A Bour

S G Kruglik

M Chabanon

P Rangamani

N Puff

S Bonneau

Lipid unsaturation properties govern the sensitivity of membranes to photo-induced oxidative stress

Unsaturated lipid oxidation is a fundamental process involved in different aspects of cellular bioenergetics; dysregulation of lipid oxidation is often associated with cell aging and death. In order to study how lipid oxidation affects membrane biophysics, we used a chlorin photosensitizer to oxidize vesicles of various lipid compositions and degree of unsaturation in a controlled manner. We observed different shape transitions that can be interpreted as an increase in the area of the targeted membrane followed by a decrease. These area modifications induced by the chemical modification of the membrane upon oxidation, were followed in situ by Raman Tweezers Microspectroscopy (RTM). We found that the membrane area increase corresponds to the lipids peroxidation and is initiated by the delocalization of the targeted double bonds in the tails of the lipids. The subsequent decrease of membrane area can be explained by the formation of cleaved secondary products. As a result of these area changes, we observe vesicle permeabilization after a time lag that is characterized in relation with the level of unsaturation. The evolution of photosensitized vesicle radius was measured and yields an estimation of the mechanical changes of the membrane over oxidation time. The membrane is both weakened and permeabilized by the oxidation. Interestingly, the effect of unsaturation level on the dynamics of vesicles undergoing photooxidation is not trivial and thus carefully discussed. Our findings shed light on the fundamental dynamic mechanisms underlying the oxidation of lipid membranes, and highlight the role of unsaturations on their physical and chemical properties

INTRODUCTION

Aerobic metabolism in cells relies on the oxidation of organic compounds such as fatty acids, allowing cells to produce energy. However, the phospholipids that constitute cell membranes are also sensitive to oxidative stress, which can lead to membrane damage, aging and potentially cell death (1)(2)(3). The molecular processes responsible for the oxidation of unsaturated lipids involve the chemical modification of their double bounds and potential cleavage of the lipid chains (4,5). Therefore, it is essential to consider the degree of unsaturation of fatty acids in order to understand the mechanisms allowing lipid membranes to withstand oxidative stress.

Biological membranes contain large amounts of mono-and polyunsaturated fatty acids, as well as traces of transition metals necessary for oxidative reactions. As they are constantly subjected to oxidation -in particular due to the respiratory chain -cellular membranes suffer from a variety of irreversible damages. Their oxidative state is defined as the balance between oxidant and antioxidant species, an imbalanced situation leading sequentially to stages of oxidative stress, cytotoxicity and pathological states (6,7). Oxidative stress is known to be involved in cell aging and, if uncontrolled, in a variety of diseases including Parkinson's and Alzheimer's neurodegenerations and cancer [START_REF] Kim | The Role of Oxidative Stress in Neurodegenerative Diseases[END_REF][START_REF] Prasad | Oxidative Stress and Cancer: Advances and Challenges[END_REF][START_REF] Winklhofer | Mitochondrial dysfunction in Parkinson's disease[END_REF][START_REF] Moreira | Mitochondrial dysfunction is a trigger of Alzheimer's disease pathophysiology[END_REF][START_REF] Zong | Mitochondria and Cancer[END_REF][START_REF] Sabharwal | Mitochondrial ROS in cancer: initiators, amplifiers or an Achilles' heel?[END_REF]. Oxidation-induced changes in membranes properties, such as fluidity and permeability [START_REF] Borst | Oxidation of unsaturated phospholipids in membrane bilayer mixtures is accompanied by membrane fluidity changes[END_REF][START_REF] Runas | Addition of Cleaved Tail Fragments during Lipid Oxidation Stabilizes Membrane Permeability Behavior[END_REF][START_REF] Runas | Low levels of lipid oxidation radically increase the passive permeability of lipid bilayers[END_REF], can result in abnormal and dysfunctional protein assemblies and in the leakage of vital molecules from organelles. Therefore, understanding membrane behavior with respect to their lipid unsaturation degree is an important biomedical challenge.

The first step of unsaturated lipid oxidation leads to the formation of hydroperoxides through a chain reaction ending when two lipid radicals react together (4,5). Due to their polarity, the peroxidized hydrocarbon tails are thought to migrate to the lipid-water interface, which increases the molecule area and changes the packing parameter [START_REF] Buettner | The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate[END_REF][START_REF] Agmon | Modeling the effects of lipid peroxidation during ferroptosis on membrane properties[END_REF][START_REF] Garrec | Lipid Peroxidation in Membranes: The Peroxyl Radical Does Not "Float[END_REF]. The second step consists of further oxidation producing secondary products, including alcohols, ketones, alkanes, aldehydes and ethers [START_REF] Buettner | Catalytic metals, ascorbate and free radicals: combinations to avoid[END_REF][START_REF] Domingues | Mass spectrometry analysis of oxidized phospholipids[END_REF]. These products are used as an index of lipid oxidation, mainly to estimate oxidation level of complex biological systems [START_REF] Janero | Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury[END_REF]. The resulting cleaved lipids have a conical shape with a larger head to tail surface area ratio, and tend to increase the membrane permeability to solutes such as sucrose and glucose [START_REF] Karatekin | Cascades of transient pores in giant vesicles: line tension and transport[END_REF].

An effective and controlled way to induce oxidation processes in a system is to use photosensitizers to generate reactive oxygen species (ROS) upon light irradiation [START_REF] Paillous | Interest of photochemical methods for induction of lipid peroxidation[END_REF]. Known as the photodynamic effect, the photochemical induction of oxidation is also involved in UV cytotoxicity and light-related ageing on tissues containing endogenous porphyrins [START_REF] Baier | Singlet Oxygen Generation by UVA Light Exposure of Endogenous Photosensitizers[END_REF]. Moreover, together with the preferential retention of certain photosensitizers by tumors as compared to normal surrounding tissues [START_REF] Bonneau | Tetrapyrrole photosensitisers, determinants of subcellular localisation and mechanisms of photodynamic processes in therapeutic approaches[END_REF], such photo-induced cytotoxicity has been applied as an anti-tumoral therapy known as the Photodynamic Therapy (PDT) [START_REF] Dolmans | Photodynamic therapy for cancer[END_REF].

Simplified models of biological membranes such as lipid vesicles are powerful tools for investigating how photo-induced oxidative species impact the phospholipid bilayer. Lipid vesicles exposed to photooxidation have been shown to exhibit a two-step behavior, corresponding to a sequential increase and decrease in membrane surface area. These modifications induce vesicle fluctuations and shape transitions [START_REF] Kerdous | Photo-dynamic induction of oxidative stress within cholesterolcontaining membranes: Shape transitions and permeabilization[END_REF][START_REF] Heuvingh | Asymmetric oxidation of giant vesicles triggers curvature-associated shape transition and permeabilization[END_REF][START_REF] Sankhagowit | The dynamics of giant unilamellar vesicle oxidation probed by morphological transitions[END_REF][START_REF] Mertins | Physical Damage on Giant Vesicles Membrane as a Result of Methylene Blue Photoirradiation[END_REF], accompanied by modifications in the membrane viscosity [START_REF] Vyšniauskas | A Molecular Rotor that Measures Dynamic Changes of Lipid Bilayer Viscosity Caused by Oxidative Stress[END_REF], bending modulus [START_REF] Bouvrais | Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers[END_REF], area expansion modulus [START_REF] Weber | Lipid oxidation induces structural changes in biomimetic membranes[END_REF], and permeability [START_REF] Kerdous | Photo-dynamic induction of oxidative stress within cholesterolcontaining membranes: Shape transitions and permeabilization[END_REF][START_REF] Heuvingh | Asymmetric oxidation of giant vesicles triggers curvature-associated shape transition and permeabilization[END_REF][START_REF] Kotova | Selective permeabilization of lipid membranes by photodynamic action via formation of hydrophobic defects or pre-pores[END_REF][START_REF] Mojzisova | Photosensitizing properties of chlorins in solution and in membrane-mimicking systems[END_REF]. Similar effects have also been reported in polymersomes [START_REF] Mabrouk | Photosensitization of polymer vesicles: a multistep chemical process deciphered by micropipette manipulation[END_REF]. Although correlations with chemical mechanisms have been proposed [START_REF] Sankhagowit | The dynamics of giant unilamellar vesicle oxidation probed by morphological transitions[END_REF], the underlying dynamic chemical scenario remains largely unexplored due to a lack of in situ measurement during the oxidation process.

In this article, we investigate how lipid oxidation affects the dynamic behavior of artificial membrane models.

We use Giant Unilamellar Vesicles (GUVs) made of controlled lipid composition with various degrees of unsaturation and expose them to photooxidation by light-activation of Chlorin e6 photosensitizer (Ce6, Fig. 1). Special attention is given to the number and position of lipid unsaturations. We first describe the time evolution of GUV morphology upon photooxidation and relate it to the membrane permeabilization. Then, we use Raman microspectroscopy combined with optical tweezers to follow in situ the membrane chemical modifications of vesicles in an aqueous environment. These results are discussed in relation to the observed mechanical and morphological changes. The electroplating method (39) was used to form giant unilamellar vesicles (GUVs) of a diameters of 20 to 30 μm. Lipid mixtures in chloroform were deposited on ITO-covered glass plates, and chloroform was evaporated in vacuum. A formation chamber was then made from two such glass plates and a Teflon spacer of 4 mm. The chamber was then filled with a solution of 300 mM sucrose and HEPES buffer solution (2 ml, pH 7.8, HEPES 0.5 mM). An AC field of 1 Volt and 8 Hz was applied between the plates for 4 hours. For experiments, the GUVs were mixed with a ~300 mM glucose solution made in the same buffer. The solution carefully adjusted ~5 mOsm/L higher than the external sucrose solution to limit the strain of the membrane. The density difference between sucrose and glucose caused the GUVs to sediment to the bottom of the chamber. The difference in optical index between sucrose inside and glucose outside the vesicle allowed phase contrast microscopy observations. Eq. 1 and is reported in Table 1. The obtained values are in good agreement with previous data obtained [START_REF] Kerdous | Photo-dynamic induction of oxidative stress within cholesterolcontaining membranes: Shape transitions and permeabilization[END_REF][START_REF] Mojzisova | Photosensitizing properties of chlorins in solution and in membrane-mimicking systems[END_REF] and depend of the membrane composition. Such an effect has been previously observed for the interactions of photosensitizers with various membrane [START_REF] Bonneau | Tetrapyrrole photosensitisers, determinants of subcellular localisation and mechanisms of photodynamic processes in therapeutic approaches[END_REF][START_REF] Kerdous | Photo-dynamic induction of oxidative stress within cholesterolcontaining membranes: Shape transitions and permeabilization[END_REF][START_REF] Mabrouk | Photosensitization of polymer vesicles: a multistep chemical process deciphered by micropipette manipulation[END_REF][START_REF] Bonneau | Tetrapyrrole-photosensitizers vectorization and plasma LDL: a physico-chemical approach[END_REF][START_REF] Kerdous | Release kinetics of an amphiphilic photosensitizer by blockpolymer nanoparticles[END_REF].

MATERIALS AND METHODS

Chemicals

Measurements of the

Model vesicles

Lipids content K B (M -1 )

18:1-GUVs DOPC (5.00±1.4)×10 France). Illumination was provided by a 120 W Metal halide lamps with a 405±10 nm bandpass filter. Neutral density filters (NDx8) were used to reduce illumination level. GUVs were simultaneously imaged in bright-field using the halogen lamp of the microscope and the fluorescence of Ce6 was cut off by using an appropriate filter.

Image acquisition (100 ms integration time) was performed with a CCD camera (Neo sCMOS, Andor Technology). The acquisition, processing and image analysis were performed with NIS-Element provided by Nikon and Image J (NIH, USA). For each membrane composition, the images of at least twenty vesicles from three sample preparations were analyzed separately. The intra-trial variability is reported in the supporting information. When photo-oxidizing the membrane, illumination was kept constant from the initial time.

Chemical scenario. To monitor the chemistry of lipid oxidation, Raman spectra of corresponding lipid vesicles in the presence of photosensitizer Ce6 were recorded using home-built Raman Tweezers Microspectroscopy (RTM) setup described elsewhere [START_REF] Tatischeff | Fast characterisation of cellderived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy[END_REF]. Briefly, RTM technique combines optical trapping [START_REF] Ashkin | Acceleration and Trapping of Particles by Radiation Pressure[END_REF] with Raman probing: the same laser radiation is used for both optical trapping of the vesicles and excitation of Raman scattering from the vesicles' constituent biomolecules. The light at 780 nm is provided by a continuous-wave Ti:Sapphire laser (Spectra Physics, model 3900S) pumped by argon-ion laser (Spectra Physics Stabilite 2017).

The laser beam is focused by a water-immersion infinity-corrected objective (Olympus LUMFL 60X, NA 1.1) brought into contact with a droplet (~100 µL) of water buffer containing the vesicles of interest. The laser power was adjusted to about 70 mW inside the sample suspension. LUVs concentration was adjusted to about 100 µg/mL in lipids and to about 0.1% in Ce6. Our RTM setup employs upright microscope configuration and the same objective is used to collect Raman signal in a back-scattering geometry and to deliver it into a spectrograph (Acton SpectraPro 2550i) coupled with a deep-depleted back-illuminated NIR CCD (Princeton Instruments SPEC-10 400BR/LN). Raman light is focused onto the spectrograph's entrance slit (semi-confocal configuration, slit width 50 µm) by an achromatic lens with f=75 mm. The Raman signal is spectrally separated by 2 Semrock RazorEdge long-pass filters (grade ''U''): one (blocking filter) with the edge wavelength of 780 nm is placed normally to the optical beam just before the focusing lens; another one with the edge wavelength of 830 nm is used as a dichroic beam splitter at an angle of incidence 45°: it reflects laser light at 780 nm and transmits all the wavelengths longer 785 nm. Spectral resolution in all Raman experiments was about 5 cm -1 . Frequency calibration was performed using Raman lines of toluene with ±2 cm -1 absolute accuracy and relative frequency position accuracy better than ±1 cm -1 .

All experiments were done in triplicates. Raman spectra were continuously acquired every 3 seconds within 10-minutes intervals using WinSpec software; further data treatment was performed using IgorPro for Windows software. The informative signal from the vesicles in the sample volume arises as additional spectral features appearing on top of water buffer Raman spectrum, as soon as the vesicles are trapped at the focus of the laser beam. Simultaneously with the event of vesicles trapping, the sample droplet was illuminated by weak light at 405 nm from the microscope white-light illumination system passing through the band-pass interference filter (Andover Corporation 405±5 nm), in order to initiate the photosensitizing effect. The details of raw Raman spectra treatment were described in detail elsewhere [START_REF] Tatischeff | Fast characterisation of cellderived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy[END_REF]. In this study, each spectra is the mean of three acquisitions.

For Raman kinetics evaluation based on spectral band areas, an additional step of spectra treatment was introduced, consisting in automatic background correction using linear functions and spectra normalization on ν(C-N+) stretch of polar heads.

RESULTS AND DISCUSSION

Preparation of the photooxidable vesicles. In order to investigate the effect of lipid unsaturation on membranes undergoing photooxidation, we use electroformed GUVs made of five distinct compositions. To vary the level of membrane unsaturation we considered (i) 18:1-GUVs, made of pure mono-unsaturated lipids DOPC, (ii) 18:2-GUVs, composed of pure polyunsaturated lipids DLPC and (iii) 18:1/18:3-GUVs, a mixture of C18:3-PC and DOPC at 50/50 molar ratio that have a mean level of unsaturation similar to DLPC. Then, to address the role of the geometry of the unsaturation, we used (iv) 18:1trans-GUVs, composed of DOPC-trans lipids and (v)

18:1∆6-GUVs, made of DOPC-∆6 lipids. All unsaturated lipids used here were C18 phosphatidylcholine, in order to address the effects of the unsaturation of the lipids tails and not that of their head group, nature or length. Control experiments were carried using GUVs made of the saturated lipid DMPC. In all cases, the photosensitizer Ce6 was added to the GUVs membranes to enable the photo-control of the membrane oxidation. Beforehand, partition experiments were carried out to quantify the propensity of the Ce6 to insert within the various membranes and determine the suitable Ce6 concentration for each membrane composition (see binding constants in Table 1).

The GUVs were prepared in a solution of sucrose and diluted in a glucose solution to produce a difference in optical index between the inner sucrose and outer glucose solutions, facilitating observations and measurements by phase contrast microscopy. GUVs were observed by bright field optical microscopy and photooxidation was induced by illumination with a 120 W Metal halide lamp with a 405±10 nm bandpass filter.

Morphological evolution of GUVs under photooxidation. In the absence of irradiation, all GUVs appeared spherical, although slightly floppy due to the small osmolarity mismatch between the inner and outer solutions (5 mOsm/L, see Materials and Methods for details). As seen from Fig. 3, upon illumination, the Ce6-labeled GUVs 34). The shape destabilization is attributed to a transient increase in membrane area, possibly coupled to a softening of the lipid bilayer mechanical properties. Most importantly, we find that the kinetics of shape transition is strongly dependent on the unsaturation properties of the membrane lipids. Although the duration of Stage 1 is of the order of 10s for both 18:1-GUVs and 18:2-GUVs, it is decreased to about 2 seconds for 18:1/18:3-GUVs.

For 18:1∆6-GUVs whose double bond is nearest the lipid head, Stage 1 lasts for up to 30s and presents shape fluctuations of higher amplitudes. In contrast, 18:1trans-GUVs seem unaffected and retain their spherical shape, initial radius and optical contrast for all the duration of the experiment (450s).

Pulsatile dynamics of photooxidized vesicles. In order to quantitatively compare the effects of lipid unsaturation on the dynamics of GUVs undergoing photooxidation, we recorded for each vesicle composition the time evolution of (i) their optical contrast and (ii) their radius. As seen Fig. 4, all photosensitive GUVs showed a dramatic decrease in optical contrast during Stage 2. Given that the optical contrast is the result of the difference in sugar composition between the interior (sucrose) and exterior (glucose) of the GUVs, the decrease in contrasts is interpreted as a combined effect of the dilution and leak-out of encapsulated sucrose due to a progressive permeabilization of the lipid membrane.

Strikingly, the vesicle radii exhibited an overall decrease, marked by pulsatile behavior of progressive swelling and abrupt drop for 18:1-GUVs, 18:2-GUVs and 18:1/18:3-GUVs. This dynamics is similar to those of lipid GUVs undergoing swell-burst cycles induced by osmotic stress [START_REF] Oglęcka | Oscillatory phase separation in giant lipid vesicles induced by transmembrane osmotic differentials[END_REF][START_REF] Su | Pulsatile Gating of Giant Vesicles Containing Macromolecular Crowding Agents Induced by Colligative Nonideality[END_REF][START_REF] Chabanon | Pulsatile Lipid Vesicles under Osmotic Stress[END_REF] or subject to surfactants [START_REF] Nomura | Capabilities of liposomes for topological transformation[END_REF][START_REF] Chabanon | Solubilization kinetics determines the pulsatory dynamics of lipid vesicles exposed to surfactant[END_REF]. Based on these observations, we propose the following mechanism for the GUVs dynamics (Fig. 5). Upon illumination, the Ce6 labeled GUVs generate reactive oxygen species (ROS), that lead to oxidative reactions of the unsaturated lipid tails. At short time scales (~10s), the oxidation produces an increase in membrane area resulting in shape destabilization of the initially spherical vesicles (Stage 1) [START_REF] Sankhagowit | The dynamics of giant unilamellar vesicle oxidation probed by morphological transitions[END_REF]. At longer time scales, the surface area of photooxidized membranes progressively reduces, resulting in a recovery of the spherical shape followed by a decrease in the GUV volume (Stage 2). Concomitantly, oxidative products are released from the membrane both in the intra-vesicular space and in the surrounding bath. However, because the volume of the bath is much larger than the vesicle volume, a positive concentration difference builds up between the inside and the outside of the GUV. Since the permeability of the membrane to oxidative product is negligible when compared to its permeability to water, the concentration difference induces an hypotonic stress driving an inflow of water through the membrane and the progressive swelling of the GUV. Lipid membranes are elastic materials able to withstand about 4% surface strain before rupturing [START_REF] Evans | Entropy-driven tension and bending elasticity in condensed-fluid membranes[END_REF]. Once this critical strained is reached, the GUV opens a micrometer sized pore through which the inner solution leaks out, relaxing the internal pressure and decreasing in turn the vesicle volume and membrane tension. This in turn allows for the pore line tensioninduced by the hydrophobic lipid mismatch at the pore edgeto drive the resealing of the pore [START_REF] Chabanon | Pulsatile Lipid Vesicles under Osmotic Stress[END_REF]. Such swelling and bursting sequence is repeated in successive cycles as long as oxidative species are released from the membrane. The 18:1∆6-GUVs showed a slightly different behavior, with no clear swell-burst cycles, although these may happen with amplitudes smaller than our setup can detect.

To confirm that the swell-burst cycles occurred in 18:1-GUVs, 18:2-GUVs and 18:1/18:3-GUVs, we tracked the vesicles with rapid video-microscopy (100 Hz) in search of a transient pore opening. As shown in Fig. 6, we observed short lived pore opening up to few microns in radius for about 50 ms, thus confirming the proposed swell-burst mechanism. We then calculated for each cycle the maximum area strain (Fig. 7, A-D). For the GUVs for which pore openings have been demonstrated, this corresponds to the critical strain  c at which membrane bursts. For 18:1-GUVs and 18:2-GUVs, the maximum strain decreases with each cycle, reaching a final value about four times lower than at the beginning of Stage 2 :  c is decreased respectively for 18:1-GUVs Contribution of membrane permeabilization to GUV contrast decay. From our observations of transient pores (Fig. 6), we did not find any significant drop in vesicle contrast with pore opening, suggesting that the short times of the pore opening events do not allow diffusion through the pore to affect the concentration imbalance in sucrose and glucose. Indeed, convection driven leak-out alone does not change the concentration of sucrose inside the vesicle [START_REF] Chabanon | Pulsatile Lipid Vesicles under Osmotic Stress[END_REF]. Therefore the main mechanisms leading to the progressive loss in contrast observed in Fig. 3 are the dilution of the inner content by the osmotic influx of water, as well as the permeabilization of the membrane to sucrose upon oxidation, in accordance with the packing defect and/or pre-pore opening scenarios, where the evolution of hydrophobic defects of the membrane involve local increase in spacing of lipid molecules [START_REF] Kerdous | Photo-dynamic induction of oxidative stress within cholesterolcontaining membranes: Shape transitions and permeabilization[END_REF][START_REF] Heuvingh | Asymmetric oxidation of giant vesicles triggers curvature-associated shape transition and permeabilization[END_REF][START_REF] Kotova | Selective permeabilization of lipid membranes by photodynamic action via formation of hydrophobic defects or pre-pores[END_REF][START_REF] Rodriguez | Indirect evidence of submicroscopic pores in giant unilamellar [correction of unilamelar] vesicles[END_REF][START_REF] Vamparys | Conical lipids in flat bilayers induce packing defects similar to that induced by positive curvature[END_REF].

In order to evaluate the permeabilization of the lipid membrane by photooxidation, we propose a mathematical model of the swell-burst process (see Supporting Information for full derivation). Based on this framework, we estimate the time evolution of the intra-vesicular sucrose concentration (c s ) during a given cycle (n):

, (Eq. 2)
where R 0n is the initial vesicle radius of the n th cycle, P s is the membrane permeability to sucrose, ∆R n is the total amplitude of the vesicle radius during that particular cycle and ∆t n is the cycle period. This equation states that the sucrose concentration in the vesicle can decrease either by permeating through the membrane (first term in parenthesis) or by dilution when the influx of water increases the vesicle volume (second term in the parenthesis). Assuming that the membrane permeability to sucrose is constant within the period of a cycle, the solution of Eq. 2 is an exponential decay of the form , (Eq. 3)

where t 0n is the time at which the cycle starts and τ is the characteristic decay time of the sucrose concentration defined as .

(Eq. 4)

The two terms in the denominator of Eq. 4 reflect the two processes involved in the progressive loss of contrast of the vesicles, i.e. respectively the permeabilization of the membrane to the sucrose and the osmotic influx of water. For each GUVs cycle, the measured quantities are ∆R n , ∆t n , R 0n and t 0n . Therefore, assuming that the optical contrast is proportional to the sucrose concentration in the GUVs, one can determine Ps, the membrane permeability to sucrose, by obtaining τ from the fit of Eq. 3 to the contrast decay during a given cycle. R n /t n is directly derived from the radii measurements (Fig. 3). We observed that the magnitude of this term represents less than 20% of Ps for 85% of the cycles and that its contribution decreases along the permeabilization (see Fig.

7, E-L). Therefore, the loss of contrast observed in Fig. 4 is essentially governed by the permeabilization of the membrane to the sucrose.

Moreover, except for 18:1/18:3-GUVs, we did not evidence any important variation in membrane permeability over the oxidation time (Fig. 7), suggesting that the oxidation of the membrane induces an increase of its permeability to sucrose in an abrupt manner, by three order of magnitude, a native phospholipid membrane being almost impermeable to sucrose (Ps* = ~10 -12 -10 -10 m/s). Interestingly, such an observation suggests a threshold effect between two distinct permeability regimes for native and oxidized membranes, regardless of their oxidation degree and the level of oxidized products [START_REF] Runas | Low levels of lipid oxidation radically increase the passive permeability of lipid bilayers[END_REF]. So, the GUVs contrast decay is characterized by two parameters: t 0 , the time of onset of contrast decay and P s , the permeability to sucrose of oxidized membranes, on which the characteristic decay time essentially depends. The measured values of t 0 for 18:1-GUVs and 18:2-GUVs (9.4±4.5 and 10.1±5.1 seconds, respectively) do not show significant differences. This indicates that the onset of the membrane permeabilization is independent of the number of double-bond in the lipid tail. However, the t 0 obtained for 18:1∆6-GUVs is 28.4±5.5 seconds, three fold higher than 18:1-GUVs and 18:2-GUVs, suggesting that the position of the unsaturation determines the initial permeability resistance to photooxidation. For 18:1/18:3-GUVs, t 0 value is 2.6±0.8 seconds, four fold smaller than for 18:1-GUVs, revealing the weak stability of this membrane upon oxidation.

In contrast, the value of P s (Fig. 7, E-H) of oxidized membranes increases with the number of unsaturation from (0.32±0.02)x10 -7 m.s -1 for 18:1-GUVs to (0.87±0.08) 10 -7 m.s -1 for 18:2-GUVs. This behavior indicates a direct correlation between the number of double bounds and the oxidized membrane permeability. For 18:1/18:3-GUV, after the abrupt increase in the value of Ps compared to that of non-oxidized membranes, we observe throughout Stage 2 a decrease of permeability to sucrose from (3.87±0.89)x10 -7 m.s -1 to (0.88±0.24)x10 -7 m.s -1 , a value of same order of magnitude than for 18:2-GUVs but that remains larger than for unoxidized membrane.

This suggests a reorganization in the lipid membrane, in parallel with its oxidation, possibly involving changes of the structural or dynamics properties such as lipid packing and order [START_REF] Van Der Paal | Effect of lipid peroxidation on membrane permeability of cancer and normal cells subjected to oxidative stress †Electronic supplementary information (ESI) available[END_REF][START_REF] Haluska | Photo-activated phase separation in giant vesicles made from different lipid mixtures[END_REF]. The fastest loss of contrast occurs for the 18:1Δ6-GUV, with a P s value of ( 1 measurements. Raman spectra of Ce6 labeled LUVs composed of unsaturated lipids exhibited bands corresponding to the vibrations of the hydrocarbon chains with some contributions of the polar head groups [START_REF] Muik | Direct monitoring of lipid oxidation in edible oils by Fourier transform Raman spectroscopy[END_REF][START_REF] Czamara | Raman spectroscopy of lipids: a review[END_REF]. The assignment of the characteristic Raman bands in spectra of lipid vesicles under study are presented in Table S1.

The characteristic photo-induced changes vary depending on the degree of unsaturation. In general, the following processes have been detected and quantified: peroxide formation, through the appearance of transient Raman band at 840-880 cm -1 ; C=C bond isomerization, via the appearance of characteristic derivative-like feature at 1650-1675 cm -1 in difference Raman spectra; conjugation of C=C bonds, through the appearance of extra intensity around 1654 cm -1 ; and a cyclization reaction, via the appearance of strong 6-membered ring bands at 1000, 1031 and 1596 cm -1 . Note that no photoinduced changes were observed for 18:1trans-LUV.

As shown in Fig. 8, all of the examined vesicles exhibit changes of the bands which were involved in C=C loss or isomerization. All photooxidized lipids also displayed an apparition of a band in the 820-860 cm -1 region corresponding to the peroxide apparition signed by an increase of the O-OH stretching mode. The oxidation process initiates through the delocalization of the double bonds (C=C), which move along the fatty chain. This results in the formation of trans and conjugated double bonds, for the mono-and poly-unsaturated lipids, respectively.

From the kinetics point of view, the rate of C=C attack is directly correlated with the number of unsaturation.

Moreover, the isomerization of 18:16-LUV is three fold faster than for 18:1-LUV (see supporting information:

the equalization of the cis and trans C=C bands intensities takes 20 seconds for DOPC6, versus 54 seconds for DOPC). This suggests an important role of the C=C bond position. In parallel to this initial step of double bonds delocalization, the peroxidation reaction with oxygen develops, leading to the primary products, the hydroperoxides. The formation of these transient compounds is visible in all spectraexcept for 18:1trans-GUVs, where no changes where evidenced. This suggests that the initial delocalization step is necessary for the reaction with oxygen, as attested by the absence of peroxidation of the 18:1trans-vesicles where the peak at 1670 cm -1 remains stable over irradiation time.

Relation between chemical reactions and observed shape transitions. In presence of oxygen and under light activation, a photosensitizer can lead to two types of oxidative reactions induced by ROS. Type I oxidations are initiated by radicals and Type II oxidations by singlet oxygen. Because the Ce6 quantum yield is important, the latter process is expected to be dominant in our experiments [START_REF] Vyšniauskas | A Molecular Rotor that Measures Dynamic Changes of Lipid Bilayer Viscosity Caused by Oxidative Stress[END_REF]. To determine the oxidative nature of the lightinduced membrane responses and to discriminate which chemical mechanism was dominant, we used sodium azide as quencher of singlet oxygen. None of the GUVs formed in 50 mM sodium azide solution showed sign of oxidation upon illumination.

These observations are in agreement with our Raman in situ analysis, highlighting that the lipid tail C=C double bonds are the privileged site of the ROS reactions. The subsequent production of hydroperoxides is manifested by an increase of the Raman peak in 840-880 cm -1 region for all the types of vesicles except trans-DOPC and DMPC. The production of such a polar group within the hydrophobic area of the membrane strongly changes the structure of the lipid tail. As this group tends to migrate near the polar zone of the lipids head, this process tends to increase the membrane surface area, leading to the dynamics observed during Stage 1 (Fig. 3) [START_REF] Kerdous | Photo-dynamic induction of oxidative stress within cholesterolcontaining membranes: Shape transitions and permeabilization[END_REF][START_REF] Heuvingh | Asymmetric oxidation of giant vesicles triggers curvature-associated shape transition and permeabilization[END_REF][START_REF] Weber | Lipid oxidation induces structural changes in biomimetic membranes[END_REF]. As expected, these two processes are very fast and temporally correlated for all lipids used in this study. Furthermore, we found that the variation of membrane surface area related to the hydroperoxidation depends on the position on the unsaturation, as seen from the large amplitude of the fluctuations during Stage 1 of 18:1∆6-GUVs photooxidation (Fig. 4). This can be explained by the fact that the 18:1∆6-GUVs double bound is positioned closer to the head group than for the other compositions, facilitating its relocation toward the lipid-water interface in the hydrophilic zone of the membrane (57).

During Stage 2 of the vesicles' photooxidation dynamics, various degradation products are produced [START_REF] Buettner | Catalytic metals, ascorbate and free radicals: combinations to avoid[END_REF][START_REF] Domingues | Mass spectrometry analysis of oxidized phospholipids[END_REF] and expelled from the hydrophobic core of the membrane because of their increased hydrophilicity. In consequence, such species are not present in the recorded Raman spectra of immobilized vesicles. However, the decrease in double bonds of the tails can be observed in some experiments. The cleaved lipids produced within the oxidized membrane are known to increase membrane permeability, as observed in our experiments during phase 2 (Fig. 4). They also weaken the membrane. Accordingly, at this stage, the optically trapped vesicles are destabilized and burst, leading to a loss of Raman signal. Moreover, cleaved lipids are known to increase membrane permeability [START_REF] Runas | Addition of Cleaved Tail Fragments during Lipid Oxidation Stabilizes Membrane Permeability Behavior[END_REF][START_REF] Karatekin | Cascades of transient pores in giant vesicles: line tension and transport[END_REF] Throughout Stage 2 the oxidation of the membrane continues, as highlighted by the successive swell-burst cycles of the vesicles resulting from the accumulation of oxidation products. The membrane composition continually changes over the oxidation. So, it would be expected that the permeability of the membrane would increase with increasing oxidation degree. This is clearly not the case and, after an abrupt increase of P s corresponding to the initiation of Stage 2, P s remains constant (18:1-and 18:2-GUVs) or even slightly decreases (18:1/18:3-GUVs). This evidences that the membrane permeability is not directly and quantitatively related to the oxidation level and that more complex phenomenon have to be taken into consideration. For example, the asymmetric distribution of oxidative damages across the bilayer has been shown to affect membrane permeability [START_REF] Heuvingh | Asymmetric oxidation of giant vesicles triggers curvature-associated shape transition and permeabilization[END_REF][START_REF] Mojzisova | Photosensitizing properties of chlorins in solution and in membrane-mimicking systems[END_REF].

Hence, structural or dynamics properties of the membrane can be modulated over oxidation [START_REF] Runas | Low levels of lipid oxidation radically increase the passive permeability of lipid bilayers[END_REF][START_REF] Van Der Paal | Effect of lipid peroxidation on membrane permeability of cancer and normal cells subjected to oxidative stress †Electronic supplementary information (ESI) available[END_REF][START_REF] Lee | Effect of low levels of lipid oxidation on the curvature, dynamics, and permeability of lipid bilayers and their interactions with cationic nanoparticles[END_REF], in addition to possible effects of cleaved tail fragments on the membrane properties [START_REF] Runas | Addition of Cleaved Tail Fragments during Lipid Oxidation Stabilizes Membrane Permeability Behavior[END_REF].

CONCLUSIONS

In this work, we studied the influence of lipid unsaturation on the dynamics evolution of the physical and chemical properties of GUVs undergoing photooxidation.

Tracking the morphology of the GUVs made of various unsaturated lipid compositions, we identified two consecutive stages upon illumination: a Stage 1 where the vesicles exhibit shape destabilizations followed by a Stage 2 associated with a rounding of the vesicles, decrease of their size and optical contrast and occurrence of swell-burst cycles. Combining quantifications of these dynamic morphological changes and theoretical analysis, we estimated the decay of membrane permeability to sucrose with time. We further showed that the GUVs lytic strain was decreased during the course of the illumination, pointing out to a weakening of photooxidized mem-branes. Interestingly, we evidenced a complete absence of oxidation of the trans fatty chains. We showed that the onset of photooxidation is independent of the number of lipid unsaturation (18:1-and 18:2-GUVs), but is faster if the position of the unsaturation is close to the head group (18:6-GUVs). However, the rate of optical contrast decayrelated to the permeabilization of the membraneis increased by both the number of double bond and the proximity of the unsaturation to the head group. Interestingly, the permeabilization of the membrane is not proportional to its oxidation level and would be governed by a threshold effect.

In order to relate the dynamic morphological changes to the chemical processes occurring in situ during the photooxidation process, we conducted the RTM experiments on optically trapped LUV. These Raman measurements allowed us to observe directly the long-scale kinetics with time resolution of 3 seconds, of the following photoinduced processes: C=C bond isomerization, conjugation of C=C bonds, peroxide formation and a cyclization reaction involving the 6-membered ring structure.

Our findings shed light on the fundamental dynamic mechanisms underlying the oxidation of lipid membranes and highlight the role of unsaturations on their physical and chemical properties. This work has important implications in the understanding of organelles and cell membrane fate under oxidative stress and has the potential to help the development of preventive treatments against premature cell aging.

ASSOCIATED CONTENT

Supporting Information.

Detailed RTS analysis

Theoretical analysis of the swell-burst cycles

Supl. Infos

S3.1 DOPC vesicles

In the case of DOPC, the most pronounced spectral changes occur in three spectral regions associated with: (i) carbon double-bond stretching (C=C), in 1650-1675 cm -1 ; (ii) rocking deformation (=CH2) at 1265 cm -1 , also involving the carbon double bond; and (iii) peroxide (O-O) stretch ( 4), which appears in difference Raman spectra within 840-880 cm -1 . The observed spectral changes reveal two major photo-induced processes that occur in lipid membrane after Ce6 photo-activation: cis-trans isomerization and peroxide formation.

Cis-trans isomerization is characterized by the decrease of (C=C)cis band intensity at 1655 cm -1 with simultaneous increase of the (C=C)trans band intensity at 1670 cm -1 which is a characteristic frequency of the trans-species (5). The corresponding difference Raman spectra contain characteristic, rather symmetric dispersive contour with the minimum at 1655 cm -1 and the maximum at 1670 cm -1 ; this contour becomes more and more pronounced with the lapse of time. The appearance of trans-DOPC species occurs immediately (within 3 s time resolution of our experiment) after photo-excitation. It takes about 54 s for (C=C)trans band to become equal in intensity with the (C=C)cis band. It is important to note that the total Raman intensity within the range 1600-1700 cm -1 , containing (C=C) stretch of both cis-and trans-DOPC, remains roughly the same with a weak tendency to the increase with time, revealing that no other, besides these two, major transient structural species is involved. The Raman intensity of rocking deformation (=CH2)cis in the 1260-1270 cm -1 range experiences kinetic changes very similar to those of (C=C)cis, but with a decreased magnitude.

Peroxide formation in DOPC vesicles has been revealed by substantial increase of Raman intensity in the characteristic (O-O) frequency range 840-880 cm -1 (4). Since this spectral region is congested by numerous Raman bands (Table S2), the kinetic changes have been analyzed from difference Raman spectra obtained by subtraction of normalized Raman spectra of vesicles with and without addition of Ce6. Peroxide species appear immediately (within our 3 s time resolution) after photo-excitation with very high amplitude (~35%) suggesting that the process of peroxide formation is very efficient from the very beginning.

The influence of the carbon double-bond position on lipids oxidation has been studied for the vesicles consisting of DOPC6. The general picture of Raman intensity changes was found to be quite similar to the previous case of DOPC; however some notable differences were observed as well. In particular, the process of cis-trans isomerization proceeds much faster in DOPC6: it takes ~20 seconds for (C=C)trans band to become equal in intensity with the (C=C)cis band, versus ~54 seconds for DOPC. At the same time, it is interesting to note that the fast components of peroxide kinetics are rather similar for DOPC and DOPC 6: in both cases it takes about 20 seconds to reach the 50% level of peroxide band intensity.

Another very interesting observation consists in the following: no photo-induced changes in Raman spectra of vesicles consisting of DOPCtrans were observed at any time delay after irradiation beginning. This finding suggests that the peroxide formation requires cis-configuration of the carbon double bond in monoene lipids in the presence of activated Ce6.

S3.2 DLPC vesicles

For DLPC vesicles, in addition to peroxide formation, we observe increase of C=C stretching band instead of cis-trans isomerization. Indeed, the (C=C)cis band at 1657 cm -1 experiences strong intensity enhancement with the lapse of time, along with the small frequency downshift to 1654 cm -1 . It is known that, in normal conditions, there exists a linear relationship between the number of C=C bonds and the intensity ratio R=I( 1667 )/ I(1444) (2,6). After normalization of this ratio R to 2 double bonds in the case of DLPC without Ce6, from normalized Raman spectra at different time delays, we obtained R=3 at t=80 s and even R~3.7 at t=280 s, Supl. Infos with the kinetics being stabilized around this value for longer time delays. Thus, our Raman data suggest that Ce6 photosensitizer activation causes the increase of the number of C=C double bonds in linoleic chain, or, more probably their delocalization and conjugation, in DLPC-vesicles. At this early stage, the intensity of the band at 1264 cm -1 remains unchanged with respect to that of non-conjugated DLPC (5,7).

It should be noted that in some experiments (data, not shown), when the experiment was extended to longer time delays, we eventually observed the reverse process of moderate intensity decrease of the (C=C)cis stretch at 1654 cm -1 with concomitant appearance of weak trans-species manifested by a shoulder at 1670 cm -1 and the intensity decrease of the (=CH2)cis deformation band at 1264 cm -1 .

The initial peroxide formation for DLPC vesicles (Fig. 5F) is again very rapid, within 1-2 time points (3-6 seconds), although being less efficient (<20%) than in the case of monoenes (30-35%, Fig. 5B,D). In the case of DLPC vesicles, it takes more than 150 seconds to reach the 50% level of peroxide band maximal intensity (Fig. 5F) in contrast to ~20 seconds in the case of DOPC vesicles (Fig. 5B).

S3.3 18:1/18:3 vesicles

Finally, in the case of the vesicles containing lipids mixture 18:3/18:1, the observed spectral changes and kinetics are rather complicated. In addition to a superposition of photoinduced processes observed for DOPC and DLPC, namely cis-trans isomerization, C=C double bond delocalization and conjugation, and peroxide formation, we observe also a cyclization reaction. Indeed, the creation of a six-membered (toluene-like) ring is manifested by the appearance of strong and narrow characteristic Raman bands at 1000, 1031, and 1596 cm -1 . Moreover, the kinetics of photoinduced changes vary from one measurement to the other reflecting the competition of simultaneously occurring processes; one set of the observed kinetics is shown in Fig. 6.

.

  All chemicals were purchased from Sigma (USA), except the lipids (Fig.1) 1,2-dilinolenoyl-snglycero-3-phosphatidylcholine (C18:3-PC), 1,2dilinoleoyl-sn-glycero-3-phosphatidylcholine (C18:2, DLPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (C18:1, DOPC), 1,2-dipetroselenoyl-sn-glycero-3phosphatidylcholine (DOPC-∆6), 1,2-dielaidoyl-snglycero-3-phosphatidylcholine (DOPC-trans), 1,2dimyristoyl-sn-glycero-3-phosphatidylcholine (C14:0, DMPC) from Avanti Polar Lipids (USA), and chlorin e6 from Frontier Scientific (USA). The lipids were handled very carefully, in vaccuum or under nitrogen atmosphere, when possible. For all lipids, the absence of oxidation is measured by Raman (spectra at t=0). Chlorin stock solution (5 mM) was prepared in ethanol and kept at -18 °C. The experimental Ce6 aqueous solutions were prepared and used without delay and handled in the dark. The osmolarity of the solutions was checked with an osmometer (Löser Messtechnik, Germany). Vesicle formation. The lipids (C18:3-PC, DLPC, DOPC, DOPC-trans or DOPC-∆6) were solubilized in chloroform. For the determination of the binding constant between lipid membranes and Ce6, Large Unilamellar Vesicles (LUVs) were prepared by extrusion method (38). After evaporation of chloroform, lipids were dispersed in PBS buffer by vortexing. Using an extruder device (Avanti Polar Lipids, USA), the liposome suspension was extruded 8-10 times through a polycarbonate membrane filter (Poretics, Livermore, CA) with pores of 200 nm.

Figure 1 .

 1 Figure 1. Formulae and abbreviations of the lipids composing the various GUVs, as well as of the photosensitizer Ce6.

  Fig. 2 (inset). These spectral changes are characteristic of the transfer of the chlorin from an aqueous to a hydrophobic environment (28, 36, 40, 42). The plot of the fluorescence intensity at 668 nm versus the lipid concentration shows a saturation profile (see Fig. 2). The binding constant value, K B , was determined by fitting the experimental data with

Figure 2 .

 2 Figure 2. Incorporation of Ce6 within lipid membranes. Evolution of the Ce6 fluorescence emission upon incorporation into vesicles (here, made with pure DLPC; Ce6 concentration is 5x10 -8 M). The excitation wavelength is 410 nm and the fluorescence intensity is recorded at 668 nm. Eq. 1 is used to fit experimental data and yields the binding constant K B . The inset shows the red shift of Ce6 fluorescence peak upon incorporation within the membrane.

Figure 3 .

 3 Figure3. Effect of lipid unsaturation on the morphological evolution of GUVs under photooxidation: Morphological changes of Ce6-labeled GUVs of various compositions exposed to photooxidation. Shortly after irradiation, GUVs enter a first stage characterized by an increase of membrane surface area leading to shape destabilization. Then, during a second stage, a decrease of membrane area allows the GUVs to recover a spherical shape, concomitant with a loss of contrast. Irradiation starts at time t=0s and time scale is adjusted according to the duration of the processes i.e. the composition of the vesicles. The figure presents images of vesicles of comparable radii (~5 µm) ; the intra-trial variability, related to the system size, is presented in the supporting information. Scale bar = 10 µm.

Figure 4 .

 4 Figure 4. Time evolution of optical contrast and radius of GUVs undergoing photooxidation. (Top) Evolution the contrast between the inside and the outside of the oxidized vesicles. The decay is the result of a decrease in concentration difference between the GUVs inner sucrose solution and the surrounding glucose solution. (Bottom) Vesicle radii exhibit cycles of progressive increases followed by abrupt drops, characteristic of GUVs swell-burst cycles. The results presented are those of the same vesicles as those in Figure 3.

Figure 6 .

 6 Figure 6. Time evolution of a transient pore opening in a 18:2-GUV under photooxidation. The pore is marked with an asterisk. Frequency of image acquisition: 100 Hz. Scale bar: 10 µm.

Figure 5 .

 5 Figure 5. Sketch of the proposed mechanism involved in the dynamics of the vesicles over oxidation.

Figure 7 .

 7 Figure 7. Evolution of physical parameters of GUVs undergoing photooxidation as a function of the swell-burst cycle number. (A-D) Evolution of the membrane critical strain, . (E-H) Permeability to sucrose, Ps. (I-L) Progressive decrease of the water influx related term of Eq. 4, . The 18:16-GUVs are analyzed as if they were undergoing a unique cycle. Dark lines are the mean of 20 vesicles per compositions, error bars are ± standard deviations.

  photoinduced oxidation. The RTM method has been described elsewhere[START_REF] Tatischeff | Fast characterisation of cellderived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy[END_REF][START_REF] Ashkin | Acceleration and Trapping of Particles by Radiation Pressure[END_REF] and is detailed in the Supporting Information. The changes in the Raman spectra of the vesicles lipids were recorded over 10 minutes, with an accumulation time of 3 seconds per spectrum effectively determining the time resolution of our RTM kinetic

Figure 8 .

 8 Figure 8. Chemical changes during vesicles oxidation. (A-D) Raman spectra corresponding to the beginning of the measurements (t=0 s, green curves), and to a specific characteristic time (red curves, A: 165 s, B: 216 s (B), C: 51s, D: 60 s). The 1:1 difference spectrum is in black. The characteristic band regions are tagged: peroxy (O-O) stretch (blue signs), carbon double-bond stretch (red signs) and 6-membered ring stretch (panel C, green signs). (E-H) Raman kinetics of characteristic band regions. The same colors than for tags in top panel are used: peroxy (O-O) stretch (blue curves), carbon double-bond stretch (red curves) and 6-membered ring stretch.

Figure 1 .

 1 Figure 1. Formulae and abbreviations of the lipids composing the various GUVs, as well as of the photosensitizer Ce6.

Figure 2 .

 2 Figure 2. Incorporation of Ce6 within lipid membranes. Evolution of the Ce6 fluorescence emission upon incorporation into vesicles (here, made with pure DLPC; Ce6 concentration is 5x10 -8 M). The excitation wavelength is 410 nm and the fluorescence intensity is recorded at 668 nm. Eq. 1 is used to fit experimental data and yields the binding constant K B . The inset shows the red shift of Ce6 fluorescence peak upon incorporation within the membrane.

Figure 3 .

 3 Figure 3. Effect of lipid unsaturation on the morphological evolution of GUVs under photooxidation: Morphological changes of Ce6-labeled GUVs of various compositions exposed to photooxidation. Shortly after irradiation, GUVs enter a first stage characterized by an increase of membrane surface area leading to shape destabilization. Then, during a second stage, a decrease of membrane area allows the GUVs to recover a spherical shape, concomitant with a loss of contrast. Irradiation starts at time t=0s and time scale is adjusted according to the duration of the processes i.e. the composition of the vesicles. The figure presents images of vesicles of comparable radii (~5 µm) ; the intra-trial variability, related to the system size, is presented in the supporting information. Scale bar = 10 µm.

Figure 4 .

 4 Figure 4. Time evolution of optical contrast and radius of GUVs undergoing photooxidation. (Top) Evolution the contrast between the inside and the outside of the oxidized vesicles. The decay is the result of a decrease in concentration difference between the GUVs inner sucrose solution and the surrounding glucose solution. (Bottom) Vesicle radii exhibit cycles of progressive increases followed by abrupt drops, characteristic of GUVs swell-burst cycles. The results presented are those of the same vesicles as those in Figure 3.

Figure 5 .

 5 Figure 5. Sketch of the proposed mechanism involved in the dynamics of the vesicles over oxidation.

Figure 6 .

 6 Figure 6. Time evolution of a transient pore opening in a 18:2-GUV under photooxidation. The pore is marked with an asterisk. Frequency of image acquisition: 100 Hz. Scale bar: 10 µm.

Figure 7 .

 7 Figure 7. Evolution of physical parameters of GUVs undergoing photooxidation as a function of the swellburst cycle number. (A-D) Evolution of the membrane critical strain,. (E-H) Permeability to sucrose,

Figure 8 .

 8 Figure 8. Chemical changes during vesicles oxidation. (A-D) Raman spectra corresponding to the beginning of the measurements (t=0 s, green curves), and to a specific characteristic time (red curves, A: 165 s, B: 216 s (B), C: 51s, D: 60 s). The 1:1 difference spectrum is in black. The characteristic band regions are tagged: peroxy (O-O) stretch (blue signs), carbon double-bond stretch (red signs) and 6-membered ring stretch (panel C, green signs). (E-H) Raman kinetics of characteristic band regions. The same colors than for tags in top panel are used: peroxy (O-O) stretch (blue curves), carbon double-bond stretch (red curves) and 6-membered ring stretch.

Figure S3 .

 S3 Figure S3. Normalized Raman spectra of various lipid vesicles in interaction with Ce6 residing within the optical trap: DMPC (panel A), 9-trans DOPC (panel B), 9-cis DOPC (panel C), and DLPC (panel D). Each panel contains 5 representative spectra recorded at various time delays after the beginning of the measurements; time delays are indicated at the right side of each panel by different colors. Upper part of each panel contains 4 difference spectra obtained by the (1:1) subtraction of the 0 s spectrum from the corresponding Raman spectra at various time delays. Accumulation time for any one Raman spectrum was 3 s.

  3

Table 1 .

 1 Lipid content and Ce6 binding constants for each type of model vesicle

	18:2-GUVs	DLPC	(6.83±0.20)×10 3
	18:1/18:3-GUVs	DOPC/C18:3-PC (50/50mol/mol)	(4.61±0.11)×10 3
	18:1∆6-GUVs	DOPC∆6	(5.36±0.13)×10 3
	18:1trans-GUVs	DOPCtrans	(3.25±0.5)×10 3
	DMPC-GUVs	DMPC	(6.1±0.6)×10 3
	Labeling of GUVs. Giant Vesicles were labeled with Ce6 as previously reported (28, 29). The Ce6 concentra-
	tion used was calculated according to the K B value determined beforehand for each lipid mixture to ensure an
	identical membrane labeling. The homogeneous distribution of the fluorescent Ce6 molecules within the vesicle
	membranes was assessed by fluorescence microscopy.
	Observation and illumination. GUVs were observed under an inverted microscope (Nikon Eclipse TE 300
	DV) equipped with a high numerical aperture phase oil objective (CFI Plan apochromat DM 60 n.a: 1.4, Nikon,

ACKNOWLEDGMENT

We thank C. Vever-Bizet and F. Sureau for helpful discussions. We are grateful for financial support by Region Ile-de-France in the framework DIM Nano-K (S.B. and A.B.). M.C. and P.R. acknowledge the support of ONR N00014-17-1-2628 award.

AUTHOR CONTRIBUTIONS

Supporting Information for:

Lipid unsaturation properties govern the sensitivity of membranes to photoinduced oxidative stress

Aurélien Bour, 1 Sergei G. Kruglik, 1 Morgan Chabanon, 2 Padmini Rangamani, 2 Nicolas Puff, 3,4 and Stéphanie Bonneau 1,* 1 Sorbonne Université, Faculté des Sciences et Ingénierie, CNRS, Laboratoire Jean Perrin, UMR 8237, 4 place Jussieu, Paris, France. 2 Department of Mechanical and Aerospace Engineering, University of California San Diego, La Jolla, CA 92093, United States 3 Sorbonne Université, Faculté des Sciences et Ingénierie, UFR 925, 4 place Jussieu, Paris, France. 4 Univ Paris Diderot, Sorbonne Paris Cité, CNRS, Laboratoire Matière et Systèmes Complexes (MSC), UMR 7057, 75013, Paris, France.

Table of Contents

S1. OXIDATION OF THE GUVs

S1. OXIDATION OF THE GUVs AND INTRA-TRIAL VARIABILITY

The light activation of the Chlorin e6 (Ce6) induces a photodynamic reaction of type II (singlet oxygen attacks). This is confirmed by the fact that all the samples treated with the Sodium Azide (50 mM) remain stables over light irradiation. Moreover, vesicles of which composition does not react with singlet oxygen do not exhibit any changes in terms of contrast as well as in terms of morphology (Fig. S1). This is the case of the saturated lipid control, the DMPC, but also of the DOPCtrans for which no oxidation signs has been measure, neither in terms of morphology and contrast nor in terms of Raman spectra modifications (see chapter S3 for the details of Raman). All the samples contains the same quantity of Ce6 embedded within the lipid membranes, and are irradiated in the same conditions of intensity, duration and fluence. Thus, the dynamics of the induced changes of the GUVs in terms of contrast and morphology is related to (1) the dynamics of the reactions of the lipid membrane of vesicles, i.e. their composition, and (2) the size of the vesicle, since the characteristic diffusion time depends on the distance to covered, i.e. the radius of the vesicle (see also Eq. S4 below).

Supl. Infos

Our aim is to study the effect of the membrane composition, and not that of the size of the vesicles. So, we choose to study parameters directly related to the membrane properties, such as permeability and critical strain. For each membrane composition, the images of about twenty vesicles from three sample preparations were analyzed separately. The intra-trial variability of the raw data are given in Fig. S2, where the radii of irradiated and treated vesicles are given, and correlated with the measured characteristic times, for all oxidable compositions of GUVs. According with the diffusion process, the characteristic time increases with the radius of the vesicle. Moreover, the smaller is the permeability the higher the characteristic time is (see Eq. 4). Interestingly, for the 18:3/18:3-GUVs, the values of τ change over time from small to high ones, as expected due to the decrease of the oxidized vesicles throughout the stage 2.

Supl. Infos

S2. THEORETICAL ANALYSIS OF THE SWELL-BURST CYCLES

In this section we aim to derive mathematical tools to gain information about the membrane properties based on the measurement of the GUVs contrast decay and radius dynamics. The theoretical framework of this model is based on Chabanon et al. (1).

S2.1. Evaluation of the membrane permeability to sucrose

We consider a spherical vesicle of volume V=4/3πRπRR 3πR with an inner sucrose concentration cs. The total amount of sucrose in the vesicle is therefore Vcs, and varies through (i) permeation through the membrane with a permeability to sucrose Ps, and (ii) by diffusion/advection through the pore of area πRr 2 when it is open. Therefore the evolution of sucrose concentration in the vesicle can be written as Supl. Infos

where Ds is the sucrose diffusion coefficient in water, ∆cs is the sucrose concentration differential, vL is the leakout velocity through the pore, and A= 4πRR 2 is the membrane surface area. Let us consider a particular swell-burst cycle, with cycle number n. Since the pore lifetime (~0.05 s) is much smaller than the cycles period (~10 s), we assume that the cycle period ∆tn is equal to the time required for the vesicle to swell from the resting radius R0n to Rfn, the radius at which the membrane ruptures. During that time, the pore is closed so that the first term proportional to the pore radius r in Equation S1 is zero. Furthermore, the surrounding bath volume is much larger that the vesicle volume and we assume the external sucrose concentration to remain at zero at all times. then Eq. S1gives us

where ∆Rn=Rfn-R0n, and ∆tn is the period of the cycle n. We can rewrite Eq. S1 for the n th cycle as

where R0n is the initial vesicle radius of the n th cycle. Assuming that the membrane permeability to sucrose is constant within one cycle, we can define the (constant) characteristic time τ=1/[3(

(Eq. S4) so that the sucrose concentration satisfying Eq. S3 follows an exponential decay such as

where t0n is the time at which the cycle starts. Given that ∆Rn, ∆tn, R0n, and t0n are constant quantities that can be measured for each cycle, Eq. S4 can be fitted to each cycle to determine τ, and therefore the membrane permeability to sucrose Ps.

S2.2 Relation to osmotic differential in oxidative products

Given that sucrose and glucose are initially at equimolar concentration and are permeating through the membrane at a similar rate, they do not contribute to the osmolarity imbalance. Therefore the only solutes responsible for the osmotic swelling of the GUVs are the products released during the oxidation process. Let ∆cp denote the concentration difference of oxidative products across the membrane responsible for the osmotic imbalance. The volume conservation of the vesicle can be written as

where Pw the membrane permeability to water, ∆pLap = 2σ/R is the Laplace pressure with σ being the membrane tension, νw is the molar volume of water, and kB, T, and NA are the Boltzmann constant, the bath temperature, and the Avogadro's number respectively. Once again, we consider the evolution of the vesicle volume during the swelling stage of a cycle number n. Furthermore, we assume that the osmotic pressure dominates the swelling phase (kBTNA∆cp >> ∆pLap) so that Laplace pressure is neglected. This assumption is supported by the fact that the radii increase are quasi-linear (see Figure 3). We get Supl. Infos

(Eq. S7)

Assuming the oxidative product concentration increases linearly during the time of a cycle, we have

where is the production rate of oxidative products (mol/m3/s), and is the initial oxidative product concentration of cycle number n. We therefore deduce from Eqs. S7 and S8

Δ R n

(Eq. S9)

Our measures show that ∆Rn/∆tn decreases with increasing cycle numbers. Two scenarios can be considered: (i) the production of oxidative products happens mainly at early time and is then zero for most of the process (αn=0). In that case ∆cpn0 decreases at each circle by leak-out through the transient pore, in a process similar to the osmotic swell-burst cycle described in Chabanon et al (1). Assuming Pwνw constant, the drop in oxidative product at each cycle can therefore be estimated from the measure of ∆Rn+1/∆tn+1 -∆Rn/∆tn. In scenario (ii), oxidative products are released during the course of the swell-burst cycles (αn>0). Given the Raman kinetics, it is likely that the release rate αn decreases at each cycle. Note that because αn∆tn is positive, this second scenario still implies that ∆cpn0 decreases at each cycle by leak-out through the pore.

S3. DETAILED RTM ANALYSIS

The characteristic photo-induced changes vary depending on the degree of unsaturation. In general, the following bands have been detected and quantified: The DMPC is an unsaturated lipid, used here as control. No changes have ever taken place, as expected. More interestingly, nothing was detected for 18:1 trans vesicles (Fig. S3) up to 600s.

Supl. Infos


Yan Li

Xavier Caumes

Matthieu Raynal
email: matthieu.raynal@upmc.fr.

Laurent Bouteiller

Modulation of catalyst enantioselectivity through reversible assembly of supramolecular helices

A multi-configurable catalyst, for which the degree of enantioinduction in successive reactions is varied between 6% e.e. and 52% e.e., is achieved by supporting copper centres at the periphery of supramolecular helices. Precise characterization of the co-assemblies corroborates the relation between helices length and catalyst selectivity.

External triggers such as light, redox potential, chemical species or mechanical energy emerged as a new strategy to control chemical processes. 1 Temporal control of chemical reactions has been achieved by toggling a catalyst back and forth between its active and inactive states. This switchable capacity of the catalyst is exploited in the realm of sequential reactions, e.g. allowing to finely control polymerization processes 2 or tandem reactions. 3 Much less effort has been devoted to the design of stimuli-responsive asymmetric catalysts. 4 Predictable control of the stereochemical direction for a single catalyst is achieved by supporting intrinsically achiral centres on a chirally-switchable platform such as overcrowded alkenes, 5 stereodynamic ligands, 6 redoxconfigurable copper complexes, 7 foldamers 8 or polymers. 9 Alternatively, chemical additives, 10 light 11 and redox potential 12 were employed to improve the degree of enantioinduction, 13 including a recent example of a rationallydesigned asymmetric auto-inductive process. 14 However, implementation of these elaborated catalytic systems in consecutive reactions or polymerization reactions will require a better control of their dynamic properties. Molecular catalysts for the preparation of atactic isotactic multi stereoblock copolymers are sparse 15 and, in that regard, establishing the design elements for a dynamic and multiconfigurable catalytic system will constitute an important advance. In parallel, the reversible and dynamic properties of supramolecular chiral assemblies 16 have been exploited in asymmetric catalysis. 17 We previously demonstrated that a significant degree of enantiocontrol can be achieved by supporting metal catalytic centres at the periphery of the helical assemblies formed by benzene-1,3,5-tricarboxamide monomers (BTA). 9i,18 Co-assembly between an achiral BTA ligand and an enantiopure BTA co-monomer into homochiral helices allows real-time control of the enantioselectivity. The stereochemical direction and the degree of asymmetric induction of the catalytic reaction are related to the handedness and the optical purity, respectively, of the helical BTA assemblies. For further development of this new class of catalysts, it is of utmost importance to probe how the enantioselectivity is affected by the average length of the helices. Herein, we show that reversible assembly of BTA monomers into helices of different length constitutes a simple method to vary the degree of enantioinduction of a catalytic reaction (Chart 1). The preparation of BTA helices of controlled sizes is hampered by the dynamic character of the assemblies. Moreover, hydrogen-bonded supramolecular polymers form onedimensional assemblies whose stability are particularly sensitive to the environment. 19 Anions have notably been used to reduce the chain length of urea-based supramolecular polymers without affecting their structure. 20 Therefore, we envisioned that the length of BTA assemblies could be modulated in a reversible manner by first adding an anion and then removing it, e.g. through a salt metathesis reaction leading to its co-precipitation with a cation (Chart 1). [START_REF] Yoon | Reversible binding of chloride anions to metal centres or thiourea functions has been exploited to control the activity but not the selectivity of switchable catalysts[END_REF] To implement our concept, we chose as a reference the catalytic mixture composed of an achiral BTA ligand BTA PPh2 (6 mol%) an enantiopure co-monomer BTA (R)-Cha (6.6 mol%), [Cu(OAc)2•H2O] (3 mol%) and PhSiH3 since it furnished the hydrosilylation product of 1-(4-nitrophenyl)ethanone (NPnone) with significant enantioselectivity (54±1% e.e. at 293 K in toluene). 9i A combination of spectroscopic analyses and catalytic experiments indicated that the chirality of the comonomer controls the handedness of the helices and thus the stereochemical direction of the hydrosilylation reaction. Two preliminary experiments now confirm that the size of the BTA assemblies also affects the level of asymmetric induction (Table S1, ESI †): (i) in THF, BTAs are molecularly dissolved and no selectivity is observed, and (ii) when the reaction is conducted with a concentration in BTA PPh2 close to its critical concentration in toluene, the selectivity is significantly decreased (19% e.e.). In the latter case, it is likely that at this concentration the assemblies are too short to arrange all the copper centers in a suitable chiral environment. We next investigated the influence of TPPCl (tetraphenylphosphonium chloride) on the performance of Chart 1 Schematic representation of the present concept. Supramolecular helices, formed by co-assembly between an achiral BTA ligand (BTA PPh2 ) coordinated to Cu and an enantiopure BTA co-monomer (BTA (R)-Cha), are shortened and lengthened reversibly by means of an appropriate combination of salts. This schematic representation of the co-assemblies is supported by analytical data but the coordination mode of Cu and the fraction of coordinated PPh2 groups in the co-assemblies are not known. S.1. Conversions (after 1h): >99% (0 and 0.3 equiv. of TPPCl), 97% (0.5 equiv.), 91% (0.75 equiv.), 75% (0.85 equiv.) and 63% (1.0 equiv.).

the catalytic system of reference (Fig. 1). [START_REF] Yoon | Reversible binding of chloride anions to metal centres or thiourea functions has been exploited to control the activity but not the selectivity of switchable catalysts[END_REF] The first (unanticipated) effect is the slowdown of the reaction rate: the higher the amount of TPPCl, the lower the rate (Table S1).

Whilst the origin of this negative effect of chloride anions on the reaction rate has not been established for the present case, inhibition of metal-catalysed reactions by soluble halogen anions has some precedent in the literature. 22 The decreased rate for the non-selective state of the supramolecular catalyst is not detrimental since good conversions can be achieved by increasing the amount of silane and the reaction time (vide infra). The second (anticipated) effect is a gradual decrease of the enantioselectivity as a function of the amount of chloride anions in the catalytic mixture: from 44% e.e. (0.3 equiv. relative to BTA PPh2 ) to 0% e.e. (1.0 equiv.). ‡ The fact that a high content of TPPCl relatively to BTA PPh2 is required to completely erase the selectivity suggests that the length of the assemblies should be shortened down to very short aggregates to be nonselective (vide infra). It is interesting to note that TPPCl does not act as dual on-off selectivity switch but rather allows programmable modulation of the degree of enantioinduction (signal output) in between fully racemic, and optimal enantiomeric preference (53% e.e.) by simply changing the concentration of TPPCl (signal input). The nature of the co-assemblies formed between BTA PPh2 and BTA (R)-Cha in toluene and their reversible assembly have been precisely characterized by means of Fourier-Transform Infrared (FT-IR), Nuclear Magnetic Resonance (NMR), Small Angle Neutron Scattering (SANS) and Circular Dichroism (CD) analyses. The "pristine" co-assemblies, i.e. in absence of any additives, are very long (DPn ≈ 250), stack with a single molecule in the cross-section and contain approximately two BTA PPh2 molecules for one BTA (R)-Cha (Figs. S1-S2). They form homochiral left-handed helices as deduced from CD analysis (Fig. 2b). 9i The coordination of BTA PPh2 to copper does not significantly change the structure of the helical co-assemblies (Fig. S4). However, both SANS (Fig. S5) and FT-IR (Figs. 2a) analyses confirm that the length of the co-assemblies is drastically decreased in presence of 1.0 equiv. of TPPCl. Notably, in FT-IR, the bonded amide band is reduced whereas the free amide band is increased in presence of TPPCl (green arrows in Fig. 2a). Deconvolution of the ester C=O and amide C=O bands yields an estimation of DPn ≈ 2 for the average length of the co-assemblies in presence of 1.0 equiv. of TPPCl (Fig. S7). The absence of an Induced CD (ICD) signal 23 for this mixture (Fig. 2b) corroborates the fact that these objects are likely too small to adopt a helical configuration and explains the absence of selectivity for the catalytic reaction (vide supra). NaNTf2 (Tf=triflimide) was selected to trigger the metathesis reaction because of the weak coordination ability and the known chemical stability of the NTf2 anion. 24 NaNTf2 is introduced in solution in acetone as we observe a good solubility of this salt in the three-solvent mixture (toluene:DCM:acetone 15:1:1). In these conditions, reassembly Please do not adjust margins Please do not adjust margins andS7). In the FT-IR spectra, both dimers of BTA (R)-Cha 25 and stack ends contribute to the free amide C=O band. The observed CD signal belongs to BTA PPh2 only and is thus an ICD signal 23 which reflects the chiral environment of the ligand. 9i For the "pristine" co-assemblies, this signal is of comparable intensity (Δ=24 L.mol -1 .cm -1 ) to that previously observed in methylcyclohexane (Δ=17 L.mol -1 .cm -1 ) inferring the presence of homochiral left-handed helices. 9i is evidenced by the increased intensity of the bonded amide band and the decreased intensity of the free amide band (wine arrows in Fig. 2a). Also, this sample exhibits a significant ICD signal (Fig. 2b) which infers that the ligand has recovered a chiral environment. The above experiments have established the use of TPPCl (1.0 equiv., added as a solution in DCM) and NaNTf2 (1.0 equiv., added as a solution in acetone) as a suitable combination of salts for the reversible assembly of the supramolecular helices. § We thus investigated whether this strategy can be used to switch the enantioselectivity of our catalytic system of reference. Sequential additions of NPnone, silane and salts (Fig. 3 top) have been performed in the following order: no salt (run 1), TPPCl (run 2), NaNTf2 (run 3), TPPCl (run 4) and NaNTf2 (run 5). An excess of silane has been engaged and the reaction times have been adapted so that a conversion superior or equal to 84% was achieved for each run (except for run 4, conversion=53%). The enantiomeric excess for each run was calculated from the cumulated e.e. obtained by chiral GC analyses of the aliquots as detailed in Table S3. As shown in Fig. 3 Modulation of the enantioselectivity of the supramolecular catalyst involved in the hydrosilylation of several equivalents of NPnone added sequentially in the reaction mixture. Top: description of the run. TPPCl and NaNTf2 are added as solutions in DCM and acetone, respectively so that the solvent nature is: toluene (run 1), toluene:DCM 15:1 (run 2), toluene:DCM:acetone 15:1:1 (run 3), toluene:DCM:acetone 15:2:1 (run 4) and toluene:DCM:acetone 15:2:2 (run 5). Bottom: enantioselectivity obtained for each run. Conversions: 99% (run 1), 86% (run 2), 89% (run 3), 53% (run 4) and 84% (run 5). Double arrows represent the selectivity obtained in the same solvent but in absence of salts. For the determination of the e.es. and the conversions, see Table S3. Fig. 3, the state of the supramolecular copper catalyst is alternatively selective (29-52% e.e.) and poorly selective (0-6% e.e.). More precisely, a significant decrease of the enantioselectivity is found for the run 2 (6±1% e.e.) and run 4 (0±5% e.e.), as a result of the disassembly of the supramolecular helices supporting the catalytic centres in presence of TPPCl. The selectivities of run 3 (43±1.5% e.e.) and run 5 (29±5% e.e.) are significantly higher than those of run 2 and run 4, which corroborates the reversible assembly of the BTA helices and validates our concept. We were intrigued by the decreased selectivity for run 3 and 5 compared to run 1 and therefore investigated its origin. After reassembly (in presence of TPPCl and NaNTf2, run 3), the coassemblies have DPn in between 6 and 15 much lower than that of "pristine" co-assemblies (run 1, DPn ≈ 250) as evidenced by FT-IR analyses (Fig. 2a). In fact, this shortening of the helices is mostly due to the presence of co-added solvents (DCM and acetone) as demonstrated by: (i) control catalytic experiments in absence of TPPCl and NaNTf2 (double red arrows, Fig. 3) which provide roughly the same selectivity for run 3 and 5, (ii) the fact that, in toluene:DCM:acetone 15:1:1, the FT-IR, NMR and CD spectra of the co-assemblies in absence or presence of TPPCl and NaNTf2 are similar (Figs. 2 andS7). This substantiates the efficiency of the metathesis salt reaction and infers that full reassembly is hampered by the competitive Please do not adjust margins Please do not adjust margins and polar nature of the solvents employed to dissolve the salts. Interestingly, the selectivity decrease (19%) observed after a single disassembly/reassembly switch (from 52% e.e. for run 1 to 43% e.e. for run 3) correlates well with the lower intensity (25%) of the ICD signal measured for co-assemblies in presence of the salt mixture and the co-solvents (Fig. 2b). There is thus a good correlation between the average length of the assemblies, the chiral environment of the ligand and the selectivity of the reaction (Table S2). For the present catalytic system, the critical length of the supramolecular helices required to locate most of the copper centres in a chiral environment suitable for asymmetric induction is thus in the range of a ten monomers. In conclusion, a multi-configurable catalyst is obtained by reversibly shortening and lengthening the supramolecular helices formed by assembly of BTA monomers. This can undoubtedly be extended to other families of hydrogenbonded supramolecular polymers and organogels with the aim of tuning their macroscopic properties. Further progress in the design of such functional supramolecular assemblies is required for the implementation of more efficient smart systems in the field of switchable catalysis. This work was supported by the French Agence Nationale de la Recherche (project ANR-13-BS07-0021 SupraCatal) and by the China Scholarship Council (CSC, PhD grant of Y.L.). Jacques Jestin (LLB, Saclay) is acknowledged for assistance with SANS experiment. Claire Troufflard and Aurélie Bernard are acknowledged for performing VT-NMR analyses.

Fig. 1

 1 Fig. 1 Enantioselectivity as a function of the amount of TPPCl. See the precise conditions in TableS.1. Conversions (after 1h): >99% (0 and 0.3 equiv. of TPPCl), 97% (0.5 equiv.), 91% (0.75 equiv.), 75% (0.85 equiv.) and 63% (1.0 equiv.).

Fig. 2

 2 Fig. 2 Characterization of the reversible assembly of supramolecular helices. FT-IR (a) and CD (b) analyses of the co-assemblies in absence and presence of salts (293 K). The concentrations in ligand and co-monomer are close to those of catalytic experiments indicated in red. The DPn values have been estimated by means of SANS and FT-IR analyses (Figs. S1and S7). In the FT-IR spectra, both dimers of BTA (R)-Cha25 and stack ends contribute to the free amide C=O band. The observed CD signal belongs to BTA PPh2 only and is thus an ICD signal23 which reflects the chiral environment of the ligand. 9i For the "pristine" co-assemblies, this signal is of comparable intensity (Δ=24 L.mol -1 .cm -1 ) to that previously observed in methylcyclohexane (Δ=17 L.mol -1 .cm -1 ) inferring the presence of homochiral left-handed helices. 9i

Conflicts of interest

There are no conflicts to declare.

Notes and references ‡ To avoid any confusion with the catalytic loading (expressed in mol% of substrate), the amount of TPPCl is expressed in number of equivalent relatively to BTA PPh2 . §TPPNTf2 and NaCl, the salts generated upon the metathesis reaction, are poor hydrogen competitors and poorly soluble, respectively (Table S1).