Polymer vesicles comprising cleavable block copolymers

Abstract

A process for the production of self-assembled polymer vesicles in an aqueous solution wherein the polymer vesicles comprise an amphiphilic diblock copolymer of the P1-b-P2 type, the process comprising the step of synthesizing an R-C-O-P1-b-P2 block copolymer comprising silane ether (-C-O-Si-) bonds, and wherein P1 is a hydrophobic polymer and P2 is a hydrophilic polymer and R is any atom or group of atoms wherein the synthesis comprises ring-opening polymerization of a cyclic monomer in the presence of a catalyst by an initiator comprising a carbon-bound hydroxyl group.

Description

Technical Field

[0001] The present disclosure relates to polymer vesicles comprising self-cleavable block copolymers, methods for preparing the same, and semipermeable separation membranes comprising the same. Background Art

[0002] Amphiphilic block copolymer materials can be used to form a wide range of self-assemblies, such as micelles, vesicles, or rods, in both organic solvents and aqueous media. Applications range from encapsulation of drugs or vitamins for stabilization and / or delivery to serving as templates for nanoscale patterning. Systems based on diblock or triblock copolymers are well known and useful because they can form polymer vesicle structures. Typically, ring-opening polymerization of hydrophobic cyclic monomers, such as ε-caprolactone or cyclosiloxane, is used, where the growth of the monomer chain is promoted by an initiator such as poly(ethylene glycol) methyl ether macroinitiator in the presence of a catalyst. The subsequent addition of a hydrophilic polymer moiety allows the acquisition of a triblock copolymer. Shuai et al. 2004 explored the synthesis of diblock copolymers of poly(ε-caprolactone) (PCL) and monomethoxy poly(ethylene glycol) (mPEG) with different compositions. In Guo et al., 2009, mPEG-PCL diblock copolymers that can self-assemble into monodisperse micelles were synthesized. Recently, Zhang et al., 2012 synthesized PEG-g-PCL diblock copolymers via an alternative “grafting onto” approach, and these diblock copolymers can self-assemble into micelle-like aggregates with different diameters in aqueous solution.

[0003] The use of amphiphilic lipids and block copolymers for forming self-assembled vesicles having a bilayer or bilayer-like structure, particularly for immobilizing amphiphilic membrane proteins such as aquaporin water channels (AQPs), is well known in the art. Vesicles comprising AQPs can then be used to make semipermeable membranes with immobilized AQPs, typically by depositing the vesicles as a layer or in a membrane on a supporting substrate, for applications such as water purification (WO2006 / 122566) or generating salinity energy (WO2007 / 033675), which allows water molecules to selectively pass through the membrane by nanofiltration, reverse osmosis, forward osmosis, or pressure-retarded osmosis.

[0004] WO2013 / 043118 discloses thin film composite (TFC) membranes in which aquaporin water channels (AQP) are incorporated into the active layer of the membrane. In addition, it discloses a method for producing thin film composite membranes and their use in filtration processes (such as nanofiltration and osmotic filtration processes). The TFC membranes include lipid-AQP / copolymer-AQP vesicles, which are incorporated into the TFC active layer. WO2010 / 146365 describes the preparation of TFC-aquaporin-Z (AqpZ) filtration membranes, which use amphiphilic triblock copolymers as vesicle-forming substances for incorporating immobilized AQP. WO2014 / 108827 discloses hollow fiber (HF) modules having fibers modified by a thin film composite (TFC) layer comprising aquaporin water channels, wherein the aquaporin water channels are incorporated into the vesicles before being incorporated into the TFC layer.

[0005] The simplest method for preparing polymer vesicles in laboratory studies is thin film hydration followed by extrusion. This method involves making a thin polymer film in a round-bottom flask by removing an organic solvent such as chloroform, for example, by vacuum drying. After adding and stirring a dispersion medium such as water, heterogeneous polymer vesicles are formed. Finally, after extrusion through, for example, a polycarbonate membrane, more uniform polymer vesicles are obtained. However, this method is not very efficient and is not particularly scalable. Therefore, the main challenge for vesicle-based transmembrane protein delivery to filtration membranes is the efficiency of vesicle formation. None of the above disclosures explain the efficiency of block copolymers in forming vesicles / polymer vesicles, nor the effect of the block copolymer synthesis route on the formation of the vesicles. Summary of the Invention

[0006] Therefore, it is an object to provide a method for forming vesicles with greater efficiency by obtaining a larger ratio of vesicles to undesirable structures (such as worm structures and rods). Another object is to provide a method for obtaining end-functionalized block copolymers suitable for vesicle formation. Another object is to provide a method for obtaining end-functionalized block copolymers suitable for crosslinking. Another object is to provide a method for obtaining vesicles of similar size.

[0007] The above and other objects are achieved by the features of the independent claims. Further forms of implementation are apparent from the dependent claims, the description and the drawings.

[0008] According to a first aspect, there is provided a method for preparing a liquid composition comprising self-assembled polymer vesicles in an aqueous solution, wherein the polymer vesicles comprise an amphiphilic diblock copolymer of the P1-b-P2 type, the method comprising the step of synthesizing an RCO-P1-b-P2 block copolymer comprising a silyl ether (-CO-Si-) bond, and wherein P1 is a hydrophobic polymer and P2 is a hydrophilic polymer and R is any atom or group of atoms, wherein the synthesis comprises ring-opening polymerization of cyclic monomers by an initiator in the presence of a catalyst, the initiator comprising a carbon-bound hydroxyl group.

[0009] In a possible implementation form of the first aspect, the cyclic monomer is a cyclic siloxane, and by using a carbon-bound hydroxyl-terminated initiator, the ring-opening polymerization reaction advantageously produces a block polymer comprising a silyl ether (-CO-Si) bond, that is, a bond in which an oxygen atom is bound to a silicone and a carbon atom, wherein the bond is unstable in an aqueous environment. Therefore, the bond will hydrolyze over time when exposed to water, and the leaving group will advantageously separate from the block polymer. Since the bond is included between the diblock copolymer of the P1-b-P2 type and the third atomic group R, R will fall off by cracking, advantageously producing a diblock copolymer and a free radical R.

[0010] In the possible implementation form of the first aspect, the diblock copolymer includes a hydrophilic part and a hydrophobic part. Therefore, after cracking, an amphiphilic diblock copolymer is produced, which can be advantageously used to synthesize polymer vesicles. Compared with the membrane rehydration method for traditional polymer vesicle synthesis, this method for producing diblock copolymers is more efficient and more scalable. Without wishing to be bound by any theory, it is believed that the concentrated polymerization produced at the interface between the dehydrated film on the surface of the round-bottom flask and the newly added dispersion medium greatly hinders the acquisition of a uniform solution that is conducive to the yield of polymer vesicles. Therefore, with this traditional method, a larger number of alternative and less ideal structures, such as rods, micelles and worm-like structures, are obtained. Therefore, the present method avoids these inefficient synthesis conditions and advantageously produces a much larger number of polymer vesicles of uniform size.

[0011] Furthermore, since the initiator is hydroxyl-terminated, a wide variety of initiators can be used, thereby generating a large number of possible R groups to bind to the P1-b-P2 block copolymer and generating the architecture (RCO-P1-b-P2) for leaving the copolymer by spontaneous hydrolysis of the silyl ether bond in the presence of water. Therefore, such molecules can be advantageously considered for various applications, such as drug delivery.

[0012] In a possible implementation form of the first aspect, the cyclic monomer is a cyclic trisiloxane.

[0013] In a possible implementation form of the first aspect, R is a hydrophilic polymer. By shedding the hydrophilic moiety R, the f value (i.e. the fraction of hydrophilicity included in the copolymer relative to the total Mn of the polymer chain) will decrease. This leads to a change in the critical packing parameter and thus changes the curvature of the self-assembly, driving the block copolymer chains to reassemble from a worm-like structure into vesicles. Thus, the presence of labile silyl ether bonds in the copolymer advantageously facilitates the efficient formation of diblock copolymer vesicles / polymer vesicles over the formation of other structures, such as rods, micelles and / or worm-like structures.

[0014] In a possible implementation form of the first aspect, the vesicle further comprises a triblock copolymer. The triblock copolymer can be a copolymer that has not undergone cleavage. The presence of a triblock copolymer in the diblock copolymer-based vesicle can advantageously allow for better integration of the transmembrane protein into the vesicle membrane.

[0015] In a possible implementation form of the first aspect, the product of the ring-opening polymerization reaction is a polymer of the type R-C-O-P1, and the method further comprises the steps of:

[0016] - reacting the R-C-O-P1 polymer with a chlorosilane for obtaining a silyl-terminated R-C-O-P1-Si-H, and

[0017] - reacting the silyl-terminated polymer of the previous step with a vinyl-terminated hydrophilic polymer (P2) for obtaining a block copolymer of the type R-C-O-P1-b-P2 comprising a silyl ether (-C-O-Si-) bond. By termination, the polymer is advantageously provided with a reactive end group in preparation for being adjoined to the second polymer P2.

[0018] In a possible implementation form of the first aspect, wherein the product of the ring-opening polymerization reaction is a polymer of the type R-P1-OH, the method can further comprise the steps of:

[0019] - reacting the R-P1-OH polymer with a chlorosilane for obtaining a silyl-terminated R-P1-C-O-Si-H, and

[0020] - reacting the silyl-terminated polymer of the previous step with a vinyl-terminated hydrophilic polymer (P2) for obtaining a block copolymer of the type R-b-P1-C-O-Si-P2 comprising a silyl ether (-C-O-Si-) bond.

[0021] In another possible implementation form of the first aspect, the chlorosilane is chlorodimethylsilane.

[0022] In another possible implementation form of the first aspect, the initiator has a Mn comprised between approximately 200 Da and 5 kDa.

[0023] In a further possible implementation form of the first aspect, the initiator has an Mn comprised between about 200 Da and 2 kDa.

[0024] In a further possible implementation form of the first aspect, the initiator has an Mn comprised between about 600 Da and 900 Da.

[0025] In a further possible implementation form of the first aspect, the initiator comprises a hydroxyl-terminated polymer, such as poly(ethylene glycol) (PEG).

[0026] In a further possible implementation form of the first aspect, the initiator comprises a hydroxyl-terminated alcohol.

[0027] In a further possible implementation form of the first aspect, the initiator comprises poly(ethylene glycol) (PEG), and PEG-O-P1-b-P2 is synthesized.

[0028] In a further possible implementation form of the first aspect, the initiator comprises methoxy-PEG (MeO-PEG-OH), and MeO-PEG-O-P1-b-P2 is synthesized.

[0029] In a possible implementation form of the first aspect, P1 is poly(dimethylsiloxane) (PDMS), and the amphiphilic diblock copolymer is of the poly(dimethylsiloxane)-b-P2 (H-PDMS-b-P2) type, and the method comprises a step of synthesizing an R-C-O-(Si(CH3)2-O)n-P2 (R-C-O-PDMS-b-P2) block copolymer comprising a siloxy ether (-C-O-Si-) bond.

[0030] In a possible implementation form of the first aspect, P1 is poly(caprolactone) (PCL), and the amphiphilic diblock copolymer is of the poly(caprolactone)-b-P2 (H-Si-PCL-b-P2) or H-Si-poly(caprolactone)-b-P3 type, and the method further comprises a step of synthesizing a P2-PCL-C-O-Si-O-PCL-b-P3 triblock copolymer comprising at least two siloxy ether (-C-O-Si-) bonds, wherein P2 and / or P3 is a hydrophilic polymer. Further, P2 = P3 or P2 ≠ P3.

[0031] In a possible implementation form of the first aspect, a coupling agent can be added to the reaction. By continuing the reaction in the presence of the coupling agent, P 2-A triblock copolymer of the PCL-CO-Si-PCL-b-P3 type, wherein P2 and P3 are hydrophilic polymers. A coupling agent, covalently attached at each end to the block polymer-b-PCL diblock copolymer, may advantageously include at least one silyl ether (-CO-Si-) bond.

[0032] In another possible implementation form of the first aspect, the coupling agent may be dichlorosilane, and a triblock copolymer of the type P2-b-PCL-CO-Si-O-PCL-b-P3 is obtained. In addition, P2=P3 or P2≠P3, and P2 and / or P3 may further be methoxy- or vinyl-terminated.

[0033] In a possible implementation form of the first aspect, the reaction is run until a certain amount of self-assembled polymer vesicles comprising amphiphilic diblock copolymers of the P1-b-P2 type are measurable in aqueous solution, such as by measurement with a cryo-TEM instrument.

[0034] In another possible implementation form of the first aspect, a cross-linking agent is further added to the reaction.

[0035] In another possible implementation of the first aspect, P2 is reacted with a thiol of the form HS-R, wherein R comprises a functional group for obtaining a functionalized diblock or triblock copolymer via a thiol-ene reaction. In another possible implementation of the first aspect, HS-R carries at least one of the following functional groups: amine, acid, alcohol, protein, peptide, or ester. In a possible implementation of the first aspect, the thiol-ene reaction is initiated by photoinitiation, thermal initiation, electrochemical initiation, or the like.

[0036] In a possible implementation form of the first aspect, one or more of P2, P3, diblock copolymer or triblock copolymer to be functionalized via a thiol-ene reaction comprises vinyl end groups. In a possible implementation form of the first aspect, a triblock copolymer of the type P2-b-PCL-CO-Si-PCL-b-P3 is functionalized to different types of functional end groups via a thiol-ene reaction.

[0037] In another embodiment of the first aspect, the HS-R thiol comprises at least two thiols in the form of HS-R1 and HS-R2, for obtaining a mixture of RCO-P1-b-P2 diblock or triblock copolymers.

[0038] In a further possible implementation form of the first aspect, the copolymer is an amine functionalized by reacting one or more of R, P2 and / or P3 with a thiol in the form of HS-R1, wherein R1 comprises an amine. According to this reaction, an R-C-O-P1-b-P2-NH2 diblock or triblock copolymer (wherein R is a polymer), or an NH2-P2-b-PCL-C-O-Si-PCL-b-P3-NH2, P2-b-PCL-C-O-Si-PCL-b-P3-NH2 or NH2-P2-b-PCL-C-O-Si-PCL-b-P3 triblock copolymer can be advantageously obtained.

[0039] In a further possible implementation form of the first aspect, the copolymer is an amine functionalized by reacting one or more of R, P2 and / or P3 with a thiol in the form of HS-R1, wherein R1 comprises an amine. According to this reaction, an R-C-O-P1-b-P2-NH2 diblock or triblock copolymer (wherein R is a polymer), or an NH2-P2-b-PCL-C-O-Si-PCL-b-P3-NH2, P2-b-PCL-C-O-Si-PCL-b-P3-NH2 or NH2-P2-b-PCL-C-O-Si-PCL-b-P3 triblock copolymer can be advantageously obtained. 2- NH2 diblock or triblock copolymer (wherein R is a polymer), or an NH2-P2-b-PCL-C-O-Si-O-C-PCL-b-P3-NH2, P2-b-PCL-C-O-Si-O-C-PCL-b-P3-NH2 or NH2-P2-b-PCL-C-O-Si-O-C-PCL-b-P3 triblock copolymer.

[0040] In a possible implementation form of the first aspect, the liquid composition comprises between 1% and 20% of the NH2-diblock or triblock amine functionalized copolymer relative to the total copolymer content. In a further possible implementation form of the first aspect, the liquid composition comprises between 2% and 5% of the NH2-diblock and / or triblock amine functionalized copolymer relative to the total copolymer content. Without wishing to be bound by any theory, it is believed that these percentage ranges allow for an optimal density of amine end groups on the surface of the vesicles which does not destroy the vesicle stability up to the point of rupture. It is further believed at present that a too high density of positive charges on the amine functionalized copolymer on the inner and outer surface can lead to vesicle rupture via charge repulsion. A maximization of the positive charge density on the outer surface of the vesicle membrane can advantageously optimize the available functional groups for further interactions, e.g. via positive electrostatic charges with negatively charged elements on the separating membrane.

[0041] In a possible implementation form of the first aspect, dissolved transmembrane proteins are added to the liquid composition. The copolymer can interact via the terminal functional groups with amino acid residues of the transmembrane proteins. Thus, in case the copolymer interacts with amino acid residues of the transmembrane proteins, amino acid negative charges can be established under certain conditions (pH, pKs, etc.) for the production of the liquid composition and / or the membrane comprising the polymeric vesicles.

[0042] In a possible implementation form of the first aspect, the method further comprises the step of producing a thin film composite (TFC) separation membrane, the method comprising the step of applying the liquid composition to a porous support membrane.

[0043] In a possible implementation form of the first aspect, the method comprises producing a TFC membrane by interfacial polymerization.

[0044] In a possible implementation form of the first aspect, the catalyst used in the method is 1,3-trimethylene-2-propyl guanidine (TMnPG). The ROP reaction catalyzed by TMnPG can advantageously allow the production of block copolymers with a narrow polydispersity, which in turn can lead to a positive influence on obtaining greater vesicle ratio undesired structures such as rods, micelles and / or worm-like structures.

[0045] In another possible implementation form of the first aspect, MeOH and NaOMe are used in the TMnPG synthesis and the ratio between MeOH and NaOMe is at least 7:1. When using this ratio or a greater ratio, a purer product can be obtained.

[0046] According to a second aspect, there is provided a polymer vesicle comprising a block copolymer of the type H-Si-P1-b-P2, wherein P1 is a hydrophobic polymer and P2 is a hydrophilic polymer.

[0047] In a possible implementation form of the second aspect, the polymer vesicle comprises a block copolymer of the type R-C-O-Si-P1-b-P2 comprising a silane ether (-C-O-Si-) bond, and wherein R is any atom or group of atoms.

[0048] In a possible implementation form of the second aspect, the block copolymer comprising a silane ether bond is a tri-block copolymer.

[0049] In a possible implementation form of the second aspect, P2 and / or P3 comprises PEG.

[0050] In a possible implementation form of the second aspect, P2 and / or R comprises PEG.

[0051] In a possible implementation form of the second aspect, P2, P3 and / or R comprises PEG.

[0052] In a possible implementation form of the second aspect, P1 comprises poly(dimethylsiloxane) (PDMS) or polycaprolactone (PCL).

[0053] In a possible implementation form of the second aspect, the triblock copolymer containing silane-based ether bonds is of the type PEG-O-PDMS-b-PEG, MeO-PEG-O-PDMS-PEG or PEG-PCL-C-O-Si-PCL-PEG.

[0054] In a possible implementation form of the second aspect, the block copolymer further comprises a functional end group allowing cross-linking, such as one selected from the group comprising primary amine (-NH2), carboxyl (-COOH), thiol (-SH) and carbonyl (-CHO).

[0055] In a possible implementation form of the second aspect, the polymersome further comprises a transmembrane protein in the membrane of the self-assembling polymersome.

[0056] According to a third aspect, there is provided an isolated membrane comprising the polymersome according to the present disclosure.

[0057] These and other aspects will be apparent from one or more of the embodiments and implementations described below. BRIEF DESCRIPTION OF DRAWINGS

[0058] In the following detailed portion of the disclosure, these aspects, embodiments and implementation forms will be explained in more detail with reference to the exemplary embodiments shown in the drawings, in which:

[0059] Figure 1 represents the ring-opening polymerization (ROP) of hexamethylcyclotrisiloxane by PEG and the synthesis of PEG-O-PDMS diblock copolymer in the presence of 1,2-trimethylene-3-propyl guanidine (TMmPG) as catalyst.

[0060] Figure 2 represents the ring-opening polymerization (ROP) of hexamethylcyclotrisiloxane by lower alcohols and the synthesis of R-O-PDMS polymers in the presence of TMmPG as catalyst.

[0061] Figure 3 represents the endcapping of PEG-O-PDMS with chlorodimethylsilane.

[0062] Figure 4 represents the addition of MeO-PEG to the chlorodimethylsilane endcapped PEG-O-PDMS.

[0063] Figure 5 represents the synthesis of the cleavable mPEG-b-PCL-b-mPEG triblock copolymer.

[0064] Figure 6 is a plot of mPEG-PDMS exposure to an aqueous medium over time.

[0065] Figure 7 Shown are cryo-TEM images of mPEG-PDMS diblock and triblock vesicle solutions after lysis.

[0066] Figure 8 It shows 1 Figure 5. Time series measurements of the ring opening of D3 from the H NMR kinetic study, showing the accumulation of PDMS with the loss of mPEG as initiator.

[0067] Figure 9 is a graph showing the conversion of D3 over time using mPEG as the initiator and assuming perfect initiation efficiency.

[0068] Figure 10 is a graph showing the Mn growth of PDMS over time in an unpurified sample (A) and a purified sample (B).

[0069] Figure 11 It shows 1 Plot of a time series measurement of the ring opening of D3 from a H NMR kinetic study, showing the accumulation of PDMS with the loss of alcohol as initiator.

[0070] Figure 12 express

[0071] FIG13 shows the self-assembled nanostructure types over time by dynamic light scattering.

[0072] Figure 14 is a graph of a UV spectrum showing that the absorbance of the self-assembled solution of cleavable mPEG-b-PDMS-b-PEG decreases over time.

[0073] Figure 15 Figure 1 is a SEC chromatogram showing the increase in mPEG 750 signal intensity due to hydrolytic cleavage of the silyl ether bond. mPEG 750 was used as a reference.

[0074] Figure 16 Represents the preparation of 1,2-trimethylene-3-propylguanidine hydroiodide TMnPG-HI. DETAILED DESCRIPTION

[0075] More specifically, the present invention relates to a method for making a liquid composition comprising self-assembled polymer vesicles, wherein the polymer vesicles comprise amphiphilic block copolymers of the P1-b-P2 type, the method comprising the steps of synthesizing a RCO-P1-b-P2 block copolymer comprising silyl ether (-CO-Si-) bonds, and wherein P1 is a hydrophobic polymer and P2 is a hydrophilic polymer and R is any atom or group of atoms, wherein the synthesis comprises the ring-opening polymerization of cyclic monomers by an initiator in the presence of a catalyst, the initiator comprising a carbon-bound hydroxyl group.

[0076] Silane ether bonds can be generated in a number of ways, for example, by a nucleophilic attack of an initiator comprising a hydroxyl end group on a cyclic siloxane monomer, such as a cyclic trisiloxane, to form a polymer chain. This process is a form of ring opening polymerization (ROP). Other forms of chain growth polymerization are known, however, the obtaining of silane ether bonds is essential to practice the invention as disclosed herein. The reaction is carried out as described in Example 1 below. See also Figure 1 and Figure 2 .

[0077] Subsequently, the obtained diblock copolymer can be end-capped with, for example, a chlorosilane, thereby making available -Si-H end groups for reaction with a selected vinyl-terminated hydrophilic polymer P2, for obtaining R-C-O-P1-b-P2 polymers. See Figure 3 and Figure 4 . The chlorosilane is of the formula Cl-Si(R1R2)-H, wherein R1= R2or R1≠ R2. Thus, R-C-O-P1-Si(R1R2)-H is obtained, wherein R1= R2or R1≠ R2. One example of a chlorosilane is dimethylchlorosilane.

[0078] Figure 5 Another method for obtaining a block copolymer comprising a silane ether bond is depicted, which comprises the step of reacting a chlorosilane coupling agent with a poly(caprolactone)-b-P2 copolymer, thereby obtaining a P2-b-PCL-C-O-Si-O-PCL-b-P3 triblock copolymer.

[0079] Yet another method for obtaining a block copolymer comprising a silane ether bond comprises the steps of ROP reaction of ε-caprolactone using mPEG as an initiator, for obtaining a polymer of the type R-b-P1-OH, followed by reaction with a chlorosilane, such as dimethylchlorosilane. Further reaction with, for example, a vinyl-terminated hydrophilic polymer results in a block copolymer of the type R-b-P1-C-O-Si-P2 comprising a silane ether (-C-O-Si-) bond.

[0080] The present invention further relates to the vesicles disclosed herein and methods for obtaining said vesicles, comprising an amphiphilic diblock copolymer of the type P1-b-P2 and a triblock copolymer of the type R-C-O-Si-P1-b-P2 or R-Si-O-C-P1-b-P2, wherein P1 is a hydrophobic polymer and P2 is a hydrophilic polymer, and wherein R is any atom or group of atoms, as a vesicle membrane forming material, and further optionally comprising a transmembrane protein.

[0081] A list of hydrophilic blocks useful in practicing the invention according to the present disclosure includes, but is not limited to, poly(oxazoline); poly(ethylene glycol); and poly(methacrylates), such as poly(dimethylamino)ethyl methacrylate) and poly(2-hydroxyethyl methacrylate).

[0082] All of these are of high interest, as all are effective in remodeling transmembrane proteins, including AQPs. Of particular interest are poly(ethylene glycol)- and poly(oxazoline)-based hydrophilic blocks.

[0083] The list of hydrophobic blocks that can be used to implement the invention according to the present disclosure includes, but is not limited to: poly(butylene oxide); poly(butadiene); poly(lactone) or more narrowly poly(caprolactone); poly(lactide); poly(siloxane) or more narrowly poly(dimethylsiloxane); and poly(methacrylates), such as poly(methyl methacrylate) and poly(butyl methacrylate).

[0084] All of these are of high interest, as all are effective in remodeling transmembrane proteins, including AQPs. Of particular interest are poly(dimethylsiloxane) and poly(caprolactone) based hydrophobic blocks.

[0085] Mixed systems that are combinations of block copolymers and lipids are also conceivable. This is probably because the lipid can be easily envisioned as the P2 moiety incorporated in the vinylsilyl reaction, i.e., a methylsilane-terminated polymer reacts with a vinyl-terminated lipid (P2) to obtain a block copolymer.

[0086] Thus, not only triblock copolymers of the ABA form are conceivable (e.g. PEG-O-PDMS-b-PEG), but also triblock copolymers of the ABC form, such as in the case of, for example, PEG-O-PDMS-b-PMOXA, where PMOXA is poly(2-methyloxazoline), and where, after cleavage, PEG is released and PDMS-b-PMOXA remains as a diblock.

[0087] In some cases, the hydrophilic blocks forming the copolymers may be end-functionalized. Examples of such functionalized hydrophilic blocks are, for example, bis(aminoalkyl) or bis(hydroxyalkyl) terminated polymers, or polymers comprising primary amines (-NH2), carboxyls (-COOH), sulfhydryls (-SH), and carbonyls (-CHO) as end groups.

[0088] Examples of transmembrane proteins are aquaporin water channels, ie aquaporins and aquaglyceroporins, such as those listed in the definitions below.

[0089] Furthermore, the present invention relates to a method for preparing the disclosed liquid composition, wherein a solution of the amphiphilic diblock or triblock copolymer according to the present disclosure is mixed as a vesicle membrane-forming material, optionally with a transmembrane protein.

[0090] Initiators suitable for ROP reactions include various types of molecules, such as hydroxyl-terminated polymers such as PEG or mPEG. In addition, lower molecular weight alcohols exhibit good ring-opening efficiency. Such alcohols may include (in decreasing order of ring-opening efficiency) methanol, 2-(methoxy)ethanol, 2-(benzyloxy)ethanol, and isopropanol.

[0091] The product of the ring-opening polymerization reaction is a polymer of the type RCO-P1, where R is the moiety contributed by the initiator species, and the method comprises the following steps:

[0092] a) reacting the RCO-P1 polymer with chlorodimethylsilane to obtain silane-terminated RCO-P1-Si-H, and

[0093] b) reacting the methylsilane-terminated polymer of step a) with a vinyl-terminated hydrophilic polymer (P2) for obtaining a block copolymer of the RCO-P1-b-P2) type containing silyl ether (—CO—Si—) bonds.

[0094] Examples of cyclic siloxanes that can be effectively formed into useful PDMS polymers by ring-opening polymerization include, but are not limited to, trisiloxanes (D3), including 1,3,5-triphenyl-1,3,5-tri-p-tolylcyclotrisiloxane, 1,3,5-trivinyl-1,3,5-trimethylcyclotrisiloxane, hexaphenylcyclotrisiloxane, and 1,3,5-triphenyltrimethylcyclotrisiloxane; tetrasiloxanes (D4); pentasiloxanes (D5), such as pentamethylcyclotrisiloxane, including (3-cyanopropyl)pentamethylcyclotrisiloxane and vinylpentamethylcyclotrisiloxane; hexasiloxanes (D6), such as hexamethylcyclotrisiloxane; heptasiloxanes (D7), and octasiloxanes (D8). In addition, any Si atom in the chain can be substituted with a methyl or phenyl group, a diphenyl group, or a methyl / vinyl group, etc. Higher polysiloxanes with such functionality also exist and are considered as starting materials, such as functionalized trisiloxanes and functionalized tetrasiloxanes.

[0095] Alternatively, the product of the ring-opening polymerization reaction is a polymer of the type R-b-P1-OH, where R is the moiety contributed by the initiator species, and the method comprises the steps of reacting the R-b-P1-OH polymer with a chlorosilane for obtaining a silane-terminated R-b-P1-C-O-Si-H, and reacting the silane-terminated polymer with a vinyl-terminated hydrophilic polymer (P2) for obtaining a block copolymer of the type R-b-P1-C-O-Si-P2 comprising silane-based ether (-C-O-Si-) linkages. A preferred way of carrying out the present application is the use of chlorodimethylsilane for obtaining the silane-terminated polymer.

[0096] The liquid composition can be applied to a support membrane or an active layer of a separation membrane. As an example, the active layer can be a thin film composite (TFC) layer formed on a support membrane. TFC membranes can be formed using alternative reaction components, for example, as described by Zhou et al. in Journal of Membrane Science, Volume 471, 1 December 2014, Pages 381-391, “Thin-film composite membranes formed by interfacial polymerization with natural material sericin and trimesoyl chloride for nanofiltration”. Highly selective active layers can also be formed on a substrate by a layer-by-layer method (see Wang et al., Membranes, 5(3):369-384, 2015).

[0097] The filtration membrane according to the present application can be prepared by adding the liquid composition comprising the diblock and / or triblock copolymer vesicles, for example, also comprising an aquaporin water channel protein as a transmembrane protein, during the membrane manufacturing process, such as adding the liquid composition to an aqueous MPD solution when forming a TFC layer.

[0098] By applying the liquid composition to a porous support membrane, it is possible to produce a thin film composite (TFC) separation membrane comprising the polymer vesicles according to the present disclosure. For membranes modified with a thin film composite (TFC) layer comprising an aquaporin water channel, the method comprises the following steps:

[0099] a) obtaining an aquaporin vesicle suspension, wherein the vesicles are polymer vesicles comprising an amphiphilic diblock or triblock copolymer in the form of any of the possible implementations according to the present disclosure, such as disclosed in Example 1 or 2 below,

[0100] b) preparing an aqueous solution of a diamine or a triamine,

[0101] c) dissolving the diacyl halide or triacyl halide in a non-polar organic solvent,

[0102] d) preparing a mixture of amine and aquaporin vesicles by dissolving / mixing the vesicle preparation from step a) with the solution from step b),

[0103] e) applying the mixture from step d) to a porous support membrane,

[0104] f) removing excess aqueous solution, for example by using an air knife,

[0105] g) applying the acid halide solution from step c) to the membrane, allowing interfacial polymerization to occur, and

[0106] h) Rinse the membrane surface with an aqueous solvent.

[0107] Definitions and Terminology

[0108] As used herein, the term "coupling agent" is a molecule that reacts with the OH-end groups of two diblock copolymers to couple them, thereby producing a triblock with (one or more) potentially unstable silyl ether bonds. The family of coupling agents includes, but is not limited to, silanes, such as dichlorosilane as used in certain embodiments disclosed herein.

[0109] As used herein, the term "transmembrane protein" refers to a class of membrane proteins that span the entire thickness of a biological membrane and are permanently attached to the biological membrane in nature. That is, in essence, transmembrane proteins span from one side of the lipid bilayer membrane to the other side of the membrane. Examples of transmembrane proteins are ammonia transporters, urea transporters, chloride channels, and aquaporin water channels.

[0110] As used herein, the term "aquaporin water channel" includes functional natural or synthetic aquaporin or aquaglyceroportin water channels, such as aquaporin Z (AqpZ), GIPf, SoPIP2;l, 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 aquaglyceroportins. Examples of water channel proteins and aquaglycerochannel proteins include: prokaryotic water channel proteins such as AqpZ; mammalian water channel proteins such as Aqpl and Aqp2; plant water channel proteins such as plasma membrane intrinsic protein (PIP), tonoplast intrinsic protein (TIP), nodule intrinsic protein (NIP) and small intrinsic protein (SIP), for example SoPIP2;1, PttPIP2;5 and PtPIP2;2; yeast water channel proteins such as AQY1 and AQY2; and aquaglycerochannel proteins such as GlpF and Yfl054. Aquaporin water channel proteins can be prepared according to the methods listed in Karlsson et al. (FEBS Letters 537:68-72, 2003) or as described in Jensen et al. US2012 / 0080377 (see, for example, Example 6).

[0111] In one embodiment of the separation membrane of the present disclosure, the transmembrane protein may be an anion channel protein, such as a voltage-dependent anion channel, which can be used to prepare an ion exchange membrane for reverse electrodialysis, see Dlugolecki et al. (Journal of Membrane Science, 319 214-222, 2008).

[0112] As used herein, the term "separation membrane" includes membranes for separating water and optionally certain small-sized solutes (including anions and cations) from other solutes, particles, colloids, and macromolecules. Exemplary separation membranes are "filtration membranes" such as nanofiltration (NF) membranes, forward osmosis (FO) membranes, and reverse osmosis (RO) membranes. One type of filtration membrane is a "thin film composite" (or TFC) membrane, which is generally divided into nanofiltration membranes and reverse osmosis membranes. TFC membranes are typically made by depositing a polyamide layer on top of a polyethersulfone or polysulfone porous layer on top of a nonwoven or woven fabric support. The polyamide rejection layer is formed by interfacial polymerization of an aqueous solution of amine with a solution of an acyl chloride in an organic solvent. TFC membranes can be produced as described in WO2013 / 043118 (Nanyang Technological University & Aquaporin A / S). Other types of filtration membranes are those formed by layer-by-layer (LbL) deposition methods, such as those described in Gribova et al. (Chem. Mater., 24:854-869, 2012) and Wang et al. (Membranes, 5(3):369-384, 2015). For example, self-assembled polymer vesicles can be embedded or incorporated into polyelectrolyte multilayer (PEM) membranes, such as those described in Gribova et al. Figure 4 As outlined.

[0113] As used herein, "thin film composite" or (TFC) membranes can be prepared using an amine reactant in an aqueous solution, preferably an aromatic amine, such as a diamine or triamine, for example 1,3-diaminobenzene (m-phenylenediamine, >99%, for example, available from Sigma-Aldrich), and an acyl halide reactant, such as a diacyl chloride or triacyl chloride, preferably an aromatic acyl halide, for example benzene 1,3,5-tricarbonyl chloride (CAS No. 84270-84-8, trimesoyl chloride (TMC), 98%, available from Sigma-Aldrich), dissolved in an organic solvent, wherein the reactants are combined in an interfacial polycondensation reaction, see Khorshidi et al. (2016) Scientific Reports 6, Article number: 22069 and U.S. Patent No.: 4,277,344, which details the formation of a composite membrane comprising a polyamide laminated on a porous membrane support on the surface of a support membrane (e.g., a polyethersulfone membrane). Dissolve benzene-1,3,5-tricarbonyl chloride (trimesoyl chloride) in a solvent such as C6-C 12 Hydrocarbons including hexane (>99.9%, Fisher Chemicals), heptane, octane, nonane, decane, etc. (straight or branched chain hydrocarbons) or other low aromatic solvents, such as Isopar TMG fluid, which is produced from a petroleum-based feedstock treated with hydrogen in the presence of a catalyst to produce a low-odor fluid, the major components of which include isoalkanes. TM G fluid: Chemical name: hydrocarbon, C 10 -C 12 , isoalkanes, <2% aromatics; CAS number: 64742-48-9, chemical name: hydrotreated heavy naphtha (petroleum) (from ExxonMobil Chemical). As known in the art, alternatives to the reactant 1,3-diaminobenzene include diamines such as hexamethylenediamine, and alternatives to the reactant benzene-1,3,5-tricarbonyl chloride include diacyl chlorides, adipoyl chloride, cyanuric acid, and the like.

[0114] As used herein, the term "diblock copolymer" means a polymer composed of two types of monomers, A and B. These monomers are arranged so that there is a chain of each monomer, and these two chains are grafted together to form a single copolymer chain.

[0115] As used herein, the term "triblock copolymer" means a polymer composed of two or three types of monomers, A, B, and / or C. These monomers are arranged so that there are chains of each monomer, and these chains are grafted together to form a single copolymer chain, such as a triblock copolymer of the form ABA or ABC.

[0116] Abbreviation M n It means the number average molecular weight. It means the total weight of the polymer divided by the number of polymer molecules. Therefore, M n It is the molecular weight weighted by the number fraction. w It refers to the weight average molecular weight. The molecular weight is weighted according to the weight fraction. Molecular mass can be measured by gel permeation chromatography (GPC) in tetrahydrofuran. The polydispersity index, defined as Mn / Mw, will be determined from the elution curve obtained in GPC.

[0117] Vesicle Size: Preferably, the vesicles of the present invention have a particle size ranging from about 10 nm in diameter to up to 200 nm in diameter, depending on the precise components of the vesicles and the conditions used to form them. It will be clear to those skilled in the art that particle size refers to a range of sizes, and that the numbers cited herein refer to the average diameter of the particles, most typically the average diameter of the range. The vesicle compositions of the present invention include vesicles having an average hydrodynamic diameter of 300 nm or less, and in some cases, an average diameter of less than 400 nm, such as less than 50 nm.

[0118] Vesicle Membrane Thickness: Preferably, the vesicles of the present invention have a membrane thickness ranging from about 6 nm in diameter to up to 18 nm in diameter, depending on the precise composition of the vesicles and the conditions used to form them. It will be clear to those skilled in the art that membrane thickness refers to a range of thicknesses, and that the numbers quoted herein refer to the average diameter, most often the average thickness, when referring to a solution of vesicles.

[0119] Examples of the molar ratio of transmembrane protein to block copolymer depend on the transmembrane protein used, the type of copolymer used, and the desired vesicle size. As an example, for amphiphilic diblock or triblock copolymers of PEG-b-PDMS or PEG-b-PCL type based vesicles and vesicles of aquaporin water channels, the molar ratio of transmembrane protein to block copolymer can be between 1:200 and 1:2000, such as 1:400 to 1:1500, such as 1:600 ​​to 1:1000.

[0120] As used herein, the term "self-assembly" refers to the process of forming vesicles through hydrophilic and hydrophobic interactions of amphiphilic substances, such as the diblock copolymers described herein having a relatively hydrophilic PEG portion and a relatively hydrophobic PCL portion.

[0121] The term hydrophilic as used herein relates to the tendency of a polymer chain to mix with, dissolve in or be wetted by water. Such a material will be relatively hydrophilic when compared to a hydrophobic polymer chain as referred to herein.

[0122] The term hydrophobic as used herein relates to the tendency of polymer chains to repel or be unable to mix with water. Such a substance will be relatively hydrophobic when compared to hydrophilic polymer chains as referred to herein.

[0123] As used herein, "hydrodynamic diameter" refers to the hydrodynamic size of a nanoparticle in an aqueous medium as measured by dynamic light scattering (DLS), defined as the size of a hypothetical hard sphere that diffuses in the same manner as the particle being measured.

[0124] Forward osmosis (FO) is an osmotic process that uses a selective permeation membrane to realize the separation of water and dissolved solutes. The driving force for the separation is the osmotic pressure gradient between the high concentration solution (referred to herein as draw) and the less concentrated solution (referred to as feed). The osmotic pressure gradient causes the net water flow through the membrane to enter the draw, thereby effectively concentrating the feed. The draw solution can be composed of a single or multiple simple salts, or can be a material specially customized for forward osmosis applications. The feed solution can be a dilute product stream, such as a beverage, a waste stream or seawater, see IFOA, http: / / forwardosmosis.biz / education / what-is-forward-osmosis /

[0125] Thus, most applications of FO fall into one of three general categories: product concentration, waste concentration, or clean water production as a byproduct of the concentration process. As used herein, the term "PAFO" describes a pressure assisted forward osmosis process. As used herein, the term "PRO" describes pressure retarded osmosis, which can be used to generate osmotic power. The membranes of the present invention can be used in all types of forward osmosis processes, and can be specifically adapted to each FO type.

[0126] As used herein, the term "reverse osmosis" (RO) refers to when a feed water pressure applied across a selectively permeable membrane is used to overcome the osmotic pressure. Reverse osmosis generally removes many types of dissolved and suspended species, including bacteria, from the feed water and is used both for industrial processes and for the production of potable water. During the RO process, solutes are retained on the pressurized side of the membrane and pure solvent (permeate) passes to the other side. The selective designation membrane does not allow larger molecules or ions to pass through its pores (holes), while allowing smaller components of the solution, such as solvent molecules, to pass freely. Low pressure reverse osmosis (LPRO) membranes are typically operated at a specific flux of 15 LMH / bar at a feed water pressure of from about <5 bar and a maximum operating pressure of up to about 25 bar. LPRO is conducted at a lower range of feed pressures, for example, 2 to 5 bar, sometimes designated as 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.

[0127] Various aspects and implementations have been described in connection with various embodiments herein. However, those of ordinary skill in the art will understand and appreciate that other variations and modifications of the disclosed embodiments can be made without departing from the claimed subject matter. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite articles "a" or "an" do not exclude a plurality.

[0128] The application is further illustrated with reference to the following non-limiting examples:

[0129] Example

[0130] Example 1.

[0131] Ring opening polymerization of D3 by using polyethylene glycol monomethyl ether mPEG as initiator.

[0132] In the general procedure (Table 1, entry 3), hexamethylcyclotrisiloxane (1.5 g, 6.7 mmol) was added to a dry round-bottom flask. The flask was then evacuated and backfilled with N2 three times. DCM (1.07 mL) was then injected, followed by the addition of THF (0.48 mL) and a THF solution of mPEG (1.14 mL, 0.5 g / mL), and the resulting solution was placed at 30°C. In addition, a DCM solution of TMnPG (1.074 mL, 0.01 g / mL) was injected into the flask, and the reaction was continued to run for 17 hours under continuous stirring. Finally, the mixture was passed through a short silica column using THF as an eluent. The solvent was then removed under reduced pressure to obtain mPEG-b-PDMS diblock as an opaque viscous liquid. Yield (1.57 g, 76%).

[0133] All mixtures and solvents were activated before use. Store over molecular sieves.

[0134]

[0135] a Poly(ethylene glycol) methyl ether, b Vinyl terminated poly(ethylene glycol).In all cases, non-stabilized HPLC grade THF was used.

[0136] Table 1: Summary of mPEG-b-PDMS diblock synthesis

[0137] Example 2.

[0138] Kinetic data for the preparation of mPEG-b-PDMS diblock copolymers using SEC.

[0139] To evaluate the efficiency of the catalyst, a kinetic study of the ROP reaction of D3 was performed using mPEG (656) as the initiating species and TMnPG as the catalyst. Two sets of parameters were tested (Table 1, entries 1 and 2). The reaction progress was followed by SEC measurements. It was noted that for lower catalyst amounts, the reaction reached near complete conversion only after 24 hours. In contrast, the reaction with 10 times higher catalyst amounts showed a similar spectrum after 3.5 hours. After 24 hours, side reactions were observed from the broadening of the polymer peak with a slight increase in molecular weight and polydispersity (D). In addition, the RI signal intensity of the mPEG species increased (at 18.3 min elution time), indicating that mPEG was slightly cracked over time.

[0140] The synthesis progress of mPEG-b-PDMS (9 / 1 / 0.09) and (9 / 1 / 0.9) was followed over time. mPEG (656) was used as an initiator. After 24 hours, Mn = 4803 g mol-1 and D = 1.15 for mPEG-b-PDMS (9 / 1 / 0.09). After 24 hours, Mn = 5253 g mol-1 and D = 1.22 for mPEG-b-PDMS (9 / 1 / 0.9).

[0141] In cryo-TEM images (not shown), no vesicles were observed to form, and instead, phase-separated droplet-like structures were observed. None of them had a double layer, which is a key characteristic of polymersomes. Over time, phase-separated oily droplet-like structures were also visible to the eye in the polymersome solution. The same observations were made with mPEG(709)-b-PDMS(1632) (Table 1, entry 3).

[0142] This behavior may be attributed to the labile silyl ether bond connecting the two blocks. Further investigation of this effect was performed by performing the same membrane rehydration method in PBS buffer and dialysis setting, see Figure 6 For dialysis, 1.0 g of material was dissolved in THF (10 mL). Dialysis was performed against DI water (1.5 L). The dialysis medium was changed twice (every 24 hours). For the membrane rehydration method, the polymersome solution was shaken for 1 day. In both cases, water was removed by performing a lyophilization procedure. The degree of degradation of the polymer was evaluated by using SEC. The results showed that long-term contact of the block copolymer with the aqueous medium resulted in a higher degree of degradation of the block copolymer material. See Figure 6 .

[0143] Example 3.

[0144] Ring-opening kinetics of D3 using mPEG as an initiator. Time series 1 H NMR measurements

[0145] In a dry vial, D3 (199 mg, 0.89 mmol) was dissolved in DCM (0.14 mL, dry). Then, mPEG (74.3 mg, 0.113 mmol, 656 g mol-1) was dissolved in THF (0.22 mL, dry). Finally, a solution of TMnPG (8 mg, 0.057 mmol) in DCM (0.14 mL, dry) was prepared. The mixture was immediately purged with N2 and transferred to a dry NMR tube. The results were analyzed on an 80 MHz SpinSolve Benchtop NMR instrument at 26°C. 1 H NMR time series measurement. Spectra were collected every 30 min for 17 h. Figure 8The resulting spectrum is given in Figure 2. The course of D3 consumption is followed by the peak at 0.04 ppm, while the growth of the PDMS chain is at -0.01 ppm. After 17 hours, the reaction reached 84% conversion.

[0146] Example 4.

[0147] As 1 The integrals of D3 and the growing PDMS chain were compared as a result of the H NMR time series measurements. From Figure 9 It can be seen in Figure 2 that the reaction proceeds slowly in the first 2 hours, after which it follows a linear growth and reaches a plateau after 13-14 hours. The solution was then extracted from the NMR tube and passed through a short silica column using THF as eluent in order to verify the correct molecular weight and account for unreacted mPEG. Figure 10 The results are given in Figure 3, presenting both the unpurified (A) and purified (B) material. After 17 hours, the 7610 g mol -1 In comparison, for the unpurified material, the expected Mn is around 1400 g mol -1 , indicating an initiation efficiency of 18.4% for the mPEG. A correction factor was applied, presenting the true molecular weight. Figure 9 and Figure 10 Further assistance for targeting more precise PDMS molecular weights. For this reason, the reaction time or the amount of D3 could be readjusted by following the same reaction temperature.

[0148] Example 5.

[0149] The ring opening polymerization of D3 by using a small molecular weight alcohol as initiator, and subsequent capping with chlorodimethylsilane.

[0150] In general procedure (table 2, entry 1), D3 (1.5g, 6.7mmol) is added to a dry flask, evacuated and backfilled 3 times with N2. Under a nitrogen atmosphere, DCM (1.6mL, dry) is injected, followed by the addition of THF (1.6mL, dry) and MeOH (0.03mL). Then, the temperature is raised to 30°C, and a DCM solution of TMnPG (0.54mL, 0.1g / mL) is injected. The reaction is carried out for 15 hours under continuous stirring. Afterwards, pyridine (0.24mL, dry) and chlorodimethylsilane (0.17mL, 1.5mmol) are added under an inert atmosphere. The reaction is run for 30min. Gas release (HCl) is observed and a white precipitate (pyridinium chloride) is formed subsequently. The mixture is then diluted with THF, filtered and solvent evaporated. The residue is diluted with heptane (10-20mL). The mixture is then filtered again and transferred to a separating funnel, where it is diluted with MeCN (10-20 mL) and washed 2-3 times. The top layer (heptane) is then collected as the main fraction, and the solvent is evaporated. The flask is filled with N2 and stored in a refrigerator (-25°C) for further use. Obtaining the final material is a clear liquid. Yield (0.9 g, 63%).

[0151]

[0152] Table 2: Conditions for the synthesis of PDMS from different low molecular weight alcohols as part of a 1H time series measurement.

[0153]

[0154] Table 3: Data for PDMS materials synthesized using low molecular weight alcohols as initiating species. a The degree of polymerization (DP) and initiation efficiency were estimated from 1H NMR measurements of the final (purified) material.

[0155] Methanol, 2-(methoxy)ethanol, 2-(benzyloxy)ethanol, and isopropanol were tested to evaluate their efficiency as ROP initiators for D3 (see Table 3). It was noted that MeOH produced PDMS with an ideal DP. In contrast, 2-(methoxy)ethanol and 2-(benzyloxy)ethanol produced approximately twice the target molecular weight. Finally, initiation from isopropanol produced a polymer that was almost five times the target. This can be explained by the presence of a more sterically hindered secondary alcohol of IPOH compared to primary alcohols such as MeOH or 2-(methoxy)ethanol or 2-(benzyloxy)ethanol. It appears that the steric hindrance and chain length of the initiating alcohol species play a key role in the reaction efficiency of D3's ROP. Therefore, it is concluded that short-chain primary alcohols are the most effective initiating species for the preparation of hydroxyl-terminated PDMS materials from cyclosiloxanes.

[0156] Example 6.

[0157] Preparation of mPEG-b-PDMS-b-PEG triblock.

[0158] D3 (0.75 g, 3.37 mmol) was added to a dry round-bottom flask. The flask was evacuated and backfilled with N2 three times. Afterwards, DCM (1.074 mL, dry) and mPEG (0.56 g, 1.52 mmol, 656 g mol) were added under N2. -1 ) in THF (1.621 mL). The reaction was set to run at 30°C with continuous stirring. Then, TMnPG (75 mg, 0.53 mmol) in DCM (1.074 mL, dry) was injected. The reaction was allowed to run for 17.5 hours. Pyridine (0.194 mL, 2.40 mmol) and chlorodimethylsilane (0.22 mL, 1.98 mmol) were injected directly into the reaction mixture. The reaction was allowed to proceed for 30 min to obtain hydride-terminated mPEG-b-PDMS diblock. The mixture was then filtered through a PTFE filter (0.45 μm) to remove the chlorinated pyridinium salt, after which it was passed through a short silica column with THF as eluent. The solvent was removed by rotary evaporation under reduced pressure. Subsequently, the material was further dried in vacuo overnight to obtain a viscous opaque liquid (0.56 g, yield: 42.8%). Mn=1930 gmol-1( 1 1H NMR).

[0159] Hydride-terminated mPEG-b-PDMS diblock (0.56 g, 0.29 mmol) and vinyl-terminated PEG (0.260 g, 0.32 mmol, 814 g mol -1 , 10% excess) was charged into a dry round-bottom flask. The flask was then evacuated and backfilled with N2 three times. Then, toluene (5 mL, dry) was injected, and the temperature of the mixture was raised to 65°C. Finally, 10 μL of Pt catalyst (2.0-2.1 wt% Pt in xylene) was introduced. The reaction was allowed to run for 18 hours under continuous stirring. The crude mixture was applied directly to a silica column with THF as eluent. The solvent was removed by rotary evaporation under reduced pressure to obtain a clear viscous liquid (0.250 g, yield: 30.5%). Total yield: 13%. Mn = 6151 g mol -1 ( 1 HNMR), Mn = 6689 g mol -1 (SEC), D = 1.29.

[0160] Example 7.

[0161] Hydrolysis study of mPEG-b-PDMS-b-PEG triblock.

[0162] PEG (1916) -b-PDMS (4235) (30 mg) was dissolved in THF (40 μL) in a small vial. The solution was slowly added to MiliQ water (2 mL) that had been previously passed through a 0.45 μm nylon filter. The suspension was stirred at 150 rpm and 30° C. throughout the study. 100 μL samples were collected and analyzed on a NanoCuvette on a UV spectrophotometer. TM The hydrolysis studies were performed in 4% CO 2 mol / L (100 mL / min). MiliQ water was used as a reference. After each measurement, the samples were collected in small vials and frozen at -30°C for further use. Table 5 gives an overview of the hydrolysis studies.

[0163]

[0164] Table 4: Data from hydrolysis studies of cleavable triblocks.

[0165] Go to Figure 14 , it can be observed that within the first 5 hours, some non-sedimented microphases are visible. After further stirring, they disappear and the suspension becomes less turbid, visible over time. Together with the decrease in absorbance, a significant change in the average particle size is observed. After 50.2 hours, the size of the self-assemblies has changed to 700 nm, with most of the worm-like structures and their aggregates present. In contrast, after 1 week and 2 days, the main species are polymer vesicles, with a difference of 2320 nm compared to the initial point. This can be explained by the cleavage of poly(ethylene glycol) methyl ether, which changes the hydrophilicity fraction of the block copolymer chains (f = 31.1% before cleavage and 22.1% after cleavage). This leads to a change in the critical packing parameter and, thereby, changes the curvature of the self-assemblies, driving the block copolymer chains to reassemble from worm-like structures into vesicles.

[0166] Example 8.

[0167] Assessment of mPEG cleavage by SEC

[0168] The frozen sample initially used for UV measurements was not thawed and was transferred to a larger vial (20 mL). The sample was then immersed in liquid N2 for a few minutes and immediately transferred to a desiccator. The water was then removed by a freeze drying step under reduced pressure. The dried material was further investigated in SEC. The results are presented in Figure 15 middle.

[0169] After 1 week and 2 days of exposure of the hydrolytically unstable triblock to aqueous media, samples were taken for cryo-TEM imaging. The samples were frozen on small copper lace grids under liquid ethane and subsequently measured on a cryo-TEM instrument. The images showed that the vesicles were uniform in size, with a diameter of approximately 100 nm. The average bilayer thickness was found to be 8.2 ± 1.46 nm. Most of the vesicles had a certain inhomogeneity in the bilayer thickness, which can be caused by the incomplete hydrolysis of the material and thus the presence of both di- and triblock species.

[0170] Figure 13 shows the results from NanoCuvette TM demonstrating the difference in average particle size over time. It can be seen that after 50 hours the main species are worm-like structures, while after 1 week and 2 days vesicles are predominantly present.

[0171] Example 9.

[0172] Synthesis of PEG-b-PDMS-b-PEG reference triblock (stable, non-cleavable)

[0173] Vinyl terminated PEG (1.44 g, 1.75 mmol, 825 g mol1) was added to a round bottom flask (100 mL). The flask was then immediately purged with N2. Then, DMS-HM15 (3.13 mL, 0.72 mmol, 4146 g mol1) was injected together with toluene (15 mL, dry). The reaction mixture was heated to 65 °C, after which 8 μΐ^of Pt catalyst (2.0-2.1 wt% Pt in xylene) was added. The reaction mixture was allowed to stir overnight. After 17.5 hours, the reaction mixture was cooled to room temperature and activated charcoal (pre-washed in toluene) was added. The mixture was allowed to stir overnight. After this, the mixture was filtered over a cellulose filter. Then, a slurry of zelite was prepared in THF, which was applied on the cellulose filter and washed with large amounts of THF. Then, the toluene mixture was passed over the zelite, washed several times with THF and the solvent was evaporated under reduced pressure, resulting in a highly viscous, opaque liquid. Yield: 86%, 3.80 g, 1 H NMR: Mn = 7489 g mol1 -1 SEC: Mn = 8887 g mol1 -1 ,

[0174] SEC of PEG-b-PDMS-b-PEG reference triblock (stable, non-cleavable)

[0175] The prepared stable reference triblock material was exposed to SEC measurements. It was noted that the hydrolytically unstable triblock had a slightly different SEC profile, suggesting that a mixture of diblock and triblock species may be present in the starting material. Both the stable and cleavable variants appeared to have very similar amounts of unreacted PEG species.

[0176] Hydrolysis study of the non-cleavable reference PEG-b-PDMS-b-PEG triblock

[0177] PEG-b-PDMS-b-PEG (30 mg) was dissolved in THF (40 μL) in a small vial. The solution was slowly added to MiliQ water (2 mL) that had been previously passed through a 0.45 μm nylon filter. The suspension was stirred at 150 rpm and 30° C. Throughout the study, 100 μL samples were collected and analyzed on a NanoCuvette on a UV spectrophotometer. TM MiliQ water was used as a reference. Table 5 gives an overview of the hydrolysis studies.

[0178]

[0179] Table 5: Data from hydrolysis studies of the non-cleavable reference triblock.

[0180] NanoCuvette showing differences in average particle size TM The diameter of the self-assemblies was shown to increase over time, indicating the aggregation behavior of the non-cleavable reference triblock (data not shown). Thus, it is clear that the non-cleavable reference triblock forms aggregates over time, which is supported by the increase in absorbance and therefore turbidity of aqueous solutions of the self-assemblies (data not shown). This behavior has the opposite effect to that of the cleavable triblock variants, in which the mPEG structure slowly rearranges from aggregates or worm-like structures to polymer vesicles after cleavage.

[0181] Other methods:

[0182] 1. Formation of vesicles from mPEG-b-PDMS diblock copolymers

[0183] 1.1 Membrane rehydration.

[0184] mPEG-b-PDMS (50 mg) was dissolved in chloroform (5 mL) in a 50 mL beaker. The chloroform was then evaporated to give a dry film. The film was rehydrated with PBS buffer (5 mL, pH = 7.5) by slow addition and left on a shaker overnight (140-150 rpm).

[0185] mPEG-b-PDMS (50 mg) was dissolved in THF (5 mL), and 50 mL of PBS buffer was slowly introduced into the mixture (5 mL hr-1) by using a syringe pump. The THF and water mixture was reduced to about 50% of the original volume by using rotary evaporation.

[0186] 1.2 Dialysis-assisted vesicle formation.

[0187] mPEG-b-PDMS (50 mg) was dissolved in THF (5 mL) and placed in a dialysis bag (1 kMW cut-off) and dialyzed against 1 L of DI water.

[0188] The NMR spectrometer was performed on an 80 MHz Spinsolve Benchtop NMR spectrometer (Magritek) with an RMX add-on module. 1 H NMR time series measurements were used to evaluate the reaction kinetics of both mPEG and low molecular weight alcohols. Measurements were performed in a DCM / THF (non-deuterated) solvent mixture at 26° C. Spectra were collected every 15 or 30 min with shimming (five measurements each) and 2 s cycling.

[0189] The purified PEG-b-PDMS diblock and triblock were evaluated on a Bruker Avance III spectrometer operating at 400.13 MHz. The spectrometer was equipped with a 5 mm Prodigy CryoProbe (Bruker). The measurements were performed in CDCl3 at 25°C.

[0190] UV measurements were performed on a UV-1900i Shimadzu UV-Vis spectrophotometer. Spectra were collected in the absorbance mode from 190 to 1100 nm with a data interval of 1.0 nm. TM The UV measurements were performed in a SpectroWorks Spectrometer with integrated photonic nanocrystals for recording light scattering. The UV measurements were first performed with a reference (fluid material - water, MiliQ) and then with a sample (granular material - polydimethylsiloxane). The UV measurements were uploaded to SpectroWorks. TM The cloud system then calculates the collected absorbance data into light scattering data.

[0191] In some cases, size exclusion chromatography (SEC) is carried out on a chromatographic system, which is composed of a Viscotek VE2001 gel permeation chromatography (GPC) solvent / sample module connected to a Viscotek TriSEC Model 302 triple detector array (refractive index, light scattering, viscometer). The column group is composed of a PL guard column and two PL gel mixed D columns (Polymer Laboratories, Britain) connected in series. Tetrahydrofuran (THF) (Merck, Germany) is used as a mobile phase. Samples are measured at a flow rate of 1 mL min-1. Molar mass is determined using a calibrator based on narrow polystyrene (PS) standards (PSS, Mainz, Germany). Samples are analyzed using OmniSEC 5.10 software.

[0192] In most cases, size exclusion chromatography (SEC) of PDMS and PEG-b-PDMS based diblock and triblock was performed on a chromatography system consisting of a Waters Acquity solvent delivery module and a column oven connected to a Waters PDA TS detector and a Malvern Omnisec Reveal triple detector array (RI, light scattering, viscometer). The columns were 150 x 4.6 mm Acquity APC TM XT 450 2.5μm, 150x 4.6mm Acquity APC TM XT 200 2.5μm and 150x 4.6mm Acquity APC TM XT 45 1.7 μm organic size exclusion chromatography column. All samples were analyzed at 35 °C in stabilized tetrahydrofuran using a flow rate of 0.7 mL min-1.

[0193] Samples for cryo-transmission electron microscopy (cryo-TEM) were vitrified on glow-discharged lace formvar film reinforced with a silicon monoxide coating and supported by a copper mesh grid (Ted Pella Inc., USA). The samples were vitrified in liquid ethane using a Vitrobot Mark IV (FEI, USA) and subsequently mounted in a Gatan cryoholder (FEI, USA). Images were acquired in cryo mode using a Tecnai G2 20TWIN 200 kV TEM equipped with a FEI high-sensitivity 4k×4k Eagle camera.

Claims

1. A method for producing self-assembled polymer vesicles in aqueous solution, wherein the polymer vesicles comprise an amphiphilic diblock copolymer of the P1-b-P2 type, the method comprising the step of synthesizing an RCO-P1-b-P2 block copolymer comprising a silyl ether (-CO-Si-) bond, wherein P1 is a hydrophobic polymer and P2 is a hydrophilic polymer and R is any atom or group of atoms, wherein the synthesis comprises the ring-opening polymerization of a cyclic siloxane by an initiator in the presence of a catalyst, the initiator comprising a carbon-bound hydroxyl group.

2. The method according to claim 1, wherein The product of the ring-opening polymerization reaction is a polymer of the RCO-P1 type, and the method further The following steps are involved: a) reacting the RCO-P1 polymer with a chlorosilane to obtain a silane-terminated RCO-P1-Si(R1R2)-H, wherein R1=R2 or R1≠R2, and b) reacting the silane-terminated polymer of step a) with a vinyl-terminated hydrophilic polymer (P2) for obtaining a block copolymer of the RCO-P1-b-P2 type containing silyl ether (—CO—Si—) bonds.

3. The method according to claim 1, wherein The product of the ring-opening polymerization reaction is a polymer of the R-P1-OH type, and the method further comprises the following steps: c) reacting the R-P1-OH polymer with a chlorosilane to obtain a silane-terminated R-P1-CO-Si(R1R2)-, and d) reacting the methylsilane-terminated polymer of step a) with a vinyl-terminated hydrophilic polymer (P2) for obtaining a block copolymer of the R—P1—CO—Si—P2 type containing silyl ether (—CO—Si—) bonds.

4. A method according to any preceding claim, wherein: The initiator has a Mn comprised between approximately 200 Da and 5 kDa.

5. The method according to claim 4, wherein The initiator has a Mn comprised between approximately 600 Da and 900 Da.

6. A method according to any preceding claim, wherein: The initiator includes a hydroxyl terminated polymer or an alcohol.

7. The method according to claim 6, wherein: The initiator includes poly(ethylene glycol) (PEG) or methoxy-PEG (MeO-PEG-OH), and PEG-O-P1-b-P2 or MeO-PEG-O-P1-b-P2 is synthesized.

8. A method according to any preceding claim, wherein: P1 is poly(dimethylsiloxane) (PDMS) and the amphiphilic diblock copolymer is of the poly(dimethylsiloxane)-b-P2 (H-PDMS-b-P2) type, the method comprising synthesizing RCO-(Si(CH3)2-O) containing a silyl ether (-CO-Si-) bond n -P2(RCO-PDMS-b-P2) block copolymer.

9. A method according to any preceding claim, wherein: The reaction is allowed to proceed until a certain amount of self-assembled polymer vesicles can be measured in the aqueous solution, such as by measuring with a cryo-TEM instrument, and / or a certain amount of cleaved free -R groups can be measured in the aqueous solution, said polymer vesicles comprising an amphiphilic diblock copolymer of the P1-b-P2 type.

10. A method according to any preceding claim, wherein A cross-linking agent is further added to the reaction.

11. A polymersome comprising a block copolymer of the H-Si-P1-b-P2 type, wherein P1 is a hydrophobic polymer and P2 is a hydrophilic polymer. 12 . The polymer vesicle according to claim 11 , further comprising a block copolymer of the RCO-Si-P1-b-P2 type containing a silyl ether (—CO—Si—) bond, wherein R is any atom or atomic group.

13. The polymer vesicle according to claim 12, wherein The block copolymer comprising silyl ether bonds is a triblock copolymer.

14. The polymer vesicle according to any one of claims 11 to 13, wherein P2 and / or R comprise PEG.

15. The polymer vesicle according to any one of claims 11 to 14, wherein P1 includes poly(dimethylsiloxane) (PDMS) or polycaprolactone (PCL). The polymer vesicle according to claim 15 , wherein The triblock copolymers containing silyl ether bonds are of the PEG-O-PDMS-b-PEG, MeO-PEG-O-PDMS-b-PEG or PEG-b-PCL-CO-Si-PCL-b-PEG type.

17. The polymersome according to any one of claims 11 to 16, wherein The block copolymer further includes functional end groups that allow crosslinking.

18. The polymer vesicle according to claim 17, wherein The functional end groups are selected from the group consisting of primary amine (-NH2), carboxyl (-COOH), sulfhydryl (-SH) and carbonyl (-CHO).

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