Tiana H Randriamamonjy

Jesús F Ontiveros
email: jesus-fermin.ontiveros@centralelille.fr

Mbolatiana Tovo Andrianjafy

Pascal Samiez

Alexandra Berlioz-Barbier

Véronique Nardello-Rataj

Jean-Marie Aubry

Voahangy Ramanandraibe

Marc Lemaire

Comparative study on the amphiphilicity, emulsifying and foaming properties of saponins extracted from Furcraea foetida

Keywords: Furcraea foetida, saponins, PIT-slope, emulsion, foam

A sustainable surfactant was obtained as a by-product of waste from the production of fibers from Furcraea foetida (FF). The extraction process is described and the chemical constituents (flavonoids, saponins and others) in the hemp of Mauritius in Madagascar were determined using ultra-performance liquid chromatography coupled to high resolution hybrid mass spectrometer. The hypothesis of surfaceactive behavior of this crude mixture was corroborated by surface tension measurements and these results were compared with 2 commercial Quillaja saponaria (QS) samples: S0019 (TCI) and Q-Naturale 200V. The critical micellar concentration of the FF saponins is 0.025 wt.%. The hydrophilicitylipophilicity radius was determined by the PIT-slope method and all saponins are more hydrophilic than the C10E4, the polyethoxylated reference surfactant. The hydrophilicity decreasing order is as follows: Q-Naturale 200V > FF > S0019. Oil in water (O/W) emulsions were formulated with isopropyl myristate as oil and their size distribution and stability during 7 days are equivalents for the three saponins. There is no coalescence, and creaming phenomena can be reduced increasing the oil fraction. Nanoemulsions were obtained with FF using ultrasounds and reducing the oil content. Foaming properties were also studied and the stability of foams is similar between the 3 saponins (higher than the foam formulated with sodium laureth sulfate). These findings show that FF is a new source of saponins that can be used as promising emulsifier and foam agent with properties equivalent to those quantified for available commercial saponins and equal or better than typical benchmark surfactants as Tween 40 (emulsifier) or sodium laureth sulfate (foaming agent).

Introduction

Mauritius hemp or Furcraea foetida (Asparagaceae) called "taretra vavy" in Madagascar, is a plant native of tropical America and found often in dry and warm areas [START_REF] Brink | Plant ressources of Tropical Africa, Fibres[END_REF]. Generally, people use this plant as a source of fiber but in some isolated localities of the island, leaves containing saponins [START_REF] Itabashi | A new bioactive steroidal saponin, furcreastatin, from the plant Furcraeafoetida[END_REF][START_REF] Da Silva | Chemical structure and biological activity of steroidal saponins from Furcraea gigantea[END_REF][START_REF] Yokosuka | Steroidal glycosides from Furcraea foetida and their cytotoxic activity[END_REF] serve as soap. The species is a non-wood plant used to make paper [START_REF] Amode | Paper Production from Mauritian Hemp Fibres[END_REF] or in composite manufacture. Totong et al. [START_REF] Totong | Extraction and characterization of natural fiber from furcraea foetida leaves as an alternative material for textile applications[END_REF] reported that from 2,5 kg of Furcraea foetida leaves, only 25 grams (1%) of fiber could be removed. Subsequently, the production of natural fibers from Furcraea foetida generates a large amount (64% wet mass [START_REF] Randriamamonjy | Contribution à l'écologie industrielle de la production de fibres naturelles issues du Chanvre de Maurice[END_REF]) of wood waste known as "chenevots" which consist of small fibers and a wide variety of chemicals, including sugar and secondary metabolites such as saponins and flavonoids among many others [START_REF] Vanitha | Phytochemical Screening and in vitro antioxidant activity of Furcraea foetida leaves[END_REF]. The renewed interest in natural fibers as alternatives to non-biodegradable or synthetic fibers induces an increase in production and requires the search for processes limiting the production of waste.

Saponins are vegetal glycosides with an aglycone moiety and one or up to 3 sugar chains (hexoses like ß-D-glucose, or pentoses like ß-D-arabinose). The aglycone moiety is the sapogenin and its structure can be a steroid or a triterpenoid [START_REF] Schwarz | Saponins[END_REF]. In most cases, saponins serve as an anti-microbial defense in plants, they have a hemolytic action, steroid-complexing properties and surfactant activity [START_REF] Schwarz | Saponins[END_REF][START_REF] Hostettmann | Chemistry and pharmacology of natural products[END_REF]. The surfactant activity of saponins is well documented and as early as 1869, a USA patent indicates the "cleansing" properties of the fluid obtained from the bulb of Phalangium pomeridianum chemically treated with borax and salt-soda [START_REF] Balcear | [END_REF]. The emulsifier properties of saponins were already mentioned in 1903 by Hillyer [12], and also by Ramsden in 1904 [START_REF] Ramsden | Separation of solids in the surface-layers of solutions and 'suspensions' (observations on surface-membranes, bubbles, emulsions, and mechanical coagulation).-Preliminary account[END_REF] and Pickering in 1907 [14]. However, the early interest on their surfactant properties declined by the exponential development of synthetic surfactants as detergents, emulsifiers, wetting or foaming agents that takes place in most of the XX century [START_REF] Oleszek | Saponin-based surfactants[END_REF].

Since the end of the XX century and until our days, there is increasing interest in replacing petro-sourced synthetic surfactants with bio-sourced compounds in most of the formulation fields. This trend is the result of both industry efforts and consumer awareness about the environment. The commercial preparation of saponin from Quillaja saponaria is generally recognized as safe (GRAS) in the USA [START_REF] Böttcher | Saponins -Self-assembly and behavior at aqueous interfaces[END_REF]. The European Union authorizes the "Quillaia extract" in food formulations (additive E-999), specially beverages; their authorities regulate its concentration at 200 mg/Kg and the origin of the saponins is clearly established as the product of "extraction of Quillaia saponaria Molina, or other Quillaia species, trees of the family Rosaceae" [START_REF] Commission | Commission regulation (EU) no 231/2012 of 9 March 2012 laying down specifications for food additives listed in annexes II and III to regulation (EC) no 1333/2008 of the European Parliament and of the Council[END_REF]. The Food and Agriculture Organization classifies the Quillaja extracts in Type 1 or 2 depending on the composition; type 1 contains over 100 triterpenoid saponins with polyphenols and tannins and type 2 is obtained after ultrafiltration of type 1, reducing the solids and polyphenols [START_REF]Evaluation of certain food additives: sixty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives[END_REF].

Since the incorporation of saponins in food, cosmetic and pharmaceutical formulations, the industrial and academic interest in these molecules has increased in order to expand their uses to other fields like enhance oil recovery [START_REF] Ahmadi | Implementation of a high-performance surfactant for enhanced oil recovery from carbonate reservoirs[END_REF], soil remediation [START_REF] Davin | Could saponins be used to enhance bioremediation of polycyclic aromatic hydrocarbons in aged-contaminated soils?[END_REF], micellar enhanced ultrafiltration [START_REF] Samal | Application of saponin biosurfactant and its recovery in the MEUF process for removal of methyl violet from wastewater[END_REF], adjuvants [START_REF] Sun | Advances in saponin-based adjuvants[END_REF], encapsulation [START_REF] Nicol | Ultrafast delivery of aggregation-induced emission nanoparticles and pure organic phosphorescent nanocrystals by saponin encapsulation[END_REF] or nanoparticles synthesis [START_REF] Karthik | Quillaja saponin: A prospective emulsifier for the preparation of solid lipid nanoparticles[END_REF]. Several works have been made to understand their interfacial properties [START_REF] Golemanov | Remarkably high surface visco-elasticity of adsorption layers of triterpenoid saponins[END_REF][START_REF] Golemanov | The role of the hydrophobic phase in the unique rheological properties of saponin adsorption layers[END_REF][START_REF] Pagureva | Surface properties of adsorption layers formed from triterpenoid and steroid saponins[END_REF][START_REF] Böttcher | Interfacial Properties of Saponin Extracts and Their Impact on Foam Characteristics[END_REF] and their behavior in complex food matrices [START_REF] Kezwon | Interaction of Quillaja bark saponins with food-relevant proteins[END_REF], and also to describe the chemical composition and applications of saponins from vegetal sources different to the well-known Quillaja saponaria [START_REF] Bozicevic | Dammarane-type saponins from leaves of Ziziphus spina-christi[END_REF][START_REF] Ralla | Oat bran extract (Avena sativa L.) from food by-product streams as new natural emulsifier[END_REF][START_REF] Thi | New furospirostane steroidal saponins from the flowers of Lilium Asiatic hybrids[END_REF][START_REF] Dabestani | A natural source of saponin: Comprehensive study on interfacial properties of Chubak (Acanthophyllum Glandulosum) root extract and related saponins[END_REF][START_REF] Schreiner | Evaluation of saponin-rich extracts as natural alternative emulsifiers: A comparative study with pure Quillaja Bark saponin[END_REF].

The main objective of this work is to study the extraction of saponins from chenevots of Furcraea foetida, to describe their chemical structure and to characterize some physicochemical properties of the saponins extract. The surface tension, the hydrophilic-lipophilic ratio quantified by the PIT-slope method, and the emulsifier and foaming properties are analyzed and compared with available commercial saponins. These properties allow evaluating the surface potential applications for these saponins that could be easily obtained from the chenevots (wastes) in Madagascar.

Material and Methods

Materials

Quillaja saponin (Q-Naturale 200V) was kindly provided by Ingredion Germany GmbH (Hamburg). This product is a brown liquid containing 14.5wt% of saponins from Quillaja saponaria Molina. Saponin S0019 is a brown powder obtained from TCI (Japan). Tetraethyleneglycol monodecyl ether C10E4 (>99%) was synthetized and distilled in the laboratory following a method described elsewhere [START_REF] Gibson | Phase-transfer synthesis of monoalkyl ethers of oligoethylene glycols[END_REF]. n-Octane (99%) and isopropyl myristate (>98%) were obtained from Sigma-Aldrich. Sodium chloride NaCl (≥99.5%) was supplied by Acros Organics. Texapon N70 (alcohols, C12-14, ethoxylated sulfates sodium salts 1-2.5EO at 70%) and Polysorbate 40 were obtained from BASF and AlfaAesar, respectively.

Extraction of saponins from Furcraea foetida

The raw material was dry chenevots from Furcraea foetida. They were collected in the village of Anjanonarivo, located 40 km northwest of the city of Antananarivo, Madagascar. They are obtained from fresh scraped leaves to separate fibers from chenevots. These chenevots were air-dried for 2 days. 200 g of dry chenevots were extracted on Soxhlet with 1250 mL of n-butanol for 6 hours. The solution was evaporated to dryness (R = 12.2%) and precipitated in ethyl acetate to remove the non-polar fraction (soluble fraction, R = 5.8%). After washing and filtration, the insoluble extract was recrystallized in isopropanol to give saponins with 2.1 % yields from dry weight. Flavonoids and other compounds were soluble in isopropanol. Extraction process is schematized in figure 1. Sample preparation. 25 mg of saponins were mixed in 2 mL of methanol and acetonitrile by using a vortex. The solution was immersed in an ultrasonic bath for 5 min and diluted in 2.5 ml of water before being centrifuged at 10000 rpm for 10 min until the saponins are completely dissolved. Chromatographic conditions. The saponin extract was analyzed on a C18 column CSH dimension 2,1 mm x 150 mm, particle size 1.7 µm (Waters), maintained at 45 °C. The mobile phase consisted of water (A) and Acetonitrile/Methanol (B), each containing 0,1 % (v/v) formic acid with the gradient of 100% of phase A for 2 min, followed by a linear increase of 0 to 100% phase B for 38 min. The flow rate was established at 400 µL/min and the injection volume was 50 µL.

Analysis by UPLC-ESI-HRMS:

Mass spectrometry detection.

The ESI-MS spectra were acquired in positive and negative ion modes. The ionization source was maintained at 200°C with a constant pressure of 45 Psi. The Acquisition mode Auto MS / MS (DDA) was used and its frequency depends on the intensity. Mass range was about 150-3000 Da. XCMS software was performed for data analysis.

2.4 Surface tension measurement. A force tensiometer "K100" with a Wilhelmy plate was used to measure the surface tension of several aqueous solutions of saponins at 25°C (±0.5°C). Highly concentrated stock solutions above the expected critical micelle concentration were serially diluted to at least 0.001 wt% saponin.

2.5 PIT-slope method.

Either the sample preparation, the heating-cooling cycles and the phase inversion temperature determination were done under the conditions previously described in the literature [START_REF] Ontiveros | A simple method to assess the hydrophilic lipophilic balance of food and cosmetic surfactants using the phase inversion temperature of C10E4/n-octane/water emulsions[END_REF][START_REF] Ontiveros | Structure-interfacial properties relationship and quantification of the amphiphilicity of well-defined ionic and non-ionic surfactants using the PIT-slope method[END_REF]. The analyzed surfactants were added to the reference system 3% wt. C10E4/n-octane/0.01M NaCl(aq) with a weight ratio water/oil of 1 (fw=0.5). The phase inversion temperature (PIT) at different concentrations of the studied surfactant "S2" was determined by the fall of the conductivity with temperature. All saponin samples were studied in a range of concentrations until 1 wt.%.

2.6 Emulsion preparation and characterization protocol Saponine/Isopropyl myristate/0.01M NaCl(aq) system were prepared 24h before the experiments at different concentrations of saponin and different mass fractions of water. In the case of 1%wt Saponin and fw=0.5, samples were prepared by pouring 4.25g of water, 4.25g of isopropyl myristate and 0.086g of saponin (or the equivalent when the saponin is in aqueous solution as in the Q-Naturale case); then the samples are gently mixed to put in contact the different phases. Samples are homogenized with IKA Ultraturrax T-10 basic with a S10N-8G dispersing tool (8mm stator diameter and 6.1mm rotor diameter) during 60 s at 16000 rpm or sonicated for 2 min using an ultrasonic probe Sonotrode S26d2 (2 mm diameter) immersed by 3 mm in the system and operated by the ultrasonic processor UP200St (Hielscher). The sonotrode pulse was fixed at 100% and the amplitude at 80%. The emulsion droplet size distribution was determined using a laser diffraction instrument (MasterSizer3000, Malvern Instruments). The emulsion stability was measured at constant temperature (25°C) by multiple light scattering using a Turbiscan Lab Expert (Formulaction).

Foaming and foam stability

The foaming properties of the saponins were studied using the DFA-100 from Krüss (Hambourg, Germany). The concentration was fixed at 2 times the CMC for all samples. 50 mL of the solution were added to the glass column CY4572. Pressurized air was sent to the column through a filter paper FL4551 (12-25 μm) at a rate of 0.2 L/min and foaming was stopped at a total height (liquid+foam) of 180 mm. The foam (hfoam), liquid (hliquid) and total height (h) were monitored for 20 min after the 180mm were reached. Bubbles size distribution was monitored by the ADVANCE software at a height of 10 cm. All experiments were done at least by triplicate. The maximum foam density (FD) and the foam stability (FS) at 1200 s are defined as follows:

𝐹𝐷 𝑡=0 = 𝑉 𝑙𝑖𝑞𝑢𝑖𝑑 𝑖𝑛 𝑡ℎ𝑒 𝑓𝑜𝑎𝑚 𝑉 𝑓𝑜𝑎𝑚 × 100 (2) 𝐹𝑆 𝑡=1200 = ℎ 𝑓𝑜𝑎𝑚 𝑡 ℎ 𝑓𝑜𝑎𝑚 𝑡=0𝑠 × 100 (3)

Results and Discussion

3.1 Structural analysis UPLC coupled with mass spectrometry was carried out to distinguish saponins in final sample. Raw formulas of different molecules were identified by comparing mass spectra with databases such as PubChem, ChemSpider and the MS/MS fragments with MetFrag database. Figure 2 shows the chromatographic profile of the isopropanol insoluble extract. Peak numbers correspond to ascending order of molecules retention times and polarity. The most polar compounds were first eluted and had low retention times. Functional groups and double bonds on genins are responsible for their polarity followed by number and nature of sugars in glycosidic moieties. The chromatograms A and B exhibit peaks grouped into different colors according to their chemical family such as saponins (red), flavonoids (black) and other compounds (blue, purple, orange). According to the LC/MS analysis, isopropanol insoluble products consist mainly of saponins and some flavonoids. Vanitha et al. [START_REF] Vanitha | Phytochemical Screening and in vitro antioxidant activity of Furcraea foetida leaves[END_REF] have revealed that other groups of compounds like carbohydrates and tannins (peaks with other colors) have also been found in the plant. Most of saponins and flavonoids have been previously isolated from leaves of Furcraea foetida [START_REF] Yokosuka | Steroidal glycosides from Furcraea foetida and their cytotoxic activity[END_REF] or found in others Furcraea species [START_REF] Da Silva | Chemical structure and biological activity of steroidal saponins from Furcraea gigantea[END_REF][START_REF] Calle | Steroidal saponins from Furcraea hexapetala leaves and their phytotoxic activity[END_REF][START_REF] El-Sayed | Isolation and identification of some steroidal glycosides of Furcraea selloa[END_REF][START_REF] Tapondjou | Flavonol Glycosides and Cytotoxic Steroidal Saponins from Furcraea Tuberosa (Agavaceae)[END_REF].

A ESI-

All the saponins identified are of the steroid type (spirostanol and furostanol ones). Two large peaks were listed and corresponded to a We note that peak N°30 has the same molecular formula C39H64O15 than cantalasaponin-1 identified previously in foetida, hexapetala and tuberosa species of the genus Furcraea but according to its retention time and polarity, we assigned the sapogenin to the crestagenin [START_REF] Kunkel | Biogeography and Ecology in the Canary Islands[END_REF], a diastereosiomer of chrysogenin, with the same sugar unities. This compound was already found in F. tuberosa and had a 25-R configuration with three hydroxyl groups (27,2α,3β) instead of 23S,3β,6α like in chrysogenin.

Some flavonoids peaks have higher retention times than saponins because of the hydroxyl groups in the aglycon skeletons inducing their polar structures. By comparing the mass spectra of each peak with those of compounds previously isolated from Furcraea genus, we have identified Kaempferol or Quercetine derivatives for the major peaks such as Camelliaside A (peak N°2), Rutin (peak N°4) [START_REF] Tapondjou | Flavonol Glycosides and Cytotoxic Steroidal Saponins from Furcraea Tuberosa (Agavaceae)[END_REF] and Nicotiflorin (Peak N°5) [START_REF] Tapondjou | Flavonol Glycosides and Cytotoxic Steroidal Saponins from Furcraea Tuberosa (Agavaceae)[END_REF]. In our knowledge, Camelliaside A is described for the first time in F. foetida. Camelliaside B was found in F. tuberosa. Figure 3 shows some of the chemical structures of different compounds found in the FF. 3.2 Amphiphilic properties 3.2.1 Self-aggregation behavior Figure 4 shows the surface tension profiles at 25°C as a function of the saponin weight concentrations between 0.0001 wt.% and 1 wt. %, not only for the Furcraea foetida but also for two saponins commercially available. The CMC and the surface tension at CMC (𝛾 𝐶𝑀𝐶 ) were determined from the breakpoint of the surface tension versus surfactant concentration profiles. The values of maximum surface excess (Γ) and the area per molecule at the interface (𝑎 1 𝑠 ) were also calculated using the Gibbs isotherm equation, expressed as follows for a nonionic surfactant:

B ESI+

𝛤 = - 1 2.303𝑅𝑇 ( 𝑑𝛾 𝑑𝑙𝑜𝑔𝐶 ) 𝑇 ( 4
)
where 𝛾 is the surface tension (mN/m), T is the absolute temperature (K), R= 8.314 J/mol•K. The area per molecule at the interface (𝑎 1 𝑠 ) is expressed in square angstroms using eq. 5:

𝑎 1 𝑠 = 10 16 𝑁𝛤 ( 5
)
where N is the Avogadro's constant and Γ is expressed in mol/cm -2 . Table 1 shows the CMC, the surface tension at the CMC, the maximum surface excess and the area per molecule at the water-air interface. The CMC of Furcraea foetida saponins is on the order of magnitude of Quillaja saponaria and its value is between that of Q-Naturale 200V and that of TCI. The CMC of saponins depends notably on their botanical origin and values from 0.008 (Aesculus hippocastanum) to 0.3 wt% (Acacia concinna) are reported in the literature [START_REF] Golemanov | Remarkably high surface visco-elasticity of adsorption layers of triterpenoid saponins[END_REF]. Previous studies for Quillaja saponaria saponins shows also CMC values from 0.05-0.08%wt./v [START_REF] Mitra | Micellar Properties of Quillaja Saponin. 1. Effects of Temperature, Salt, and pH on Solution Properties[END_REF], 0.025%wt. [START_REF] Stanimirova | Surface Rheology of Saponin Adsorption Layers[END_REF], 0.017%wt./v [START_REF] Wojciechowski | Surface activity of saponin from Quillaja bark at the air/water and oil/water interfaces[END_REF], 0.005%wt. [START_REF] Tippel | Interfacial properties of Quillaja saponins and its use for micellisation of lutein esters[END_REF] and 0.008%wt. [START_REF] Böttcher | Interfacial Properties of Saponin Extracts and Their Impact on Foam Characteristics[END_REF].

The differences in these values indicate that not only the origin but also the purification steps in the extraction process change the chemical composition and the surface properties of saponins. The surface tension at the CMC for the two commercial saponins are similar to those reported by Stanimirova et al. [START_REF] Stanimirova | Surface Rheology of Saponin Adsorption Layers[END_REF] (38-40mN/m). The value for FF is higher than others, indicating a lower surface activity in aqueous solution. The area per molecule is an average value of different molecules present in the samples and the obtained value for FF extract is close to the reported for other saponins as Gypsophila (triterpenoid aglycone) or Tribulus terrestris (steroid aglycone) which are 77 and 66𝐴 2, respectively. Molecular modeling have been reported [START_REF] Stanimirova | Surface Rheology of Saponin Adsorption Layers[END_REF] and several configurations at the interface are proposed for triterpenoid aglycone depending on the "average" value of 𝑎 1 𝑠 [START_REF] Pagureva | Surface properties of adsorption layers formed from triterpenoid and steroid saponins[END_REF]. However, there is no available molecular modeling data about steroidal saponins present in FF extract to conclude about their spatial configuration.

Amphiphilic behavior in Surfactant/Oil/Water systems

The PIT-slope method allows evaluating the hydrophilic-lipophilic behavior of a surfactant thought a temperature dynamic scan. Aliquots of the studied surfactant are added into a reference system 3 % wt. C10E4/n-octane/10 -2 M NaCl(aq) and the linear variation on the phase inversion temperatures allows calculate the dPIT/dC or "PIT-slope", a comparative criterion that indicates if the surfactant is more or less hydrophilic than C10E4. Figure 5A shows the conductivity profile of the reference system when adding saponins from Furcraea foetida.

B

that ionic compounds (salts/acids) are present inside the saponin extract. This behavior is obtained for all studied saponins and it is more pronounced for the Saponine S 0019 and Q-Naturale 200V (see Supplementary Information).

Figure 5B shows the evolution of the PIT at several concentrations of saponins. In all cases, a higher temperature than the reference C10E4 is needed to inverse the emulsion indicating that added saponins are more hydrophilic than C10E4. From the figure 5B, the PIT-slope can be calculated for all saponins as shown in table 1 and classified in decreasing order of hydrophilicity: Q-Naturale 200V > Furcraea foetida > S0019 (TCI). Using these values, their PIT-slope can be compared with other technical-grade or well-defined surfactants published in the literature [START_REF] Ontiveros | A simple method to assess the hydrophilic lipophilic balance of food and cosmetic surfactants using the phase inversion temperature of C10E4/n-octane/water emulsions[END_REF][START_REF] Ontiveros | Structure-interfacial properties relationship and quantification of the amphiphilicity of well-defined ionic and non-ionic surfactants using the PIT-slope method[END_REF].

The value of dPIT/dC = 10.2°C /%wt. for Furcraea foetida saponin matches with the value of Tween 40 which is a commercial polyethoxylated sorbitan monopalmitate surfactant used as an additive in the food industry (E-434). The PIT-slope of saponins are comprised in the C12E6-C12E10 hydrophilicity interval.

From 0.54% by mass for saponin Q-Naturale 200V, the emulsion does not inverse in the temperature range studied, indicating a highly hydrophilicity compared with the other saponins. No matter the temperature this system remains as an O/W emulsion at this concentration as reported previously for well-defined ionic surfactants at lower concentrations [START_REF] Ontiveros | Structure-interfacial properties relationship and quantification of the amphiphilicity of well-defined ionic and non-ionic surfactants using the PIT-slope method[END_REF]. The presence of acid groups in some saponins has been established by FTIR, their influence studied in several works [START_REF] Böttcher | Interfacial Properties of Saponin Extracts and Their Impact on Foam Characteristics[END_REF][START_REF] Ulaganathan | The influence of pH on the interfacial behaviour of Quillaja bark saponin at the air-solution interface[END_REF] and the question about the non-ionic or ionic behavior of saponins is still open. Böttchet and Drush [START_REF] Böttcher | Interfacial Properties of Saponin Extracts and Their Impact on Foam Characteristics[END_REF] determined the CMC of 6 saponins with a conductivity titration, common for ionic surfactants, and the characteristic break was observed for 4 saponins (Quillaja saponaria Molina, Gypsophila, Aescumus hippocastanum and Glycyrriza glabra) that were considered as "ionic compounds". The "acidity" of Q-Naturale 200V and the influence of pH on its interfacial behavior at air-liquid interface have been described by Ulaganathan et al. [START_REF] Ulaganathan | The influence of pH on the interfacial behaviour of Quillaja bark saponin at the air-solution interface[END_REF]. The conductivity and pH of FF saponins were evaluated as a function of the concentration (see Figure SI.1 in the supplementary information). Increasing the concentration of FF saponins diminishes the pH and increases the conductivity. A solution 1 wt% has a pH of 5.1 and a conductivity of 0.65mS/cm, verifying the presence of ionic compounds and their slightly acid character.

Saponins in dispersed systems 3.3.1 Emulsifier properties

The ability of the three saponins to stabilize emulsions of water and isopropyl myristate (IPM) (50/50 w/w) was then investigated. Equal weight fractions of aqueous and oily phase were chosen in order to privilege the nature of the surfactant on the final morphology of the emulsion. All systems were oil-in-water emulsions (O/W), in agreement with the hydrophilic nature of saponins as quantified by the PIT-slope method. Indeed, Q-Naturale 200V and Tween 80 emulsions have been compared by Yang et al. [START_REF] Yang | Formation and stability of emulsions using a natural small molecule surfactant: Quillaja saponin (Q-Naturale®)[END_REF] in the formulation of O/W food emulsions, showing that they have similar interfacial properties. Figure 6A indicates the influence of 1wt.% saponin surfactant on the droplet distribution of model emulsion formulated under the same stirring conditions (2min at 16000rpm Ultraturrax).

At first sight, all systems present a monomodal distribution. However a small population (indicated by the corresponding color arrow in Figure 6A) is always present with droplet diameters smaller than those of the major distribution, explaining the lower calculated values of the Sauter mean diameters (d(3,2)) compared with the apparent mean volume diameter (dv0.5). FF saponin shows a slightly lower d(3,2) than the two commercial saponins (17.1 m versus 23.9 and 24.4 m). Figure 6B and 6C shows the photo of emulsions formulated with Q-Naturale 200 and FF observed by optical microscopy confirming the distribution size obtained using the Mastersizer 3000. Although the droplet size is relatively large, no coalescence was observed in any of the samples during the 7-days period. However, all samples presented, as expected, a slow creaming phenomenon as indicated by the transmittance profiles from Turbiscan (see the FF profile on Figure SI3 in the supplementary information). The absence of coalescence confirms the good emulsifying properties of saponins as reported in the literature [START_REF] Böttcher | Saponins -Self-assembly and behavior at aqueous interfaces[END_REF]. All these findings show that FF saponins also have very good emulsifying properties. The concentration of surfactant is an important parameter into the formulation of emulsions. Figure 7 shows the influence of saponin FF concentration on influence on d(3,2) and dv(0.5). As expected, when increasing the FF concentration the droplet size decreases by a large and fast coverage of the oil/water interface. An asymptotic behavior is reached in the vicinity of 0.5%, under the emulsification conditions, this concentration is enough to cover the interface and the excess of saponin remains in the the continuous phase. In order to compare the role of FF as emulsifier with a surfactant of same PIT-slope, Figure 7A shows the droplet size for emulsions formulated with different concentrations of Tween 40. At 1 wt.%, the size obtained with the polyethoxylated surfactant is slightly lower. However, when the concentration diminishes until 0.05 wt.% the difference increases and the size is higher for the emulsions with Tween 40. Figure 7B show the delta transmittance (ΔT) measured at each height at different times for emulsions formulated with saponins FF and Tween 40 at 0.05wt.%. ΔT indicates the difference of the transmittance of the sample compared to the signal at t=0. Figure 7B show that both emulsions present a clarification phenomenon with time (high ΔT signal in the bottom zone of the tube). Comparing the profiles at the same time, the clarification is faster for the Tween than for the FF saponin, as expected for the higher size of their droplets at this concentration. Even if both emulsions have the same water fraction, the height of the clarified aqueous phase is slightly lower in the FF saponin emulsion. No coalescence is observed nor in transmittance nor in backscattering profiles.

The influence of the water fraction was also studied, changing from fw = 0.5 to more "common" formulations namely fw = 0.3, 0.7 and 0.9. The emulsion with a higher proportion of oil (fw =0.3) does not present nor coalescence nor creaming for 7 days (figure SI4 on the supplementary information). The high content of internal phase increases the viscosity of the emulsion [START_REF] Briceño | Rheology of suspensions and emulsions[END_REF], decreasing the creaming phenomena observed for the other emulsions with lower content of oil. The size of these emulsions are similar to the system at fw = 0.5 represented in Figure 7A (with d(3,2)=15.1µm for fw = 0.3, 14.5µm for fw = 0.7 and 15.3 µm for fw=0.9). The fact that the droplet sizes are equivalents for the sample with the higher and the lower amount of dispersed oil, indicates that the concentration of 1 wt.% of saponin is enough to fully cover the surface of the generated droplets at fw = 0.3, and that it is largely in excess when the oil content is lower as in the samples at fw = 0.7 and fw = 0.9. This result confirms the asymptotic behavior observed in Figure 7A when fw = 0.5.

In order to prevent the creaming phenomena, the emulsification protocol could be optimized to reduce the droplet. At a water fraction of 0.9, and the same concentration of FF saponins (1 wt.%), a nanoemulsion (d(3,2)=200nm) can be obtained using ultrasonic sonotrode for 2 minutes. The size is clearly lower that the obtained with Ultraturrax (see figure SI5 on the supplementary information) and there is no creaming nor coalescence in 7 days. At this concentration using other stirring methods (highpressure homogenizer [START_REF] Zhu | Comparison of natural and synthetic surfactants at forming and stabilizing nanoemulsions: Tea saponin, Quillaja saponin, and Tween 80[END_REF] or microfluidizer [START_REF] Yang | Formation and stability of emulsions using a natural small molecule surfactant: Quillaja saponin (Q-Naturale®)[END_REF]) nanoemulsions with the approximately the same size (200-250 nm) have been also reported in literature with QS, Tea saponin and Q-Naturale 200V.

Foaming properties

The ability of the 3 saponins to produce and stabilize the foam was investigated. In addition, Tween 40 and a commercial sodium laureth sulfate (Texapon N70) were also studied. The Tween 40 by its PITslope identical to the FF and the Texapon N70 by its well-known foaming properties that make it a typical ingredient in shampoo formulations [START_REF] Barel | Handbook of cosmetic science and technology[END_REF]. All surfactants were tested at a concentration equal to 2 times its CMC (the foaming is maximum at concentrations near or higher than CMC [START_REF] Salager | Mousses -Formation, formulation et propriétés[END_REF]). The CMC values for Tween 40 and Texapon N70 are those reported by Ghosh [START_REF] Ghosh | Surface chemical and micellar properties of binary and ternary surfactant mixtures (cetyl pyridinium chloride, Tween-40, and Brij-56) in an aqueous medium[END_REF] and Fieber [START_REF] Fieber | Towards a general understanding of the effects of hydrophobic additives on the viscosity of surfactant solutions[END_REF], respectively (the weight concentrations for all experiments are indicated in table 3). Figure 7 shows the foaming profile of the FF saponin and Texapon N70. This profile includes the initial period of time when the flow of air passes into the 50mL of surfactant solution until the total height (foam+liquid) of 180mm is reached. Then the stability is followed for 1200 seconds. The others saponins and Tween 40 profiles are not shown in the same figure to maintain its clarity. The kinetics of the foam formation is quantified by the kf parameter, the speed in which the maximum height of foam is reached, as schematized in Figure 8. Table 2 contains all the parameters for the 3 saponins. The time required to reach the initial total height are comparable for the three saponins and the kf constants are similar. For the Tween 40 the value is lower and for the Texapon N70 slightly higher, indicating that the foamability decreasing order is: Texapon N70 > Saponin S0019 ≈ Q-Naturale 200 ≈ Saponin FF > Tween 40 However, their foam densities are different and the Texapon N70 and the Saponin from TCI incorporate much more liquid than others surfactants and these foams present the smaller size of bubbles at the beginning of the study. Indeed, when the total height of 180mm was reached, the difference on the foam density is easily pointed out in the liquid height of Texapon N70 that present deepest minima compared to the FF saponin (red dotted and continuous line in Figure 8 for Texapon and FF saponin, respectively). Concerning the stability, the 85.1% of the initial FF foam still remains after 1200s, a slightly high value than obtained with the anionic surfactant (80%) and considerably higher than the obtained with Tween 40 (8%). The decreasing order of stability is: Q-Naturale 200 ≈ Saponin S0019 > Saponin FF > Texapon N70 >> Tween 40 The average size of the bubbles was followed at a height of 100 mm and it evolves from 116 (at t=0) to 270 μm (at t=1200s) for the FF saponin. The increase on the size of bubbles is slightly higher than for the other two saponins but it is considerably lower than the change quantified to the Texapon N70. For Tween 40, the measure at 1200s was impossible because there is no foam at the fixed height. The big difference on the mechanism of stabilization in foam surfaces between typical ionic and non-ionic surfactants is well stablished in literature [START_REF] Petkova | Foamability of aqueous solutions: Role of surfactant type and concentration[END_REF]. For ionics, the stability is due to the electrostatic repulsion and for non-ionics via steric repulsion, needing different levels of surface coverage to get the same effect. The good foam stability properties of some saponins have been ascribed to their "high resistance to gas transfer of the saponin adsorption layers" [START_REF] Tcholakova | Role of surface properties for the kinetics of bubble Ostwald ripening in saponin-stabilized foams[END_REF]. Once again, the diversity of origin of the different saponins (and compositions) shows different behaviors [START_REF] Böttcher | Interfacial Properties of Saponin Extracts and Their Impact on Foam Characteristics[END_REF]. However, the properties of foam formulated with FF are similar to those measured for the other two commercial saponins and the slightly ionic behavior determined by the PIT-slope could also be at the origin of the good foaming behavior.

Conclusion

The interest on new sustainable surfactants is growing in order to substitute petrochemical synthetic surfactants. Saponins are one of the most interesting alternatives and this work study the extract from Furcraea foetida as a new source of these surfactants from as a by-product of waste from the production of fibers. The described extraction process is easily reproducible on a large scale for future industrial applications. The solvents can be recycled after each extraction and reused. The most important saponins and flavonoids in the samples were described. Surface tension of FF extract allows determining the CMC (0.025 wt.%) in the expected range of those reported in literature. The hydrophilicity-lipophilicity behavior for FF and two commercial saponins (QS) was determined by the PIT-slope method and all saponins are hydrophilic: PIT slope Q-Naturale 200V > FF > S0019 (TCI). This method confirms also the ionic character of some compounds present in these samples, and for Q-Naturale 200V the conductivity-temperature profiles, when mixed with the C10E4/n-octane/0.01M NaCl system, presented a behavior comparable to well defined ionic surfactants, even if its PIT-slope value is close to high hydrophilic commercial polyethoxylated surfactants as C12E10. The PIT-slope of FF is identical to the value reported for Tween 40. The increasing conductivity and the decrease of pH when increasing FF saponin concentration indicate the presence of ionic compounds of acid character.

The surface properties of saponins were studied in dispersed systems: O/W emulsions and aqueous foams were formulated with all samples and compared with common benchmarks in these applications. Increasing the concentration of the FF saponin diminishes the size droplet of the emulsion at fw=0.5 but for concentrations higher than 0.5% wt. the size remains identical indicating a saturation at the interface. At low concentrations (0.05% wt.), the size droplet of the FF saponin emulsion is lower than the Tween 40 and the clarification is slower. The good properties, at very low concentrations, indicate the high potential of FF saponins used as emulsifier. If all saponins are compared at the same concentration and water fraction (1% wt. / fw=0.5) their stability and size are similar. When the water fraction changes until 0.3, very stable emulsions without clarification are obtained. Foaming properties of FF saponins are similar to those of other saponins and even more stables than those obtained with sodium laureth sulfate (Texapon N70). The decreasing order of foam stability is Q-Naturale 200 ≈ Saponin S0019 > Saponin FF > Texapon N70 >> Tween 40. The smaller initial size of foam bubbles is obtained with Saponin S0019 (comparable to Texapon N70), probably due to the higher foam initial density. FF is a promising source of saponins with emulsifier and foaming properties as good as those described for commercial saponins. Moreover, toxicity and organoleptic properties must be studied to extend the application of these saponins in food and cosmetic domains.

Figure 1 .

 1 Figure 1. Extraction procedure of saponins from Furcraea foetida.

Figure 2 .

 2 Figure 2. Chromatographic profiles (LC/MS) of insoluble isopropanol extract from Furcraea foetida leaves. A) Chromatogram in negative mode (ESI-). B) Chromatogram in positive mode (ESI+).

  crestagenin 3-O-β-D-glucopyranosyl-(1→4)-O-β-D-glucopyranoside (peak N°30) and a chlorogenin 6-O-[β-D-glucopyranosyl]-(1→4)-O-β-D-glucopyranoside (peak N°47).

Figure 3 .

 3 Figure 3. Structures of flavonoids and two major saponins in Furcraea foetida

Figure 4 .

 4 Figure 4. Surface tension profiles as a function of saponin concentrations (wt.%). Dotted lines indicate the logarithmic fit. Empty cercles indicate the CMC. Confidence limits of CMC are calculated as recommended by Miller [42].

Figure 5 .

 5 Figure 5. A) Conductivity-Temperature profile at several weight percentages of saponins from Furcraea foetida. (Data from the second heating cycle). B) Phase inversion temperature of the system 3 wt.% C10E4/n-octane/0.01M NaCl(aq) at fw=0.5 versus weight percentage of added saponins. Data from the second heating cycle.

Figure 6 .

 6 Figure 6. A) Droplet size distribution of prepared emulsions using different saponins in the 1wt.% Saponin/Isopropyl myristate/0.01M NaCl(aq) system (fw=0.5). B) Observation of Q-Naturale and FF emulsion (C) using an optical microscope. Red bar indicates 20μm.

Figure 7 .

 7 Figure 7. A) Influence of Saponins FF and Tween 40 concentration on the mean droplet diameter (d(3,2)) and volume diameter dv(0.5) for Surfactant/IPM/Water emulsion at fw=0.5. B) Delta Transmittance (ΔT) versus sample height and time at 25°C and for 0.05%Surfactant/IPM/Water emulsions at fw=0.5.

Figure 8 .

 8 Figure 8. Foaming profile, height (mm) vs. time (s) for FF saponin (0.048 wt.%) and TexaponN70 (0.054 wt.%) at 2CMC.

200 g Dried chevenots from Furcraea foetida

	Equipment. Structural identification was performed in high pressure liquid chromatography coupled
	with mass spectrometry (LC/MS) at the Institut de Chimie, de Biochimie Moléculaire et
	Supramoléculaire (ICBMS), University of Claude Bernard Lyon 1 using ultra-performance liquid
	chromatography (UPLC-U3000, Thermo Fisher Scientific) coupled to a high resolution hybrid mass
	spectrometer of four pole time flight type.
		Soxhlet
		95-110°C, 6 hours
		Filtration
	n-butanol extract
	R = 12.2 %
		Precipitation in Ethyl
		acetate 0.1 Kg/L
		Depigmentation
	Ethyl acetate soluble extract	Ethyl acetate insoluble extract
	(chlorophyll and pigments)	(Flavonoids and saponins)
	R = 5.8 %	R = 6.0 %
		Recrystallization with isopropanol 0.1 Kg/L
	Isopropanol soluble extract	Isopropanol insoluble extract
	(Flavonoids)
	R = 1.8 %	Drying
		Saponins
		R = 2.1 %

Table 1 .

 1 Critical Micellar Concentration (CMC), surface tension at CMC (mN/m), surface excess concentration of saponins (mol/cm 2 ), area per molecule (𝐴 2), PIT-slope (°C/wt.%)

	Surfactants	CMC (wt.%)	γCMC (mN/m)	Γmax•10 10 (mol/cm 2 ) *a	𝑠 𝑎 1 (𝐴 2)	PIT-slope (°C/wt.%)
	Saponins from Furcraea foetida (FF)	0.024	49.2	2.2±0.1	75±4	10.2±0.8
	Saponin S 0019 (TCI)	0.043	39.1	2.2±0.1	77±4	9.5±0.2
	Q-Naturale 200V (Ingredion)	0.0026	43.5	3.9±0.4	43±5	18±1

a Note that the surface excess can be calculate without knowing the molecular weight of the sample because the units of the concentration on equation 4 are irrelevant 𝑑𝑙𝑜𝑔𝐶 ≅ ΔlogC=log(C1/C2).

Table 2 .

 2 Foam characterization. Foaming speed (kf), foam density, foam stability at 1200s and average size of droplets when the height of foam is maximal (t=0) and at the end of the 1200s.

	Surfactants	Concentration (wt.%)	kf (mm/s)	Foam density at hmax	Foam stability FS1200	R0 (μm 2 )	R1200 (μm 2 )
	Saponins
	Furcraea foetida	0.048	3.1±0.1	9±1	85.1±0.8	116±9	270±60
	(FF)
	Saponin S0019	0.086	3.32±0.06	15±1	89.1±0.5	76±14	124±26
	Q-Naturale 200	0.0052	3.15±0.02	6±1	92±1	174±40 264±75
	Tween 40	0.0059	2.6±0.2	3.1±0.4	8±2	236±21	n.d.

ACKNOWLEDGMENTS

L'association Universitaire Francophone (AUF) is thanked by financing the travel of TR in Lille, France. Chevreul Institute (FR 2638), Ministère de l'Enseignement Supérieur et de la Recherche, Région Hautsde-France, and FEDER are acknowledged for supporting and funding partially this work. We are grateful to Yves Koidjane who formulated and characterized the emulsions by ultrasound during his Master internship.


Yandong Ma

Zhihua Zhang

Fan Sun

Pierre Mesdom

Xin Ji

Pierre Burckel

Gilles Gasser

Min-Hui Li
email: min-hui.li@chimieparistech.psl.eu

Red-light Responsive Polypeptoid Nano-assemblies Containing a Ruthenium(II) Polypyridyl Complex with Synergistically Enhanced Drug Release and ROS Generation for Anticancer Phototherapy

Keywords: Polymer vesicle, polypeptoid, Ru(II) complex, light-activated cleavage, photoresponsive release, phototherapy

Polymer micelles/vesicles made of a red-light-responsive Ru(II)-containing block copolymer (PolyRu) are elaborated as a model system for anticancer phototherapy. PolyRu is composed of PEG and a hydrophobic polypeptoid bearing thioether side chains, 40% of which are coordinated with [Ru(2,2′:6′,2′′-terpyridine)(2,2′-biquinoline)](PF6) 2 via Ru-S bond resulting in 67wt% of Ru complex loading capacity. Red-light illumination induces the photocleavage of Ru-S bond and produces [Ru(2,2′:6′,2′′-terpyridine)(2,2′-biquinoline)(H2O)](PF6)2. Meanwhile, ROS are generated under the photosensitization of Ru complex and oxidize hydrophobic thioether to hydrophilic sulfoxide, causing the disruption of micelles/vesicles. During the disruption, ROS generation and Ru complex release are synergistically enhanced. PolyRu micelles/vesicles are taken up by cancer cells, while they exhibit very low cytotoxicity in the dark. In contrast, they show much higher cytotoxicity under red-light irradiation. PolyRu micelles/vesicles are promising nano-assembly prototypes that protect metallodrugs in the dark but exhibit light-activated anticancer effects with spatiotemporal control for photoactivated chemotherapy and photodynamic therapy.

INTRODUCTION

Polymer nano-assemblies as drug delivery systems have drawn an increasing attention for cancer therapy, since they can circumvent some pharmacological limitations of small molecular medicine such as poor water solubility, nonspecific distribution, systemic toxicity, etc. [START_REF] Mitchell | Engineering precision nanoparticles for drug delivery[END_REF][START_REF] Sun | Engineered nanoparticles for drug delivery in cancer therapy[END_REF] Moreover, stimuliresponsive polymer nano-assemblies further improve the bioavailability of drugs through controllable drug release in response to endogenous stimuli (such as pH variation, redox gradient, enzymes, etc) 3- 5 or exogenous stimuli (such as light, temperature change, ultrasound, magnetic field, etc). [START_REF] Boissenot | Ultrasound-triggered drug delivery for cancer treatment using drug delivery systems: From theoretical considerations to practical applications[END_REF][START_REF] Gohy | Photo-responsive block copolymer micelles: design and behavior[END_REF][START_REF] Liu | Thermoresponsive Polypeptoids[END_REF][START_REF] Tombacz | Magnetic iron oxide nanoparticles: Recent trends in design and synthesis of magnetoresponsive nanosystems[END_REF][START_REF] Uppalapati | Conducting polymers with defined micro-or nanostructures for drug delivery[END_REF] Among these stimuli, light-responsive drug release systems have been actively studied due to their convenient remote-control and exceptional spatiotemporal resolution. [START_REF] Sun | Engineered nanoparticles for drug delivery in cancer therapy[END_REF] This work aims to construct lightresponsive polymer micelles and vesicles based on an amphiphilic polypeptoid containing a Ru(II) complex using red-light as stimulus (Scheme 1). The polypeptoid, composed of N-thioether substituted amino acids, [START_REF] Gangloff | Peptoids and Polypeptoids at the Frontier of Supra-and Macromolecular Engineering[END_REF] is chosen because of its exceptional biocompatibility and biodegradability. [START_REF] Li | Synthesis and Characterization of Cleavable Core-Cross-Linked Micelles Based on Amphiphilic Block Copolypeptoids as Smart Drug Carriers[END_REF][START_REF] Ulbricht | On the biodegradability of polyethylene glycol, polypeptoids and poly(2-oxazoline)s[END_REF] The Ru(II) complex with cleavable Ru(II)-thioether coordination bond (noted as Ru-S hereinafter) is chosen as prodrug model since Ru-S bond can be cleaved by long-wavelength lights and the released Ru complex can be used in both photoactivated chemotherapy and photodynamic therapy. [START_REF] Bonnet | Why develop photoactivated chemotherapy?[END_REF][START_REF] Knoll | Control and utilization of ruthenium and rhodium metal complex excited states for photoactivated cancer therapy[END_REF][START_REF] Liu | Functionalization and cancer-targeting design of ruthenium complexes for precise cancer therapy[END_REF][START_REF] Mahmud | Ruthenium Complexes: An Alternative to Platinum Drugs in Colorectal Cancer Treatment[END_REF][START_REF] Mari | Combination of Ru(ii) complexes and light: new frontiers in cancer therapy[END_REF] The long-wavelength lights like red or near-infrared (NIR) light show higher tissue penetration and cause less photodamage to biological systems than UV light frequently reported in light-responsive systems. [START_REF] Wu | Near-infrared-responsive functional nanomaterials: the first domino of combined tumor therapy[END_REF] Ru(II) complexes bearing terpyridyl-like or other sterically hindered ligands can selectively photo-substitute one of the ligands by solvent molecule(s). [START_REF] Bahreman | Spontaneous formation in the dark, and visible light-induced cleavage, of a Ru-S bond in water: a thermodynamic and kinetic study[END_REF][START_REF] Laemmel | Efficient and Selective Photochemical Labilization of a Given Bidentate Ligand in Mixed Ruthenium(II) Complexes of the Ru(phen)2L2+ and Ru(bipy)2L2+ Family (L = Sterically Hindering Chelate)[END_REF] Such reactivity is explained by their low-lying, metal centered ( 3 MC) excited states with dissociative character, which are thermally populated from the photochemically generated metal-to-ligand charge-transfer ( 3 MLCT) excited states under visible and NIR light illumination. These light-induced substitution reactions have been used as an interesting way to activate caged bioactive ruthenium complexes or ligands both in small molecular systems [START_REF] Meijer | Diastereoselective Synthesis and Two-Step Photocleavage of Ruthenium Polypyridyl Complexes Bearing a Bis(thioether) Ligand[END_REF][START_REF] Goldbach | N-Acetylmethionine and Biotin as Photocleavable Protective Groups for Ruthenium Polypyridyl Complexes[END_REF][START_REF] Howerton | Strained Ruthenium Complexes Are Potent Light-Activated Anticancer Agents[END_REF][START_REF] Wachter | Light-activated ruthenium complexes photobind DNA and are cytotoxic in the photodynamic therapy window[END_REF][START_REF] Van Rixel | Photo-Uncaging of a Microtubule-Targeted Rigidin Analogue in Hypoxic Cancer Cells and in a Xenograft Mouse Model[END_REF] and in polymer self-assembly drug delivery systems. [START_REF] Sun | Ruthenium-Containing Block Copolymer Assemblies: Red-Light-Responsive Metallopolymers with Tunable Nanostructures for Enhanced Cellular Uptake and Anticancer Phototherapy[END_REF][START_REF] Sun | An Amphiphilic Ruthenium Polymetallodrug for Combined Photodynamic Therapy and Photochemotherapy In Vivo[END_REF][START_REF] Chen | Red-Light-Responsive Metallopolymer Nanocarriers with Conjugated and Encapsulated Drugs for Phototherapy Against Multidrug-Resistant Tumors[END_REF][START_REF] Zeng | Amphiphilic Metallodrug Assemblies with Red-Light-Enhanced Cellular Internalization and Tumor Penetration for Anticancer Phototherapy[END_REF] The Ru complex used in this work is [Ru(tpy)(biq)(H2O)](FP6)2 (Ru = ruthenium, tpy = 2,2′:6′,2′′-terpyridine and biq = 2,2′-biquinoline) since it has been reported previously in small molecular systems and in polymer nano-assemblies for anticancer application. [START_REF] Van Rixel | Photo-Uncaging of a Microtubule-Targeted Rigidin Analogue in Hypoxic Cancer Cells and in a Xenograft Mouse Model[END_REF][START_REF] Sun | Ruthenium-Containing Block Copolymer Assemblies: Red-Light-Responsive Metallopolymers with Tunable Nanostructures for Enhanced Cellular Uptake and Anticancer Phototherapy[END_REF][START_REF] Askes | Activation of a Photodissociative Ruthenium Complex by Triplet-Triplet Annihilation Upconversion in Liposomes[END_REF] The different manners to encapsulate Ru complexes into nanomaterials were recently reviewed. [START_REF] Karges | Encapsulation of Ru(II) Polypyridine Complexes for Tumor-Targeted Anticancer Therapy[END_REF][START_REF] Soliman | Incorporation of Ru(II) Polypyridyl Complexes into Nanomaterials for Cancer Therapy and Diagnosis[END_REF][START_REF] Villemin | Polymer encapsulation of ruthenium complexes for biological and medicinal applications[END_REF][START_REF] Zeng | The development of anticancer ruthenium(ii) complexes: from single molecule compounds to nanomaterials[END_REF] For example, in reported Ru-containing polymer micelles and vesicles, [START_REF] Sun | Ruthenium-Containing Block Copolymer Assemblies: Red-Light-Responsive Metallopolymers with Tunable Nanostructures for Enhanced Cellular Uptake and Anticancer Phototherapy[END_REF] a non-biodegradable polymethacrylate was used as polymer backbone, to which Ru complexes were coordinated via cyano-groups. Here, biocompatible and biodegradable PEGylated polypeptoid, PEG-b-PMeSPG (PEG = poly(ethylene glycol), PMeSPG = poly(N-3-(methylthio)propyl glycine)), [START_REF] Deng | Synthesis and self-assembly of poly(ethylene glycol)-block-poly(N-3-(methylthio)propyl glycine) and their oxidation-sensitive polymersomes[END_REF] with a thioether-bearing polypeptoid PMeSPG is used and [Ru(tpy)(biq)](PF6)2 groups are attached to the hydrophobic block PMeSPG via Ru-S bond.

Concretely, [Ru(tpy)(biq)(thioether)](PF6)2 is formed via the exchange reaction between aqua ligand in [Ru(tpy)(biq)(H2O)](PF6)2 and thioether ligand in the dark. Nearly 40% of PMeSPG side chains are coordinated with Ru complexes resulting in 67wt% Ru complexes in the final amphiphilic block copolymer PolyRu. Depending on the co-solvent used, PolyRu is self-assembled into micelles and vesicles by nanoprecipitation. Amphiphilic polypeptoids bearing thioether groups and their polymersomes are oxidation responsive, as demonstrated previously by our group. [START_REF] Deng | Synthesis and self-assembly of poly(ethylene glycol)-block-poly(N-3-(methylthio)propyl glycine) and their oxidation-sensitive polymersomes[END_REF][START_REF] Deng | Oxidation-Sensitive Polymersomes Based on Amphiphilic Diblock Copolypeptoids[END_REF] The hydrophobic thioether groups in the hydrophobic polypeptoid block can be transformed into more polar sulfoxide or sulfone groups under the action of oxidants like H2O2 generated enzymatically or reactive oxygen species (ROS) generated photochemically in the presence of a photosensitizer. Consequently, porous structures appeared in the vesicular membranes and polymersome bursting or disruption occurred eventually, because a part of amphiphilic block copolymers become entirely hydrophilic. Actually, as many Ru complex, [Ru(tpy)(biq)(L)](PF6)2 (L = H2O or -S-) is also a photosensitizer that can produce ROS to kill cancer cells in photodynamic therapy (PDT), [START_REF] Knoll | Control and utilization of ruthenium and rhodium metal complex excited states for photoactivated cancer therapy[END_REF][START_REF] Albani | Marked improvement in photoinduced cell death by a new tris-heteroleptic complex with dual action: singlet oxygen sensitization and ligand dissociation[END_REF][START_REF] Zhang | Combining Ruthenium(II) Complexes with Metal-Organic Frameworks to Realize Effective Two-Photon Absorption for Singlet Oxygen Generation[END_REF] and to also oxidize hydrophobic thioether to hydrophilic sulfoxide. The change of hydrophobic/hydrophilic balance within amphiphilic copolymers then result in the disassembly or dissolution of their nano-assemblies and may synergistically facilitating the Ru complexes release despite the low biological ROS concentration (50 µM -100 µM). [START_REF] Wang | Self-Strengthened Oxidation-Responsive Bioactivating Prodrug Nanosystem with Sequential and Synergistically Facilitated Drug Release for Treatment of Breast Cancer[END_REF][START_REF] Dai | Photosensitizer enhanced disassembly of amphiphilic micelle for ROS-response targeted tumor therapy in vivo[END_REF] In this work, the photocleavage of the Ru-S coordination bond and their influence on the PolyRu nano-assemblies were studied. Red-light activated ROS generation and the Ru complexes release synergistically boosted by ROS were investigated. Finally, cellular uptake and cytotoxicity assay of PolyRu micelles and vesicles were performed. Interestingly, although the PEGylation exhibited less non-specific cellular uptake, [START_REF] Schottler | Protein adsorption is required for stealth effect of poly(ethylene glycol)-and poly(phosphoester)-coated nanocarriers[END_REF][START_REF] Zhang | Polymersomes with Red/Near-Infrared Emission and Reactive Oxygen Species Generation[END_REF] the introduction of Ru complexes in the PEGylated polypeptoid nano-assemblies could increase the cancer cellular uptake. PolyRu micelles and vesicles showed very low cytotoxicity in dark (IC50 > 280 µM) and high cytotoxicity under red-light activation (IC50 ~ 20 -30 µM) which was even higher than that of the free aqua Ru complexes (IC50 ~ 45 µM). Therefore, PolyRu nano-assemblies reported here represent an interesting prototype for ruthenium-based photoactivated chemotherapy and photodynamic therapy. Porous micelles and vesicles improve the oxygen access and enhance further ROS generation. The whole process causes rapid release of Ru complexes for anticancer therapy.

EXPERIMENTAL SECTION

Synthesis of Ru complex and amphiphilic diblock copolymers PEG-b-PMeSPG and PolyRu

[Ru(tpy)(biq)(H2O)](PF6)2 was synthesized as reported in the litterature. [START_REF] Sun | Ruthenium-Containing Block Copolymer Assemblies: Red-Light-Responsive Metallopolymers with Tunable Nanostructures for Enhanced Cellular Uptake and Anticancer Phototherapy[END_REF][START_REF] Bahreman | Spontaneous formation in the dark, and visible light-induced cleavage, of a Ru-S bond in water: a thermodynamic and kinetic study[END_REF][START_REF] Boyer | Effects of a proximal base on water oxidation and proton reduction catalyzed by geometric isomers of [Ru(tpy)(pynap)(OH2)]2+[END_REF] The monomer N-3-(methylthio)propyl glycine N-thiocarboxyanhydride (MeSPG-NTA) was synthesized according to our previous work. [START_REF] Deng | Oxidation-Sensitive Polymersomes Based on Amphiphilic Diblock Copolypeptoids[END_REF] The polymer PEG-b-PMeSPG was prepared by ring opening polymerization of MeSPG-NTA using PEG-NH2 as macro-initiator. [START_REF] Deng | Synthesis and self-assembly of poly(ethylene glycol)-block-poly(N-3-(methylthio)propyl glycine) and their oxidation-sensitive polymersomes[END_REF] The detailed procedures and characterizations are described in the Supporting Information.

To synthesize the Ru complex-functionalized amphiphilic diblock copolymer, PolyRu, PEG-b-PMeSPG (35 mg, 0.0075 mmol) and Ru complex [Ru(tpy)(biq)(H2O)](PF6)2 (182 mg, 0.2025 mmol) were dissolved into acetone (8 mL) in a 25 mL Schlenk tube. After being degassed for 30 min, the mixture was stirred at room temperature under argon for 72h in the dark. The reaction mixture was participated from ethanol to obtain the crude polymer, which was further purified twice by precipitation using acetone/ethanol as solvent/non-solvent pair. The final polymer was dried in vacuum (68 mg, yield: 85.07%). Its characterization is described in the Supporting Information.

Self-Assembly of PolyRu and of PEG 45 -b-PMeSPG 18

PolyRu (2.0 mg) was dissolved in 0.2 mL DMF or 0.2 mL THF/acetone mixture (with different ratio) and stirred for 0.5 h. A total of 1.8 mL of milli-Q water was slowly injected into the solution at a speed of 0.6 mL/min with slight shaking. Then, the colloidal dispersion was further stirred for 0.5 h and dialyzed against Milli-Q water for 8 h to remove organic solvents using a dialysis tube (Mw cutoff, 3.5 kDa). In the dialysis process, Milli-Q water was replaced approximately every 1 h. The preparation of PEG45-b-PMeSPG18 nanoparticles was conducted in the same way using DMF as organic solvent.

The loading capacity of Ru complex in the polymer nano-assemblies can be calculated by the following equation:

Characterization of Polymer nano-assemblies

Dynamic light scattering (DLS). The hydrodynamic diameters of the self-assembles and their surface Zeta potentials were measured by Zetasizer Nano Series (Malvern Instruments) at 25 °C. For size analysis, the samples were measured at a fixed angle at 90° and a wavelength of 657 nm.

Loading capacity (wt%) = Weight of Ru complex in the PolyRu nano-assembly Weight of PolyRu nano-assembly UV-visible absorbance and spectrofluorometers. UV spectra were measured by a Milton Ray Spectronic 3000 Array spectrophotometer. Photoluminescence (PL) spectra were collected on a Horiba FluoroMax Spectrofluorometers.

Attenuated Total Reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The measurements were conducted with a Nicolet Magna-IR 550 FTIR spectrometer with 50 scans.

Scanning electron microscope (SEM). SEM images were observed by a Zeiss Leo 1530 FEG-SEM.

Briefly, the silicon wafers were treated in 100 mL mixture of H2SO4/H2O2 (v/v, 3/1) at 90 ℃ for 2h.

After hydrophilizing, the silicon wafers washed by Milli-Q water, acetone, and ethanol successively, then dried with liquid nitrogen before use. To prepare SEM sample, a drop of dispersion of polymer nano-assemblies was added onto silicon wafer and freeze-dried.

Transmission electron microscopy (TEM).

The morphology of the polymer nano-assemblies was imaged by a JEOL 2100 Plus transmission electron microscope operated at 200kV. To prepare TEM grid, 5 μL of dispersion of polymer nano-assemblies was added on a carbon-covered 200-mesh holey copper grid (Ted Pella Inc., U.S.A.) and dried under vacuum.

Cell uptake, intracellular ROS detection and cytotoxicity assays

Cell Culture. The A549 cell line was incubated in F-12k media (Gibco) supplemented with 10% fetal calf serum (Gibco) and 1% Penicillin-Streptomycin antibiotic (Gibco). Cells were used in the assays one week after the end of the treatment to avoid interfering with the results.

Cell uptake tests by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). A549 cells were

incubated at a density of 1 × 10 6 cells/dish. After 24 h, cells were treated with 10 μM of PolyRu micelles and vesicles as well as 70 μM of Ru complexes (all at 70 μM equivalent concentration of Ru complexes) in the cell culture medium, respectively. Cells were collected after different time of treatment (2 h, 4 h, 6 h and 8 h), counted, and stored at -80 °C. For the analysis by ICP-MS, all cellular samples were digested in 70% HNO3 (0.5 mL, 70 °C, overnight) and subsequently diluted 1:100 (1% HCl solution in Mili-Q water). ICP-MS measurements were performed on an Agilent 7900 Quadrupole ICP-MS located at the Institut de Physique du Globe de Paris (France). The Ru content of each sample was detected using 101 Ru as monitored isotope without collision gas for higher sensitivity. The uncertainties were calculated by error propagation equations, considering the combination of standard deviation (SD) on the replicated samples (n = 3), internal standard ratio and blank subtraction. The amount of metal detected in the cell samples was transformed from ppb to μg o1f metal. Data were then normalized to the number of cells and expressed as nanograms of metal/number of cells.

Intracellular ROS detection by flow cytometry. A549 cells (5×10 5 cells well-1) were seeded in the 6-well plates and cultured for 24 h. After treatment with medium alone, Ru complexes, PolyRu micelles and vesicles (70 µM of Ru complexes) for 6 h, the cells were washed with PBS and then incubated with 1 mL fresh medium containing 10 μM dichloro-dihydro-fluorescein diacetate (DCFH-DA) for 30 min at 37 ℃. After washing thrice with PBS, the cells were exposed to red light irradiation (λ = 625 nm, 11.57 mW/cm 2 ) for 30 min. Subsequently, the cells were trypsinized and collected, and their mean fluorescence was analyzed by flow cytometry (λex = 488 nm, λem = 525 nm) to evaluate the intracellular ROS level.

Cytotoxicity Assay in the dark and under red-light irradiation. The cytotoxicity of Ru complex, PolyRu micelles or PolyRu vesicles was accessed by a fluorometric cell viability assay using Resazurin (Acros Organics). Briefly, A549 cells were seeded in triplicate in 96-well plates at a density of 5×10 3 cells/well in 100 μL. After incubation at 37 ℃, 5% CO2 for 24 h, the cell medium was replaced by a series of solutions of Ru complex, PolyRu micelles or PolyRu vesicles with increasing concentrations (100 µL/well). Ru complex solution with concentrations from 0.03 µM to 280 µM were diluted with media from 10 mM stock solution of Ru complex in DMSO. PolyRu micelles and vesicles with concentrations from 0.01 µM to 40 µM (polymer PolyRu concentration) were diluted with media from their initial dispersion in water. The cells were then incubated with different Ru samples for 6h. For phototoxicity assay, cells plates were irradiated under red light (625 nm, 11.57 mW/cm 2 ) for 1 h. For dark toxicity assay, cells plates were kept in the dark. After another 24 h of incubation, the medium was removed and 100 μL of complete medium containing resazurin (0.2 mg/mL final concentration) was added in each well. After 4 h of incubation at 37 °C, the fluorescence signal of the resorufin product transformed from resazurin was read (ex 540 nm, em 590 nm) by Infinite 200 PRO Microplate Reader from TECAN. IC50 values were then calculated using GraphPad Prism software.

RESULTS AND DISCUSSION

Synthesis of amphiphilic block copolymer PolyRu

The synthesis of PolyRu with Ru complexes-coordinated polypeptoid block is shown in Scheme 2.

[Ru(tpy)(biq)(H2O)](PF6)2 was first synthesized as described in the literature (see SI and Scheme S1, Figure S1 andS2). [START_REF] Sun | Ruthenium-Containing Block Copolymer Assemblies: Red-Light-Responsive Metallopolymers with Tunable Nanostructures for Enhanced Cellular Uptake and Anticancer Phototherapy[END_REF] The block copolymer PEG45-b-PMeSPG was prepared by the ring-opening polymerization (ROP) of MeSPG-NTA (Figure S3) using amino-terminated PEG45-NH2 (molecular weight Mn = 2000 Da and degree of polymerization DP = 45) as a macromolecular initiator according to the procedures published previously. [START_REF] Deng | Synthesis and self-assembly of poly(ethylene glycol)-block-poly(N-3-(methylthio)propyl glycine) and their oxidation-sensitive polymersomes[END_REF] The obtained PEG45-b-PMeSPG was characterized by [START_REF] Mitchell | Engineering precision nanoparticles for drug delivery[END_REF] H NMR and Size Exclusion Chromatography (SEC) (Figure S4, Figure S5 and Table S1), which showed a polymer of Mn = 4.6 kDa with narrow molecular weight distribution (Đ =1.08) corresponding to PEG45-b-PMeSPG18 (DP = 18 for PMeSPG). [Ru(tpy)(biq)] 2+ was then coordinated with the thioether groups of the PMeSPG block in PEG45-b-PMeSPG18 (ratio Ru/thioether = 1/1) by ligand exchange between aqua and thioether to get PolyRu (Mn = 10900 Da). The block copolymer PolyRu was finally obtained by precipitating thrice in ethanol to remove unreacted [Ru(tpy)(biq)(H2O)](PF6)2. The composition of the final PolyRu was calculated according to the characteristic signals in [START_REF] Mitchell | Engineering precision nanoparticles for drug delivery[END_REF] H NMR spectrum (Figure S6). The signal "b" of the HC8 in biquinoline spatially close to the coordination bond Ru-S is upfield-shifted compared to the signal "a" of HC8 in biquinoline spatially close to Ru-H2O bond (Figure S6 andS2). However, the signal "a" still co-exists with "b" in the final PolyRu despite the purification, which corresponds approximatively to one free [Ru(tpy)(biq)(H2O)](PF6)2 with nine [Ru(tpy)(biq)(-S-)](PF6)2 connected to PMeSPG, probably because the coordination exchange between Ru-H2O and Ru-S is a reversible reaction and reaches a certain equilibrium. [START_REF] Bahreman | Spontaneous formation in the dark, and visible light-induced cleavage, of a Ru-S bond in water: a thermodynamic and kinetic study[END_REF] Evaluation of the percentage of Ru-S coordination in the block copolymer PolyRu was done by comparing the integration of signal "b" to the integration of -CH2-CH2-O-signals of PEG. Only a part of thioester groups is coordinated with [Ru(tpy)(biq)] 2+ ; Ru-S percentage is of 39%, namely 7 monomers over 18 per chain being coordinated.

PolyRu micelles and vesicles self-assembled by nanoprecipitation

The nano-assemblies of PolyRu were fabricated through nanoprecipitation method. The hydrophilic block PEG weight ratio over the copolymer weight of PolyRu, fPEG, is 22%. The control of morphology was achieved by using different organic cosolvent including THF/acetone mixture and DMF. Briefly, PolyRu was first solubilized in the organic solvent at a concentration of 1wt %. To 0.2 mL of this PolyRu solution was added dropwise 1.8 mL Milli-Q water at a rate of 0.2 mL/min with slight shaking. The dispersion was then dialyzed thrice against a large volume of water within 8 h to eliminate the organic solvent. The nano-assemblies were subsequently characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS). The loading capacity of Ru complexes in polymer micelles and vesicles was 67%, which was a relative high value due to the coordination binding of Ru complex to PMeSPG18 in PolyRu.

As revealed by SEM images (Figure 1A), micelles were the major morphology of nanoassemblies using DMF as the organic solvent. An average diameter of 13 ± 2 nm was obtained by analysing 30 micelles in the SEM images by ImageJ. By DLS (Figure 1B and Figure S7), an average hydrodynamic diameter (Dh) of 21± 1 nm (number profile) was measured. Dh is higher than the diameter measured by SEM, because DLS gave the hydrodynamic diameter of micelles in hydrated state in water, while SEM measured the diameter of micelles in dried state.

Different ratios of THF/acetone from 0/3 to 3/1 were also tested as organic cosolvent for PolyRu nanoprecipitation. There, THF is a good solvent for PEG45-b-PMeSPG18, but a bad solvent for Ru complex, while acetone is a good solvent for both PEG45-b-PMeSPG18 and Ru complex. No selfassemblies were obtained using pure acetone as cosolvent. Increasing the THF/acetone ratio, the selfassemblies could be detected by DLS, as shown in Figure S8 and Table S2. The average hydrodynamic diameters of self-assemblies increased (from 49 ± 2 nm to 68 ± 1 nm) with the THF/acetone ratio's increasing. Notably, the self-assemblies fabricated using THF/acetone ratio = 1/1 exhibited the narrowest size distribution with PDI of 0.32. Therefore, THF/acetone (1/1) was chosen as organic cosolvent to prepare the self-assemblies for the following investigation. SEM and TEM (Figure 1C and Figure S9A) images reveal the vesicular structures for these PolyRu selfassemblies, where collapsed vesicles with membranes of high contrast are visible. An average hydrodynamic diameter (Dh) of 59 ± 2 nm (number profile) with PDI of 0.32 was measured by DLS (Figure 1D and Figure S9B).

Photocleavage of Ru-S coordination bond in PolyRu and PolyRu nano-assemblies

Before the study of Ru complex photo-substitution, the stability of PolyRu and PolyRu nanoassemblies in aqueous medium in the dark needs to be first checked. As in the case of Ru complex coordinated with small thioether, [START_REF] Deng | Oxidation-Sensitive Polymersomes Based on Amphiphilic Diblock Copolypeptoids[END_REF] the UV-visible spectrum of PolyRu showed a typical metal-toligand charge transfer (MLCT) band of Ru-S with maximal absorption around 520 nm while extending to around 700 nm (Figure S10). However, the precursor, [Ru(tpy)(biq)(H2O)](PF6)2 with aqua ligand showed the MLCT band of Ru-H2O with maximal absorption around 550 nm (Figure S10). Therefore, the MLCT band can be used to follow the change of coordination in PolyRu aqueous solution and in PolyRu nano-assemblies dispersed in the water. As shown in Figure 3A, over 6 h, the MLCT absorption band of PolyRu solution in water/acetone (volume ratio 1/1) shifted from 520 nm to 550 nm with the increase of its intensity, indicating the thermal cleavage of Ru-S band to get uncaged Ru complex with aqua ligand. An equilibrium between Ru-S and Ru-H2O coordination bonds exists at room temperature. Ru-H2O can be produced in abundant water environment. [START_REF] Bahreman | Spontaneous formation in the dark, and visible light-induced cleavage, of a Ru-S bond in water: a thermodynamic and kinetic study[END_REF] Nevertheless, the MLCT band of PolyRu micelles and vesicles dispersed in the water or PBS had no remarkably change over 24 h in the dark (Figure 2B, 2C and S11). These interesting observations indicated that self-assemblies enhanced the stability of Ru moiety in PolyRu copolymer, which can be explained since water cannot easily access the hydrophobic core or layer of polymer colloids. The dark stability of micelles and vesicles is the key point in the construction of photocontrolled drug delivery systems. It is known that the Ru-S bond in the Ru complex-coordinated small thioether molecule can be cleaved in water under visible light to obtain Ru complex with aqua ligand Ru-H2O. [START_REF] Bahreman | Spontaneous formation in the dark, and visible light-induced cleavage, of a Ru-S bond in water: a thermodynamic and kinetic study[END_REF][START_REF] Meijer | Diastereoselective Synthesis and Two-Step Photocleavage of Ruthenium Polypyridyl Complexes Bearing a Bis(thioether) Ligand[END_REF] Here, this visible light-induced cleavage was first tested for block copolymer PolyRu aqueous solution, then studied in the PolyRu micelles and vesicles, especially for their photo-induced Ru complex release.

Interestingly, when 625 nm-light irradiation was applied to PolyRu solution in water/acetone (1/1) mixture (30 mW/cm 2 , 240 s), the UV-visible spectrum of the solution quickly showed a blue-shifted MLCT band similar to that of Ru-H2O (Figure 2D and Figure S10). Therefore, a photo-cleavage of Ru-S occurred and photo-induced ligand-substitution took place within 4 min in Ru complex to obtain the uncaged [Ru(tpy)(biq)(H2O)](PF6)2. 625 nm light was used since long wavelengths exhibit better tissue penetration compared to short wavelengths. [START_REF] Mallidi | Beyond the Barriers of Light Penetration: Strategies, Perspectives and Possibilities for Photodynamic Therapy[END_REF] The time evolution of the photo-cleavage was followed every 30 s from 0 to 240 s (Figure 2D) and showed the photo-substitution conversion from Ru-S to Ru-H2O reached maximum after 210 s (3.5 min) of light illumination (see also Figure S12A). This photo-substitution reaction was also confirmed by 1 H NMR spectra. As shown in Figure S13, when the irradiation time increased, the signals of the Ha near Ru-H2O bond at 6.7 ppm increased but those of the Hb near Ru-S bond at 6.37 ppm decreased.

Under 625 nm light irradiation, red shifts of MLCT band were also observed in the UV-visible absorption spectra of PolyRu micelles and vesicles as shown in Figure 2E and2F. However, the time to reach the maximal conversion took more time in the case of PolyRu micelles (tmicelle = 24 min) and vesicles (tvesicle = 36 min) than in the PolyRu polymer solution (tpolymer = 3.5 min) (see also Figure S12B andC). These phenomena were also reported in other nano-assemblies made of Ru complexcontaining polymer with Ru-cyano (Ru-CN) coordination bonds. [START_REF] Sun | Ruthenium-Containing Block Copolymer Assemblies: Red-Light-Responsive Metallopolymers with Tunable Nanostructures for Enhanced Cellular Uptake and Anticancer Phototherapy[END_REF][START_REF] Chen | Nanostructured polymer assemblies stabilize photoactivatable anticancer ruthenium complexes under physiological conditions[END_REF] Under red-light irradiation (625 nm, 30 mW/cm 2 ), the shift of MLCT band of Ru-CN reached the maximum after tpolymer(Ru-CN) = 2.3 min in block copolymer solution, [START_REF] Chen | Nanostructured polymer assemblies stabilize photoactivatable anticancer ruthenium complexes under physiological conditions[END_REF] while in polymer micelles or vesicles (nano-assemblies, NS) it reached the steady state after much longer time (tNS(Ru-CN) = 180 min). [START_REF] Sun | Ruthenium-Containing Block Copolymer Assemblies: Red-Light-Responsive Metallopolymers with Tunable Nanostructures for Enhanced Cellular Uptake and Anticancer Phototherapy[END_REF] These observations can also be explained by the stabilization effect of nano-assemblies because at the beginning only the Ru-S or Ru-CN at the hydrophilic-hydrophobic interface were in contact with water, and the access of aqua ligand to the hydrophobic core or membrane was progressive from the hydrophilic-hydrophobic interface. Note interestingly that tNS/tpolymer is 78 in the case of Ru-CN polymer nano-assemblies, [START_REF] Sun | Ruthenium-Containing Block Copolymer Assemblies: Red-Light-Responsive Metallopolymers with Tunable Nanostructures for Enhanced Cellular Uptake and Anticancer Phototherapy[END_REF][START_REF] Chen | Nanostructured polymer assemblies stabilize photoactivatable anticancer ruthenium complexes under physiological conditions[END_REF] while tNS/tpolymer is only 7 -10 in PolyRu micelles or vesicles with Ru-S coordination here. The relative photocleavage efficiency of Ru polymer micelles and vesicles with Ru-S coordination is much higher than those with Ru-CN one. This observation is attributed to the simultaneous oxidation of thioether groups by ROS generated in-situ by Ru complexes that are also known as photosensitizer and capable of generating ROS under illumination. The oxidation transformed thioether to sulfoxide and made the hydrophobic block partially or totally hydrophilic. Consequently, the access of water molecules to the Ru-S coordination bonds became easier along with light illumination (see below for details).

Light-Activated ROS Generation and Boosted Release of Ru complex from PolyRu Micelles and Vesicles

To assess the ROS generation capacities of PolyRu self-assemblies, DCFH was used as indicator (Figure 3A). The fluorescence of DCFH aqueous solution alone (λex = 488 nm) was almost negligible after 625 nm-light illumination (30 mW/cm 2 ) of 0 -24 min, as shown in Figure S14. However, the solution fluorescence exhibited a sharp enhancement with peak around 521 nm as the 625 nm-light illumination time progressed until 24 min in the presence of all samples including Ru complexes, PolyRu micelles and vesicles at an equivalent concentration of Ru of 70 µM, which clearly indicated ROS generation. Moreover, these ROS included 1 O2, as confirmed by a separate test in PolyRu organic solution using 1, 3-diphenylisobenzofuran (DPBF) as indicator (see Figure S15 for detail). Interestingly, after 24 min of irradiation, the emission intensity of DCFH in the presence of PolyRu micelles and vesicles reached twice of that with free Ru complexes (Figure 3B, 3C, 3D and3E). This observation demonstrated superior ROS production efficiency of PolyRu self-assemblies, which could be associated with the morphological alterations occurring in the PolyRu micelles and vesicles upon irradiation caused by the oxidation of thioether groups. Indeed, the transformation from thioether groups to sulfoxide groups on PolyRu after irradiation was confirmed by [START_REF] Mitchell | Engineering precision nanoparticles for drug delivery[END_REF] H NMR and ATR-FTIR spectra (see Figure S16 and Figure S13). According to previous works, [START_REF] Deng | Oxidation-Sensitive Polymersomes Based on Amphiphilic Diblock Copolypeptoids[END_REF][START_REF] Vasdekis | Precision intracellular delivery based on optofluidic polymersome rupture[END_REF] the oxidization of thioether groups into hydrophilic sulfoxide groups by ROS in the hydrophobic block of PolyRu could provoke the disruption of self-assemblies. Porous structures with higher specific surface area occur in vesicles and micelles during the disruption process, which could improve the oxygen and promote the oxygen contact with the Ru-based photosensitizers to further enhance ROS generation efficiency. [START_REF] Zhang | Combining Ruthenium(II) Complexes with Metal-Organic Frameworks to Realize Effective Two-Photon Absorption for Singlet Oxygen Generation[END_REF] Effectively, the disruption of PolyRu micelles and vesicles were followed by their count rate ratio α measured by DLS (α = (derived count rate at the irradiation time t)/(derived count rate at t = 0)), since the derived count rate represented the scattering intensity and demonstrated the size and/or quantities of particles in the solution. The decline of α as a function of irradiation time (Figure 3F) supported the decreasing tendency of concentration/size of the self-assemblies through the photoactivated oxidation. The symbols are the experimental points. The lines are the fits with the function α = (α0 -αmin) e -t/τ + αmin, where αmin = 0.184 (PolyRu micelles) and 0.325 (PolyRu vesicles) correspond to the minimum count rate ratio nearly unchanged with time, and τ is the characteristic time when (α -αmin) declines to 36.8% of (α0 -αmin). τ is 19.05 min for PolyRu micelles, and 45.03 min PolyRu vesicles.

The photoactivated release of Ru complexes from PolyRu self-assemblies under 625 nm-light illumination were then studied in vitro. [START_REF] He | A Sequential Dual-Model Strategy Based on Photoactivatable Metallopolymer for On-Demand Release of Photosensitizers and Anticancer Drugs[END_REF] 1 mL of PolyRu micelles or vesicles (1 mg/mL) were irradiated under red-light (625 nm, 30 mW/cm 2 ) for 30 min and then placed into a dialysis bag (Mw cutoff of 3500 Da). The dialysis bag was immersed in 40 mL PBS solution at 37 °C and stirring at 250 rpm, and an aliquot 2mL of the PBS solution was withdrawn after 1 h. The same procedure was performed for the PolyRu micelles and vesicles in the dark without irradiation. The amount of [Ru(tpy)(biq)(H2O)](PF6)2 released out of the dialysis bag was wRu = (40 mL/2 mL)cRu, where the concentration cRu was determined according to the calibration curve established beforehand using UV-vis spectra (absorbance at 550 nm) (Figure S17 and Table S3). The results showed a relative release wRu(after irradiation)/wRu(in the dark) of 13 and 4 for PolyRu vesicles and micelles, respectively. Clearly, the releases from irradiated samples were significantly higher than those in the dark, because of the photocleavage of Ru-S and the photoactivated disruption of micelles and vesicles through the enhanced ROS generation as discussed above. Interestingly, the difference of the relative light/dark release between micelles and vesicles was not caused by their photoactivated release difference, but by the difference of their background release in the dark (see Table S3). Therefore, PolyRu vesicles are more interesting systems for dark protection and photocontrolled release.

Cellular Uptake, intracellular ROS detection and Cytotoxicity Assay of PolyRu micelles and vesicles

In the above sections, we have demonstrated the photocleavage of Ru complexes from PolyRu selfassemblies, ROS generation by PolyRu self-assemblies under red-light irradiation, and the release of Ru complexes promoted by the disruption of PolyRu self-assemblies through the simultaneous ROS generation. The cellular uptake, intracellular ROS generation and cytotoxicity of PolyRu micelles and vesicles were then studied for their potential in anticancer therapy.

PEGylation of anticancer agents has been extensively utilized to prolong their blood circulation time and to enhance their tumor accumulation. [START_REF] Gref | The controlled intravenous delivery of drugs using PEG-coated sterically stabilized nanospheres[END_REF] However, PEGylation could also reduce their cell uptake due to their nearly neutral surface charge and poor protein adsorption. [START_REF] Schottler | Protein adsorption is required for stealth effect of poly(ethylene glycol)-and poly(phosphoester)-coated nanocarriers[END_REF][START_REF] Zhang | Polymersomes with Red/Near-Infrared Emission and Reactive Oxygen Species Generation[END_REF] Here, the introduction of Ru complexes might change the situation. The zeta potential of PolyRu micelles and vesicles were first measured as shown in Figure 4A. Effectively, both PolyRu micelles and vesicles exhibited a significant positive surface zeta potential (+16.4 mV for micelles and +18.3 mV for vesicles), in contrast to the zeta potential of +0.96 mV measured for nanoparticles formed by the processor of PolyRu, PEG45-b-PMeSPG18 (Figure S18). According to the litterature, [START_REF] Donahue | Concepts of nanoparticle cellular uptake, intracellular trafficking, and kinetics in nanomedicine[END_REF][START_REF] Hald Albertsen | The role of lipid components in lipid nanoparticles for vaccines and gene therapy[END_REF] the endocytosis of cationic nanoparticles is promoted through the interaction between the positive charge of nanoparticles and the negative charge of cell membrane. The cellular uptakes of PolyRu micelles and vesicles as well as Ru complexes alone were then tested using human non-small cell lung cancer cell line (A549). A549 cells were incubated with PolyRu micelles, PolyRu vesicles or Ru complexes in the cell culture medium, collected at different time (2 h, 4 h, 6 h and 8 h), and digested in 70% HNO3 for analysis by ICP-MS using 101 Ru as monitored isotopes (see SI for details). As illustrated in Figure 4B, Ru was effectively detected inside the cells, and the intracellular Ru content of both PolyRu micelles and vesicles increased as the incubation time prolonged. Notably, the intracellular Ru content of PolyRu vesicles was approximately twice as much as that of PolyRu micelles, which may be explained by the larger positive zeta potential (+ 18.3 mV, see Figure 6A) and larger size (Dh = 59 nm, see Figure 2) of vesicles. Moreover, the intracellular Ru content of both PolyRu vesicles and micelles were higher than that of Ru complexes alone, which may be caused by the different endocytosis pathway of nanoparticles. Further, the intracellular ROS generation upon red light irradiation was investigated using DCFH-DA. After the diffusion of DCFH-DA into the cell, DCFH-DA is then deacetylated by cellular esterases to the non-fluorescent DCFH, which is later oxidized by ROS into the highly fluorescent DCF (see Figure 3A). The intracellular ROS production is proportional to the fluorescence intensity of DCF measured by flow cytometry (Figure 4C). The results indicated clearly that PolyRu micelles and vesicles produced more ROS inside the cells than The IC50 values of PolyRu micelles and vesicles expressed in concentration of Ru complexes were even lower than IC50 value of free Ru complexes because of the enhanced ROS generation efficiency during the disruption of nano-assemblies. Of course, the Ru complex used in this work was not a highly efficient metallodrug for chemotherapy and photodynamic therapy. Other chemical drug could be attached to the Ru complexes [START_REF] Sun | Red-Light-Controlled Release of Drug-Ru Complex Conjugates from Metallopolymer Micelles for Phototherapy in Hypoxic Tumor Environments[END_REF] and more efficient prodrugs based on other Ru(II) complexes or other thioether ligands like dithioether ligand [START_REF] Meijer | Diastereoselective Synthesis and Two-Step Photocleavage of Ruthenium Polypyridyl Complexes Bearing a Bis(thioether) Ligand[END_REF] could be used in the future. In conclusion, PolyRu

Scheme 1 .

 1 Scheme 1. (A) Amphiphilic diblock copolymer PEG-b-PMeSPG, Ru complex [Ru(tpy)(biq)H2O]

  LED source. A high intensity LED spotlight with λ = 625 nm (device type: 86-432, Edmund Optics Ltd) was used for all light illumination studies. Detection of reactive oxygen species (ROS) and singlet oxygen ( 1 O 2 ) DCFH was used to detect the ROS produced by PolyRu copolymer and PolyRu nano-assemblies. The commercially available DCFH-DA was first activated by NaOH solution to DCFH. DCFH solution (1 μM) was added into [Ru(tpy)(biq)(H2O)] 2+ (70 μM) solution, and PolyRu micelles and vesicles aqueous dispersion (containing 70 μM of Ru complexes), respectively. Afterward, the mixtures were irradiated by 625 nm-light (30 mW/cm 2 ) for different length of time. ROS generated could transform non-fluorescent DCFH to hyperfluorescent oxidated DCFH. The fluorescence intensity of the oxidated DCFH (with peak at 521 nm, λex = 488 nm) was measured as a function of red-light irradiation time. The 1 O2 generated from [Ru(tpy)(biq)(H2O)] 2+ and PolyRu copolymer were monitored by the transformation of fluorescent 1, 3-diphenylisobenzofuran (DPBF) to non-fluorescent oxidized DPBF under red-light irradiation. 38, 45 Briefly, the methanol solution of DPBF (25 μM) in the presence of [Ru(tpy)(biq)(H2O)] 2+ (55 μM) and PolyRu copolymer (containing 55 μM of Ru complexes), respectively, was irradiated by red light (λ = 625 nm, 30 mW/cm 2 ) for different length of time. The fluorescence intensity (emission around 446 nm, λex = 405 nm) was subsequently recorded as a function of irradiation time. As a control experiment, the solution of DPBF (25 μM) was irradiated in the same way but without Ru samples.

Scheme 2 .

 2 Scheme 2. Synthetic route to PEG45-b-PMeSPG18 and Ru complexes-coordinated PEG45-b-

Figure 1 .

 1 Figure 1. Characterization of PolyRu self-assemblies. (A) SEM image of PolyRu micelles formed

Figure 2 .

 2 Figure 2. (A-C) UV-vis absorption spectra of samples incubated in aqueous solution in the dark for

Figure 3 .

 3 Figure 3. ROS generation detected by DCFH for samples under red-light irradiation (625 nm, 30 mW/cm 2 ). (A) ROS transform non-fluorescent DCFH to hyperfluorescent oxidated DCF. The

Figure 4 .

 4 Figure 4. (A) Zeta potentials of PEG45-b-PMeSPG18 nanoparticles (PMeSPG NPs), PolyRu micelles

Table 1 . 7 [

 17 phototoxicity index PI of 9.1 and 13.7, respectively, in contrast to the PI of free Ru complexes of 1.5.

Ru complexes after 30 min of red-light irradiation. Moreover, the mean fluorescence intensity of PolyRu vesicles was higher than that of PolyRu micelles, which could be explained by the more efficient cellular uptake of vesicles compared to micelles (see Figure 4B).

Finally, the cytotoxicity of PolyRu micelles and vesicles was investigated using A549 cells in the dark and under 625 nm light illumination (11.57 mW/cm 2 , 1 h). The results are summarized in Table 1 (see also Figure S19 and SI for experimental details). As reference, the free Ru complex was first tested with A549 cells in the same conditions. The Ru complex displayed some degree of photocytotoxicity and dark-cytotoxicity: the concentration necessary to kill 50% of cancer cells (IC50 value) of 68.5 µM in the dark and IC50 value of 44.9 µM after irradiation. The photo-cytotoxicity of Ru complexes was higher than that of dark-cytotoxicity because of the photo-generated ROS. As for PolyRu micelles and vesicles, their IC50 values in the dark are 280.3 µM and 319.7 µM, respectively, concentrations corresponding to the Ru complexes contents in the nano-assemblies. Therefore, PolyRu micelles and vesicles showed very low cytotoxicity in the dark and the vesicles are still less toxic than micelles. This result is consistent with the protecting role of polymer micelles and vesicles for Ru complexes as above discussed. In contrast, both PolyRu micelles and vesicles exhibited much superior cytotoxicity under red-light irradiation, with IC50 values of 27.9 µM and 23.3 µM, respectively, which are even lower than IC50 value of free Ru complexes (44.9 µM). This observation could be explained by the higher efficiency of ROS production of PolyRu micelles and vesicles than that of free Ru complexes (see Figure 3E anddiscussion). The phototoxicity index (PI, defined as the ratio between IC50 in the dark and the IC50 with light irradiation) of PolyRu micelles (PI = 9.1) and vesicles (PI=13.7) were significantly higher than PI of free Ru complexes (PI = 1.5) (Table 1); moreover, vesicles showed higher photocytotoxicity than micelles. Therefore, PolyRu micelles and vesicles reported here are promising drug delivery systems that can protect Ru complex drugs in the dark and obtain light-activated anticancer effects with spatiotemporal control for photoactivated chemotherapy and photodynamic therapy.

vesicles and micelles presented here are promising nano-assembly prototypes that can efficiently protect Ru complex drugs in the dark and achieve red-light-activated anticancer effects with spatiotemporal control for photoactivated chemotherapy and photodynamic therapy.

ASSOCIATED CONTENT

Supporting Information.

The following files are available free of charge.

Materials, and supplemental methods, experimental details, and characterization data (PDF)

AUTHOR INFORMATION

Corresponding Author * Dr. Min-Hui LI : min-hui.li@chimieparistech.psl.eu

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT

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