | Composition comprising a quaternary ammonium compound, a cationic polysaccharide and a nonionic polysaccharide, Patent number WO 2015/192971 Al[END_REF][START_REF] Zhang | Compositions Comprising Quat and Polysaccharides, Patent number WO 2017/101798 Al[END_REF]. TEQ was first melted at 60 °C and was added dropwise in water. The final pH was adjusted at 4.5. The dispersions containing guar polymers were produced by first adding the guars in 60 °C water and then the melted TEQ. The pH was adjusted at 4.5 after the guar addition and at the final step. Five formulations were prepared: TEQ 10.5 wt. %, TEQ 6 wt. %, TEQ 6 wt. % + C-Guar 0.4 wt. %, TEQ 6 wt. % + HP-Guar 0.4 wt. % and TEQ 6 wt. % + C-Guar 0.3 wt. % + HP-Guar 0.3 wt. %. Also, for SAXS studies TEP : HP-Guar : C-Guar = 4 : 0.3 : 0.3 were prepared. The investigated dispersions were produced by dilution down to the desired TEQ concentration using MilliQ filtered water (𝑇𝑇 = 25 °C). Finally, for polymer characterization, aqueous dispersions of each guar (pH 4.5) were prepared by diluting a stock aqueous solution of 1 wt. % prepared at 60 °C water. Unfiltered and 0.45 µm filtered (cellulose acetate) diluted dispersions were used for characterization.

Mixing protocol. The interactions between CNC and guars or CNC and TEQ/guar mixtures were investigated using the direct mixing formulation pathway [START_REF] Fresnais | Nanoparticle aggregation controlled by desalting kinetics[END_REF][START_REF] Torrisi | Preventing Corona Effects: Multiphosphonic Acid Poly(ethylene glycol) Copolymers for Stable Stealth Iron Oxide Nanoparticles[END_REF][START_REF] Vitorazi | Evidence of a two-step process and pathway dependency in the thermodynamics of poly(diallyldimethylammonium chloride)/poly(sodium acrylate) complexation[END_REF]. Batches of CNC, guars or TEQ/C-Guar (dilution from TEQ 6 wt. % + C-Guar 0.4 wt. % at TEQ 0.01 wt. %) were prepared in the same conditions of pH (pH 4.5) and concentration (𝑐𝑐 = 0.01 and 0.1 wt. %) and then mixed at different ratios, noted 𝑋𝑋 = 𝑉𝑉 !"#$ 𝑉𝑉 !"! , where 𝑉𝑉 !"#$ and 𝑉𝑉 !"! denote the volumes of the softener formulation (either guar or TEQ + guar) and CNC dispersion, respectively. The mixed dispersions were prepared at room temperature (25 °C). After mixing, the dispersions were stirred rapidly, let to equilibrate for 5 minutes and the scattered intensity and hydrodynamic diameter were measured in triplicate. As the concentrations of the stock solutions are identical, the volumetric ratio 𝑋𝑋 is equivalent to the mass ratio between constituents. In the figures, the concentration of the stock solutions are set at 𝑋𝑋 = 10 -3 and 𝑋𝑋 = 10 3 .

II.2 -Experimental techniques

Light scattering. The scattering intensity 𝐼𝐼 ! and hydrodynamic diameter 𝐷𝐷 ! were measured using the Zetasizer Nano ZS spectrometer (Malvern Instruments, Worcestershore, UK). A 4 mW He-Ne laser beam (𝜆𝜆 = 633 nm) is used to illuminate the sample dispersion, and the scattered intensity is collected at a scattering angle of 173°. The Rayleigh ratio ℛ was derived from the intensity according to the relationship: ℛ = (𝐼𝐼 ! -𝐼𝐼 ! )𝑛𝑛 ! ! ℛ ! /𝐼𝐼 ! 𝑛𝑛 ! ! where 𝐼𝐼 ! and 𝐼𝐼 ! are the water and toluene scattering intensities respectively, 𝑛𝑛 ! = 1.333 and 𝑛𝑛 ! = 1.497 the solution and toluene refractive indexes, and ℛ ! the toluene Rayleigh ratio at 𝜆𝜆 = 633 nm (ℛ ! = 1.352× 10 !! 𝑐𝑐𝑐𝑐 !! ) [START_REF] Mousseau | Polyelectrolyte assisted charge titration spectrometry: Applications to latex and oxide nanoparticles[END_REF]. The second-order autocorrelation function 𝑔𝑔 (!) (𝑡𝑡) is analyzed using the cumulant and CONTIN algorithms to determine the average diffusion coefficient 𝐷𝐷 ! of the scatterers. The hydrodynamic diameter is then calculated according to the Stokes-Einstein relation, 𝐷𝐷 ! = 𝑘𝑘 ! 𝑇𝑇/3𝜋𝜋𝜋𝜋𝐷𝐷 ! , where 𝑘𝑘 ! is the Boltzmann constant, 𝑇𝑇 the temperature and 𝜂𝜂 the solvent viscosity. Measurements were performed in triplicate at 25 °C after an equilibration time of 120 s.

Zeta Potential. Laser Doppler velocimetry (Zetasizer, Malvern Instruments, Worcestershore, UK) using the phase analysis light scattering mode and detection at an angle of 16° was performed to determine the electrophoretic mobility and zeta potential of the different dispersions studied. Measurements were performed in triplicate at 25 °C, after 120 s of thermal equilibration.

Single angle X-ray scattering. Samples were loaded into quartz capillaries (diameter 1.5 mm, Glass Müller, Berlin, Germany). The top of the capillaries was sealed with a drop of paraffin to prevent water evaporation. The temperature of the sample holder was set to 25°C. The scattered intensity was reported as a function of the scattering vector 𝑞𝑞 = 4𝜋𝜋𝜋𝜋𝜋𝜋𝜋𝜋𝜋𝜋/𝜆𝜆, where 2𝜃𝜃 is the scattering angle and 𝜆𝜆 the wavelength of the incident beam. Small-Angle X-ray Scattering (SAXS) experiments were performed on the SWING beamline at the SOLEIL synchrotron source (Saint-Aubin, France). SAXS patterns were recorded using a two-dimensional AVIEX CCD detector placed in a vacuum detection tunnel. The beamline energy was set at 12 keV and the sample-to-detector distance was fixed to cover the 0.08 -8 nm -1 𝑞𝑞-range. The calibration was carried out with silver behenate. The acquisition time of each pattern was 250 ms; 5 acquisitions were averaged for each sample. Intensity values were normalized to account for beam intensity, acquisition time and sample transmission. Each scattering pattern was then integrated circularly to yield the intensity as a function of the wave-vector. The scattered intensity from a capillary filled with water was subtracted from the sample scattering curves.

Optical Microscopy. Phase-contrast images were acquired on an IX73 inverted microscope (Olympus) equipped with 20×, 40×, and 60× objectives. Seven microliters of TEQ dispersion were deposited on a glass plate and sealed into a Gene Frame (Abgene/ Advanced Biotech) dual adhesive system. An Exi-Blue camera (QImaging) and Metaview software (Universal Imaging Inc.) were used as the acquisition system. Fluorescent Microscopy. For fluorescent microscopy, 100 µl of TEQ 0.1 wt. % dispersion were mixed with 100 µl of 1 µM PKH67 (Sigma) solution. PKH67 is used in cellular biology as a green fluorescent molecular linker developed for cell membrane labeling. It is characterized by a absorption maximum at 490 nm and an excitation maximum at 502 nm. The solution was then stirred and let equilibriate for 1 h before use. The fluorescent vesicles were then mixed with C-Guar and CNC at the adequate mixing ratios.

Cryogenic transmission electron microscopy (Cryo-TEM).

A few microliters of the samples were deposited on a lacey carbon coated 200 mesh (Ted Pella). The drop was blotted with a filter paper on a VitrobotTM (FEI) and the grid was quenched rapidly in liquid ethane, cooled with liquid nitrogen, to avoid the crystallization of the aqueous phase. The membrane was finally transferred into the vacuum column of a TEM microscope (JEOL 1400 operating at 120 kV) where it was maintained at liquid nitrogen temperature thanks to a cryo holder (Gatan). The magnification was selected between 3000× and 40000×, and images were recorded with a 2k-2k Ultrascan camera (Gatan).

Rheology. The rheological studies were performed with a Physica RheoCompass MCR 302 (Anton Paar) using a cone-and-plate geometry (diameter 50 mm, cone angle 1°) and a solvent trap to prevent water evaporation. The linear viscoelastic regime was determined by performing a strain sweep test at a frequency of 1 rad s -1 . The frequency dependent storage and loss moduli, 𝐺𝐺′(𝜔𝜔) and 𝐺𝐺′′(𝜔𝜔), were determined by applying an oscillatory shear stress of 1 rad s -1 .

III -Results and Discussion

III.1 -Partial replacement of TEQ surfactant with natural guar polymers

III.1.1 -Guar polymer characterization

Here we investigate the guar polymers used in the patented formulation described in the Introduction section [START_REF] Zhang | Composition comprising a quaternary ammonium compound, a cationic polysaccharide and a nonionic polysaccharide, Patent number WO 2015/192971 Al[END_REF]. Two different guar polymers were used in this study, a cationic (C-Guar) and a hydroxypropyl (HP-Guar) guar. The C-Guar is believed to enhance the deposition on fabrics thanks to its positive charges, while HP-Guar was chosen for its rheological properties. For the characterization, aqueous guar dispersions were prepared at different concentrations (0.001 -1 wt. %) and were studied by light scattering and 𝜁𝜁-potential. The second-order autocorrelation functions 𝑔𝑔 ! 𝑡𝑡 of filtered C-Guar and HP-Guar dispersions at 0.02 wt. % are shown as a function of delay time in Figs. 1a and1b respectively. The intensity distributions in the insets reveal a single peak around 200 nm for C-Guar and a double peak for HP-Guar at 50 nm and 350 nm. These hydrodynamic diameters are larger than those expected from polymers of molecular weight 𝑀𝑀 ! = 0.5 and 2×10 6 g mol -1 respectively [START_REF] Rubinstein | Polymer physics[END_REF], suggesting that the chains may by associated in water and form hydrocolloid particles [START_REF] Saha | Hydrocolloids as thickening and gelling agents in food: a critical review[END_REF]. Electrophoretic mobility measurements on dilute dispersions give zeta potential of +30 mV and 0 mV respectively.

III.1.2 -Effect of guars on the vesicle structure at low concentration

Mixed surfactant/guar dispersions were first studied by phase-contrast optical microscopy in the diluted regime. The dispersions were obtained by diluting 6 wt. % TEQ formulations with DI-water at pH 4.5. For the sample containing the polysaccharides, the C-Guar and HP-Guar concentrations were 0.3 wt. %. Fig. 2a and2b show optical microscopy images of diluted TEQ dispersions in the conditions of use (𝑐𝑐 !"# = 0.01 wt. %) without and with guar polymers, respectively. Both figures exhibit well-contrasted spherical particles of average size 800 nm in both cases (Fig. 2b and2d). These objects were identified as TEQ vesicles (see Supplementary Movies 1 and 2 illustrating the Brownian motion of the vesicles). This finding shows that at the micron scale there is no apparent difference between vesicles with or without guar polymer and that TEQ vesicles are stable upon guar addition. These observations were further confirmed by cryo-TEM. Representative images from the dilute regime (0.1 wt. %) for both formulations are shown in Fig. 3a and3b. It is found that the TEQ surfactants self-assemble locally into a bilayer structure. The bilayers close on themselves to form unilamellar, doublets and multivesicular vesicles of size in the range of 100 -300 nm. Multivesicular vesicles are defined as a membrane compartment that encapsulates several smaller vesicles [START_REF] Mousseau | Biophysicochemical interaction of a clinical pulmonary surfactant with nanoalumina[END_REF]. It should be noted that in contrast to earlier studies [START_REF] Basavaraj | Nanovesicle formation and microstructure in aqueous ditallowethylesterdimethylammonium chloride (DEEDMAC) solutions[END_REF][START_REF] Saha | Impact of nearly water-insoluble additives on the properties of vesicular suspensions[END_REF] multi-lamellar vesicles in the form of "onions" were not observed for this surfactant (see Supplementary Fig. S2). Electrophoretic mobility measurements show that the TEQ vesicles are positively charge, with a zeta potential of 𝜁𝜁 = + 65 mV. The average size and zeta potential data determined for the different species studied in this work, including the C-Guar and HP-Guar polymers, the TEQ vesicles and the cellulose nanocrystals are summarized in Table I. In conclusion, cryo-TEM reveals similar structures for the two dilute formulations and confirms that the TEQ-bilayers with and without polymers are stable upon dilution in the conditions of use.

Table I: List of the characteristic features obtained for the polymers, vesicles and celluloses

used in this study. The characteristic size was determined from light scattering, optical microcopy and transmission electron microcopy. * The cellulose nanocrystals are anisotropic in the form of laths with dimensions 180×17×7 nm 3 .

III.1.3 -Effect of guars on the vesicle structure in formulations in the concentrated regime

The effect of polymer guars at the formulation concentration is first examined by optical microscopy. Fig. 4 displays a series of 40× magnification images of TEQ dispersions at concentration 10.5 wt. % (Fig. 4a) and 6 wt. %, this later being without and with guar polymers (Figs. 4c and 4d respectively). The TEQ 10.5 wt. % dispersion exhibits highly contrasted and densely packed vesicles characterized by broad dispersity, with sizes comprised between 200 nm to 10 µm (Fig. 4b). Some large vesicles are also not spherical and exhibit facets (Fig. 4a), a phenomenon attributed to mechanical stresses generated during preparation and related with the liquid-crystalline order of the alky chains [START_REF] Nagai | Microscopic investigation on morphologies of bilayer gel structure in the mixed polyoxyethylene-type nonionic surfactant systems[END_REF][START_REF] Seth | Dilution technique to determine the hydrodynamic volume fraction of a vesicle suspension[END_REF][START_REF] Seth | Origins of microstructural transformations in charged vesicle suspensions: The crowding hypothesis[END_REF]. In Supplementary Movie 3, the large vesicles appear to be immobile and stuck to the glass surface. Numerous smaller vesicles are visible however and animated of rapid Brownian motion. At 6 wt. %, the TEQ vesicles are less packed and exhibit again Brownian motion (see Supplementary Movie 4). The appearance of the guar/surfactant mixed dispersions in Fig. 4d is different: neither large faceted nor single vesicles are observed and the previous patterns are replaced by a heterogeneous micron sized texture. This texture appears moreover to be frozen at the time scale of the observations (> minutes, see Supplementary Movie 5). To get a better insight into these dispersions, SAXS and rheology measurements were performed. Figs. 5a, 5b and 5c display the SAXS patterns for 4 wt. % TEQ aqueous dispersions without and with polymers respectively, the guar concentrations being 0.2 wt. % and 0.6 wt. % for each. Data obtained from pure polymer dispersions (0.6 wt. %) are also included for comparison. The large oscillation in the q-range 0.4 -2.5 nm -1 observed in Fig. 5a is characteristic of the TEQ bilayer form factor and arises from the electronic density modulation across the bilayer [START_REF] Kawabata | Bilayer structure of ester-amide-type cationic surfactants in a dilute aqueous solution[END_REF][START_REF] Kucerka | Scattering density profile model of popg bilayers as determined by molecular dynamics simulations and small-angle neutron and x-ray scattering experiments[END_REF]. The bilayer form factor is modeled using a two-level electronic density profile (inset), one for the head groups (0.8 nm) and the other for the alkyl chains (3.0 nm). As a result, the overall layer thickness is estimated at 4.6 nm, in good agreement with the cryo-TEM determination [START_REF] Oikonomou | Fabric softener-cellulose nanocrystal interaction: A model for assessing surfactant deposition on cotton[END_REF]. The continuous line labeled Model in Fig. 5a proves that this approach is well founded. For the polymers, the scattering cross-section exhibits a decrease consistent with that of polymer material, in particular the observation of 𝑞𝑞 !! power law at large wave-vector [START_REF] Rubinstein | Polymer physics[END_REF]. The range studied does not allow however to determine the polymer gyration radius. Upon addition of 0.2 wt. % of guar, the TEQ form factor is not altered, suggesting no significant change in the TEQ bilayer. At 0.6 wt. % however, low amplitude Bragg peaks superimposed to the bilayer form factor are observed [START_REF] Kawabata | Bilayer structure of ester-amide-type cationic surfactants in a dilute aqueous solution[END_REF]. For TEQ + C-Guar these peaks are located at 𝑞𝑞 ! = 0.6, 𝑞𝑞 ! = 0.9 and 𝑞𝑞 ! = 1.18 nm -1 and at 𝑞𝑞 ! = 0.62, 𝑞𝑞 ! = 0.91 and 𝑞𝑞 ! = 1.14 nm -1 for TEQ + HP-Guar. These peaks are reminiscent of the maxima observed in TEQ concentrated dispersions at 8, 12 and 15 wt. %. In our previous study, these peaks were interpreted in terms of a structure factor arising from stacked bilayers [START_REF] Oikonomou | Fabric softener-cellulose nanocrystal interaction: A model for assessing surfactant deposition on cotton[END_REF]. Note here that the first diffraction order at 𝑞𝑞 ! is absent, probably because it is down modulated by the bilayer form factor oscillation at 0.4 nm -1 . In terms of inter-lamellar distances, one finds 20.9 nm for TEQ + C-Guar and 20.3 nm for TEQ + HP-Guar. Interestingly, the positions of the Bragg peaks found here are similar to those observed in pure TEQ dispersion at 8 wt. %. So, adding 0.6 wt. % of guar to a 4 wt. % TEQ dispersion or doubling the TEQ concentration has a similar effect on the vesicular structure. In summary we have found that for concentrated dispersions the guars have a significant impact on the vesicle microstructure. The presence of Bragg peaks suggests a transformation from multi-vesicular to multilamellar vesicles upon polymer addition. We surmise that crowding effects are triggered by the increase in overall volume fraction and/or by polymer induced depletion interactions [START_REF] Seth | Dilution technique to determine the hydrodynamic volume fraction of a vesicle suspension[END_REF][START_REF] Seth | Origins of microstructural transformations in charged vesicle suspensions: The crowding hypothesis[END_REF]. Adding polymers to a colloidal dispersion is known to induce short-range attractive forces between particles above a threshold volume fraction [START_REF] Anderson | Direct measurement of doublelayer, van der waals, and polymer depletion attraction forces between supported cationic bilayers[END_REF][START_REF] Das | Interaction of adsorbed polymers with supported cationic bilayers[END_REF]. It is also possible that other interaction types are present and also modify the vesicular organization.

III.1.4 -Effect of guars on the surfactant formulation rheology

The elastic and loss moduli 𝐺𝐺 ! (𝜔𝜔) and 𝐺𝐺"(𝜔𝜔) were determined experimentally for TEQ surfactant solutions at 10.5 wt. % (Fig. 6a) and 6 wt. % (Fig. 6b). It is observed that the concentrated TEQ solution exhibits gel-like behavior, as 𝐺𝐺 ! (𝜔𝜔) > 𝐺𝐺"(𝜔𝜔) . This structural arrangement imparts to the fluid the rheological properties of a soft solid characterized by a gellike elastic complex modulus and a non-zero yield stress. From the stress versus rate flow curve, the yield stress was estimated at 0.1 Pa [START_REF] Oikonomou | Fabric softener-cellulose nanocrystal interaction: A model for assessing surfactant deposition on cotton[END_REF], that is typically 40 times lower than the elastic modulus at 1 rad s -1 (see Supplementary information S3). In contrast, the 6 wt. % TEQ solution exhibits a liquid-like behavior (𝑖𝑖. 𝑒𝑒. 𝐺𝐺"(𝜔𝜔) > 𝐺𝐺 ! (𝜔𝜔)) with values for storage and loss moduli around 0.01 Pa. These features indicate that upon dilution the formulation has lost its soft solid character. These results are in agreement with the optical microscopy where for TEQ 6 wt. % vesicles undergo rapid Brownian motion (see movie#2). Upon guar addition, the rheological behavior of the 6 wt. % dispersion changes drastically. As shown in Figs. 6c and6d, the moduli are both increased and 𝐺𝐺′ is now higher than 𝐺𝐺". We also notice that the effect is stronger for HP-guar than for C-Guar. With the combination of the two polymers, the rheological properties are further enhanced and similar to the initial 10.5 wt. % formulation (Fig. 6e). At the concentration used (0.4 wt. %), the aqueous guar dispersions alone are viscous polymeric fluids with a slight viscoelastic behavior (see Supplementary Fig. S4). The high viscosity of the guars arises from H-bonding intermolecular association between the hydrocolloids [START_REF] Cheng | homme, Characterization and intermolecular interactions of hydroxypropyl guar solutions[END_REF]. In conclusion, we interpret the guar viscosifying effects as a combination of two factors. On one side, in the concentrated phase, the guars induce vesicular crowding and/or aggregation driven by depletion interaction. In addition to this structural change, the HP-Guar hydrocolloid network enhances the background viscosity endowing the system with the appropriate rheological properties.

III. 2. Cellulose nanocrystal/guar interaction

In our previous report, we propose a method to evaluate cellulose/surfactant interactions with increased sensitivity [START_REF] Oikonomou | Fabric softener-cellulose nanocrystal interaction: A model for assessing surfactant deposition on cotton[END_REF]. The method is based on the use of cellulose nanocrystals instead of micron-sized fibers or yarns and of a combination of different bulk characterization techniques.

As a result, we could show that cellulose nanocrystals interact strongly with the TEQ vesicles via electrostatic charge complexation leading to the formation of large-scale aggregates in which the vesicles remain intact and adsorbed at the cellulose surface [START_REF] Oikonomou | Fabric softener-cellulose nanocrystal interaction: A model for assessing surfactant deposition on cotton[END_REF]. Here we extend this approach and investigate the interaction of CNCs with guar polymers using the continuous variation method [START_REF] Job | Studies on the formation of complex minerals in solution and on their stability[END_REF][START_REF] Renny | Method of continuous variations: Applications of job plots to the study of molecular associations in organometallic chemistry[END_REF][START_REF] Facchiano | Modification of Job's method for determining the stoichiometry of protein-protein complexes[END_REF][START_REF] Berret | Controlling electrostatic co-assembly using ion-containing copolymers: From surfactants to nanoparticles[END_REF]. C-Guar/CNC dispersions were prepared by mixing stock solutions at different volumetric ratios between 10 -3 and 10 3 . In Fig. 7a, the Rayleigh ratio obtained at 0.01 wt. % exhibits a marked maximum centered around 𝑋𝑋 !"# = 0.2. The continuous red line is calculated assuming that ℛ 𝑋𝑋 is the sum of the Rayleigh ratios ℛ !"! and ℛ !!!"#$ weighted by their respective concentrations, where ℛ !"! and ℛ !!!"#$ are the Rayleigh ratios of the CNCs and polymer dispersions [START_REF] Berret | Stoichiometry of electrostatic complexes determined by light scattering[END_REF][START_REF] Fresnais | Nanoparticle aggregation controlled by desalting kinetics[END_REF]. ℛ 𝑋𝑋 is found to be higher than the prediction for non-interacting species. The occurrence of a maximum suggests that C-Guar/CNC aggregates are characterized by a fixed stoichiometry, likely related to the point of zero charge [START_REF] Mousseau | Polyelectrolyte assisted charge titration spectrometry: Applications to latex and oxide nanoparticles[END_REF][START_REF] Mousseau | Biophysicochemical interaction of a clinical pulmonary surfactant with nanoalumina[END_REF]. The scattered intensity is high (ℛ > 10 !! cm -1 ) and indicates a strong interaction behavior. These results were confirmed by the hydrodynamic diameter measurement that also displays a characteristic peak at 𝑋𝑋 !"# (see Supplementary Fig. S5).

The distribution intensities versus zeta potential obtained from electrophoretic mobility measurements between 𝑋𝑋 = 0 and 𝑋𝑋 = 100 are presented in Fig. 7b. Well-defined intensity peaks are found to shift progressively from a negative value (𝜁𝜁 = -38 mV) for pure CNC to a positive one (𝜁𝜁 = + 30 mV) for C-Guar solutions (see Table I). The point of zero charge is found at 𝑋𝑋 = 0.5, close to the scattering maximum (Fig. 7a) [START_REF] Vitorazi | Evidence of a two-step process and pathway dependency in the thermodynamics of poly(diallyldimethylammonium chloride)/poly(sodium acrylate) complexation[END_REF][START_REF] Mousseau | Polyelectrolyte assisted charge titration spectrometry: Applications to latex and oxide nanoparticles[END_REF]. The results suggest an interaction based on electrostatic association between opposite charged species [START_REF] Berret | Stoichiometry of electrostatic complexes determined by light scattering[END_REF][START_REF] Berret | Controlling electrostatic co-assembly using ion-containing copolymers: From surfactants to nanoparticles[END_REF][START_REF] Courtois | Probing oppositely charged surfactant and copolymer interactions by isothermal titration microcalorimetry[END_REF]. There, the cationic guar particles are found to adsorb onto the CNCs, eventually reversing the sign of the electrophoretic mobility when added in excess and inducing aggregation at charge ratio close to unity (Fig. 7d). In contrast, weak interaction was found between CNC and HP-Guar, as exemplified in Supplementary Fig. S6. So far, we have evaluated the interaction between the different species in a pairwise manner: surfactant and guar, surfactant and nanocellulose and in the previous section guar and nanocellulose. We now investigate ternary dispersion containing the three components. To this aim, the guar to surfactant ratio is fixed at its actual formulation value, 6.0, 0.30 and 0.30 for TEQ, C-Guar and HP-Guar respectively. The ternary stock solutions were prepared by dilution at a TEQ concentration of 0.01 wt. % and were mixed with a 0.01 wt. % nanocellulose aqueous solution at different volumetric ratios. The results for TEQ + C-Guar mixed with CNC are shown in Fig. 8. It is found that at a ratio of about 0.5 the dispersions precipitate (green shaded area in Fig. 8a). Similar results were found for TEQ/HP-Guar mixed dispersions (see Supplementary Fig. S7). From this result it is concluded that both guars enhance the interaction of TEQ vesicles with nanocellulose. We recall that TEQ/CNC solutions prepared in the same conditions do not display a phase separation [START_REF] Oikonomou | Fabric softener-cellulose nanocrystal interaction: A model for assessing surfactant deposition on cotton[END_REF]. These findings indicate synergistic effects between TEQ and the guars and suggest that in real conditions guar polymers not only adsorb on cotton but also increase the TEQ adsorption. Fluorescent microscopy studies on the mixed aggregates were conducted to evaluate the size and morphology of the separated phases and to test whether the phase separation is of liquid-liquid (coacervation) or of liquid-solid type [START_REF] Vitorazi | Evidence of a two-step process and pathway dependency in the thermodynamics of poly(diallyldimethylammonium chloride)/poly(sodium acrylate) complexation[END_REF]. TEQ bilayers were labeled with the fluorescent dye PKH67, a lipid molecule used in cellular biology and known to insert into biological membranes. In Figure 8b, a mixture of TEQ+ C-Guar/CNC (𝑋𝑋 = 1, 𝑐𝑐 = 0.1 wt. %) is observed by phase contrast and fluorescent optical microscopy. Micron-sized aggregates reveal the coexistence of bright spots identified as TEQ vesicles (arrows) and a green fluorescence continuum and confirm the liquid-solid character of the transition.

IV -Conclusion

On the basis of the previously reported preparative approaches [START_REF] Oikonomou | Fabric softener-cellulose nanocrystal interaction: A model for assessing surfactant deposition on cotton[END_REF][START_REF] Zhang | Composition comprising a quaternary ammonium compound, a cationic polysaccharide and a nonionic polysaccharide, Patent number WO 2015/192971 Al[END_REF][START_REF] Zhang | Compositions Comprising Quat and Polysaccharides, Patent number WO 2017/101798 Al[END_REF], this work demonstrates that fabric conditioner formulations containing reduced amounts of surfactants, typically from 10-15 wt. % to 4-6 wt. % can be produced while preserving their overall physicochemical properties. To this aim, part of the surfactant is replaced by minute amount of natural guar polymers obtained from the seeds of cyamopsis tetragonalobus plants. In the formulation, the guars are chemically modified with cationic charges or with hydroxypropyl groups, each function having its own specific effect in the dispersion. In previous reports, it was shown that TEQ esterquat dispersions formulated with guar polymers around 4-6 wt. % exhibited excellent softening and fragrance delivery performances, with softness rated from testers at 4.4 on a 1-to-5 scale in double-blinded tests. These results have led to the filing of several patents for this invention [START_REF] Zhang | Composition comprising a quaternary ammonium compound, a cationic polysaccharide and a nonionic polysaccharide, Patent number WO 2015/192971 Al[END_REF][START_REF] Zhang | Compositions Comprising Quat and Polysaccharides, Patent number WO 2017/101798 Al[END_REF]. Here, we investigate the same dispersions as in the patents focusing on their structural and rheological properties. The guar effects are monitored by a combination of different techniques including optical microscopy, cryo-TEM, SAXS and rheology. The dispersions are studied both in the concentrated and dilute regimes of concentration, mimicking the storage and use conditions of these products. The main result of this study is that at low concentration, i.e. around 0.01 wt. % the polymers do not alter the local structure of the cationic esterquat vesicles. At 4-6 wt. % however the polymers modify the phase and rheological behaviors. Concerning the structure, SAXS suggests that the cationic guars induce a local crowding associated to depletion interactions and lead to the formation of a local lamellar order. Adjusting the polymer concentration around 0.2 wt. % for each guar is sufficient to compensate the decrease in storage modulus associated with the surfactant reduction. Recently, we also proposed a method to evaluate cellulose/surfactant interactions with increased sensitivity [START_REF] Oikonomou | Fabric softener-cellulose nanocrystal interaction: A model for assessing surfactant deposition on cotton[END_REF].

This method is applied here to assess the effect of the guar polymers related to the surfactant deposition on cellulose. Interaction studies confirm the high affinity of the surfactant vesicles with the nanofibers. The interaction with the cellulose surface is mainly driven by the oppositely charged surfaces and leads clear phase separations. Concerning the mechanism, it is proposed that in solution the vesicles adsorb spontaneously on cellulose nanofibers and that their structures remain intact. For the next step, further experiments dealing with the deposition and drying of the formulation on actual cotton fabrics should be performed to confirm these assumptions. In conclusion, we have shown that it is possible to reduce the concentration of cationic surfactant present in fabric softeners by half and to preserve the overall physicochemical properties of the formulations, a result that should pave the way for the development of more environmental friendly softeners.

Figure 1 :

 1 Figure 1: Second-order auto-correlation functions and size distribution of a) C-Guar and b) HP-Guar aqueous filtered dispersions at 0.02 wt. %.

Figure 2 :

 2 Figure 2: a) Phase-contrast microscopy image of a 0.01 wt. % TEQ dispersion and b) associated vesicular size distribution. c and d), same as a) and b) for a 0.01 wt. % TEQ + guars dispersion. The dispersions without polymers was obtained by diluting 6 wt. % TEQ formulation with DI-water at pH 4.5. The dispersion containing the polysaccharides was prepared at 𝑐𝑐 !"# = 6 wt. % with 0.3 wt. % for each guar. Scale bars are 10 µm.

Figure 3 :

 3 Figure 3: Representative cryogenic transmission electron microscopy (cryo-TEM) images of 0.1 wt. % TEQ surfactant aqueous dispersions a) without guar polymers and b) with C-Guar and HP-Guar at 0.01 wt. %. Yellow and red arrows indicate the unilamellar and multi-vesicular vesicles respectively. Scale bars are 100 nm.

Figure 4 :

 4 Figure 4: TEQ surfactant dispersions studied by phase-contrast microscopy: a) image at 10.5 wt. % and b) the corresponding vesicular size distribution, c and d) microscopy images at TEQ 6 wt. % without and with 0.6 wt. % of guar polymers (0.3 wt. % of C-Guar and 0.3 wt. % of HP-Guar). Scale bars are 10 µm. Blue arrows: large faceted vesicles. Red arrows: micrometric size vesicles.

Figure 5 :

 5 Figure 5: Small-angle X-ray scattering from TEQ 4 wt. % (a) and TEQ 4 wt. % in presence of 0.2 wt. % or 0.6 wt. % guar polymers (b, c). In a) the bilayer form factor is adjusted using a 4.6 nm membrane with a two-level scattering length density (SLD) displayed in the inset. The spectra in b) and c) show the effect upon C-Guar and HP-Guar addition respectively. The arrows indicate the values of wave-vectors where a Bragg peak is present.

Figure 6 :

 6 Figure 6: Elastic and loss modulus 𝐺𝐺 ! (𝜔𝜔) and 𝐺𝐺 !! (𝜔𝜔) as a function of the angular frequency for TEQ surfactant at a) 10.5 wt. %, b) 6 wt. %, c) 6 wt. % in presence of C-Guar or HP-Guar 0.4 wt. % and d) 6 wt. % in presence of both C-Guar 0.2 wt.% and HP-Guar 0.2 wt.%. e) 𝐺𝐺 ! (𝜔𝜔) values of the formulations at 1 rad s -1 .

Figure 7 :

 7 Figure 7: a) Rayleigh ratio ℛ 𝑋𝑋 and b) zeta potential results 𝜁𝜁 𝑋𝑋 of mixed C-Guar / cellulose nanocrystal dispersions as a function of X. The continuous line in red in a) is calculated from the non-interacting model (see text). The continuous lines in black and red in a) and b) respectively are guides to the eyes. c) Intensity distributions as a function of the zeta potential 𝜁𝜁 for samples between 𝑋𝑋 = 0 and 𝑋𝑋 = 100 showing a displacement of the peak from negative (pure cellulose nanocrystals) to positive (pure C-Guar) zeta values. c = 0.01 wt. %. d) Optical microscopy images of mixed C-Guar / cellulose nanocrystal dispersions at different mixing ratios X (here c = 0.1 wt. % and the scale bars are 20 µm).

Figure 8 :

 8 Figure 8: a) Rayleigh ratio ℛ 𝑋𝑋 for TEQ+C-Guar / cellulose nanocrystal dispersions (𝑐𝑐 = 0.01 wt. %) as a function of the mixing ratio 𝑋𝑋 and b) Phase-contrast and fluorescence optical microscopy mixed TEQ+C-Guar / cellulose nanocrystal aggregates (𝑐𝑐 = 0.1 wt. %). The scale bar is 10 µm.

Acknowledgements

We thank Chloé Puisney, Victor Baldim and Fanny Mousseau for fruitful discussions. ANR (Agence Nationale de la Recherche) and CGI (Commissariat à l'Investissement d'Avenir) are gratefully acknowledged for their financial support of this work through Labex SEAM (Science and Engineering for Advanced Materials and devices) ANR 11 LABX 086, ANR 11 IDEX 05 02. We acknowledge the ImagoSeine facility (Jacques Monod Institute, Paris, France), and the France BioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04, « Investments for the future »). This research was funded by Solvay, and in part by the Agence Nationale de la Recherche under the contract ANR-13-BS08-0015 (PANORAMA), ANR-12-CHEX-0011 (PULMONANO) and ANR-15-CE18-0024-01 (ICONS).

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