EP4676634A2

EP4676634A2 - Cellulosic filtration membrane, methods of formation, and methods of use

Info

Publication number: EP4676634A2
Application number: EP24767784.2A
Authority: EP
Inventors: Jamie A. Hestekin; Christa HESTEKIN; John P. Moore; Efecan PAKKANER; Peter A. Crooks; Soma Shekar Dachavaram; Roland G. LUDLOW
Original assignee: United States Kidney Research Corp; University of Arkansas at Fayetteville; University of Arkansas at Little Rock
Current assignee: United States Kidney Research Corp; University of Arkansas at Fayetteville; University of Arkansas at Little Rock
Priority date: 2023-03-06
Filing date: 2024-03-06
Publication date: 2026-01-14
Also published as: WO2024186907A3; WO2024186907A2

Abstract

An ultra-filtration membrane comprising cellulose nano-material, which may include cellulose nano-fibers. The ultrafiltration membrane may have non-spherical or slit-shaped pores. The membrane may be an ionogel. A method for forming the ultra-filtration membrane may be a phase-inversion process that uses an ionic liquid. The ultra-filtration membrane may be used in ultrafiltration methods, including hemodialysis. The ultra-filtration membrane exhibits high protein rejection (%) as well as high blood filtration rate (L/H m²), which is unique especially among ultrafiltration membranes for hemodialysis.

Description

CELLULOSIC FILTRATION MEMBRANE, METHODS OF FORMATION, AND METHODS OF USE

RELATED APPLICATION DATA

The present application claims priority pursuant to Article 8 of the Patent Cooperation Treaty to United States Provisional Application Serial Number 63/450,166 filed March 6, 2023, which is incorporated herein by reference in its entirety.

FIELD

The technology described herein generally relates to cellulosic filtration membranes, including cellulosic membranes for filtration processes, including ultra-filtration processes.

BACKGROUND

Ultra-filtration (UF) is a membrane filtration process where membrane modules of filters are available in, for example, plate-and-frame, spiral-wound, and tubular configurations with pore sizes in the range of 0.001 to 0.1 microns. In UF, forces like pressure or concentration gradients lead to separation through a membrane. Suspended solids and solutes of high molecular weight are retained in the retentate, while water and low molecular weight solutes pass through the membrane in the permeate or filtrate. UF is typically used for purifying and concentrating macromolecular (10³-10⁶ Da) solutions, particularly protein solutions.

UF and UF membranes have had a wide-range of successful applications including drinking water treatment, endotoxin and pyrogenic material removal, separation of micro-pollutants, and hemodialysis. Ding, Y., Ma, B., Liu, H. & Qu, J. Effects of protein properties on ultrafiltration membrane fouling performance in water treatment. Journal of Environmental Sciences 77, 273-281 (2019), Difelice, A. et al.

Ultrafiltration and Endotoxin Removal from Dialysis Fluids, Kidney International 43, S201-S204 (1993); Boleda, M., Galceran, M. & Ventura, F. Behavior of pharmaceuticals and drugs of abuse in a drinking water treatment plant (DWTP) using combined conventional and ultrafiltration and reverse osmosis (UF/RO) treatments. Environmental Pollution 159, 1584-1591 (2011); Lin, X. C. et al. Porous diffusion dialysis membranes for rapid acid recovery. J Membrane Sci 502, 76-83 (2016). However, fouling of membranes is a challenge that affects membrane performance adversely and remains as the main limitation. In dialysis, blood creates even more significant challenge, where proteins and clotting can significantly affect the ultra-filtration properties. Biran, R. & Pond, D. Heparin coatings for improving blood compatibility of medical devices. Advanced Drug Delivery Reviews 112, 12-23 (2017); Huang, X. J., Guduru, D., Xu, Z. K., Vienken, J. & Groth, T. Blood compatibility and permeability of heparin- modified polysulfone as potential membrane for simultaneous hemodialysis and LDL removal. Macromol Biosci 11, 131-140 (2011). Since bio-foulants are typically hydrophobic in nature, to boost the anti-fouling properties of a typical ultra-filtration membrane, changes to its surface properties including hydrophilicity, negative surface charge density, and coupling with anticoagulants are typically attempted. A variety of approaches have been utilized including modified surface coatings using bio-polymers such as polydopamine, heparin, chitosan, poly L-lysine and mucin. Kumar, R. & Ismail, A. F. Fouling control on microfiltration/ultrafiltration membranes: Effects of morphology, hydrophilicity, and charge. J Appl Polym Sci 132 (2015); Jin L., Z. F., Gao Y., Sui S., Zhang D. Purification of pectin by ultrafiltration in combination with sodium citrate. Journal of Food Engineering 335 (2022); Cheng, C. et al. The hydrodynamic permeability and surface property of polyethersulfone ultrafiltration membranes with mussel-inspired polydopamine coatings. Journal of Membrane Science 417, 228-236 (2012); Cheng, C. et al. The hydrodynamic permeability and surface property of polyethersulfone ultrafiltration membranes with mussel-inspired polydopamine coatings. Journal of Membrane Science 417, 228-236 (2012); Yang, M. & Lin, W. Protein adsorption and platelet adhesion of poly sulfone membrane immobilized with chitosan and heparin conjugate. Polymers For Advanced Technologies 14, 103-113 (2003); Liu, T. et al. Immobilization of heparin/poly-L-lysine nanoparticles on dopamine-coated surface to create a heparin density gradient for selective direction of platelet and vascular cells behavior. Acta Biomaterialia 10, 1940-1954 (2014); Winkeljann, B. et al. Covalent Mucin Coatings Form Stable Anti-Biofouling Layers on a Broad Range of Medical Polymer Materials. Adv Mater Interfaces 7 (2020). Although these have been used, a long-term remedy to increase the biocompatibility of membranes in contact with blood has thus far not been achieved.

Hemodialysis (HD) involves UF. Hemodialysis (HD) plays an important role in providing life support for patients with end-stage renal syndrome (ESRD). Currently in the USA, there are -600,000 patients undergoing hemodialysis on a chronic basis. In addition, patients are treated with hemodialysis when they have acute kidney injury (AKI). In HD, blood, and a dialysate solution flow through a dialyzer where dialysis takes place between two fluid streams. Typically, the membrane used in the dialyzer is configured in a hollow fiber format. Various polymeric materials have been used as dialysis membranes including cellulose acetate (CA), polyacrylonitrile (PAN), and poly-sulfone (PS). Idris, A. & Yet, L. K. The effect of different molecular weight PEG additives on cellulose acetate asymmetric dialysis membrane performance. Journal of membrane science 280, 920-927 (2006); Pascual et al., Adsorption of Complement Factor-D by Polyacrylonitrile Dialysis Membranes, Kidney International 44, 260-260 (1993); Vanommeslaeghe, F. et al. A randomized cross-over study with objective quantification of the performance of an asymmetric triacetate and a polysulfone dialysis membrane using different anti coagulation strategies (vol 14, pg 398, 2021). Clinical Kidney Journal 14, 463-464 (2021). Whether a membrane is acceptable for dialysis is based on its biocompatibility and ability to mediate the flow of ions, urea, and uremic toxins between the patient’s bloodstream and the dialysate solution within the timeframe of a typical treatment session.

In a conventional dialyzer, two different processes are utilized. The membrane can act as an ultrafilter. In an ultra-filtration process, a driving force (in this case blood pressure) is applied and that separates two phases. In this mode of operation, various amounts of water can be removed depending on the patient’s volume status. The second mode of operation is dialysis which is a diffusion-driven process. In this process, a chemical potential gradient (p) in the absence of a pressure gradient between the two streams induces the permeation of a solute through the membrane. Various ions, urea and uremic toxins permeate in this manner.

Cellulose, which is a naturally abundant, easy to handle biopolymer has been found to be an effective material to be utilized in ultra-filtration membranes. Membranes including cellulose nanomaterials have been successfully used for ultra-filtration processes, including water purification and hemodialysis. See Moore, John P. et al., Oxone-Mediated TEMPO -oxidized Cellulose Nanomaterial Ultra-filtration and Dialysis Mixed-Matrix Hollow Fiber Membrane , Polymers 2020, 12(6), 1348, which is incorporated herein by reference in its entirety. However, a desire for cellulosic membranes with improved properties, including improved flux (L/h/m²), high protein rejection (%), and improved antifouling, among other things, are desirable.

SUMMARY

Disclosed herein is an improved cellulosic membrane for use in various ultra-filtration processes, including hemodialysis. In some embodiments, the membrane comprises a cellulose nano-material (CNM) and exhibits one or more of the following properties: a filtration rate (L/H m²) greater than 80, greater than 90, or greater than 100; a protein rejection rate greater than 95%, 98%, or 99%; and/or greater than 5 hours permeation with blood with no anti-coagulant. Notably, the cellulosic membranes described herein have at least both of the following: a blood filtration rate (L/H m²) greater than 80 and a protein rejection rate greater than 95%, greater than 98%, or greater than 99%. This is notable because prior ultra-filtration membranes exhibiting high protein rejection rates have typically had very low filtration rates. Rejections of target proteins is important for certain ultra-filtration applications such as hemodialysis where you want to minimize loss of certain proteins from the blood. Thus, to be able to maintain a high rejection rate and a high filtration rate is notable. Additionally, having a greater than 5-hour permeation of blood with no anti-coagulant indicates that the membrane has superior anti-fouling properties. Comparatively, poly sulfone membranes exhibit zero flux. Finally, these membranes show no rejection characteristics in ultra-filtration and dialysis experiments with rats indicating that they have many applications in dialysis and other blood applications.

In some preferred embodiments, the membrane described herein is an ultra-filtration membrane that comprises a cellulose nano-material (CNM). The CNM, in some embodiments, may comprise cellulose nano-fibers (CNFs). CNFs are not so limited, and individual CNFs may have a width of from lnm-9nm or from lnm-5nm. In some embodiments, the CNM or CNFs may have one or more carboxylate functional groups.

In some embodiments described herein the ultra-filtration membrane may have non-spherical pores. In some embodiments, these non-spherical pores may be slit-shaped. The membrane with non- spherical pores may be an ionogel. In some embodiments, the active layer of the ultra-filtration membrane may have a thickness less than 0.01 microns or 0.001 um to 0.01 pm.

In some embodiments, the ultra-filtration membrane is formed from a solution comprising a CNM and an ionic liquid. In such embodiments, at least one functional group of the CNM (e.g., and ROH' group or a RCOO ) may be associated (e.g., ionic bond, covalent bond, or Van der Waals bond) with an ion of the ionic liquid (e.g., a cation of the ionic liquid). In some embodiments, a covalent bond may be formed between the at least one functional group and the ion of the ionic liquid. In such embodiments, the ion of the ionic liquid may be a cation of an imidazolium-based ionic liquid. For example, the ion may be ethylmethylimidazoluium (EMIM) when the ionic liquid comprises l-ethyl-3-methylimidazolium acetate (EMIMAc).

In another aspect, a method for manufacturing an ultra-filtration membrane having properties described herein is provided. The method comprises at least a step of providing a solution that comprises the CNM and an ionic liquid. As mentioned above, the CNM may comprise CNFs, and individual CNFs may have a width from Inm to 9 nm wide or from 1 nm to 5 nm wide. The CNM or CNFs may comprise one or more carboxylate functional groups. The ionic liquid, in some embodiments, may comprise an imidazolium-based ionic liquid, e.g., l-Ethyl-3-methylimidazolium acetate (EMIMAc).

In some preferred embodiments, the method for manufacturing is a phase-inversion process. For example, the method may involve providing the solution by casting to form a cast film, and then immersing the cast film in de-ionized water.

In another aspect, an ultra-filtration method is disclosed. The method comprises at least a step of providing an ultra-filtration membrane as described hereinabove. In some embodiments, the ultrafiltration method may be hemodialysis. The method may comprise a step of providing an ultra-filtration membrane as described herein.

The subject matter of aspects of the present disclosure is described with specificity herein to meet statutory requirements. However, the description itself is not intended to limit the scope of this patent. Rather, the inventors have contemplated that the claimed subject matter might also be embodied in other ways, to include different steps or combinations of steps similar to the ones described in this document, in conjunction with other present or future technologies. Moreover, although the terms “step” and/or “block” can be used herein to connote different elements of methods employed, the terms should not be interpreted as implying any particular order among or between various steps disclosed herein unless and except when the order of individual steps is explicitly described.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. l is a schematic showing an atypical phase-inversion process utilized to form the membranes described herein, e.g., the NC-ILM product.

Fig. 2A is an SEM picture of a final ultra-filtration membrane (NC-ILM product) as described herein with a 5-micron scale.

Fig. 2B is a WAXS at the SLAC beamline showing anisotropic property of a final ultra-filtration membrane (NC-ILM product).

Fig. 2C is FT-IR spectra of TEMPO Oxidized cellulose, l-ethyl-3-methylimidazolium acetate (EMIMAc) ionic liquid and a final membrane product (NC-ILM product) described herein.

Fig. 2D includes sieving coefficients plotted for experimental (dots) and theoretical (lines) for a final membrane product (NC-ILM product) described herein for varying molecular weight proteins.

Fig. 3A is a graph comparing different membrane performance for commercial membranes, e.g., Millipore PIBC, and experimental membranes described herein, e.g., the NC-ILM product. Fig. 3B is a graph showing change in average permeate flux of heparinized whole porcupine blood through an experimental membrane described herein, e.g., the NC-ILM product, and polysulfone (PSf) membranes (n=3).

Fig. 3C is a graph showing change in average permeate flux of citrated whole porcupine blood through an experimental membrane described herein, e.g., the NC-ILM product, and polysulfone (PSf) membranes (n=3).

Fig. 3D is a graph showing change in permeate flux of a non-coagulated whole porcupine blood through an experimental membrane described herein, e.g., the NC-ILM product, and polysulfone (PSf) membranes (n=3).

Fig. 4A is a schematic diagram of a dialysis (hemodialysis) experimental setup.

Fig. 4B includes sieving curves comparison for different membranes, experimental (e.g., NC-ILM product) and commercialized. The NC-ILM product has the tightest molecular weight cut-off.

Fig. 4C is a graph of urea and lysozyme clearance performance by a silicon nanopore membrane (left bar), a commercialized high flux membrane (middle bar), and the experimental membrane, e.g., the NC-ILM product (right bar).

Fig. 5 A is a schematic of an ultra-filtration ex vivo experiment as described herein.

Fig. 5B is a graph showing permeation of BUN through an experimental membrane as described herein, e.g., the NC-ILM product.

Fig. 5C is a graph showing initial and final concentration of complement component C3(C3, left bar) and rat soluble terminal complement complex (SC5B-9, right bar) in mg/dL.

Fig. 5D is a graph of vital measurements from one dialysis experiment described herein.

Fig. 6A is a picture of an entire ultra-filtration set-up according to some embodiments described herein.

Fig. 6B is a picture of membrane placement in a stirred cell unit as described herein.

Fig. 7 is a schematic showing an interaction between the ions of the ionic liquid and the cellulose nano-material according to some embodiments described herein.

DETAILED DESCRIPTION

A novel ultra-filtration membrane is described herein. Notable characteristics of this membrane include one or more of the following: a filtration rate (L/H m²) greater than 80; a protein rejection rate greater than 95%, and greater than 5 hours permeation with blood with no anti-coagulant. In some preferred embodiments, the ultra-filtration membranes described herein have both a filtration rate (L/H m²) greater than 80 and a protein rejection rate greater than 95%. This is exceptional compared to prior membranes where in order to achieve an acceptable protein rejection rate, filtration rate decreased. Rejections of target proteins is important for certain ultra-filtration applications such as hemodialysis where it is desirable to minimize loss of certain proteins from the blood. This can be seen looking at Fig. 3A where commercial membranes and experimental membranes with comparable protein rejection (%) are compared. Only the experimental membrane (NC-ILM product) has a filtration rate above 20, let alone above 80 as claimed. Thus, the ultra-filtration membranes described herein are shown to work quickly and effectively, particularly for hemodialysis.

In some particularly preferred embodiments, the ultra-filtration membranes described herein have at least all three of the following properties: a filtration rate (L/H m²) greater than 80; a protein rejection rate greater than 95%, and greater than 5 hours permeation with blood with no anti-coagulant. Additionally, having a greater than 5-hour permeation of blood with no anti-coagulant (see Fig. 3D) indicates that the membrane has superior anti-fouling properties. Comparatively, commonly used polysulfone membranes exhibit zero flux.

Filtration rates (L/H m²), including blood filtration rates, of the ultra-filtration membranes described herein may be greater than 20, greater than 25, greater than 30, greater than 35, greater than 40, greater than 45, greater than 50, greater than 55, greater than 60, greater than 65, greater than 70, greater than 75, greater than 80, greater than 85, greater than 90, greater than 95, or greater than 100.

Protein rejection rates (%) of ultra-filtration membranes described herein may be greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.

In some embodiments, the ultra-filtration membranes described herein may have a protein rejection rate greater than 95% and a filtration rate greater than 20. In preferred embodiments, the protein rejection rate may be greater than 98% or greater than 99%, and the filtration rate may be greater than 20. In even more preferred embodiments, the protein rejection rate may be 95% or more and the filtration rate may be higher than 80, 85, 90, or 100. In even more preferred embodiments, the protein rejection rate may be 98% or more and the filtration rate may be higher than 80, 85, 90, or 100.

With further regard to the novel ultra-filtration membrane described herein, a 1-2 order of magnitude increase in flux was observed when compared to the polysulfone (PSf) and other commercial membranes with increase rejection of target protein. Second, these ultra-filtration membranes have a sharper molecular weight cut-off than other polymeric membranes, which is believed to be due to a slit pore formation as shown with scattering experiments. This is a unique structure, unlike the structure of other membrane formed using a typical phase-inversion process. Third, in dialysis applications, this membrane shows high passage of urea, low passage of proteins, and high passage of surrogate uremic toxins. Fourth, anti-fouling properties have been shown to be superior for the NC-ILMs, including greater than 5-hours permeation with blood with no anti-coagulant as compared to zero flux for PSf membranes. Finally, these membranes show no rejection characteristics in ultra-filtration and dialysis experiments with rats indicating that they have many applications in dialysis and other blood applications.

Ultra-filtration Membrane

The ultra-filtration membranes described herein may comprise, consist of, or consist of a cellulose nano-material (CNM). In some embodiments, the CNM may comprise, consist of, or consist essentially of cellulose nanoparticles/cellulose nanocrystals (CNCs), cellulose nano-fibers (CNFs), or combinations thereof. In some preferred embodiments, the CNM may comprise, consist of, or consist essentially of CNFs. Typically, the width of CNFs may be in a range from Inm to lOOnm, Inm to 90nm, 1 nm to 80 nm, 1 nm to 70 nm, 1 nm to 60 nm, 1 nm to 50 nm, 1 nm to 40 nm, 1 nm to 30 nm, 1 nm to 20 nm, or 1 nm to 10 nm. In some particularly preferred embodiments, the width may be 1 nm to 10 nm, 1 nm to 9 nm, 1 nm to 8 nm, 1 nm to 7 nm, 1 nm to 6 nm, 1 nm to 5 nm, 1 nm to 4 nm, 1 nm to 3 nm, or 1 nm to 2nm. Preferably, the CNFs also have a high aspect ratio (length to width), which is preferably greater than 50, greater than 60, greater than 70, greater than 80, greater than 90, or greater than 100.

In some preferred embodiments, the cellulose nano-material (CNM) may be an oxidized CNM where oxidation of cellulose primary hydroxyl groups to carboxylate groups has occurred. One such example is TEMPO-oxidized cellulose. In some embodiments, the TEMPO-oxidized cellulose is produced in accordance with the methods and oxidants disclosed in United States Patent Application Serial Number 16/634,703 which is in incorporated herein by reference in its entirety. In some embodiments, for example, the oxidant component comprise oxone.

The ultra-filtration membrane described herein may have a thickness of from 50 microns to 500 microns, from 50 microns to 400 microns, from 50 microns to 300 microns, from 50 microns to 200 microns, or from 50 microns to 100 microns.

The active layer of the ultra-filtration membrane described herein is extremely thin. It is less than 0.02 microns, and preferably less than 0.01 microns. Typical ultra-filtration membranes (e g., polysulfone membranes that may be used for hemodialysis) have an active layer in the range of 0.027 microns to 17.5 microns. As understood by those skilled in the art, in an SEM, the active layer may appear as a dense layer on a sponge-like support.

The ultra-fdtration membrane described herein may have an average pore size in a range from 10 nm to 20 nm, from 10 nm to 19 nm, from 10 nm to 18 nm, from 10 nm to 17 nm, from 10 nm to 16 nm, from 10 nm to 15 nm, from 11 nm to 15nm, from 12 nm to 15 nm, from 13 nm to 15 nm, or from 14 nm to 15 nm. In some particularly preferred embodiments, the ultra-filtration membrane may have an average pore size in the range from about 11 microns to about 14 microns.

The ultra-filtration membranes described herein have a unique non-spherical pore shape. In some preferred embodiments, the pores are slit-shaped, and average pore size is measured as the width of the slit-shaped pores.

The ultra-filtration membrane described herein may have an extremely tight molecular weight (mw) cut-off with 100% sieving at 15 kDa and 0% sieving at 66 kDa. This finding is further evidence that this material has non-circular or slit-shaped properties rather than circular pore structures.

In some preferred embodiments, the ultra-filtration membranes described herein are ionogels. An ionogel is a composite material including an ionic liquid immobilized by an inorganic or a polymer matrix. As described herein, a method to produce ionogels is to employ imidazolium-based ionic liquids (ILs), which potentially provide high nucleophilicity and alkaline-stability to the desired membrane product. Dissolving and recovering cellulose with ILs induce intriguing and unique transformative properties, where resulting cellulose based ionogels have shown interesting ordered self-assembly properties. In this process the anionic residues of the ILs (i.e., acetates, halides, etc.) disrupt the structure of the biopolymer. As shown, the mechanism of ordered TEMPO modified cellulosic membranes can lead to a high- performance ultra-filtration membrane with unique properties.

The unique structure results at least in part due to the unique method used to make it. In some preferred embodiments described herein the ultra-filtration membrane is formed from a solution comprising, consisting of, or consisting essentially of a CNM and an ionic liquid. In the final membrane, at least one functional group of the CNM (e.g., and -ROH’ group or a RCOO’) may be associated with an ion of the ionic liquid (e.g., a cation of the ionic liquid). The association may be an ionic bond, a covalent bond, or a Van der Waals bond. In some embodiments, a covalent bond may be formed between the at least one functional group and the ion of the ionic liquid. In such embodiments, the ion of the ionic liquid may be a cation of an imidazolium-based ionic liquid. For example, the ion may be ethylmethylimidazoluium (EMIM) when the ionic liquid comprises or is l-Ethyl-3-methylimidazolium acetate (EMIMAc). Thus, the ionic liquid is believed to create space between the nanofibular structure. The cationic imidazolium group can covalently bind to the cellulosic backbone and surface anion groups that were present in the ionic liquid are now exposed to create a negative surface charge. See the dotted box in Fig. 1 reproduced in Fig. 7.

Method of Membrane Formation

The method of forming the ultra-filtration membranes described herein is unique, and is believed to be responsible for the unique membrane structures and properties described hereinabove. In some embodiments, the method may comprise at least a step of providing a solution that comprises, consists of, or consists essentially of a CNM as described hereinabove and an ionic liquid. In preferred embodiments, the method may be a phase inversion process that uses an ionic liquid instead of the organic solvents used in typical phase inversion processes, e.g., n-methyl-2-pyrrolidone (NMP). The phase inversion process may comprise at least a step of providing a solution that comprises, consists of, or consists essentially of a CNM as described hereinabove and an ionic liquid.

In preferred embodiments, the amount of CNM in the solution may be from about 5% to about 20%, from about 5% to about 15%, or from about 5% to about 10%.

The ionic liquid is not so limited, and in preferred embodiments it may be an imidazolium-based ionic liquid. For example, an exemplary imidazolium based ionic liquid may be -Ethyl-3- methylimidazolium acetate (EMIMAc).

The method of providing the solution is not so limited and may be a casting process, a spin-coating process, a blade-coating process or the like. In some preferred embodiments, the solution is provided on a support or substrate to form a film. For example, the solution may be cast on a support or substrate to form a cast film.

Cast films were then immersed in a de-ionized water bath, and fully soaked for 3 minutes to complete the phase inversion.

After the film is formed, a phase-inversion process may be performed. The phase-inversion process may comprise, consist of, or consist essentially of immersing the films in de-ionized water for an amount of time sufficient to complete phase inversion. This time may be from 1 minute to 1 hour, or more.

Further steps of solvent evaporation, a de-ionized water rinse, or a combination of both may be performed to obtain the final ultra-filtration membrane product. Method of Use

In some embodiments, the ultra-filtration membranes described herein may be used for ultrafiltration. In some preferred embodiments, the membranes may be used for hemodialysis.

Accordingly, embodiments described herein can be understood more readily by reference to the following detailed description, examples, and figures. Elements, apparatus, and methods described herein, however, are not limited to the specific embodiments presented in the detailed description, examples, and figures. It should be recognized that the exemplary embodiments herein are merely illustrative of the principles of the invention. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the invention.

In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1.0 to 10.0” should be considered to include any and all subranges beginning with a minimum value of 1.0 or more and ending with a maximum value of 10.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9.

All ranges disclosed herein are also to be considered to include the end points of the range, unless expressly stated otherwise. For example, a range of “between 5 and 10” or “5 to 10” or “5-10” should generally be considered to include the end points 5 and 10.

Further, when the phrase “up to” is used in connection with an amount or quantity; it is to be understood that the amount is at least a detectable amount or quantity. For example, a material present in an amount “up to” a specified amount can be present from a detectable amount and up to and including the specified amount.

Additionally, in any disclosed embodiment, the terms “substantially,” “approximately,” and “about” may be substituted with “within [a percentage] of’ what is specified, where the percentage includes 0.1, 1, 5, and 10 percent.

Many different arrangements of the various components and/or steps depicted and described, as well as those not shown, are possible without departing from the scope of the claims below. Embodiments of the present technology have been described with the intent to be illustrative rather than restrictive. Alternative embodiments will become apparent from reference to this disclosure. Alternative means of implementing the aforementioned can be completed without departing from the scope of the claims below. Certain features and subcombinations are of utility and can be employed without reference to other features and subcombinations and are contemplated within the scope of the claims. EXAMPLES

Biocompatibility and the ability to mediate the flow of ions, urea, and uremic toxins between a bloodstream and dialysate solution are key parameters for membranes used in dialysis. Oxone® mediated TEMPO-oxidized cellulose nano-materials have shown to be an excellent additive in the production tunability of flat sheet ultra-filtration and dialysis membranes. Nano-cellulose ionic liquid membranes (NC-ILMs) were tested in vitro and ex vivo in this study.

Materials

Oxone® mediated TEMPO-oxidized cellulose in powder form was received from University of Arkansas Medical Sciences department. Poly sulfone pellets (Mw: 75000) was purchased from Acros Organics (ThermoFisher Scientific, Geel, Belgium). 1-methyl 2-pyrrolidone (NMP) and l-Ethyl-3- methylimidazolium acetate (EMIMAc) were acquired from Millipore Sigma (Merck KGaA, Darmstadt, Germany). Bovine serum albumin (BSA) was purchased from VWR USA (Radnor, PA, USA). Heparinized whole porcine blood was purchased from Pel-Freez, LLC (Rogers, AR, USA). All aqueous solutions were prepared using Milli-Q water.

Membrane casting

Experimental Membrane (NC-ILM product) using phase-inversion with an ionic liquid: 10% solution of Form I Oxone® mediated TEMPO-oxidized cellulose was prepared by dissolving the cellulose residues in l-Ethyl-3-methylimidazolium acetate (EMIMAc) ionic liquid. The cast mixture was uniformly mixed on a turning roller for 7 days, making sure no cellulose lumps were present in the mixture.

Polysulfone Comparative Membrane using typical phase-inversion with NMP: The same procedure was applied for poly sulfone control membranes as 10% poly sulfone solution was prepared in 1-methyl 2-pyrrolidone (NMP) instead of the ionic liquid.

The experimental and comparative mixtures were filtered under vacuum and air bubbles were removed. The homogeneous cast mixtures where then cast onto glass plates, via a casting blade which was adjusted to cast a membrane with a thickness of 200pm. Cast films were then immersed in a deionized water bath, and fully soaked for 3 minutes to complete the phase inversion. Next, a solvent evaporation step was performed followed by a de-ionized water rinse. See Fig. 1, which includes a schematic drawing of the process to form the NC-ILM experimental product.

Average pore radius and porosity were calculated using the Guerout-Elford-Ferry method. Briefly, porosity of the membrane samples was calculating using a gravimetric method, with the equation given below: where, wi and W2 are the wet and dry weight of the membrane sample, and D_w and D_P are the density of water and the polymer, respectively.

Calculated porosity values were then placed in the following equation: f 2.90 — 1 .75f?}^]hQ

R f = , 1 ^{: 1}

\ i-'A where, r| is water viscosity, h is the thickness of the membrane coupon, Q is the permeated water flux per time, P is the operating pressure and A is the membrane area.

Scanning Electron Microscopy (SEM)

Pictures of the membrane samples were taken in e-SEM mode. The samples were cut into fragments in millimeter size range and sputter-coated with gold layer in rarefied argon. An SEM of the NC-ILM product is provided in Fig. 2A.

Fourier Transform Infrared Spectroscopy (FT-IR)

FTIR spectra of the ionic liquid, raw cellulose and the membrane samples were drawn using Perkin Elmer Frontier FT-IR Spectrometer (PerkinElmer, Waltham, MA, USA). The absorption spectra were taken between wavenumbers 4000 cm’¹ and 600 cm’¹ with a scan number of 32 and a resolution of 4 cm’ Fig. 2C includes an FT-IR spectra of TEMPO Oxidized cellulose and l-ethyl-3-methylimidazolium acetate (EMIMAc) ionic liquid, which are components used to from the NC-ILM product. An FT-IR spectrum of the NC-ILM product is also provided in Fig. 2C.

In vitro experiments and sample analyses

To test the filtration performance of the membranes, a Sterlitech HP4750 stirred cell was utilized with a membrane area of 20 cm² (Fig. 6-B) (Sterlitech, Kent, WA, USA). 250 mL of the filtration solution was placed inside the cell chamber, with a magnetic stirrer attached inside, continuously stirring at 200 rpm. The unit was pressurized with N2 inert gas, and the pressure was monitored by a digital flow meter. Operating pressure was kept at 30 psig. Permeate was collected in measure flasks and flux was manually recorded with a timer. Two filtration media was used for the experiments: a 1 mg/mL bovine serum albumin (BSA) solution for the protein bio-fouling tests and a whole porcine blood solution, anticoagulated with heparin. BSA content of the solution samples were measured using a standard bicinchoninic acid (BCA) assay. Complete blood count (CBC) was performed on the blood samples to determine the blood cell concentrations.

Method for spin-coating and blade-coating for X-Ray Experiments

Solutions of NC in ILM (5 or 10%) were spin coated on a silicon wafer at 6000 rpm for 60s. Water was spin coated on top after spin coating the first layer. (Figure S5). Blade-coated solutions of NC in ILM (5 or 10%) were cast on a silicon wafer at a gate height of 250um. Drops of water were added post bladecoating to induce the phase inversion process.

Wide-Angle X-Ray Scattering (WAXS) Characterization

Grazing-Incidence Wide-Angle X-Ray Scattering (GIWAXS) was performed at beamline 11-3 at the Stanford Synchrotron Radiation Light source (SSRL) at SLAC National Accelerator Laboratory with an X-ray beam energy of 12.7 keV (0.976 A). The beam defining slits were set to 150um(horizontal)x50um(vertical). 2D GIWAXS images were acquired on a Rayonix MX225 CCD area detector comprising 3072 x 3072 pixels with a pixel size of 73.2 x 73.2 pm2. The sample to detector distance was set at 300 mm and Lanthanum Hexaboride (LaBe) was used to calibrate the detector orientation. All samples were placed in a helium-filled chamber. Detector images were processed using a combination of pyFAI⁵⁰ , pygix, and a custom Python script. The perpendicular scattering was obtained by processing a 15-degree cake slice offset 5 degrees from the true out-of-plane direction. The parallel scattering was obtained by processing a 15-degree cake slice offset 5 degrees from the true in-plane direction. X-Ray imaging was performed at beamline 10-2 at the Stanford Synchrotron Radiation Light source (SSRL) at SLAC National Accelerator Laboratory. The X-ray beam energy of 9 keV using a homebuilt full-field X-Ray camera.

Ex vivo filtration experiments and sample analysis

The animal studies were conducted in a surgical suite in the Central Laboratory Animal Facility (CLAF), at the University of Arkansas. Sprague-Dawley rats pre-implanted with femoral vein and artery catheters were ordered from Envigo (MA, USA). All animals were kept under anesthesia during the whole filtration process. CODA tail-cuff monitoring device (CT, USA, purchased from Kent Scientific Corporation) was used to continuously measure their vitals such as blood pressure, heart rate, and temperature. Initially, the animal was slowly heparinized with 0.5 mL of the 100 lU/mL heparin on each port adapter. Once the vitals were considered stable, the extra corporeal circuit was connected to the animal through the arterial port. Then the dialysate (if dialysis experiment) and blood pumps were started at approximately 10 mL/min and 1 mL/min, respectively. After 2 or 3 drops of saline at the end of the circuit, the femoral port was connected to the animal (Figure SA). For ultra-filtration experiments, a pressure of 2 - 3 psi was applied on the membrane. Changes in the anesthetic levels or saline administration were done based on the vitals measurements. At the end of each experiment, euthanasia was performed before the animal woke up.

Enzyme-linked Immunosorbent Assay Kit for Complement Component 3 (C3) and Rat Soluble Terminal Complement Complex (SC5B-9) ELISA Kit were used to test the complement activation.

Results

The NC-ILM were made via an aty pical phase inversion (utilizing ionic liquid) and the process is shown in Fig. 1. Thus, as shown in Fig. 2A, this material has aligned fibers which make up the membrane “pores,” which are non-circular or slit-shaped pores. This is a unique property that exists when the membrane is cast in the presence of ionic liquids (ILs) as opposed to other solvents. Typical phase inversion membranes, which use solvents like NMP instead of ionic liquids, have a different structure. This NC-ILM membrane was analyzed using wide angle X ray scattering (WAXS) at the Stanford Linear Accelerator Center (SLAC) beamline (Fig. 2B) and found that it is anisotropic in nature as compared to polysulfone which shows no higher structure. Prior to the introduction of water, a very broad, isotropic peak around q=1.6 A’¹ is the dominant feature with no presence of the conventional cellulose crystals detected. The peak around q=1.6 A'¹ is consistent with the cellulose being well dissolved by the ionic liquid and no preferred orientation of the cellulose chains induced during blade-casting either parallel or perpendicular to the substrate. Upon the introduction of water, a broad peak around q=0.5-0.6 A'¹ appears, corresponding to a periodicity of approximately 10.5-12.5 A. This feature is anisotropic as evidenced by the stronger relative intensity perpendicular (blue) to the membrane surface.

X-ray full-field imaging was performed on the NC-ILM membranes and compared to polysulfone membranes made by the same non-solvent induced phase inversion process. The 2D cross-section and normal incidence micrographs of the polysulfone phase inverted material showed fine features of small pores visible near the top surface of the membrane with larger, coarser features evolving towards the bottom (left side of the image) of the membrane. This structure is absent in images of the NC-ILM membranes in normal incidence. The membrane edge shows no obvious large-scale structure that is associated with typical phase inverted membranes. Qualitatively, the NC-ILM membrane is less tough with a consistency that is reminiscent of a gel. Except for a few small cracks, there is no measurable internal structure of the NC-ILM membrane from imaging, suggesting that the internal structure has very little electron density fluctuations on a scale of >3 microns. These observations are consistent with the optical clarity of the NC-ILM membrane compared with the white appearance of the polysulfone membrane due to the strong scattering.

Using the Guerout-Elford-Ferry method, the average pore size of the NC-ILM is 12.5 +/- 1.5 nm which is larger than a typical dialysis membrane as shown in Table 1 below:

Table 1

Previous studies have reported active layer thicknesses that were very constant and ranged from 0.027 pm to 17.5 pm with polysulfone depending on the casting conditions. Pinnau et al., Journal of Applied Polymer Science 43, 1491-1502 (1991). This membrane, however, has an extremely thin active layer (estimated to be less than 0.01 pm), not traditionally seen in phase inversion membranes. The extremely thin active layer has a spongy support. Fig. 2C shows that acetate groups are present in the final membrane structure indicating that this stability mechanism has taken place. Fig. 2D shows the sieving coefficient of this membrane versus solute molecular weight as compared to previously characterized porous membrane. Moore, J. et al. Oxone (R)-Mediated TEMPO-Oxidized Cellulose Nanomaterials form I and form II. Molecules 25 (2020); Fissell, W. H. et al. High-Performance Silicon Nanopore Hemofiltration Membranes. J Memb Sci 326, 58-63 (2009); Kanani, D. M. et al. Permeability - Selectivity Analysis for Ultrafiltration: Effect of Pore Geometry. J Memb Sci 349, 405 (2010). This NC- ILM has an extremely tight molecular weight (MW) cut-off with 100% sieving at 15 kDa and 0% sieving at 66 kDa. This finding is a further evidence that this material has non-circular or slit pore structures rather than what would be expected with circular pore structures. The ability of this new membrane to reject bovine serum albumin (BSA) is shown in Fig. 3A. Its rejection properties (99.6%) are superior to both in-house made poly-sulfone membrane (94.5% rejection) and the published performance of commercial membranes. However, despite the improvement in BSA rejection, the membrane still had a superior water flux that was 21 times greater than poly-sulfone and 75- 350 times greater than commercial membranes. This finding indicates that there is no performance tradeoff between protein rejection and water flux as is seen in other synthetic polymeric membranes. Data on the rejection of blood cell components including red blood cells, white blood cells, and platelets demonstrates that the NC-ILM is comparable or superior in each instance to other commercial membranes. Middle molecule uremic toxins vary from >500 Da to <12000 Da in MW. Using lysosome as a surrogate for the middle molecule uremic toxin 02M (beta-2-microglobulin), NC-ILM allowed lysozyme to freely permeate compared to 70% rejection with polysulfone. This finding suggests again that the gaps between the fibers most likely function as slits rather than circular pores accounting for the sharp molecular weight cut-offs. Fig. 3B and Fig. 3C show the performance of this membrane with anticoagulated (heparin & citrate, respectively) porcine blood. In both cases the NC-ILM has at least an order of magnitude higher initial water flux than polysulfone and at the end of the experiment (5-10 hours) had a higher water flux than the initial flux of polysulfone. Fig. 3D shows an experiment where the citrate was removed and so the blood was without an anticoagulant. In this case polysulfone had zero water flux after 2 hours and the NC-ILM was still operational after 5 hours. Therefore, this new membrane demonstrates more than 10 times higher water flux than poly sulfone under all operating conditions with blood indicating that it is the highest flux polymeric membrane reported for blood ultra-filtration to date. Further, the fouling characteristics are superior to polysulfone in every case.

Dialysis was performed with this membrane using the schematic shown in Fig. 4A. Table 2 shows the two different dialysis conditions tested to determine the appropriate dialysate flow.

Table 2

In these experiments we tested the dialysis of urea and lysozyme. The removal of urea is typically assessed clinically in patients on dialysis and we used lysozyme is a surrogate for uremic toxins. The urea clearances are shown in Fig. 4B. It is interesting to compare this membrane to a previously characterized silicone slit membrane with a much higher urea clearance because of its enhanced membrane surface area. Kim, S. et al. Diffusive Silicon Nanopore Membranes for Hemodialysis Applications. PLoS One 11, e0159526 (2016). This membrane has a theoretical treatment time of 3.44 hours as compared to 3.17 hours for commercial membrane and is much lower than the silicone slit membrane (8 hours). When comparing lysozyme (Fig. 4B) the results are even more interesting. The NC-ILM has a much higher passage of lysozyme than the silicone nanopore membrane indicating that uremic toxins with a ~14 kD MW should permeate the NC-ILM freely. Fig. 4C shows this data along with the study of Boschetti-de- Fierro et al. (2015) showing that in the dialysis configuration this membrane has a very tight flux profile. Boschetti-de-Fierro, A., Voigt, M., Storr, M. & Krause, B. MCO Membranes: Enhanced Selectivity in High-Flux Class. Sci Rep 5, 18448 (2015).

To study the ex vivo behavior of the NC-ILM, a rat animal model was developed where the membrane could be tested in both ultra-filtration (Fig. 6A and Fig. 6B) and dialysis operations. Fig. 5A shows a schematic of the rat experiment. Sprague-Dawley rats pre-implanted with an access button for femoral vein and artery catheterization were used. The animals were kept under anesthesia during the entire procedure. The blood was pumped from the femoral artery to flow through the membrane device placed outside of the animal’s body and returned to the animal through the femoral vein. The complete flow (single pass) through the extracorporeal circuit took approximately 4 minutes. Fig. 5B shows the blood urea nitrogen (BUN) in the blood and in the permeate, indicating that BUN permeates through the membrane. The average water flux for the ultra-filtration experiments was 7.0 ± 2.1 L/m2.h at 3 psig. This is far superior to commercial membranes which have a flux of 0.3-1.4 at 30 psig. In a live animal trial, complement activation can potentially foul the membrane. As shown in Fig. 5C, complement component 3 (C3) and rat soluble terminal complement complex (SC5B-9) did not have significant changes before and after permeation, indicating that the NC-ILM is biocompatible and does not activate complement measurably. In addition, necropsy studies did not reveal any evidence of systemic inflammatory responses induced by the NC-ILM. Vitals measurements were monitored during the experiments. Fig. 5D shows vitals data from a dialysis ex vivo experiment. Anesthesia level changes and saline administration were provided according to the vitals measurements to keep the animal stable. Discussion

Cellulosic nano-materials have the potential for high biocompatibility and increased hydrophilicity. Habibi, Y, Chanzy, H. & Vignon, M. TEMPO-mediated surface oxidation of cellulose whiskers. Cellulose 13, 679-687 (2006). As described herein cellulose nano-materials have been successfully made into ionogels. One effective way to produce ionogels is to use imidazolium-based ionic liquids (ILs), which potentially provide high nucleophilicity and alkaline-stability to the desired membrane product. Dissolving and recovering cellulose with ILs have shown to have intriguing transformative properties, where resulting cellulose based ionogels have shown interesting ordered selfassembly properties. In this process the anionic residues of the ILs (i.e., acetates, halides, etc.) disrupt the structure of the biopolymer. As shown, the mechanism of ordered TEMPO modified cellulosic membranes can lead to a high-performance ultra-filtration membrane with unique properties.

The ordered structure of the NC-ILM (see Fig. 2A and Fig. 7 ) is thought to be a function of the ionic liquid creating space between the nanofibular strcuture. This is because the cationic imidazolium group covalently binds to the C-l carbon of the cellulosic backbone and surface anion groups that were present in the ILs and are now exposed to create a negative surface charge. Habibi, Y, Chanzy, H. & Vignon, M. TEMPO-mediated surface oxidation of cellulose whiskers. Cellulose 13, 679-687 (2006).

The feature from scattering figure is hypothesized to be a signature of an ordered, IL-mediated self-assembly of cellulose fibers. Endo, T., Hosomi, S., Fujii, S., Ninomiya, K. & Takahashi, K. Anion Bridging-Induced Structural Transformation of Cellulose Dissolved in Ionic Liquid. J Phys Chem Let 7, 5156-5161; Samayam, I. P, Hanson, B. L., Langan, P. & Schall, C. A. Ionic-Liquid Induced Changes in Cellulose Structure Associated with Enhanced Biomass Hydrolysis. Biomacromolecules 12, 3091-3098 (2011). At 10%, the solutions are viscous, yet well below the solubility limit. Upon the introduction of water during the phase inversion process, water competes with the cellulose phase for EMIMAc, thereby effectively increasing the nano-cellulose concentration relative to the ionic liquid within regions of solvated cellulose. Studies of the phase behavior of EMIMAc- water and EMIMAc-cellulose-water show the viscosity within the self-assembled aggregates of the EMIMAc-cellulose rapidly increases, particularly with the introduction of water, before coagulation. Le, K. A., Rudaz, C. & Budtova, T. Phase diagram, solubility limit and hydrodynamic properties of cellulose in binary solvents with ionic liquid. Carbohyd Polym 105, 237-243 (2014).

It has been hypothesized that during the phase inversion process, this self-aggregation kinetically traps a structure like the concentrated state (-25-70%) observed by Endo, et al. Endo, T., Hosomi, S., Fujii, S., Ninomiya, K. & Takahashi, K. Anion Bridging-Induced Structural Transformation of Cellulose Dissolved in Ionic Liquid. J Phys Chem Lett 7, 5156-5161 (2016). After soaking in excess water, the majority of remaining IL may be exchanged slowly, but an ordered state remains.

NC-ILM membranes were found to be highly porous with a significantly low deviation in between the samples which were gathered from different batches. However, PSf membranes were found to be less porous, with almost 10 nm wide pores left as the casting solvent (NMP) solvent inverses through the dense polymeric layer into the non-solvent (water) phase. The literature supports the calculated pore diameter, as previously published, for the same solvent-non solvent system, the flat sheet membrane pore diameters were reported to be in between 7-11 nm. Tan, X. & Rodrigue, D. A Review on Porous Polymeric Membrane Preparation. Part I: Production Techniques with Polysulfone and Poly (Vinylidene Fluoride). Polymers (Basel) 11 (2019). On the other hand, pore size of NC-ILM was found to be slightly higher, when compared to PSf, having an average diameter of 12.5 nm. This can be attributed to the rate of transport of the EMIMAc to be slightly lower when compared to NMP, as it was previously stated that the increased velocity of the solvent towards the non-solvent coagulation bath yields to a tighter membrane with a thicker dense layer. Kim, I. C., Yoon, H. G. & Lee, K. H. Formation of integrally skinned asymmetric polyetherimide nanofdtration membranes by phase inversion process. J Appl Polym Set 84, 1300-1307 (2002). Moreover, considering the viscosity of EMIMAc-cellulose complex being higher than the NMP-PSf casting solution a slower transport of EMIMAc through the polymeric layer could be causing a higher pore size in the membrane structure. Gericke, M., Schlufter, K., Liebert, T, Heinze, T. & Budtova, T. Rheological properties of cellulose/ionic liquid solutions: from dilute to concentrated states. Biomacromolecules 10, 1188-1194 (2009). Le, K. A., Sescousse, R. & Budtova, T. Influence of water on cellulose-EMIMAc solution properties: a viscometric study. Cellulose 19, 45-54 (2012).

Claims

1. An ultra-filtration membrane comprising: a cellulose nano-material (CNM) having non-spherical pores, wherein the ultra-filtration membrane has one or more of the following properties: a blood filtration rate (L/H m2) greater than 80; and a protein rejection rate greater than 90%.

2. The ultra-filtration membrane of claim 1, wherein the non-spherical pores are slit-shaped.

3. The ultra-filtration membrane of claim 1, wherein the non-spherical pores have an average size of 10-20 pm.

4. The ultra-filtration membrane of claim 1, wherein the CNM comprises cellulose nano-fibers (CNFs).

5. The ultra-filtration membrane of claim 4, wherein individual CNFs have a width of 1-9 nm.

6. The ultra-filtration membrane of claim 1, wherein the CNM comprises TEMPO-oxidized cellulose.

7. The ultra-filtration membrane of claim 1 further comprising a cationic species of an ionic liquid associated with one or more functional groups of the CNM.

8. The ultra-filtration membrane of claim 7, wherein the cationic species is an imidazolium cation.

9. The ultra-filtration membrane of claim 8, wherein the imidazolium cation resides between cellulose nano-fibers (CNFs) of the CNM.

10. The ultra-filtration membrane of claim 7, wherein the one or more functional groups comprise carboxylate groups, hydroxyl groups, or mixtures thereof.

11. The ultra-filtration membrane of claim 1 having molecular weight (mw) cut-off with 100% sieving at 15 kDa and 0% sieving at 66 kDa.

12. The ultra-filtration membrane of claim 7 further comprising an anionic species of an ionic liquid associated with one or more functional groups of the CNM.

13. The ultra-filtration membrane of claim 7, wherein the anionic species is an acetate.

14. The ultra-filtration membrane of claim 1 having a thickness of 50 pm to 500 pm.

15. The ultra-filtration membrane of claim 1 having an active layer thickness of 0.001 pm to 0.01 pm.

16. An ultra-filtration membrane comprising: a cellulose nano-material (CNM) having non-spherical pores; and a cationic species of an ionic liquid associated with one or more functional groups of the CNM.

17. The ultra-filtration membrane of claim 16, wherein the cationic species is an imidazolium cation.

18. The ultra-filtration membrane of claim 17, wherein the imidazolium cation resides between cellulose nano-fibers (CNFs) of the CNM.

19. The ultra-filtration membrane of claim 16, wherein the one or more functional groups comprise carboxylate groups, hydroxyl groups, or mixtures thereof.

20. The ultra-filtration membrane of claim 16 having molecular weight (mw) cut-off with 100% sieving at 15 kDa and 0% sieving at 66 kDa.

21. The ultra-filtration membrane of claim 16 further comprising an anionic species of an ionic liquid associated with one or more functional groups of the CNM.

22. The ultra-filtration membrane of claim 21, wherein the anionic species is an acetate.

23. A method of making an ultra-filtration membrane comprising: providing a solution comprising a cellulose nano-material (CNM) disposed in an ionic liquid solvent; casting a film from the solution; and performing phase inversion to form the ultra-filtration membrane.

24. The method of claim 23, wherein the phase inversion comprises removing the ionic liquid with water.

25. The method of claim 23, wherein the CNM of the formed ultra-filtration membrane has non- spherical pores.

26. The method of claim 23, wherein the CNM comprises cellulose nano-fibers (CNFs).

27. The method of claim 23, wherein the CNM comprises TEMPO-oxidized cellulose.

28. The method of claim 23, wherein a cationic species of the ionic liquid associated with one or more functional groups of the CNM.

29. The method of claim 28, wherein the cationic species is an imidazolium cation.

30. The method of claim 29, wherein the imidazolium cation resides between cellulose nano-fibers (CNFs) of the CNM.

31. The method of claim 25, wherein the non-spherical pores are slit-shaped.

32. The method of claim 25, wherein the non-spherical pores have an average size of 10-20 pm.

33. The method of claim 23, wherein the formed ultra-filtration membrane has a thickness of 50 pm to 500 pm.

34. The method of claim 23, wherein the formed ultra-filtration membrane has an active layer thickness of 0.001 pm to 0.01 pm.

35. The method of claim 23, wherein the formed ultra-filtration membrane has one or more of the following properties: a blood filtration rate (L/H m2) greater than 80; and a protein rejection rate greater than 90%.