SELF-ASSEMBLED POLYMERIC VESICULAR STRUCTURES WITH FUNCTIONAL MOLECULES

Abstract

Disclosed is a vesicle comprising polystyrene-polyacrylic acid (PS-PAA) block copolymer and an amphiphilic functional molecule. The vesicle is stable even at elevated temperatures and the amphiphilic functional molecule remains active. Also discloses is a selectively permeable membrane comprising a support layer and a selective layer incorporating the vesicles.

Claims

1. A vesicle comprising polystyrene-polyacrylic acid (PS-PAA) block copolymer and an amphiphilic functional molecule.

2. The vesicle according to claim 1, wherein said block copolymer has a molecular weight of from about 7500 Da to about 25000 Da

3. The vesicle according to claim 1 or 2, wherein said PS-PAA block copolymer is selected from block copolymers having the molecular weights 8000 Da, 13000 Da and 23300 Da.

4. The vesicle according to any of the claims claim 1 or 2, wherein the PS-PAA block copolymer has a hydrophilic to hydrophobic ratio in the range of from about 0.4 to about 3.6.

5. The vesicle according to any one of claims 1 to 4, wherein the PS-PAA block copolymer has an end functionalization.

6. The vesicle according to claim 5, wherein the end functionalization is selected from an azide group, a carboxyl group, or a DDMAT group exhibiting a thiol moiety.

7. The vesicle according to any of the claims 1 to 6, which has a hydrodynamic diameter of from about 50 nm to about 300 nm at room temperature.

8. The vesicle according to any of the claims 1 to 7, which is present in the form of an emulsion or a mixture composition prepared by direct dissolution in an aqueous medium in the presence of a detergent.

9. The vesicles according to an of the claims 1 to 8, wherein the detergent is selected among lauryldimethylamine-N-Oxide (LDAO) and octyl glucoside (OG).

10. The vesicles according to claim 8 or 9, wherein the detergent is used in a concentration in the range of 0.05 to 2.5% v/v.

11. The vesicle according to any one of claims 1 to 10, wherein the molar ratio of copolymer:detergent:AqpZ is in the range of from about 1:0.017:0.0008 to 1:0.19:0.0047.

12. The vesicle according to any one of the claims 1 to 11, wherein the amphiphilic functional molecule is selected from the group of amphiphilic peptides and transmembrane proteins.

13. The vesicle according to claim 12, wherein said transmembrane protein is an aquaporin water channel.

14. The vesicle according to claim 13, wherein said aquaporin water channel is selected from aquaporin Z, aquaporin-1, aquaporin-2 or SoPIP2;1.

15. The vesicle according to any of the claims 1 to 14, which is stable in admixture with a 3% aqueous m-phenylene-diamine (MPD) solution for at least about 12 h.

16. The vesicle according to any one of the claims 1 to 15, wherein the emulsion or mixture composition substantially does not include organic solvents, such as dioxane or dimethylformamide.

17. A selectively permeable membrane comprising a support layer and a selective layer, wherein the membrane comprises vesicles according to any one of claims 1 to 16 incorporated in the selective layer.

18. The selectively permeable membrane according to claim 17, wherein the selective layer is a thin-film composite (TFC) layer.

19. The selectively permeable membrane according to claim 17, wherein the selective layer has a layer-by-layer (LBL) structure.

20. The membrane according to claim 18 or claim 19, wherein the vesicles are fully negatively charged at pH>5 offering an increased incorporated vesicle packing density in the selective layer.

21. The membrane according to any one of claims 17 to 20 in the form of a flat sheet membrane, a hollow fiber membrane or a tubular membrane.

22. A method of preparing PS-PAA block copolymer vesicles having incorporated an amphiphilic functional molecule, comprising the steps of providing an aqueous composition comprising PS-PAA block copolymer vesicles and incorporating the amphiphilic functional molecule in the presence of a detergent.

23. The method according to claim 22, wherein the incorporation of the amphiphilic functional molecule in the polystyrene-polyacrylic acid (PS-PAA) block copolymer vesicle is effected by direct dissolution in an aqueous medium in the presence of a detergent.

24. The method according to claim 22 or 23, wherein the composition substantially does not include organic solvents, such as dioxane or dimethylformamide.

25. The method according to any one of the claims 22 to 24, wherein said amphiphilic functional molecule is a peptide or protein, such as a transmembrane protein, such as an aquaporin water channel.

26. Use of the membrane according to any one of claims 17 to 20 in a low pressure reverse osmosis (LPRO) process.

27. The use according to claim 26 wherein said process is a water purification process.

28. A low pressure reverse osmosis apparatus for water purification comprising the selectively permeable membrane according to any one of claims 17 to 20.

29. The apparatus according to claim 28 which is a household water purifier operating at a pressure below about 5 bar.

30. A brackish water reverse osmosis (BWRO) apparatus comprising the selectively permeable membrane according to any one of claims 17 to 20.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0025] More specifically, the present invention describes a novel approach for PS-PAA self-assembly by direct dissolution in a phosphate saline buffer, such as having a pH=7.2 or the like, in the presence of various ratios of a detergent, such as lauryldimethylamine N-oxide (LDAO) or octyl glucoside (OG), and with and without transmembrane protein incorporation as obtained by addition of an AqpZ dispersion having a concentration of from 1 to 100 mg/L or such as from 5 to 50 mg/L, and where the components are stirred or shaken up to 24 hours. The PS-PAA self-assembled structures of the present invention having vesicle sizes, e.g. as measured hydrodynamic diameter (Dz), in a desired range of from above 40 nm to 300 nm, such as from about 90 to 100 nm to about 200 to 250 nm, may also be obtained in the same way by varying the PS-PAA block copolymer's molecular weight and its hydrophilic to hydrophobic ratio and the detergent (e.g. LDAO, OG etc.) concentration, cf. Example 4 below. The successful reconstitution of AqpZ inside the formed PS-PAA structures was also obtained, and suitable conditions for the AqpZ reconstitution are disclosed in Example 3 below. Furthermore, the stability of the formed structures was established in the temperature range of from 30 to 100 C., which renders the self-assembled structures useful and suitable for incorporation in biomimetic membranes that may have to withstand various temperatures while still preserving their functionality and especially the water transporting functionality of incorporated aquaporin water channels.

Definitions

[0026] The term PS-PAA as used herein includes poly(styrene)-block-poly(acrylic acid), also known as PS-PAA amphiphilic diblock copolymers and polystyrene-polyacrylic acid polymersome forming polymer having the linear formula Ha[(C.sub.6H.sub.5)CHCH.sub.2].sub.x [(CO.sub.2H)CHCH.sub.2].sub.y C(CH.sub.3).sub.2C(O)OCH.sub.2CH.sub.3, cf. http://www.sigmaaldrich.com/catalog/search?term=PS-PAA&interface=All&N=0&mode=mode%20matchall&lang=en&region=DK&focus=product. wherein Ha=halogen such as F, Cl or Br, x=28 and y=70. Examples of PS-PAA diblock copolymers useful herein include Polystyrene-block-poly(acrylic acid) 130000 Da P19511-SAA PolymerSource; Polystyrene-block-poly(acrylic acid) 128000 Da P19513-SAA PolymerSource; Polystyrene-block-poly(acrylic acid) 230000 Da P3001-SAA PolymerSource.

[0027] The PS-PAA diblock copolymers may be terminally functionalized, such as having a DDMAT group, where DDMAT is 2-(Dodecylthiocarbonothioylthio)-2-methylpropanoic acid, S-Dodecyl-S-(,-dimethyl--acetic acid)trithiocarbonate. http://www.sigmaaldrich.com/catalog/product/aldrich/723010?lang=en&region=DK. The DDMAT terminated PS-PAA block copolymer may be useful due to the presence of an SS bond that can easily be used for functionalization with any functional molecule that contains an SH group, such as 2-Propene-1-thiol which is useful for further cross linking and 5-Fluorobenzoxazole-2-thiol being useful for spectroscopic characterisation. Other types of terminal functionalization include azide and carboxyl terminated PS-PAA polymers.

[0028] The term transmembrane protein as used herein includes any protein that occurs naturally in monomeric or multimeric form as inserted in a biological bilayer membrane, such as a cell or an organelle membrane. Transmembrane proteins are typically amphiphilic. Transmembrane proteins tend to aggregate and precipitate in aqueous solutions and it may therefore be suitable that the transmembrane protein is solubilized in a detergent. While a number of detergent may be used, generally the detergent is selected from the group consisting of lauryldimethylamine N-oxide (LDAO), octyl glucoside (OG), dodecyl maltoside (DDM) or combinations thereof. Preferred examples are aquaporin and aquaglyceroporin proteins, e.g. prokaryotic Aquaporin Z (AqpZ) and eukaryotic aquaporins, such as human aquaporin 1 and 2, and spinach SoPIP2;1. Further aquaporin water channels include bacterial aquaporins and eukaryotic aquaporins, such as yeast aquaporins, plant aquaporins and mammalian aquaporins, as well as related channel proteins, such as aquaglyceroporins. Examples of aquaporins and aquaglyceroporins include: prokaryotic aquaporins such as AqpZ; mammalian aquaporins, such as Aqp1 and Aqp2; plant aquaporins, such as plasma intrinsic proteins (PIP), tonoplast intrinsic proteins (TIP), nodulin intrinsic proteins (NIP) and small intrinsic proteins (SIP), e.g. SoPIP2;1, PttPIP2;5 and PtPIP2;2; yeast aquaporins, such as AQY1 and AQY2; and aquaglyceroporins, such as GlpF and Yfl054. Aquaporin water channel proteins may be prepared according to the methods described herein or as set out in Karlsson et al. (FEBS Letters 537: 68-72, 2003) or as described in Jensen et al. US 2012/0080377 A1 (e.g. see Example 6).

[0029] Hydrodynamic diameter as used herein represents the hydrodynamic size of nanoparticles in aqueous media measured by dynamic light scattering (DLS) defined as the size of a hypothetical hard sphere that diffuses in the same fashion as that of the particle being measured, for example using a ZetaSizer NanoZs from Malvern and stopped-flow using a Bio-Logic SFM 300.

[0030] In a preferred aspect of the invention, the active layer comprises the vesicles incorporated in a thin film composite layer formed on a porous substrate membrane. Without wishing to be bound by any particular theory, it is believed that the vesicles containing carboxylic acid groups on the surface will be not only physically incorporated or immobilized in (adsorbed), but, in addition, chemically bound in the TFC layer, because the reactive acid groups, will participate in the interfacial polymerization reaction with the acyl chloride, such as a trimesoyl chloride (TMC). In this way, it is believed that vesicles will be covalently bound in the TFC layer, leading to relatively higher vesicle loading and thus higher water flux through the membranes. In addition, it is believed that the covalent coupling of vesicles in the TFC layer results in higher stability and/or longevity of the aquaporins and aquaporin-incorporated vesicles when incorporated in the selective membrane layer.

[0031] Furthermore, when said transmembrane protein comprises an ion channel or an aquaporin or the like, and said vesicles comprising said transmembrane protein are immobilized or incorporated in said active or selective layer, it becomes feasible to manufacture separation membranes or filtration membranes having diverse selectivity and transport properties, e.g. ion exchange membranes when said transmembrane protein is an ion channel, or water filtration membranes when said transmembrane protein is an aquaporin. Because the transmembrane protein maintains its biologically active folded structure when complexed into the self-assembled vesicles wherein it may be shielded from degradation. Even sensitive amphiphilic proteins may become sufficiently stable and, thus, preserve their desired functionality when processed into separation membranes in lab and industrial scale.

[0032] The present invention further relates to a method of preparing a thin film composite layer immobilizing vesicles incorporating a transmembrane protein on a porous substrate membrane, comprising the steps of

[0033] a. Providing an aqueous solution comprising the vesicles prepared as mentioned above and a di-amine or tri-amine compound,

[0034] b. Covering the surface of a porous support membrane with the aqueous solution of step a,

[0035] c. Applying a hydrophobic solution comprising an acyl halide compound, and

[0036] d. Allowing the aqueous solution and the hydrophobic solution to perform an interfacial polymerization reaction to form the thin film composite layer.

[0037] The di-amine compound may be selected among a range of compounds including for example, phenylenediamines, such as m-phenylenediamine (MPD), p-phenylenediamine, 2,5-dichloro-p-phenylenediamine, 2,5-dibromo-p-phenylenediamine, 2,4,6-trichloro-m-phenylenediamine, 2,4,6-tribromo-m-phenylene-diamine, etc; diaminobiphenyls, such as 2,2-diamino-biphenyl, 4,4-diaminobiphenyl, 3,3-dichloro-4,4-diamino-biphenyl, 3,5,3,5-tetrabromo-4,4-diaminobiphenyl, etc; diaminodiphenylmethanes, such as 4,4-diaminodiphenylmethane, 3,3-diaminodiphenylmethane, 2,2-diaminodiphenyl-methane, 3,3-dichloro-4,4-diaminodiphenylmethane, 2,2-dichloro-4,4-diaminodiphenylmethane, 3,5,3,5-tetrachloro-4,4-diaminodiphenylmethane, 3,5,3,5-tetrabromo-4,4-diaminodiphenylmethane, etc.; diaminobibenzyls, such as 4,4-diaminobibenzyl, 3,5,3,5-tetrabromo-4,4-diamino-bibenzyl, etc.; 2,2-bisaminophenylpropanes, such as 2,2-bis(4-aminophenyl)propane, 2,2-bis(3,5-dichloro-4-amino-phenyl)propane, 2,2-bis(3,5-dibromo-4-aminophenyl)-propane, etc.; diaminodiphenylsulfones, such as 4,4-diamino-diphenylsulfone, 3,5,3,5-tetrachloro-4,4-diamino-diphenylsulfone, 3,5,3,5-tetrabromo-4,4-diaminodiphenyl-sulfone, etc.; diaminobenzophenones, such as 4,4-diamino-benzophenone, 2,2-diaminobenzophenone, 3,3-dichloro-4,4-diaminobenzophenone, 3,5,3,5-tetrabromo-4,4-diamino-benzophenone, 3,5,3,5-tetrachloro-4,4-diaminobenzo-phenone, etc.; diaminodiphenyl ethers, such as 3,3-diamino-diphenyl ether, 4,4-diaminodiphenyl ether, 3,3-dibromo-4,4-diaminodiphenyl ether, etc. piperazine, N-phenyl-benzene-1,3 diamine, melanine, and mixtures of such compounds. In a preferred aspect, the diamine is selected as m-phenylenediamine (MPD) also known as 1,3-diaminobenzene.

[0038] The tri-amine compound may be selected among a range of compounds including for example, diethylene triamine, dipropylene triamine, phenylenetriamine, bis(hexamethylene)-triamine, bis(hexamethylene)triamine, bis(3-aminopropyl)-amine, hexamethylenediamine, N-tallowalkyl dipropylene, 1,3,5-triazine-2,4,6-triamine, and mixtures of these compounds.

[0039] The acyl halide compound usually has two or three acyl halide groups available for reaction with the di- or triamine compound. Suitable examples of diacyl halide or triacyl halide compounds include trimesoyl chloride (TMC), trimesoyl bromide, isophthaloyl chloride (IPC), isophthaloyl bromide, terephthaloyl chloride (TPC), terephthaloyl bromide, adipoyl chloride, cyanuric chloride and mixtures of these compounds.

[0040] The amine groups of the di-amine or tri-amine compound will compete with the acid chloride groups of the acyl halide compound for reaction. Generally, the proportion by weight of the di-amine or tri-amine compound to acyl halide compound is from 0:1 to 30:1. When a high density of vesicles on the surface is required the amount of di-amine or tri-amine groups is usually in the lower part of the range, i.e. 0:1 to 1:1, such as between 0:1 to 0.5:1. In other embodiments of the invention, a more rigid TFC layer is desired and a selection of the reactants are in the higher end of the range, such as 1:1 to 30:1, preferably 1:1 to 5:1.

[0041] The porous support membrane may be formed by a number of materials. The specific choice of material is not essential as long as the support membrane is able sufficiently to support the TFC layer and to withstand decomposition during operation condition, i.e. able to withstand the pressure and/or the chemical environment on either side of the membrane. Specific examples of materials for the porous support membrane include polysulfone or a polyethersulfone polymer. The support may be symmetrical or asymmetrical. In the case the porous support membrane is asymmetrical, the TFC layer is suitably formed on the skin layer face.

[0042] The porous support membrane may further be supported by a woven or non-woven mechanical support in some embodiments to increase the mechanical construction and reduce the risk of fractures during operation.

[0043] The porous support membrane may any physical appearance known in the art, such as flat sheet membrane, tubular membrane, or hollow fiber membrane. In a certain aspect of the invention a hollow fiber membrane is preferred as it provides for higher packing density, i.e. the active membrane area is higher for a certain volume. The membranes may be grouped together or assembled into a module as known in the art. Thus, a plurality of flat sheet membranes may be assembled into a plate-and-frame membrane configuration. Plate-and-frame membrane systems utilize membranes laid on top of a plate-like structure, which in turn is held together by a frame-like support.

[0044] Flat sheet membranes may also be assembled into spiral-wound filter modules. In addition to the flat sheet membranes, the spiral-wound membrane modules include feed spacers, and permeate spacers wrapped around a hollow tube called the permeate tube. Spiral wound elements utilize cross flow technology, and because of its construction, can easily be created in different configurations with varying length, diameter, and membrane material. A spiral-wound filter module may be produced by first laying out a membrane and then fold it in half with the membrane facing inward. Feed spacer is then put in between the folded membranes, forming a membrane sandwich. The purpose of the feed spacer is to provide space for water to flow between the membrane surfaces, and to allow for uniform flow between the membrane leaves. Next, the permeate spacer is attached to the permeate tube, and the membrane sandwich prepared earlier is attached to the permeate spacer using glue. The next permeate layer is laid down and sealed with glue, and the whole process is repeated until all of the required permeate spacers have been attached to the membranes. The finished membrane layers then are wrapped around the tube creating the spiral shape.

[0045] Tubular membrane modules are tube-like structures with porous walls. Tubular modules work through tangential cross-flow, and are generally used to process difficult feed streams such as those with high dissolved solids, high suspended solids, and/or oil, grease, or fats. Tubular modules consist of a minimum of two tubes; the inner tube, called the membrane tube, and the outer tube, which is the shell. The feed stream goes across the length of the membrane tube and is filtered out into the outer shell while concentrate collects at the opposite end of the membrane tube.

[0046] The hollow fiber membranes may be assembled into a module. Thus, the present invention provides the step of producing a hollow fiber module by assembling a bundle of hollow fibers in a housing, wherein an inlet for passing a first solution is connected to the lumen of the hollow fibers in one end and an outlet is connected to the lumen in the other end, and an inlet is provided in the housing for passing a second solution to an outlet connected to the housing.

[0047] The membrane modules produced in accordance with the present invention may be used in various configurations, including forward osmosis configurations and reverse osmosis configurations.

[0048] Forward osmosis (FO) is an osmotic process that uses a selectively-permeable membrane to effect separation of water from dissolved solutes. The driving force for this separation is an osmotic pressure gradient between a solution of high concentration, herein referred to as the draw and a solution of lower concentration, referred to as the feed. The osmotic pressure gradient induces a net flow of water through the membrane into the draw, thus effectively concentrating the feed. The draw solution can consist of a single or multiple simple salts or can be a substance specifically tailored for forward osmosis applications. The feed solution can be a dilute product stream, such as a beverage, a waste stream or seawater, cf. IFOA, http://forwardosmosis.biz/education/what-is-forward-osmosis/

[0049] Most of the applications of FO, thus fall into three broad categories: product concentration, waste concentration or production of clean water as a bi-product of the concentration process. The term PAFO when used herein describes a pressure assisted forward osmosis process. The term PRO when used herein describes pressure retarded osmosis which is useful in the generation of osmotic power. Membranes of the present invention are useful in all types of forward osmosis processes and may be specifically adapted for each FO type.

[0050] The term reverse osmosis (RO) is used herein refers to when an applied feed water pressure on a selectively permeable membrane is used to overcome osmotic pressure.

[0051] Reverse osmosis typically removes many types of dissolved and suspended substances from feed water, including bacteria, and is used in both industrial processes and in the production of potable water. During the RO process, the solute is retained on the pressurized side of the membrane and the pure solvent, the permeate, passes to the other side. Selectivity specifies that the membrane does not allow larger molecules or ions through its pores (holes), while allowing smaller components of the solution (such as solvent molecules) to pass freely. Low pressure reverse osmosis (LPRO) membranes typically operates at a feed water pressure of from about <5 bar and up to a maximum operating pressure of about 25 bar 15 specific flux LMH/bar. LPRO performed at the lower feed pressure ranges, e.g. 2 to 5 bar is sometimes designated ultra-low pressure reverse osmosis. LPRO membranes known in the art have typical operating limits for feed water temperature of about 45 C., feed water pH in the range of 2 to 11, and chemical cleaning in the range of pH 1 to 12.

[0052] The present invention relates more specifically to an aqueous composition comprising PS-PAA block copolymer vesicles having incorporated an amphiphilic functional molecule in the presence of a detergent. In certain embodiments, the aqueous composition is essentially free of apolar solvents. Examples of said functional molecule include an amphiphilic peptide or protein, such as a transmembrane protein, such as an aquaporin water channel, e.g. aquaporin Z, or SoPIP2;1 and other plant aquaporins, or aquaporin-1, or aquaporin-2. More specifically said copolymer is selected from PS-PAA block copolymers having a hydrophilic to hydrophobic ratio in the range of from 0.4 to 3.6; and the molar ratio of polymer:detergent:AqpZ is in the range of from about 1:0.017:0.0008 to 1:0.19:0.0047.

[0053] In addition, examples of said PS-PAA copolymer are selected from block copolymers having a molecular weight (Mw) of from about 8000 Da to about 25000 Da, such as block copolymers having the molecular weights 8000 Da, 13000 Da and 23300 Da.

[0054] In the composition of the invention, the detergent may be selected from LDAO and OG and said detergent may be present in a concentration of from about 0.05 to about 2.5% v/v.

[0055] In addition, the invention relates to a vesicle comprising PS-PAA block copolymer and an amphiphilic functional molecule. In certain embodiments, the vesicle has a hydrodynamic diameter of from about 50 nm to about 200 nm, such as from about 55 nm to about 100 nm; and the vesicle further comprises a detergent, such as LDAO or OG; and the amphiphilic functional molecule is selected from the group of amphiphilic peptides and transmembrane proteins, such as aquaporin water channels.

[0056] In exemplary embodiments of the present invention, the vesicle is stable in admixture with MPD for at least about 6 h, more preferably at least about 12 h, more preferably at least about 18 hours and most preferably up to about 24 hours.

[0057] The present invention further relates to a selectively permeable membrane comprising a porous support layer and a dense or non-porous selective layer wherein the vesicles of the invention are incorporated. The membrane ma y be in the form of a flat sheet membrane or a hollow fiber membrane or a tubular membrane. The membrane of the invention is useful for filtration of water using forward osmosis or reverse osmosis. Low pressure reverse osmosis (LPRO) membranes typically operates at a feed pressure of from about 5 to 10 bar and a specific flux of about 15 LMH/bar. The lower feed pressure ranges, e.g. <5 bar are sometimes designated ultra-low pressure reverse osmosis. Thus, an aspect of the present invention relates to the use of the selectively permeable water membrane of the invention in a low pressure reverse osmosis (LPRO) process, such as a water purification process utilizing a natural water source or a surface or ground water source as feed water.

[0058] In some embodiments, the selectively permeable membranes of the present invention may further be subjected to a surface treatment on the selective layer or the separation layer, for example to provide a coating layer over the selective layer and/or the separation layer. By way of example, this may take the form of a thin coating comprising hydrophilic polydopamine, cf. Environ. Sci. Technol. Lett., 2016, 3 (9), pp 332-338 for antifouling purposes, or as a PVA coating, cf. U.S. Pat. No. 6,413,425, for the improvement of parameters such as salt rejection, fouling tolerance etc.

[0059] In a further aspect, the present invention provides a low pressure reverse osmosis apparatus for water purification comprising the selectively permeable membrane of the invention, where an example of said apparatus is a household water purifier operating at a pressure below about 5 to 10 bar, such as between 2 to 5 bar.

[0060] An additional aspect of the present invention is a brackish water reverse osmosis (BWRO) apparatus comprising the selectively permeable membrane of the invention. The selectively permeable membranes of the present invention may be used for brackish water desalination, where the incorporation of active aquaporin water channels in the selective layer provides for improved flux and reduced energy consumption compared to traditional systems.

[0061] The present invention is versatile in that it provides PS-PAA self assembled vesicles or polymersomes that may encapsulate or incorporate a range of functional molecules having both amphiphilic, hydrophilic or hydrophobic nature. For this purpose, the functional molecule may be mixed directly with the mixture of PS-PAA and suitable aqueous detergent to ensure encapsulation inside the vesicle for hydrophilic compounds or inside the PS block for the hydrophobic compound or aligned in the amphiphilic vesicle membrane for amphiphilic compounds, e.g. certain peptides (e.g. insulin) or transmembrane molecules or proteins, and the encapsulated molecules may then be released in controlled conditions.

EXAMPLES

[0062] The present invention is further illustrated by the following examples which should not be construed as further limiting.

General Protocols

Example 1. Direct Dissolution of PS-PAA in Phosphate Buffer pH=7.2 in Presence of LDAO to Form Self-Assembled Vesicles

[0063] Materials and Methods

[0064] Polystyrene-block-poly(acrylic acid), DDMAT terminated (MW 8000 Da) (PS-PAA 3000:5000, PDI<1.1) was purchased from Sigma-Aldrich (HOCOC(CH.sub.3).sub.2(CH.sub.2CHC.sub.6H.sub.5).sub.m(CH.sub.2CHCOOH).sub.nSCSSC.sub.12H.sub.25) wherein m=28 and n=70, and was used as received without any other purification. 10 mM phosphate saline solution (PBS) (pH 7.2, 136 mM NaCl, 2.6 mM KCl) was prepared by dissolving 8 g NaCl, 0.2 g KCl, 1.44 g Na.sub.2HPO.sub.4 and 0.24 g of KH.sub.2PO.sub.4 in 800 mL MiliQ purified H.sub.2O, adjusting the pH to 7.2 with HCL and completing the volume to 1 L. N,N-Dimethyldodecylamine N-oxide BioXtra (Lauryldimethylamine N-oxide) (99% purity), LDAO was purchased from Sigma Aldrich.

[0065] PS-PAA incorporating AqpZ vesicles were prepared by LDAO mediated direct dissolution method. For that 200 mg PS-PAA powder were mixed with 0.5 mL 5% LDAO stock solution and 195 mL PBS.

[0066] The PS-PAA, LDAO mixture was stirred overnight at 170 rotations per minute, overnight not more than 24 hours (but not less than 12 h). After stirring next day, the mixture was transferred in 100 mls glass bottle and kept at room temperature. After transfer to the storage glass bottle the size and the permeability of the PS-PAA self-assembled structures (vesicles) and zeta potential were determined by dynamic light scattering using a ZetaSizer NanoZs from Malvern and stopped-flow using a Bio-Logic SFM 300.

[0067] The hydrodynamic diameter of PS-PAA structures was determined as 78 nm (in average). The zeta potential was determined for the PS-PAA self-assembled structures as 13 mV, indicating the expected negative charge of the structures.

[0068] The permeability data obtained from stopped-flow measurements in 0.5 M NaCl as the osmolyte lead to a fast diffusion coefficient K.sub.i of 100 s.sup.1.

Example 2. General Purification of Aquaporin and Preparation of Aquaporin Stock Solution

[0069] Recombinant Production of Aquaporin Z

[0070] All types and variants of aquaporin water channel proteins, including aquaglyceroporins, may be used in the manufacture of membranes and compositions according to this invention, cf. methods described in WO2010/146365. Representative examples include the spinach aquaporin SoPIP2;1 protein and the bacterial aquaporin-Z from E. coli.

[0071] Functional aquaporin-Z was overproduced in E. coli strain BL21(DE3) bacterial cultures as His-tagged protein with a tobacco etch virus cleavage site. The fusion protein has 264 amino acid and a Mw of 27234 Da. Genomic DNA from E. coli DH5 was used as a source for amplifying the AqpZ gene. The AqpZ gene was amplified using gene specific primers with the addition of a tobacco etch virus cleavage site (TEV); ENLYFQSN at the N-terminus of AqpZ. The amplified AqpZ was digested with the enzyme NdeI and BamHI and then ligated to the similarly digested 6-His tagged expression pET28b vector DNA. The positive clones were verified by PCR-screening. The authenticity of the constructs was then confirmed by DNA sequencing.

[0072] The E. coli strain BL21(DE3) was used for expression of the protein. Luria Broth cultures containing 50 g/ml kanamycin were incubated for 13-16 hours at 37 C, diluted 100-fold into fresh LB broth and propagated to a density of about 1.2-1.5 (OD at 600 nm). Expression of recombinant protein was induced by addition of 1 mM IPTG for 3 hour at 35 C. before centrifugation. Harvested cells were resuspended in ice-cold binding buffer (20 mM Tris pH 8.0, 50 mM NaCl, 2 mM -mercaptoethanol, 10% glycerol) in the presence of 0.4 mg/ml lysozyme, 50 units Bensonase and 3% n-octyl -D-Glucopyranoside. The sample was subjected to five times lysis cycles in a microfluidizer at 12,000 Pa. Insoluble material was pelleted by 30 minutes centrifugation at 40,000g. The supernatant was passed through a Q-Sepharose fast flow column (Amersham Pharmacia), and the flow through was 10 collected. The flow though fraction was topped up with NaCl to 300 mM before loaded onto a pre-equilibrated Ni-NTA column. The column was washed with 100 column volumes of a wash buffer (20 mM Tris pH 8.0, 300 mM NaCl, 25 mM imidazole, 2 mM -mercaptoethanol, 10% glycerol) to remove non-specifically bound material. Ni-NTA agarose bound material was eluted with five bed volumes of elution buffer (20 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole, 2 mM -mercaptoethanol, 10% 15 glycerol, containing 30 mM n-octyl -D-Glucopyranoside). AqpZ was further purified with anion exchange chromatography; monoQ column (GE healthcare). The sample mixture was diluted and concentrated to bring the salt and imidazole concentration to approximately 10 mM with Amicon concentrator, membrane cut off 10,000 Da before loading to MonoQ column. The buffer used during anion exchange chromatography were (A) 20 mM Tris pH 8.0, 30 mM OG, 10% glycerol and (B) 20 mM 20 Tris pH 8.0, 1 M NaCl, 30 mM OG, 10% glycerol. The eluted peak fractions containing AqpZ from the ion exchange column was pooled. The purified AqpZ extract was kept frozen at 80 C.

[0073] Procedure for Purification of Aquaporin Protein

[0074] A batch of frozen extract of aquaporin protein, such as aquaporin Z, AQPZ, e.g. from an E. coli fermentation, was obtained and treated as follows for use in the experiments to produce and characterise membranes comprising protein-PAI nanostructures of the present invention.

[0075] One day before the purification experiment, the AQP extract (stored at 80 C. freezer) was thawed on ice or in a 4 C. refrigerator. Portions of the buffers and ddH2O were readied at 4 C. The AQP extract was stirred in an adequate chilled beaker on ice bath by a magnetic stick to dissolve any precipitate. 1.5 volumes of pre-chilled LDAO-free AQP binding buffer was gradually added into 1 volume of the solubilized extract (using a further 0.5 volume buffer for rinsing the extract tubes and filtration cup), mixed well and filtered through a sterile 0.45 M vacuum filter cup. Vacuum was applied to the filter cup to avoid excess foaming and the filtrate was placed on ice to use within 2 hours.

[0076] A Histrap column was equilibrated with sterile water followed by AQP Binding buffer at RT. The flow rate was set at lml/min (for 1 mL prepacked column) or 2.5 ml/min (for 5 ml prepacked column and self-packed column). The 3 times diluted extract (on ice water bath) was loaded onto the Histrap column using AKTA program. The flow rate was set at 1 ml/min (for 1 mL prepacked column) or 2.5 ml/min (for 5 mL prepacked column and self-packed column). The loading volume was less than 30 ml/ml resin. The extract flow-through on ice-water bath was collected and stored at 4 C. for further use. The column was washed with 10 CV ice cold AQP binding buffer. The flow rate was set at 2.5 ml/min (for 5 ml prepacked column and self-packed column) or set at 1 ml/min for 1 ml prepacked column. The AQP protein was eluted with ice cold AQP elution buffer (10 column volume) at flow rate 2.5 ml/min using AKTA program. The fraction volume was set to 10 ml and collection started in 15 mL PP tubes after 0.5-1CV.

[0077] Eluted fractions were capped and stored on ice or 4 C. The AQP purity and conformation was examined by denaturing and native PAGE analysis respectively. Protein concentration was measured by Nanodrop. The extract flow-through may be processed a second and a third time as needed to produce an AQP composition of suitable quality.

[0078] When AQP quality analyses are passed, the protein concentration may be adjusted to 5 mg/ml by adding ice cold imidazole-free AQP binding buffer containing 2% LDAO. Finally the AQP was sterilized by filtration through 0.45 M sterilized cup and stored at 4 C. in refrigerator for use within a month or else stored at 80 C. in a freezer.

Example 3. Preparation of PS-PAA Vesicles Having AqpZ Incorporation Using LDAO as a Detergent

[0079] Materials and Methods

[0080] Polystyrene-block-poly(acrylic acid), DDMAT terminated (MW 8000 Da) (PS-PAA as in Example 1) was purchased from Sigma Aldrich and was used as received without any other purification. 10 mM phosphate saline solution (PBS) (pH 7.2, 136 mM NaCl, 2.6 mM KCl) was prepared by dissolving 8 g NaCl, 0.2 g KCl, 1.44 g Na.sub.2HPO.sub.4 and 0.24 g of KH.sub.2PO.sub.4 in 800 mL MilliQ purified H.sub.2O, adjusting the pH to 7.2 with HCL and completing the volume to 1 L. N,N-Dimethyldodecylamine N-oxide BioXtra (Lauryldimethylamine N-oxide) (99% purity), LDAO was purchased from Sigma Aldrich). 5 mg/mLAqpZ purified stock solution in 2% LDAO, see general preparation in Example 2 above.

[0081] PS-PAA incorporating AqpZ vesicles were prepared by LDAO mediated direct dissolution method. For that 200 mg PS-PAA powder were mixed with 0.5 mL 5% LDAO stock solution and 194.9 mL PBS and 0.5 mg (0.1 mL) AqpZ purified stock solution in 2% LDAO to achieve a 1/330 AQPZ/polystyrene-block-poly(acrylic acid), DDMAT terminated molar ratio.

[0082] The PS-PAA, LDAO, AqpZ mixture was stirred overnight at 170 rotations per minute, overnight not more than 24 hours (but not less than 12 h). After stirring next day, the mixture was transferred in 100 mL glass bottle and kept at room temperature. After transfer to the storage glass bottle the size and the permeability of the PS-PAA AqpZ self-assembled structures and zeta potential were determined by dynamic light scattering using a ZetaSizer NanoZs from Malvern and stopped-flow using a Bio-Logic SFM 300.

[0083] The hydrodynamic of PS-PAA AqpZ structures was determined as 69 nm (in average). The zeta potential was determined for the PS-PAA AqpZ self-assembled structures as 14 mV, indicating the expected negative charge of the structures.

[0084] The permeability data obtained from stopped-flow measurements in 0.5 M NaCL as the osmolyte lead to a fast diffusion coefficient K.sub.i of 1000 s.sup.1.

[0085] The temperature stability of the PS-PAA AgpZ based self-assembled structures was determined by warming up 5 mL for 10 min at various temperatures ranging from 30 to 100 C. and their size and water permeability was further determined by dynamic light scattering and stopped-flow measurements. The size decrease with the temperature increase at 60 C. (reaching 39 nm), while the fast diffusion coefficient K.sub.i values do not change.

[0086] The same type of experiment was performed by using PS-PAA copolymers with Mw of from 8000 Da to 23300 Da and having hydrophilic to hydrophobic ratios from 0.4 to 3.6, and using LDAO concentrations ranging from 0.05 to 2.5% v/v.

Example 4. Preparation of PS-PAA Vesicles Having AqpZ Incorporation Using OG as a Detergent

[0087] Materials and Methods

[0088] Polystyrene-block-poly(acrylic acid), DDMAT terminated (MW 8000 Da) (PS-PAA as in Example 1) was purchased from Sigma Aldrich and was used as received without any other purification. 10 mM phosphate saline solution (PBS) (pH 7.2, 136 mM NaCl, 2.6 mM KCl) was prepared by dissolving 8 g NaCl, 0.2 g KCl, 1.44 g Na.sub.2HPO.sub.4 and 0.24 g of KH.sub.2PO.sub.4 in 800 mL MiliQ purified H.sub.2O, adjusting the pH to 7.2 with HCL and completing the volume to 1 L. N,N-Octyl -D-glucopyranoside (98% purity), OG was purchased from Sigma Aldrich. 5 mg/mL AqpZ purified stock solution in 1% OG.

[0089] PS-PAA incorporating AqpZ vesicles were prepared by OG mediated direct dissolution method. For that 200 mg PS-PAA powder were mixed with 0.25 mL 10% OG stock solution and 195.15 mL PBS and 0.5 mg (0.1 mL) AqpZ purified stock solution in 1% OG to achieve a 1/330 AQPZ/polystyrene-block-poly(acrylic acid), OG terminated molar ratio.

[0090] The PS-PAA, OG, and AqpZ mixture was stirred overnight at 170 rotations per minute, overnight not more than 24 hours (but not less than 12 h). After stirring next day, the mixture was transferred in 100 mls glass bottle and kept at room temperature. After transfer to the storage glass bottle the size and the permeability of the PS-PAA AqpZ self-assembled structures and zeta potential were determined by dynamic light scattering using a ZetaSizer NanoZs from Malvern and stopped-flow using a Bio-Logic SFM 300.

[0091] The hydrodynamic diameter size of PS-PAA AqpZ vesicular structures was determined as 50 nm (in average) with peaks at 56 nm (84%) and 71 nm (16). The zeta potential was determined for the PS-PAA AqpZ self-assembled vesicular structures as 14 mV, indicating the negative charge of the structures. The permeability data obtained from stopped-flow measurements in 0.5 M NaCl as the osmolyte lead to a fast diffusion coefficient K.sub.i of 1350 s.sup.1.

Example 5. Handmade FO Filtration Membranes Using PS-PAA 8000,/Polystyrene-Block-Poly(Acrylic Acid), DDMAT Terminated from Sigma-Aldrich

[0092] A TFC layer was formed on a PES support membrane using a manual protocol;

[0093] a) An MPD/water solution was made by dissolving 1.5 g MPD in about 55 mL of MilliQ water to get app. 2.5-3% (W/W) concentration;

[0094] b) An aqueous PS-PAA-aquaporin Z solution is mixed with the MPD solution prepared in step a): mix 6 mL PS-PAA/aqpZ solution with about 54 mL of MPD aqueous solution;

[0095] c) TMC was dissolved in Isopar to a final concentration of 0.15% W/V;

[0096] d) Cover a rectangular shaped membrane, e.g. 5.5 cm11 cm filter membrane of 0.1 m in pore size (MICRO PES 1FPH, manufactured by Membrana Co.) with about 20 mL/m.sup.2 membrane of PS-PAA-aqpZ/MPD solution prepared in step b), and leave for 30 seconds under gentle agitation;

[0097] e) Dry the non-active side (back side) with lab drying paper (e.g. Kim-Wipe) for 5-10 seconds;

[0098] f) Put the membrane on a glass plate and dry gently with N.sub.2 until the surface turns from shiny to dim;

[0099] g) Apply tape around the edges of the membrane (1 mm);

[0100] h) Put the glass plate with the taped membrane into a glass or metal container, add about 155 mL/m.sup.2 membrane TMC-Isopar to one end and rock gently back and forth for 30 seconds;

[0101] i) Remove glass plate from reservoir and dry with N.sub.2 for 10 to 15 seconds.

[0102] After removal of the tape the membrane can be transferred to MilliQ with the newly formed active side up and keep wet during handling in subsequent steps if necessary.

[0103] Five coated membranes of 5.5 cm11 cm shapes were separately fitted into a Sterlitech CF042 FO cell and run for 200 minutes with a 5 M calcein in DI water feed solution and a 1 M NaCl draw solution. Mean results with standard deviation are shown in Table 1 for membranes obtained by using the vesicles obtained in example 3. From the results it can be seen that the FO water flux (Jw) performance is both satisfactory and highly reproducible as shown by a low standard deviation. At the same time the reverse sodium chloride flux is low, and the ratio Js/Jw is excellent, i.e. below 0.20. The obtained calcein rejection of more than 99% is a measure characterizing a tight membrane without pin holes or other faults.

TABLE-US-00001 TABLE 1 Number of FO Calcein membranes Jw SD Js SD rejection tested [L/m.sup.2h] [g/m.sup.2h] Js/Jw SD % SD 5 7.3 0.73 1.37 0.36 0.19 0.04 99.86 0.04

Example 6. Pilot Machine Made FO Filtration Membranes Using PS-PAA 8000,/Polystyrene-Block-Poly(Acrylic Acid), DDMAT Terminated from Sigma-Aldrich

[0104] A TFC layer is formed on a PES support membrane using a pilot coating machine.

[0105] a) An MPD/water solution is made by dissolving MPD in MilliQ water to get a 2.5% (W/W) concentration;

[0106] b) A PS-PAA/aquaporin-Z/MPD/water solution is prepared as in steps a) and b) of Example 5;

[0107] c) TMC is dissolved in Isopar to a final concentration of 0.15% W/V;

[0108] d) A roll of filter membrane of MICRO PES 1FPH (0.1 m in pore size;

[0109] manufactured by Membrana Co.) is installed on the unwinding unit of the machine;

[0110] e) The membrane is threaded through the coating;

[0111] f) The washing bath is filled with DI water;

[0112] g) The coating process is run (at 0.6 m/min): [0113] membrane is unrolled from unwinder; [0114] then soaked in MPD/water in foulard bath; [0115] surface water is removed by air knife (0.5 bar air); [0116] the PA-PAA/Aquaporin/MPD/water solution of step b) is applied via slot die at pump rate of 1.2 mL/min; [0117] surface water is removed via air knife to ensure a droplet free surface before interfacial polymerization (0.75 bar); [0118] TMC/Isopar is applied via slot die at 4.2 mL/min to start interfacial polymerization; [0119] Isopar is dried off the surface of the membrane at ambient air; [0120] leftover chemicals are removed in wash bath; [0121] coated membrane is rolled up with active side pointing towards the roll;

[0122] h) The coated membrane is run through a final drying step.

[0123] Five coated membranes are cut in 5.5 cm11 cm shapes and separately fitted into a Sterlitech CF042 FO cell and run for 200 minutes with a 5 M calcein in DI water feed solution and a 1 M NaCl draw solution. Mean results with standard deviation of the FO water flux (Jw) performance are obtained.

Example 7. Handmade TFC PS-PAA-AQPZ Filtration Membranes for RO Low Pressure Using PS-PAA 8000,/Polystyrene-Block-Poly(Acrylic Acid), DDMAT Terminated from Sigma-Aldrich

[0124] The membranes were made according to the steps outlined below:

[0125] a) Provide a support membrane, e.g. a PES non-woven having fingerlike structure, size 5.5 cm11 cm (e.g. a MICRO PES 1FPH having 0.1 m in pore size; manufactured by Membrana Co.);

[0126] b) Mix 3 wt % MPD, and 93.5 wt % DI water to obtain a solution;

[0127] c) Add 0.1 mg/mL of PS-PAA-AQPZ proteopolymersomes (self-assembled nanovesicles) prepared according to Example 3, to obtain a suspension;

[0128] d) Incubate the suspension from c) for 2 hours;

[0129] e) Prepare TMC solution from 0.09 wt % TMC, 99.01 wt % Isopar, and optionally less than about 1 wt % of an apolar solvent, such as acetone;

[0130] f) Dip coat the support membrane in the suspension d) for about 30 seconds;

[0131] g) Apply drying with air knife;

[0132] h) Add the TMC solution from e) for interfacial polymerization;

[0133] i) Follow with 2 min drying in fume hood.

[0134] Eight membranes were made and mounted in a Sterlitech CF042 RO cell, www.sterlitech.com, operated at 5 bar using 500 ppm NaCl as feed for 60 minutes. The results are indicated in table 2 below and shows that the RO water flux (Jw) performance is both satisfactory and highly reproducible, while at the same time the sodium chloride rejection is high.

TABLE-US-00002 No. of Permeability Rejection Samples (LMH/bar) (%) PS-PAA Aqp 8 9.7 0.5 95.6 0.5 vesicles

Example 8. Preparation of a Layer-by-Layer Membrane Using the PS-PAA Self-Assembled Vesicles of the Invention

[0135] LbL polyelectrolyte assembly has been employed for membrane separations, for many porous membrane substrates with different sizes and topology that can adsorb the initial polyelectrolyte layer such as poly(ether sulfone), poly(vinylamine), poly(4-methyl-1-pentene), polyamide, polyacrylonitrile (PAN), poly(vinyl pyrrolidone), anodic alumina in flat sheet, tubular or hollow fiber structures [Duong, P. H. H., Zuo, J., Chung, T-S., J. Memb. Sci. 427 (2013), 411-421].

[0136] We propose using LBL polyelectrolyte technique to prepare ultrafiltration membranes based on non-woven PES substrate and PEI/PAA polyelectrolyte layer incorporating PS-PAA AqpZ nanostructures. In order to prepare the membranes, the following steps will be employed.

[0137] Step 1. Select and prepare the negatively charged PES on the non-woven support;

[0138] Step 2. Adsorb PEI on the negatively charged surface of the substrate due to the electrostatic attraction; by just immersion in PEI solution;

[0139] Step 3. Wash the substrate surface with de-ionized water in order to remove excess PEI molecules which are not strongly adsorbed on the surface;

[0140] Step 4. Immerse the PES covered with PEI into PS-PAA Aqpz solution, where the negative charges will be adsorbed onto the surface;

[0141] Step 5. Wash the substrate surface with de-ionized water in order to remove excess PS-PAA AqpZ structures which are not strongly adsorbed on the surface;

[0142] Step 6. Immerse the PES covered with negative charges from PS-PAA Aqpz solution into PEI solution;

[0143] Step 7. Wash PES surface covered with PEI and PAA AgpZ structures with de-ionized water in order to remove excess PEI molecules;

[0144] Step 8. Adsorb PS-PAA on the positively charged surface by direct immersion in 2 mg/mL PS-PAA 8000 Da solution;

[0145] Step 9. Wash PES surface covered with PS-PAA and PEI structures with de-ionized water in order to remove excess PS-PAA molecules;

[0146] Step 6. Repeat steps 6-9 until reaching the targeted number of multilayers2;

[0147] Step 8. Wash with deionized water

[0148] In case other pairs of electrolytes are preferred similar procedure will be used in order to prepare the membranes. PS-PAA AqpZ based nanostructures will be used to replace the polyanion used to assembly the electrolyte multilayers.

Example 9: Preparation of a Layer-by-Layer (LBL) Membrane Incorporating Aquaporin Vesicles

[0149] Materials:

[0150] PAHPolyallylamine 40 wt % in water; Mw=150,000 g/mol; Manufactured by Nittobo; grade: PAA-HCL-10L.

[0151] PSSPoly(sodium 4-styrenesulfonate) solution 30 wt % in water; Mw=200,000 g/mol; Manufactured by Sigma-Aldrich.

[0152] NaClSodium chloride; Manufactured by Akzo Nobel.

[0153] FibersUltrafiltration membranes made by TWENTE University from sulfonated polyethersulfone with poly(diallyldimethylammonium chloride). Inner diameter is 0.68 mm and the outer diameter is 0.88 mm. The fiber has a standard permeability of around 200 Lmhb (L*m.sup.2*h.sup.1*bar.sup.1).

[0154] Preparation of LBL

[0155] The polyelectrolyte multilayer (PEM) was prepared by dipping the fibers in a 0.5 M of NaCl and 0.1 g/l of polyelectrolyte solution. The polyelectrolytes were PAH (polyallylamine) and PSS (polystyrene sulfonate) and all solutions were made with deionized water. The fibers were first put into the PSS solution for 15 minutes, then were rinsed in three separate cylinders for 5 minutes each. Subsequently, the fibers were put into the PAH solution also for 15 minutes. This was repeated until a 7 bilayer system ([PSS/PAH].sub.7) was made.

[0156] Since the vesicles have a negative charge, they should be attach onto the positively charged surface. A module was made from the membrane where one side was closed off like a dead-end filtration. The module was constructed from a PE tube with a hole in the bottom. The PS-PAA Aqpz vesicles solution prepared in example 4 were put into a syringe and then connected to the membrane module. The vesicle solution was subsequently allowed to flow through the inside of the membrane until all the air is out. Then one side is closed off like a dead end filtration and the PS-PAA solution is pushed through, from the inside to the outside, until the membrane starts dripping.

[0157] After this, the membrane is dried for at least 4 hours in 15/85 wt % glycerol/water and then dried overnight before any further measurements were done.

[0158] For this specific case a single salt concentration for building the PEM was used. This can, however, be varied from 5 to 1000 mM (0.005 to 1.0 M) of NaCl.

TABLE-US-00003 Number of RO Jw SD Rejection % membranes tested [L/m.sup.2h/bar] [NaCl] SD 4 6.46 0.3 44.1 4

[0159] Similar results for the rejection of salt were described by Zhang T et al 2013.

Example 10. Characterization of the PS-PAA Vesicles of the Invention Using Laser Scanning Microscopy and Scanning Electron Microscopy

[0160] The morphology as well the size of the formed PS-PAA AqpZ and PS-PAA vesicles will characterized by transmission electron microscopy on a Tecnai T20 G2 electron microscope which was operated at 100 kV. Vesicles dispersions will be deposited on a carbon-coated copper grid and negatively stained with 2% uranyl acetate solution.

[0161] Scanning electron microscopy of the cross section of prepared TFC and LBL membranes will be performed on a FEI Inspect S microscope.

Example 11. Characterization of Various PS-PAA-AqpZ Handmade FO Membranes Using 5 M Calcein as Feed and 1 M NaCl Standard Solutions as Draw in a Standard Test Setting Using the Sterlitech CF042 Flow Chamber

[0162] All Membranes were Prepared Using the Protocol of Example 6 Above.

[0163] Equipment: [0164] Flexible silicone tubing (Tygon L/S25di=4.8 mm) [0165] Conductivity meter (Thermo Scientific Orion 3 star+data logging software (StarPlus Navigator, LabSpeed Navigator) [0166] Conductivity probe (Thermo Scientific 013016MD cell constant 0.1/cm working range 0.1-300 S) [0167] Pumps (Longer BT100-1L with 3 roller pumps head YZ1515x) [0168] Magnetic stirrers (Assistent Magnetmix 2070) [0169] Kern Scale 572+software Balance connection 4 [0170] CF042 FO-cell [0171] 1 CF042 sized membrane prepared as described above [0172] 2 bottles (feed/draw reservoir, plastic or glass) Draw: 2 L and feed 1 L volume [0173] Invitrogen Qubit Flourometer Q 32857 Gonotec Osmomat 030 Cryoscopic Osmometer [0174] Lab boy or similar to raise draw reservoir.

[0175] Summary of Standard FO Test Setup: [0176] membrane orientation: AS-FS [0177] run-time: >215 min/analysis time: 200 min (15 min run-in time not included in recording) [0178] flow speed on pump: 50 mL/min [0179] Draw: 1M NaCl [0180] Feed: 5 M calcein in DI water [0181] Same height of feed and draw top surface at start

[0182] Preparation of the FO Station:

[0183] 1. Fill the draw reservoir with 1 L of a 1M NaCl solution and note down the weight on the report sheet.

[0184] 2. Fill the feed reservoir with 1 L of a 5 M calcein solution and note down the weight on the report sheet.

[0185] 3. Ensure that the height of the water level in draw and feed are on the same level (use a lab boy or similar to raise the magnetic stirrer)

[0186] 4. Fill the entire system (feed and draw) via the pump (high speed)

[0187] 5. Set the pump speed to 50.03 mL/min (tube inner diameter 4.8 mm)

[0188] Results are given in Tables 2 to 5 below where desirable Jw/Js ratios ranging from the very low 0.11 and up to 0.37 are obtained in all experimental runs. In addition, very high calcein rejections of more than 99.7% were found in all runs proving the faultless nature of the tfc layer.

TABLE-US-00004 TABLE 2 200 min run time PS-PAA 8000* Jw Js Calcein 2-0.1-5** [L/m.sup.2h] [g/m.sup.2h] Jw/Js rej. Rca 7.91 1.63 0.21 99.88 7.28 1.75 0.24 99.78 6.79 1.42 0.21 99.77 5.06 0.60 0.12 99.96 6.17 1.24 0.20 99.82 5.89 0.66 0.11 99.84 *Purchased from Sigma-Aldrich, cf. Ex. 3 **2 mG/mL PS-PAA:0.1 mG/mL LDAO:5 mg/L AqpZ

TABLE-US-00005 TABLE 3 900 min run time PS-PAA 8000* Jw Js Calcein 2-0.1-5** [L/m.sup.2h] [g/m.sup.2h] Jw/Js rej. Rca 7.21 1.3 0.18 99.87 6.73 2.46 0.37 99.75 6.16 1.36 0.22 99.71 *Purchased from Sigma-Aldrich, cf. Example 3 **2 mG/mL PS-PAA:0.1 mG/mL LDAO:5 mg/L AqpZ

TABLE-US-00006 TABLE 4 200 min run time PS-PAA 23300* Jw Js Calcein 2-0.2-5** [L/m.sup.2h] [g/m.sup.2h] Jw/Js rej. Rca 7.43 2.11 0.28 99.81 7.33 1.87 0.26 99.76 7.58 1.43 0.19 99.83 7.71 1.65 0.21 99.95 7.43 2.11 0.28 99.81 *Purchased from Sigma-Aldrich, cf. Example 3 **2 mG/mL PS-PAA:0.2 mG/mL LDAO:5 mg/L AqpZ

TABLE-US-00007 TABLE 5 200 min run time PS-PAA 13000* Jw Js Calcein 2-0.25-5** [L/m.sup.2h] [g/m.sup.2h] Jw/Js rej. Rca 8.89 1.9 0.21 99.72 8.73 1.32 0.15 99.78 6.25 0.99 0.16 99.77 6.95 1.18 0.17 99.89 6.84 1.35 0.20 99.87 *Purchased from Sigma-Aldrich, cf. Example 3 **2 mG/mL PS-PAA:0.25 mG/mL LDAO:5 mg/L AqpZ

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