Helene Pillet
email: helene.pillet@ensam.eu

Xavier Bonnet

Amandine Boos

Lucas Sedran

Bruno Watier
email: bruno.watier@laas.fr

Seeking for a better Human-Prosthesis energetic gait efficiency by quantifying both propulsion power and instability control *

The present study aims at quantifying propulsion and dynamic balance through biomechanical parameters issued from theoretical modeling and analysis of locomotion during the gait of people using prosthetic devices. An experimental protocol combined motion capture and oxygen consumption quantification during gait on a treadmill. The mechanical work produced and dissipated by the lower limbs and the evolution of a biomechanical indicator of balance were used and the estimation of the metabolic cost of walking was made from oxygen consumption. To test the relevance of the chosen parameters, the experiments were performed on six ablebodied volunteers successively equipped with two prosthetic ankle-feet (elastic vs rigid) mounted on a femoral prosthetic simulator. For each participant, the parameters were computed and compared in three configurations: i/ without prosthesis, ii/ with rigid prosthetic ankle-foot iii/ with elastic prosthetic ankle-foot. The results put in evidence an increase of energy consumption in both prosthetic configurations compared to the configuration without prosthesis. However, no differences could be observed between the elastic and rigid prosthetic configurations. The analysis of mechanical work performed by each lower limb, which confirmed the energy delivered by the elastic foot during the propulsion, did not explain by its own this discrepancy. The maintenance of balance that seems to be more challenging during the double support in the elastic configuration could be involved in this counter-intuitive result. Finally, this preliminary study shows the importance to consider simultaneously propulsion and balance objectives during gait as they must both require muscular actions involved in the production of energy by the prosthesis user.

I. INTRODUCTION

Poor human-prosthesis adaptation has been identified as a key factor of the relative inefficiency of robotic lower limb prostheses to restore a normal metabolic cost of transport of individuals with lower limb amputation [START_REF] Helen) Huang | Taking both sides: seeking symbiosis between intelligent prostheses and human motor control during locomotion[END_REF]. Indeed, if considered as an isolated parameter, ankle push-off is clearly related to the energetic efficiency of walking in humans [START_REF] Huang | Mechanical and energetic consequences of reduced ankle plantarflexion in human walking[END_REF]. However, powered prosthetic ankles failed in restoring both normal energetic consumption and preferred gait speed of people with transtibial amputation [START_REF] Walker | Lower limb amputation following injury: a survey of long-term functional outcome[END_REF] . A possible explanation lies in the independent control of the prosthesis and the human musculo-skeletal system. In normal locomotion, the joints are actuated by muscles including biarticular ones that ensure consistent actions under the control of the central nervous system. While wearing a prosthesis, both prosthetic and residual musculo-skeletal system are not directly connected anymore, which can hinder their correct interaction. Considering people with transfemoral amputation, this should be emphasized as they have to use simultaneously a prosthetic knee and a prosthetic ankle-foot, which behavior are generally independent from each other. On another hand, dynamic balance during locomotion is also known as challenging due to the inherent instability of biped walking. Besides, the control of instability is intuitively involved in the overall energetic cost of walking as it requires muscle activations to prevent falling. Again, the challenge is all the more important for persons wearing prosthetic devices that are known to have more asymmetrical and perturbed kinematics and dynamics. However, very few studies have sought to evaluate simultaneously 'balance-related effort' and push-off work control [START_REF] Wen | Wearer-Prosthesis Interaction for Symmetrical Gait: A Study Enabled by Reinforcement Learning Prosthesis Control[END_REF]- [START_REF] Houdijk | Energy storing and return prosthetic feet improve step length symmetry while preserving margins of stability in persons with transtibial amputation[END_REF].

A. State of the art

The study of the energetics of gait has been the topic of numerous biomechanical studies. To this aim, the estimation of the metabolic energy through oxygen consumption quantification is considered as a gold standard to analyze the effect of different configurations for a given individual at a given time [START_REF] Ding | Control and Optimization of Soft Exosuit to Improve the Efficiency of Human Walking[END_REF] even if the results are known to be sensitive to the well-known biases associated to great interindividual variations, influence of nutrition and fatigue. To better understand the link with muscle functions, several teams have tried to link the metabolic energy consumed by the individuals to mechanical works performed by the joints [START_REF] Donelan | Mechanical work for step-to-step transitions is a major determinant of the metabolic cost of human walking[END_REF]- [START_REF] Arones | Musculoskeletal Model Personalization Affects Metabolic Cost Estimates for Walking[END_REF]. The definition of mechanical work that should be correlated to metabolic energy is also subject to debate. The definition depends on the underlying modeling, of which the complexity can vary. Work of the Individual Limb Method (WILM) [START_REF] Donelan | Simultaneous positive and negative external mechanical work in human walking[END_REF] seems a good compromise being simple to assess and closely related to other methods such as summed-joint-power classically considered as a standard in biomechanics [START_REF] Bonnet | Distribution of joint work during walking on slopes among persons with transfemoral amputation[END_REF]. To date, more complex models such as complete musculoskeletal modeling of the body have not led to significantly more accurate estimation of the metabolic cost [START_REF] Umberger | Stance and swing phase costs in human walking[END_REF]. As concerns gait instability assessment, several indicators have also been proposed [START_REF] Bruijn | Assessing the stability of human locomotion: a review of current measures Assessing the stability of human locomotion: a review of current measures[END_REF]. Among them, the variation of the angular momentum at the body center of mass (BCoM) during the motion appears as one of the most promising due to its ability to link the rotations of the segments to the external mechanical action at the center of mass [START_REF] Popovic | Angular momentum regulation during human walking: biomechanics and control[END_REF]. However, both its determination and interpretability can be complex. In a recent work, we propose to use a novel mechanical indicator of the instability of gait that partly solve these issues [START_REF] Bailly | A Mechanical Descriptor of Human Locomotion and its Application to Multi-Contact Walking in Humanoids[END_REF], [START_REF] Al-Abiad | A Mechanical Descriptor of Instability in Human Locomotion: Experimental Findings in Control Subjects and People with Transfemoral Amputation[END_REF]. It is based on a distance computed between the Minimal Moment Axis of external mechanical actions to the Body Center of Mass (MMA/BCoM). To our knowledge, no study has analyzed for the same subjects, the quantification of mechanical work, of gait instability and of the consumed metabolic energy in a population of people with transfemoral amputation. One reason of this lack is the difficulty to recruit such volunteers. To solve the issue of recruitment, some authors have proposed to use prosthetic simulators that can be worn by non-amputee persons [START_REF] Caputo | Prosthetic ankle push-off work reduces metabolic rate but not collision losses in non-amputee walking experiment[END_REF], [START_REF] Adamczyk | Mechanisms of Gait Asymmetry Due to Push-Off Deficiency in Unilateral Amputees[END_REF]. They have been widely used for prosthetic ankle-foot evaluation but never for knee and ankle-foot replacement.

B. Contributions

In the present work, we propose to combine both complementary approaches of mechanical work quantification and gait instability assessment and to analyze then in regards of the quantification of the metabolic energy. We therefore proposed to determine ILM mechanical work (WILM) together with the MMA/BCoM indicator described in the above section. Both parameters determination relies on the computation of body center of mass kinematics and external mechanical actions during gait. Metabolic energy was assessed independently from oxygen consumption uptake during walking. In order to test the relevance and sensitivity of the parameters, we designed an original experimental framework involving volunteers equipped with prosthesis simulator including knee and ankle-foot joints. Three conditions could be compared without and with the simulator equipped with two different prosthetic foot. Finally, the implications of the experimental results for prosthesis design and control have been extracted.

II. THEORETICAL BACKGROUND

A. Mechanical work and energetic considerations

There exist different ways of quantifying mechanical power and work performed by the joints when walking. The kinetic energy theorem links the variation of kinetic energy E kin of a system (here the entire body) relatively to a referent frame (here R o ) to the sum of the powers of external and internal mechanical actions (P ext and P int ) [START_REF] Helen) Huang | Taking both sides: seeking symbiosis between intelligent prostheses and human motor control during locomotion[END_REF].

dE kin (body/R o ) dt = P ext + P int (1)
Considering the entire body as the mechanical system of interest and modeling the body segments as articulated rigid bodies, the sources of internal power originate from the mechanical actions in the anatomical joints, which themselves result from the muscles mechanical actions passing through the joints. Therefore, at steady state, for example during a gait cycle the metabolic energy that is consumed for the functioning of muscles generating joint motions should theoretically be inferred from the computation of mechanical work performed inside the body [START_REF] Riddick | Mechanical work accounts for most of the energetic cost in human running[END_REF]. Mechanical work quantification over a given period of time can be done by integrating mechanical power. However, as this computation stands on an algebraic sum of powers, it implicitly assumes that the mechanical energy produced in a joint can be absorbed by another one whether it is in the same limb or between limbs [START_REF] Willems | External, internal and total work in human locomotion[END_REF]. If this is correct in a mechanical system, the physiologic reality could be different as no evidence that such types of transfer exist between muscles [START_REF] Willems | External, internal and total work in human locomotion[END_REF] and the current level of knowledge does not allow to integrate refined modeling of muscle functioning in an analysis including the whole body. Taking a more pragmatic approach, some authors have proposed to sum the joint power no more in an algebraic sense but by using absolute values either considering individual joints [START_REF] Willems | External, internal and total work in human locomotion[END_REF], [START_REF] Devita | Muscles do more positive than negative work in human locomotion[END_REF], [START_REF] Montgomery | The contributions of ankle, knee and hip joint work to individual leg work change during uphill and downhill walking over a range of speeds[END_REF] or individual limbs [START_REF] Zelik | Mechanical work as an indirect measure of subjective costs influencing human movement[END_REF]. In the individual limbs paradigm, the underlying modeling of gait is a succession of pendulum-like phases where power is mainly generated or dissipated during the step-to-step transition [START_REF] Houdijk | The energy cost for the step-to-step transition in amputee walking[END_REF], [START_REF] Bonnet | Mechanical work performed by individual limbs of transfemoral amputees during step-to-step transitions: Effect of walking velocity[END_REF]. The internal mechanical power performed by the body can therefore be computed from the scalar product of the ground reaction forces (equal to the internal force in the pendulum) and the velocity of the center of mass. The comparison of this estimation of the internal power with the one made from the multibody modeling has already been performed from experimental data [START_REF] Zelik | Human walking isn't all hard work: evidence of soft tissue contributions to energy dissipation and return[END_REF], [START_REF] Zelik | Six degreeof-freedom analysis of hip, knee, ankle and foot provides updated understanding of biomechanical work during human walking[END_REF] and from a theoretical approach [START_REF] Bonnet | Mechanical work performed by individual limbs of transfemoral amputees during step-to-step transitions: Effect of walking velocity[END_REF] for level walking.

B. Control of the instability during locomotion

In addition to provide the necessary energy to achieve a given displacement of the body center of mass, the human musculo-skeletal system must be controlled in a way to avoid falling. In the case of generic biped walking, the external mechanical actions that act on the system are gravity and ground contact forces, knowing that contacts are regularly broken (toe off) and recovered (heel strike) [START_REF] Wieber | On the stability of walking systems[END_REF]. In mechanics, stability refers to a given equilibrium position of the system (where the movement parameters remain constant) and can be defined as the ability of the system to recover from a perturbation around that equilibrium position. Thus, walking is not a succession of equilibrium positions and cannot be considered as stable from this point of view [START_REF] Wieber | On the stability of walking systems[END_REF]. From such mechanical considerations, it does not appear straightforward to relate mechanical modeling to comprehensive parameters of balance capacity. The dynamic laws directly relate the acceleration of the center of mass of the system to the external resulting forces acting on it and the variations of the angular momentum at the body center of mass (which includes the effect of inertia and the rotational and translational accelerations of the individual body segments) to the resulting moment from mechanical actions at the center of mass. A variety of parameters have been derivated from this type of analysis involving more or less complex modeling of the human body used in both biomechanics and robotics among which Zero Moment Point (ZMP)/Center of Pressure (COP) is the most popular [START_REF] Sardain | Forces acting on a biped robot. Center of pressure -Zero moment point[END_REF]. Its relative position to the support basis has been used as an objective for robot control and for biomechanical evaluation of gait stability. However, this criterion has revealed limited and not applicable in several conditions because it assumes that the contact points are on the same horizontal surface and internal muscles or motor actions alleviate immediate fallings. Therefore, alternative parameters involving the kinematics of the center of mass (Margin of Stability [START_REF] Hof | The 'extrapolated center of mass' concept suggests a simple control of balance in walking[END_REF], Foot Rotation Index [START_REF] Goswami | Postural stability of biped robots and the foot-rotation indicator (FRI) point[END_REF], . . . ) have been developed. At the same time, a thorough analysis of the amplitude of the whole-body angular momentum at the center of mass during human gait [START_REF] Herr | Angular momentum in human walking[END_REF]- [START_REF] Neptune | Dynamic Balance during Human Movement: Measurement and Control Mechanisms[END_REF] showed that it is controlled during steady-state gait and increases in impaired populations [START_REF] Herr | Angular momentum in human walking[END_REF], [START_REF] Neptune | Dynamic Balance during Human Movement: Measurement and Control Mechanisms[END_REF]. The variations of the wholebody angular momentum can be also captured from the ground reaction forces and moments. This is the basis for the definition of the Centroidal Moment Point developed by Herr and Popovic [START_REF] Herr | Angular momentum in human walking[END_REF] or the Virtual Pivot Point proposed by Maus [START_REF] Maus | Upright human gait did not provide a major mechanical challenge for our ancestors[END_REF]. However, both consider a simplified representation of the mechanical actions of contact between the feet and the ground by neglecting in particular the free moment which remains at the center of pressure around the vertical axis. On the contrary, our team proposed a parameter called MMA/BCoM index which consists in the computation of the distance between the center of mass (BCoM) of the system and the minimum moment axis (MMA) of the external mechanical actions of the ground on the system [START_REF] Bailly | A Mechanical Descriptor of Human Locomotion and its Application to Multi-Contact Walking in Humanoids[END_REF], [START_REF] Al-Abiad | A Mechanical Descriptor of Instability in Human Locomotion: Experimental Findings in Control Subjects and People with Transfemoral Amputation[END_REF]. Indeed, the theory of mechanical actions modeling shows that there is an axis called the central axis or minimum moment axis along which the ground reaction moment is minimal [START_REF] Herr | Angular momentum in human walking[END_REF]. The distance of this axis relatively to the center of mass d BCoM/MMA is therefore directly related to the moment of external actions acting at the center of mass M BCoM and the ground reaction force F , with the advantage of being easy to understand [START_REF] Huang | Mechanical and energetic consequences of reduced ankle plantarflexion in human walking[END_REF]. The greater the distance, the greater the angular acceleration of the body around the center of mass is.

d BCoM/MMA = ||F × M BCoM || ||F 2 || (2)

III. EXPERIMENTAL ASSESSMENT

A. Prosthetic configurations

Starting from the statements that (i) most of the power is delivered at the ankle joint during normal gait and (ii) larger increase of energy consumption has been observed for transfemoral amputee, we have chosen to compare two femoral prostheses in which only the prosthetic ankle-foot varied. The prosthetic knee was a magneto-rheologic microprocessorcontrolled knee (Rheo T M , Ossur). The two prosthetic anklefeet were a carbon-leaf elastic foot, referred thereafter as ESR (Dynatreck T M , Proteor) and a rigid 3D printed foot, referred thereafter as RF Fig. 1. The stiffness of the ESR foot was chosen according to the mass of each person in accordance with prosthetist recommendations. The rigid foot was designed to reproduce a similar evolution of the center of pressure in the tibial local frame for the RF as compared to the ESR [START_REF] Hansen | Roll-over characteristics of human walking on inclined surfaces[END_REF].

B. Participants and prosthesis simulator

To avoid the difficulties associated to the inclusion of persons with amputation (recruitment, security), we proposed to use a prosthesis simulator that can be worn by nonamputee persons (Fig. 1). It also allows to compare the two prosthetic configurations to the non-amputee configuration where the same individuals walked without the simulator. Six able-bodied volunteers (2 females and 4 males) participated in the study (mean age 31 years (std 8.6), mean height 174 cm (std 5.8), mean mass 68 kg (std 10.9). They were trained (at least one hour a day during one week) to walk with the simulator before the experiments.

C. Experimental protocols

The protocol (2020-A02369-30) was approved by ethical committee and each participant signed an informed consent. The protocol was divided in two sessions in which they successively underwent a quantitative gait analysis from motion capture system (ViconTM, Oxford Metrics®) and force plates acquisition (AMTI®) and an analysis of metabolic energy consumption using a respirometry system (K5TM, Cosmed®).During the first session, they were asked to walk on a 10 m flat walkway including three separate force plates (two 586*464cm and one 600*800 cm) in series. The contact reaction mechanical actions between each foot and the ground could therefore be acquired at 1000 Hz during three successive stance phases. In the same time, subjects were equipped with 58 reflective markers that were captured synchronously by the optoelectronic system at 200 Hz. The subjects chose their own self-selected speed with the prosthesis simulator equipped with the rigid foot and reproduced it for all other configurations (elastic foot and without the simulator). In the second session, the same subjects walked in the same three configurations at the velocity chosen in the first session on a treadmill for 6 min per configuration. Before starting the experiment, the resting metabolic energy consumption was measured for 3 min for baseline while the subject stood quietly. Then, oxygen consumption was continuously recorded during walking.

D. Data acquisition and processing

From the first session, only the trials with three successive foot contacts on the force plates were considered. Force plates data and markers' trajectories were filtered using a 4th order low-pass Butterworth filter with a 15 Hz cutoff frequency. A 15-segment hybrid inertial model defined according to [START_REF] Pillet | Evaluation of force plate-less estimation of the trajectory of the centre of pressure during gait. Comparison of two anthropometric models[END_REF] was used to obtain body segmental inertial parameters, and the BCoM instantaneous position was computed as the weighted sum of the segments' centers of mass. Then, the mechanical power of each individual limb was assessed by the dot product of the velocity of the body center of mass (by derivation of its position) and the resulting ground reaction forces on the considered lower limb. Positive and negative works (WILM+ and WILM-) were calculated by numerical integration of positive and negative powers respectively over time and normalized according to the body mass. The MMA along which moment of the external contact forces is minimal was computed from GRF. was then obtained at each instant of time using: From the second session, the metabolic cost was derived from the oxygen consumption (VO2 (mL/min)) for each trial. In order to exclusively obtain the metabolic cost for walking the metabolic resting values for standing were subtracted from the metabolic energy consumed during walking. The values were normalized according to the body mass.

IV. RESULTS AND DISCUSSION

A. Comparison of configurations

The average self-selected walking velocity across subject was 1.08 m/s (std: 0.14 km/h). The stiffness of the carbon leaf foot was 203 Nm/rad when participant body mass was inferior to 60kg and 297 Nm/rad when it was superior to 60 kg. The Fig. 2, Fig. 3 and Fig. 4 gives the oxygen consumption, the mechanical work produced by each individual limb (prosthetic and contralateral WILM) during one gait cycle and the evolution of the distance during the gait cycle of the prosthetic limb respectively for the two configurations with the simulator compared to the configuration without prosthesis.

B. Energetic considerations

From the analysis of the oxygen consumption in the three studied configurations, it can be observed that the gait with the simulator induced an increased expense of metabolic energy whatever the ankle-foot used. This trend Average distance between the Body Center of Mass and the Minimal Moment Axis (BCoM-MMA) for the three configurations : without prosthesis in grey solid line, with rigid foot in grey dotted line and with elastic foot in black dashed line is consistent with the one observed when comparing the energy consumption of people with transfemoral amputation and non-amputee people. However, the oxygen consumption was doubled for all the subjects between the configuration with versus without prosthesis whereas it was reported to be between 30% and 50% between transfemoral amputee and able-bodied persons [START_REF] Van Schaik | Metabolic costs of activities of daily living in persons with a lower limb amputation: A systematic review and meta-analysis[END_REF]. In contrast the variation of oxygen consumption between the two prosthetic configurations (elastic and rigid foot) was very limited (2% increase for the elastic foot vs the rigid one). This result can seem counter intuitive considering the property of the elastic foot to store and return energy. Indeed, the elastic foot embeds a carbon leaf that stores mechanical energy by deformation at the beginning of the stance which can be released at the end of the stance when the foot is progressively unloaded during the step-to-step transition. On the contrary, the rigid foot can only roll-over the shape described by the center of pressure in the tibial frame when walking but without any energy storing and return property. The computation of the mechanical work performed by the individual limbs confirmed that positive energy is delivered by the elastic ESR foot (0.16 J/kg) over the gait cycle which is less than the one produced by the ankle of the non-amputee subject (0.25 J/kg) but far more than the one quantified with rigid RF foot (0.05 J/kg). This energy that is provided to the person should theoretically reduce the necessary energy to be produced by the residual joints of the person. However, the analysis of the mechanical work performed by the contralateral limb revealed that an increase of the work performed when wearing the elastic ESR foot (0.53 J/kg) compared to the one performed when wearing the RF foot (0.45 J/kg). Even if the value of the mechanical work cannot be directly extrapolated to explain the increase of the metabolic cost of walking when wearing the simulator, the results of our study are in favor of a role of the contralateral limb in the production of energy in the configuration with the elastic ESR foot. In addition, the analysis of the evolution of over the prosthetic cycle shows an asymmetry of the profile with a significant increase of the distance just before the step-to-step transition at the end of the stance phase. This increase is not retrieved neither at the end of the contralateral stance phase nor for non-amputee people. The values of maximal distance found in our study are consistent with previous studies for both non-amputee persons and people with transfemoral amputation [START_REF] Al-Abiad | A Mechanical Descriptor of Instability in Human Locomotion: Experimental Findings in Control Subjects and People with Transfemoral Amputation[END_REF]. The increase of the distance in this phase of gait can result from the necessity to unlock the prosthetic knee which requires a muscular action at the residual hip. Even considering the necessity to unlock the knee, it must be also noted that the distance, can be seen as a proxy of the variation of angular momentum, which increase is itself limited by the mechanical actions that can be transmitted at the contact between the foot and the ground (i.e. through friction forces). Finally, it can be hypothesized that it exists a compromise between limiting the instability and unlocking the knee for the swing phase. This compromise is made possible from the muscular actions at different levels of the body that could participate to the increase of the energy consumption and partly explain the relative inefficiency of the releasing of elastic energy in late stance with the ESR foot.

C. Limitations

Several limitations exist in the present study. The representativity of the simulator has not been evaluated. The generalization of results will necessitate studies including people with amputation. For example, the added mass of the simulator contributes to the increase of the oxygen uptake compared to the "without" configuration. However, it does not explain the overall difference. In the same idea, even if not fully representative of the results that should be obtained with people with amputation, the study shows that energy restitution provided by elastic foot is not sufficient to induce a reduction of metabolic cost of walking. Finally, metabolic cost could not be quantified simultaneously with the biomechanical parameters in the absence of an instrumented treadmill.

V. CONCLUSION

To conclude, the present work is, to our knowledge, the first contribution which aimed to compare elastic energy storing and release foot versus rigid foot from the energetic point of view in femoral prosthetic simulator including a microprocessor-controlled knee. To perform this comparison, three complementary analyses were conducted including metabolic analysis of energy consumption, quantification of the mechanical work performed by the limbs and the estimation of the variation of angular momentum through an indicator developed in the team. The results obtained in the present study show that the mechanical work delivered by the prosthetic foot is not the only determinant of the metabolic energy consumed by the prosthetic user. Considering that the knee is passive and does not provide any energy, this study tends to demonstrate that the overconsumption of energy observed in people with transfemoral amputation also comes from the modification of the dynamics of the overall body. Thus, from our work, it is possible to define targets to be reached by the design and control of the prosthesis and by the rehabilitation process. In particular, the challenge is to manage simultaneously propulsion at the ankle and control of the knee to decrease compensatory motions of the residual limb and the upper body which increase overall instability. This should also result in a decrease of the demand on the contralateral side. The indicators used in the present study are both based on BCoM kinematics and ground reaction forces and moments. One perspective of this work could be to compute them from on board sensors embedded in the device. If the analysis must be deepened to understand the interaction between the foot design and the performed compensation, the results should in the future be also extended to other assistive devices such as exoskeleton and more largely to a better understanding of anthropomorphic action.

Fig. 1 .

 1 Fig. 1. Prosthetic feet used (A) Energy Storing and Release and (B) rigid and prosthesis simulator (C)

Fig. 2 .

 2 Fig. 2. Average oxygen consumption (VO2: mL/min/kg) for the 3 configurations (elastic foot : ESR, rigid foot : RF and non amputee : NA) and comparison to existing literature including transfemoral amputee

ACKNOWLEDGMENT

The authors thank the volunteers who participated in the study.

*Research supported by the Fondation de l'Avenir (grant number AP-RM-20-001), DGA-RAPID AAFAPE and ANR Hobis (grant number 18CE27-0010) and COBOT (grant number 18CE10 -0003).


Chengying Li

Zi Lin

Xingyu Jiang

Nicolas Martin

Liangfei Tian
email: liangfei.tian@zju.edu.cn

Engineering the Coacervate Microdroplet Interface via Polyelectrolyte and Surfactant Complexation

Complex coacervate microdroplets, which are formed via spontaneous liquid-liquid phase separation by mixing two oppositely charged polyelectrolytes in water, have emerged as a new paradigm in the fields of origin of life, membraneless subcellular compartmentalization, bioreactors, and drug delivery. However, how to further improve its stability and enhance its selectivity in one particular coacervate system remains a challenge. By generating a membrane-like layer at the surface of coacervate microdroplets via electrostatic interactions between oppositely charged surfactants and polyelectrolytes, we here achieve tunable permeability and enhanced stability of the coacervates at the same time. Depending on the surfactants used, membrane-like layer-coated coacervate microdroplets exhibit different selectivity over solute sequestration and can promote or inhibit DNA hybridization. Our approach provides a practical tool to engineer functional bioinspired microcompartments with potential applications in the fields of controlled drug release and microreactor technology.

Introduction

Over the past few decades, coacervates have been recognized as an emerging research area in various fields, such as origin of life, (1-4) membraneless cellular compartments, [START_REF] Gomes | The Molecular Language of Membraneless Organelles[END_REF][START_REF] Shin | Liquid Phase Condensation in Cell Physiology and Disease[END_REF][START_REF] Yewdall | Coacervates as Models of Membraneless Organelles[END_REF][START_REF] Banani | Biomolecular Condensates: Organizers of Cellular Biochemistry[END_REF](9)[START_REF] Donau | Active Coacervate Droplets as a Model for Membraneless Organelles and Protocells[END_REF][START_REF] Rumyantsev | Polyelectrolyte Complex Coacervates: Recent Developments and New Frontiers[END_REF] bioreactors [START_REF] Wang | Spatiotemporal Organization of Coacervate Microdroplets[END_REF][START_REF] Liu | Hydrogel-Immobilized Coacervate Droplets as Modular Microreactor Assemblies[END_REF][START_REF] Leathers | Reaction-Diffusion Patterning of DNA-based Artificial Cells[END_REF][START_REF] Mu | Membrane-Confined Liquid-Liquid Phase Separation toward Artificial Organelles[END_REF] and drug delivery. [START_REF] Mi | Thermostabilization of Viruses via Complex Coacervation[END_REF][START_REF] Singh | Coacervation of Elastin-Like Recombinamer Microgels[END_REF][START_REF] Vieregg | Oligonucleotide-Peptide Complexes: Phase Control by Hybridization[END_REF][START_REF] Sun | Phase-Separating Peptides for Direct Cytosolic Delivery and Redox-Activated Release of Macromolecular Therapeutics[END_REF][START_REF] Liu | High-Order Assembly toward Polysaccharide-Based Complex Coacervate Nanodroplets Capable of Targeting Cancer Cells[END_REF] Complex coacervate microdroplets are formed via spontaneous liquidliquid phase separation by mixing two oppositely charged polyelectrolytes in water, resulting in a dense, polyelectrolyte-rich phase (the coacervate phase) dispersed in a dilute continuous phase. [START_REF] Chen | Optothermally Programmable Liquids with Spatiotemporal Precision and Functional Complexity[END_REF] Due to their all-aqueous nature, these droplets have been described as water-in-water (w/w) emulsion systems, which are colloidal dispersions consisting of two immiscible aqueous phases and exhibit good biocompatibility and environmental friendliness. [START_REF] Esquena | Water-in-Water (W/W) Emulsions[END_REF][START_REF] Gucht | Polyelectrolyte Complexes: Bulk Phases and Colloidal Systems[END_REF][START_REF] Keating | Aqueous Phase Separation as a Possible Route to Compartmentalization of Biological Molecules[END_REF] However, due to their lack of a membrane, coacervate droplets typically lack selective permeability to solutes and are susceptible to coalescence and pH/salt-induced disassembly, which limits their applications.

To date, different approaches have been developed to overcome these restrictions through engineering the interface of the coacervate microdroplet by either interfacial self-assembly of block copolymers, [START_REF] Mason | Hierarchical Self-Assembly of a Copolymer-Stabilized Coacervate Protocell[END_REF] protein nanoconjugate, [START_REF] Li | Spatial Organization in Proteinaceous Membrane-Stabilized Coacervate Protocells[END_REF] molecular amphiphiles, [START_REF] Tang | Fatty Acid Membrane Assembly on Coacervate Microdroplets as a Step towards a Hybrid Protocell Model[END_REF][START_REF] Zhou | Fatty Acid-Based Coacervates as a Membrane-free Protocell Model[END_REF][START_REF] Chang | Membrane-Mediated Transport in a Non-Equilibrium Hybrid Protocell based on Coacervate Droplets and a Surfactant[END_REF][START_REF] Zhang | Giant Coacervate Vesicles as an Integrated Approach to Cytomimetic Modeling[END_REF][START_REF] Tian | Artificial Morphogen-Mediated Differentiation in Synthetic Protocells[END_REF][START_REF] Coudon | Stabilization of All-Aqueous Droplets by Interfacial Self-Assembly of Fatty Acids Bilayers[END_REF] nanoparticles, [START_REF] Gao | Triggerable Protocell Capture in Nanoparticle-Caged Coacervate Microdroplets[END_REF][START_REF] Williams | Spontaneous Structuration in Coacervate-based Protocells by Polyoxometalate-Mediated Membrane Assembly[END_REF] or supramolecular entities [START_REF] Liu | Enzyme-Mediated Nitric Oxide Production in Vasoactive Erythrocyte Membrane-Enclosed Coacervate Protocells[END_REF][START_REF] Pir Cakmak | Lipid Vesicle-Coated Complex Coacervates[END_REF] on coacervate microdroplets or generation of a membrane-like layer around the coacervate microdroplet core. [START_REF] Yin | Spontaneous Membranization in a Silk-Based Coacervate Protocell Model[END_REF][START_REF] Abbas | Peptide-Based Coacervate-Core Vesicles with Semipermeable Membranes[END_REF][START_REF] Merindol | Pathway-Controlled Formation of Mesostructured all-DNA Colloids and Superstructures[END_REF] Although these previous investigations showed that the selective permeability towards the external medium and the inhibition of coalescence could be achieved to some extent after the treatment, there is still no effective way to simultaneously improve selective permeability and stability against coalescence and pH/salt-induced disassembly.

Herein, we report a general approach to engineering the interface of coacervate microdroplets using attractive electrostatic interactions between oppositely charged surfactants and polyelectrolytes (Scheme 1). We show that various positively charged surfactants, such as cetyltrimethylammonium bromide (CTAB), didecyldimethylammonium bromide (DDAB), and tetradecyltrimethylammonium bromide (TTAB), or negatively charged polyelectrolytes, such as double-strand deoxyribonucleic acid (dsDNA) and polyacrylic acid (PAA), can form a membrane-like layer via the polyelectrolyte-micelle interaction at the interface of the different coacervate microdroplets. This membrane-like layer can concurrently improve selective permeability and stability against coalescence and pH-/salt-induced disassembly of the coacervate microdroplets, and can be used to promote or inhibit DNA hybridization by selective partitioning or exclusion of functional biomolecules. Scheme 1. Schematic Representation of the Preparation of Surfactant-Coated Coacervate Microdroplets a a A two-step procedure was developed for the preparation of surfactant-coated coacervate microdroplets. In the first step, the coacervate micro-microdroplets were formed by direct mixing of aqueous solutions of different polycations, such as PEI and polydimethyl diallyl ammonium chloride (PDDA) and polyanions, such as dsDNA and PAA. Then, different surfactants, such as CTAB, DDAB, and TTAB, were added to the corresponding suspension of coacervate microdroplets, leading to the formation of different surfactant-coated coacervate microdroplets.

Experimental section Preparation of Surfactant-Coated Coacervate Microdroplets

DNA/PEI coacervate microdroplets were produced by mixing polyethylenimine (PEI, monomer concentration 50 mM, 50 μL, pH 8) and DNA (average monomer concentration 50 mM, 100 μL, pH 8) aqueous solutions in water (850 μL) to reach final monomer concentrations of 2.5 mM for PEI and 5 mM for DNA. Then, surfactant-coated DNA/PEI coacervate microdroplets were produced by the addition of CTAB (50 mM, 5 μL), DDAB (50 mM, 5 μL), or TTAB (50 mM, 5 μL) to DNA/PEI coacervate microdroplet suspension (95 μL) to reach final concentrations of 2.5 mM CTAB, DDAB, or TTAB. Similarly, PDDA/DNA and PDDA/PAA coacervate microdroplets were prepared by mixing poly(diallyldimethylammonium chloride) (PDDA 10 mg/mL, 100 μL, pH 7) and DNA (32.5 mg/mL, 100 μL, pH 7) or PDDA (10 mg/mL, 100 μL, pH 12) and PAA (10 mg/mL, 100 μL, pH 12) in water (800 μL) or NaCl solution (250 mM, 800 μL, pH 12). The CTABcoated PDDA/DNA coacervate microdroplets were prepared by the addition of CTAB (50 mM, 5 μL) to the PDDA/DNA coacervate microdroplet suspension (95 μL) to reach a final concentration of 2.5 mM CTAB. In comparison, CTAB-coated PDDA/PAA coacervate microdroplets were prepared by the addition of CTAB (10 mM, 5 μL) to PDDA/PAA coacervate microdroplet suspension (95 μL) to reach a final concentration of 0.5 mM CTAB.

Sequestration Properties of Coacervate Microdroplets

Different fluorescent dyes (Nile red: 1 μL, 10 μM, final concentration 0.1 μM; methylene blue: 1 μL, 10 μM, final concentration 0.1 μM; rhodamine B: 1 μL, 10 μM, final concentration 0.1 μM, calcein: 1 μL, 10 μM, and final concentration, 0.1 μM), polymers with different molecular weights (fluorescein isothiocyanate-dextran (FITC-dextran): 4 kDa, 40 kDa, 150 kDa, 1 μL, 10 μM, final monomer concentration 0.1 μM), or biomacromolecules (rhodamine isothiocyanate-bovine serum albumin (RITC-BSA) 66 kDa, RITC-horseradish Peroxidase (RITC-HRP) 40 kDa, 5-carboxytetramethylrhodamine-single-stranded DNA (TAMRA-ssDNA), 1 μL, 10 μM, final concentration: 0.1 μM) were added to freshly prepared non-coated and CTAB, DDAB, or TTAB-coated DNA/PEI coacervate microdroplet suspension (100 μL). The mixtures were incubated for 30 min and then loaded on a custom-made capillary slide and left to settle for 10 min for confocal fluorescence microscope imaging. Partition coefficients were determined from the ratio of fluorescence intensities in the droplets and in the surrounding dilute aqueous phase. Measurements were performed on 10 droplets from three different fields of view of the same sample, and the average value and standard deviation were calculated.

Stability Studies of Coacervate Microdroplets

To investigate the stability of the non-coated and CTAB, DDAB, or TTAB-coated DNA/PEI coacervate microdroplets in different ionic strengths, a series of coacervate microdroplet suspensions were prepared in the presence of different concentrations of NaCl (50, 100, 150, 200, 250, 400, and 800 mM). Typically, NaCl stock solution (5 μL; 1, 2, 3, 4, and 5 M) was added into freshly prepared CTAB, DDAB, or TTAB-coated and non-coated coacervate microdroplet solutions (95 μL) to prepare different microdroplet suspensions with a final NaCl concentrations of 50, 100, 150, 200, and 250 mM. To prepare the mixture with final NaCl concentration of 400 and 800 mM, NaCl stock solution (50 μL; 0.4 and 1.6 M) was added into freshly prepared CTAB, DDAB, or TTAB-coated and non-coated coacervate microdroplet solutions (950 μL) in an Eppendorf tube. All the samples were incubated for 60 min at the desired salt concentration and then imaged by microscopy. To study the stability of the noncoated and CTAB, DDAB, or TTAB-coated DNA/PEI coacervate microdroplets at different pH, CTAB, DDAB, or TTAB-coated and non-coated coacervate microdroplets were prepared as described previously at pH 8. HCl or NaOH solutions (1 M) were used to adjust the pH from 1 to 11, and the mixtures were incubated for 120 min for further measurements.

Confocal Microscopy

Confocal images were taken using a Leica TCS SP5 laser scanning confocal inverted microscope (LSCM) with Leica LAS AF software and an HCX PL APO CS 63.0×/1.40 oil UV objective. Nile red, Methylene blue, Calcein, Rhodamine B, FAM-, TAMRA-, RITC-, and FITC-were excited at 559, 665, 494, 570, 488, 561, 543, and 488 nm, respectively. The corresponding emissions were 635, 690, 517, 595, 525, 650, 580, or 515 nm. Fluorescence intensity data was acquired from raw fluorescence images using ImageJ.

Results

Preparation of CTAB-Coated Coacervate Microdroplets

We employed a two-step procedure [START_REF] Mason | Hierarchical Self-Assembly of a Copolymer-Stabilized Coacervate Protocell[END_REF][START_REF] Li | Spatial Organization in Proteinaceous Membrane-Stabilized Coacervate Protocells[END_REF][START_REF] Tang | Fatty Acid Membrane Assembly on Coacervate Microdroplets as a Step towards a Hybrid Protocell Model[END_REF][START_REF] Zhang | Giant Coacervate Vesicles as an Integrated Approach to Cytomimetic Modeling[END_REF][START_REF] Coudon | Stabilization of All-Aqueous Droplets by Interfacial Self-Assembly of Fatty Acids Bilayers[END_REF][START_REF] Gao | Triggerable Protocell Capture in Nanoparticle-Caged Coacervate Microdroplets[END_REF][START_REF] Williams | Spontaneous Structuration in Coacervate-based Protocells by Polyoxometalate-Mediated Membrane Assembly[END_REF][START_REF] Liu | Enzyme-Mediated Nitric Oxide Production in Vasoactive Erythrocyte Membrane-Enclosed Coacervate Protocells[END_REF] to prepare surfactant-coated coacervate microdroplets (Scheme 1). In the first step, coacervate microdroplet suspensions were produced by mixing two oppositely charged polyelectrolytes, and then various surfactants were added to the suspension of coacervate microdroplets, leading to the formation of different surfactant-coated coacervate microdroplets. As a proof of principle, we first prepared DNA/polyethylene imine (PEI) complex coacervate microdroplets with a final monomer concentration of 5/2.5 mM. The gradual addition of CTAB (critical micelle concentration (CMC) of CTAB, CMC = 1 mM) to the coacervate microdroplet suspension resulted in a progressive increase of surface charge of the DNA/PEI coacervate microdroplets from -25.6 to 32.5 mV with an increase in the final CTAB concentration from 0 to 2.5 mM (Figure 1A), which could be due to the electrostatically mediated adsorption of the cationic CTAB molecules on the microdroplet surface. To further verify this, the surface charge of the coacervate microdroplets was modified by changing the DNA/PEI molar ratio (Figure S1). The increase of final monomer concentrations of PEI from 0.5 to 10 mM led to an increase in surface charge from─50.4 to 33.6 mV with a constant final average DNA monomer concentration (5 mM). The addition of positively charged CTAB to the negatively charged coacervates resulted in a similar increase in the surface charge of the microdroplets from -25.6 to 32.5 mV with an increase of the CTAB concentration, while there was no significant change of the surface charge when CTAB was added to positively charged coacervates (Figure S1), confirming that the adsorption of cationic CTAB molecules was driven by electrostatic interactions.

Small-angle X-ray scattering (SAXS) experiments were performed to examine the mesoscale structure after the addition of CTAB to the coacervate microdroplets suspension. The scattering pattern showed a long-range mesostructured order (with a peak at q = 1.48 nm -1 ) between CTAB micelle and dsDNA, (40) which was also observed in coacervate droplets suspensions when the final concentration of CTAB (2.5 mM) was above the CMC of CTAB (Figure 1B). On the opposite, such a longrange ordered structure was neither found when the final concentration of CTAB was below the CMC (Figure S2) nor when the surface charge of coacervate microdroplets was near neutral or positive (Figure S3). This was also in agreement with cryo-transmission electron microscopy (cryo-TEM) measurements (Figures 1C,D andS4). A membrane-like layer around the coacervate microdroplet core with a thickness of 400 ± 150 nm was found when the final concentration of CTAB was 2.5 mM, suggesting the formation of polyelectrolyte-micelle complexes at the interface of the coacervate microdroplet. The confocal microscopy image and associated fluorescence intensity line profile of CTAB-coated DNA/PEI coacervate microdroplets, in which 0.1% of PEI was labeled with Rhodamine-B, showed almost the same intensity through the droplet (Figure S5), indicating PEI was not depleted in the membrane. The fluidity of the membrane-like layer was then examined by fluorescence recovery after photobleaching (FRAP) experiments (Figures 1E,F andS6). To this end, the DNA/PEI coacervate microdroplets were labeled with the DNA staining dye 4′ and 6-diamidine-2′-phenylindole dihydrochloride, and the photobleached area was chosen as a square spot of 0.5 μm width in the center or at the interface of a single droplet. Photobleaching a small section of the interface of CTABcoated coacervate microdroplets gave rise to a relatively slow recovery of fluorescence intensity compared with similar measurements recorded for the internal part. In contrast, the recovery of fluorescence intensity was nearly the same when a similar photobleaching measurement was performed in non-coated DNA/PEI coacervate microdroplets, indicating that the DNA mobility was greatly inhibited in the membrane-like layer in CTAB-coated coacervate microdroplets. The impact of salt concentration on the formation of surfactant polyelectrolyte complexes at the surface of coacervate microdroplets was explored by preparing different coacervate microdroplet suspensions in the presence of NaCl (200 and 400 mM) (Figure S7). It was found that coacervate microdroplets were stable in the presence of 200 mM NaCl. The increase in salt concentration can also lead to an increase of electrostatic screening effect, resulting in a decrease in the CMC of CTAB in the presence of 200 mM NaCl (CMC = 0.35 mM, Figure S8). The long-range mesoscale ordered structure (with a peak at q = 1.28 nm -1 ) was found when the final CTAB concentration was 0.8 mM, indicating that the increase of salt concentration will facilitate membrane-like layer formation (Figure S7). However, the further increase of final concentration of NaCl to 400 mM completely destroyed the coacervate microdroplets (Figure S7).

Influence of the Membrane-like Layer on the Uptake or Exclusion of Guest Molecules

Taken together, the above observations showed that a membrane-like structure was formed at the interface of coacervate microdroplets upon addition of surfactant micelles, presumably attributed to the electrostatic complexation between micelles and oppositely charged polyelectrolyte chains. We then explored the influence of this membrane on the uptake or exclusion of guest molecules present in the external medium, such as different charged molecular dyes, polymers with different molecular weights, and various biomacromolecules, using confocal fluorescence microscopy (Figure 2A). The corresponding plots of partitioning coefficients demonstrated that CTAB-coated microdroplets showed a strong preference in the uptake of low molecular weight, negatively charged calcein and TAMRA-ssDNA and in the exclusion of positively charged methylene blue (Figure 2B-D). The uptake of low molecular weight neutral and zwitterionic Nile red and rhodamine B and high molecular weight polymers (FITC-dextran, 4, 40, 150 kDa) and biomacromolecules (RITC-BSA 66 kDa, RITC-HRP 40 kDa) were essentially the same for both CTAB-coated and non-coated coacervate microdroplet. The different uptake properties could be due to the change in surface charge from -25.6 to 32.5 mV after the addition of CTAB. Plots of the change of partitioning coefficients in CTAB-coated and non-coated coacervate microdroplets revealed that the membrane-like layer had a strong impact on the sequestration of charged low molecular weight dyes but was not noticeably selective toward low molecular weight neutral and zwitterionic molecular dyes or high molecular weight polymers (Figure 2E-G).

Influence of the Membrane-like Layer on the Stability of Coacervate Microdroplets

The stability against coalescence was then explored by mixing two populations of fluorescently labeled surfactant-coated or non-coated coacervate microdroplets. To this end, fluorescence resonance energy transfer (FRET)-paired complementary single strands of TAMRA-ssDNA (red) or 5carboxyfluorescein-ssDNA (FAM-ssDNA, green) were pre-sequestered in CTAB-coated or non-coated coacervate microdroplets, and then these two populations were mixed with a volume ratio of 1:1. The CTAB-coated coacervate microdroplets were stable and displayed no content mixing (Figure 3A). While the uncoated coacervate microdroplets readily coalesced within 10 min, FRET was observed due to the hybridization of these two complementary ssDNA (Figure 3B), indicating that the membrane like layer could inhibit the coalescence of the coacervate microdroplets. Due to the high viscosity of the droplet, the mobility of fluorescently labeled DNA was reduced, requiring a longer incubation time for complete mixing. They did coalesce after a longer incubation time (Figure S9). To verify the results if mix-coated and uncoated coacervate microdroplets together, we supplemented the experiments with FAM-ssDNA (green) or TAMRA-ssDNA (red)-labeled CTAB-coated and non-coated coacervate microdroplets. After mixing, there was no coalescence within 10 min, while they did coalesce after a longer incubation time (Figure S10). Moreover, the impact of the membrane-like layer on the stability of the microdroplets was investigated by preparing CTAB-coated and non-coated coacervate microdroplets in different concentrations of NaCl solution (Figures 4 andS11 andS12). After incubating the droplets with 200 mM NaCl solution for 1 h, fluorescence-activated cell sorting (FACS) measurements showed that the number of coacervate microdroplets was significantly reduced for both CTAB-coated and non-coated samples, with an increase of their average size from 2.3 to 3.7 μm for CTAB-coated samples and 2.4 to 4.3 μm for non-coated samples (Figure 4A-D). Plots of the number of remaining coacervate microdroplets in solutions containing different concentrations of NaCl showed that the number of CTAB-coated microdroplets was higher than that of non-coated ones (Figures 4E and S11 and 12), revealing the enhanced stability after CTAB-coating. The increase of average size of CTAB-coated and non-coated coacervate microdroplets was attributed to the change of surface charges due to the electrostatic screening effect after the addition of NaCl. For instance, the surface charges for both CTAB-coated and non-coated coacervate microdroplets became neutral (Figures S13 andS14) after the addition of NaCl, leading to an increased tendency of microdroplets to coalesce. Similar results were also found using optical microscopy (Figure 4F-P). The non-coated microdroplets almost disappeared when the final concentration of NaCl reached 250 mM, while a few CTAB-coated microdroplets were still observed when the final NaCl concentration was increased to 600 mM. The membrane persisted even after the disintegration of coacervate droplets inside. The improvement of stability could be due to the fact that the addition of membrane could reduce coalescence and the occurrence of Ostwald ripening between different coacervate microdroplets. [START_REF] Li | Ostwald-Like Ripening of the Anomalous Mesoscopic Clusters in Protein Solutions[END_REF] The influence of pH on the coacervate microdroplets prepared from DNA/PEI microdroplets with or without CTAB-coating was also studied (Figures S15 andS16). ( 42) Both CTAB-coated and non-coated coacervate microdroplets showed a similar trend in the basic condition, and the microdroplets disappeared when the pH was increased to 11. However, CTAB-coated DNA/PEI microdroplets were more stable in acidic conditions, and a few microdroplets were still present when the pH was decreased to 1, while noncoated coacervate microdroplets disappeared when the pH was decreased to 4. The mean coacervate microdroplet counts were calculated from optical microscopy images using Image J software, and the standard deviation was calculated accordingly. The fit lines serve only for eye guidance.

General Applicability of the Surface Engineering Approach

We further extended the current approach to prepare coacervate microdroplets coated with different surfactants, such as DDAB and TTAB (Figures S17 andS18). After the addition of DDAB (2.5 mM, CMC = 0.1 mM) and TTAB (2.5 mM, CMC = 1.5 mM) to the DNA/PEI coacervate microdroplet suspension, a similar increase of surface charge and appearance of scattering patterns in the SAXS profiles were found for both samples, suggesting comparable membrane-like layers were formed at the interface of DNA/PEI coacervate microdroplets. Moreover, different polyelectrolytes were also used to examine the general applicability of membrane formation at the interface of coacervate microdroplets. Another coacervate microdroplet with different polyanion and polycation made by PAA/PDDA was also prepared in the presence of NaCl (200 mM) (Figure S19). Due to the increase of ionic strength, the CMC of CTAB is reduced from 1 to 0.35 mM (Figure S8). ( 43) Comparable results were also found after adding CTAB (final concentration: 0.5 mM) to PAA/PDDA coacervate microdroplet suspension in the presence of NaCl (200 mM) via complexation of CTAB micelles and PAA [START_REF] Niu | Complexation of DNA with Cationic Surfactants as Studied by Small-angle X-ray Scattering[END_REF] at the interface of the droplet (Figure S19).

The permeability of the membrane-like layers was accordingly explored (Figures S20 andS21), and observations showed that the low molecular weight neutral dye Nile red was preferentially enriched within, while the charged methylene blue, calcein, and rhodamine B were excluded from DDAB-coated coacervate microdroplets. After the addition of TTAB, there was no obvious difference in the uptake of Nile red and rhodamine B and an exclusion of calcein and methylene blue. In comparison, high molecular-weight FITC-dextran (4, 40, and 150 kDa) and biomacromolecules (RITC-BSA, RITC-HRP, and T-ssDNA) tended to be sequestered in DDAB-and TTAB-coated microdroplets. In general, the DDAB or TTAB-coated coacervate microdroplets showed less selectivity toward different charged small molecules compared to CTAB-coated coacervates, which might have been due to the different surface charges of the DDAB-or TTAB-coated microdroplets (around 0 mV) compared to CTAB-coated microdroplets (+30 mV). The fact that DNA/PEI coacervate microdroplets showed different permeabilities after the addition of different surfactants (CTAB, DDAB, and TTAB) revealed that the addition of different surfactants can be used to alter the permeability of the coacervate microdroplets.

Given that the permeability of coacervate microdroplets could be regulated by engineering their interface using various surfactants, we sought to investigate the efficiency of ssDNA hybridization using fluorescence resonance energy transfer (FRET)-paired complementary single strands of TAMRA-ssDNA (red) or FAM-ssDNA (green). After hybridization, the emission of the donor ssDNA (FAM-ssDNA) can excite the acceptor ssDNA (TAMRA-ssDNA) due to FRET, leading to a red fluorescence output with an excitation wavelength of 488 nm. To this end, the donor ssDNA (FAM-ssDNA) was pre-sequestered in surfactant-coated or non-coated coacervate microdroplets (Figure 5A), and confocal fluorescence microscopy revealed a green fluorescence and no red fluorescence after exposure to blue light (488 nm) (Figures 5B,C andS22). After the addition of acceptor ssDNA to the suspension, an immediate increase of red fluorescence and a continuous decrease of green fluorescence were found under blue light (488 nm), indicating complementary ssDNA hybridization (Figure 5B,C). Plots of the normalized fluorescence intensities against time showed that there was more DNA hybridization in CTAB-coated coacervate microdroplets than that in non-coated droplets (Figure 4D), which was in good agreement with the increased uptake of ssDNA in CTAB-coated coacervate microdroplets (Figure 2D). The same experiments were also performed for DDAB-and TTAB-coated samples. The normalized maximum intensities (Figure 5E) and corresponding deduced apparent rate constants (Figure 5F) showed that DNA hybridization was promoted after the formation of the CTAB/TTAB membrane-like layer. In contrast, the apparent rate constant was decreased by a factor of 10 for the DDAB-coated sample, which could be due to the double-chained molecular architecture of DDAB, leading to the formation of a lamellar structure. [START_REF] Zhou | Nanostructures of Complexes Formed by Calf thymus DNA Interacting with Cationic Surfactants[END_REF] The apparent rate constants increase as the ssDNA partitioning coefficients increase. In addition, there were no noticeable differences in local viscosity for various surfactantcoated coacervate microdroplets (Figure S23). Taken together, we speculate that the change of crossmembrane rate could be the main driving force behind the change in the hybridization rate. The stability of coacervate microdroplets against ionic strength was also explored after the addition of different surfactants. After incubation in the presence of varying concentrations of NaCl for 1 h, the stability of TTAB-and DDAB-coated coacervate microdroplets was greatly improved (Figure 6A-C), which was similar to the case of CTAB-coated droplets (Figure 4F-J). One interesting finding was that the DDAB-coated microdroplets deformed (Figure 6B) when the final salt concentration reached 200 mM. We also found that water-filled vacuoles formed in these non-spherical DDABcoated coacervate microdroplets with an increase of mean diameter from 2.2 to 5.6 μm (Figure 6D-G and Movie S1). The whole process was completely reversible; the vacuoles disappeared and the DDABcoated microdroplets adopted a spherical shape after re-dispersing the sample in the salt-free supernatant of DDAB-coated DNA/PEI coacervate microdroplets solution for 5 min (Figure 6D-G and Movie S2). The reversible vacuolization within DDAB-coated coacervate microdroplets could be associated with osmotic pressure differences after the addition or removal of NaCl. Vacuoles were not observed when the other surfactants were used, so they could be associated with the double-chained molecular architecture of DDAB, resulting in a much more stable membrane that could accommodate the osmotically-driven volume expansion.

Conclusion

In conclusion, we have shown that attractive electrostatic interactions between oppositely charged surfactants and polyelectrolytes can be used as a general approach to engineer the interface of coacervate microdroplets by generating a membrane-like layer at the droplet surface. Depending on the surfactants, membrane-like layer coated coacervate microdroplets exhibit different solutes sequestration selectivity and can promote or inhibit DNA hybridization. The surfactant-coated coacervate microdroplets can simultaneously improve their stability against coalescence and pH/saltinduced disassembly and are more resistant to a broader range of ionic conditions (up to 600 mM NaCl) or pH environments (pH 1 to pH 10). Our approach opens the possibility of developing a novel model of w/w emulsion system with tunable permeability and enhanced stability and provides a practical tool to engineer functional bioreactors which can promote or inhibit particular biochemical reactions by regulating the enrichment and exclusion of functional biomolecules, with potential applications in the fields of controlled drug release such as gene delivery [START_REF] Ahn | DNA/PEI Nanoparticles for Gene Delivery of Rat Bone Marrow Stem Cells[END_REF][START_REF] Pack | Design and Development of Polymers for Gene Delivery[END_REF] and microreactor technology.

Figure 1 .

 1 Figure 1. Preparation of CTAB-coated DNA/PEI coacervate microdroplets. (A) Zeta potentials of CTABcoated DNA/PEI coacervate microdroplets at different CTAB concentrations (0-2.5 mM). (B) SAXS profiles of DNA/PEI coacervate microdroplets (red line), CTAB-coated DNA/PEI coacervate microdroplets (2.5 mM, black line), and CTAB/DNA complex (blue line). (C,D) Cryo-TEM images of CTAB-coated (C) and non-coated (D) coacervate microdroplets. Scale bars, 1 μm. (E,F) Corresponding FRAP measurements of CTAB-coated (E) and non-coated (F) coacervate microdroplets. The blue and black plots correspond to the FRAP measurements recorded at the internal part and at the interface of CTAB-coated (E) and non-coated (F) coacervate microdroplets. Error bars indicate the standard deviation of three repeated measurements.

Figure 2 .

 2 Figure 2. Sequestration properties of CTAB-coated and non-coated coacervate microdroplets. (A) Confocal fluorescence microscopy images of the sequestration of molecular dyes (calcein, rhodamine B, methylene blue, Nile red), FITC-dextran with average molecular weights of 4, 40, and 150 kDa, and different biomacromolecules, such as RITC-BSA, RITC-HRP, and T-ssDNA, in CTAB-coated DNA/PEI coacervate microdroplets ([CTAB] = 2.5 mM) and DNA/PEI coacervate microdroplets (without CTAB). Scale bars, 1 μm. (B-D) Corresponding plots of partitioning coefficients of molecular dyes (B), FITCdextran with different molecular weights (C), and different biomacromolecules (D) in CTAB-coated (K1) and non-coated (K2) coacervate microdroplets. The partitioning coefficients are 11/9.4 (nile red), 970/5100 (calcein), 174/7.2 (methylene blue), 31/59 (rhodamine B), 1.3/1.5 (FITC-dextran, 4 kDa), 1.6/1.2 (FITC-dextran, 40 kDa), 1.6/0.8 (FITC-dextran, 150 kDa), 1.5/2.6 (RITC-BSA), 1.4/2.0 (RITC-HRP), and 15/50 (T-ssDNA) for non-coated and CTAB-coated coacervate microdroplet. (E-G) Plots of the change of partitioning coefficients in CTAB-coated and non-coated coacervate microdroplets for different molecular dyes (E), FITC-dextran with different molecular weights (F) and different biomacromolecules (G). The difference in the change of partitioning coefficient was obtained by K1/K2 when dyes were more sequestered in CTAB-coated coacervate microdroplets compared to non-coated droplets, or by K2/K1 when dyes were more sequestered in non-coated coacervate microdroplets compared to the coated ones.

Figure 3 .

 3 Figure 3. Stability against coalescence of CTAB-coated coacervate microdroplets. (A,B) Confocal microscopy images of a mixture of FAM-ssDNA (green) or TAMRA-ssDNA (red) labeled CTAB-coated (A) and non-coated (B) coacervate microdroplets. After mixing, there was no coalescence for the CTABcoated samples. While the uncoated coacervate microdroplets readily coalesced within 10 min. Scale bars, 1 μm for (A), 10 μm for (B, left), and, 1 μm for (B, right).

Figure 4 .

 4 Figure 4. Stability against ionic strength of CTAB-coated and non-coated coacervate microdroplets. (A-D) Two-dimensional (2D) dot plots of side-scattered (SSC) versus forward-scattered (FSC) light for noncoated (A) and CTAB-coated (B) PEI/DNA coacervate microdroplets in aqueous solution and noncoated (C) and CTAB-coated (D) PEI/DNA coacervate microdroplets in the presence of 200 mM NaCl obtained by FACS. (E) Plots of the FACS-derived histograms for non-coated (red) and CTAB-coated (black) PEI/DNA coacervate microdroplets with different concentrations of NaCl (0, 50, 100, 150, 200, and 250 mM). Error bars indicate the standard deviation of three repeated measurements. (F-O) Optical microscopy images of CTAB-coated (F-J) and non-coated (K-O) coacervate microdroplets at different concentrations of NaCl (0, 50, 200, 250, and 600 mM). Scale bars, 10 μm. (P) Plots of the mean coacervate microdroplet counts for non-coated (red) and CTAB-coated (black) PEI/DNA coacervate microdroplet with different concentrations of NaCl (0, 100, 150, 200, 400, 600, and 800 mM). The mean coacervate microdroplet counts were calculated from optical microscopy images using Image J software, and the standard deviation was calculated accordingly. The fit lines serve only for eye guidance.

Figure 5 .

 5 Figure 5. Tunable permeability of various surfactant-coated coacervate microdroplets. (A) Schematic representation of the complimentary single-strand DNAs (ssDNAs) hybridization inside coacervate microdroplets. The donor ssDNA (FAM-ssDNA) was pre-sequestered in the surfactant-coated or noncoated coacervate microdroplets, and then the acceptor ssDNA (TAMRA-ssDNA) was added to the suspension. These two complimentary ssDNAs were hybridized inside the coacervate microdroplets due to the sequestration of acceptor ssDNA. (B,C) Time-dependent confocal fluorescence microscopy images of single FAM-ssDNA containing CTAB-coated PEI/DNA coacervate microdroplets after the addition of TAMRA-ssDNA in the green (γex = 488 nm, γem = 500-550 nm, FAM-ssDNA) (B) and red (γex = 488 nm, γem = 575-700 nm, TAMRA-ssDNA) (C) channels. Time in seconds. Scale bar, 1 μm. (D) Plots of the normalized fluorescence intensities in green (FAM-ssDNA) and red (TAMRA-ssDNA) channels against time after the addition of TAMRA-ssDNA for CTAB-coated (empty circle) and non-coated (solid square) PEI/DNA coacervate microdroplets. (E) Corresponding normalized maximum intensities in red (TAMRA-ssDNA) channels for non-coated and DDAB, CTAB, and TTAB-coated PEI/DNA coacervate microdroplets. (F) Corresponding plots of the slopes of the initial change of fluorescence intensities (apparent rate constant, k) in green (FAM-ssDNA) and red (TAMRA-ssDNA) channels for non-coated and DDAB-, CTAB-, and TTAB-coated PEI/DNA coacervate microdroplets. The apparent rate constants were deduced from the linear fit of the plots of the change of fluorescence intensity from 0 to 120 s. Errors bars represent the standard deviation values for measurements on 10 individual droplets in the same field of view.

Figure 6 .

 6 Figure 6. Stability against ionic strength of various surfactant-coated coacervate microdroplets (A,B) Optical bright field images of TTAB-(A) and DDAB-coated (B) PEI/DNA coacervate microdroplets with different concentrations of NaCl (0, 200, 400, and 800 mM). Scale bars, 10 μm. (C) Plots of the mean coacervate microdroplet counts for TTAB-coated (green) and DDAB-coated (blue) PEI/DNA coacervate microdroplets with different concentrations of NaCl (0, 100, 200, 250, 400, and 800 mM). The mean coacervate microdroplet counts were calculated from optical microscopy images using Image J software, and the standard deviation was calculated accordingly. The fit lines serve only for eye guidance. (D-G) Confocal fluorescence microscopy images (D-F) and mean diameter (G) of a single DDAB-coated PEI/DNA coacervate microdroplet without salt, with 200 mM NaCl and recovery upon the addition of salt-free supernatant. Diameter measurements were performed on 20 droplets from the same sample, and the average value and standard deviations calculated.


Mireille Le Guen

Clémence Schantz

Arnaud Régnier-Loilier

Elise De La

Why some women went off the pill: Adverse side-effects and claims of naturalness as reasons for rejecting hormonal contraception in Western countries

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

1 Le Guen Mireille, Schantz-Inguenault Clémence, Régnier-Loilier Arnaud, La Rochebrochard Elise (de), « Why some women went off the pill: Adverse side-effects and claims of naturalness as reasons for rejecting hormonal contraception in Western countries ». PopDigest, 2022.

Why some women went off the pill:

Adverse side-effects and claims of naturalness as reasons for rejecting hormonal contraception in Western countries

Mireille Le Guen 1,2 , Clémence Schantz 1,3 , Arnaud Régnier-Loilier 1 , Elise de La Rochebrochard and Elise de La Rochebrochard (Ined, Inserm-Cesp) conducted a systematic review of the scientific literature in order to build a typology of arguments given by female users and their male partners against hormonal contraceptive methods.

Although the contraceptive pill remains a widely used method, its use has declined in recent years in many Western countries, such as Australia, Belgium, France, Germany and Switzerland. This has not been in favour of the new contraceptive methods marketed in the 2000s (implant, patch and ring) but in favour of condoms and IUDs. Although most health professionals attributed these changes to a latent 'hormonophobia', i.e. an irrational fear of hormones, there may be other reasons that lead women to adopt new contraceptive behaviours, as recent testimonials on social networks seem to suggest.

Mireille Le Guen (Ined and UCLouvain), Clémence Schantz (IRD-Ceped), Arnaud Regnier Loilier (Ined) and Elise de La Rochebrochard (Ined, Inserm-Cesp) conducted a systematic review of the scientific literature (original research published since the 2010s using data collected in Western countries) in order to build a typology of arguments given by female users and their male partners against hormonal contraceptive methods. Their research, which involved the analysis of 42 articles, enabled them to identify major arguments for rejecting hormonal contraception.

Women and men interviewed in the selected articles mentioned they have experienced adverse sideeffects attributed to the use of hormonal contraception. They cite a large range of physical side-effects with two of the most recurrent being weight gain and headaches. Women also reported side effects affecting their mental health like irritability and mood swings as well as sadness and depression.

Women and men also mentioned an impact on their sexuality due to a reduced or a lacking libido among women and some discomforts during sex due to vaginal dryness or breast pain. Women also reported that hormonal contraception negatively impacted their menstrual flow: abnormal blood loss or no blood loss at all, which made them anxious because they utilised regular periods to ensure they were not pregnant. More generally, the authors found a deep anxiety associated with the use of hormonal contraception due to a distrust and a fear of experiencing short-and long-term side effects.

Particularly, women and men wonder about the impact of hormonal contraception on women's future fertility, leading some of them to not want to use it.

These experiences and fears about the adverse side-effects of hormonal contraception were often perceived by users as being ignored by health care providers who did not take their concerns about hormonal contraception seriously. Women also described how they managed the adverse side-effects of hormonal contraception on their bodies by themselves. As a consequence, women and men appealed for more 'natural' contraception arguing that hormones are unnatural or chemical and that they damage women's bodies. Some of them were already using a hormone-free methods like sterilisation, condoms or withdrawal.

The findings suggest that the recent changes in contraceptive use in Western countries do not seem to be exclusively due to excessive fear of hormones, but they testify to the diversity of reasons that can lead individuals to not want to use hormonal contraception (anymore).

Référence :

Le Guen M., Schantz C., Régnier-Loilier A., de La Rochebrochard E. (2021). Reasons for rejecting hormonal contraception in Western countries: A systematic review. Social Science & Medicine, 284. https://doi.org/10. 1016/j.socscimed.2021.114247

  de recherche en épidémiologie et santé des populations Mireille Le Guen (Ined and UCLouvain), Clémence Schantz (IRD-Ceped), Arnaud Regnier Loilier (Ined)