Andrei Diaconu

Tom Coenye

Dr Mihail Barboiu
email: mihail-dumitru.barboiu@umontpellier.fr

Stéphane P Vincent
email: stephane.vincent@unamur.be

Prof. Dr ] A Diaconu

Prof. Dr Coenye

Dynamic Constitutional Frameworks as Antibacterial and Antibiofilm Agents

Keywords: biofilms, Dynamic Constitutional Chemistry, Antibacterial agents, antibiofilm agents, crystal violet

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

Biofilms are communities of microbial cells that are embedded in a self-produced extracellular polymeric matrix and are either attached to a surface or occur as non-surface attached aggregates. [1] Biofilms are frequently encountered in nature; they can be beneficial (e.g. waste water treatments) or have a negative impact, e.g. when causing fouling in industrial settings or when they form on medical devices like catheters. [2] Microorganisms in biofilms are protected against the activity of antibiotics [3] and a lot of efforts are being made to develop novel compounds with antibiofilm activity. [4] Although many bactericidal molecules or materials have been developed to date, only few inhibit biofilm formation. While a bactericide is a molecule or a material that kills bacteria, a biofilm formation inhibitor blocks bacterial attachment to surfaces and/or formation of aggregates. Molecules or materials that can disturb the structure of a mature biofilm, reduce or remove its biomass are named biofilm disruptors, and are also very difficult to develop or design. [5] A wide range of nanofabrication techniques and materials have been used to generate antibacterial materials based on nanopillars that induce mechanical stretching of the bacterial membrane, leading to bursting of the cells upon contact. [6] Enzymatic degradation of the extracellular matrix (including extracellular DNA) produced by cells in a biofilm can weaken its structure and can lead to dispersal of cells from the biofilm. However, bacteria produce extracellular exoenzymes that apparently prevent excessive degradation of the biofilm matrix. [6c, 7] By interfering with these self-controlling mechanisms, specific molecular binders could represent a potent strategy for controlling biofilm growth. Positively charged multivalent platforms [6b, 8] have been used as inhibitors for biofilm formation, as many components of the biofilm are negatively charged in physiological conditions. [9] For instance, Haag et al. synthesized multifunctional zwitterionic pillar [5]arenes, which formed self-assembled positively charged nanoaggregates displaying antibacterial and biofilm disrupting activities. [6b] Dynamic constitutional frameworks (DCFs) are adaptive 3D dynamic polymeric -dynameric networks that have been used as multivalent platforms for DNA recognition and transfection as well as other biomedical applications. [10] Linear or cross-linked connecting building blocks and polyfunctional core centers, linked together by reversible bonds were used to form cross-linked 3D scaffolds on which cationic or H-bonding head building blocks have been attached as terminal components, giving the DCF scaffolds the ability to bind proteins or nucleic acids. Importantly, these species display low cytotoxicity with eukaryotic cells. [13b] The dynamic covalent and supramolecular bonds play an important role in the ability to adaptively implement reversible and constitutional rearrangements of the components within DCFs, controlling their highly competitive multivalent synergetic interactions with the components of the biofilm in order to create effective networks for biofilm disruption (Figure 1). In this study we are targeting two biofilm producing bacteria, Pseudomonas aeruginosa and Staphylococcus aureus. P. aeruginosa is a Gram-negative opportunistic human pathogen involved in a wide variety of infections, including respiratory, urinary tract and skin infections [9,11] S. aureus is a Gram-positive opportunistic human pathogen that can cause a range of illnesses, from minor skin infections to life-threatening diseases such as pneumonia, meningitis, osteomyelitis and endocarditis. It is frequently associated with hospital-acquired infections and is often the cause of wound infections following surgery.

In the present study, we propose an unprecedented and easily processable strategy based on dynamic constitutional chemistry for the self-construction of DCFs which are screened for their antibacterial and antibiofilm activity. We use simple building blocks and an easy parallel screening of complex mixtures via cooperative and competitive binding effects. This approach allows the rapid identification of potential highly active candidates, generated from a large set of building blocks. The methodology to generate DCFs is based on imine bond formation and exchanges between three types of building blocks (Figure 2): i) The core center which is the connection point of the network (1,3,5benzenetrialdehyde, BTA); ii) The poly-(ethyleneglycol)-bis(3aminopropyl) terminated-PEG is used as the cross-linking connector of the network and as enhancer of the solubility in water; iii) The amino or acyl hydrazone functional heads are the most important molecular subunits, that determine the interactional affinity and the selectivity of the DCFs for the biological targets.

Results and Discussion

In the present study, the trialdehyde BTA core 1 and the PEG diamine linker 2 are present in all DCFs in a 1:1 molar ratio, leaving free aldehydes for further reactions with amino or acylhydrazone functional heads 3, to form DCFs in water (Figure 2). Beside cationic functional heads 3, we also selected several neutral ones that may bind proteins playing a key role in biofilms. For instance, lectins LecA and LecB selectively bind uncharged molecules and are important in the virulence and the biofilm formation of P. aeruginosa [12] Treatment of 1 with 2 (1:1, mol:mol) in acetonitrile (r.t., 48-72h) afforded, as previously reported, [13] two mixtures of linear and cross-linked neutral DCF0 assembled via reversible amino-carbonyl/imine chemistry (Figure 2). Importantly, the 1 H-NMR spectra, recorded in CD3CN and D2O are similar and remain unchanged for months at neutral pH. As previously observed, the PEG chains may have a protecting effect against the hydrolysis of the imine bonds, favouring the imine formation, leading to linear and crosslinked species in solution. [13] 1 H-NMR spectra show the presence of unreacted aldehydes in the resulting DCF0. DCF0 (2 mg/mL of 1) was further reacted in water with functional heads 3A-W at a molar ratio 1: 2: 3 mol:mol:mol of 1:1:2 generating the final DCF3A-DCF3W (Figure 2, see Supplementary information for the synthetic details). Their 1 H-NMR spectra are reminiscent with the formation of completely condensed frameworks with an optimal molar ratio of 1:1:2, that will be used for biofilm inhibition screening. The molar ratio between the functional heads and the core subcomponents will be further optimized after the biological screening To gain insight into the morphology and aggregation of DCFs in aqueous solution we investigated their self-assembly behaviours by Dynamic Light Scattering (DLS) and zeta potential experiments (Figure 3). [14] When dissolved in water, the measured hydrodynamic diameters show that most of the DCFs are highly dispersed, suggesting that they are not aggregating in solution, as expected for hydrophilic charged species. However, some particle aggregation can be observed in several cases: a) DCF3P (polydispersity index [PDI]=0.256) and DCF3Q (PDI=0.157) have a good PDI with an average diameter of nanoparticles of 122.0 and 36.5 nm, respectively. b) DCF3H (PDI=0.350) and DCF3S (PDI=0.421) have a high PDI with an average diameter of nanoparticles of 400.0 nm and 2000.0 nm respectively, while DCF3K, DCF3V and DCF3W show a high PDI and a broad distribution of the diameter of nanoparticles, indicating that several groups of nanoparticles of different dimensions are present in aqueous solution (Figure 3b). Zeta potential experiments for which a large distribution of potential values was observed (Figure 3c) suggest that most of the DCFs have a low colloidal stability. These data are in agreement with the DLS analysis, since the aggregation processes of DCFs results in large structures and indicates that the hydrophilic PEG connectors within the DCFs participate in the charge distribution effect in aqueous solution. The dispersion of DCFs within biofilm may represent an advantage as they cover large surfaces within biofilm space as a source of enhanced activity. The non-aggregating behaviours observed in aqueous solution by DLS and zeta potential data were confirmed by Atomic Force Microscopy (AFM) experiments. The AFM micrographs (Figure 4) show a large variety of morphologies: a) the formation of nanoparticles of DCF0 confirm the formation of highly polydisperse interconnected aggregates from 10 nm to 150 µm. (Figure 4a); b) dendritic structures have been observed for dried solid samples of DCF3A (Figure 4b), while the PDI higher than 0.7 in the DLS analysis is confirming the occurrence of highly dispersed PEI800 heads in water; c) replacing PEI800 with PEI2000 in DCF3B results in spherical particles with diameter from ~100-200 nm to 5-10 µm and height up to 95 nm are observed by AFM (Figure 4c), while in solution particles with polydisperse size can be observed by DLS; d) different spherical, rod-like, and dendritic 100 nm to 15-20 µm interconnected particles can be observed for DCF3F (Figure 4d), confirming the large distribution observed by DLS; e) DCF3J has a complex size distribution with highly aggregated particles from 100 nm to 100 µm (Figure 4e); f) AFM analysis of DCF3V shows a homogenous distribution of particles from 10 to 300 nm, with a height up to 38 nm. The AFM data show that the studied DCFs present unpredictable highly aggregated structures in the solid state (Figure 4). These diverse morphological behaviours confirm the conclusions of DLS studies which lay to the formation of the extended DCF networks having a large polydispersity in solution. The dimensions of DCF particles strongly depend on their solubility which is mostly dictated by the nature of the terminal heads.

The antibacterial and antibiofilm activities of the library of DCFs were subsequently tested to identify the best combinations and compositions. Our methodology was to 1) screen the library at a single DCF components' ratio 1:1:2 using a standard P. aeruginosa biofilm assay to identify the best functional modules; 2) optimize the components ratio on the best DCFs; 3) characterize the antibiofilm activity using three distinct biological assays; and 4) test the antibiofilm activity on S. aureus. The assay used to identify the active DCFs was the standard crystal violet biofilm biomass staining of biofilms grown in 96-well microtiterplates. [2a, 15] Bacterial biofilms were grown in the presence of increasing concentrations of DCFs in 96-well plates. After rinsing the plates, the remaining biofilm biomass was stained with crystal violet and the amount of biofilm biomass was directly assessed by UV-Vis absorbance. Tobramycin 3D was used as a positive control. [9,11,16] The antibiofilm activities of the best DCFs against P. aeruginosa biofilms are shown in Figure 5 (See Figure S6, Supporting Information, for the entire series). DCF containing cationic functional heads 3A, 3B and 3C have the strongest antibiofilm activities while DCF containing functional heads 3F, 3V and 3W have lower activities. All the other functional heads screened in this DCF library did not provide any antibiofilm activity. This initial screening clearly evidenced that both the nature (ammoniums, pyridiniums, guanidiniums etc…) and the structure of the functional heads play a central role in the observed antibiofilm activities. Transamination reactions might occur at the surface of the cell membranes. As we already mentioned, previous studies demonstrated that the presence of cationic heads and PEG connectors are useful for the formation of cyto-friendly DNA polyplexes and DCFs as DCF3A and DCF3B are well tolerated by cells. 13b The fact that the very best DCFs have been identified within the subgroup of cationic functional heads can potentially be explained by the fact that P. aeruginosa biofilm cells are embedded in negatively charged polymeric biomolecules like alginate [9,17] , other exopolysaccharides or extracellular nucleic acids. [18] All these negatively charged molecules could thus strongly bind the positive charges of the functional heads, thus giving a better biofilm inhibition compared with the neutral and negatively charged DCFs. Having identified the best functional modules, the next step was to evaluate whether the relative ratios of each component of the DCFs would affect their capability of inhibiting biofilm formation.

We prepared new DCFs containing different amounts of functional heads 3A, 3B and 3C at four molar ratios (1.5, 2.0, 2.5 and 3) relative to the core BTA 1 (Figure 6, plots A, B, and C for functional modules 3A, 3B, 3C, respectively). As for the initial DCF library, the molar ratio between core 1 and linker 2 was 1:1. The general trend was that increasing the proportion of cationic functional module relative to 1 increased the antibiofilm activity. For modules 3A and 3C the ratio 2.5 gave the best inhibition results. At this ratio the concentration of 31µM (relative to 1) gives a biofilm inhibition higher than 50%. This series of experiments indicate that the charge density at the surface of these dendritic materials play an important role in the antibiofilm activity. As a control, we also measured the antibiofilm activity of the functional reference heads (Figure 6D). Functional heads 3A and 3B show antibiofilm activity on their own. Interestingly, functional module 3C does not show any antibiofilm activity by itself while its DCFs show powerful biofilm inhibition. Noteworthy, the control 1:1 mixture of core BTA 1 and linker PEG diamine 2 without functional module (compound named DCF0) did not display any antibiofilm activity. Globally, the best DCFs present an antibiofilm activity at concentrations of 2 to 123 µM of 1 (or 5 µg/mL to 1 mg/mL for the whole material).

D C F 3 A 2 . 0 D C F 3 B 2 . 0 D C F 3 C 2 . 0 D C F 3 F 2 . 0 D C F 3 V 2 . 0 D C F 3 W
To further quantify the antibiofilm activity, the concentrations at which the increase of biofilm biomass is inhibited by at least 50% (BBIC50) were determined for the best DCFs and their corresponding functional modules (Table 1). As usual in microbiology and material sciences, we specified the mass concentrations (µg/mL) of the whole material. To allow comparison of the molar concentrations of the different DCFs we also expressed them as the µmolar concentration (µM) of 1 that is present in the DCF. We also determined the minimal biofilm eradication concentration (MBEC50, Table 1), which is a measure of the ability of a material to disrupt an existing biofilm. MBEC50 represents the lowest concentration of DCF that eradicates at least 50% of the biofilm biomass of a mature biofilm (supplementary information for detailed protocol). Here, bacteria were allowed to form biofilm during 24 hours, and after rinsing the planktonic bacteria, fresh culture medium and increasing concentrations of DCFs were added and incubated for another 24 hours. The remaining biofilm biomass was compared with the control experiment (untreated PAO1 biofilm formation after 24h).

In general, MBEC50 values were found to be in the same range as the MBIC50 values.

Globally, the three most-active DCFs are DCF3A, DCF3B and DCF3C. Their antibiofilm activities are in the same range but DCF3B had a better profile, especially for its ability to eradicate a pre-established biofilm. However, the most remarkable result of the whole series is the fact that DCF3C displays good biofilm inhibition properties despite the fact that its subcomponents have neither antibiofilm nor bactericidal activities.

The results obtained are encouraging as the activity range obtained against P. aeruginosa is similar to the literature data on positively charged pillararenes, bridged bicyclic peptides or glycopeptide dendrimers, [8b, 19] while the synthetic work to generate our DCF materials is significantly more simple and less expensive, thanks to the dynamic chemistry approach.

To assess whether this pronounced antibiofilm activity is due to bacterial growth inhibition, we determined the MIC50 of the most active DCFs (Table 1). The MIC50 is the minimal concentration that reduces the growth of planktonic bacteria with at least 50% compared to untreated controls. 3A and 3B and DCF3B have a BBIC50 that is lower than the MIC50 which indicates that this material might prevent P. aeruginosa biofilm formation at concentrations that do not inhibit growth. a) Concentration (µM) in pure functional module b) value higher than 1 mM c) value higher than 0.5 mM.

To shed light on the origin of the antibiofilm activity, growth curves have been measured in presence of 0.63 µg/mL, 10 µg/mL and 80 µg/mL of compounds DCF3A, DCF3B and DCF3C (ESI, Figures S14-S16). With a full growth inhibition at 10 µg/ml DCF 3C proved to be the most potent material for this assay (DCF3C > DCF3B > DCF3A). Our data suggest that the antibiofilm effect of the three DCFs is at least partially due to their concentrationdependent inhibition of bacterial growth. Such a feature has been already observed among antibiofilm agents that often proceed through bacterial killing [6b, 20] . In general the MBEC50 concentrations are equal or higher than the MIC50 or BBIC50 for all the DCFs, except for DCF 3C. This result was not surprising as it is clear from the literature that eradicating a pre-existing biofilm is more difficult than inhibiting biofilm formation.

Next, we evaluated the antibiofilm properties of the best materials using an in vitro biofilm model of chronic wound infection. [21] In brief, biofilms were grown on an artificial dermis and the antibiofilm properties of DCF 3A-3C were evaluated by CFU (colony forming units) numbering (See SI Figure SI17). Interestingly, DCF 3C was from far the most potent material with this assay (DCF3C > DCF3B > DCF3A), a result in good correlation with the growth inhibition experiments described above.

To broaden the scope of this investigation, we also tested the best DCFs against a relevant Gram-positive bacterium, S. aureus (strain Mu50, Table 2). Globally, the selected DCFs are slightly less active against S. aureus than against P. aeruginosa, except for DCF3B that displayed the best activities at similar concentrations for both strains. Interestingly, the two functional modules 3A and 3B did not show any antibiofilm activity (BBIC50 > 1 mM) whereas their corresponding DCFs inhibited S. aureus biofilm formation. Even more interesting was the fact that the BBIC50 of DCF3A was lower than its MIC50 suggesting an antibiofilm mechanism that is not due to growth inhibition. Hemolytic activities of the 3 best DCFs have been measured on human red blood cells in the same concentration range than the antibiofilm assays realized in our study. These experiments show that DCF3A and DCF3B have no detectable hemolytic activities (3 independent experiments on blood samples of 3 different donors). DCF3C displayed only 2% hemolytic activity at the highest tested concentration (0.5 mg.mL -1 ), which is much higher than the antibiofilm activity.

Conclusion

In this study, we generated a large number of Dynamic Constitutional Frameworks (DCFs), thanks to an easy synthesis method based on dynamic chemistry. The DCFs were tested against two bacterial strains (P. aeruginosa PAO1 and S. aureus Mu50) for their antibacterial, antibiofilm and biofilm disruption effects.

The main findings of this study are:

1) The functional heads play a crucial role in DCF's bioactivity as antibacterial agents and biofilm disruptors. Positively charged multivalent DCFs gave the best results. The nature of the cation or polycation has a dramatic impact on the antibiofilm activity.

2) DCF3B gave the best antibiofilm activity for both P. aeruginosa and S. aureus using the standard crystal violet assay. Using a more advanced in vitro biofilm model of chronic wound infection, DCF3C was found significantly superior than the other DCFs. 3) Some of the functional heads did not display any or only poor activity by themselves but their corresponding DCFs had significant antibiofilm activity. These results showed that the central core of the DCFs (DCF0), although biologically inactive, could play a key role, probably by properly displaying the functional heads. This feature was particularly important for DCF3C whose functional module 3C had neither antibiofilm nor bacterial growth inhibition activities, which showed that the DCF itself displayed the antibiofilm activity, and not its subcomponents, even if released under acidic conditions. 4) The mode of action of the other cationic materials was mainly bactericidal, although some of them displayed antibiofilm activities at concentrations lower than those required for growth inhibition. The latter feature is rare and demonstrates that the specific structures of the best DCFs could interact with the biofilm elements themselves and prevented the biofilm growth.

Our study shows that dynamic combinatorial chemistry can enable the quick identification of novel materials with highly challenging biological properties such as biofilm inhibition growth or mature biofilm disruption. Of course, further studies are still required in view of the development of materials as commercially available antibiofilm agents: For instance, the bioavailability and the stability in biological media have still to be determined. Such a discovery approach could also be extrapolated for the identification and development of dynamic materials for other important biomedical applications.

Figure 1 .

 1 Figure 1. Dynamic Constitutional Frameworks (in red) acting as potential disruptors of bacterial biofilms as adaptive recognition platforms and effective barriers against the diffusion of biomolecules (i.e. DNA, proteins, etc.) between different bacteria (in green) of the biofilm community.

Figure 2 -

 2 Figure 2 -Synthesis of DCFs combining 1,3,5-benzenetryaldehyde core 1, (blue) with PEG connector 2, (black) and functional heads 3, (red) in water to generate DCFs. The names of the synthetized DCFs take into account both the functional module 3X and its molarity compared to 1 and 2. For instance, for functional module 3A, we used 4 different molar ratios 1:3, and their corresponding names are: DCF 3A1.5, DCF 3A2.0, DCF 3A2.5 and DCF 3A3.0. (SeeTable 1SI in supporting information).

Figure 3 -

 3 Figure 3 -DLS analysis a) Polydispersity index indicating the dispersity of the samples: values higher than 0.7 indicate that sample has a very broad size distribution and b) Hydrodynamic diameter (PDI = 0.05 and 0.7) showing several particle size dimension with different low and high distributions and c) Zeta potential-ZP of the DCFs samples, indicating the stability of colloidal samples related to electrically stabilized colloids for high ZP values or low dispersed flocculate particles for low ZP values.

Figure 4 -

 4 Figure 4 -The AFM micrographs of DCF0, DCF3A, DCF3B, DCF3F, DCF3J and DCF3V.

Figure 5 -

 5 Figure 5 -Biofilm biomass evaluated after the treatment of ~ 5 × 10 6 CFU of P. aeruginosa PAO1 with positively charged DCFs at increasing concentrations. The concentration range is 4 -493 µM of 1 for all DCFs. Untreated bacteria are used as positive control (Pos C set to 100%). The experiments were performed a single time with 3 biological replicates each with 2 technical replicates. Data shown are average, error bars indicate standard deviation. * ANOVA with a Dunnett post hoc test statitical test with P>0.0001.

Figure 6 -

 6 Figure 6 -Biofilm biomass evaluated after the treatment of ~ 5 × 10 6 CFU of P. aeruginosa PAO1 with DCFs composed of variable functional module ratio (1.5; 2.0; 2.5; 3.0 relative to 1) at increasing DCFs concentrations and compared to the untreated controls Pos.C; A) DCFs with functional module 3A B) DCFs with functional module 3B C) DCFs with functional module 3C D) functional modules alone and DCF0 (the network DCF0 between 1 and 2 at a 1:1 molar ratio) at the concentration range of the DCFs. The experiments were repeated 2 times, each with 6 technical replicates. * ANOVA with a Dunnett post hoc test statitical test with P<0.0001.

  ) in pure functional module b) value higher than 1 mM c) value higher than 0.5 mM d) ntnot tested; Table 2 -MIC50 and MBIC50 results for active DCFs and functional module against S. aureus Mu50.

Table 1 -

 1 MIC50, MBIC50 and MBEC50 values for the most active DCFs and functional modules against P. aeruginosa PAO1

	DCF	BBIC50	MBEC50 µg.mL -1 (µM of 1)	MIC50
	DCF 3A2.0	201 (62)	808 (246)	201 (62)
	3A	49.3 (62 a )	98.7 (123 a )	98.7 (123 a )
	DCF 3B2.0	87.3 (15)	174.6 (62)	174.6 (32)
	3B	62 (31 a )	123 (62 a )	123 (62 a )
	DCF 3C2.0	116 (62)	-c	116 (62)
	3C	-b	-b	-b
	DCF 3D2.0 (control)	10 (3.9)	20 (7.8)	5 (1.92)
	3D (control antibiotic)	3.6 (7.8 a )	3.6 (7.8 a

Acknowledgements

We thank Mihaela Balan for measuring part of the NMR spectra, Laura Ursu for AFM experiments and Lilia Clima and Mariana Pinteala for the continuous support of our work. This research was funded by of Ministry of Research and Innovation, CNCS-UEFISCDI, project number SupraChem Lab PN-III-P3-3.6-H2020-2016-0011 and UNamur FSR PhD grant to AC.

Entry for the Table of Contents

Dynamic constitutional frameworks are expressed in the presence of bacteria templating from dynamic covalent libraries made of aromatic cores, PEGylated connectors and ionic heads. This templated DCFs further translates into the multivalent presentation of cationic heads, subsequently promoting antibacterial and antibiofilm activities.

Institute and/or researcher Twitter usernames: Synergistic multivalent dynamic constitutional frameworks with antibacterial activity and antibiofilm disruption.

Fruitfull collabaoration between @MihailBarboiu @CNRS_OccitaniE, @tcoenye @ipm_ugent and Stephane Vincent, Universite de Namuur


Maria Di Vincenzo

Alberto Tiraferri

Valentina-Elena Musteata

Stefan Chisca

Mihai Deleanu

Francesco Ricceri

Didier Cot

Suzana P Nunes

Mihail Barboiu
email: mihail-dumitru.barboiu@umontpellier.fr

Tunable membranes incorporating artificial water channels for high-performance brackish/low-salinity water reverse osmosis desalination

Keywords: artificial water channels, self-assembly, bio-inspired membranes, reverse osmosis, desalination

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

Introduction

Competition over increasingly contaminated freshwater resources during the last decades has made the availability to safe drinking water a serious global challenge for the 21 st century. Desalination is one of the solutions to the problem of freshwater scarcity, along with environmental remediation technologies and sustainable conservation policies. 1,[START_REF] Vincenzo | Biomimetic artificial water channels membranes for enhanced desalination[END_REF] Currently, about 100 million m 3 /day of desalinated water is being produced, with an annual increase of more than 10 %.Traditional reverse osmosis (RO) membranes are thin-film composites (TFC), which rely on a supported polyamide layer to perform water/solute separations. [START_REF] Fane | Synthetic membranes for water purification: Status and future[END_REF][START_REF] Lau | A review on polyamide thin film nanocomposite (TFN) membranes: History, applications, challenges and approaches[END_REF] Synthetic membranes act by dissolving and diffusing solutes with scale selectivity through their polymeric matrix: a material property that leads to a permeability-selectivity trade-off behavior. [START_REF] Imbrogno | Membrane Desalination: Where Are We, and What Can We Learn from Fundamentals?[END_REF] Numerous approaches have been proposed to overcome this limitation, including membrane grafting, chemical functionalization, and processes optimization. [START_REF] He | Promoted water transport across graphene oxide-poly(amide) thin film composite membranes and their antibacterial activity[END_REF][START_REF] Kim | High-performance reverse osmosis nanocomposite membranes containing the mixture of carbon nanotubes and graphene oxides[END_REF][START_REF] Safarpour | High flux and fouling resistant reverse osmosis membrane modified with plasma treated natural zeolite[END_REF][START_REF] Chan | Zwitterion functionalized carbon nanotube/polyamide nanocomposite membranes for water desalination[END_REF][START_REF] Vincenzo | Polyol-functionalized thin-film composite membranes with improved transport properties and boron removal in reverse osmosis[END_REF] Despite several elegant design strategies, membrane-based technologies always report an improved water permeability at the expense of lower selectivity, or vice versa. [START_REF] Yang | The upper bound of thin-film composite (TFC) polyamide membranes for desalination[END_REF] Although membrane-based separation offers energy efficiency, excellent water quality, and capability for implementation at industrial scale, there is a pressing need for membranes with both improved selectivity and enhanced permeability, especially for increasing the efficiency of the 0 purification technology. [START_REF] Vincenzo | Biomimetic artificial water channels membranes for enhanced desalination[END_REF][START_REF] Lau | A review on polyamide thin film nanocomposite (TFN) membranes: History, applications, challenges and approaches[END_REF] Recent advances have made materials prepared under molecular control increasingly more attractive to researchers, offering the possibility of stable and high efficiency bio-inspired membranes. 1,[START_REF] Vincenzo | Biomimetic artificial water channels membranes for enhanced desalination[END_REF] Due to their unique functional design, biological aquaporins and artificial water channels (AWC), with proven high performances at the Å-scale, when embedded in lipid bilayers, have inspired their incorporation into desalination membranes. [START_REF] Barboiu | From natural to bio-assisted and biomimetic artificial water channel systems[END_REF][START_REF] Giwa | Biomimetic membranes: A critical review of recent progress[END_REF] Self-assembled [START_REF] Leduc | Imidazole-Quartet Water and Proton Dipolar Channels[END_REF][START_REF] Barboiu | Artificial water channels Angew[END_REF] and unimolecular [START_REF] Hu | Single-Molecular Artificial Transmembrane Water Channels[END_REF] AWCs have been developed during the last decade with the hope to mimic the complex biological machinery. [START_REF] Murail | Water permeation across artificial I-quartet membrane channels: from structure to chaos[END_REF][START_REF] Gong | Artificial water channels: Inspiration, progress, and challenges[END_REF][START_REF]Artificial Water Channels: Faraday Discuss[END_REF] These approaches have shown that AWCs are mostly active when embedded in lipid bilayers, but upscaling methods toward biomimetic RO polymeric membrane that can be prepared at industrial standards is possible. [START_REF] Vincenzo | Biomimetic artificial water channels membranes for enhanced desalination[END_REF] The defects created at the interface between active AWCs and surrounding matrices are critical for the performances of RO membrane for desalination. When fabricating a high-performance RO membrane for desalination, a number of necessary characteristics need to be achieved to maximize the productivity, including suitable mechanical resistance, high density of the functional channels per unit area and, perhaps the most ambitious of all, the absence of defects. 1 We postulated that one of the creative strategies for addressing the scale-up challenges and improved performances is to combine the established polyamide (PA) material prepared via interfacial polymerization (IP) and known for its easy scalability via the integration within a typical roll-to-roll processing system, with both permeable and selective AWCs. [START_REF] Vincenzo | Biomimetic artificial water channels membranes for enhanced desalination[END_REF] We previously reported the fabrication of a biomimetic membranes, containing I-quartet AWCs embedded in a classical polyamide PA. [START_REF] Vincenzo | Biomimetic artificial water channels membranes for enhanced desalination[END_REF] We know that compositional diversity of PA components and additives have a tremendous impact on membrane structure and performances. 1 In the present report, our novel strategy leads to a greater fundamental understanding of how AWC incorporation influence the performance of PA/AWC membranes. The novel reported hybrid AWC-PA materials can be optimized at the nanoscale with different morphologies and chemical properties by the optimal mixing of various recipes exploiting variable amounts of AWCs and PA components. Specifically, we investigate valuable insights into fabrication-structure-performance relationship and discuss the effect of composition of AWC and m-phenylenediamine components for improving and discovering new performances of bioinspired membranes. The novelty here is related to the straightforward quantification of specific compositions, that allow obtainment of seamless active layers in which AWCs are gently and optimally incorporated without creating defects, in all cases. Homogeneous sponge-like superstructures of hybrid PA-AWC materials are thus achieved and applied under new filtration conditions real brackish water and low-salinity-water RO desalination conditions.

Results

Formation pathways of control and bioinspired reverse osmosis membranes. The imidazolequartet, I-quartet, [START_REF] Leduc | Imidazole-Quartet Water and Proton Dipolar Channels[END_REF][START_REF] Barboiu | Artificial water channels Angew[END_REF] comprises stacks of four molecules HC6 and two water molecules that form a pore diameter of 2.6 Å filled with oriented single water wires (Fig. 1a). [START_REF] Licsandru | Salt-Excluding Artificial Water Channels Exhibiting Enhanced Dipolar Water and Proton Translocation[END_REF] The water orientation [START_REF] Kocsis | Oriented chiral water wires in artificial transmembrane channels[END_REF] is of prime importance for enhancing its translocation in the range of 10 6 -10 7 water molecules•s -1 per channel, with a perfect salt rejection via a single wire [START_REF] Licsandru | Salt-Excluding Artificial Water Channels Exhibiting Enhanced Dipolar Water and Proton Translocation[END_REF] or sponge cluster 17 mechanism. The crystal packing reveals parallel sheets of the alternative I-quartets self-assembled in a lamellar phases of parallel single water channels (Fig. 1c,d) and matching well with the ordered rows observed in the TEM micrograph of a crystalline powder obtained from the evaporation of an ethanolic aqueous casting solution of HC6 (Fig. 1b).

The RO membranes in this study were prepared by interfacial polymerization (IP) of mphenylenediamine (MPD) and trimesoylchloride (TMC), resulting in the formation of polyamide (PA) layers onto a commercial polysulfone (PSf) support. For the synthesis of bioinspired PA layers, the conventional IP procedure was preceded by the incorporation of AWCs (Fig. 1e). In a first step, an ethanolic aqueous solution of HC6 results in the formation of stable colloidal aggregates (Dh = 200 nm), observed by dynamic light scattering DLS when water was added to ethanolic solutions of HC6, without any visible precipitation. Please note that compared with other previously studied channels HC4-HC8, HC6 with an optimal solubility, avoid precipitation under described IP experimental conditions leading to the formation of free defect membranes. This aqueous solution was poured onto the PSf support which was then immersed in an aqueous MPD solution, resulting in the formation of hybrid AWC/MPD nanosized colloidal aggregates. The DLS analysis shows that smaller aggregates (Dh = 70 nm) (See SI Appendix, Fig. S1) were obtained when an aqueous solution of MPD was used instead of pure water, reminiscent of H-bonding interactions between AWC and MPD. [START_REF] Vincenzo | Biomimetic artificial water channels membranes for enhanced desalination[END_REF] The integration of AWC into PA hybrid layers is firstly related to a nucleation process of such colloidal structures; then, a heterogeneous nucleation is promoted by H-bonding interactions between MPD and colloidal AWC nano-aggregates, as shown by DLS, while any precipitation process was observed. Based on this information, different HC6/MPD ratios were investigated to explore the effect of the composition on the material morphology that control and tune the membrane transport performances (See SI Appendix, Table S1). We know that, over the period of incipient formation of the PA film, the polymer growth remains nearly constant, being determined by the diffusion rate of the MPD monomers in the organic phase. [START_REF] Freger | Kinetics of film formation by interfacial polycondensation[END_REF] An excess of MPD is required to promote a significant diffusion into the organic phase during film formation; therefore, an equal or sub-stoichiometric amount of MPD may result in a lower polymerization degree. [START_REF] Freger | TFC polyamide membranes modified by grafting of hydrophilic polymers: An FT-IR/AFM/TEM study[END_REF][START_REF] Liu | Study on a novel polyamide-urea reverse osmosis composite membrane (ICIC-MPD). I. Preparation and characterization of ICIC-MPD membrane[END_REF] After the immersion of the support layer into the MPD solution, the excess solution was removed with an air gun from the surface, and the HC6-MPD-saturated support was immersed into a TMC solution to form the PA layer. In this step, the AWC/MPD amphiphilic colloidal aggregates might help the migration of the MPD to the organic phase, favoring the IP reaction, while they may synergistically interact with the growing PA segments via H-bonding. We surmise that this process results in the formation of cross-linked hybrid AWC-PA materials. Following the IP procedures to prepare both the control and the bioinspired membranes, they were cured in DI water at 95 °C, dipped in a 200 ppm NaOCl aqueous solution, and then in a 1000 ppm NaHSO3 aqueous solution. The membranes were finally rinsed and stored in DI water at 4 °C before testing or characterization.

Morphological / structural properties of the bioinspired membranes. Evidence of AWCs incorporation. Fourier Transform Infrared (FTIR) spectra demonstrate the formation of a hybrid PA-AWC networks (See SI Appendix, Fig. S2 a,b). Vibration bands associated with amide bonds of the PA are visible in all IR spectra, namely, the peaks at 1502 cm -1 (amide-II, N-H in-plane bending) and 1666 cm -1 (amide-I, C=O stretching vibration). [START_REF] Freger | Kinetics of film formation by interfacial polycondensation[END_REF] Evidence for AWCs incorporation within hybrid PA membranes was obtained from the vibration shifts at 1585 cm -1 and 1609 cm -1 , assigned to the C=C stretching of the imidazole and to the C=O stretch of the urea groups, as well as the -CH2,as and -CH2,sym stretch modes of the alkyl chains of HC6 visible in the range 2750 and 2957 cm -1 , respectively. The sharp band centered around 3400 cm -1 is attributed to the O-H stretching vibration of strongly H-bonded water within I-quartet channels. This band became broad for the TFC-HC6 membrane, due to the presence of more mobile less dense H-bonded water within the matrix of the bioinspired layer. This new type of PA-AWC hybrid material generated via a nucleation/IP mechanism, is reminiscent of the emergence of self-assembled I-quartets within a porous sponge-like membrane that theoretically present enhanced water permeation as showed by our previous molecular modeling studies. [START_REF] Murail | Water permeation across artificial I-quartet membrane channels: from structure to chaos[END_REF] They are suggestive of dynamic superstructures to provide cooperatively interconnected membrane-spanning pathways through water networks.

Fig. S2b, See SI Appendix, presents the average elemental mass fraction X% obtained from energy dispersive X-ray spectroscopy (EDX) analysis for bioinspired membranes prepared by keeping constant the amount of HC6 and varying MPD concentration. When compared to the control TFC membranes presenting a C % of 61-67 w % as previously reported in literature for traditional PA membranes, [START_REF] Freger | TFC polyamide membranes modified by grafting of hydrophilic polymers: An FT-IR/AFM/TEM study[END_REF][START_REF] Liu | Study on a novel polyamide-urea reverse osmosis composite membrane (ICIC-MPD). I. Preparation and characterization of ICIC-MPD membrane[END_REF] the C % of 80-85 w % was significantly higher in all the bioinspired PA-AWC layers. This result is expected because the C/O mass ratio of HC6 is significantly higher (~10) than that of PA (~3.5-4), thus providing additional evidence of AWCs incorporation in hybrid AWC-PA layers. By reducing the MPD molar ratio, thereby decreasing the density of amine moieties and amide bonds, the N % decreased and the relative C % increased by roughly 4%.

Scanning electronic microscopy (SEM) micrographs (Fig. 2 and See SI Appendix, Fig. S3) suggest that the morphology of the layer and the level of HC6 incorporation can be tuned by adjusting the MPD/HC6 ratio during membrane fabrication. All the bioinspired layers appeared continuous and free of microscopic defects. The nano-and micro-structures of PA-layers governing mass transport is strongly dependent on the cross-linking degree of PA, influencing the density and distribution of AWCs within the PA matrix. In general, with a constant HC6 load, as the concentration of MPD was reduced in the fabrication solution, the top surface evolved from a "ridge-and-valley" morphology (an effect of the rapid diffusion of concentrated MPD into the organic phase), typical of conventional PA layers, to a "flake-like" morphology (See SI Appendix, Figs S4, S5) observed for lower MPD concentrations. In addition to a change in the surface roughness, the bioinspired membranes with higher HC6 loads and medium to high MPD concentrations displayed a distinct, highly porous core structure, with a relatively large thickness ranging from 150 to 300 nm. Even more interestingly, all the layers prepared in the presence of HC6 further comprised a diffuse distribution of sponge likepacked superstructures that spanned the whole active layer (Fig. 2a,b and See SI Appendix, Fig. S6). These innovative inner nanostructures and overall morphology appeared to be pronounced for the membrane referred to as 0.8-HC6 (0.8% w/w MPD and the optimal load of 1.5% w/w HC6), shown in Fig. 2c,d. Clearly, the cross-section of this representative bioinspired membrane was characterized by: (i) a smoother upper surface compared to the control TFC membrane, (ii) a highly porous multi-layer structure comprising large voids, (iii) a greater overall thickness and (iv) the presence of high-density and homogeneously distributed nanoparticles embedded in the PA matrix. The best combination of productivity and selectivity was obtained with this membrane in experimental brackish water reverse osmosis tests (vide infra). When bioinspired PA-AWC layers were instead prepared with a sub-stoichiometric MPD molar ratio (≤ 0.4 % w/w), the concentration of this monomer became the dominant factor governing the final film structure: these layers were generally free of underlying large voids and were characterized by a much smaller average thickness of 50 nm; see the representative samples in Fig. 2e-f. The sponge-like nanostructures embedded in the layer were still observed, although with apparently lower density, possibly due to a more difficult incorporation in thin-films with smaller polymerization degree. This result suggests that the complex core structure of the selective layer embedding self-aggregated AWC-PA spongelike superstructures is a strong function of both the HC6/MPD ratios and the absolute monomer concentrations employed during fabrication, and that the layer architecture can ultimately be controlled by these parameters to tune and improve the RO performance, which is described in detail below.

The surface morphology of the bioinspired PA layers was further investigated by atomic force microscopy-AFM (See SI Appendix, Fig. S7). In agreement with the SEM analysis, revealing the disappearance of the rough ridge-and-valley features, values of RMS <100 nm roughness were found for membranes fabricated with MPD concentrations below 2% w/w. These RMS values are lower than typical values of 100 -300 nm often observed for classical PA membranes. [START_REF] Mansourpanah | Using different surfactants for changing the properties of poly(piperazineamide) TFC nanofiltration membranes[END_REF] The average roughness, indicative of the overall thickness of the layer, increased when the bioinspired membranes were fabricated using 1.5 % w/w HC6 and 0.8-1.2 % w/w MPD (Fig. 3a-f), in accordance with the greater internal porosity and thickness of the morphologies observed in SEM micrographs.

Transmission electronic microscopy (TEM) cross-sectional images in Fig. 3 also evidence that by increasing the MPD concentration from 0.2 to 2.0% w/w while keeping constant the amount of HC6 (1.5% w/w) leads to an increase of membrane thickness; the maximum height of the PA layer correlates with the amount of MPD, with alternating thinner / thicker regions on the top of the PSf support. For 0.2 and 0.4 w/w % MPD (Fig. 3a,b), the PA layer has a more irregular aspect, encompassing larger portions (up to 1 μm, as observed in TEM cross-sections of the 0.2-HC6 membrane) with 20 -40 nm thickness, portions with more regular aspect of ridge and valley and areas with higher thickness (up to 240 nm and 170 nm for 0.2 and respectively, 0.4-HC6). Starting with 0.8-1.8 w/w % MPD, (Fig. 3c-e) the IP layer has a more uniform aspect with ridge and valley morphology. In addition, more developed areas with branches more equally grown in all dimensions (with flower/rose-like morphology) are being observed. For these membranes, the PA layer has a more tortuous appearance and therefore, a higher surface area. It is also clear that the IP layer has a complex internal structure when the AWC are added, with specific nanoparticulate domains present over its whole thickness. AWC/PA nanoparticles, corresponding to the brighter spots and having a diameter of ~15-20 nm in the middle of the PA layer for 0.2 and 0.4 w/w % MPD, while higher density of nanoparticles are observed for 0.8-1.8 w/w % MPD. In addition, they are more equally distributed across the membranes thickness, from the top to the bottom of the layer. These less dense white regions are filled by porous nanoparticles of AWC/PA hybrids during the PA formation. This apparent lower layer density may lead to a lower resistance to transport that would in part explain larger water permeance values observed for the TFC-HC6 films. In summary, a low concentration of MPD promoted the formation of a smoother surface, while the presence of HC6 during IP produced thicker and more porous selective layers compared to their reference TFC counterparts free of HC6. In addition, appropriate loadings of MPD above 0.4 % w/w and of HC6 below 1.8% w/w, together with an appropriate ratio of HC6/MPD of 0.7-1.9 (w/w) during fabrication, allowed suitable embedding of self-assembled AWC/PA nanoparticles within the PA layer, which are hypothesized to promote faster and selective water passage during membrane filtration.

Performance of the membranes in low-pressure RO filtration of brackish water. The filtration performance of bioinspired membranes was measured in brackish water reverse osmosis (BWRO) and tap water reverse osmosis (TWRO) desalination. [START_REF] Yan | The porous structure of the fully-aromatic polyamide film in reverse osmosis membranes[END_REF] First, the BWRO performances (i.e., water permeance, A, and the solute permeability coefficient, B) were evaluated for membranes prepared by varying the HC6 loading from 0 % (control TFC) to 1.8% w/w, while keeping the MPD concentration constant (3.4 % w/w) (Fig. 4a,b). By increasing the HC6 loading, membranes exhibited progressively higher water permeance increasing from 1.5 to 3.5 L m -2 h -1 bar -1 , with up to 185% increase with respect to the control TFC membrane, while maintaining very low and near uniform salt fluxes up to around 1.3% w/w HC6 of initial loading. Above this concentration, a drop of performance was observed. This result is rationalized with the HC6 concentration at the highest loading of 1.8 % w/w, which inhibited the formation of a defectfree selective layer during IP, also suggested by the large error bars related to erratic filtration performance of different samples fabricated using the same procedure. This set of filtration results translated into values of A/B with a peak for concentrations of 0.9 % w/w HC6. Then, we observed that by lowering the MPD concentration to 2 % w/w, higher values of water permeances were obtained with an average A of 4.5 L m -2 h -1 bar -1 and low salt passage: 99.8 % real NaCl average rejection were observed up to 1.5 % w/w HC6 loading (empty symbols in Fig. 4 a,b).

We know that the MPD concentration control its diffusion into the organic phase when IP reaction is occurring, [START_REF] Freger | TFC polyamide membranes modified by grafting of hydrophilic polymers: An FT-IR/AFM/TEM study[END_REF] but this parameter also interferes with the HC6 aggregation through H-bonding. Therefore, the HC6/MPD ratio clearly determines the final layer morphology and transport properties. With these assumptions in mind, further experiments aimed at investigating lower MPD concentrations, from 2 % to 0.2 % w/w, combined with an optimal HC6 loading, fixed at 1.5 % w/w (Fig. 4c,d). The best samples prepared with this loading showed a consistent value of A/B close to ~35 1/bar, thus associated with an overall remarkable permeability-selectivity combination to be applied in BWRO desalination, whereby membranes with a permeance of >3 L m -2 h -1 bar -1 are required. Under BWRO conditions, the water permeance increased significantly and reached a peak of ~6.9 L m -2 h -1 bar -1 for the MPD concentration of 0.8-1.2 % w/w, while maintaining very low associated salt fluxes rates. This result represents an exceptional ~360 % increase in water permeance at equivalent A/B ratio, with respect to the best membrane fabricated using 3.4% w/w MPD concentration and a ~130% increase in water permeance compared to commercial BW30 membranes (permeance of ~3.0 L m -2 h -1 bar -1 and 99.5 % NaCl rejection), with comparable observed NaCl rejection of ≥99.4%. We noted that the membrane performance dropped rapidly by using sub-stoichiometric MPD concentrations lower than 0.8 % w/w. The data suggest that a maximum loading of HC6 of 1.8% w/w and minimum loading of MPD of 0.4% w/w are needed to construct the optimal porous sponge-like AWC/PA structures and to prevent the formation of defects in the selective layer. Also, there is a clearly optimal combination of 0.8-1.2% w/w MPD with 1.5% w/w HC6, for a resulting HC6/MPD ratio of roughly 1.2 w/w to achieve high-performance defect-free bioinspired membranes.

Performance of the membranes in low-pressure RO filtration of Tap water. This study also aimed at evaluating the performances of bioinspired membranes for TWRO desalination. The water flux followed the same trend measured under BWRO desalination, decreasing from 34 to 24 L m -2 h -1 by increasing the MPD concentration from 0.8% w/w to 1.8% w/w with a constant optimal HC6 load of 1.5% w/w (applied pressure of 6 bar, Table S5, S6). Simultaneously, the observed rejections based on global conductivity measurements grew accordingly, from 97.5% to 99.3% (Fig. 4e). These values are in the same range, and in some cases slightly higher, of those measured under the same experimental conditions with commercial TWRO membranes (31±1 L m -2 h -1 ; 97.5 % observed rejection), such as the TW30 from DuPont. Furthermore, multivalent cations including Ca 2+ were rejected at a higher rate of 99.8 % compared to Na + and K + cations, due to size exclusion mechanism, [START_REF] Matsumoto | Membrane potential across reverse osmosis membranes under pressure gradient[END_REF] specifically indicating the potential of these membranes for water softening applications (Fig. 4f).

Discussion

Relationship between structural behavior and transport performance of the biomimetic membranes. The present study leads to the discovery of an unexplored mechanistic strategy allowing homogeneous AWC incorporation starting from their in-situ colloidal self-assembled superstructures leading to the identification of sponge-like particles present within the hybrid PA-AWC materials. During IP, the MPD is interacting with soft self-assembled HC6 colloidal nanoparticles via H-bonding. Due to their amphiphilic properties, the AWC aggregates may also contribute to enhance the diffusion of the MPD into the organic phase to react with the TMC as revealed by previous studies. [START_REF] Mansourpanah | Using different surfactants for changing the properties of poly(piperazineamide) TFC nanofiltration membranes[END_REF] The formation of the hybrid layers may be further generated via the interaction of the amphiphilic AWC/MPD nanoparticles with nascent PA oligomers via H-bonding. These interactions depend strongly on the HC6/MPD ratio and under favorable conditions, they effectively promote the PA formation and facilitate the gentle incorporation of AWC aggregates, whereby preventing the formation of defects, which are commonly observed when solid state nanoparticles are directly incorporated within the PA. 1,[START_REF] Vincenzo | Biomimetic artificial water channels membranes for enhanced desalination[END_REF] Other than the seamless in-situ adaptive incorporation of distributed AWC/PA-sponge like nanostructures into the PA layer, a morphological result observed in this study suggests the homogeneous formation of unique highly porous AWC/PA structures with reduced water transport resistance at the interface of the selective layer with the PSf support. Such membranes provided the best performances in terms of water transport and selectivity, and the porous structure did not affect the mechanical resistance or the membrane properties under RO filtration, unlike previous results reported for PA membranes prepared with additives. [START_REF] Yan | The porous structure of the fully-aromatic polyamide film in reverse osmosis membranes[END_REF][START_REF] Hermans | Study of synthesis parameters and active layer morphology of interfacially polymerized polyamide-polysulfone membranes[END_REF] It can be concluded that the improved transport performances stem from a combination of higher porosity of the overall PA-based layer, the absence of defects, and the occurring fast transport through the HC6 nanostructures, whose excellent supramolecular adaptive properties confirm their ability to selectively translocate water while rejecting small ions even when incorporated into hybrid PA-AWC membranes. A significant loss in the perm-selectivity was instead observed for the membranes fabricated with too low MPD concentration or too high HC6 loadings. For example, membranes fabricated with insufficient MPD exhibited a dense, more symmetric, and extremely thin selective layer. These results suggest that the presence of an excess of MPD seems to be imperatively needed for synthetizing defect-free bioinspired layers. [START_REF] Wei | Influence of monomer concentrations on the performance of polyamide-based thin film composite forward osmosis membranes[END_REF] We know from literature data that the use of a spray coated CNT layer on the PES support before the IP, provides an interface that enables the generation of a highly permeable and selective PA layer with a large effective surface area for water transport. [START_REF] Zhou | High-performance thin-film composite membrane with an ultrathin spray-coated carbon nanotube interlayer[END_REF] Some similarities may explain the formation mechanism related to this study, whereby the incorporation of HC6 in the PA layer leads to combined effects, such as the formation of a gutter layer or the generation of higher surface roughness and/or a leaf-like morphology that might be related to the amphiphilic properties of the HC6 (in contrast to the use of additives, such as SDS). Nonetheless, if we look at the experimental results in the upper bound graph (Fig. 5c), our membranes show to be highly permeable and selective, lying at the limit that exists between BWRO and SWRO regions. High recovery and fouling experiments (See SI Appendix, Figs S8, S9) confirm the high-quality features of our biomimetic membranes. Therefore, in the range/area that delimitates the BWRO membranes, our membranes are highly selective, more selective than the other membranes prepared on laboratory scale, including thin-film nanocomposites (TFN) obtained using nanofillers (i.e., zeolites, carbon nanotubes, graphene oxide), additives (i.e., proton acceptors, surfactants, acids), or optimized by using salts/hydrophilic additives, different solvent or co-solvent, or by varying other experimental conditions (i.e., pH, T or concentrations in the casting solutions) during the membrane fabrication. [START_REF] Yang | The upper bound of thin-film composite (TFC) polyamide membranes for desalination[END_REF] Of particular interest is the potential ability of such PA films to present directional pathways for water transport. Herein, microscopy studies revealed that such hybrid PA-AWC materials are composed by AWC crystalline nanoparticles, randomly dispersed in the PA matrix (Fig. 5b). For 1.2-1.8 % w/w MPD these particles are homogeneously and densely distributed within all the thickness of the PA layer, while for lower concentration of 0.2-0.8 % w/w MPD the are less dense and are situated the middle part of the PA layer. For the sponge like nanoparticles 17 of 20-40 nm the permeability of water can be theoretically estimated up to PAWC = 131 L•m -2 h -1 bar -1 (see SI Appendix). Independently of what is possible by microscopy or not, a high density of channels percolating from one side to the other of the membranes would be ideal, but we are not there yet. It is not about alignment which is clearly proved to occur along nanometric distances of crystals of AWC that are showing order. It is about percolation and high density of channels that for the moment is achieved at this nanometric scale. Although these nanoparticles do not merge to cross the µmetric PA films (Fig. 5a), they are randomly distributed within the PA layer (PPA=1 LMH/bar). The AWC crystals locally contribute to enhance translocation of water, as PAWC>>PPA (Fig. 5b). We do not intent to prove anything in a definite or exactly mathematical way here it is only an intuitive way to explain our results on AWC-PA membrane permeabilities that are experimental and unquestionable. The hybrid PA-AWC materials are reminiscent with previous hybrid ion-channels siloxanes, providing high ionic conduction through their nanometric self-organization of binding sites in hybrid materials. [START_REF] Barboiu | Ion-driven ATP-pump by Self-Organized Hybrid Membrane Materials[END_REF][START_REF] Michau | Self-organized proton conductive layers in hybrid proton exchange membranes, exhibiting high ionic conductivity[END_REF][START_REF] Barboiu | Constitutional hybrid materials-toward selection of functions[END_REF] The assumption is that, beside 1. the tunable formation of colloidal nanoparticles within the PA, 2. the formation of inner porosity/voids (as a consequence of the effect of the HC6 "interlayer"), and 3. the gutter layer-like structure, there is the diffusion of hydrophilic and selective sponge-like AWCs-PA (which we surmise are also in the form of I-quartets). Thus, we assume that this performance is achieved thanks to the combined effect of all these aspects. It is also true that the AWCs are intrinsically "nanofillers" but, by comparison, our membranes are more selective and permeable, possibly due to the compatibility of the HC6. Obviously, a high density of channels is desired to promote high permeance. Alignment is not essential, but the percolation of AWC particles is important and should be optimized to have the water transport mainly taking place through the channels. Homogeneous distribution with particle percolation should be preferred. High density of particles without aggregation should be obtained. Achieving these conditions was a main goal of the optimization in this work.

In this work, bioinspired membranes for low salinity BWRO and TWRO water desalination were fabricated by incorporating I-quartet AWC in the classical PA layer. Specifically, the performance of hybrid AWC-PA membranes was tuned by studying the effect of the optimal AWC loads and that of MPD monomer concentration during IP, thus optimizing the selective layer. It may be concluded that self-aggregated AWC colloidal nanoparticles were incorporated by means of their supramolecular interactions with MPD monomer and their presence altered the IP process, and thus the final layer properties. These dynamic self-assembly processes amount to adaptive colloidal entities with the nascent PA oligomers.

This study illustrates a complete interplay of supramolecular aggregation and IP processes: a) related to supramolecular aggregation, once the AWC load is increasing, the selective PA-AWC layers became more porous and AWC nanoaggregates were homogeneously distributed within the hybrid PA, resulting in high-performance membranes. However, overly high concentrations of AWC nanoaggregates led to the formation of defects, resulting in the poorest performances.

b) The MPD monomer concentration had also an important effect during membrane fabrication and seamless biomimetic layers were synthetized by adjusting this parameter reaching the optimal HC6/MPD ratio between 0.7 and 1.9 w/w. In general, a minimum concentration of MPD (~0.8% w/w) was required to obtain high selectivity. However, even this concentration should not be too high in order to maintain high water fluxes. In particular, the membranes fabricated with 1.2 % w/w MPD and 1.5% w/w HC6 provided a ~360% increase in water permeance at equivalent water to salt flux ratio with respect to the membrane fabricated using 3.4 % w/w MPD.

The best membranes displayed excellent productivity of 110 L m -2 h -1 under 15.5 bar applied pressure and 35 L m -2 h -1 under 6 bar applied pressure, while maintaining high selectivity properties, outperforming current BWRO and TWRO commercial membranes. Ultimately, we demonstrated that bioinspired membranes incorporating I-quartet AWCs own the potential for improving existing low-pressure RO applications and their composition can be adjusted to tune their superstructures and performances to target different applications.

Methods Section

Chemicals, Membrane morphological and physio-chemical characterization, Dynamic light scattering (DLS) measurements, Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), Scanning electron microscopy (SEM), Transmission Electronic Microscopy (TEM) and Membrane performance evaluation are presented in supporting information -see SI appendix Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) spectra were recorded on a NEXUS spectrometer. The ATR accessory contained a monolithic diamond crystal at a nominal angle of incidence of 45°, yielding 1 internal reflection at the sample surface. All spectra were recorded at 25 °C in the region 625-3500 cm -1 . Elemental analysis of the active layers was conducted by energy dispersive X-ray spectroscopy (EDX) using an AZTEC System (Oxford Instruments, UK), at an accelerating voltage of 10 kV and a working distance of 8.5 mm. At least three spectra were captured at different positions to calculate an average atomic percentage of C, N, and O.

Scanning electron microscopy (SEM).

The incorporation of HC6 artificial water channels in polyamide layers was verified by a high-resolution scanning electron microscope (SEM, HITACHI S-4800), at an accelerating voltage of 2 kV. For cross-sectional studies, membrane coupons were prepared by freeze-fracturing in liquid nitrogen and then dried in dry air for at least 24 h. A 5-nm thick coating of chromium was sputtered (SC7620 Mini Sputter Coater, Quorum Technologies Ltd.) under Ar atmosphere (10 -1 mbar) to achieve a minimum conductivity for reliable SEM information. The roughness of the membrane surfaces was investigated by atomic force microscopy (AFM) using a Nanoman, with Nanoscope5 instrument (Bruker Instruments). The tips were from Nanosensors, PPP NCSTR, with a nominal spring constant of 7 N/m and a typical radius of less than 5 nm. All images were acquired in tapping mode with a sampling resolution of at least 512 pts/512 lines using a scan rate of 5.5 Hz for 5 µm² areas, and 0.65 Hz for smaller areas.

Transmission Electronic Microscopy (TEM). The membranes were stained by immersion in 1% zinc nitrate aqueous solution for 15 min, followed by rinsing in large amount of distilled water. Zinc salt stained more selectively the imidazole and amide groups in the polyamide layer. Membrane pieces were embedded in epoxy resin (EMbed812, Fisher Scientific), which was thermally cured overnight at 70 °C. Ultrathin sections (100 nm) were cut with an ultramicrotome (Leica EM UC7) and deposited on 200 mesh copper grids. The TEM imaging was performed using a Titan CT microscope (FEI company) operating at 300 kV equipped with a 4k × 4k CCD camera (Gatan, Pleasanton, CA, USA). For HC6 imaging, a drop of solution (1.5 w/w% in ethanol/water 85:15 v/v%) was deposited on carbon coated copper film, blotted with filter paper and let it dry in the lab atmosphere. Theoretical membrane performance evaluation.

We have calculated the theoretical membrane permeability considering the theoretical values of single permeabilities of single channels and of sponge like structures of HC6 as reported in reference 17. The HC6-I4 single channel permeabilities are 3.8 x 10 6 molecules / s / channel for single channel and 5.25 x 10 7 molecules / s / channel for the sponge-like water channels. [START_REF] Murail | Water permeation across artificial I-quartet membrane channels: from structure to chaos[END_REF] Our results on AWC-PA membrane permeabilities are experimental and unquestionable. These calculations are performed just to support some discussion as the stability of the channels is clearly different as they a present in bilayer membranes or in crystalline lamellar structures confined within PA matrix. We do not intent to prove anything in a definite or exactly mathematical way here.

We note that the stability of the channels in bilayer is strongly dependent on the size of the tail, [START_REF] Licsandru | Salt-Excluding Artificial Water Channels Exhibiting Enhanced Dipolar Water and Proton Translocation[END_REF] while within the crystals the I-quartet pillars are almost similarly stabilized between lamellar hydrophobic phases that are not much altered in the solid state by the dimension of the tail. These values are superior to those observed with conventional BWRO membranes (1-8 L•m -2 h -1 bar -1 ) [START_REF] Yang | The upper bound of thin-film composite (TFC) polyamide membranes for desalination[END_REF] , usually operated at slightly higher 15-18 bars and for higher saline concentrations.

Intrinsic transport properties of control TFC and bioinspired membranes were evaluated using a laboratory-scale cross-flow unit, comprising a high-pressure pump, a feed vessel, and a flat membrane housing cell. The effective membrane active area was 28.9 cm 2 , the crossflow velocity was fixed at 0.9 m/s, and the temperature was constant at 25 ± 0.5 °C. Prior to each experiment, the membrane was immersed in water overnight. Two sets of filtration tests were conducted, referred to as "brackish water" and "tap water" tests. The membranes were compacted with DI water as feed at 18 bar (261 psi) of applied pressure, ΔP, until the permeate flux reached a steadystate. The pressure was then lowered to 15.5 bar (217 psi) or 6 bar (87 psi) for brackish water and tap water tests, respectively.

The pure water flux, Jw,0, was calculated by dividing the volumetric permeate rate, obtained at steady-state, by the membrane active area. The pure water permeability coefficient, A, was determined by dividing the water flux, Jw,0, by the applied pressure, 𝐴 = 𝐽 𝑤,0 𝛥𝑃 ⁄ .

Subsequently, for brackish water tests, NaCl was added from a 5 M stock solution to reach a final concentration of 100 mM, and salt concentrations in the feed and permeate streams were measured using a calibrated conductivity meter (pHenomenal® CO 3100 H, VWR Instruments). The rejection was always measured at steady state (more than one hour after steady state was reached and within this time the selectivity always increased of few decimals). Therefore, the rejection was always constant in time; three separate rejection measurements were performed, distanced 30-60 min from each other In the tap water tests, real tap water was taken from the European Institute of Membranes (IEM) and used as feed stream; salt concentrations were determined by both ion and conductivity measurements. Upon reaching steadystate, the permeate flux, Jw, was calculated by dividing the volumetric permeate rate by the membrane area. Observed rejection, R, was then computed from the concentrations determined in bulk feed, cf, and in the permeate stream, cp, for each ionic species or for global salinity (using conductivity as a proxy for salinity):

𝑅 = 1 - 𝑐 𝑝 𝑐 𝑓 ⁄ .
The rejection values reported for each sample are the average of three different measurements. The solute permeability coefficient, B, was computed by taking into account the external concentration polarization, as:

𝐵 = 𝐽 𝑤 ( 1-𝑅 𝑅 ) 𝑒 (-𝐽 𝑤 𝑘 ) ,
where k is the mass transfer coefficient of the cross-flow cell.

Table S4. TWRO performances of bioinspired active layers; experimental conditions: 6 bar of applied pressure and 25 °C; pure water as feed solution for the measurement of the water permeance A; real tap water from the European Institute of Membranes (IEM) for the measurement of the permeate flux with complex real solution; conductivity measurements for the evaluation of the observed global rejection; ionic chromatography measurements for the estimation of the bivalent Ca +2 rejection. "HC6" stands for optimized precursor concentration (1.5% w/w) and the number before for the actual MPD concentration (% w/w) used during the interfacial polymerization.

Membrane

Figures and Tables Fig. 1 .

 Tables1 Figures and Tables

Fig. 2 .

 2 Fig. 2. Representative cross-section SEM micrographs of (a,b) H-0.9; (c,d) 0.8-HC6 and (e,f) 0.2-HC6. MPD concentrations in the casting solutions were (a,b) 3.4%; (c,d) 0.8%; (e,f) 0.2% (w/w). HC6 concentrations in the casting solutions were (a,b) 0.9%; (c,f) 1.5% (w/w).

Fig. 3 .

 3 Fig. 3. TEM cross-sectional image of a) 0.2-HC6, b) 0.4-HC6, c) 0.8-HC6, d) 1.2-HC6, and e) 1.8-HC6 membranes, % w/w MPD, 0.1 w/w% TMC and 1.5% w/w HC6 stained with Zn(NO 3 ) 2 ) showing the detail of the top hybrid layer containing AWC/PA white nanoparticles embedded with high precision in darker PA matrix.

Fig. 4 .

 4 Fig. 4. BWRO desalination performances at 15.5 bar as a function of (a,b) HC6 and (c,d) MPD concentrations. Experimental conditions: pure water for the measurement of A; 100 mM NaCl in the feed solution for the measurement of B. TWRO desalination performance at 6 bar e) Permeate flux and observed rejection at steady-state as a function of MPD concentration. f) Observed rejection of different cations and anions at steady-state of 1.6-HC6 membranes under TWRO conditions. Lines connecting the data points are only intended as a guide for the eye.

Fig. 5 .

 5 Fig. 5. Enhancement permeation mechanism. a) the ideal membrane with perfectly aligned channels though the whole thickness of the PA membrane (blue); b) uniform aleatory distribution of AWC nanoparticles within the layer. Enhanced permeation at the nanometric level relates to AWCs nanoparticles regions-see text for the details; c) Water permeance and selectivity of commercial seawater reverse osmosis (SWRO, red), brackish water RO (BWRO, blue) and NF membranes (green). Blue dash lines correspond to the NaCl rejection of 99% and 90%, respectively, at a flux of 20 L•m -2 •h -1 . The yellow star highlights the perm-selectivity performance of our optimized BWRO membranes prepared using AWCs, while the empty symbols represent all the other laboratory scaled membranes 11 Fig. adapted from ref. 11 Copyright Elsevier 2019.

Fig. S2 .

 S2 Fig. S2. a) FTIR-ATR spectra of control TFC (blue), HC6 I-quartet (black) and bioinspired membranes (red) (1.8% w/w MPD and 1.5% w/w HC6). b) Atomic mass fraction of % C, N and O from EDX measurements: average values and standard deviations as determined from three different samples for each membrane. The results refer to control TFC membranes (white bar) and to bioinspired membranes (colored bars) containing the optimal concentration of 1.5% w/w HC6, with variable MPD concentrations of 0.2-1.8% w/w.

Fig. S3 .

 S3 Fig. S3. Representative SEM micrographs of the surface morphology of a) 2.0-HC6; b) 1.8-HC6; c) 1.6-HC6; d) 1.4-HC6; e) 1.2-HC6; f) 0.8-HC6; g) 0.4-HC6; and h) 0.2-HC6 bioinspired membranes prepared with different MPD concentrations during interfacial polymerization (IP). "HC6" stands for optimized precursor concentration (1.5% w/w) and the number before for the actual MPD concentration (% w/w) used during the IP reaction.

Fig. S4 .

 S4 Fig. S4. Representative SEM micrographs of surface top view of, a)-c), control TFC and, b)-d), HC6 bioinspired membrane, showing different morphology and diffused HC6 nanostructures.

Fig. S5 .

 S5 Fig. S5. Representative SEM micrographs of detailed surface top view of a) 1.2-HC6 and b) 0.8-HC6 showing compact HC6 nanostructures. Cross-sectional view of c) 1.2-HC6 and d) 0.8-HC6showing the detailed distribution of diffused nanoparticles spanning the whole active layers. "HC6" stands for optimized precursor concentration (1.5% w/w) and the number before for the actual MPD concentration (% w/w) used during interfacial polymerization (IP).

Fig. S6 .

 S6 Fig. S6. Representative SEM micrographs of cross-sectional view of a) 0.4-HC6 and b) 0.2-HC6showing very thin and symmetric dense layers free of inner porosity; "HC6" stands for optimized precursor concentration (1.5% w/w) and the number before for the actual MPD concentration (% w/w) used during the interfacial polymerization reaction. c)-d) Details of nanoaggregates spill from the active layer observed on the surface of biomimetic membranes prepared with substoichiometric MPD concentration, indicating failing in suitable HC6 incorporation.

Fig. S7 .

 S7 Fig. S7. AFM measurements. a) Experimental and guide to the eye profiles of average and maximum roughness of membranes versus MPD concentrations in the casting solutions. The projection area scanned during the measurements was 5 µm × 5 µm. Representative AFM surface images with a projection area of 2 µm × 2 µm of b) 0.2-HC6, c) 0.4-HC6, d) 0.8-HC6, e) 1.2-HC6, and f) 1.8-HC6 membranes).

Assuming a 3 . 5

 35 nm thickness (lipid bilayer) and 𝝅= 8.3 bar (200 mM NaCl) HC6-I4 single channel permeability: 3.8 × 10 6 H 2 O / s / channel From the single crystal structure I4 density: 3.3 × 10 17 channels / m 2 The water flux (Jw) is: J w = 1.25 × 10 24 H 2 O m -2 s -1 = 136.8 Lm -2 h -1 Water permeability: 𝐖𝐏 = 𝟏𝟔. 𝟒𝟖 𝐋𝐦 -𝟐 𝐡 -𝟏 𝐛𝐚𝐫 -𝟏 HC6-I4 sponge like channel permeability: 5.25 × 10 7 H 2 O / s / channel From the single crystal structure I4 density: 3.3 × 10 17 channels / m 2 The water flux (Jw) is: J w = 1.73 × 10 25 H 2 O m -2 s -1 = 1890 Lm -2 h -1 Water permeability: 𝐖𝐏 = 𝟐𝟐𝟕 𝐋𝐦 -𝟐 𝐡 -𝟏 𝐛𝐚𝐫 -𝟏 In the same way we can calculate the water permeation rate assuming a 20-40 nm thickness (crystal) and 𝝅= 1.68 bar (34 mM NaCl, same as during the experiments) HC6-I4 single channel permeability: 3.8 × 10 6 H 2 O / s / channel From the single crystal structure I4 density: 3.3 × 10 17 channels / m 2 The water flux (Jw) is: J w = 1.46 × 10 23 H 2 O m -2 s -1 = 15.96 Lm -2 h -1 Water permeability: 𝐖𝐏 = 𝟗. 𝟓 𝐋𝐦 -𝟐 𝐡 -𝟏 𝐛𝐚𝐫 -𝟏 HC6-I4 sponge like channel permeability: 5.25 × 10 7 H 2 O / s / channel From the single crystal structure I4 density: 3.3 × 10 17 channels / m 2 The water flux (Jw) is: J w = 2.02 × 10 24 H 2 O m -2 s -1 = 220 Lm -2 h -1 Water permeability: 𝐖𝐏 = 𝟏𝟑𝟏 𝐋𝐦 -𝟐 𝐡 -𝟏 𝐛𝐚𝐫 -𝟏

Table S1 .

 S1 Concentrations of HC6 and MPD in the aqueous phase and TMC in the organic phase for the preparation of different membranes by interfacial polymerization. "H" and "L" stands for high (3.4% w/w) and low (2% w/w) MPD concentration in the aqueous phase

	MEMBRANE	HC6	MPD	TMC
		w/w % w/w % w/w %
	control TFC	-	3.4	0.1
	H-0.9	0.9	3.4	0.1
	H-1.3	1.3	3.4	0.1
	H-1.5	1.5	3.4	0.1
	H-1.8	1.8	3.4	0.1
	L-1.3	1.3	2.0	0.1
	L-1.5	1.5	2.0	0.1
	1.8-HC6	1.5	1.8	0.1
	1.6-HC6	1.5	1.6	0.1
	1.4-HC6	1.5	1.4	0.1
	1.2-HC6	1.5	1.2	0.1
	0.8-HC6	1.5	0.8	0.1
	0.4-HC6	1.5	0.4	0.1
	0.2-HC6	1.5	0.2	0.1

Table S2 .

 S2 Energy-dispersive X-ray (EDX) spectroscopy of pristine PA and bioinspired reverse osmosis active layers prepared by varying the MPD monomeric concentration in the casting solutions used during interfacial polymerization (IP). "HC6" stands for optimized precursor concentration (1.5% w/w) and the number before for the actual MPD concentration (% w/w) used during the IP reaction. Atomic mass percentage of C, N, and O were obtained as the average mass fraction value from three different positions of each membrane sample.

	Membrane	C (%)	N (%)	O (%)
	pristine PA	67.50 ± 0.1	3.50 ± 0.2	18.4 ± 0.10
	0.2-HC6	85.15 ± 0.2	0.83 ± 0.1	11.4 ± 0.10
	0.8-HC6	84.23 ± 0.2	1.54 ± 0.2	11.6 ± 0.01
	1.2-HC6	83.77 ± 0.3	2.01 ± 0.3	11.8 ± 0.08
	1.8-HC6	81.47 ± 0.3	4.93 ± 0.4	11.3 ± 0.19

Table S3 .

 S3 Surface roughness parameters of bioinspired reverse osmosis membranes obtained by AFM measurements (projection area of the samples: 5 µm x 5 µm).

	Membrane AVG [nm]	Max [nm]	RMS [nm]
	0.2-HC6	44±2	155±45	56±8
	0.4-HC6	45±2	200±65	56±13
	0.8-HC6	53±2	147±43	55±6
	1.2-HC6	95±6	145±90	57±10

Acknowledgments

This work was supported by Agence Nationale de la Recherche ANR-18-CE06-0004-02, WATERCHANNELS and ERANETMED 2-72-357 IDEA.

Supplementary Information for

Tuneable bio-inspired membranes incorporating artificial water channels for high-performance brackish/tap water reverse osmosis desalination Maria Di Vincenzo, 1 Alberto Tiraferri, [START_REF] Vincenzo | Biomimetic artificial water channels membranes for enhanced desalination[END_REF] Mihai Deleanu, 1 Didier Cot, 1 Valentina-Elena Musteata, [START_REF] Fane | Synthetic membranes for water purification: Status and future[END_REF] Stefan Chisca, [START_REF] Fane | Synthetic membranes for water purification: Status and future[END_REF] Suzana P. Nunes, [START_REF] Fane | Synthetic membranes for water purification: Status and future[END_REF] Mihail Barboiu 1 1Institut Européen des Membranes, Adaptive Supramolecular Nanosystems Group, University of Montpellier, ENSCM, CNRS, Place Eugène Bataillon, CC 047, F-34095, Montpellier, France. 2Department of Environment, Land and Infrastructure Engineering (DIATI), Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Turin, Italy. 3King Abdullah University of Science and Technology (KAUST), Biological and Environmental Science and Engineering Division, Advanced Membranes and Porous Materials Center, 23955-6900, Thuwal, Saudi Arabia.

Mihail Barboiu

Email: mihail-dumitru.barboiu@umontpellier.fr

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Supplementary text

METHODS

Chemicals.

Commercial flat-sheet polysulfone (PSf) ultrafiltration membranes, PS35-GPP (Solecta, USA), were used as support layers for the fabrication of all the polyamide-based membranes. Trimesoyl chloride (TMC, 98%), m-phenylenediamine (MPD, flakes 99%), histamine (97%), hexyl isocyanate (97%), sodium chloride (NaCl) (≥99.5%), sodium metabisulfite (Na2S2O5, 98%), were purchased from Sigma-Aldrich, France. Artificial water channels precursor HC6 has been synthesized as previously reported.14 All solvents used in this study were HPLC grade. Methanol (MeOH), ethanol (EtOH), tetrahydrofuran (THF), ethyl acetate (EtOAc), dimethylacetamide (DMA), acetonitrile (ACN), and hexane were purchased from VWR International Ltd. Unless specified, all chemicals were dissolved in DI water obtained from a Milli-Q ultrapure water purification system (Millipore, France). All the reagents and solvents were used without any further purification.

Membrane morphological and physio-chemical characterization.

Dynamic light scattering (DLS) measurements were performed using a Malvern Zetasizer with a 173° backscatter measurement angle and a quartz cuvette with a square aperture. The samples were prepared by dissolving the HC6 in ethanol to achieve 0.5, 1 and 2.5% w/w. In order to investigate the formation of AWC colloidal aggregates, different amount of water was added to the HC6/ethanol solution under various conditions. We investigated the formation of AWC colloidal aggregates by dynamic light scattering (DLS). Fig. S1a shows the size distribution of hydrodynamic diameter (Dh) after the addition under vigorous stirring of pure water and 2 w/w% aqueous solution of MPD to 1% HC6 in ethanol to reach EtOH/H2O and EtOH/H2O+MPD =1/3. The Dh of AWC colloidal aggregates is 200 nm when pure water was added, whereas, when 2 w/w% of an aqueous solution of MPD was added, the Dh is reduced to 70 nm and the size distribution became slightly larger (Fig. S1b). This indicate that HC6 interacts with the MPD through hydrogen bonding, leading to stabilized AWC colloidal aggregates. When a larger amount of water was added to 1% HC6 in ethanol (to reach EtOH/H2O =1/6), we observed a decrease of the Dh of the AWC colloids from 200 nm to 80 nm, and by adding a larger amount of MPD aqueous solution a further reduction of Dh is observed (from 70 nm to 60 nm). Therefore, we believe that during the IP synthesis, when a large amount of MPD aqueous solution is added, nanometer colloidal AWC is formed at the alcohol/water interface in the polysulfone pores. Table S5. Ionic chromatography measurements of ionic concentrations (ppm) of both feed and permeate samples to evaluate the specific ionic separation performances of bioinspired active layers for TWRO desalination. Real tap water was taken from the European Institute of Membranes (IEM) and used as feed stream. The membranes presented in this study were selected because of their higher water flux obtained at an applied pressure of 6 bar, values that are comparable or slightly higher compared to marketed state-of-the-art tap water reverse osmosis membranes, such as TW30 (DuPont). "HC6" stands for optimized precursor concentration (1.5% w/w) and the number before for the actual MPD concentration (% w/w) used during the interfacial polymerization. Table S6. Ionic chromatography measurements to evaluate the specific ionic rejection performances of bioinspired active layers for TWRO desalination. Real tap water was taken from the European Institute of Membranes (IEM) and used as feed stream. The membranes presented in this study were selected because of their higher water flux obtained at an applied pressure of 6 bar, values that are comparable or slightly higher compared to marketed state-of-the-art tap water reverse osmosis membranes, such as TW30 (DuPont). "HC6" stands for optimized precursor concentration (1.5% w/w) and the number before for the actual MPD concentration (% w/w) used during the interfacial polymerization.

Protocol for high-recovery experiment

The membranes were compacted at 17 bar until steady-state flux was measured. The pressure was lowered to 15.5 bar and NaCl was added into the feed solution to reach a concentration of 34 mM (2 g/L). The system was thus opened (the permeate stream was not recirculated into the feed tank but collected separately), and the flux constantly monitored until a recovery rate of 75% was obtained. The observed rejection was determined by measuring the conductivity of the feed and the permeate streams throughout the test.

Protocol for fouling experiment

The membranes were compacted at 17 bar until steady-state flux was measured. The pressure was lowered to 15.5 bar and NaCl was added into the feed solution to reach a concentration of 34 mM (2 g/L). At steady-state and in closed-loop mode (the permeate stream recirculated into the feed tank), the observed rejection was determined by measuring the conductivity of the feed and the permeate streams three times at intervals of roughly 30 min between each sampling, resulting in values of 99.20 ± 0.04 % and 98.07 ± 0.02 % for the biomimetic membrane and the XLE membrane, respectively. Subsequently, CaCl2, alginate, and BSA were added into the feed solution and the applied pressure was adjusted for each membrane to obtain an initial flux of 42 ± 2 L m -2 h -1 and to initiate the accelerated fouling test, which was carried out for approximately 18 h.