Stabilized filtration device

Abstract

The polymeric membrane structure (2) includes a membrane (3) having a water-permeable integral membrane protein (IMP) (1) coated on a first surface with a silica layer (4). The silica layer (4) stabilizes the polymeric membrane structure (2) and the water-permeable IMP (1) while maintaining the water-permeable function of the IMP (1). As a result of this stabilization, the polymeric membrane structure (2) can be used in a filtration device (5) for various filtration operations, including water purification.

Description

[Technical Field]

[0001] The present invention relates generally to polymeric membrane structures and filtration devices including such polymeric membrane structures and their use, for example, in water filtration. [Background technology]

[0002] Access to clean water has been defined by the United Nations as a human right, yet hundreds of millions of people lack access to clean drinking water and are at immediate risk of being exposed to toxic levels of contaminants in their drinking water.

[0003] Water treatment includes drinking water purification and wastewater treatment, both of which typically involve several stages of treatment to reach a desired level of purity. Filtration is commonly used in these processes, for example, to prevent undesirable compounds from appearing in drinking water and to limit the amount of pollution caused by wastewater discharges to marine ecosystems.

[0004] Filters used in water treatment can be divided into two main categories: size-exclusion filters and solution-diffusion filters. Size-exclusion filters have specific pore sizes and therefore prevent substances larger than the pores from entering the filtrate, also known in the art as permeate. A limitation of these filters is that the pore size cannot be adjusted to a diameter small enough to stop, for example, sodium and chloride ions from passing through the filter, which is necessary for the production of drinking water from seawater, i.e., desalination. The production of drinking water from seawater is becoming increasingly popular due to the decreasing amount of available sufficiently pure freshwater. Solution-diffusion filters are crucial in desalination, which is typically performed by reverse osmosis (RO). The selective layer of RO filters typically consists of thin-film composite (TFC) polymer membranes formed by interfacial polymerization. Water filtration through TFC membranes is based on a solution-diffusion mechanism: water first molecularly dissolves in the polymer matrix at the higher chemical potential side, thereby diffusing through the polymer down the chemical potential gradient and ultimately desorbing at the lower chemical potential side. To overcome the osmotic pressure involved, a pump is used to drive the filtration process. The rate-limiting step is diffusion through the polymer matrix, which can be improved at the expense of reduced selectivity. Thus, solution-diffusion filters suffer from suboptimal water diffusion rates and require large amounts of energy to purify water.

[0005] Advances in protein engineering and nanotechnology have opened up the possibility of utilizing biomimetic techniques to perform highly selective water treatment. The primary driving force behind this approach is the potential to combine high selectivity with high flux, which is not possible with the use of synthetic TFC membranes. The development of biomimetic-based filters has proven problematic due to the limited stability of biological components, such as aquaporins, outside their natural environment. Therefore, various approaches have been explored to fabricate biomimetic filters stable enough for real-world use. One approach explored to stabilize aquaporins is to deposit supported lipid bilayers (SLBs) or proteoliposomes containing aquaporins onto a porous support. The most common approach to date, judging by scientific results, is based on the insertion of aquaporins into amphiphilic block copolymer (BCP) bilayers. Aquaporins have also been stabilized using other organic molecules, such as bolaamphiphiles, and in a two-step method using amphiphilic peptides for initial aquaporin stabilization followed by polymer stabilization.

[0006] U.S. Pat. No. 9,943,812 relates to a filtering structure for enhancing stability and durability, comprising a porous support in which polymeric membrane structures containing membrane proteins are fixed to each other and to the supporting pore walls via linkers.

[0007] WO 2010 / 040353 relates to a method for producing membranes or thin fabrics for selectively transporting and / or filtering compounds between fluids. The document discloses natural or genetically engineered proteins incorporated into polymer vesicles that bind to the threads to form vesicle-thread complexes.

[0008] WO 2015 / 144724 discloses a filtration membrane comprising a porous support and a layer containing a plurality of vesicles covalently bound to its surface, the vesicles having incorporated therein a transmembrane protein. The vesicles are formed from an amphiphilic block copolymer, and the vesicles are covalently bound to each other to form a viscous mass.

[0009] WO 2010 / 091078 discloses a nanoengineered membrane containing polymerized proteoliposomes, which is a bio-nanofusogenic selective membrane using protein-incorporated UV-crosslinkable liposomes with a chemically reactive biocompatible interstitial matrix.

[0010] Ji et al., Recent developments in nanofiltration membranes based on nanomaterials, Chinese Journal of Chemical Engineering 2017, 25:1639-1652, is a review of research on nanofiltration membranes containing metal and metal oxide nanoparticles, carbon-based nanomaterials, metal-organic frameworks (MOFS), water channel proteins, and organic nanoparticles.

[0011] Despite the above approaches, the commercial potential of nanoscale biomimetic water filtration remains largely unexplored. The main reasons for limited commercial implementation are manufacturing difficulties, particularly related to the formation of macroscopic defects in the selective layer and the lack of stability necessary for practical use in membrane designs incorporating aquaporins. Therefore, despite the efforts invested in developing biomimetic water filters through various creative approaches, this research field has yet to deliver real-world performance on par with its predicted potential. Summary of the Invention

[0012] It is a general objective to provide polymeric membrane structures with improved mechanical properties.

[0013] It is a particular object to provide such polymeric membrane structures that are stable enough to be used in filtration devices.

[0014] These and other objectives are met by the embodiments disclosed herein.

[0015] The invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0016] One aspect of the embodiment relates to a polymeric membrane structure comprising a membrane comprising an integral membrane protein that allows water to pass through, the membrane having a first surface coated with a silica layer.

[0017] Another aspect of the embodiment relates to a filtration device comprising a porous support comprising a plurality of pores and the polymeric membrane structure described above.

[0018] A further aspect of the embodiment relates to a method for preparing a water filtrate, the method comprising filtering an aqueous solution through the polymeric membrane structure described above or the filtration device described above to obtain a water filtrate.

[0019] Yet another aspect of the embodiment relates to a method for concentrating a compound dissolved or dispersed in an aqueous solution, the method comprising filtering the aqueous solution through the polymeric membrane structure described above or the filtration device described above to obtain an aqueous filtrate free of the compound and a retentate containing the compound at a higher concentration than the aqueous solution.

[0020] One aspect of the embodiments relates to a method for preparing a polymeric membrane structure, the method comprising contacting a membrane containing a water-permeable integral membrane protein with a silica precursor to form a silica layer coated on a first surface of the membrane.

[0021] Another aspect of the embodiment relates to a method for preparing a filtration device, the method comprising depositing a membrane comprising a water-permeable integral membrane protein on and / or in a porous support, the method also comprising contacting the membrane comprising the water-permeable integral membrane protein deposited on and / or in the porous support with a silica precursor to form a silica layer coated on a first surface of the membrane.

[0022] The present invention provides for the stabilization of polymeric membrane structures, such as proteoliposomes, incorporating water-permeable integral membrane proteins to enable the use of these structures in filtration devices and for various filtration applications. Stabilization is achieved by coating the membrane having the water-permeable integral membrane protein with a silica layer while still maintaining the water-permeable function of the water-permeable integral protein.

[0023] The embodiments, together with further objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0024] [Figure 1] Schematic showing the silicification of proteoliposomes. After the addition of tetraethyl orthosilicate (TEOS), a silica shell is deposited onto proteoliposomes consisting of human aquaporin 4 (hAQP4) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids. [Figure 2A] 1 shows SANS profiles showing the silicification method of hAQP4-containing proteoliposomes. [Figure 2B] 1 shows CD spectra of hAQP4 during silicification of proteoliposomes. [Figure 3A] Partially opened silicified hAQP4-containing proteoliposomes with assigned geometric parameters: ti represents the thickness of the inner leaflet lipid headgroups, Dc represents the thickness of the hydrophobic lipid tails from both leaflets, t0 represents the thickness of the outer leaflet lipid headgroups, and tSiO2 represents the thickness of the silica shell. [Figure 3B] 1 is a comparison of deposited silica volume as a function of time for the silicification of liposomes (squares) and proteoliposomes (circles). The solid line shown is a fit to an Avrami-type equation. [Figure 3C] Schematic of the silicification method: hAQP4-containing proteoliposomes are mixed with the silica precursor TEOS, which forms a silica shell on the outside of the proteoliposomes. [Figure 4A] TEM micrograph of silicified hAQP4-containing POPC proteoliposomes at low magnification. The dark lines indicate the silicified aquaporin-containing lipid bilayer, and the lighter areas surrounded by or associated with the aquaporin-containing lipid bilayer indicate silica. [Figure 4B] TEM micrograph of silicified hAQP4-containing POPC proteoliposomes at high magnification. The dark lines indicate the silicified aquaporin-containing lipid bilayer, and the lighter areas surrounded by or associated with the aquaporin-containing lipid bilayer indicate silica. [Figure 4C] FIG. 1 shows a STEM micrograph of silicified proteoliposomes, with silica deposits shown as bright features. [Figure 4D] FIG. 1 shows a STEM micrograph of silicified proteoliposomes, with silica deposits shown as bright features. [Figure 5] Scanning transmission electron microscopy-energy dispersive X-ray spectroscopy (STEM-EDX) imaging was performed on dried silicified vesicles containing aquaporins. Elemental maps were obtained for sulfur (present only in aquaporins), silicon, carbon, phosphorus (present only in POPC lipids), and oxygen. The elemental maps of individual elements identified the locations of these elements in the silicified vesicles containing aquaporins. All of these elements were shown to be preferentially located in the bilayer and its associated silica coating. A STEM high-angle annular dark-field (HAADF) micrograph of the same sample area is inserted at the top left. [Figure 6] Energy-filtered transmission electron microscopy (EFTEM) was used as a complementary method to STEM-EDX for the localization of specific elements in dried silicated vesicles containing aquaporins. EFTEM, due to its increased sensitivity to lighter elements, was able to map both nitrogen and hydrogen in addition to the elements mapped by STEM-EDX. EFTEM confirmed the STEM-EDX results in that all of the relevant elements were preferentially located in the bilayer and its associated silica coating. A plasmon-filtered EFTEM image of the same sample area is inserted at the top left. [Figure 7]1 is a schematic diagram of a filtration device according to one embodiment. [Figure 8] FIG. 2 is a schematic diagram of a filtration device according to another embodiment. [Figure 9] FIG. 10 is a schematic diagram of a filtration device according to a further embodiment. [Figure 10] FIG. 10 is a schematic diagram of a filtration device according to yet another embodiment. [Figure 11] Time-resolved dynamic light scattering (DLS) was used to study the aggregation behavior of liposomes and proteoliposomes during the silicification process. DETAILED DESCRIPTION OF THE INVENTION

[0025] In general, all terms used herein should be interpreted according to their ordinary meaning in the relevant technical field unless a different meaning is clearly given and / or implied from the context in which they are used. All references to elements, apparatus, components, means, steps, etc. should be openly interpreted as referring to at least one instance of the element, apparatus, component, means, step, etc., unless expressly stated otherwise. The steps of any method disclosed herein need not be performed in the order disclosed, unless a step is explicitly described as following or preceding another step and / or unless it is implicit that a step must follow or precede another step. Any feature of any of the embodiments disclosed herein may be applied to any other embodiment, as appropriate. Similarly, any advantage of any embodiment may be applied to any other embodiment, and vice versa. Other objects, features, and advantages of the accompanying embodiments will become apparent from the following description.

[0026] The present invention relates generally to polymeric membrane structures and filtration devices including such polymeric membrane structures and their use, for example, in water filtration.

[0027] The present invention is based on the finding that the stability of polymeric membrane structures incorporating integral membrane proteins (IMPs), such as transmembrane proteins (TPs), can be enhanced by coating the membrane surface with a silica layer. Such surface coating with silica can be performed while also maintaining the functionality of the IMPs or TPs, and preferably the native conformation of the IMPs and TPs. Thus, silicification constitutes an efficient method for achieving sufficient stability in biomimetic membranes and filters, enabling the practical implementation of such polymeric membrane structures in practical applications, for example, in filtration devices and water filtration.

[0028] Accordingly, the present invention relates to a polymeric membrane structure comprising a membrane that contains an IMP or TP that allows water to pass through and is coated with a silica layer on a first surface of the membrane.

[0029] 1 shows a schematic diagram of an example of such a polymeric membrane structure in the form of a lipid bilayer vesicle 2 (also referred to in the art as a protein-containing liposome or proteoliposome 2) containing water-permeable IMP1. The proteoliposome 2, in this example, has a silica layer or shell 4 on its outer surface.

[0030] The membrane 3 of the polymer membrane structure 2 is preferably a bilayer membrane 3, i.e. a bilayer membrane 3 comprising two layers. The layers of the bilayer membrane 3 are preferably composed of amphiphilic molecules, i.e. molecules having a hydrophilic part and a lipophilic, i.e. hydrophobic part.

[0031] In certain embodiments, the membrane 3 of the polymeric membrane structure 2 is a lipid bilayer membrane 3. Thus, the amphiphilic molecules are amphiphilic lipids. Non-limiting, but illustrative, examples of such amphiphilic lipids include phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, cardiolipin, cholesterol, sphingomyelin, asolectin, diphytanoylphosphatidylcholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dihex ... dilauroylphosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(l-glycerol)] (DMPG), 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (14:0 lyso-PCs such as 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 lyso-PC), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (16:0 lyso-PE) or 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:1 Lyso-PEs such as 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dierucoyl-sn-glycero-3-phosphate (DEPA), 1,2-erucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dierucoyl-sn-alicero-3-phosphoethanolamine (DEPE), 1,2-linoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-dilauroyl-sn-glycero-3-phosphate (DLPA), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dilauro ...2-Dilauroyl-sn-glycero-3-phosphoserine (DLPS), 1,2-Dimyristoyl-sn-glycero-3-phosphate (DMPA), 1,2-Dimyristoyl-sn-glycero-3-phosphoserine (DMPS), 1,2-Dioleoyl-sn-glycero-3-phosphate (DOPA), 1,2-Oleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine ( DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycerco-3-phosphoserine (DPPS), 1,2-distearoyl-sn-glycero-3-phosphate (DSPA), 1,2-distearoyl-sn-glycero 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diostearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine These include glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), and mixtures thereof. Currently, a preferred example of an amphiphilic lipid is POPC.

[0032] Amphipathic lipids may also or alternatively be selected from natural sources, such as lipids from cell membranes and / or organelles, such as the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, and lysosomes.

[0033] In fact, the membrane of the polymeric membrane structure may actually be an intact cell membrane, such as a yeast or bacterial cell, containing a water-permeable IMP and coated with a silica layer. Furthermore, organelles containing such water-permeable IMPs can be coated with a silica layer to form the polymeric membrane structure of the present invention. In these cases, the cell or organelle membrane typically contains other membrane proteins besides the water-permeable IMP.

[0034] Alternatively or additionally, the membrane 3 may contain a cross-linkable lipid, i.e., an amphiphilic lipid having a cross-linkable chemical structure in the hydrophobic and / or hydrophilic portions of the amphiphilic lipid molecule. Examples of such cross-linkable lipids are disclosed in WO 2010 / 091078, and include, inter alia, 1-palmitoyl-2-(10Z,12Z-tricosdiynoyl)-sn-glycero-3-phosphocholine, 1-palmitoyl-2-(10Z,12Z-tricosdiynoyl)-sn-glycero-3-phosphoethanolamine, 1,2-di-(10Z,12Z-tricosdiynoyl)-sn-glycero-3-phosphocholine, and 1-2-(10Z,12Z-tricosdiynoyl)-sn-glycero-3-phosphoethanolamine. The cross-linkable lipid is preferably UV-cross-linkable, i.e., cross-linking is induced by UV exposure.

[0035] The membrane may comprise a single type or species of amphipathic lipid or a mixture of multiple, ie, at least two different types or species of amphipathic lipid.

[0036] Alternatively or additionally, the amphiphilic molecules of membrane 3 can be amphiphilic copolymers, such as amphiphilic AB, ABA, and / or ABC block copolymers. Illustrative, but non-limiting, examples of such amphiphilic copolymers include poly(methyloxazoline)-poly(dimethylsiloxane)-poly(methyloxazoline) (PMOXA-PDMS-PMOXA), poly(2-ethyl-2-oxazoline)-b-poly(dimethylsiloxane)-b-poly(2-ethyl-2-oxazoline) (PEtOz-PDMS-PEtOz), and mixtures thereof. More generally, amphiphilic copolymers include (poly)2-C 1-3 At least one hydrophilic block comprising alkyl-2-oxazoline and at least one hydrophobic block comprising PDMS, e.g., ((poly)2-C 1-3 alkyl-2-oxazoline a -PDMS b -((poly)2-C 1-3 alkyl-2-oxazoline a wherein each a is independently a number from 5 to 100, and b is a number from 5 to 140. In the case of a membrane 3 having an amphiphilic copolymer, the polymeric membrane structure 2 is a proteopolymersome.

[0037] The polymeric membrane structure 2 is preferably in the form of a vesicle, proteoliposome, or proteopolymersome 2 as shown in Figure 1. In such cases, the membrane 3 is a proteoliposome or proteopolymersome 2, the outer surface of which is coated with a silica layer 4.

[0038] In another embodiment, the polymeric membrane structure 2 is a substantially flat or 2D membrane structure having the membrane 3 as a substantially flat or planar structure. The silica layer 4 is then applied to one of the surfaces of the flat or planar membrane 3.

[0039] In one embodiment, the water channel IMP1 incorporated into the membrane 3 of the polymeric membrane structure 2 is aquaporin 1.

[0040] Aquaporins, also known as water channels, are a family of major endogenous proteins that form pores in the membranes of living cells and primarily facilitate the transport of water between cells. The cell membranes of various different bacterial, fungal, animal, and plant cells contain aquaporins, allowing water to flow through them more rapidly into and out of cells than by diffusion through the phospholipid bilayer. Aquaporins contain six transmembrane alpha-helical domains with both carboxylate and amino termini on the cytoplasmic side. Two hydrophobic loops contain the conserved asparagine-proline-alanine NPA motif.

[0041] In one embodiment, the aquaporin is selected from the group consisting of human aquaporin (hAQP), bovine aquaporin (bAQP), fish aquaporin, yeast aquaporin, plant aquaporin and bacterial aquaporin, and mixtures thereof.

[0042] There are 13 human aquaporins, which can be divided into three subgroups: water-selective (orthodox) aquaporins (hAQP0, hAQP1, hAQP2, hAPQ4, hAPQ5, hAQP6, hAQP8), which transport only water; aquaglyceroporins (hAQP3, hAQP7, hAQP9, hAQP10), which transport small uncharged solutes such as glycerol in addition to water; and superaquaporins (hAQP11, hAQP12), whose transport properties remain to be further elucidated. In one embodiment, the hAQP is selected from the group consisting of hAQP0, hAQP1, hAQP2, hAQP3, hAQP4, hAQP5, hAQP6, hAQP7, hAQP8, hAQP9, hAQP10, hAQP11 and hAQP12, preferably selected from the group consisting of hAQP0, hAQP1, hAQP2, hAQP4, hAQP5, hAQP6 and hAQP8, i.e., water-selective human aquaporins, more preferably hAQP4.

[0043] A preferred bovine aquaporin is bAQP1, and a preferred fish aquaporin is cpAQP1aa. A preferred example of a yeast aquaporin is Aqy1, and a suitable bacterial aquaporin is AqpZ. Examples of plant aquaporins that can be used include SoPIP2;1, AtTIP2;1, and AtPIP2;4.

[0044] In one embodiment, the membrane 3 comprises a single type or species of aquaporin 1. In another embodiment, the membrane 3 comprises multiple types or species of aquaporin 1.

[0045] In one embodiment, the silica layer 4 has an average thickness selected within the range of 0.1 to 1000 nm, preferably 1 to 100 nm, more preferably 1 to 10 nm. In a specific embodiment, the silica layer has an average thickness within the range of 2 to 6 nm, preferably 3 to 5 nm, more preferably 3 to 4 nm.

[0046] As previously described herein, a membrane 3 comprising a water-permeable IMP1 can be coated with a silica layer 4 as disclosed herein while maintaining the water-permeable function of the water-permeable IMP1. Thus, the water channel IMP1 remains functional in the polymeric membrane structure 2 in that it can transport water through the membrane 3 in the presence of the silica layer 4. Thus, in certain embodiments, the silica layer 4 does not prevent or block the water-permeable or transport function of the water-permeable IMP1.

[0047] In one embodiment, the silica layer 4 is a functionalized silica layer 4. Thus, the silica layer 4 includes functional molecules that can exert a desired function on the polymeric membrane structure 2. One example of such a function is a linking function (binding function). Thus, the functional molecules of the silica layer 4 can be used to fix, attach or link, e.g., covalently bond, the polymeric membrane structure 2 to a support.

[0048] In certain embodiments, the functionalized silica layer 4 comprises a silane, such as fluoroalkylsilane (FAS), an alkoxysilane, such as hexamethyldisilazane (HMDZ), or a combination thereof. These functional molecules can then be used to covalently bond the polymeric membrane structure 2 to a support using silane chemistry. For example, FAS can be used to form a covalent bond between the silica layer and an alumina support or a support having an alumina coating.

[0049] The polymeric membrane structure 2 can be prepared by a method that includes contacting a membrane 3 containing a water-permeable IMP 1 with a silica precursor to form a silica layer 4 coated on a first surface of the membrane 3.

[0050] In one embodiment, the silica precursor is selected from the group consisting of silicon alkoxides, silanes, silicates, silanols, silazanes, N-sec-butyl(trimethylsilyl)amine, and combinations thereof.

[0051] In one embodiment, the silicon alkoxide is selected from the group consisting of tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS), tetrapropyl orthosilicate, tetrabutyl orthosilicate, methyltriethoxysiloxane (MTES), dimethyldiethoxysiloxane (DMDES), tetrakis(glycerol) orthosilicate (TGS), tetrakis-(2-hydroxyethyl)-orthosilicate (THEOS), and combinations thereof.

[0052] In one embodiment, the silane is allyltrimethoxysilane, (3-aminopropyl)triethoxysilane, butyltrichlorosilane, chloropentamethyldisilane, 1,2-dichlorotetramethyldisilane, diethoxydiphenylsilane, [3-(diethylamino)propyl]trimethoxysilane, dimethoxydimethylsilane, dimethoxy(methyl)octylsilane, (3-glycidyloxypropyl)trimethoxysilane, hexamethyldisilane, isobutyl(trimethoxy)silane, methyltrichlorosilane, pentamethyldisilane , n-propyltriethoxysilane, tetraethylsilane, 1,1,2,2-tetramethyldisilane, tetramethylsilane, triethoxymethylsilane, triethoxyoctylsilane, trimethoxyphenylsilane, triethoxyphenylsilane, triethoxyvinylsilane, trimethoxymethylsilane, gamma-aminopropyltriethoxysilane, silicon tetrachloride (tetrachlorosilane), silicon tetrabromide (tetrabromidosilane), gamma-aminopropylsilanetriol (APSTOL), and combinations thereof.

[0053] In one embodiment, the silicate is sodium silicate (water glass).

[0054] In one embodiment, the silanol is selected from the group consisting of tris(tert-pentoxy)silanol, tris(tert-butoxy)silanol, and combinations thereof.

[0055] In one embodiment, the silica precursor is TEOS.

[0056] In one embodiment, the membrane 3 is contacted with the silica precursor in a buffer solution containing a buffering agent. Non-limiting, but illustrative, examples of buffering agents that can be used in the buffer solution include tris(hydroxymethyl)aminomethane (Tris), phosphate-buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 2-(N-morpholino)ethanesulfonic acid (MES), and 3-(N-morpholino)propanesulfonic acid (MOPS). One example of a suitable buffering agent is Tris. The buffer solution can then be an aqueous Tris-HCl solution.

[0057] In one embodiment, the buffer solution contains a salt to adjust or set the ionic strength of the buffer solution. Non-limiting, illustrative examples of salts that can be used include chloride salts, sulfate salts, carbonate salts, and mixtures thereof. Chloride salts that can be used according to the embodiment include NaCl, KCl, CaCl2, and MgCl2. MgSO4 and CaSO4 are suitable examples of sulfate salts, while Na2CO3 is a preferred example of carbonate salts.

[0058] The pH of the buffer solution is determined at least in part based on one or more buffering agents. The pH of the buffer solution can be acidic, i.e., less than 7, neutral, i.e., about 7, or basic, i.e., greater than 7. The pH of the buffer solution affects the properties of the formed silica layer 4. For example, an acidic pH, such as pH 2, produces a denser silica layer 4 compared to a neutral or basic buffer solution. Correspondingly, the formation of the silica layer under slightly basic conditions (pH 8) proceeded by the initial placement of silicic acid around the membrane 3, followed by densification.

[0059] The water-permeable IMP1 in the water-selective Membrane 3 effectively excluded certain reagents used in the silicification process from entering or passing through the membrane containing the water-permeable IMP1. This was an important and highly unexpected advantage of the present invention, as the reagents would otherwise adversely affect the Membrane 3 and polymeric membrane structure 2 and potentially contaminate the filtrate passing through the Membrane 3.

[0060] Polymeric membrane structures 2 of embodiments, such as those prepared according to the methods described above, may include a thin layer of water between the membrane 3 and the silica layer 4. Such a water layer may be encapsulated during the preparation method and typically has a thickness in the nm or sub-nm range.

[0061] In one embodiment of the method for producing silica-coated proteoliposomes 2 containing aquaporin 1, phosphatidylcholine lipids, such as 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine (POPC), are first uniformly dispersed in an aqueous buffer solution, which may contain, for example, either Tris or PBS to set the pH and a salt such as NaCl to set the ionic strength. The lipids may or may not be dissolved in a solvent less polar than water, such as chloroform (CHCl), and the CHCl may then be removed before dispersion in the aqueous buffer solution. A mild detergent, such as n-octyl-β-D-glucoside (β-OG) or n-nonyl-β-D-glucoside (β-NG), can be added to solubilize the liposomes. Purified aquaporin stabilized with a mild detergent, such as β-OG or β-NG, is then added to the liposome mixture. The detergent is then preferably removed from the mixture, for example, by adsorption onto polystyrene beads followed by removal, or by dialysis using cellulose acetate dialysis membranes with a molecular cutoff of 1000 or 2000 Da. This procedure results in multilamellar and polydisperse vesicles 2 containing aquaporin 1.

[0062] In one embodiment, vesicles 2 may be further processed prior to siliconization to reduce the degree of multilamellarity and polydispersity. For example, vesicles 2 containing aquaporin 1 may be extruded through the pores of a polycarbonate extrusion membrane or a nylon centrifugal filter having a diameter ranging from 30 nm to 1000 nm to reduce the degree of multilamellarity and polydispersity.

[0063] A layer of silica (4) ranging in thickness from 0.1 nm to 1000 nm can then be formed on the aquaporin-1-containing vesicles. In an exemplary silica coating procedure, a silicon alkoxide, such as TEOS, or cation-exchanged sodium silicate (water glass) is added to the aquaporin-1-containing vesicles (2). The silicon alkoxide can be pre-hydrolyzed in a solution free of vesicles (2) before addition to the aquaporin-1-containing vesicles (2), or added directly to the aquaporin-1-containing vesicles (2). Hydrolysis of the silicon alkoxide results in the formation of orthosilicic acid and silicon alkoxide Q1, Q2, and Q3 species, where 0, 1, 2, and 3 alkoxide groups are covalently bonded to silicon due to incomplete hydrolysis of the silicon alkoxide. Condensation of these species in the presence of the aquaporin-1-containing vesicles (2) results in the formation of a silica layer (4) on the exterior of the aquaporin-1-containing vesicles (2). Silica-coated vesicles 2 containing aquaporin 1 may aggregate during condensation, which may result in the formation of a second silica coating on the aggregates ranging in thickness from 0.1 nm to 300 nm.

[0064] In one embodiment, the formation of the silica layer 4 may be followed by surface modification of the vesicles 2 containing aquaporin 1. In an exemplary procedure, functional groups, such as silanes, are introduced into the silica layer 4 of the vesicles 2 containing aquaporin 1. In an exemplary procedure, the coated vesicles 2 containing aquaporin 1 are placed in hexane containing 1% (volume / volume) hexamethyldisilazane (HMDZ). Available silanol groups (Si-OH) on the silica surface are partially replaced with methylated groups. Other examples of silanes that can be used include, but are not limited to, fluoroalkylsilanes (FAS) and alkoxysilanes.

[0065] Another aspect of the present invention relates to a filtration device 5. See Figures 7 to 9. The filtration device 5 includes a porous support 6 including a plurality of pores 7 and a polymeric membrane structure 2 according to an embodiment.

[0066] In one embodiment, the polymer membrane structure 2 is provided on the surface of a porous support 6. In an exemplary embodiment, vesicles 2 containing a water-permeable IMP are introduced into a porous support 6 having surface-accessible pores 7, e.g., with pore widths in the range of 0.5 to 50 nm. The vesicles 2 are adsorbed onto the porous support 6 and collapse into a supported lipid bilayer containing a single pore-piercing aquaporin or a stack of supported lipid bilayers containing multiple aquaporins.

[0067] Alternatively or additionally, the polymer membrane structure 2 is disposed within the pores 7 of a porous support 6. Depending on the material of the porous support 6, the pores 7 may be regular or irregular, have clearly defined pores 7 with specific diameters, such as 50 nm to 5000 nm, or may be vaguely defined by a network structure. The pore width may be the same throughout the thickness of the porous support 6, or may be wider at one end of the porous support 6 compared to the other end. The pore width may also vary throughout the porous support 6 regardless of the position in the thickness. Vesicles 2 containing IMP1, which are permeable to water, can be introduced into the pores 7 by placing the porous support 6 in a solution of vesicles 2. External forces, such as those introduced by applying pressure or suction, may or may not be used to successfully introduce the vesicles. In one example, the porous support 6 is placed in a filter holder connected to a syringe pump via tubing.

[0068] The porous support 6, together with the polymeric membrane structure 2, forms a filtration device 5 or filter that can be used to filter a variety of liquids, as further disclosed herein. Coating the membrane 3 with a silica layer 4 improves the stability of the polymeric membrane structure 2 and the water-permeable IMP 1, allowing them to be used in such filtration operations while still having a sufficient operational or shelf life, which is otherwise a significant problem with prior art biomimetic filtration devices.

[0069] The porous support 6 can be any support that is semi-permeable, i.e., allows filtrate to pass through the porous support 6 and the polymeric membrane structure 2 present therein or thereon. The porous structure 6 supports the polymeric membrane structure 2 but should also be durable to the operation or method to be performed, such as being able to withstand a particular pressure or chemical environment. The porous support can take any shape, including a flat membrane, a circular membrane, etc., to suit the operation or method to be performed.

[0070] In one embodiment, the porous support 6 can be made from a polymer, a metal, a metal oxide, silicon dioxide, glass fiber, or a mixture thereof. Non-limiting, but illustrative, examples of polymeric materials for the porous support 6 include polysulfone, polyethersulfone, polyphenylsulfone, polyetherethersulfone, polyetherketone, polyetheretherketone, polyphenylene ether, polydiphenylphenylene ether, cellulose, polyvinylene cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose nitrate, polyphenylene sulfide, nitrocellulose, acetylated methyl cellulose, polyacrylonitrile, polyvinyl alcohol, polycarbonate, organosiloxane carbonate, polyester carbonate, organopolysiloxane, polyethylene oxide, polyamide, polyimide, polyamideimide, polybenzimidazole, polyolefin, polyacrylonitrile, nylon, and mixtures thereof. Non-limiting, but illustrative examples of metal (oxide) materials for the porous support 6 include aluminum, aluminum oxide (alumina), titanium, titanium dioxide, zirconium, zirconium dioxide (zirconia), iron, iron oxide, and mixtures thereof.

[0071] The polymeric membrane structure 2 may be provided on the porous support 6 and / or within the pores 7 of the porous support 6 in the form of a single layer, as shown in FIG. 8, or may comprise a thicker layer of polymeric membrane structure 2 as shown in FIG. 7, which typically comprises multiple layers of polymeric membrane structure 2.

[0072] 10 shows an embodiment of a filtration device 5 in which a membrane 3 containing an IMP 1 that allows water to pass through is in the form of a planar structure deposited on a porous support 6 and contains pores 7. The side of the membrane 3 facing the porous support 6 and the opposite side of the membrane 3 are coated with a silica layer 4.

[0073] When the silica layer 4 of the polymeric membrane structure 2 has functional groups as described herein above, the functional groups of the functionalized silica layer may link the polymeric membrane structure 2 to the porous support 6, such as by covalent bonding. For example, the functional groups may anchor, attach, and secure the polymeric membrane structure 2 to the walls of the pores 7 of the porous support 6 and / or to the top surface of the porous support 6.

[0074] In one embodiment, the polymeric membrane structure 2 is coated with a silica layer 4 before being deposited on and / or in the porous support 6 of the filtration device 5. In another embodiment, a membrane 3 containing IMPs 2 that allow water to pass through, for example in the form of proteoliposomes 2, is first deposited on and / or in the porous support 6, and then a silicification process is carried out to coat the proteoliposomes 2.

[0075] In this latter embodiment, the method for preparing the filtration device 5 includes depositing a membrane 3, e.g., proteoliposomes 2, comprising a water-permeable IMP1 on and / or within a porous support 6, and contacting the water-permeable IMP1-comprising membrane 3 deposited on and / or within the porous support 6 with a silica precursor to form a silica layer 4 coated on a first surface of the membrane 3.

[0076] In one embodiment, membranes 3, such as in the form of proteoliposomes 2, can be fixed or attached to a porous support 6 prior to the siliconization process. In such an embodiment, proteoliposomes 2 can be attached to the upper surface of the porous support 6 and / or to the walls within the pores 7 of the porous support 6. Attachment and fixation can be achieved by linkers such as those disclosed in U.S. Pat. No. 9,943,812. Such linkers can be selected from the group consisting of primary amine crosslinkers, sulfhydryl crosslinkers, carbohydrate crosslinkers, carboxyl crosslinkers, and photoreactive crosslinkers. Primary amine crosslinkers can be imidoesters, N-hydroxysuccinimide esters, or glutaraldehydes, while sulfhydryl crosslinkers can be maleimides, haloacetyls, or pyridyl disulfides. Carbohydrate-reactive crosslinkers can be 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or 1,3-dicyclohexylcarbodiimide, and photoreactive crosslinkers can be aryl azides or diazirines.

[0077] The polymer membrane structure 2 and the filtration device 5 of the embodiment can be used for water purification to prepare a water filtrate. In such an application, an aqueous solution is filtered through the polymer membrane structure 2 or the filtration device 5 to obtain a water filtrate.

[0078] The aqueous solution may be, for example, contaminated or polluted water, salt water, or indeed any aqueous solution to be filtered or purified by removing contaminants or contamination such as salt ions from the water to produce a pure water filtrate.

[0079] The polymeric membrane structure 2 and filtration device 5 of the embodiments can also be used in a method for concentrating a compound dissolved or dispersed in an aqueous solution, which method includes filtering the aqueous solution through the polymeric membrane structure 2 or filtration device 5 to obtain a compound-free aqueous filtrate and a retentate, also referred to in the art as a retentate, which contains the compound at a higher concentration than the aqueous solution.

[0080] Thus, filtering an aqueous solution through the polymeric membrane structure 2 or filtration device 5 provides an aqueous filtrate that enriches the retentate with respect to compounds dissolved or dispersed in the retentate.

[0081] Thus, once the polymeric membrane structure 2 comprising the water-permeable IMP 1 is deposited and, optionally, immobilized on and / or within the porous support 6, the resulting filtration device 5 may be useful in methods of water filtration to provide a pure water filtrate. This may be done using methods such as forward osmosis, reverse osmosis, or pressure-retarded osmosis. The same configuration may also be used to concentrate a solution of a target molecule by removing excess water from the solution containing the target molecule.

[0082] Aquaporin-1-containing water-permeable IMP1-containing membranes 3 or vesicles 2 can be formed by incorporating water-permeable IMP1 into membranes 3 or vesicles 2 by a process called reconstitution, which generally involves solubilizing water-permeable IMP1 using specific detergents, which aid in the removal of water-permeable IMP1 from their source membranes while maintaining the integrity and biological function of water-permeable IMP1. Once solubilized, water-permeable IMP1 can be reinserted into the target membrane 3 or vesicle 2 of interest.

[0083] Water-permeable membranes 3 or vesicles 2 containing IMP1 can also be formed by converting source membranes into membranes 3 and vesicles 2, thus preserving as much of their natural environment as possible. Such conversion is performed by extruding the source membrane through a porous filter with a predetermined pore size, thus reducing the source membrane to small fragments and vesicles 2 of a specific size. [Example]

[0084] Example This example describes a method for coating proteoliposomes with a thin layer of silica. More specifically, the passive water transporter human aquaporin 4 (hAQP4) was reconstituted in phosphatidylcholine (POPC) liposomes, which were then coated with a thin layer of silica. The proteoliposome silicification process was closely monitored to elucidate the mechanism of silica shell growth. The secondary structure of hAQP4 was also monitored throughout the silicification process to provide an assessment of protein compatibility in this type of silicification.

[0085] hAQP4 production in Pichia pastoris and protein purification Protein production in P. pastoris was performed according to a protocol based on the production of genetically optimized hAQP4 [Nyblom et al., Protein Expr. Purif. 2007, 56(1):110-120], resulting in a yield of more than 300 g of wet cells per L of culture. Cells were harvested by centrifugation (6000 g, 45 min, 4°C) and stored at -20°C. For membrane preparation, 85 g of cells were thawed at 4°C and resuspended in 200 mL of disruption buffer (50 mM Tris-HCl pH 7.4 (Sigma-Aldrich) containing 150 mM NaCl (Sigma-Aldrich), 1 mM 2-mercaptoethanol (Fluka AG), and two EDTA-free cOmplete protease inhibitor cocktail tablets (Roche)). Cells were disrupted with 0.5 mm glass beads (Scientific Industries) using a Bead Beater (BioSpec) for 12 30-second periods, with a 60-second cooling period between runs. Undisrupted cells were collected by centrifugation (6000 g, 10 min, 4°C), and crude membranes were then recovered from the supernatant by ultracentrifugation (19000 g, 60 min, 4°C). The resulting membranes were washed with urea buffer (4 M urea, 5 mM Tris-HCl pH 7.4, 2 mM EDTA, 2 mM EGTA) using a homogenizer and centrifuged again (19000 g, 60 min, 4°C). The resulting pellet was homogenized and subjected to a sodium hydroxide wash (20 mM NaOH) followed by a subsequent centrifugation step (19000 g, 60 min, 4°C). A final wash was performed to remove traces of NaOH by homogenizing the membranes in membrane resuspension buffer (20 mM Tris-HCl pH 7.4, 250 mM NaCl, 1 mM 2-mercaptoethanol, 10% (wt / vol) glycerol). After a final centrifugation (19000 g, 60 min, 4°C), the washed membrane pellet was resuspended in resuspension buffer at a concentration of approximately 400 mg membranes / ml.

[0086] hAQP4 membrane solubilization was performed by mixing the membranes with solubilization buffer (25 mM Tris-HCl pH 7.4, 250 mM NaCl, 1 mM 2-mercaptoethanol, 10% (wt / vol) glycerol, 400 mM n-octyl-β-glucopyranoside (OG, analytical grade, Anatrace)) supplemented with an EDTA-free protease inhibitor cocktail tablet at a 1:1 volume ratio. After gentle stirring at 4 °C for 90 min, the insolubilized material was removed by ultracentrifugation (19,000 g, 60 min, 4 °C), and imidazole was added to the supernatant to a final concentration of 50 mM imidazole.

[0087] For purification of hAQP4, the supernatant was loaded onto a pre-equilibrated 5 ml Ni-NTA HisTrap HP column (GE Healthcare) and circulated for at least 2 hours. Prior to loading the supernatant, the column was equilibrated for 3 CV (20 mM Tris-HCl pH 7.4, 300 mM NaCl, 10% glycerol, 40 mM OG, 50 mM imidazole). Nonspecifically bound proteins were removed by washing the column matrix with 20 ml of equilibration buffer. The protein was eluted with 20 ml of elution buffer (20 mM Tris-HCl pH 7.4, 300 mM NaCl, 10% glycerol, 40 mM OG, 300 mM imidazole) and collected in fractions. Protein fractions were analyzed using SDS-PAGE, and protein-containing fractions were pooled. Subsequent buffer exchange into storage buffer (25 mM citrate pH 6.0, 50 mM NaCl, 5% (wt / vol) glycerol, 40 mM OG, 2 mM DTT) was then performed immediately. Finally, hAQP4 was concentrated to a final concentration of 9.7 mg / ml using a 50 kDa cutoff concentrator (Merck Millipore) and stored at -80°C.

[0088] Preparation of proteoliposomes Proteoliposomes were formed from POPC liposomes and purified hAQP4 by a reconstitution method. Chloroform (Sigma-Aldrich) containing POPC (Avanti Polar Lipids Inc.) was rotary evaporated at 40°C for 3 hours, followed by removal of residual chloroform under nitrogen. Lipid films were prepared at 10 mg ml -1 The lipids were resuspended in reconstitution buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0) at a concentration of 4 mg ml, and then adjusted with DO (99.8 atom % D, Sigma-Aldrich) for the SANS experiments (pH 8.4) and with Milli-Q water for the remaining experiments. -1 For 1 ml of proteoliposomes, 10 mg ml -1 400 μl of reconstitution buffer containing POPC was mixed with 30 μl of 1 M NaCl (Sigma-Aldrich), 30 μl of 1 M Tris-HCl pH 8 (Sigma-Aldrich), and 430 μl of Milli-Q water. 99 μl of 10% (wt / vol) n-octyl-β-D-glucoside was then added with mixing, followed by a 5-minute incubation. 9.7 mg ml -1 Purified hAQP4 was added in an amount that resulted in a final protein-to-lipid ratio (PLR) of 1 part hAQP4 to 6 parts POPC lipid (1:6) by mass for CD measurements and 1:50 for the remaining experiments. The solution was gently mixed and incubated at 20°C for 10 min. Biobeads SM2 adsorbent (Bio-Rad Laboratories) was equilibrated with reconstitution buffer and then added at a wet fraction of 30% (weight / volume) of the sample volume, followed by incubation for 6–10 h at 20°C on a rotating table in the dark. Biobeads were removed from the sample by centrifugation at 11,000 rpm through a 0.2 μm spin column filter (WVR) before use.

[0089] Characterization of proteoliposomes by DLS DLS analysis was performed using disposable UVette® cuvettes (Eppendorf) on a Malvern Zetasizer Nano ZS (Malvern) instrument at a fixed detection angle of 173°, with a concentration of 0.05 mg ml -1 Liposome and proteoliposome samples containing 150 μl of lipid were tested at 20° C. Data presented are the average of three replicates performed consecutively.

[0090] Silicification of proteoliposomes For all experiments except time-resolved DLS and stopped-flow light scattering, 4 mg ml -1 Silicated liposomes and proteoliposomes were formed using 7.6 μl of tetraethyl orthosilicate (TEOS, 98%, Sigma-Aldrich) added to 800 μl of reconstitution buffer containing 2 mg ml of liposomes and proteoliposomes, and 3.8 μl of TEOS was added to 2 mg ml of reconstitution buffer. -1 The liposomes and proteoliposomes were added to 800 μl of reconstitution buffer. Siliconization was performed in glass vials without stirring at 25 °C for 4 to 16 h in all cases except for the in situ SANS siliconization kinetics experiments.

[0091] Characterization by TEM, STEM, SANS, DLS, stopped-flow light scattering and CD For transmission electron microscopy (TEM) analysis, 4 mg ml -1 A 2 μl droplet of reconstitution buffer containing silicified liposomes and proteoliposomes was placed on a Lacey carbon 300 mesh copper grid (Ted Pella Inc.) and allowed to dry in ambient air. TEM analysis was performed using an FEI Titan 80-300 operated at 300 kV and an FEI Tecnai TF20 operated at 200 kV. Samples for scanning TEM (STEM) were prepared in the same manner as for TEM, and STEM analysis was performed using an FEI Titan 80-300 operated at 300 kV.

[0092] Small-angle neutron scattering (SANS) experiments were performed on the D11 instrument at ILL (Institut Laue-Langevin, Grenoble, France) and on the KWS-1 instrument at FRMII (Research neutron source Heinz-Maier Leibnitz, Garching, Germany).

[0093] In FRMII, a narrow quartz cell was used. No rotation was applied. 4 mg / ml -1 Non-silicified and silicified samples were prepared in both Milli-Q water and DO. These samples were contrast-matched, with silica having an SLD of 3.47 and silicon having an SLD of 2.07. They were studied as is, and a "kinetic" study was also performed analyzing proteoliposome samples before, during, and after silicification. λ = 7 Å was used. Detectors were positioned at 1.5 m, 8 m, and 20 m to span a wide q range with good overlap between data sets. Data were reduced and modeled using QtiKWS software.

[0094] At the ILL, Hellma Analytics 120-QS quartz cells with a 2 mm optical path were used. They were mounted in a motorized holder that rotated the sample at 5.5 revolutions per minute to prevent settling during the silicidation process. 4 mg ml -1 The proteoliposome samples were diluted in DO to match the contrast with silica at an SLD of 3.47. Pre-silicified proteoliposomes and the silicification method for proteoliposomes were studied. λ = 5 Å was used to obtain the highest possible intensity. Detectors were positioned at 1.4 m, 8 m, and 39 m to span a wide q range with good overlap between data sets. The temperature was 25°C for both SANS experiments. Data were reduced using BerSANS software [Keiderling, Appl. Phys. A-Mater. Sci. Process 2002, 74:S1455-S1457], while data were modeled using QtiKWS software.

[0095] Disposable UVette® cuvettes (Eppendorf) were used to measure 2 mg ml of HCl at a fixed detection angle of 173° in a Malvern Zetasizer Nano ZS (Malvern) instrument. -1 Time-resolved DLS silicification analysis was performed on 150 μl of liposome and proteoliposome samples containing 2 mg ml lipid. Following the initial DLS recording, -1 To 800 μl of reconstitution buffer containing 100 μl of liposomes and proteoliposomes, 3.8 μl of TEOS was added. 150 μl of sample was analyzed by DLS every 30 minutes, at which point the sample was returned to the siliconized vial. The temperature was 20°C, and the data presented are the average of three consecutive replicates.

[0096] 2mgml -1 Time-resolved stopped-flow light scattering experiments were performed by rapidly mixing reconstitution buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0) containing silicified samples of liposomes and proteoliposomes with a hyperosmotic solution (reconstitution buffer containing 300 mM sucrose). Following the initial recording, 2 mg ml -1 3.8 μl of TEOS was added to 800 μl of reconstitution buffer containing liposomes and proteoliposomes. Experiments were performed at 1-h intervals on an SFM2000 (BioLogic Science Instruments), with each rapid mix requiring 80 μL of sample and 80 μL of hyperosmolar buffer. Scattering was monitored at a fixed angle of 90°, and data were collected at a wavelength of 438 nm. To obtain the rate constant for water transport, the collected data were fitted to a biexponential function, which is shown as the k value.

[0097] Circular dichroism (CD) spectra were obtained on a Chirascan™ circular dichroism spectrometer equipped with a Peltier temperature controller. The lamp, monochromator, and sample chamber were purged with N2(g) for 5 minutes before analysis at flow rates of 1 L / min, 3 L / min, and 1 L / min, respectively. Spectra were recorded from 190 nm to 250 nm in a 1 mm pathlength quartz cuvette using a 1 nm bandwidth and a 10 second time constant. Data below 200 nm were omitted because buffer absorbance below 198 nm would cause a high voltage (HV) exceeding the 700 V threshold. Data presented are the average of three scans recorded at 25°C. Data were subjected to background (pure buffer) subtraction before presentation. 1 mg / ml -1 Measurements were performed in reconstitution buffer at the lipid concentration, mass of LPR6. To account for the increase in absorption caused by the formation of the silica shell, CD data were normalized to the shift in applied HT voltage.

[0098] Derivation of a SANS model for silicified proteoliposomes STEM analysis of silicified proteoliposomes shows a core-shell structure in which the proteoliposomes are covered by a thin silica shell. The geometric model parameters of the bilayer and silica shell used for SANS data analysis are shown in Figure 3A. Each proteoliposome has a radius R core The proteoliposome consists of a spherical aquaporin-containing lipid bilayer that encases a liquid-filled core of 1000 sq m. The protein-containing lipid bilayer that constitutes the shell of the proteoliposome is divided into three radial layers based on the composition, with a thickness of 1000 sq m. i The inner hydrophilic lipid head region layer, containing the protein, has a thickness of D c of hydrophobic lipid tail layer, and thickness t o The total radius of the proteoliposome is therefore the total radius at the equator R tot is defined by R tot is the core radius R core and the thickness of the three layers of the lipid bilayer, t i , D c and t oThis model is the sum of the thickness t silica The silica layer also includes:

[0099] Therefore, the total scattered intensity I(q) can be written as:

number

[0100] The scattering amplitude of each concentric spherical layer can be described by:

number

number

number

number

number

number

number

[0101] To illustrate the method of silica deposition onto proteoliposomes, silica The parameter f was introduced to indicate the fraction of proteoliposomes covered by a silica shell. protein was similarly used to indicate the volume fraction of protein in the sample. Both silica and protein are separated by the inner lipid head layer (f sil,i、 f prot,i ), lipid tail layer (f sil,c、 f prot,c ), and the outer lipid head layer (f sil,o、 f prot,o ) in f x,i +f x,c +f x,o =1 where the subscript x represents sil or prot. The number of lipids in each proteoliposome, P, is

number

[0102] The contrast between the different layers is Δp i =p head (1-f prot,i f protein -f sil,i f silica -f w,i )+p prot f prot,i f protein +p silica f sil,i f silica +p solvent f w,i -p solvent Δp c =p tail (1-f prot,c f protein -f sil,c f silica )+p prot f prot,c f protein +p silica f sil,c f silica -p solvent Δp o =p head (1-f prot,o f protein -f sil,o f silica -f w,o )+p prot f prot,o f protein +p silica f sil,o f silica +p solvent f w,o -p solvent Δp silica =p silica (1-f w,silica )+p solvent f w,silica -p solvent is defined by In the formula, p head , p tail , p silica and psolvent are the scattering length densities (SLDs) of the lipid head group, lipid tail, silica, and bulk solvent, respectively.

[0103] Finally, the dry volume of silica deposited on each liposome / proteoliposome was calculated as

number

[0104] result The structural characteristics of liposomes and proteoliposomes were evaluated using DLS and SANS. The polydispersity index (PDI) determined by DLS was 0.15 for liposomes and 0.27 for proteoliposomes. The vesicle size (approximately 2 × 10 -3 Å -1 The broadness of the SANS peaks corresponding to the vesicles (located at ) also suggested polydispersity in sample size. The liposomes and proteoliposomes had Z-avg diameters of 124.8 nm ± 2.9 nm and 132.9 ± 7.0 nm, respectively. The data were also weighted by number to compensate for the non-proportional light scattering contribution from large vesicles, which were apparently present due to the polydispersity of both samples. The weighted average diameters were then 78.0 nm ± 5.1 nm for liposomes and 66.0 nm ± 3.2 nm for proteoliposomes.

[0105] SANS characterization of the samples before addition of the silica precursor was performed using two contrasts that matched the scattering length density (SLD) of DO and silica (CMSiO). Simultaneous fitting of these contrasts to a custom-designed core-multishell model yielded diameters of 45 nm, internal lipid headgroup thicknesses of 9 Å, and lipid tail layer thicknesses of 34 Å for both liposomes and proteoliposomes. The thickness of the external lipid headgroups was 10 Å for liposomes and 12 Å for proteoliposomes.

[0106] SANS was used to study the silicification process and the evolution of the silica shell to elucidate how liposomes and proteoliposomes are affected in the process. The size and composition of the bilayer compartments and silica shell were obtained. SANS profiles obtained during the silicification of proteoliposomes are shown in Figure 2A. A reasonable fit to the silicification process was achieved by adjusting five fitting parameters: the concentration of species in the neutron beam (conc.), the fraction of silica-coated liposomes / proteoliposomes (f silica ), thickness of the outer lipid head group (t o ), the thickness of the silica shell (t silica ), and the water content of the silica shell (f w,silica The dry volume of the silica shell (V silica,dry , derived using Equation 1) along with the initial and final fitted values ​​of the selected parameters are shown in Table 2.

number

[0107] V silica,dry is also shown as a function of time in Figure 3B. The data shown in Figure 3B was fitted to the following Avrami-type growth function [Avrami, J. Chem. Phys. 1940, 8:212-224, and Avrami, J. Chem. Phys. 1941, 9(2):177-184]:

number

number

[0108] Time-resolved DLS was used to study the aggregation behavior of liposomes and proteoliposomes during the siliconization process. The scattered light intensity began to increase in both liposome and proteoliposome samples after approximately 3 hours of siliconization (Figure 11). This increase in scattering is attributed to the aggregation of the siliconized liposomes and proteoliposomes.

[0109] The functionality of aquaporins during and upon silicification was assessed using time-resolved stopped-flow light scattering. As can be seen in Table 1, proteoliposomes highly retain their water transport capacity upon silicification, as evidenced by the small changes in k values. The lipid membrane itself appears to become slightly water-permeable. The relatively low R obtained from a 10-hour fit 2 The values ​​are due to the lower signal-to-noise ratio of vesicle shrinkage due to the presence of large aggregates. [Table 1]

[0110] The effect of silicification on hAQP4 secondary structure was assessed using CD (Figure 2B). The results show that the predominantly α-helical native secondary structure of the protein was largely preserved upon silicification. [Table 2] [Table 3]

[0111] Upon complete siliconization and drying, silica-coated liposomes and proteoliposomes were studied using TEM. Results collected using TEM (Figure 4A, Figure 4B) and STEM (Figure 4C, Figure 4D) further described the geometric and compositional characteristics of the siliconized samples. STEM characterization indicates that the siliconized liposomes and proteoliposomes primarily adopt a core-shell architecture.

[0112] The silicified proteoliposomes were also subjected to elemental mapping. As can be seen in Figure 5, STEM energy-dispersive X-ray spectroscopy (EDX) was used to localize different elements. A STEM high-angle annular dark-field (HAADF) micrograph of the same sample area is also inserted. The results suggested that the visualized elements were more abundant along the silicified aquaporin-containing lipid bilayer lining the proteoliposomes.

[0113] Additional elemental mapping was performed using energy-filtered (EF) TEM, which is more sensitive to lighter elements compared to EDX and was therefore able to detect both nitrogen and hydrogen in addition to sulfur, silicon, carbon, phosphorus, and oxygen (Figure 6).

[0114] SANS data modeling revealed that most of the parameters used to describe the system remained constant during silicification of both liposomes and proteoliposomes. In fact, the same five parameters (t o , f silica ,conc.,t silica , and f w,silica The change in σ was sufficient to accurately describe the changes in both samples. In this model, the concentration (conc.) was used as a floating parameter and directly interconnected with the other parameters. The silica-coated liposomes / proteoliposomes (f silicaThe fraction of SiO2 remained constant at 0 for the first 1 and 3.5 h of silicification of liposomes and proteoliposomes, respectively. This "lag phase" is associated with the hydrolysis of the alkoxide precursor, during which the TEOS ester bond is cleaved, ultimately forming silicic acid and ethanol. At pH 8, the process is expected to proceed slowly, as the hydrolysis reaction is catalyzed by acid and base.

[0115] After hydrolysis of the precursor, f silica increased to 1 over a 1.5-hour and 3-hour period for liposomes and proteoliposomes, respectively. This indicated that silica deposition occurred within a limited time frame, rather than continuously throughout the entire duration of silicification. An interesting observation was that the thickness of the silica shell did not increase linearly with time. Instead, a lipid bilayer approximately 4 nm thick (composed of silicic acid and buffer) was placed on the liposomes and proteoliposomes as soon as deposition began. Liposomes and proteoliposomes (without the silica shell) remained unchanged except for the outer lipid head layer, which experienced swelling. The outer leaflet of the lipid bilayer is in direct contact with the deposited silicic acid and is therefore susceptible to changes in the surrounding environment. According to this model, the outer lipid head layer of the template samples consisted of 82% and 86% buffer for liposomes and proteoliposomes, respectively. Especially considering the high proportion of buffer in the initially deposited silica layer (96% buffer in liposomes and 98% in proteoliposomes by the start of deposition), silica interacted closely with the outer lipid head layer. o The overall thickening of may be due to the presence of an interfacial water layer between the lipid bilayer and the silica shell.

[0116] t o Also silica Neither sample showed a consistent increase or decrease throughout the method, with slight deviations interrupting the overall pattern. Therefore, employing Equation 1, t o and t silica the remaining geometric parameters and f w,silicaThe growth rate of the silica shell was studied by coupling the volume of the buffer-free silica shell, V, as a function of time. silica,dry The change in silica shell volume over time follows a sigmoidal pattern, which was fitted to an Avrami-type growth function (Equation 2), originally derived for phase transformation processes, particularly crystallization. Different lengths of lag phases resulted in different Avrami parameters β for the silicification of liposomes and proteoliposomes. Therefore, comparison of reaction rates k was not straightforward. Therefore, the half-life of silicification (t) was adjusted to accommodate the difference in β. 1 / 2 ) was calculated (Equation 3). As shown in Table 3, the silicification of proteoliposomes was significantly higher than that of liposomes. 1 / 2 This means that the silicification of liposomes reached half of the plateau volume in slightly less time than the silicification of proteoliposomes. 1 / 2Because ρ is a composite measure of the lag and exponential phases, we also utilized another method for comparison. We plotted both shell growth processes from the onset of exponential growth, excluding the initial precursor hydrolysis phase. The growth phases were remarkably similar. Therefore, we concluded that the difference in silica formation between liposomes and proteoliposomes occurred during the first part of the method, i.e., precursor hydrolysis. Since the only difference between the samples was protein, the presence of protein altered the mechanism of silica shell formation. Two major protein properties can affect silica shell formation: electrostatic repulsion and steric hindrance. POPC lipids are zwitterionic and therefore neutral at pH 8. On the other hand, hAQP4 has a slight negative net charge due to its isoelectric point of 7.6. When TEOS is hydrolyzed to silicic acid, a small portion of the silicic acid becomes ionized and therefore negatively charged. Therefore, the slight negative net charge of proteins introduces electrostatic repulsion that may prevent some of the silicic acid from accessing the lipid bilayer head groups. However, this is unlikely to be the only explanation, especially since the silica shell interacts with the lipid head groups via hydrogen bonding to the negatively charged phosphatidyl moiety rather than the positively charged choline moiety. Therefore, steric hindrance, possibly in combination with electrostatic repulsion, presents itself as a more likely option. The C-terminal domain of hAQP4 reaches into the bulk, which may prevent direct contact between the proteoliposomes. This also seemed to be true for silicified proteoliposomes, which did not aggregate to the same extent as silicified liposomes. Steric hindrance may be the reason for the long lag period in the silicification of proteoliposomes, due to the low accessibility of silica to the lipids.

[0117] A similarity between the silicification of liposomes and proteoliposomes was the thickness of the silica shell, which was approximately 4 nm using these formation conditions. Interestingly, smaller silica particles were present in the silicified and dried samples, which was clearly visible by TEM (Figure 4B). This indicated that more material was available than was consumed in forming the silica shell. For both samples, the majority of silica formation appeared to occur in the bilayer (Figure 4). It was also interesting to study the disposition of other elements during silicification to obtain an indication of how the original proteoliposome composition changed. STEM-EDX showed that the detected elements were most abundant along the silica backing of the proteoliposomes, indicating that the aquaporin-containing lipid bilayer was intact. Because the mapping was performed in transmission mode, the map represents a 2D projection of the 3D sample; therefore, elements were detected in other parts of the silicified proteoliposomes as well, albeit at lower concentrations. Complementary elemental mapping was performed using EFTEM, which is more sensitive to lighter elements. Therefore, in addition to the elements detected by EDX, nitrogen and hydrogen, common components of biological materials, could also be detected. EFTEM elemental mapping confirmed the indications provided when using EDX, with elements associated with aquaporin-containing lipid bilayers being enriched in the silicified regions lining the proteoliposomes.

[0118] CD studies revealed that the protein remained intact throughout the entire siliconization process (Figure 2B). Changes in the helicity of the protein structure are typically assessed by changes in the absorption band at 222 nm, which corresponds to the n→π* transition in peptide bonds. Judging from the CD data, the absorption at 222 nm was nearly identical, which means that the transmembrane portion of hAPQ4 was intact. Therefore, the hydrophobic tail region of the lipid bilayer remained the same, and as a result, the protein secondary structure was preserved. The reason for the small change in amplitude at 209 nm could be minor modifications to the disordered portions of the protein, which would be plausible since some of those portions are exposed to silica.

[0119] This example demonstrated the stabilization of hAQP4-containing proteoliposomes by coating them with a thin silica shell. Importantly, the native conformation of the protein was maintained upon silicification.

[0120] The above-described embodiments should be understood as a few illustrative examples of the present invention. Those skilled in the art will understand that various modifications, combinations, and changes can be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in different embodiments can be combined in other configurations, where technically possible. Aspects or embodiments that may be included in the present invention are summarized as follows. [1]. A polymeric membrane structure (2) comprising a membrane (3) containing an integral membrane protein (1) that allows water to pass through, the first surface of the membrane (3) being coated with a silica layer (4). [2]. 2. The polymer membrane structure according to item 1, wherein the membrane (3) is a bilayer membrane (3) containing amphiphilic molecules. [3]. The membrane (3) is a lipid bilayer membrane (3), and the amphipathic lipid of the lipid bilayer (3) is preferably phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, cardiolipin, cholesterol, sphingomyelin, asolectin, diphytanoylphosphatidylcholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dihexanoyl-sn- Glycero-3-phosphoethanolamine (DHPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dimyristoylphosphatidylserine (DMPS), dimyristoylphosphatidylglycerol, dilauroylphosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG), 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (14:0 lyso-PCs such as 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 lyso-PC), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (16:0 lyso-PE) or 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:1 Lyso-PEs such as 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dierucoyl-sn-glycero-3-phosphate (DEPA), 1,2-erucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dierucoyl-sn-alicero-3-phosphoethanolamine (DEPE), 1,2-linoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-dilauroyl-sn-glycero-3-phosphate (DLPA), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dilauroyl-sn-glycero-3-phosphoserine (DLPS), 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), 1,2-Dimyristoyl-sn-glycero-3-phosphoserine (DMPS), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), 1,2-oleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dipalmitoyl-sn-glycero-3-phosphate 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), 1,2-distearoyl-sn-glycero-3-phosphate (DSPA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diostearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-myristoyl 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC) The polymer membrane structure according to item 2, wherein the lipid is selected from the group consisting of 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), lipids from cell membranes, lipids from organelles, cross-linkable lipids, and mixtures thereof, and is preferably POPC. [4]. Item 3. The polymeric membrane structure according to item 2, wherein the amphiphilic molecules are selected from amphiphilic AB, ABA and ABC copolymers and mixtures thereof, such as poly(methyloxazoline)-poly(dimethylsiloxane)-poly(methyloxazoline) (PMOXA-PDMS-PMOXA), poly(2-ethyl-2-oxazoline)-b-poly(dimethylsiloxane)-b-poly(2-ethyl-2-oxazoline) (PEtOz-PDMS-PEtOz) and mixtures thereof. [5]. The polymer membrane structure according to any one of items 1 to 4, wherein the membrane (3) is a proteoliposome or a proteopolymersome, and the outer surface of the proteoliposome or the proteopolymersome is covered with the silica layer (4). [6]. The integral membrane protein (1) that allows water to pass through is an aquaporin, preferably hAQP0, hAQP1, hAQP2, hAQP3, hAQP4, hAQP5, hAQP6, hAQP7, hAQP8, hAQP9, hAQP10, hAQP11, hAQP12, preferably hAQP0, hAQP1, hAQP2, hAQP4, hAQP5, hAQP6, or hAQP8, more preferably hAQP11. 6. The polymer membrane structure according to any one of items 1 to 5 above, wherein the polymer membrane structure is selected from the group consisting of human aquaporins (hAQPs) such as APQ4; bovine aquaporins such as bAQP1; fish aquaporins such as cpAQP1aa; yeast aquaporins such as Aqy1; plant aquaporins such as SoPIP2;1, AtTIP2;1, or AtPIP2;4; bacterial aquaporins such as AqpZ; and mixtures thereof. [7]. 7. The polymer membrane structure according to any one of items 1 to 6, wherein the silica layer (4) has an average thickness selected within the range of 0.1 nm to 1000 nm, preferably 1 nm to 100 nm, more preferably 1 nm to 10 nm. [8]. 8. The polymer membrane structure according to any one of items 1 to 7, wherein the water-permeable integral membrane protein (1) is capable of passing water through the membrane (3) in the presence of the silica layer (4). [9]. 9. The polymer membrane structure according to any one of items 1 to 8, wherein the silica layer (4) is a functionalized silica layer (4) preferably containing a silane such as fluoroalkylsilane (FAS), an alkoxysilane such as hexamethyldisilazane (HMDZ), or a combination thereof.

[10] . A filtration device (5), a porous support (6) containing a plurality of pores (7); The polymer membrane structure (2) according to any one of items 1 to 9 above, A filtration device (5).

[11] . Item 11. The filtration device according to item 10, wherein the polymer membrane structure (2) is provided on the surface of the porous support (6) and / or within the pores (7) of the porous support (6).

[12] . Item 12. The filtration device according to item 10 or 11, wherein the porous support (6) is formed from a material selected from the group consisting of polymers such as polysulfone, polyethersulfone, polyphenylsulfone, polyetherethersulfone, polyetherketone, polyetheretherketone, polyphenylene ether, polydiphenylphenylene ether, cellulose, polyvinylene cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose nitrate, polyphenylene sulfide, nitrocellulose, acetylated methyl cellulose, polyacrylonitrile, polyvinyl alcohol, polycarbonate, organosiloxane carbonate, polyester carbonate, organopolysiloxane, polyethylene oxide, polyamide, polyimide, polyamideimide, polybenzimidazole, polyolefin, polyacrylonitrile, nylon, and mixtures thereof; metals such as aluminum, titanium, zirconium, iron, or oxides thereof, or oxides of said metals; silicon dioxide; glass fiber; and mixtures thereof.

[13] . The filtration device (5) comprises the polymer membrane structure (2) described in item 9 above, and The functional groups of the functionalized silica layer (4) connect the polymer membrane structure (2) to the porous support (6); The filtration device according to any one of items 10 to 12 above.

[14] . A method for preparing an aqueous filtrate, comprising filtering an aqueous solution through the polymer membrane structure (2) described in any one of Items 1 to 9 or the filtration device (5) described in any one of Items 10 to 13 to obtain the aqueous filtrate.

[15] . A method for concentrating a compound dissolved or dispersed in an aqueous solution, comprising filtering the aqueous solution using the polymer membrane structure (2) described in any one of Items 1 to 9 or the filtration device (5) described in any one of Items 10 to 13, to obtain a water filtrate that does not contain the compound and a retentate that contains the compound at a higher concentration than in the aqueous solution.

[16] . A method for preparing a polymeric membrane structure (2), comprising contacting a membrane (3) containing a water-permeable integral membrane protein (1) with a silica precursor to form a silica layer (4) coated on a first surface of the membrane (3).

[17] . A method for preparing a filtration device (5), comprising the steps of: depositing a membrane (3) containing a water-permeable integral membrane protein (1) on and / or within a porous support (6); contacting the membrane (3) containing the water-permeable integral membrane protein (1) deposited on and / or within the porous support (6) with a silica precursor to form a silica layer (5) coated on a first surface of the membrane (3); A method comprising:

[18] . The silica precursor is selected from the group consisting of silicon alkoxides such as tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS), tetrapropyl orthosilicate, tetrabutyl orthosilicate, methyltriethoxysiloxane (MTES), dimethyldiethoxysiloxane (DMDES), tetrakis(glycerol) orthosilicate (TGS) and tetrakis-(2-hydroxyethyl)-orthosilicate (THEOS); allyltrimethoxysilane, (3-aminopropyl)triethoxysilane, butyltrichlorosilane, chloropentamethyldisilane, 1,2-dichlorotetramethyldisilane, diethoxydiphenylsilane, [3-(diethylamino)propyl]trimethoxysilane, dimethoxydimethylsilane, dimethoxy(methyl)octylsilane, (3-glycidyloxypropyl)trimethoxysilane, hexamethyldisilane, isobutyl(trimethoxysilane), 18. The method according to item 16 or 17, wherein the silane is selected from the group consisting of silanes such as methyltrichlorosilane, pentamethyldisilane, n-propyltriethoxysilane, tetraethylsilane, 1,1,2,2-tetramethyldisilane, tetramethylsilane, triethoxymethylsilane, triethoxyoctylsilane, trimethoxyphenylsilane, triethoxyphenylsilane, triethoxyvinylsilane, trimethoxymethylsilane, γ-aminopropyltriethoxysilane, silicon tetrachloride (tetrachlorosilane), silicon tetrabromide (tetrabromidosilane) or γ-aminopropylsilanetriol (APSTOL); silicates such as sodium silicate; silanols such as tris(tert-pentoxy)silanol or tris(tert-butoxy)silanol; silazanes, N-sec-butyl(trimethylsilyl)amine, and combinations thereof, and preferably TEOS.

Claims

1. A polymeric membrane structure (2) comprising a membrane (3), The membrane (3) comprises a water-permeable integral membrane protein (1), and the entire first surface of the membrane (3) is directly coated with a silica layer (4) having an average thickness selected within the range of 1 nm to 100 nm while maintaining the water-permeable function of the water-permeable integral membrane protein (1); The polymer membrane structure (2) is characterized in that the membrane (3) is a proteoliposome or a proteopolymersome, and the first surface of the membrane (3) is the outer surface of the proteoliposome or the proteopolymersome.

2. 2. The polymeric membrane structure according to claim 1, wherein the membrane (3) is a bilayer membrane (3) comprising amphiphilic molecules.

3. The membrane (3) is a lipid bilayer membrane (3), and the amphipathic lipid of the lipid bilayer membrane (3) is phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, cardiolipin, cholesterol, sphingomyelin, asolectin, diphytanoylphosphatidylcholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dihex ... cerole-3-phosphoethanolamine (DHPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dimyristoylphosphatidylserine (DMPS), dimyristoylphosphatidylglycerol, dilauroylphosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(l-glycerol)] (DMPG), 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (14:0 lysoPC selected from 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 lysoPC), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (16:0 lysoPE), or 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:1 Lyso-PE selected from 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dierucoyl-sn-glycero-3-phosphate (DEPA), 1,2-erucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dierucoyl-sn-alicero-3-phosphoethanolamine (DEPE), 1,2-linoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-dilauroyl-sn-glycero-3-phosphate (DLPA), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dilauroyl-sn-glycero-3-phosphoserine (DLPS), 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), 1,2-Dimyristoyl-sn-glycero-3-phosphoserine (DMPS), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), 1,2-oleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycerco-3-phosphoserine (DPPS), 1,2-distearoyl-sn-glycero-3-phosphate (DSPA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diostearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-myristoyl-2-stearoyl-sn-glycero -3-phosphocholine (MSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-stearoyl-2-myristoyl-sn-glycero-3 1-Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), lipids derived from cell membranes, lipids derived from organelles, crosslinkable lipids which are amphiphilic lipids having a crosslinkable chemical structure in the hydrophobic and / or hydrophilic portions of the amphiphilic lipid molecule, and mixtures thereof.

4. 3. The polymeric membrane structure of claim 2, wherein the amphiphilic molecules are selected from amphiphilic AB, ABA and ABC copolymers and mixtures thereof.

5. 5. The polymeric membrane structure of claim 4, wherein the amphiphilic molecule is selected from poly(methyloxazoline)-poly(dimethylsiloxane)-poly(methyloxazoline) (PMOXA-PDMS-PMOXA), poly(2-ethyl-2-oxazoline)-b-poly(dimethylsiloxane)-b-poly(2-ethyl-2-oxazoline) (PEtOz-PDMS-PEtOz), and mixtures thereof.

6. 6. The polymer membrane structure according to claim 1, wherein the integral membrane protein (1) that allows water to pass through is an aquaporin selected from the group consisting of human aquaporin (hAQP); bovine aquaporin; fish aquaporin; yeast aquaporin; plant aquaporin; bacterial aquaporin; and mixtures thereof.

7. The polymeric membrane structure of claim 6, wherein the human aquaporin (hAQP) is selected from the group consisting of hAQP0, hAQP1, hAQP2, hAQP3, hAQP4, hAQP5, hAQP6, hAQP7, hAQP8, hAQP9, hAQP10, hAQP11, and hAQP12.

8. The polymer membrane structure according to any of the preceding claims, wherein said silica layer (4) has an average thickness selected in the range of 1 nm to 10 nm.

9. The polymer membrane structure according to any one of claims 1 to 8, wherein the silica layer (4) is a functionalized silica layer (4) having silane, alkoxysilane, hexamethyldisilazane (HMDZ), or a combination thereof introduced as a functional group.

10. A filtration device (5), a porous support (6) comprising a plurality of pores (7); The polymer membrane structure (2) according to any one of claims 1 to 9. A filtration device (5).

11. 11. The filtration device according to claim 10, wherein the polymer membrane structure (2) is provided on the surface of the porous support (6) and / or within the pores (7) of the porous support (6).

12. 12. The filtering device according to claim 10 or 11, wherein the porous support (6) is made of a material selected from the group consisting of polymers; metals or oxides of said metals; silicon dioxide; glass fibers; and mixtures thereof.

13. The filtration device (5) comprises a polymeric membrane structure (2) according to claim 9, and The functional groups of the functionalized silica layer (4) connect the polymeric membrane structure (2) to the porous support (6). The filtration device according to any one of claims 10 to 12.

14. A method for preparing a water filtrate, comprising filtering an aqueous solution through the polymeric membrane structure (2) according to any one of claims 1 to 9 or the filtration device (5) according to any one of claims 10 to 13 to obtain the water filtrate.

15. A method for concentrating a compound dissolved or dispersed in an aqueous solution, the method comprising filtering the aqueous solution with a polymeric membrane structure (2) according to any one of claims 1 to 9 or a filtration device (5) according to any one of claims 10 to 13, to obtain a water filtrate that does not contain the compound and a retentate that contains the compound at a higher concentration than the aqueous solution.

16. A method for preparing a polymeric membrane structure (2), comprising:

1. A method comprising contacting a membrane (3) containing a water-permeable integral membrane protein (1) with a silica precursor to form a silica layer (4) coated directly on the entire first surface of the membrane (3) while maintaining the water-permeable function of the water-permeable integral membrane protein (1), wherein the silica layer (4) has an average thickness selected within the range of 1 nm to 100 nm, the membrane (3) is a proteoliposome or a proteopolymersome, and the first surface of the membrane (3) is the outer surface of the proteoliposome or proteopolymersome.

17. A method for preparing a filtration device (5), comprising the steps of: depositing a membrane (3) containing a water-permeable integral membrane protein (1) on and / or within a porous support (6); contacting the membrane (3) containing the water-permeable integral membrane protein (1) deposited on and / or within the porous support (6) with a silica precursor to form a silica layer (4) coated directly on the entire first surface of the membrane (3) while maintaining the water-permeable function of the water-permeable integral membrane protein (1); Including, wherein the silica layer (4) has an average thickness selected within the range of 1 nm to 100 nm, the membrane (3) is a proteoliposome or a proteopolymersome, and the first surface of the membrane (3) is the outer surface of the proteoliposome or proteopolymersome.

18. 18. The method of claim 16 or 17, wherein the silica precursor is selected from the group consisting of silicon alkoxides; silanes; silicates; silanols; silazanes, N-sec-butyl(trimethylsilyl)amine, and combinations thereof.

19. 19. The method of claim 18, wherein the silicon alkoxide is selected from the group consisting of tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS), tetrapropyl orthosilicate, tetrabutyl orthosilicate, methyltriethoxysiloxane (MTES), dimethyldiethoxysiloxane (DMDES), tetrakis(glycerol) orthosilicate (TGS), or tetrakis-(2-hydroxyethyl)-orthosilicate (THEOS).

Images