METHOD OF PREPARING A THIN FILM COMPOSITE LAYER

Abstract

The present disclosure relates to a method of preparing a thin film composite layer immobilizing vesicles incorporating a transmembrane protein on a porous substrate membrane, comprising providing an aqueous solution comprising the vesicles and a di-amine or tri-amine compound, covering the surface of a porous support membrane with the aqueous solution, applying a hydrophobic solution comprising an acyl halide compound, and allowing the aqueous solution and the hydrophobic solution to perform an interfacial polymerization reaction to form the thin film composite layer.

Claims

1. A method of preparing a thin film composite layer immobilizing vesicles incorporating a transmembrane protein on a porous substrate membrane, comprising a) providing an aqueous solution comprising a di-amine or tri-amine compound and the vesicles prepared by mixing the transmembrane protein and vesicle forming material comprising a mixture of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) and polyetheramine, b) covering the surface of a porous support membrane with the aqueous solution of a), c) applying a hydrophobic solution comprising an acyl halide compound, and d) allowing the aqueous solution and the hydrophobic solution to perform an interfacial polymerization reaction to form the thin film composite layer.

2. The method according to claim 1, wherein the transmembrane protein is an aquaporin water channel.

3. The method according to claim 1, wherein the transmembrane protein is solubilized in a detergent.

4. The method according to claim 3, wherein the detergent is selected from the group consisting of lauryldimethylamine N-oxide (LDAO), octyl glucoside (OG), dodecyl maltoside (DDM) or combinations thereof.

5. The method according to claim 1, further comprising addition of a buffer having a pH in the range of 7 to 9.

6. The method according to claim 1, wherein the mixture is continually agitated for 12-16 hours.

7. The method according to claim 1, wherein the di-amine compound is 1,3-diaminobenzene.

8. The method according to claim 1, wherein the proportion by weight of the di-amine or tri-amine compound to acyl halide compound is from 0:1 to 30:1 by weight.

9. The method according to claim 1, wherein the porous support membrane is formed by a polysulfone or a polyethersulfone polymer.

10. The method according to claim 1, wherein the poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) is a substantially linear polymer having an average molecular weight of between about 1,000 Da to about 15,000 Da.

11. The method according to claim 1, wherein the poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) has the chemical formula: ##STR00003## in which x denotes an integer between 10-30 y denotes an integer between 50-100 z denotes an integer between 10-30.

12. The method according to claim 1, wherein the polyetheramine is of the general structure ##STR00004## in which m is an integer of 1-15 n is an integer of 5-50 R═CH.sub.3.

13. The method according to claim 1, wherein the proportion by weight between the poly(ethylene glycol)-block-poly(propylene glycol)-block-poly-(ethylene glycol) and the polyetheramine is 5 to 1.

14. The method according to claim 1, wherein the porous support membrane is a hollow fiber.

15. The method according to claim 14, further comprising producing a hollow fiber module by assembling a bundle of hollow fibers in a housing, wherein a first solution is passed through an inlet to the lumen of the hollow fibers in one end of the housing to an outlet fluidly connected to the lumen of the hollow fibers in the other end of the housing, and a second solution is passed through a second inlet in one end of the housing to a second outlet in the other end of the housing.

16. The method according to claim 1, wherein the porous support membrane is a flat sheet.

17. The method according to claim 16, further comprising producing a spiral wound membrane module by winding the flat sheet membrane.

18. A method for embedding or incorporating vesicles comprising a transmembrane protein on a porous substrate membrane, comprising a) providing an aqueous solution comprising the vesicles prepared by mixing the transmembrane protein and vesicle forming material comprising a mixture of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) and polyetheramine, and b) covering the surface of a porous support membrane with the aqueous solution of a) using layer-by-layer deposition method to form an active layer incorporating the vesicle.

19. The method according to claim 18, wherein the vesicles are embedded or incorporated in a polyelectrolyte multilayer (PEM) film.

Description

DETAILED DESCRIPTION

[0076] Broadly, the present disclosure relates to the use of a mixture of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) and polyetheramine, to form self-assembled vesicles with transmembrane proteins, such as aquaporin water channels. The vesicles having incorporated the transmembrane protein may then be used in the production of separation membranes in which the transmembrane proteins are incorporated or immobilized, for example for allowing water molecules to pass through the membrane. For example, for the production of separation membranes comprising the transmembrane proteins, the vesicles may be added to an aqueous liquid composition comprising an aromatic amine, such as a diamine or triamine, e.g. 1,3-diaminobenzene (MPD) applied to the surface of a porous support structure, which when brought into contact with a solution of an acid chloride in an organic solvent will participate in an interfacial polymerization reaction to form a thin film composite active or selective layer on said support thus forming a separation membrane, wherein said vesicles have become immobilized or incorporated.

[0077] Without wishing to be bound by any particular theory, it is believed that the vesicles containing free available NH.sub.2 reactive 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 NH.sub.2 reactive 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. Furthermore, it is believed that by proper adjustment of the reactive components, a high vesicle loading can be obtained and thus higher flux through a certain area of the membrane. In addition, it is believed that the covalent coupling of vesicles in the TFC layer results in higher stability and/or longevity of the vesicles comprising the transmembrane proteins when incorporated in the selective membrane layer.

[0078] 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 novel 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 nanostructures 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.

[0079] The separation membrane of the present disclosure is useful in an industrial or domestic setting for preparing a pure water filtrate, such as filtering an aqueous solution through a separation membrane in a nanofiltration process or in a reverse osmosis process. For the purposes herein the term “separation membrane” includes selectively permeable membranes and semipermeable membranes for water filtration and water separation, such as asymmetric membranes comprising a porous support membrane having a selective layer formed on one side, such as a thin crosslinked aromatic polyamide layer or film or a layer of alternately charged polyelectrolytes (L-B-L). The other side may be reinforced by a woven or non-woven layer or mesh typically made of polyester fibers.

[0080] In addition, the separation membrane of the present disclosure is useful in a method for the concentration of a product solution, said method comprising utilizing a separation membrane of the present disclosure mounted in a filter housing or module to extract water from the product solution, e.g., by forward osmosis.

[0081] In an aspect of the present disclosure, it includes a hollow fiber (HF) module having a bundle of hollow fiber membranes modified with a selective layer comprising the vesicle formulation of the present disclosure. Preferably, the selective layer comprises a thin film composite (TFC) layer formed on the inside surface of the fibers through an interfacial polymerization reaction, wherein said TFC layer comprises aquaporin water channels incorporated in vesicles composed of a mixture of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly-(ethylene glycol) and polyetheramine.

[0082] The separation membrane of the present disclosure may additionally be useful in a method for the production of salinity power using pressure retarded osmosis, said method comprising utilizing said separation membrane to increase hydrostatic pressure, and using the increase in hydrostatic pressure as a source of salinity power, cf. WO2007/033675 and WO2014128293 (A1).

[0083] The term “aquaporin water channel” as used herein includes a functional natural or synthetic aquaporin or aquaglyceroporin water channel, such as aquaporin Z (AqpZ), GIPf, SoPIP2;1, aquaporin 1 and/or aquaporin 2. 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 Yf1054. 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).

[0084] The term “separation membrane” as used herein includes membranes useful for separating water and, optionally, certain small size solutes including anions and cations, from other solutes, particles, colloids and macromolecules. Examples of separation membranes are “filtration membranes” such as nanofiltration (NF) membranes, forward osmosis (FO) membranes and reverse osmosis (RO) membranes. One type of filtration membranes is a “thin film composite” (or TFC) membrane, often classified as nanofiltration and reverse osmosis membranes. Flat sheet TFC membranes are typically made by depositing a polyamide layer on top of a polyethersulfone or polysulfone porous layer on top of a non-woven or woven fabric support. The polyamide rejection layer is formed through interfacial polymerization of an aqueous solution of an amine with a solution of an acid chloride in an organic solvent. TFC membranes may be produced as described in WO 2013/043118 (Nanyang Technological University & Aquaporin A/S). Other types of filtration membranes are those formed by the layer-by-layer (LbL) deposition method, such as described in Gribova et al. (Chem. Mater., 24: 854-869, 2012) and Wang et al. (Membranes, 5(3): 369-384, 2015). For example, the vesicles of the present disclosure may be embedded or incorporated in the polyelectrolyte multilayer (PEM) films, as outlined in FIG. 4 of Gribova et al.

[0085] “Thin-film-composite” or (TFC) membranes as used herein may be prepared using an amine reactant, preferably an aromatic amine, such as a diamine or triamine, e.g.,1,3-diaminobenzene (m-Phenylenediamine, >99%, e.g. as purchased from Sigma-Aldrich) in an aqueous solution, and an acyl halide reactant, such as a di- or triacid chloride, preferably an aromatic acyl halide, e.g. benzene-1,3,5-tricarbonyl chloride (CAS No. 84270-84-8, trimesoyl chloride (TMC), 98%, e.g. as purchased from Sigma-Aldrich) dissolved in an organic solvent where said reactants combine in an interfacial condensation polymerization reaction, cf. Khorshidi et al. (2016) Scientific Reports 6, Article number: 22069, and U.S. Pat. No. 4,277,344 which describes in detail the formation of a composite membrane comprising a polyamide laminated to a porous membrane support, at the surface of the support membrane, e.g. a polyethersulfone membrane. Benzene-1,3,5-tricarbonyl chloride (trimesoyl chloride) is dissolved in a solvent, such as a C.sub.6-C.sub.12 hydrocarbon including hexane (>99.9%, Fisher Chemicals), heptane, octane, nonane, decane etc. (straight chain or branched hydrocarbons) or other low aromatic hydrocarbon solvent, e.g. Isopar™ G Fluid which is produced from petroleum-based raw materials treated with hydrogen in the presence of a catalyst to produce a low odour fluid, the major components of which include isoalkanes. Isopar™ G Fluid: Chemical Name: Hydrocarbons, C10-C12, isoalkanes, <2% aromatics; CAS No: 64742-48-9, chemical name: Naphtha (petroleum), hydrotreated heavy (from ExxonMobil Chemical). Alternatives to the reactant 1,3-diaminobenzene include diamines such as hexamethylenediamine etc., and alternatives to the reactant benzene-1,3,5-tricarbonoyl chloride include a adipoyl chloride, cyanuric acid etc. as known in the art.

[0086] The vesicles of the present disclosure may be referred to “self-assembled” to describe the process by which vesicles are formed through hydrophilic and hydrophobic interaction of the amphiphilic substances, i.e. the mixture of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) and polyetheramine.

[0087] “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.

[0088] Forward osmosis (FO) or direct osmosis is an osmotic process that uses a selective and 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/.

[0089] 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 disclosure are useful in all types of forward osmosis processes and may be specifically adapted for each forward osmosis type.

[0090] The term “reverse osmosis” (RO) as used herein refers to when an applied feed water pressure on a selectively permeable membrane is used to overcome osmotic pressure. 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.

[0091] The present disclosure is further illustrated with reference to the following non-limiting examples:

EXAMPLES

Example 1

[0092] Preparation of vesicles from Pluronic® P-123 triblock copolymer (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) Mn 5800 Da) and Jeffamine® M-2005 (polyetheramine) having a nominal 2000 molecular weight) and preparation of water membrane using said vesicles.

[0093] Materials:

[0094] Pluronic® P-123 triblock copolymer (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) having a composition of PEG20-PP070-PEG20 with molecular weight of 5800 Da was purchased from Sigma Aldrich and was used as received.

[0095] Jeffamine® M-2005 is a polyetheramine with the ratio polyethylene oxide polypropylene oxide of 29 to 6 and molecular weight of 2000 Da and was purchased from Huntsman and was used as received.

[0096] Phosphate buffer 10 mM (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 Na2HPO4 and 0.24 g of KH2PO4 in 800 mL MiliQ purified H2O, adjusting the pH to 7.2 with HCL and completing the volume to 1 L.

[0097] Aquaporin Z 5 mg/mL stock solution was prepared as disclosed below. 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.

[0098] The E. coli strain BL21(DE3) was used for expression of the protein. Luria Broth cultures containing 50 μg/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 hours 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 β-mercaptoethanol, 10% glycerol) in the presence of 0.4 mg/ml lysozyme, 50 units Bensonase and 3% n-octyl β-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 40,000×g. 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 β-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 β-mercaptoethanol, 10% 15 glycerol, containing 30 mM n-octyl β-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.

[0099] One day before the purification, 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 μM 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.

[0100] A Histrap column was equilibrated with sterile water followed by AQP Binding buffer at RT. 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 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 mil/mil 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 (column volume) 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.

[0101] 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.

[0102] When AQP quality analyses are passed, the protein concentration was 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 μM sterilized cup and stored at 4° C. in refrigerator for use within a month or else stored at −80° C. in a freezer.

[0103] Preparation method: [0104] 1. Prepare a fresh solution of Pluronic® P-123 by dissolving the polymer in PBS to a final concentration of 10 mg/mL in a glass cylinder. [0105] 2. Weight in a flask 15 mg/mL Jeffamine® M-2005. [0106] 3. Add Aquaporin Z stock solution to a final concentration of 1/200 AQPZ/polymer molar protein ratio, in which polymer is the combined amount of Pluronic® P-123 and Jeffamine M-2005. [0107] 4. Add Pluronic® P-123 solution prepared in step 1 to the mixture of Jeffamine® M-2005 and Aquaporin Z to reach 9.9 mg. [0108] 5. Stir the mixture overnight at 170 rotations per min (not more than 20 hours) at room temperature. [0109] 6. Next morning take the vesicle formulation obtained in the sequence of steps 1 to 5, transfer it to the storage flask and keep it at room temperature.

[0110] The vesicle formulation was tested for size, water permeability and zeta potential point of view by DLS, Zeta potential and stopped flow measurements in 0.5 M NaCl. The results are measured 5 times for 5 different batches.

TABLE-US-00001 TABLE 1 Formulation Dh/nm (DLS) 200 nm ± 24 (90% ± 10%) 28 nm ± 2 (0% ± 10%) Zeta potential/mV 3 ± 2 Ki/s.sup.−1 1702 ± 200 

[0111] Temperature stability and thermal behavior were tested by warming up 5 mL of vesicle formulation for 10 min at various temperatures ranging from 30° C. to 100° C. and their size and water permeability was further determined by DLS and stopped-flow measurements.

[0112] Thermal treatment does not affect significantly the stability of the formulation, resulting in the diameter shrinkage of the larger size structures from around 200 nm at room temperature to around 1800 nm. From water permeability point of view no changes can be observed up to 100° C. Ki values from 1700 to 1687 s-1 were recorded.

[0113] The pH behavior shows the disassemble of vesicle formulation at pH varying from 1 to 7 to micelles with a diameter up to 20 nm and reassembling at basic pH values (from 9 to 13 showing the same size 180 nm and Ki values around 1700 s-1.

Example 2

[0114] Preparation of BWRO (Brackish Water Reverse Osmosis) Membranes

[0115] These membranes were made according to the process outlined below: [0116] a) Dissolve MPD in MilliQ water to get a 2.5%(W/W) concentration, see below [0117] b) Dissolve TMC in Isopar to a final concentration of 0.15% W/V [0118] c) Cover a rectangular shaped membrane, e.g. 5.5 cm×11 cm Membrana 1 FPH PES membrane with about 20 mL/m2 membrane of MPD solution and leave for 30 seconds under gentle agitation [0119] d) Dry the non-active side (back side) with lab drying paper (e.g. Kim-Wipe) for 5-10 seconds [0120] e) Put the membrane on a glass plate and dry gently with N2 until the surface turns from shiny to dim [0121] f) Apply tape around the edges of the membrane(≈1 mm) [0122] g) Put the glass plate with the taped membrane into a glass or metal container, add about 155 mL/m2 membrane TMC-Isopar to one end and rock gently back and forth for 30 seconds [0123] h) Remove glass plate from reservoir and dry with N2 for 10 to 15 seconds.

[0124] 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.

[0125] MPD Solution Calculation:

[0126] Weigh off 1.05 g of MPD and dissolve in 35 mL of MilliQ. Add 7 mL of liquid AQPZ composition prepared as described in example 1. Keep the solution topped with inert gas (Ar or N2) as much as possible.

[0127] TFC membranes with liquid AQPZ formulation of 5.5 cm×11 cm sizes was then be mounted in a Sterlitech CF042 FO cell (www.sterlitech.com) and subjected to tests of 60 minutes (5 membranes) and tests of 900 minutes (4 membranes) duration in FO mode using deionised (MilliQ) water as feed and 1 M NaCl aqueous solution as draw and feed and draw speeds of 268 mL/min.

[0128] Results are given in tables 4.

TABLE-US-00002 TABLE 4 Vesicle formulation tested on the RO low pressure membranes. Applied Vesicle No. of Jw Rejection pressure, formulation Samples (L/m.sup.2h) NaCl (%) bar 6 7.15 ± 0.5 90.5 ± 0.1 5

Example 3

[0129] Preparation of Handmade TFC FO (Forward Osmosis) Filtration Membranes

[0130] The membranes were made according to the process outlined below: [0131] a) Provide a support membrane, e.g. a PES non-woven having fingerlike structure, size 5.5 cm×11 cm [0132] b) Mix 3 wt % MPD with 3 wt % ε-caprolactam, 0.5 wt % NMP, and 93.5 wt % DI water to obtain a solution [0133] c) Add 0.1 mg/mL of liquid AQPZ formulation of example 1 to obtain a suspension [0134] d) Incubate the suspension from c) for 2 hours [0135] e) Prepare TMC solution from 0.09 wt % TMC, 0.9 wt % acetone, and 99.01 wt % Isopar [0136] f) Dip coat the support membrane in the suspension d) for 30 seconds [0137] g) Apply drying with air knife [0138] h) Add the TMC solution from e) for interfacial polymerization [0139] i) Follow with 2 min drying in fume hood

[0140] Three 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.

[0141] Results are given in table 5.

TABLE-US-00003 TABLE 5 Vesicle formulation tested on the FO handmade membranes Vesicle No. of Jw Js formulation Samples (L/m.sup.2h) (gmh) Js/Jw 3 10.84 ± 1.2 2.03 ± 0.2 0.18 ± 0.1

Example 4

[0142] Preparation of FO (Forward Osmosis) Membranes

[0143] Vesicles incorporating AqpZ were prepared by firstly mixing the aqueous solution of transmembrane protein (Aquaporin Z stock solution as prepared above) with polyetheramine (15 mg/mL Jeffamine® M-2005) to obtain a final concentration of 1/200 AQPZ/polymer molar protein ratio. Subsequently, adding PEO-PPO-PEO aqueous solution (Pluronic® P-123 having a molecular weight of 5800 Da in PBS to a final concentration of 10 mg/mL), and agitating the mixture overnight at 170 rotations per min at room temperature.

[0144] Such prepared vesicles were incorporated into the polyamide thin-film composite (TFC) membrane, by interfacial polymerization on the porous support. Aqueous solution was prepared comprising the vesicle mixture (6 ml of the mixture prepared above) and m-phenylenediamine solution (prepared by dissolving 1.5 g MPD in 52.5 ml MilliQ). The organic solution comprised of trimesoyl chloride (TMC) and Isopar™ E in a concentration of 0.15% W/V.

[0145] As detailed above, the coating protocol comprised soaking the porous support with aqueous solution, followed by gentle removal of its excess. Subsequently, organic solution was applied and polyamide layer was formed, excess of organic solution was gently dried. The membranes were stored in mili-Q water prior testing.

[0146] Vesicle properties: Ki 14125.sup.−1, pH 9.83, Zeta potential −0.339 (average), size: 204 nm (average), 100% population. The dimensions of the extruded vesicles (hydrodynamic diameter) were determined by dynamic light scattering using ZetaSizer NanoZs from Malvern. The water flux through AQP channels was tested using a Bio-Logic SFM 300 stopped-flow device, using a monochromator at 517 nm and a cut off filter at 530 nm. For each individual stopped-flow test, 0.13 ml extruded polymersome or AQP inserting polymersomes was quickly mixed with 0.13 ml NaCl 0.5 M, which caused water efflux from vesicles that resulted in vesicle shrinkage. The kinetic data were fitted with a double exponential equation, and the rate constant (s.sup.−1) that is directly proportional with the water flux through polymeric membrane was determined.

[0147] The test was made on the Forward Osmosis setup with 1M salt as the draw solution and 5 μM calcein as feed. The draw and feed solution were pumped counter-currently, with the active side of the membrane facing feed solution. The results are presented in table 6 below.

TABLE-US-00004 TABLE 6 Vesicles incorp. Jv Js Calcein R Js/Jv Yes 11.17 ± 1.61  1.23 ± 0.25 99.80 ± 0.14 0.14 ± 0.02 No 4.85 ± 1.02 0.82 ± 0.74 99.85 ± 0.02 0.15 ± 0.10 Commer- >10 <3 >99% <0.3 cially available

[0148] Membranes containing amino modified vesicles incorporating AqpZ protein, resulted in improved performance when compared to the membranes without the vesicles. The average water flux through the membrane (J.sub.v) was improved by 84% (J.sub.v=11.17±1.61 Lm.sup.−2h.sup.−1 vs J.sub.v=4.85±1.02 Lm.sup.−2h.sup.−1), while rejection of calcein (R) stayed on the comparable level (R>99%). Reverse salt flux increased on average by 50% when incorporating vesicles (J.sub.s=1.23±0.25 gm.sup.−2h.sup.−1 vs J.sub.s=0.82±0.74 gm.sup.−2h.sup.—1), nevertheless the overall performance by means of specific salt flux J.sub.s/J.sub.v stayed on comparable level (0.14±0.02 for membrane with vesicles, compared to 0.15±0.10 for membrane without vesicles).

[0149] The data show that amino decorated vesicles incorporating aquaporin proteins are an efficient way to incorporate AqpZ protein into the polyamide membranes, resulting in improvement of the water flux, without compromising the specific salt flux. Without wishing to be bound by any particular theory, it can be explained that vesicles containing amine groups on the surface will be not only physically incorporated, but, in addition chemically bound in the polyamide layer of the TFC membrane, because of the presence of the reactive amine groups. These amine groups will participate in the interfacial polymerization reaction with acyl chloride to obtain vesicles being covalently bound to the layer. The covalent bonding opens the possibility for higher vesicle loading and thus higher water flux through the membranes.

Example 5

[0150] Vacuum-Mediated Coating

[0151] The porous support was mounted in a suction cell with the active layer facing upwards, and a vacuum pump applied underneath, facing the inactive layer. The support for used for the TFC layer was MicroPES 1F PH microporous support from Membrana GmbH. 50 mL of aqueous solution containing MPD and Formulation 10-2-10 in RO water was poured into the suction cell, covering the porous support. Afterwards, a suction of 100 mBar was applied for 5 minutes, sucking the MPD and formulation onto the support. Vacuum was turned off and 50 mL of organic solution containing TMC and Isopar-E was applied and given 1 minute of reaction time to facilitate the interfacial polymerization. The organic solution was then flushed out, and the membrane was left to dry for 3 minutes and was then transferred to a petri dish with RO water until ready for QC testing.

[0152] Formulation 10-2-10: 10 mg/ml Pluronic f127 (poloxamer 407)-2 mg/ml Jeffamine M2005-10 mg/ml aquaporin stock solution. in PBS buffer (137 mM NaCl, 2,7 mM KCl, 10 mM Na.sub.2HPO.sub.4 and 2 mM of KH.sub.2PO.sub.4).

TABLE-US-00005 TABLE 7 Quality testing of forward osmosis membranes by vacuum-mediated coating Average Formulation Series 1 Series 2 Series 3 and std Control - aqueous Jw: 1.45 Jw: 2.40 — Jw: 1.92 ± 0.5 solution w/o Js: 311.02 Js: 262.98 — Js: 287 ± 24 formulation 100% 99.69% 99.9% ± 0.2 2% v/v Jw: 2.72 Jw: 2.32 Jw: 2.00 Jw: 2.35 ± 0.3 formulation Js: 1.39 Js: 0.48 Js: 3.48 Js: 1.78 ± 1.3 99.93% 99.75% 99.89% 99.9% ± 0.1 5% v/v Jw: 1.69 Jw: 1.64 Jw: 1.99 Jw: 1.77 ± 0.2 formulation Js: 0.50 Js: 0.34 Js: 1.08 Js: 0.64 ± 0.3 99.95% 99.51% 99.87% 99.8% ± 0.2 7% v/v Jw: 1.38 Jw: 1.44 Jw: 1.79 Jw: 1.54 ± 0.2 formulation Js: 0.86 Js: 0.40 Js: 0.81 Js: 0.69 ± 0.2 99.76% 99.56% 99.64% 99.7% ± 0.1 10% v/v Jw: 1.98 Jw: 1.37 Jw: 2.46 Jw: 1.94 ± 0.4 formulation Js: 1.61 Js: 0.83 Js: 2.09 Js: 1.51 ± 0.5 99.82% 99.96% 99.99% 99.9% ± 0.1 Results of Quality testing of Forward Osmosis membranes prepared by vacuum-mediated coating Flux, here denoted as Jw, is measured in L/m{circumflex over ( )}2 h Salt flux, here denoted as Js, is measured in g/m{circumflex over ( )}2 h Calcein rejection is measured in %

[0153] While the water flux and the calcein rejection did not differ substantially from the control coupons, the salt flux was significantly lower in the coupons coated with solutions containing formulation as opposed to controls coated with a TFC layer without formulation. This indicates that the presence of the formulation decreased the salt flux (and thus increased salt rejection). A student's unpaired t-test showed a strong trend when comparing salt flux of control membranes with formulations, with p-values ranging from 0.053191 (2%) to 0.053386 (10%). It did not seem that an increase in percentage wise concentration of formulation in the aqueous solution used for coating influenced the level of salt rejection.

Example 6

[0154] Amino Decorated Vesicle Incorporating Transmembrane Protein

[0155] Preparation method of vesicles that incorporate Aquaporin Z: [0156] 1. Prepare a fresh solution of Pluronic® P-123 by dissolving the polymer in PBS to a final concentration of 10 mg/mL in a glass cylinder. [0157] 2. Weight in a flask 15 mg/mL Jeffamine® M-2005. [0158] 3. Add Aquaporin Z stock solution to Jeffamine® M-2005 bottle, to obtain a final concentration of 1/200 AQPZ/P-123-Jeffamine molar protein ratio. [0159] 4. Add Pluronic® P-123 solution prepared in 1. to the mixture of Jeffamine® M-2005 and Aquaporin Z to reach 9.9 mg/mL. [0160] 5. Stir the mixture overnight at 170 rotations per min (not more than 20 hours) at room temperature. [0161] 6. Next morning take the vesicle formulation obtained in the sequence of 1 to 5, transfer it to the storage flask and keep it at room temperature.

[0162] Preparation method of control vesicles which does not incorporate Aquaporin Z: [0163] 1. Prepare a fresh solution of Pluronic® P-123 by dissolving the polymer in PBS to a final concentration of 10 mg/mL in a glass cylinder. [0164] 2. Weight in a flask 15 mg/mL Jeffamine® M-2005. [0165] 3. Add Pluronic® P-123 solution prepared in 1. to the mixture of Jeffamine® M-2005 to reach 9.9 mg/mL. [0166] 4. Stir the mixture overnight at 170 rotations per min (not more than 20 hours) at room temperature. [0167] 5. Next morning take the vesicle formulation obtained in the sequence of 1 to 4, transfer it to the storage flask and keep it at room temperature.

[0168] The vesicle formulations were both tested for size, water permeability and zeta potential point of view by DLS, Zeta potential and stopped flow measurements in 0.5 M NaCl.

[0169] Characterization of Vesicles:

[0170] The dimensions of the vesicles (hydrodynamic diameter) are determined by dynamic light scattering using ZetaSizer Nano ZS from Malvern. The water flux through vesicle membrane is tested using a Bio-Logic SFM 300 stopped-flow (SF) device, using a monochromator at 517 nm and a cut-off filter at 530 nm. For each individual SF test, 0.13 ml polymersomes or AqpZ embedding polymersomes samples, were quickly mixed with 0.13 ml NaCl 0.5 M, which caused water efflux from vesicles resulting in vesicle shrinkage.

TABLE-US-00006 TABLE 8 Hydrodynamic diameter (nm) - % Intensity Zeta Osmotic Population Population potential coefficient Formulation 1. 2. (mV) pH k.sub.i (s.sup.−1) Pluronic ® 158 ± 63 − 93% 34 ± 8 − 7% +3 9.91 1700 vesicles reconstituting Aquaporin Z Pluronic ® 147 ± 49 − 95% 33 ± 6 − 5% +3 9.92 200 vesicles without Aquaporin Z

[0171] Table 8 shows the osmotic coefficient k., which is calculated based the exponential growth of the stopped-flow light scattering results for the vesicles incorporating Aquaporin Z and blank ones. The analysis of exponential growth is made on the first population of the structures showing the most rapid shrinkage. The osmotic coefficient k.sub.i (s.sup.−1) is directly proportional with the water flux through polymeric membrane and the results show that the presence of aquaporins in the vesicles significantly increases the water flux through polymeric membrane. The other properties of the vesicles are substantially unaffected by the presence of aquaporin Z, i.e. the hydrodynamic diameter, zeta potential and the pH remain at the same level.

Example 7

[0172] Preparation Method of TFC FO Membranes Incorporating Vesicles Reconstituting Aquaporin Z: [0173] a) Provide a support membrane, e.g. PES non-woven having fingerlike structure, size 5.5 cm×11 cm. [0174] b) Prepare MPD solution in MiliQ water, to obtain 2.5% (W/W) concentration. If Aquaporins are to be incorporated to the membrane, add the solution of vesicles. The final concentration of MPD solution can contain from 10 g/L to 100 g/L solution of vesicles. [0175] c) Prepare TMC solution in Isopar E, to obtain 0.15% (W/V) [0176] d) Soak rectangle shaped membrane in MPD solution to completely cover the membrane surface [0177] e) Transfer rectangle shaped membrane from MPD solution to dry the side which will be non-active side on the lab drying paper (e.g. Kim-Wipe) for 5-10 seconds [0178] f) Put the membrane on a glass plate and dry gently with N2 until the surface turns from shiny to dim [0179] g) Apply tape around the edges of the membrane(≈1 mm) [0180] h) Transfer a glass plate with the taped membrane into a glass container and cover the membrane with TMC solution, to completely cover the membrane surface [0181] i) Remove the glass plate from reservoir and dry with N2 until the surface turns shiny to dim [0182] j) Put the membrane on a glass plate in horizontal position for about 10 seconds, and remove the tape [0183] k) Transfer the membrane to the first container filled with MilliQ for 5 minutes [0184] l) Transfer the membrane to the second to the container filled with MilliQ for storage, prior testing described in subsequent steps.

Testing of TFC FO Membranes

[0185] TFC FO membranes with Aquaporin Z formulation of 5.5 cm×11 cm sizes were then mounted in a Sterlitech CF042 FO cell (www.sterlitech.com) and subjected to tests of 200 minutes duration in FO mode, using 5 μM calcein in deionised (MilliQ) water as feed and 1 M NaCl aqueous solution as draw and feed and draw speeds of 50 mL/min.

TABLE-US-00007 TABLE 9 Membrane Jv Js Calcein number calculated calculated rejection Js/Jv in batch Vesicles [Lm.sup.−2h.sup.−1] [gm.sup.−2h.sup.−1] [%] [gL.sup.−1] 1 1% of vesicles 11.17 1.45 99.88 0.13 2 incorporating 7.79 1.32 99.78 0.17 3 Aquaporin Z 9.04 1.27 99.61 0.14 4 7.73 0.87 99.91 0.11 1 Without 3.90 0.32 99.84 0.08 2 vesicles 5.01 0.59 99.84 0.12 3 6.21 1.91 99.86 0.31 4 4.27 0.44 99.87 0.10

[0186] Table 9 shows the results of FO experiment with membranes incorporating vesicles incorporating Aquaporin Z proteins and comparison to the blank ones (control membranes). It may be concluded that Jv increases by the incorporation of the vesicles incorporating Aquaporin Z and that the Js/Jv remains at the same level.