Hierarchical Optimization of High-Performance Biomimetic and

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Invited Feature Article

Hierarchical optimization of high performance biomimetic and bioinspired membranes Woochul Song, Yu-Ming Tu, Hyeonji Oh, Laxmicharan Samineni, and Manish Kumar Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03655 • Publication Date (Web): 21 Dec 2018 Downloaded from http://pubs.acs.org on December 25, 2018

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Hierarchical optimization of high performance biomimetic and bioinspired membranes Woochul Song1, Yu-Ming Tu1, Hyeonji Oh1, Laxmicharan Samineni1 and Manish Kumar*1,2,3 1Department

of Chemical Engineering, 2Department of Biomedical Engineering, 3Department of Civil and Environmental Engineering, The Pennsylvania State University, University Park, PA, 16802 USA *To whom correspondence should be addressed Tel.: (814)-865-7519 E-mail: [email protected]

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Abstract Biomimetic and bioinspired membranes have emerged as an innovative platform for water purification and aqueous separations. They are inspired by the exceptional water permeability (~109 water molecules per second per channel) and perfect selectivity of biological water channels, aquaporins. However, only few successes have been reported for channel-based membrane fabrication due to inherent challenges of realizing coherence between channel design at the angstrom level and development of scalable membranes that maintain these molecular properties at practice-relevant scales. In this article, we feature recent progress towards practical biomimetic membranes, with the review organized along a hierarchical structural perspective that biomimetic membranes commonly share. These structures range from unitary pore shapes and tubular hydrophobic channel geometries to self-assembled bilayer structures and finally to macro-scale membranes covering a size range from the angstrom, to the micrometer scale and finally to the centimeter and larger scales. To maximize the advantage of water channel implementation into membranes, each feature needs to be optimized in an appropriate manner that provides a path to successful scale-up to achieve high performance in practical biomimetic and bioinspired membranes.


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Introduction Water scarcity is a major challenge facing the world today. More than one third of all countries suffer from lack of access to safe water supplies and, paradoxically, the population growth in these affected areas is particularly rapid.1-2 Innovations in water treatment technologies have resulted in dramatic energy reduction in processes used for advanced water purification such as membrane-based seawater desalination and wastewater reuse, providing enhanced access to new water sources.2-4 For example, the power consumption to desalinate seawater has been consistently lowered from ~17 kWh/m3 in the 1960s to close to the thermodynamic energy limit of ~1 kWh/m3 for the reverse osmosis (RO) process.5 These dramatic reductions have resulted from overall process optimization and the development of effective membrane materials.5 Further development of effective membrane materials could have a large impact on membrane-based water separations as several applications of membranes including wastewater reuse and brackish water desalination are still not as close to the thermodynamic limit as seawater reverse osmosis, and development of highly selective and permeable membranes can significantly reduce the footprint of water treatment equipment.6 Also, limited success has been achieved during last few decades in moving beyond the standard polyamide thin film composite membranes currently common in RO applications. This is due to the convoluted microstructures of these membranes and the lack of known chemistry-structure-function relationships7 that make these current materials efficient. So innovation in membrane materials could provide mode options for such applications. Biomimetic and bioinspired membranes (BBMs) have emerged as an innovative platform to address some challenges of current aqueous separation membranes by providing a simple bioinspired structure with an efficient water transport mechanism.8-9 This work has been accelerated since groundbreaking work in 1990s, which provided insights into the molecular structure and function of biological membranes.10-11 Aquaporins (AQPs) were identified as a class of biological membrane proteins that act as molecular water channels in cell membranes, and were demonstrated to have exceptionally high water permeability (~109 water molecules per second per channel, H2O/s/channel). At the same time these channels have very high water selectivity (for example ~109:1 water over K+, for AQP1) over any other solutes including protons.12-14 They have precisely defined pores of ~3Å diameter that are responsible for size-based selective water permeation. Sub-nm confinement in a mostly hydrophobic pore directs water flow as a single water wire. This results in high permeability as a result of favorable energy compensation of one-dimensional water diffusion inside the channels compared to macroscopic bulk diffusion.15 In addition, self-assembly of channels within biological membranes can lead to high channel packing density within the ~4 nm thickness of lipid bilayer membranes in many instances.9 The distinctive features of biological membranes such as defined pore structures of angstrom size, high pore (channel) density, and thin membrane layers have inspired researchers to mimic them in developing efficient membranes. However, successful development of biomimetic membranes has been limited by the fact that many biomimetic membrane studies have been focused on maximizing water channel function but not on understanding the importance of other membrane components in maintaining coherence across scales.8-9, 16-17 We propose that, to successfully optimize the performance of biological membranes, it is essential to develop an understanding of membrane design details from the molecular to practical membrane scale and to incorporate this understanding while designing biomimetic membrane components (Figure 1). In this feature article, we describe optimization of each step of developing high performance BBMs as illustrated in our work, starting from tuning pore structures of a pore-forming membrane protein, outer membrane protein F (OmpF), for designing efficient size selective biological water channels. Then block copolymers (BCPs) are introduced as a robust membrane matrix platform, followed by considerations for maximizing channel insertion efficiency into biomimetic membranes membranes such as physicochemical compatibility between channels and membranes and controlling selfassembly kinetics. Next a relatively new class of water channels, artificial water channels, will be


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featured as promising alternative nanoarchitectures that mimic biological water channels. Finally, strategies for successful water channel implementation into BCP membranes will be described with as an example of progress towards the ultimate goal of development of defect-free membrane fabrication with practical scalability.

PoreDesigner: A framework for (re)designing biological water channels AQPs are archetypal biological water channels with outstanding water transport properties while maintaining selectivity. However, recent advances in biophysics of water transport through membrane protein channels have strongly suggested that AQPs may not have the ideal pore environment for efficient water transport as far as permeability is concerned.18 The formation of hydrogen bonds (Hbonds) between water molecules and channel inner walls is a major impeding factor which reduces the water permeability during sterically confined single file water flow in sub-nm pore size channels. Even though AQPs show exceptionally fast water permeation, protein backbones near channel lumen surface serve as H-bond donors and acceptors, significantly interacting with diffusing water wires and retarding water permeation. To address this issue, we have built a framework to redesign efficient biological water channel pores, termed PoreDesigner. This framework has been tested using the ~1 nm pore size protein channel Outer Membrane Protein F (OmpF) as a modular template. We have redesigned its pore to have similar to or more favorable environments for water permeation than AQPs, while providing a way to target different solute selectivities (Figure 2).19 PoreDesigner. PoreDesigner is a systematic workflow to precisely tune any angstrom-scale membrane protein channel structure spanning 3 - 10 Å in pore size, to achieve high water permeability (≥109 H2O/s/channel) and desired molecular solute selectivity based on size.19 The example protein used in our work, OmpF porin, is a trimeric integral membrane protein channel. Wild type (WT) OmpF has a hydrophilic pore (7 × 11 Å) that allows ions, antibiotics, and amino acids to permeate through cellular membranes, while rejecting solutes larger than molecular weights (MW) of ~600 Da.20 The stability and mutation tolerance makes OmpF an excellent candidate for a template for protein redesign and applications. To redesign the pore structure to allow fast single file transport, single file water traces of AQP1 from molecular dynamics (MD) simulations was isolated and computationally placed inside the OmpF pore (Figure 2A). After that, through iterative computational calculations, the pore constricting residues were redesigned (computationally mutated) to fill up void spaces around water wires, constructing a water-selective internal geometry, which is reminiscent of the AQP1 internal structure but reduced interacting hydrogen bonding residues. To minimize water-pore wall interactions, amino acids with relatively long side-chain and hydrophobicity such as tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr) were selected as pore constriction residues to occupy and occlude the OmpF pore lumen. As a result, three distinct groups of pore geometries were created by PoreDesigner (Figure 2B): off-center pore closure design (OCD), uniform pore closure design (UCD), and cork-screw design (CSD). Among more than 40 created mutant structures, one representative mutant channel was selected from each pore geometry group for experimental evaluation of permeability and selectivity. Mutant OmpF channel experimental evaluation. Selected OmpF mutant channels had following predicted ellipsoidal pore dimensions; OCD (3.54×3.25 Å), CSD (3.18×3.12 Å), and UCD (3.05×3.01 Å). These mutants were selected with the criterion that their pore dimensions should be smaller than ~4 Å, as the separation in this size range may be considered as the most challenging for membranebased separations. These channels were then over-expressed in an Escherichia coli (E. coli) expression system and purified using standard membrane protein isolation techniques. To measure single mutant channel water permeability, purified mutants were reconstituted into L-α-phosphatidylcholine/L-αphosphatidylserine (PC/PS) lipid vesicular membranes and subjected to stopped-flow light scattering experiments for bulk vesicle permeability measurements and fluorescence correlation spectroscopy (FCS) experiments to evaluate average number of proteins inserted per unit area.19 Experimental


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permeability measurements revealed that all tested OmpF mutants had water permeabilities (1.4−4.4(±0.93) × 10-12 cm3/s/channel) exceeding the permeability of E. coli aquaporin AqpZ (2.4(±0.47) × 10-13 cm3/s/channel) by more than one order of magnitude (Figure 3B). Also, they demonstrate single channel permeability higher than wild type (WT) OmpF, which is consistent with the pore design strategy of increasing permeability by minimizing water-pore wall interactions of WT OmpF scaffold. Qualitative analysis of stopped-flow scattering traces indicated that selected mutants have different solute rejection properties, depending on their pore sizes; OCD, CSD, and UCD have rejection limits around ~342 Da, ~180 Da, and ~58 Da (NaCl) respectively (Figure 3B). Importantly, UCD OmpF mutant channels seem to effectively reject NaCl while having higher water permeability than AqpZ, indicating its possible use as a component for desalination membranes. MD simulations MD simulations were used to evaluate experimental single channel permeability measurements and pore structures during water permeation (Figure 3A). The calculated permeability of three mutants and WT OmpF channels followed the same trend of single channel permeability as seen in experiments (Figure 3C). The highest permeability of the CSD mutant could also be explained by scoring hydrophobicity of pore inner walls, confirming the importance of channel interior hydrophobicity in determining water permeability through channels. In terms of reliability of pore structures, MD trajectories analysis shows that mutant OmpF pores have lower fluctuat ions in terms of pore diameters during MD simulation, compared to WT OmpF pore structure (Figure 3D). This indicates that mutant pores became mechanically rigid after redesign due to packing of hydrophobic amino acids within the pore lumen. Overall, PoreDesigner provides an excellent workflow for redesigning specific sub-nm pore structure in the range of 3 to 10 Å which is ideal for angstrom scale membrane-based separations. This redesigned pore sizes and shapes provide an opportunity to develop highly selective channel-based separation membranes with high permeability.

Block copolymers as robust platform for functional biomimetic membranes The relatively low stability of lipid membranes is one of the major challenges for direct use of biological membranes for engineering applications. For example, the phosphoric head groups of naturally occurring phospholipids are highly sensitive to oxidation and this can lead to formation of non-water selective transient pores across the membrane,21 which are detrimental to membrane performance. Meanwhile, amphiphilic block copolymers (BCPs) are an excellent alternative for lipids in biomimetic membrane matrices. They are chemically and mechanically stable, while providing controllable amphiphilic bilayer structures as robust membrane matrices for water channel incorporation.22 There have been several studies of protein water channel incorporation into BCP membranes,22-25 but there have been no demonstration of controlling water permeability through macro-scale BCP membranes by water channel incorporation, until our recent work discussed later in this review. We demonstrated that E. coli AqpZ can be successfully inserted into poly(2-methyloxazoline)-blockpoly(dimethylsiloxane)-block-poly(2-methyloxazoline) (PMOXA15-PDMS110-PMOXA15) BCP membranes (ABA110) with more than an order of magnitude improvement in water permeability and effective water selectivity for water desalination (Table 1).26 This ABA type tri-BCPs can selfassemble into vesicular membranes (polymersomes) with channels incorporated in the PDMS hydrophobic middle domains similar to that seen in lipid bilayers. Successful formation of polymersomes and their physical dimensions were characterized by light scattering spectrometry, cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM) (Figure 4A). The hydrodynamic radius of vesicles was estimated at ~160 nm, which was consistent with previously reported PMOXA-PDMS based polymersome formation with different block ratios,27 and this radius was used for calculating water permeability of channel incorporated polymeric membranes.


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Water permeability of AqpZ reconstituted polymeric membranes was measured using stopped-flow light scattering experiments. Significantly improved water permeability of AqpZ reconstituted BCP (AqpZ-ABA110) membranes was demonstrated, indicating successful AqpZ incorporation into BCP membrane matrices and effective water permeation through the channels. In particular, AqpZ-ABA110 membranes with initial molar ratio of AqpZ to BCP of 1:200 showed 74 µm/s of net water permeability which is 90 times higher than native ABA110 membranes without AqpZ channels (0.8 µm/s) (Figure 4B). Static light scattering experiments were used to determine approximate vesicle molecular weights. AqpZ-ABA110 vesicles were estimated to have, at a maximum, 25 AqpZ monomers and the calculated AqpZ tetramer permeability was approximated to be ~13 × 10-14 cm/s, which corresponds to AqpZ permeability in lipid bilayer systems, confirming the functionality of AqpZs inside BCP membranes.28 Also, activation energy for water permeation through the membranes decreased from 8.7 kcal/mol to 3.4 kcal/mol, after AqpZ incorporation. This low activation energy (< 5 kcal/mol) is a proof of singlefile water transport across the membranes which is a characteristic water transport in AQPs.15 Insights into water selectivity of AqpZ-ABA110 membranes can be obtained from measuring solute reflection coefficients.29 Using glucose (180 Da) as reference solutes, the reflection coefficients of NaCl (58.4 Da), urea (60 Da) and glycerol (92 Da) are calculated to be ~1, implying that these solutes are effectively rejected by AqpZ-ABA110 membranes.26 High NaCl solute reflection coefficients demonstrated the potential of AqpZ rich polymeric membranes for desalination applications. Consequently, this was the first demonstration that, by water channel incorporation into BCP membranes, great control over the BCP membrane’s water permeability can be achieved, which could be leveraged towards developing energy efficient selective desalination processes.

Understanding compatibility of channels with biomimetic membrane matrices Achieving high channel packing density is another challenge when we consider practical channelbased membrane development. An intuitive method to realize this is to develop strategies to enhance channel insertion efficiency into BCP membranes. Recently we showed that single channel water permeability is consistent regardless of how many channels are inserted into membranes for AqpZs.30 Hence, it is important to understand underlying principles governing channel compatibility with membrane matrices and use this information to increase membrane channel insertion efficiency. Since hydrophobic self-assembly is the main driving force for channel insertion, we focused on hydrophobic compatibility to seek out relationships between channel and membrane material hydrophobicity and insertion. Hydrophobic compatibility was assessed from both physical and chemical perspectives. First, AQPs from Rhodobacter sphaeroides (RsAqpZ) and two ABA type tri-BCP membranes (ABA22 and ABA60) were chosen to study how hydrophobic physical mismatch affects channel insertion behavior (Table 1). RsAqpZs’ hydrophobic columnar height is estimated to be ~3 nm from its crystal structure,31 and hydrophobic domain thickness of two BCP membranes were reported as 4.4 and 9.0 nm for ABA22 and ABA60 membranes, respectively.32 To evaluate physical hydrophobic mismatch effect, RsAqpZs were reconstituted into ABA22 and ABA60 BCP membranes, and their channel insertion efficiencies were compared to the reference value, which is obtained from RsAqpZ insertion into naturally occurring PC/PS lipid membranes. As shown in Figure 5B, a clear trend is seen between channel insertion behavior and hydrophobic thickness mismatches; smaller mismatches in hydrophobic lengths result in more favorable channel incorporation into the membranes. This is in accordance with previous assertions that there would be higher energy-penalty for channel incorporation in hydrophobic length mismatched matrices.33 Next, we studied the effect of chemical compatibility as another potential factor affecting channel insertion behavior into membranes. Based on an analogy with the Flory-Huggins theory for polymersolvent and polymer-polymer interactions,34-35 it is postulated that smaller chemical hydrophobicity differences between membranes and channels would yield lower Flory-Huggins interaction parameters


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(𝑋𝐴𝐵 in equation (1)) and thus lower Gibbs free energies of mixing (∆𝐺𝑚𝑖𝑥 in equation (2)), leading to energetically favorable channel insertion into such membranes. 𝑉𝑟

𝑋𝐴𝐵 = 𝑅𝑇(𝛿𝐴 ― 𝛿𝐵)2 (1) where 𝑋𝐴𝐵 is Huggins interaction parameter, 𝑉𝑟 is reference volume, 𝑅 is ideal gas constant, 𝑇 is temperature (K) and 𝛿 is solubility parameter. ∆𝐺𝑚𝑖𝑥 𝑉𝑅𝑇

∅𝐴

∅𝐵

= 𝑉𝐴ln ∅𝐴 + 𝑉𝐵ln ∅𝐵 +

𝑋𝐴𝐵∅𝐴∅𝐵 𝑉 𝐴𝑉 𝐵

(2)

where ∆𝐺𝑚𝑖𝑥 is Gibbs free energy of mixing, 𝑉 is total volume, ∅ is volume fraction, 𝑉𝑖 is molar volume of i. An approach to quantify this chemical hydrophobic mismatch was developed to verify this conjecture by using the membrane-intercalating solvatochromic fluorophore DSSN+ (4,4’-bis(4’-(N,N-bis(6”(N,N,N-trimethylammonium)hexyl)amino)-styryl)stilbene tetraiodide) (Figure 5A).36 DSSN+ has been widely used to measure organic solvent polarity. The dielectric property change of a medium rearranges the excited electron status of DSSN+ fluorophore, releasing less energy (red shifted emission wavelength peak) upon relaxation of electron levels from excited to ground states in more polar solvents. Therefore, inspired by the membrane intercalation property of DSSN+, we exploited this fluorophore to measure the polarity change of the hydrophobic domain of membranes before and after channel incorporation (Figure 5C and D). It was hypothesized that, if chemical mismatches between channels and membranes are greater, the emission shift after channel incorporation would be larger due to significant polarity change. To test this hypothesis, artificial water channel peptideappended pillar[5]arenes (PAPs, which will be introduced in a later section) were chosen as a simplified water channel model and inserted into different membranes with same molar ratios; PC/PS membranes, ABA membranes and poly(butadiene)-poly(ethyleneoxide) (PB-PEO) di-BCP membranes. As expected, DSSN+ emission wavelength peaks were shifted for all cases after channel incorporation, possibly due to polarity change induced by chemical mismatches (Figure 5D). The relative wavelength shift (%), which is defined in equation (3), was compared to PAP channel insertion efficiency to derive relationship between hydrophobic mismatches and channel insertion (Figure 5D). 𝑟𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑠ℎ𝑖𝑓𝑡 (%) =

|∆𝜆𝑒𝑚, 𝑐ℎ𝑎𝑛𝑛𝑒𝑙 𝑟𝑒𝑐𝑜𝑛𝑠𝑡𝑖𝑡𝑢𝑡𝑒𝑑 𝑚𝑒𝑚𝑏𝑟𝑎𝑛𝑒 ― ∆𝜆𝑒𝑚,𝑛𝑎𝑡𝑖𝑣𝑒 𝑚𝑒𝑚𝑏𝑟𝑎𝑛𝑒| ∆𝜆𝑒𝑚, 𝑛𝑎𝑡𝑖𝑣𝑒 𝑚𝑒𝑚𝑏𝑟𝑎𝑛𝑒

(3)

where ∆𝜆𝑒𝑚 is DSSN+ emission wavelength shift of membranes compared to control DSSN+ emission wavelength which is obtained in membrane free HEPES (4-2-hydroxyethyl)-1piperazineethanesulfonic acid) buffer solutions (Figure 5C and D). The results showed that lower relative shift values correlate with higher channel insertion (Figure 5E), confirming our hypothesis that similar polarities between channels and membranes are desirable for higher channel insertion efficiency and, therefore, hydrophobic chemical compatibility should also be considered to maximize channel density in biomimetic membranes.

Maximizing pore density of channel-incorporated BCP membranes Currently, most channel-incorporated macro-scale membranes have adopted simple mixing, grafting or embedding of channel-reconstituted vesicles into selective layer of thin film composite membranes to improve water transport across membranes.8, 37-42 However, these approaches still have limited channel-loading capacities in terms of both: (1) channel density in vesicular membrane, and (2) the volume fraction of reconstituted vesicles inside the selective layer of the scalable membranes. As a first step towards overcoming these challenges, a strategy of achieving high channel packing density inside either vesicular or planar BCP membranes will be described in this section. The second step of the proposed solution (resulting in macro-scale membranes) will be discussed in the membrane fabrication section of this article.


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High-density channel reconstitution into BCP membranes with membrane morphology transition One of the most widely used methods for making channel-reconstituted vesicular membranes is film rehydration.43 However, the membranes made by this method result in relatively low channel loading capacity due to difficulties in controlling channel reconstitution kinetics over more favorable selfassembly of BCPs with themselves. The maximum channel loading amount is reported to be limited by the molar ratio of polymer to protein (mPoPR) of ~100 when film rehydration technique is used (note that lower mPoPR numbers indicate higher channel amounts incorporated into polymersomes).26 Therefore, an alternative technique is required to maximize channel packing densities in BCP membranes. We adopted a dialysis-based reconstitution method based on reported work on twodimensional crystallization of membrane proteins in lipids.43 This method uses high concentrations of membrane-protein compatible detergents to solubilize water channels and BCP molecules resulting in formation of ternary micelles containing proteins, polymers, and detergents. By targeting a low mPoPR and thus high protein concentration relative to polymer in preparation of these ternary micelles and then slowly removing the detergent through a dialysis process, channels and BCPs can be assembled together in a manner that results in flat films, large micron-scale collapsed vesicles, and in some cases two–dimensional crystals of membrane proteins in BCPs. Compared to traditional film rehydration methods, this slow dialysis method not only provides a dramatic increase in channel reconstitution density but also induces a morphology change of channel-BCP aggregates from vesicular to planar membrane structures. To study various scenarios and suggest a general self-assembly trend of protein-BCP aggregate formation, two commonly used PB-PEO di-BCPs and PMOXA-PDMS-PMOXA tri-BCPs were selected with various block lengths for this study conducted in 2012 (Table 1). Also, lens-specific aquaporin-0 (AQP0) was chosen as a model channel protein since it is well known to form array structures upon reconstitution into lipids, simplifying comparisons between reconstitution properties of AQP0-BCP systems and AQP0-lipid systems. Successful AQP0 incorporation into BCP matrices is dependent on several key factors including complete initial dissolution of BCPs at the detergent concentrations used and detergent removal rate by dialysis. To prevent any structural changes of protein channels occurring from ionic amphiphiles, nonionic detergents such as octyl-β,D-glucoside (OG) are used for dialysis process. TEM examination revealed that relatively high OG concentrations (> 4%) are required for complete dissolution of all tested BCPs in aqueous solution compared to lipids, which require ~2% OG. More importantly, the optimal dialysis rate for OG removal was established to be ~5 (mg/ml)/day, particularly during OG concentration transitioned through its critical micelle concentration (cmc). Upon successful dialysis, all tested BCPs shows a similar trend of AQP0-BCP aggregate morphology transition with increasing amounts of AQP0 (Figure 6). In the absence of channel proteins, the BCPs used formed predicted polymeric structures such as network structures, vesicles with attached tubes or small vesicles, depending on intrinsic polymer properties as previously reported.43 As the concentration of channels increased, AQP0-BCP aggregates started to form mono-dispersed vesicles, and finally planar membranes with high channel packing densities. AQP0-PB12 and AQP0-ABA42 aggregates were found to be organized into 2D crystals at low mPoPRs. The most dramatic morphology transition was seen with the AQP0-PB12 system (Figure 6A). Without any channels, PB12 BCPs assembled into network structures which correspond to a previous study of PB-PEO BCP phase separation, reported by Jain and Bates.44 As AQP0s were gradually added, at mPoPR ranges between ~250 and ~50, the aggregate morphology transformed from the native network structures to vesicle structures. At a mPoPR of ~15.5, vesicular membranes with diameters of 200 – 300 nm were formed, and these vesicular membrane structures started to evolve into 2D planar membranes around a mPoPR of ~3.9. At a mPoPR of ~1.3, AQPs and PB12 BCPs were crystallized into 2D arrays with tetragonal crystal unit cells (Figure 7A).


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AQP0-ABA42 system shows similar morphology transition within a narrower mPoPR range than the AQP0-PB12 system (Figure 6B). The native self assembled structure of ABA42 BCPs is small vesicular membranes as shown in a previous AqpZ functional study.26 With the incorporation of AQP0s, the size of the vesicles became larger (mPoPR of ~43.2) and the morphology started to transform into planar membrane structures around a mPoPR of ~8.6. At lower mPoPR than ~2.2, the aggregates formed planar membrane patches and, eventually, 2D crystals with the same unit cell dimensions as the AQP0-PB12 system (Figure 7B) and previously reported AQP0-lipid systems. We tested WT OmpF protein channels for 2D crystal formation with PB12 BCPs to test adaptability of the slow dialysis method to additional protein channel systems. At a weight molar ratio of polymer to protein (wPoPR) of ~0.2, OmpF-PB12 aggregates formed 2D crystals with characteristic hexagonal crystal structures with unit cell constant of a = b = 19 nm and γ = 120° (Figure 7C). The Fast Fourier transforms (FFT) of TEM images of 2D crystals, prepared from PB12 and ABA42 systems show clear tetragonal electron diffraction patterns with unit cell lattice constants of a = b = 6.5 nm, identical to that in AQP0 crystals prepared with lipids (Figure 7A and B). This structural identity between synthetic BCPs and natural lipid systems reinforces our previous conclusion that BCPs can be reasonably adopted as excellent replacements for lipids in robust biomimetic membrane platforms while providing similar degrees of channel incorporation in highly packed crystalline structures. Phase segregation behavior of amphiphilic molecules, especially BCPs, have been extensively studied in polymer physics,45 and established frameworks are widely used for analyses. Their phase segregation behavior has been correlated with physical and chemical BCP properties such as volumetric ratios of hydrophilic to hydrophobic block lengths, different chain geometries, and symmetries. In a similar manner, biomimetic membranes would have great benefits from seeking out morphology transition trends for channel-amphiphile systems. To achieve this objective, dioleoylphosphatidylethnolamine (DOPE) lipids were chosen as a model system of natural lipids and the morphology transitions of AQP0-DOPE aggregates were compared to the PB12 and ABA42 BCP cases. DOPE lipids assemble into lipid vesicles (liposomes) in their native state, with addition of AQP0s, the aggregates form planar membranes and 2D crystal structures at the lipids to protein molar ratio (mLPR) of ~13 and ~12. These mLPR ratios at which structural transition occurs are within the reported mLPR range for various AQP-lipid crystals (about from ~50 to ~8),46-51 but they significantly deviate from the transition ratios of BCP systems. Given that AQP0 concentrations seem to determine the final aggregate morphologies, converting molar ratios (mLPR or mPoPR) to hydrophobic volume fractions of channels provided us a platform to compare phase transition behavior of natural lipids and synthetic BCP systems (Figure 6C and D). From the atomistic structure of AQP0, with a molecular volume of 18.91 nm3,43 hydrophobic volume fractions (fAQP0) were calculated in each aggregate and then the morphology transition phases were plotted along with hydrophobic volume fractions (Figure 6C). This plot indicates a clear trend between membrane morphology and hydrophobic volume fraction. Most importantly, it shows that planar membranes and 2D crystals form at hydrophobic volume fractions between ~0.6 and ~1, with significantly high channel reconstitution densities inside the membranes. From an engineering perspective, 2D planar structures are much more favorable than vesicular membranes since they can be implemented into sheet-like membrane structures with an order of magnitude higher pore packing densities than vesicles. Therefore, extending this understanding of channel-polymer self-assembly to other biomimetic membrane systems would be of great importance for development of channel-based membranes.

Biomimetic artificial water channels Although biological water channels are proving to be highly advantageous for biomimetic membranes,


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there are intrinsic drawbacks of these biomaterials, such as protein stability and limited usable crosssectional pore area, that need to be addressed to fully realize their potentail.8 Proteins are comprised of tertiary and quaternary structures, which require exquisite protein folding processes within cells, and these structures have been optimized in living systems through evolution. This results in challenges with predicting protein stability as well as function during industrial membrane applications where conditions may be harsh compared to cellular physiological environments. These conditions could include high salinity (≤ ~35,000 ppm), high hydraulic pressure (~80 bars) and presence of cleaning chemicals such as sodium hydroxide and acids, which are commonly used to clean reverse-osmosis membranes.5, 9 Also, from the engineering perspective, protein channels usually have very limited usable cross-sectional area of pores.52 For example, AQPs’ functional pores have diameters of ~3 Å but the surrounding protein scaffold diameters could be ~3 nm in diameter, indicating that even when AQPs are tightly packed into 2D crystals with a tetrameric unit cell size of ~95 Å × 95 Å, the net porosity of the membrane would be only ~0.3 %.52 To overcome these challenges, we have explored artificial water channels, which are chemically synthesized nanoarchitectures that mimic several structural and functional features of biological water channels. Two types of water channels, unimolecular peptide-appended pillar[5]arene channels and supramolecular imidazole-quartet channels, have been characterized in our lab in terms of water permeability and selectivity. Each of these channels possesses distinctive characteristics in terms of function, geometry, and processability for membrane applications as described in the following subsections. Supramolecular Imidazole-quartet (I-quartet) channels I-quartet channels were first proposed and synthesized by Barboiu and coworkers.53-54 The structure of these channels was inspired by the histidine quartet selectivity filter of influenza A M2 proton channels (Figure 8A). This channel is comprised of ureido imidazole units which self-assemble into columnar structures spanning lipid bilayers (Figure 8B and C). Under appropriate assembly conditions, one I-quartet motif is stabilized with two dipolar water molecules and this stabilized structure is stacked with each other in the perpendicular direction, forming columnar assemblies with binary dipolar water wires inside the columnar structure (Figure 8C). Due to the presence of continuous dipolar water wires spanning the entire membrane, I-quartets were reported to be the first artificial water channel system which can reject monovalent ions such as Na+ and Cl-, while selectively allowing water molecules to permeate through.53 Also, since these compounds can be very easily prepared by one-step azole-based chemistry, they have potential to be applied for large-scale membrane production.8, 55 One limiting factor for I-quartet channels is that their low single channel water permeability (~106 H2O/s/channel) compared to biological water channels. In our work we tested the hypothesis that, since supramolecular configuration can be influenced by molecular state of the channel elements in the membrane, assembly state optimization of I-quartet motif would result in enhanced water permeability. A series of I-quartet derivatives were synthesized and their water transport properties were investigated as a part of this work.55 Supramolecular columnar assembly Successful assembly of I-quartet motifs into columnar configuration is critical for efficient water transport through this channel.53 A series of ureido imidazole unit compounds were synthesized by extending different types of alkyl chains on the ureido imidazole templates (Figure 8B) to test the effect of changes in structure of each subunit on overall self assembly and function. Each compound was designed to have different alkyl chain lengths (HC4, HC6 and HC8) and chirality (S-HC8 and R-HC8) so that they can be aligned in different configurations within the hydrocarbon domain of lipid bilayers. Each compound was first analyzed in bulk crystal phases to evaluate assembled structures. X-ray crystallography of these compounds after recrystallizing from water confirmed that, except for HC4, the other four channels assembled into properly stacked columnar structures with stabilized continuous water wires (Figure 8E - H). All the H atoms of water molecules participated in polar interactions with adjacent imidazole quartets,


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stabilizing the entire assembly. Interestingly, dipolar water wires showed different configurations depending on chirality of unit channel compounds. Water wires have centrosymmetric structures for the achiral channels (HC6 and HC8, Figure 8E and F) whereas chiral channel compounds (S-HC8 and R-HC8) have non-centrosymmetric water wires (Figure 8G and H). I-quartet structure-permeability relationships in biomimetic membranes Each I-quartet compound was reconstituted into lipid vesicles and subjected to stopped-flow experiments to measure water permeability. As shown in Figure 8D, apparent water permeability was shown to depend on grafted alkyl chain geometries. Permeability exponentially increased as chain length increased and noncentrosymmetric dipolar water wires (R-HC8 and S-HC8) resulted in higher permeability than centrosymmetric water wires (HC8). Based on channel insertion efficiency, actual lipid concentrations, and channel configurations, single channel permeability was estimated as 1.4(±0.4) × 106, 7.9(±2.1) × 105, 1.5(±0.1) × 106 H2O/s/channel for HC8, R-HC8, and S-HC8, respectively. Even though these permeability values are two orders of magnitude lower than AQPs (~108 – 109 H2O/s/channel), all tested I-quartets shows effective rejection of monovalent ions (except for protons), suggesting I-quartet channels could be promising materials for scalable desalination membranes. These results suggest two correlations between channel structure to water transport functionality. First, greater hydrophobic volume fraction of channel compounds results in higher water permeability. This might be attributed to the fact that, through synergistic interaction between hydrocarbon tails of lipid membranes, hydrophobic channel structure can be stabilized into columnar assembly of channels as well as water wires, and therefore can mediate water permeation across the membranes more efficiently. Secondly, chiral R-HC8 and S-HC8 channels perform better than other non-chiral channels and, specifically, S-HC8 channels show higher permeability than R-HC8 channels. This is probably due to very specific compatibility of S-chiral I-quartet structures with naturally occurring L-chiral lipids. These structure-activity relationships, which originate from compatibility between channels and membrane matrices once again highlights the importance of the viewpoint that we need to consider biomimetic membranes more systemically rather than just focus on channel performance. Unimolecular peptide-appended pillar[5]arene (PAP) channels PAP channels were first synthesized by Hou and coworkers in an attempt to develop transmembrane architectures that have a uniform sub-nm inner pore structure.56-57 The PAP channel geometry resembles characteristic features of biological water channels such as a uniform and rigid pore size of angstrom size and a cylindrical shape with hydrophobic outer surfaces. These properties are critical to size-exclusion based water selectivity over larger solutes and favorable channel insertion of biological water channels into membranes, respectively. This architecture leads to its recognition as an unimolecular synthetic water channel after extensive characterization of water transport through these channels in lipid bilayer membranes.58 PAP channels are constructed by extending hydrophobic tripeptide phenylalanine chains (D-Phe-LPhe-D-Phe) from the molecular template of pillar[5]arene macrocycles (Figure 9A). Pillar[5]arenes have a rigid and uniform internal cavity of ~5 Å diameter, which can function as a selective filter for water molecules (~3 Å). Tripeptides have outwardly facing hydrophobic benzoyl ligands that can interact with hydrophobic membrane matrices so that channels can be favorably inserted into lipid bilayers due to hydrophobic compatibility between channel outer surfaces and the bilayer core (Figure 9B). Also, the ends of tripeptide chains are terminated with carboxylic acid groups (-COOH) to confer hydrophilicity at the channel entrance rims to enhance hydrophilic compatibility between channel ends and bilayer membrane interfaces and to reduce the energy barrier for entry of water molecule into the channels. PAP channel water permeability in biomimetic membranes To confirm favorable PAP channel insertion into membranes and quantify water permeation rates, PAP-reconstituted PC/PS vesicles were prepared and net water permeability of vesicular membranes was measured using stopped-flow light


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scattering experiments. The vesicles were exposed to outwardly and inwardly directed osmotic gradients, and the water permeability was measured in both the “shrinking” and “swelling” modes. These measurements were made by obtaining the kinetic constants from double exponential fitting of the rise rates in accordance with analysis methods reported in recent literature.28, 59 Interestingly, the net water permeability of PAP reconstituted vesicles demonstrated different permeability values in each mode. In the shrinking mode, where outwardly directed osmotic pressure was imposed, no apparent change in net water permeability is observed when the larger exponential constant k1 was considered, indicating that the PAP channels’ contribution to net permeability is not significant compared to that of intrinsic lipid membranes. Therefore, smaller exponential constant k2 (which had a measurable value) was used for calculating channel-induced water permeation through the membranes. This procedure had been previously adopted for characterizing water channels which have relatively low permeabilities.60 PAP channel-induced water permeability increased proportionally to the molar ratio of PAP channels to lipids (mCLR, note that higher mCLR indicates a higher number of channels per lipid vesicle), implying successful functional channel insertion into vesicular membranes (Figure 10A). In the swelling mode, an increase in net water permeability is observed with an increase in the larger exponential constant k1 with increasing mCLRs, showing that PAP channels mediate water transport more efficiently in swelling mode compared to shrinking mode (Figure 10B). To accurately evaluate the water transport property of PAP channels, the single channel permeability was numerically calculated based on average PAP channel number per vesicles estimated using time correlated single photon counting (TCSPC) implemented in FCS mode.61 PAP channels demonstrated a water permeability of 3.7(±1.2) × 106 and 3.5(±1.0) × 108 H2O/s/channel in shrinking and swelling modes, respectively. Introducing artificial water channels into a BCP membrane system would provide additional advantages, particularly in terms of stability, because all biological membrane components would then be replaced with bioinspired synthetic materials. Therefore, in another study we reconstituted PAP channels into PB-PEO BCP membranes (PB23, Table 1) and tested water channel functionality (Figure 10C).62 Compared to the PC/PS membrane system, PAP channel insertion efficiency decreased from ~35% to ~10%, in accordance with results from our previous study that PB23 matrices are less compatible for PAP insertion than PC/PS membranes due to hydrophobic chemical mismatch.30 In this system, single PAP channel permeability was measured at 1.6(±0.4) × 108 H2O/s/channel in swelling mode, which is similar to PAP permeability measured in the PC/PS membrane system (Figure 10D). MD simulations Computational MD simulations provided us a more detailed understanding of water transport phenomena through PAP channels. A simulation system was built with a 1-palmitoyl-2oleoyl-sn-glycero-3-phosphatidylcholine (POPC) model lipid bilayer membrane. Initially, 25 channels were embedded in a uniformly spaced pattern within the membrane. MD simulations revealed that PAP channels were repeatedly filled with water molecules and emptied, showing wetting-dewetting transitions, which take place within 3 ns (Figure 10E). Regardless of whether simulation started with water-filled or empty channels, after the system reaches equilibrium (after ~30 ns), the fraction of water-filled channels stabilized at ~40 % on average, showing non-continuous single file water permeation through the channels (Figure 10F). MD simulation snapshots show the transition states inside the channels, previously seen in other biological and artificial channels (Figure 10E).63-65 The collective diffusion model was used to calculate single PAP channel permeability from MD simulations and compared to experimentally obtained permeability values. A permeability of 1.5 – 1.9 × 10-14 cm3/s/channels was obtained, which is in good agreement with experimental values from swelling mode (Figure 10D). Thus, both experimental and simulation results indicate single PAP channel permeability (3.5(±1.0) × 108 H2O/s/channel) is within the range of reported AQP permeabilities (3.4 – 40.3 × 108 H2O/s/channel). PAP channels were the first artificial water channel comprehensively demonstrated to have comparable water transport rate to AQPs.


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Molecular selectivity Patch-clamp experiments were performed to measure ion selectivity of PAP channels by measuring ionic current across PAP reconstituted lipid bilayers. Apparent ionic permeation through membranes was monitored using current to voltage (I-V) curves. The results indicated that ions were not effectively rejected by inserted PAP channels. Based on the I-V curves of different ionic pairs, ionic selectivity was calculated using the Goldman-Hodgkin-Katz equation66 and the resulting selectivity order was NH4+ > Cs+ > Rb+ > K+ > Na+ > Li+ > Cl-. This order matches with expected phenomena, because PAP entrance is functionalized with negatively charged carboxylic acid groups preferentially allowing cations to pass through depending on the degree of energy compensation for dehydration of the cations.66 Additionally, reflection coefficients of various solutes were measured from stopped-flow experiments to estimate solute rejection efficiency of PAP channels. The molecular weight cut-off (MWCO) was calculated to be ~420 Da for solutes with a reflection coefficient of 0.9, which is consistent with PAP pore size (~5Å). At this pore size there was no water selectivity over ions compared to biological water channels indicating that pore sizes smaller than 5Å are needed for rejection of monovalent ions. High-density PAP reconstitution into membranes As discussed in the earlier section, achieving high channel packing density is of high interest for practical membrane applications. Therefore, the slow dialysis method for high-density protein channel assembly was tested to see if artificial water channels can be reconstituted into membranes with high packing density in both lipid and BCP membrane systems. A PAP-lipid morphology transition was similar to that seen with membrane proteins and lipids and in our previous work with membrane proteins and BCPs. The native self assembled structure of PC lipids is vesicles with diameter around 100 – 200 nm. The size of vesicles increased upon addition of PAPs to system reaching a vesicle diameter of ~ 1 µm at a mCLR of ~0.5. A morphology transition from vesicles to flat sheet membranes took place at mCLR of ~0.714. At a higher mCLR of 0.909, PAPs and lipids aggregated into tightly packed 2D structures with hexagonal unit cell constants of a = b = 2.1 nm and γ = 120° (Figure 11A - C). This transition ratio was higher than transition ratios of other protein-lipid aggregate cases (0.02 – 0.125), due to smaller cross-sectional area of PAP (~300 Å) than protein channels. Such a transition is also seen in the PAP-PB12 system. PB12 BCPs were chosen as a model system due to their hydrophobic chain length compatibility with PAP channels (Table 1). The most abrupt transition from vesicles to planar structures occurred at similar transition ratios as (molar ratio of channels to polymers, mCPR) PAP-lipid system (Figure 11D). However, at high mCPRs, PAP-PB12 aggregates show unique microphase separated structures instead of assembling into highly ordered 2D crystals (Figure 11E and F). In the large flat sheet structure of PAP-PB12 aggregates, PAPs were segregated from PB12 matrices, forming raft like PAP domains. The presence of these rafts was confirmed by energy filtered TEM and energy dispersive spectroscopy (EDS) elemental mapping. This work demonstrated that slow dialysis could be used to create highly packed fully synthetic bioinspired membrane patches consisting of BCPs and artificial water channels.

Defect free integration of biomimetic membrane components into scalable membranes Achieving better permeability and selectivity has been an eagerly sought goal in all membrane fields. One of the most commonly exploited strategies to realize this is to make membranes which have thin, porous, and uniform pore size distribution in selective layers. Accordingly, many 2D porous materials such as graphene oxides (GO), zeolites or metal-organic framework nanosheets have been proposed as promising candidates for effective membrane development. These materials were shown to demonstrate desirable properties at the sub-micron or few micron scales.67-70 Nonetheless, one major challenge to implementation of these materials into membranes is scaling them up to practical membrane sizes without detrimental defects which occur most commonly at the boundaries of the 2D materials. Similar issues were encountered when water channel based 2D aggregates were used to make scalable membranes. To address this challenge, we recently introduced a modified layer-by-layer


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technique to integrate PAP-BCP 2D aggregates into scalable membranes. We also propose a newly conceptualized BCP based lamellar structure as a next generation biomimetic membrane platform. Layer-by-layer construction of highly porous PAP-BCP planar aggregates As described before, a number of channel-BCP systems have demonstrated the capability of assembling into planar membranes with high channel density. These planar materials are more suitable for practical membrane development compared to alternative channel enhanced materials such as spherical vesicles. To explore membrane fabrication using these 2D materials, PAP-PB12 was chosen as model system since it is entirely comprised of synthetic materials and provides greater flexibility in terms of chemical modifications as well as chemical and biological stability when compared to analogous biological or biohybrid systems. To successfully build a PAP-PB12 based selective layer, a modified layer-by-layer technique was introduced (Figure 12A).62, 71 Briefly, PEO blocks of PB12 BCPs were terminated with carboxylic acid groups (PB12COOH, Table 1) so that PAP-PB12 nanosheet surfaces were fully negatively charged and these reactive groups could be used for chemical crosslinking. PAP-PB12COOH aggregates consisting of planar sheets and large collapsed vesicles were prepared with an mCLR of 0.5. A polyether sulfone (PES) support membrane was first functionalized by oxidation under UV/Ozone irradiation to make the surface negatively charged. Then a cationic polymer, polyethyleneimine (PEI), was deposited on the support membrane surfaces via electrostatic interactions though simple application to reverse the surface charge of the assembly to a positive charge. In the next step, PAPPB12COOH nanosheets were deposited on top of the positively charged PEI layer under pressure. Since one layer of PAP-PB12COOH deposition was not sufficient to cover the entire membrane surface, repeating PEI/PAP-PB12COOH layers were deposited until no defects remained, especially around boundaries of nanosheets. It turned out that after 4 layers of deposition, ~100% coverage was achieved as seen from SEM images (Figure 12B). Finally, each PEI and PAP-PB12COOH layer was crosslinked with each other using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide·HCl chemical crosslinker, in order to confer enough mechanical strength to channel-based selective layers. PAP membranes were tested using dead-end stirred filtration cells to characterize them in terms of water permeability and selectivity. Water permeability gradually decreased with increase in number of deposition layers and finally reached up to 64.8 ± 11.3 LMH/bar (liter·m-2·hour-1·bar-1) for 4 layer membranes (Figure 12C). The MWCO of this membrane assembly was determined to be ~450 Da (Figure 12C), which is in great agreement with the measured MWCO (~420 Da) of the designed water channels as mentioned in the earlier section.58 Also, the PAP membrane permeability (~65 LMH/bar) corresponds to theoretical value (~22 LMH/bar) within a ~3 fold discrepancy range. This theoretical permeability is calculated based on single PAP channel permeability and channel density per unit area (~4.2 × 105 µm-2) of PAP-PB12 aggregates.62 Given that these two values come from very different experimental conditions and scales (single channel permeability at molecular level of nm scale and membrane permeability at cm2 scale), we consider this as good agreement. Overall this membrane is a successful demonstration of highly functional bioinspired membrane that maintains its molecular level properties at the macro-scale. PAP membranes show significantly higher water permeability compared to all other commercial nanofiltration (NF ) membranes which have MWCO in the range of 200-1000 Da. (Figure 12E).62 For a more accurate comparison, two commercial NF membranes with similarly rated MWCO values (400 – 500 Da), N30F and NDX, were tested under exactly same conditions along with PAP membranes. As shown in Figure 12C, PAP membranes exhibit an order of magnitude higher water permeability than the two test NF membranes. To evaluate pore size distributions, molecular rejection profiles were fitted to sigmoidal function of which the derivative equation can be expressed as a probability function of pore size distribution. It turned out that PAP membranes have narrower pore size distribution than others (Figure 12D inset), indicating that PAP membranes have more idealized membrane features than commercial membranes.


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One question regarding PAP membranes is whether the molecular rejection observed in these membranes arise from designed channel structures (size exclusion) or from electrostatically multi layered membrane structures (electrostatic exclusion) formed to stabilize channel nanosheets. The tested solutes (small organic dyes) have innate charges, which can possibly interact with membranes resulting in electrostatic repulsive forces and dominate molecular rejection behavior over channelbased size exclusion through Donnan exclusion.71 To test if this hypothesis is valid in this scenario, PAP membranes were tested with different ionic strength feed solutions so that electrostatic interaction could be screened out, as indicated by the calculated Debye length (λD). Previous studies have indicated that, if λD is smaller than pore diameter of porous membranes even at sub-nm scale, the Donnan exclusion effect becomes negligible.72 Rose Bengal (RB, MW 1017) and Acid Fuchsin (AF, MW 585) dyes were selected and tested under various ionic strengths resulting in various λD environments. As shown in Figure 12E, no significant change in rejection was observed even when λD become smaller than pore size of PAP channels (~5 Å). Also, X-ray diffraction spectroscopy revealed that domain spacing (d-spacing) of layered structure of PAP membranes is around ~1.5 nm, which indicated that the interlayer spacing was much larger than the size of solutes used for testing. These results provide confidence that that electrostatic interaction arising from both channel structure as well as interlayer filtration do not play a large role in exclusion of the molecular solutes tested with PAP membranes. Consequently, the molecular selectivity of PAP membranes can be confidently ascribed to the size exclusive property of PAP channel pore structures. Other supporting information including control experiments and MD simulations to prove channel-based separation of PAP membranes are available in our recent publications.62, 71 Overall, even though some knowledge gaps still remain, originating from multi-layered membrane structures such as water flow between each layer, PAP membranes were successfully demonstrated as high performance bioinspired membranes with molecularly designed membrane structures using synthetic membrane components. Block copolymer lamellar structure as robust and efficient platform for scalable biomimetic membranes Even though we demonstrated highly porous and scalable channel-based membranes, several aspects should be further addressed to improve the membrane fabrication process with regard to scalability. For example, the previously suggested layer-by-layer technique might not be the most optimal from the perspective of membrane processability, because channel reconstituted nanosheets require few days to be prepared and membranes should be built up throughout single “layer-by-layer” steps. Also, the current BCP membrane systems that mimic biological membranes have adopted di-BCPs or ABA type tri-BCPs in which hydrophilic blocks face the solution phase, once they are assembled into membrane structures in the aqueous solution. Although they are considered as favorable materials for channel insertion, when it comes to scalable membrane fabrication where 2D nanosheets need to be laterally stacked with each other to form a continuous selective layer, there could be stability challenges. For example, in the absence of significant crosslinking between the hydrophilic blocks of alternate membrane layers, each 2D membrane layer can be delaminated due to swelling of hydrophilic domains in aqueous solutions (Figure 13A) destabilizing selective layers and resulting in defects. For PAP-PB12COOH membrane systems, inter-polymeric PEI layers were introduced and each layer was crosslinked with the other to resolve this problem, but it makes resulting membrane structures and formation complicated. To overcome this challenge, we suggest lamella forming layered BAB (hydrophobic-hydrophilichydrophobic) type tri-BCPs as more stable and easily processible materials for biomimetic membranes.73 Note that BAB type tri-BCPs have hydrophilic (A) middle blocks with extended hydrophobic (B) blocks at both ends. From this polymeric structure, we can envision that BAB type lamellar structures will have more resistance against delamination in aqueous solutions since hydrophilic domains are rigidly held together by covalently connected hydrophobic domains (Figure 13A). Poly(isoprene)-poly(ethylene oxide)-poly(isoprene) (BAB45) tri-BCPs were synthesized and


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processed to form lamellar 2D nanofilms, to assess BAB type tri-BCPs as a platform for BBMs (Table 1). Poyisoprene (PI) was chosen as model hydrophobic block chains since it can be readily crosslinked via UV-triggered thiol-ene click chemistry, which can confer additional stability to the membranes. Each block of BCPs was designed to have MWs of 3,000 and 6,000 g/mol for PI and PEO blocks respectively, targeting a volume fraction of PEO blocks to be 46 %. This hydrophilic volume fraction is needed for lamellar structure formation.45 The self-assembly behavior of BAB45 BCPs were first analyzed at bulk phase using transmission small-angle X-ray scattering (SAXS) spectroscopy (Figure 13C). The SAXS patterns indicate lamellar (q/q* = 1, 4, 9, 16) domains of assembled BAB45s with d-spacing value of 16.1 nm which corresponds to designed domain lengths (~8 nm for hydrophilic PEO middle block domain and hydrophobic PI bilayer domains, respectively), implying successful lamellar structure formation. An advantage of the BAB type BCP system from engineering perspective is that lamellar nanofilms can be easily prepared through wet film coating process such as spin coating (Figure 13B). First, to confer hydrophilic property on substrate, silicon wafer substrate surface was either treated with UV/Ozone or a poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) sacrificial layer via spin coating. This allowed the BAB45 BCPs to be assembled into parallel-stacked structures over the substrate plane (Figure 13B), based on hydrophilic interaction between substrate and PEO block chains. Then, BAB45 polymer solution in toluene, containing photo initiators and chemical crosslinkers, was spin coated on top of the substrate and annealed under toluene vapor for 45 minutes. After that, films were exposed to UV irradiation to complete thiol-ene polymerization in hydrophobic PI domains. Finally, to prepare free standing nanofilm, the substrate was immersed in pure water to dissolve sacrificial PEDOT: PSS layer and make nanofilms float into water without imposing any mechanical stress, which may damage the films. (Figure 13D). Successful lamellar nanofilm formation was confirmed by AFM analysis. Although different film morphology was obtained in terms of thickness and surface coverage depending on film preparation conditions such as polymer concentration and spin coating speed, every film showed designed lamellar structures with stepwise film thickness of ~16 nm step heights, which corresponds to d-spacing measured at bulk phase analysis (Figure 14A and B). Also, first layer step height was observed to be ~8 nm which is identical to half folded BAB45 polymer length due to hydrophilic interaction between hydrophilic surface of silicon substrate and PEO middle blocks (Figure 14A). One limiting factor is that nanofilms formed were partially noncontinuous, as seen from the substrate surface in AFM images (Figure 14A and B). This could be attributed to high crystallinity of PEO domains, hampering continuous first layer formation over the entire substrate surface due to polymeric rigidity. Hence, further optimization is needed to mitigate this strong PEO interaction in order to develop defect free nanofilms. Meanwhile, scalable and stable free standing lamellar nanofilms were successfully prepared by introducing hydrophilic PEDOT: PSS sacrificial layer between substrate and BCP films, which can be readily transferred to porous support membranes and subjected to actual filtration applications (Figure 13D and, 14 C and D).74-75 In conclusion, it was demonstrated that scalable BCP lamellar films can be successfully prepared by simple two step processes of wet-film coating of BCP containing organic solvent (doping) solution and then drying out, which is expected as favorable for continuous and scalable process development. Both lamellar film formation of BCP membrane matrix and channel insertion into hydrophobic layer is driven by molecular self-assembly based on phase segregation of hydrophilic and hydrophobic properties. Also, artificial water channel containing dope solutions can be easily prepared since they are highly stable and soluble in various organic solvents. Therefore, we can envision that very similar process could provide channel incorporated lamellar film formation, even though several optimization process would be required such as hydrophobic compatibility between channels and matrix as


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discussed in earlier sections. Given the efficient processability as well as desirable structural properties, BAB type BCP-based lamellar structures could provide a rational platform for foreseeable future biomimetic membrane development. Conclusion and Future Perspective Current progress of our work has been summarized in terms of structural and functional optimization of high performance BBMs. As a key membrane component, research into water channels has resulted in several advances during last few years, resulting in biological (mutant OmpF) and artificial (Iquartet and PAP) channels with better performance or structural advantages compared to AQPs, respectively.19, 55, 58 Also, efficient channel insertion into amphiphilic membrane matrices have been achieved through research into compatibility and novel assembly techniques for channels and matrices. In this article, we have featured o potential applications of biomimetic and biological membranes mainly focusing on water treatment, since water channel AQPs are most archetypal and widely studied channel structures. However, hierarchical structural design makes BBMs a promising platform for various membrane based sub-nm separations in various application fields, because specialized separation property can be readily conferred to the membranes by replacing water channels to others. For example, biomimetic ion channels have orders of higher selectivity to specific ions over other ion species, such as sodium, potassium or chloride ions.76, 78-81 It implies that ion channel based membranes can be used to specific ion separations such as development of ion-sensitive electrode,82 disease prognosis and diagnosis kits,83 or separators in batteries.84 Additional applications such as chiral separations57 or antibiotic purifications85 could be included, being attributed to functional channel properties. Beyond the functionality of channel pores, solvent resistant and mono-dispersed pore shapes of bioinspired membranes provide further application opportunities in organic solvent based industrial process developments.86 For example, a number of chemical and pharmaceutical products are being produced using homogeneous catalyst reactions.87-91 Recovery of catalysts is crucial in terms of industrial as well as environmental aspects, since usually they are costly and contain rare heavy metals. One challenge is that conventional separation methods such as thermal distillation is not recommended for homogeneous catalyst recovery due to their temperature sensitivity.92 Also, in many cases, molecular size gaps between catalysts, organic solvents, and chemical products are quite narrow within sub-nm range and conventional NF polymeric membranes’ molecular selectivity is not satisfactory to efficiently separate these molecules due to broad free volume void size distributions. Considering these aspects, enhanced selectivity of bioinspired membranes at sub-nm scale would be able to provide significant process improvements in chemical industries.62 Despite promising potential applications, efficient integration of BBM components into scalable membranes is still a significant challenge. We believe that the proposed BAB type BCPs will pave a way to overcome this challenge, as they are feasible in terms of structural and chemical modifications and can provide designed lamellar structures, which are favorable for channel insertion. We have also demonstrated that they can be easily processed to form scalable nanofilms throughout wet-film coating process. BCP based lamellar structures have favorable aspects for development of biomimetic membranes, even though several aspects need to be optimized. Firstly, currently demonstrated lamellar structures have ~8 nm hydrophobic domains which will not be suitable for channel insertion due to hydrophilic thickness mismatches with channels (~4 nm). Secondly, PEO polymers are known to have high crystallinity and this resulted in partial defects on scalable nanofilms, implying that we need to find out a solution to mitigate this strong PEO interactions or another hydrophilic block chains to resolve this problem. Lastly, current lamellar films are prepared on non-porous silicon substrates via spin-coating. If we can design a continuous wet-film coating process onto porous membrane substrates for BCP lamellar nanofilm formation, it would allow us to take significant strides towards extensive applications of biomimetic and bioinspired membranes.


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Figure 1. Overview of hierarchical design of structures of biomimetic and bioinspired membranes with critical considerations for high performance membrane development at each scale. Central molecular modeling figure is reproduced with permission from ref 62.


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Figure 2. PoreDesigner was developed as a design work flow to computationally redesign sub-nm pore structures in the OmpF scaffold protein. (A) A single-file water wire template was isolated from MD simulation frames of AQP1 and computationally placed into OmpF internal structure. Pore constriction amino acid residues were then redesigned to fill up void spaces around water wires using a protein redesign algorithm to approximately mimic internal structure of AQP1. (B) Three distinctive pore geometries were created: 1) OCD pores resulting from off-center combination of bulky and nonbulky side chain residues, 2) UCD pores resulting from orderly distribution of bulky and non-bulky side chains, and 3) CSD pores which were a combination of alternatively stacking long and short sidechain residues, forming screw twist in the pore structures. Reproduced with permission from ref 19.


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Figure 3. Molecular transport properties of designed mutant OmpF channels were confirmed using simulations and experiments and found to surpass biological water channels and provide a larger design space. (A) Snapshots of MD simulation set up. The OmpF monomer and model lipid bilayer are colored with purple and turquoise, respectively. Water molecules are depicted with red and white spheres. Na+ and Cl- ions are represented with yellow and green spheres, respectively. (B) Experimentally observed single channel permeability and solute rejection property of WT and mutant OmpF membrane proteins. (C) Comparison of calculated (simulation) and observed (experiment) water permeability of various OmpF channels. (D) Pore size fluctuation during MD simulations. Reproduced with permission from ref 19.


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Figure 4. Structural and functional characterization of AqpZ-ABA110 polymeric membrane vesicles. (A) Cryo-TEM image of single and cluster (inset) of polymersomes confirm successful membrane reconstitution of AqpZs into BCP matrix. (B) Stopped-flow scattering traces of control ABA110 and AqpZ-ABA110 (molar ratio of AqpZ to ABA110 is 1:200) membranes. Abrupt scattering intensity increase of AqpZ-ABA110 reflects significantly enhanced water permeability compared to control ABA110 membranes due to functional AqpZ incorporation. Reproduced with permission from ref 26. Copyright 2007 National Academy of Sciences.


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Figure 5. Physical and chemical hydrophobic mismatch can both affect channel insertion behavior into biomimetic membranes. (A) Schematic explanation of principle of membrane hydrophobicity measurements using DSSN+ fluorophore. (B) RsAqpZ insertion efficiency change along with physical (thickness) hydrophobic mismatches (nm). (C) DSSN+ emission wavelength shifts after intercalation into hydrophobic domains of various biomimetic membranes (PC/PS, PB-PEO, ABA) indicating varying polarity of each membrane hydrophobic interior. Emission wavelength at HEPES aqueous buffer is used as reference value. (D) DSSN+ emission peak shift of PB-PEO BCP membranes before and after PAP channel insertion. (E) Channel insertion efficiency was found to be inversely proportional to relative wavelength shift indicating that minimizing hydrophobic chemical mismatch indicated by minimal wavelength shift leads to higher insertion. Reproduced with permission from ref 30. Copyright 2017 John Wiley and Sons.


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Figure 6. Morphology transitions of AQP0-BCP aggregates created using slow dialysis with increasing density of AQP0. A series of schematic drawings show representative morphology structure transitions. (A) AQP0-PB12 aggregates at mPoPRs of ∞ (1), 15.5 (2), 3.9 (3), and 1.3 (4). (B) AQPABA42 aggregates at mPoPRs of ∞ (1), 43.2 (2), 2.2 (3), and 0.6 (4). (C) Morphology transition plot for all tested BCP and lipid aggregates depending on hydrophobic fractional volume of AQP0 (fAQP0); N: native structures, V: vesicles, M: planar membranes, C: 2D crystals. (D) Morphology transition diagram of AQP0-amphiphile aggregates with variables of fAQP0 and MW of the aggregate unit, which is defined as MW of one amphiphile (lipid or BCP) molecule with associated fractional MW of AQP0. Reproduced with permission from ref 43 (https://pubs.acs.org/doi/10.1021/ja304721r). Copyright 2012 American Chemical Society.


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Figure 7. 2D crystals of different protein channel-amphiphile aggregates. (A) AQP0-PB12 crystals with mPoPR of 1.3. (B) AQP0-ABA42 crystals with mPoPR of 0.6. (C) OmpF-PB12 crystals with wPoPR of ~0.2. Insets are electron diffraction pattern from FFT. Scale bars are 100 nm and 5 nm-1 for main images and insets, respectively. Reproduced with permission from ref 93 (Copyright 2016 American Chemical Society) and ref 43 (https://pubs.acs.org/doi/10.1021/ja304721r, Copyright 2012 American Chemical Society).


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Figure 8. Supramolecular I-quartet channels were proposed as the first artificial water channels based on their crystal structure and then shown to have high water transport rates and high selectivity. (A) Molecular structure of influenza A M2 proton channel selectivity filter with histidine (orange) quartet (dashed red box). Water molecules are represented with red spheres. H-bond formation between water molecules and protein channel backbones are depicted by black lines. Reproduced with permission from ref 94. Copyright 2010 National Academy of Sciences. (B) Chemical structure of unit compounds of I-quartet motifs with different alkyl chain derivatives. (C) Schematic illustration of columnar assembly of I-quartet motifs spanning hydrophobic compartment of lipid bilayers. (D) Net water permeability of I-quartet channel reconstituted lipid vesicular membranes. Crystal structure of (E) HC6, (F) HC8, (G) S-HC8, and (H) R-HC8 compounds, containing dipolar water wires. Reproduced with permission from ref 55. Copyright 2016 American Chemical Society.


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Figure 9. Molecular structure of unimolecular peptide-appended pillar[5]arene (PAP) channel. (A) PAP channels are constructed by extending tripeptide phenylalanine chains (D-Phe-L-Phe-D-PheCOOH) upon pillar[5]arene template. (B) Molecular modeling of PAP shows structural similarities with AQP1 such as rigid pore shape at angstrom scale for single file water permeation, hydrophobic outer surface, and compatible channel height (~4 nm) for favorable insertion into biomimetic membranes. Reproduced with permission from ref 58. Copyright 2015 National Academy of Sciences.


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Figure 10. PAP water channel permeability measurements in lipid and BCP membrane systems. Stopped-flow scattering traces of PAP reconstituted vesicular membranes under (A) shrinking and (B) swelling modes of PC/PS vesicles, and (C) swelling mode of PB23 vesicles. (D) Single channel permeability measured in PC/PS and PB23 membranes, respectively demonstrates similar values that are within one order of magnitude of aquaporins. Reproduced with permission from ref 62. (E) MD simulation snapshots show noncontinuous single file water flow through PAP channels, undergoing frequent wetting-dewetting transitions. (F) The average fraction of water filled PAP channels along with simulation time (ns). Average fraction converged to around 40% whether the simulation started with water filled channels or empty channels. Reproduced with permission from ref 58. Copyright 2015 National Academy of Sciences.


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Figure 11. Planar PAP-amphiphile aggregates can be prepared by slow dialysis method with high channel density. (A) TEM image of PAP-lipid aggregates (mCLR 0.714) and (B) molecular modeling of their crystal structure with hexagonal array pattern. Central pores and extended chains of PAPs are colored with blue and translucent violet, respectively. Lipids are represented with gray color. (C) Hexagonal electron diffraction pattern of PAP-lipid 2D crystal (mCLR of 0.909). (D) TEM image of PAP-PB12 aggregates (mCPR 0.67). (E) Schematic drawing of phase separated PAP-PB12 aggregates. PAPs and PB12 BCPs are colored by purple and gray, respectively. (F) TEM image of raft-like microphase segregation between channels (dark black area) and PB12 BCPs (gray area), which is identified by energy dispersive spectroscopy (EDS) elemental mapping (not shown). Reproduced with permission from ref 58 (Copyright 2015 National Academy of Sciences) and 62.


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Figure 12. Highly porous PAP water channel based membranes. (A) Schematic description of layerby-layer technique to make porous channel based membranes using PAP-PB12COOH 2D sheets. (B) SEM images of membrane surfaces before and after selective layer formation on porous PES support membrane by lateral deposition of PAP-PB12COOH aggregates using layer-by-layer technique. Scale bars are 1µm. (C) Water permeability and (D) molecular rejection profile comparison of PAP membranes to similarly rated commercial NF membranes N30F and NDX. Inset for panel (D) shows pore size distribution probability function of PAP, N30F and NDX membranes analyzed by sigmoidal curve fitting. (E) PAP membrane performance comparison to all commercial NF membranes at present time. (F) Solute rejection tests of PAP membranes under increasing ionic strength of feed solution. RB (1017 Da) and AF (585 Da) charged dyes did not show significant changes in terms of rejection, excluding electrostatic repulsive interaction as possible separation mechanism. Reproduced with permission from ref 62.


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Figure 13. BAB type tri-BCPs for lamellar nanofilm formation. Schematic (A) comparison of lipid bilayer, AB type di-BCP, and BAB type tri-BCP membranes (lamellar structure) and (B) illustration of wet-film spin coating for lamellar nanofilm formation. (C) Bulk phase SAXS pattern of assembled BAB45 BCPs at 25 °C, showing designed lamellar structures. (D) Free-standing lamellar nanofilm floating on water separated from silicon substrate owing to water soluble sacrificial PEDOT:PSS layer. Reproduced with permission from ref 73. Copyright 2018 The Royal Society of Chemistry.


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Figure 14. AFM images of BAB45 lamellar nanofilms. (A and B) Nanofilms were prepared directly on the silicon substrate surface. Stepwise film thicknesses are shown with corresponding step height with designed BCP domain thickness. Different coating condition resulted in different substrate surface coverages; (A) 2 mg/mL BCP solution at1000 rpm and (B) 5 mg/mL BCP solution at 2000 rpm. High crystallinity of PEO blocks may induce defects shown in AFM images (white arrows). AFM images of (C) sacrificial PEDOT:PSS layer on silicon substrate and (D) lamellar nanofilms on top of PEDOT:PSS layer. Surface morphology change indicates successful lamellar film formation on top of sacrificial layer. Reproduced with permission from ref 73.Copyright 2018 The Royal Society of Chemistry.


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Table 1. Block copolymer identification used in this article. Polymer ID

Block composition

Type

PB12 PB12COOH PB23 ABA22 ABA42 ABA55 ABA60 ABA110 BAB45

PB12-PEO10 PB12-PEO8-COOH PB23-PEO14 PMOXA5-PDMS22-PMOXA5 PMOXA20-PDMS42-PMOXA20 PMOXA12-PDMS55-PMOXA12 PMOXA12-PDMS60-PMOXA12 PMOXA15-PDMS110-PMOXA15 PI45-PEO135-PI45

diblock diblock diblock ABA ABA ABA ABA ABA BAB


MW (g·mol1) 1,089 1,000 1,806 2,478 6,508 6,110 6,480 10,690 12,000

Hydrophobic volume fraction 0.68 0.71 0.70 0.66 0.49 0.67 0.69 0.74 0.54

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Acknowledgements The authors would like to acknowledge financial support from National Science Foundation (NSF) CAREER grant (CBET-1552571) to MK for this work. Support was also provided through NSF CBET- 1512099, US Army CERL W9132T-16-2-0004-P00003, NSF DMR- 1709522 for various aspects of this work.


and

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Graphical Table of Contents


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Author Biographies

(Left to right: Laxmicharan Samineni, Yu-Ming Tu, Manish Kumar, Woochul Song, Hyeonji Oh) Woochul Song is a PhD candidate in Chemical Engineering at Pennsylvania State University. He received BS and MS degrees in Chemical Engineering from Sungkyunkwan University, South Korea. He worked at Korea Institute of Science and Technology for about one year as a research scientist, conducting research on development of nanoparticles as efficient intracellular drug delivery platform. His current aims to reveal microstructure-to-molecular transport property of membranes ranging from bioinspired to polymeric TFC membranes and use it for efficient membrane development. Yu-Ming Tu is a PhD student in Chemical Engineering at Pennsylvania State University. He received his B.S. in Chemical Engineering from the National Cheng Kung University in Taiwan. He completed his M.S. in the area of lipid bilayers and membrane protein dynamics in Chemical Engineering at the National Taiwan University in Taiwan. His research interests include biomaterials, membrane protein biophysics, and biomimetic membranes. His current research is focused on the development of scalable membranes using outer membrane protein channels. Hyeonji Oh is a senior undergraduate student in Chemical Engineering at Pennsylvania State University. Currently, she is working on fabricating TFC (Thin-Film Composite) membranes using biomimetic water channels. She intends to pursue graduate studies in the bio-inspired materials/separation area after graduation. Laxmicharan Samineni is currently pursuing his Ph.D in Chemical Engineering at Pennsylvania State University. He received his bachelors degree in Chemical Engineering from the National Institute of Technology in Warangal, India. He finished his Masters degree in Chemical Engineering from Indian Institute of Technology, Kanpur, India and then worked for about four years as a Process Scientist in the area of crystallization process development at a pharmaceutical company in India. He is currently working on developing protein coated sand filters for enhanced pathogen removal. His research interests include depth filtration, clean bed filtration models, and plant based antimicrobial proteins. Manish Kumar is an associate professor of Chemical Engineering, Environmental Engineering, and Biomedical Engineering at Penn State. He received his bachelor's degree in Chemical Engineering


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from the National Institute of Technology in Trichy, India. He completed an MS in environmental engineering at the University of Illinois, and then worked for approximately seven years in the consulting industry on applied research projects on membrane water and wastewater treatment. Manish returned to Illinois to complete a PhD in the area of biomimetic membranes and then conducted postdoctoral research at the Harvard Medical School on the structure of water channel proteins, aquaporins. He works in the areas of desalination and water purification membranes, membrane protein structure and biophysics, membrane protein enhanced synthetic block copolymer membranes, and on developing artificial membrane proteins and biocompatible electrical interfaces. He has received the NSF CAREER award and the Della and Rustom Roy award for outstanding materials research.


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Figure 1 159x85mm (600 x 600 DPI)


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Figure 2 86x88mm (300 x 300 DPI)


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FIgure 5 159x79mm (600 x 600 DPI)


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FIgure 6 119x147mm (600 x 600 DPI)


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Figure 7 158x50mm (300 x 300 DPI)


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Figure 8 158x66mm (300 x 300 DPI)


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Figure 9 139x49mm (600 x 600 DPI)


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Figure 10 159x68mm (600 x 600 DPI)


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Figure 11 80x117mm (300 x 300 DPI)


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Figure 12 159x79mm (600 x 600 DPI)


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Figure 13 85x114mm (600 x 600 DPI)


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Figure 14 85x94mm (600 x 600 DPI)


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Hierarchical Optimization of High-Performance Biomimetic and

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