Synthetic network materials and methods of making and use thereof

Abstract

The present invention provides a method of making a novel synthetic material comprising a plurality of aqueous compartments and a bilayer network comprising amphiphilic molecules, the method comprising the steps of: providing an emulsion comprising a plurality of aqueous compartments encompassed by amphiphilic molecules dispersed within a hydrophobic liquid; applying a force to the emulsion; and removing the hydrophobic liquid to isolate the synthetic material.

Description

[0001] TITLE OF THE INVENTION

[0002] SYNTHETIC NETWORK MATERIALS AND METHODS OF MAKING AND USE THEREOF

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under Grant No. CMMI2119716 awarded by the National Science Foundation and Grant No. DE-SC0023265 awarded by the Department of Energy. The government has certain rights in the invention.

[0005] BACKGROUND OF THE INVENTION

[0006] Soft tissue-like materials, which emulate the functionality, responsiveness, and reconfigurability inherent in biological tissues, may lead to transformative advancements across various domains such as robotics, sensing, computing, biomanufacturing, and separations (Dahiya, R. et al., 2015, Biomimetic Technologies, 69; Markovic, D. et al., 2020, Nature Reviews Physics, 2, 499; Rus, D., 2015, Nature, 521, 467; Shen, Y.-X. et al., 2014, Journal of Membrane Science, 454, 359). The successful replication of natural tissue structures and functions involves the capacity to generate aqueous membrane-bound protocells organized into three-dimensional (3D) networks, while achieving seamless communication with their surrounding environment (Bayley, H. et al., 2019, Emerging Topics in Life Sciences, 3, 615; Lin, A. J. et al., 2023, ACS Synthetic Biology, 12, 1889; Mukwaya, V. et al., 2021, Communications Chemistry, 4, 161). However, creating synthetic tissues using bottom-up approaches remains a challenging task (Wang, X. et al., 2021, Advanced Materials, 33, 2002635).

[0007] Thus, there is a need in the art for methods and technologies that produce synthetic tissues that can mimic the mechanical and electrical properties of biological tissues. The present invention satisfies this unmet need.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention relates to, in part, a method of making a synthetic material comprising a plurality of aqueous compartments and a bilayer network comprising amphiphilic molecules, the method comprising the steps of providing an emulsion comprising a plurality of aqueous compartments encompassed by amphiphilic molecules dispersed within a hydrophobic liquid; applying a force to the emulsion; and removing the hydrophobic liquid to isolate the synthetic material. In some embodiments, the plurality of aqueous compartments has a normal distribution of size. In some embodiments, at least one aqueous compartment within the plurality of aqueous compartments is polyhedral. In some embodiments, the amphiphilic molecule is a lipid. In some embodiments, the amphiphilic molecule is DPhPC. In some embodiments, the amphiphilic molecule is a polymer. In some embodiments, the amphiphilic molecule is a polymer selected from polybutadiene (PB), polyisoprene (PI), polyethylene (PE), polypropylene (PP), polystyrene (PS), polyacrylonitrile (PAN), poly(ethylene terephthalate) (PET), poly(tetrafluoroethylene), polypropylene carbonate), and copolymers thereof. In some embodiments, the amphiphilic molecule is a polymer selected from polyethylene oxide (PEO), polyvinylpyrrolidone (PVP), poly(acrylic acid) (PAA), poly(vinyl alcohol) (PVA), poly(acrylamide) (PAAm), poly(N,N-dimethylacrylamide) (PDMA), and copolymers thereof. In some embodiments, the amphiphilic molecule is a block copolymer of at least one hydrophilic polymer and at least one hydrophobic polymer. In some embodiments, the amphiphilic molecule is a block copolymer of polybutadiene polyethylene oxide (PB-PEO), a block copolymer of polyisoprene polyethylene oxide (PI-PEO), or a combination thereof.

[0010] In some embodiments, the hydrophobic liquid is selected from the group consisting of a lubricant, a long-chain hydrocarbon, a vegetable oil, a fatty acid ester, and combinations thereof. In some embodiments, the hydrophobic liquid comprises squalane, hexadecane, silicone oil, or a combination thereof. In some embodiments, the step of applying a force compacts the plurality of aqueous compartments. In some embodiments, the step of applying a force to the emulsion comprises centrifuging the emulsion.

[0011] In some embodiments, the ratio of the volume of the plurality of aqueous compartments to the volume of the hydrophobic liquid in the emulsion is about 1 : 1. In some embodiments, the step of removing the hydrophobic liquid comprises the step of decanting off the hydrophobic liquid above the synthetic material.

[0012] In some embodiments, the emulsion further comprises a channel-forming molecule, a membrane protein, an artificial channel, an ionophore, or any combination thereof. In some embodiments, the method further comprises the step of extruding the synthetic material onto a surface open to air. In some embodiments, the method further comprises the step of extruding the synthetic material into an aqueous solution. In some embodiments, the method further comprises the step of extruding the synthetic material into water.

[0013] The present invention further relates to, in part, a synthetic material comprising a plurality of aqueous compartments and a bilayer network comprising amphiphilic molecules, wherein each aqueous compartment is entirely encompassed by amphiphilic molecules. In some embodiments, the plurality of aqueous compartments has a normal distribution of size. In some embodiments, at least one aqueous compartment within the plurality of aqueous compartments is polyhedral. In some embodiments, the amphiphilic molecule is a lipid. In some embodiments, the amphiphilic molecule is DPhPC. In some embodiments, the amphiphilic molecule is a polymer. In some embodiments, the amphiphilic molecule is a polymer selected from polybutadiene (PB), polyisoprene (PI), polyethylene (PE), polypropylene (PP), polystyrene (PS), polyacrylonitrile (PAN), polyethylene terephthalate) (PET), polytetrafluoroethylene), polypropylene carbonate), and copolymers thereof. In some embodiments, the bilayer network comprises a polymer selected from polyethylene oxide (PEO), polyvinylpyrrolidone (PVP), poly(acrylic acid) (PAA), poly(vinyl alcohol) (PVA), poly(acrylamide) (PAAm), poly(N,N- dimethylacrylamide) (PDMA), and copolymers thereof. In some embodiments, the amphiphilic molecule is a block copolymer of at least one hydrophilic polymer and at least one hydrophobic polymer. In some embodiments, the amphiphilic molecule is a block copolymer of polybutadiene polyethylene oxide (PB-PEO), a block copolymer of polyisoprene polyethylene oxide (PI-PEO), or a combination thereof.

[0014] In some embodiments, the synthetic material has a storage modulus of at least 40 Pa. In some embodiments, the synthetic material is a hydrogel. In some embodiments, the synthetic material is self-healing.

[0015] In some embodiments, the bilayer network further comprises a channel -forming molecule, a membrane protein, an artificial channel, an ionophore, or any combination thereof. In some embodiments, the bilayer network further comprises a molecule selected from a porin, a pore-forming toxin, an aquaporin, alamethicin, gramicidin A (gA), outer membrane protein F (OmpF), outer membrane protein X (OmpX), hemolysin toxin (aHL), ferrichrome outer membrane transporter (FhuA), AquaporinO, aquaporin Z (AqpZ), magainin, cecrophin, melittin, maculatin, or combinations thereof. In some embodiments, the bilayer network is water permeable. In some embodiments, the aqueous compartments further comprise a drug, a therapeutic agent, an ion, or any combination thereof.

[0016] The present invention further relates to, in part, applications of the synthetic material, including a bioink comprising the synthetic material, a soft robotic component comprising the synthetic material, a permeable membrane comprising the synthetic material, and a biomimetic memristor comprising the synthetic material.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following detailed description of various embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0019] Fig. 1, comprising Fig. 1A through Fig. IF, depicts stable biomimetic tissues created using Jammed Interconnected by Bilayers Emulsion (JIBE) presented in this work, which are analogs of biological tissues that can be 3D printed, have biological tissue-like mechanical properties, and can be functionalized with biological channels to create ion conductive membrane barriers with ion selectivity and emergent memristance. Fig. 1A depicts the formation of a droplet interface bilayer between two aqueous droplets coated with a lipid monolayer assembled at the water / oil interface. Fig. IB depicts a schematic of a random jammed packing of hard spheres. Fig. 1C depicts an image of a JIBE made with PBu-PEOe after oil removal. The material has a gel-like consistency, a characteristic of jammed materials. Fig. ID depicts an image showing how JIBEs can be molded into self-supporting 3D shapes by using a commercial bioprinter due to the material rheological properties such as yield stress, shear thinning behavior, and self-healing post-shear. The scale bar represents 5 mm. Fig. IE depicts ion channels utilized in this work to functionalize JIBE and imprint different properties such as selectivity, conductance, and memristance. Fig. IF depicts a schematic of JIBE formation in a process that combines elements from DIBs and jammed systems. The centrifugation of droplets coated by monolayers made with a bilayer-forming amphiphile results in a jammed packing of polyhedral droplets that create a bilayer network. The incorporation of ion channels results in random ion paths that can be leveraged to tune material properties and communication with its surroundings when the oil phase is excluded.

[0020] Fig. 2, comprising Fig. 2A through Fig. 21, depicts the rapid and scalable formation of JIBEs through the rapid emulsification-centrifugation process, which can be employed with lipids and block copolymers and the resulting tissue-like materials with tissue-reminiscent properties including selective water permeability, capacitance, and channel induced ion transport. Fig. 2A depicts JIBE formation by emulsification-centrifugation. Water, oil and amphiphiles combine to make a W / O emulsion. Water and oil are originally present at a ratio ~1 : 1. The amphiphile can be incorporated in the oil or water phase (as vesicles). Mixing at low- speed (3000 rpm in this work) creates monolayer enclosed droplets. The droplets are jammed by centrifugation, originating bilayers between contacting droplets. Over 95% of the oil is excluded in this process, resulting in a network of interconnected droplets that create a gel-like material. Fig. 2B depicts a microscopy picture of DPhPC W / O emulsion (before centrifugation). At this point, droplets spontaneously form bilayers when particles collide, however, excess oil prevents the construction of functional networks. Scale bar = 100 pm. Fig. 2C depicts a microscopy picture of a DPhPC W / O emulsion after centrifugation (JIBE). Droplets change from spherical to polyhedral shape. Poly dispersity favors a tight packing as small droplets can accommodate in the Plateau border between bigger droplets. Scale bar = 100 pm. Fig. 2D depicts a microscopy picture of PBis-PEOe W / O emulsion before centrifugation. Scale bar = 100 pm. Fig. 2E depicts a PB 15-PEOe JIBE. Scale bar = 100 pm. Fig. 2F depicts a 1.5 mL centrifuge tube containing DPhPC vesicles : hexadecane : silicone oil AR20 = 4:3:2. Before mixing the phases separate. After 3 min vortexing at 3000 rpm a W / O emulsion is formed. After centrifugation JIBEs are formed between the oil layers. Fig. 2G depicts how JIBEs demonstrate selective water permeability. Two osmotically unbalanced lines of PBis-PEOe JIBEs fold as a unified structure over 24 hours because of water transport through bilayers, also highlighting the modularity of the system. Scale bar = 5 mm. Fig. 2H demonstrates how DPhPC and PBis-PEOe JIBEs are non- conductive despite its high-water content and demonstrate the capacitive behavior that characterizes bilayers. Plot represents current response to a triangular voltage waveform of + 10 / - 10 mV. Fig. 21 depicts how the conductance of JIBEs can be stunned by incorporation of ion channels, and presents DPhPC JIBE with OmpF at an LPR=200 which exhibits >4000 times higher conductance than the control. Error bars represent average of three independent measurements.

[0021] Fig. 3, comprising Fig. 3A through Fig. 3H, depicts rheological characterization of the JIBE material. Fig. 3 A through Fig. 3D depict data for the characterization of JIBE containing 5 mg / mL DPhPC. Fig. 3A depicts frequency sweeps from 0.1-100 rad / s at 1% strain demonstrate linear viscoelastic properties at low frequencies. Fig. 3B depicts strain sweeps of JIBE from 0.5- 500% strain at a frequency of 0.5 rad / s demonstrate strain yielding behavior of material. Fig. 3C depicts the reduction in JIBE viscosity with increasing shear rate 0.01-100 s’1demonstrates shear thinning behavior of material. Fig. 3D depicts how subjecting JIBE to alternating periods of low strain (0.5%, below yield strain) and high strain (300%, above yield strain) demonstrates self- healing properties of the material. Fig. 3E through Fig. 3H depict data for the characterization of JIBEs containing 5 mg / mL PB-PEO. Fig. 3E depicts frequency sweeps from 0.1-100 rad / s at 1% strain demonstrate linear viscoelastic properties at low frequencies. Fig. 3F depicts strain sweeps of JIBE from 0.5-500% strain at a frequency of 0.5 rad / s demonstrate strain yielding behavior of material. Fig. 3G depicts the reduction in JIBE viscosity with increasing shear rate 0.01-100 s’1demonstrates shear thinning behavior of material. Fig. 3H depicts how subjecting JIBE to alternating periods of high strain (above yield strain) and low strain (below yield strain) demonstrates self-healing properties of the material.

[0022] Fig. 4, comprising Fig. 4A through Fig. 4Q, depicts how JIBEs can be 2D / 3D printed in air and aqueous solutions and maintain stability. Fig. 4A depicts a schematic of the 3D-printing process of bulk JIBEs in air, using a pneumatic commercial bioprinter. A fdament is obtained by regulating printer speed and pressure. The fdament is compatible with air since lipid / BCP monolayer with hydrophobic tails out stabilizes the interface air / water. Fig. 4B depicts the original design of a star of 10 mm height and width fed to the G-code operated 3D printer. Fig. 4C depicts a representation of 3D printing process of JIBEs in solution. The fdament is stabilized in water through the self-assembly of a bilayer at the interface with the hydrophilic head / block out, conferring long-term aqueous stability. Fig. 4D through Fig. 4F depicts photographs of the design depicted in Fig. 4C, printed with PBn-PEOe JIBEs in air. JIBEs demonstrate shape fidelity and the ability to self-support structures up to 10 mm in height. Printer settings, air pressure = 45 kPa, speed = 10 mm / s, and nozzle gauge = 27. Scale bar = 5mm. Fig. 4G depicts a photograph of the construct shown in Fig. 4D through Fig. 4F, 4 hours after printing. The initially stable structure sagged because of the lateral spreading of the material observed in air prints, and the drying of the JIBEs. Fig. 4H depicts a microscopy picture of a filament printed in air in a side view. Droplets closer to the air interface appear smaller because of the drying of the filament. Fig. 41 depicts a picture of filaments printed with PBis-PECE JIBE in air at varying air pressures. From top to bottom pressures correspond to 6, 9, and 12 psi. Filament diameter is inversely proportional to air pressure. Scale bar = 5 mm. Fig. 4J depicts a picture of filaments 2 hours after printing. Fig. 4K through Fig. 4M depict pictures of the design depicted in Fig. 4C, printed with PBis-PEOe JIBEs in an osmotically balanced solution. The structure is self- supporting. Printer settings, air pressure = 25 kPa, speed = 10 mm / s, and nozzle gauge = 27. Scale bar = 5mm. Fig. 4N depict a picture of the construct shown in Fig. 4K through Fig. 4M, 48 hours after printing demonstrating high stability in water. Fig. 40 depicts a microscopic side view image of a filament printed in solution. Droplet size is homogenous since moisture is locked in the material. Fig. 4P depicts a picture of filaments printed with PBis-PEOe JIBE in solution at varying air pressures. From top to bottom pressures correspond to 6,9 and 12 psi. Fig. 4Q depicts a picture of filaments printed in Fig. 4P, after 24 hours. The original shape is preserved.

[0023] Fig. 5, comprising Fig. 5A through Fig. 5F, depicts the representative fabrication of the present self-healing, biomimetic membrane for electrically driven ion separations. Fig. 5A depicts an electrophysiology setup used to measure electrically driven transport in a droplet interface bilayer, the inset shows bilayer formation between two 300 nL droplets coated by a DPhPC lipid monolayer formed in a hexadecane bath. Scale bar = 100 pm. Fig. 5B depicts a schematic representation of the system depicted in Fig. 5A. Gramicidin peptide self-inserts in the lipid bilayer allowing the transport of monovalent cations. Fig. 5C depicts a picture of the membrane assembly used in this work, a 3D-printed ABS container with a spacing of 4.5 mm for the JIBEs and 2 mm for the hydrogel. The volume of the solution was kept at 1 mL and the bilayer area is ~ 0.72 cm2. Fig. 5D depicts a schematic representation of the membrane prototype used in electrically driven ion separations. A JIBE doped with a selective ion channel is supported by two hydrogel layers that separate two aqueous compartments. Applying a DC voltage across the membrane results in the transport of the ions that are permeable to the channel (potassium in this example). The magnified inset depicts the tissue-like organization inside the JIBE containing gramicidin A channels, which are selective to monovalent cations only. As a result, random conducting pathways are created inside the network allowing the transport under potential. Fig. 5E depicts single salt permeability studies of gramicidin doped DIB, the peptide concentration was set to 100 nM. Fig. 5F depicts a representative single salt permeability transport study in JIBE membrane containing 2 pM of gA. In the absence of ion channels, the membranes have almost negligible cation permeability, demonstrating the nonexistence of defects in the JIBE network. A y-axis break was made on the plot to observe the values which are two orders of magnitude smaller than the membranes containing gramicidin. In the presence of gA, the membrane exhibits the same trends of selectivity reported for the protein in traditional electrophysiology studies.

[0024] Fig. 6A, comprising Fig. 6A through Fig. 6D, depicts how Alamethicin-doped JIBEs demonstrate memristive behavior. Fig. 6A depicts an illustration of voltage-gated insertion reported for Alamethicin peptides in DIBs. Multiple units of the peptide assemble into a channel conformation above a voltage threshold. The insertion is reversible, and when the potential falls the channels leave the membrane returning to an insulating state. As a result, a memristive behavior is observed per the pinched current-voltage hysteresis. Fig. 6B depicts a representative picture of the Alamethicin doped JIBE used in this work. The JIBE is mounted on top of a glass slide and supported with a 2 mm thick PDMS substrate. JIBE had a square shape of 0.25 cm2as defined by the support. Two Ag / AgCl electrodes were placed on the opposite sides of the square diagonal. Fig. 6C depicts an IV plot obtained for JIBEs made with 300 mM of NaCl and no channels when a triangular wave of + / -150 mV was applied at a scanning rate of 10 mV / s. The control displays minimal transport. Fig. 6D depicts an IV plot obtained for JIBEs made with 300 mM of NaCl and 6 pM Alamethicin when a triangular wave of + / -150 mV was applied at a scanning rate of 10 mV / s. As the JIBE represents a complex network of bilayers this behavior was observed after repetition of the stimulus for 10 - 20 min. The pinched curve is the signature that defines a memristor, thus demonstrating that JIBEs can be functionalized with membrane proteins preserving the properties observed at the single-bilayer nanoscale.

[0025] DETAILED DESCRIPTION

[0026] The present invention is based, in part, on the discovery of a novel synthetic material and a method of making said novel material. The synthetic material provided by the present invention is uniquely suited for use in applications including, but not limited to, drug screening and testing, platforms for drug screening and testing, systems to investigate cellular signaling pathways, sensors for toxins and environmental pollutants, membranes for separations, ion separations, electrically driven ion separations, water purification and treatment, nutrient recovery, mining industry separations, biomedical devices, tissue engineering, biomimetic tissues, development of artificial organs, drug delivery, soft robotics, biocomputing, and neuromorphic computing.

[0027] Definitions

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0029] As used herein, each of the following terms has the meaning associated with it in this section.

[0030] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0031] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass non-limiting variations of ±40% or ±20% or ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate.

[0032] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Description

[0033] The present invention is based, in part, on the discovery of a novel synthetic material comprising a plurality of aqueous compartments, wherein the aqueous compartments are each surrounded by a bilayer of amphiphilic molecules that form a continuous network. Thus, the present invention is directed, in part, to methods of a novel biomimetic network material and applications comprising the material thereof.

[0034] In some aspects, the present invention also provides a method of making a synthetic material comprising a plurality of aqueous compartments, the method comprising the steps of: providing an emulsion comprising a plurality of aqueous droplets surrounded by amphiphilic molecules dispersed within a hydrophobic liquid; applying a force to the emulsion; and removing the hydrophobic liquid to isolate the synthetic material.

[0035] Network Materials

[0036] The present invention provides, in part, a synthetic material comprising a plurality of aqueous compartments and a bilayer network comprising amphiphilic molecules, wherein each aqueous compartment is entirely encompassed by amphiphilic molecules. The present invention further provides compositions comprising the synthetic material.

[0037] In certain embodiments, an aqueous compartment within the plurality of aqueous compartments is polydisperse in size. In certain embodiments, the plurality of aqueous compartments comprises at least one aqueous compartment. In one embodiment, the plurality of aqueous compartments has a normal distribution of size. In one embodiment, at least one aqueous compartment within the plurality of aqueous compartments is polyhedral. In one embodiment, the aqueous compartments are polydisperse in size. In one embodiment, the aqueous compartments have a normal distribution of size. In one embodiment, the aqueous compartments are polyhedral.

[0038] As used herein, the terms “normal distribution” or “Gaussian distribution” refers to a continuous probability distribution, also known as the bell-shaped curve.

[0039] In one embodiment, the aqueous compartments have a range of diameters ranging from about 0.01 pm to about 500 pm. In one embodiment, the aqueous compartments have a range of diameters ranging from about 0.10 pm to about 500 pm. In one embodiment, the aqueous compartments have a range of diameters ranging from about 1 pm to about 500 pm. In one embodiment, the aqueous compartments have a range of diameters ranging from about 0.01 pm to about 400 pm. In one embodiment, the aqueous compartments have a range of diameters ranging from about 0.10 pm to about 400 pm. In one embodiment, the aqueous compartments have a range of diameters ranging from about 1 pm to about 400 pm. In one embodiment, the aqueous compartments have a range of diameters ranging from about 0.01 pm to about 300 pm. In one embodiment, the aqueous compartments have a range of diameters ranging from about 0.10 pm to about 300 pm. In one embodiment, the aqueous compartments have a range of diameters ranging from about 1 pm to about 300 pm. In one embodiment, the aqueous compartments have a range of diameters ranging from about 0.01 pm to about 200 pm. In one embodiment, the aqueous compartments have a range of diameters ranging from about 0.10 pm to about 200 pm. In one embodiment, the aqueous compartments have a range of diameters ranging from about 1 pm to about 200 pm. In one embodiment, the aqueous compartments have a range of diameters ranging from about 0.01 pm to about 100 pm. In one embodiment, the aqueous compartments have a range of diameters ranging from about 0.10 pm to about 100 pm. In one embodiment, the aqueous compartments have a range of diameters ranging from about 1 pm to about 100 pm.

[0040] As used herein, the term “amphiphilic” or “amphipathic” describes a compound or molecule which possesses both hydrophilic and hydrophobic portions. “Hydrophilic” typically means a portion that interacts intermolecularly and intramolecularly with water and other polar molecules. “Hydrophobic” typically means a portion that interacts preferentially with oils, fats or other non-polar molecules rather than aqueous media.

[0041] In some embodiments, the amphiphilic molecule is a lipid. In one embodiment, the bilayer network comprises a lipid. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids. Lipids suitable for use can be obtained from commercial sources. In certain embodiments, the lipid is a membrane forming lipid, or a lipid that, in an aqueous environment, assemble in a lipid bilayer structure that consists of two opposing layers of amphipathic molecules know as polar lipids. Each polar lipid has a hydrophilic moiety, i.e., a polar group such as, a derivatized phosphate or a saccharide group, and a hydrophobic moiety, i.e., a long hydrocarbon chain. Exemplary polar lipids include phospholipids, sphingolipids, glycolipids, ether lipids, sterols and alkylphosphocholines. Amphipathic lipids include but are not limited to membrane lipids, i.e. amphipathic lipids that are constituents of a biological membrane, such as phospholipids.

[0042] In one embodiment, the lipid is a synthetic lipid. In one embodiment, the lipid is a natural lipid. In one embodiment, the lipid is soy-PC (soy-derived L-a-phosphatidylcholine).

[0043] In one embodiment, the lipid is a neutral lipid. The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Neutral lipids useful in the invention do not include PEG-phospholipids (e.g. polyethylene oxide-containing phospholipids). Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides. In one embodiment, the neutral ligand is a phosphatidylethanolamine (PE) lipid.

[0044] Exemplary lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-0-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1- stearioyl-2-oleoyl-phosphatidy ethanol amine (SOPE), and l,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE).

[0045] In certain embodiments, the lipid can be a cationic lipid, or a lipid which is capable of having a net positive charge at a selective pH, a stabilizing lipid, including neutral lipids and pegylated lipids, or combinations thereof. Lipids which may be included in the present invention include, but are not limited to, diphytanoylphosphatidyl choline (DPhPC), N,N-dioleyl-N,N- dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N — (N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l -(2,3-dioleoyloxy)propyl)-N-2- (sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dioleoyl-3 -dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), N-(l,2-dimyristyloxyprop- 3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), and combinations thereof. In some embodiments, the bilayer of amphiphilic molecules comprises DPhPC.

[0046] In one embodiment, the lipid further comprises a functional group such as a fluorescent or phosphorescent group. In one embodiment, the lipid comprises a tethering group. In one embodiment, the lipid comprises a functional agent that is covalently attached to the lipid.

[0047] In some embodiments, the amphiphilic molecule is a polymer. In one embodiment, the bilayer network comprises a polymer. In one embodiment, the polymer is a biodegradable polymer, biocompatible polymer, edible polymer, food grade polymer, plant-based polymer, animal-derived polymer, human-derived polymer, or any combination thereof.

[0048] The term “polymer” as used herein refers to the product of a polymerization reaction, and is inclusive of homopolymers, copolymers, terpolymers, etc.

[0049] Unless otherwise specified, the term “copolymer(s)” as used herein refers to polymers formed by the polymerization of at least two different monomers. For example, the term “copolymer” includes the copolymerization reaction product of ethylene and an alpha-olefin (a- olefin), such as by way of example only propylene or 1 -hexene.

[0050] The term “biodegradable”, as used herein, refers to polymers that degrade fully (i.e., down to monomeric species) under physiological or endosomal conditions. In preferred embodiments, the polymers and polymer biodegradation byproducts are biocompatible. Biodegradable polymers are not necessarily hydrolytically degradable and may require enzymatic action to fully degrade.

[0051] The term “biocompatible”, as used herein, generally refers to a condition of being compatible with a living tissue or a living system by not being toxic, injurious, or physiologically reactive and / or not causing immunological rejection.

[0052] In one embodiment, the polymer is an organic polymer. In one embodiment, the bilayer network comprises an organic polymer. Examples of such organic polymers include, but are not limited to, polyalkenes (e.g., polyethylene, poly isobutene, polybutadiene), polyacrylics (e.g., polyacrylate, poly(methyl methacrylate), poly cyanoacrylate), polyvinyls (e g., poly(vinyl alcohol), poly(vinyl acetate), poly(vinyl butyral), poly(vinyl chloride)), polystyrenes, polycarbonates, polyesters, polyurethanes, polyamides, polyimides, polysulfone, polysiloxanes, polyheterocycles, cellulose derivative (e.g., methyl cellulose, cellulose acetate, nitrocellulose), polysilanes, fluorinated polymers, epoxies, polyethers, phenolic resins (e.g., Cognard, J. Alignment of Nematic Liquid Crystals and Their Mixtures, in Mol. Cryst. Liq. Cryst. 1 : 1-74 (1982)), poly dimethyl siloxane, polyethylene, polyacrylonitrile, cellulosic materials, polycarbonates, poly(vinyl pyridinium), zein, alginate, hyaluronic acid, mycelium, or any combination thereof.

[0053] In one embodiment, the polymer is a synthetic polymer. In one embodiment, the bilayer network comprises a synthetic polymer. A synthetic polymer material can be any material prepared through a method of artificial synthesis, processing, or manufacture. In one embodiment, the polymer is a biocompatible polymer.

[0054] In some embodiments, the polymer is a biocompatible synthetic polymer. In one embodiment, the bilayer network comprises a biocompatible synthetic polymer. Examples of biocompatible synthetic polymers include, but are not limited to, poly(urethanes), poly(siloxanes) or silicones, poly(ethylene), poly(vinyl pyrrolidone), poly(2-hydroxy ethyl methacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate), polyvinyl alcohol) (PVA), poly(acrylic acid), polyvinyl acetate), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylic acid), polylactic acid (PLA), polyglycolic acids (PGA), poly(lactide-co-glycolides) (PLGA), nylons, polyamides, polyanhydrides, poly(ethylene-co-vinyl alcohol) (EVOH), poly caprolactone, poly(vinyl acetate)), polyvinylhydroxide, zein, alginate, hyaluronic acid, mycelium, polyethylene oxide) (PEO) and polyorthoesters or co-polymers thereof.

[0055] In one embodiment, the polymer is a biodegradable polymer. In one embodiment, the bilayer network comprises a biodegradable polymer. Examples of suitable biodegradable materials include, but are not limited to collagen, poly(alpha esters) such as poly(lactate acid), poly(glycolic acid), polyorthoesters, polyanhydrides polyglycolic acid and polyglactin, and copolymers thereof. Other suitable biodegradable polymers include cellulose ether, cellulose, cellulosic ester, fluorinated polyethylene, phenolic, poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether, polyester, polyestercarbonate, polyether, polyetheretherketone, poly etherimide, polyetherketone, polyethersulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, poly oxadi azole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, poly(vinylidene fluoride), regenerated cellulose, silicone, urea-formaldehyde, zein, alginate, hyaluronic acid, mycelium, or copolymers or thereof.

[0056] In some embodiments, the polymer is a hydrophobic polymer. In one embodiment, the bilayer network comprises a hydrophobic polymer. Examples of suitable hydrophobic polymers include, but are not limited to, polybutadiene (PB), polyisoprene (PI), polyethylene (PE), polypropylene (PP), polystyrene (PS), polyacrylonitrile (PAN), polyethylene terephthalate) (PET), poly(tetrafluoroethylene), polypropylene carbonate), and copolymers thereof.

[0057] In some embodiments, the polymer is a hydrophilic polymer. In one embodiment, the bilayer network comprises a hydrophilic polymer. Examples of suitable hydrophilic polymers include, but are not limited to, polyethylene oxide (PEO), polyvinylpyrrolidone (PVP), poly(acrylic acid) (PAA), poly(vinyl alcohol) (PVA), poly(acrylamide) (PAAm), poly(N,N- dimethylacrylamide) (PDMA), and copolymers thereof.

[0058] In some embodiments, the polymer is a straight chain polymer (i.e., linear polymer) or a branched chain polymer (i.e., branched polymer), including hyperbranched polymers. In some embodiments, the polymer is a neutral polymer, ionic polymer, anionic polymer, or cationic polymer.

[0059] In some embodiments, the polymer is a homopolymer, copolymer, or block copolymer. In some embodiments, the block copolymer is a triblock, tetrablock, pentablock, or at least six block copolymer. In one embodiment, the block copolymer has at least one distinct hydrophilic region and at least one distinct hydrophobic region, thus making the block copolymer amphiphilic. In some embodiments, the bilayer of amphiphilic molecules comprises a block copolymer of at least one hydrophilic polymer and at least one hydrophobic polymer. Thus, in some embodiments, the bilayer of amphiphilic molecules comprises a block copolymer comprising a hydrophilic polymer block and a hydrophobic polymer block.

[0060] The hydrophobic and hydrophilic polymer blocks may each independently have a range of molecular weights. For example, the block may have a molecular weight of from about 0.5 kDa to about 50 kDa, from about 1 kDa to about 45 kDa, from about 2 kDa to about 40 kDa, from about 3 kDa to about 30 kDa, from about 4 kDa to about 20 kDa, or from about 5 kDa to about 10 kDa. In embodiments, the block may have a molecular weight of at least about 1 kDa, at least about 2 kDa, at least about 3 kDa, at least about 4 kDa, at least about 5 kDa, at least about 6 kDa, at least about 7 kDa, at least about 8 kDa, at least about 9 kDa, at least about 10 kDa, at least about 15 kDa, at least about 20 kDa, at least about 25 kDa, at least about 30 kDa, at least about 35 kDa, at least about 40 kDa, or at least about 45 kDa. In embodiments, the block may have a molecular weight of less than about 50 kDa, less than about 45 kDa, less than about 40 kDa, less than about 35 kDa, less than about 30 kDa, less than about 25 kDa, less than about 20 kDa, less than about 15 kDa, less than about 10 kDa, less than about 9 kDa, less than about 8 kDa, less than about 7 kDa, less than about 6 kDa, less than about 5 kDa, less than about 4 kDa, less than about 3 kDa, or less than about 2 kDa.

[0061] In one embodiment, the bilayer network comprises polybutadiene. In one embodiment, the bilayer network comprises polyethylene oxide. In one embodiment, the bilayer network comprises polyisoprene. In one embodiment, the bilayer network comprises a block copolymer of polybutadiene polyethylene oxide (PB-PEO), a block copolymer of polyisoprene polyethylene oxide (PI-PEO), or a combination thereof. In one embodiment, the bilayer network comprises PB15-PEO6.

[0062] In some embodiments, the polymer has molecular weight of 0.5 kDa-3000 kDa. For example, in one embodiment, the polymer has a molecular weight of 0.5 kDa-2000 kDa, 0.5 kDa-1500 kDa, 0.5 kDa-1000 kDa, 0.5 kDa-800 kDa, 0.5 kDa-500 kDa, 0.5 kDa-300 kDa or 0.5 kDa-200 kDa or 800 kDa-3000 kDa. In some embodiments, the bilayer of amphiphilic molecules comprises a polymer having molecular weight of 0.5 kDa-3000 kDa. For example, in some embodiments, the bilayer of amphiphilic molecules comprises a polymer having a molecular weight of 0.5 kDa-2000 kDa, 0.5 kDa-1500 kDa, 0.5 kDa-1000 kDa, 0.5 kDa-800 kDa, 0.5 kDa- 500 kDa, 0.5 kDa-300 kDa, 0.5 kDa-200 kDa, or 800 kDa-3000 kDa.

[0063] In one embodiment, the synthetic material demonstrates solid-like behavior, in other words, the synthetic material has a storage modulus which exceeds its loss modulus at lower frequencies. In one embodiment, the synthetic material has a storage modulus of at least 40 Pa, at least 50 Pa, at least 60 Pa, at least 70 Pa, at least 80 Pa, at least 90 Pa, at least 100 Pa, at least 200 Pa, at least 300 Pa, or at least 400 Pa.

[0064] In one embodiment, the synthetic material is a hydrogel forming polymer, or a polymer participating in the formation of a hydrogel. It may be a naturally occurring polymer or a synthetic polymer capable of forming a hydrogel. The hydrogel forming polymer may include polymer(s) making a contribution to hydrogel formation. In one embodiment, the synthetic material is a hydrogel. The term “hydrogel”, as used herein, generally refers to a gel or gel-like structure comprising one or more polymers suspended in an aqueous solution (e.g., water). Water may be present in a composition of the present invention in any suitable amount. In some embodiments, water may be present in an amount of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,

[0065] 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,

[0066] 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,

[0067] 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,

[0068] 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% by weight of the synthetic material. In some embodiments, the synthetic material or a composition comprising the synthetic material is an extrudable hydrogel.

[0069] In one embodiment, the synthetic material has a non-zero yield stress. In one embodiment, the synthetic material is self-healing, in other words, the synthetic material automatically recovers, heals, or repairs itself after physical impact, damage, or cracks.

[0070] In certain embodiments, the bilayer network further comprises a channel-forming molecule, a membrane protein, a carbon nanotube, an artificial channel, an ionophore, or any combination thereof. In one embodiment, the bilayer network further comprises a molecule selected from a porin, a pore-forming toxin, an aquaporin, alamethicin, gramicidin A (gA), outer membrane protein F (OmpF), outer membrane protein X (OmpX), hemolysin toxin (aHL), ferrichrome outer membrane transporter (FhuA), AquaporinO, aquaporin Z (AqpZ), magainin, cecrophin, melittin, maculatin, or combinations thereof. In one embodiment, the bilayer network is water permeable. In one embodiment, the bilayer network is permeable to gases, liquids, molecules in solution, or any combination thereof. In one embodiment, the bilayer network is permeable to ions, including but not limited to, sodium ions, potassium ions, ammonium ions, and combinations thereof.

[0071] In one embodiment, the bilayer network further comprises an ionophore selected from the group consisting of gramicidin, valinomycin, nonactin, crown ethers, crown ether derivatives, podands, coronands, cryptands, A-23187 (Calcimycin), ionomycin, and combinations thereof. Exemplary membrane proteins include proteins that can be associated with the membrane of a cell or an organelle, such as integral membrane proteins (a protein including at least one transmembrane domain which indicates any protein segment which is thermodynamically stable in a membrane, as will be understood by a skilled person and comprise a protein (or assembly of proteins) that are stably attached to the biological membrane), or peripheral membrane proteins (proteins including at least one transmembrane domain that are reversibly attached to the biological membrane to which they are associated). Membrane proteins utilized in the present invention can be separated from the biological membranes using detergents, nonpolar solvents, or some denaturing agents as will be understood by a skilled person. In some instances, peripheral membrane proteins attach to integral membrane proteins, or penetrate the peripheral regions of a lipid bilayer with a reversible attachment.

[0072] Artificial channels can be synthesized using simple chemistry, and are solvent compatible, thus allowing manufacturing techniques common in polymer processing to be applied. More importantly, flexibility in design of the chemical structures of artificial channels further allows for specific functionalization to tailor their permeability and selectivity. These precisely designed pore structures are ideal for membranes that can overcome the aforementioned permeability-selectivity trade-off of current commercial membranes. Relative to state-of-the-art commercial membranes, biomimetic membranes incorporating these pore structures are expected to exhibit high permeability and selectivity because they possess a high density of channels with a well-defined pore geometry and functionality designed to exclude or pass specific components from complicated mixtures. Exemplary artificial channels include, but are not limited to, carbon nanotube (CNT) porins and peptide-appended pillar[5] arenes (PAP). As used herein, the term “carbon nanotube” refers to a hollow tube comprising graphene sheets having a diameter ranging from 1 nm to 100 nm.

[0073] In certain embodiments, the aqueous compartments further comprise a cargo such as a drug, a therapeutic agent, an ion, or any combination thereof. Exemplary cargo include, but are not limited to, biologically relevant cations, including but not limited to, sodium ions, potassium ions, ammonium ions, one or more stem cells (e.g., adipose tissue-derived stem cells and / or bone marrow-derived mesenchymal stem cells), steroids (e.g., steroids to prevent edema), agents to prevent post-inflammatory skin hyperpigmentation (e.g., hydroquinone, azelaic acid, kojic acid, mandelic acid, or niacinamide); one or more painkillers (e.g., paracetamol / acetaminophen, aspirin, dextropropoxyphene napsylate, co-codamol, opioids (e.g., morphine, codeine, oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine, tramadol, etc.). , or methadone), fentanyl, procaine, lidocaine, tetracaine, dibucaine, benzocaine, p- butylaminobenzoic acid 2-(diethylamino)ethyl ester HC1, mepivacaine, piperocaine, dyclonine, or venlafaxine; one or more antibiotics (e.g., cephalosporins, bacitracin, polymyxin B sulfate, neomycin, bismuth tribromocarbonate, or polysporin); one or more antifungal agents (e.g., nystatin), one or more anti-inflammatory agents (e.g., nonsteroidal anti-inflammatory drugs (NSAIDs), e.g., ibuprofen, ketoprofen, flurbiprofen, piroxicam, indomethacin, diclofenac, sulindac, naproxen, aspirin, ketorolac, or tacrolimus), cyclooxygenase and / or cyclosporine (COX-2)-specific inhibitors (COX-2 inhibitors, e.g., rofecoxib. etoricoxib, and celecoxib, glucocorticoid drugs, specific cytokines targeting T-lymphocyte function, steroids (e.g., glucocorticoids (e.g., corticosteroids such as aldosterone, beclomethasone, betamethasone, cortisone, deoxycorticosterone acetate, dexamethasone, fludrocortisone acetate, hydrocortisone, methylprednisolone, prednisone, prednisolone, or triamcinolone) or mineralocorticoid drugs (e.g., aldosterone, corticosterone, or deoxycorticosterone)), or immunoselective antiinflammatory derivatives (e.g., phenylalanine, one or more antimicrobial agents (e.g., chlorhexidine gluconate, iodine (e.g., tincture of iodine, povidone iodine, or Lugol's iodine), or silver, such as silver nitrate (e.g., as a 0.5% solution), silver sulfadiazine (e.g., as a cream), or Ag in one or more useful carriers (e.g., alginates or a mixture of alginate, carboxymethylcellulose, and silver-coated nylon fibers).+; foams (e.g. soft hydrophilic polyurethane foam and silver,); hydrocolloids (e.g., ionic silver and hydrocolloids); or hydrogels, one or more germicides (e.g., alcohols such as ethanol (e.g., 60-90%), 1-propanol (e.g., 60-70%), and mixtures of 2- propanol / isopropanol); boric acid; calcium hypochlorite; hydrogen peroxide; manuka honey and / or methylglyoxal; phenolic (carbolic acid) compounds (e.g., sodium 3,5-dibromo-4- hydroxybenzenesulfonate, tri chlorophenyl methyl iodosalicyl, or triclosan); polyhexanide compounds (e.g., polyhexam ethylene biguanide (PHMB)); quaternary ammonium compounds (e.g., benzalkonium chloride (BAC), benzethonium chloride (BZT), bromide cetyltrimethylammonium (CTMB), cetylpyridinium chloride (CPC), chlorhexidine (e.g., chlorhexidine gluconate) or octenidine (e.g., octenidine dihydrochloride); sodium bicarbonate; sodium chloride; sodium hypochlorite (e.g., optionally in combination with boric acid in Dakin's solution); or triarylmethane dyes (e.g., brilliant green); one or more antiproliferative agents (e.g., sirolimus, tacrolimus, zotarolimus, biolimus, or paclitaxel); one or more emollients; one or more hemostatic agents (e.g., collagen, such as microfibrillar collagen, chitosan, calcium-containing zeolite, cellulose, anhydrous aluminum sulfate, silver nitrate, potassium alum, titanium dioxide, fibrinogen, epinephrine, calcium alginate, poly-N-acetate, tilglucosamine, thrombin, clotting factor(s) (e.g., II, V, VII, VIII, IX, X, XI, XIII, or von Willebrand factor, and its activated forms), procoagulants (e.g., propyl gallate), antifibrinolytics (epsilon aminocaproic acid or tranexamic acid), etc.); one or more procoagulants (e g., any of the hemostatic agents described herein, desmopressin, clotting factor(s) (e.g., II, V, VII, VIII, IX, X, XI, XIII, or von Willebrand factor, and its activated forms), procoagulants (e.g., propyl gallate), antifibrinolytics (e.g., epsilon aminocaproic acid), etc ); one or more anticoagulants (e.g., heparin or a derivative thereof, such as low molecular weight heparin, fondaparinux, or idraparinux; aspirin antiplatelet agents such as dipyridamole, ticlopidine, clopidogrel, or prasugrel; factor Xa inhibitors such as direct acting factor Xa inhibitors (e.g., apixaban or rivaroxaban); thrombin inhibitors such as direct acting thrombin inhibitors (e.g., argatroban, bivalirudin, dabigatran, hirudin, lepirudin, or ximelagatran; or coumarin derivatives or vitamin K antagonists (e.g., warfarin (Coumadin), acenocoumarol, atromentin, phenindione, or phenprocoumon); one or more immunomodulators including corticosteroids and nonsteroidal immunomodulators (e.g., NSAIDS as described herein); one or more proteins; one or more vitamins (e.g., vitamin A, vitamin C, and / or vitamin E); or one or more growth factors (e.g., vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-0), fibroblast growth factor (FGF), epidermal growth factor (EGF), and keratinocyte growth factor).

[0074] There is no particular limit to the size (e.g. mass / volume) of the synthetic material produced using this method. In one embodiment, the synthetic material has a total volume of about 1 mL. In one embodiment, the synthetic material has a volume comparable to greater than 64 billion droplets. In one embodiment, the synthetic material has a volume greater than 1 mL.

[0075] Methods of Using Network Materials

[0076] The present invention further provides applications using and / or comprising the synthetic material. In some embodiments, the synthetic material is used for separations. In some embodiments, the synthetic material is used for separations. In some embodiments, the synthetic material is used as a bioink or in a bioink composition. In some embodiments, the synthetic material is used in a synthetic tissue. In some embodiments, the synthetic material is used in a biomimetic material. In some embodiments, the synthetic material is used in a memristor and / or in a biomimetic memristor.

[0077] In some embodiments, the synthetic material is biomimetic, in other words, the synthetic material bears resemblance to a substance that occurs naturally in a human body and which is not substantially rejected by (e.g., does not cause an unacceptable adverse reaction in) the human body. When used in connection with biological systems such as tissue scaffolds, biomimetic means that the scaffold is substantially biologically inert (i.e., will not cause an unacceptable immune response / rej ection) and is designed to resemble a structure (e.g., soft tissue anatomy) that occurs naturally in a mammalian, e.g., human, body and that promotes healing when implanted into the body. In some embodiments, the synthetic material is used as a synthetic tissue such as an artificial organ.

[0078] In some embodiments, the synthetic material is permeable to gases, liquids, molecules in solution, or any combination thereof, and the present invention provides a permeable membrane comprising the synthetic material. In some embodiments, the synthetic material is used in a selectively permeable membrane. In some embodiments, the synthetic material is used for ion separation. In some embodiments the synthetic material is used to remove solutes and / or pollutants in water. In some embodiments, the synthetic material is used for nutrient recovery from wastewater. In some embodiments, the synthetic material is used for electrically driven ion separation. In some embodiments, the synthetic material is microporous. In some embodiments, the synthetic material preferentially or selectively permits passage of monovalent and / or multivalent ions, including but not limited to, ions of potassium, sodium, magnesium, zinc, calcium, iron, and combinations thereof. In some embodiments, the synthetic material preferentially or selectively permits passage of a cargo as described elsewhere herein. In some embodiments, the synthetic material is used in drug screening and / or testing. In some embodiments, the synthetic material is used to investigate cellular signaling. In some embodiments, the synthetic material is used in a sensor for toxins and / or pollutants.

[0079] In some embodiments, the synthetic material can be extruded through additive manufacturing or three-dimensional (3D) printing to construct a three-dimensional object, thus, the present invention provides a bioink comprising the synthetic material or a bioink composition comprising the synthetic material. In some embodiments, the bioink comprising the synthetic material is extruded layer-by-layer onto a surface on which the synthetic material self-joins or self-fuses to form a cohesive object. In some embodiment, the synthetic material is stable in air. In some embodiments, the synthetic material has an elastic modulus similar to a natural tissue.

[0080] The synthetic material of the present invention and / or compositions comprising the synthetic material may have an elastic modulus (i.e., stiffness), at room temperature and atmospheric pressure, that is sufficiently low such that the composition can be manipulated and / or deposited onto a substrate by whatever deposition method is employed (e g., extrusion deposition, bioprinting, etc.). The elastic modulus, again at room temperature and atmospheric pressure, of the composition may be sufficiently high so that the composition will substantially retain the shape and / or configuration in which it is deposited.

[0081] In some embodiments, a synthetic material of the present invention may have an elastic modulus (E') from about 0.01, 0.025, 0.05, 0.1, 1, or 5 kiloPascals to about 10, 15, 20, 25, 50, or 100 kiloPascals (kPa). In some embodiments, a synthetic material of the present invention may have an elastic modulus (i.e., stiffness) from about 0.01, 0.025, 0.05, or 0.1 kiloPascals to about 0.5, 1, 5, 10, 15, 20, or 25 kiloPascals, or more, at room temperature and atmospheric pressure. In some embodiments, the synthetic material prior to deposition has a stiffness of from about 10 or 25 Pascals (Pa) to about 500 Pa at room temperature and atmospheric pressure. In some embodiments, the synthetic material has a stiffness of about 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 Pascals at room temperature and atmospheric pressure. In some embodiments, the synthetic material after deposition has a stiffness from about 0.1 kPa to about 25 kPa at room temperature and atmospheric pressure. In some embodiments, the synthetic material after deposition has a stiffness of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 kPa at room temperature and atmospheric pressure. In some embodiments, the synthetic material of the present invention may mimic the elastic modulus of a tissue in vivo. In some embodiments, a synthetic material of the present invention has an elastic modulus value that is substantially the same as (i.e., within ±20% of) the elastic modulus value of a tissue in vivo.

[0082] A synthetic material of the present invention may have an elastic modulus after extrusion and / or bioprinting that varies by less than about ±20% compared to an elastic modulus of the synthetic material prior to extrusion and / or bioprinting. In some embodiments, the synthetic material may have an elastic modulus (G') and a loss modulus (G") that are within about ±20%, 15%, 10%, or 5% of each other or less. In some embodiments, a synthetic material of the present invention is thixotropic. In some embodiments, during the application of a stress, a synthetic material of the present invention has an elastic modulus that decreases and then, after removal of the stress, the synthetic material returns to an elastic modulus that is similar to the elastic modulus prior to the stress (e.g., within about ±20%).

[0083] In some embodiments, a synthetic material of the present invention has one or more properties and / or features (e.g., aqueous compartments and / or transient intermolecular forces) that allow for and / or provide shifts in elastic moduli in response to a physical stress. For example, the shear stress levels encountered for a synthetic material when driven through a printhead, upon reaching some stress threshold, the transient bonds may break and the shear elastic modulus (G') may be reduced to a level below the shear loss modulus (G"), at which point the synthetic material is effectively a fluid and is easily extruded. Immediately following deposition, the stress from the printing is no longer present and the internal bonds reform, returning the synthetic material to a gel state that holds its 3D shape. In some embodiments, a synthetic material of the present invention, upon application of a stress, has an elastic modulus (G') that is below the loss modulus (G"), but, upon removal of the stress, the elastic modulus (G') is above the loss modulus (G").

[0084] A synthetic material of the present invention may be extrudable. For example, in some embodiments, the synthetic material may be extrudable from a syringe and / or bioprinter. In some embodiments, the synthetic material may be extruded with an applied mechanical stress in a range from about 5 kPa to about 80 kPa. In some embodiments, the synthetic material may be extruded with an applied mechanical stress of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 kPa. In some embodiments, the synthetic material is extrudable through a 19.5, 20, 22, 23.5, or 30.5 Gauge needle, optionally at room temperature and / or pressure. The synthetic material may be liquid upon extrusion and after extrusion may be solid. A synthetic material of the present invention may be stable and solid under normal conditions, but is printable and can transition between a solid-like behavior at rest, and a liquid-like behavior during printing. In some embodiments, a synthetic material of the present invention is thixotropic. Thixotropism is a material characteristic that is a special case of shear thinning, and refers to the material's ability to behave like a solid under low shear conditions and behave like a liquid under high shear conditions. In some embodiments, a synthetic material of the present invention remains a solid while in a syringe, behaves like a liquid when subjected to the high shear environment of extrusion through the nozzle and / or needle of the syringe, and then regains its solid like characteristics upon deposition (e.g., onto a surface of a substrate and / or into a synthetic material). In some embodiments, a synthetic material of the present invention is thixotropic, can retain its shape following deposition (e.g., extrusion from a syringe and / or printer), and can support two or more layers of the synthetic material.

[0085] In some embodiments, the synthetic material is highly compliant, i.e. flexible, bendable, and / or elastic, and can be incorporated into a soft robotic component and / or a soft actuator, such as a joint, sensor, muscle, or skin. In some embodiments, the synthetic material is operably linked to a soft robotic device. In some embodiments, the synthetic material is capable of expansion or collapse on change of pressure.

[0086] In some embodiments, the synthetic material demonstrates memristive behavior. Thus, the present invention further provides a memristor or a biomimetic memristor comprising the synthetic material. In some embodiments, the synthetic material is used in a memristive neuromorphic hardware. In some embodiments, the synthetic material behaves as an RC circuit. In some embodiments, the synthetic material can be used in biocomputing.

[0087] Methods of Preparing Network Materials

[0088] The present invention also relates, in part, to methods, techniques, and strategies for fabricating and characterizing the materials described herein. In one aspect, the present invention relates, in part, to methods of generating the synthetic material described herein. Thus, the present invention provides a method of making a synthetic material comprising a plurality of aqueous compartments, the method comprising the steps of: providing an emulsion comprising a plurality of aqueous compartments encompassed by amphiphilic molecules dispersed within a hydrophobic liquid; applying a force to the emulsion; and removing the hydrophobic liquid to isolate the synthetic material.

[0089] The provided emulsion is generally a mixture of an aqueous phase, which, in certain embodiments, forms the plurality of aqueous compartments, a hydrophobic liquid, and any amphiphilic molecule described throughout the present disclosure, which has been agitated to disperse the aqueous phase throughout the hydrophobic liquid. In certain embodiments, the mixture is agitated using vortexing.

[0090] In some embodiments, the hydrophobic liquid is a lubricant, a long-chain hydrocarbon, a vegetable oil, a fatty acid ester, or combinations thereof. Exemplary hydrophobic liquids include, but are not limited to, hydrocarbons, including hexadecane, fluorous materials, ionic liquids, non-Newtonian fluids, lubricating powders or beads, silicone oils, glycols, vegetable oils, monoglycerides, diglycerides, triglycerides, and fatty acid methyl esters. In one embodiment, the hydrophobic liquid comprises squalane, hexadecane, silicone oil, or a combination thereof.

[0091] In some embodiments, the step of applying a force compacts the plurality of aqueous compartments. In one embodiment, compacting the plurality of aqueous compartments results in a continuous network of the amphiphilic molecules. In one embodiment, compacting the plurality of aqueous compartments results in a bilayer network comprising amphiphilic molecules. In one embodiment, the continuous network of amphiphilic molecules is a continuous bilayer, as shown in Fig. 2A. In one embodiment, the step of applying a force to the emulsion comprises centrifuging the emulsion. In one embodiment, the emulsion is centrifuged at a force of at least 1000g, at least 2000g, at least 3000g, at least 4000g, at least 5000g, at least 6000g, at least 7000g, at least 8000g, at least 9000g, or at least 10000g. In one embodiment, the step of applying a force is performed for at least 1 second, at least 30 seconds, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, or at least 10 minutes.

[0092] In one embodiment, the ratio of the volume of an aqueous compartment within the plurality of aqueous droplets to the volume of the hydrophobic liquid in the emulsion is about 1 : 1. In one embodiment, the ratio of the volume of an aqueous compartment within the plurality of aqueous droplets to the volume of the hydrophobic liquid in the emulsion is about 2: 1. In one embodiment, the ratio of the volume of an aqueous compartment within the plurality of aqueous droplets to the volume of the hydrophobic liquid in the emulsion is about 1:2.

[0093] In one embodiment, the step of removing the hydrophobic liquid comprises the step of decanting off the hydrophobic liquid above the synthetic material. In one embodiment, the step of removing the hydrophobic liquid comprises the step of filtering the hydrophobic liquid off of the synthetic material.

[0094] In one embodiment, the emulsion further comprises a channel-forming molecule, a membrane protein, an artificial channel, an ionophore, or any combination thereof. In one embodiment, emulsion further comprises a plurality of aqueous droplets surrounded by amphiphilic molecules and at least one molecule selected from a channel -forming molecule, a membrane protein, an artificial channel, an ionophore, or any combination thereof.

[0095] In some embodiments, the synthetic material can be further extruded to produce a three- dimensional material. Thus, in one embodiment, the method comprises the step of extruding the synthetic material onto a surface open to air or into an aqueous solution (i.e. water). In one embodiment, the aqueous solution is a buffer solution.

[0096] EXPERIMENTAL EXAMPLES

[0097] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0098] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0099] Example 1 : Network Materials and Methods of Making and Using Thereof

[0100] This application relates to, in part, processes for the rapid and scalable production of network materials (e.g., artificial tissues, soft robotic components, membranes, etc.) using droplet interface bilayers, and the use of such tissues for various applications in separations, biotech, and healthcare.

[0101] For example, described herein are processes for making network materials (e.g., artificial tissues, soft robotic components, membranes, etc.) using the droplet interface bilayer technique, as well as applications of the resulting tissues. The processes can comprise forming a water-in- oil emulsion using bilayer-forming amphiphilic molecules as emulsifiers. Lipids and polymers can be used as emulsifiers; examples include amphiphilic molecules such as 1,2-diphytanoyl-sn- glycero-3 -phosphocholine (DPhPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), polybutadiene polyethylene oxide (PB-PEO), polyisoprene polyethylene oxide (PLPEO) and other block copolymers. Emulsification allows for the formation of aqueous droplets enclosed by a monolayer.

[0102] After the droplets are formed, a force can be applied to the emulsion to create a tightly packed jammed system. The hydrophobic (oil) phase can then be separated. For example, the emulsion can be centrifuged to create a tightly packed jammed system, and the excess oil can be discarded. The contact of the droplets creates a gel-like substance that mimics natural tissues since each droplet within the gel is enclosed by a bilayer, thus creating a network of communicating water compartments. This bilayer is known as a droplet interface bilayer (DIB).

[0103] The droplet size can be controlled through the tuning of the surfactant concentration, and the mixing energy supplied during emulsification. The process enables the formation of small tissues (volumes in the pL scale) up to industrial-scale tissues (volumes in the liter scale). The resulting material has outstanding properties compared to the initial building blocks. The material exhibits viscoelasticity, self-healing behavior, and it is nonconductive despite its high water content (over 99%). The material can also behave as an RC-circuit. Slight modifications of the process can be applied to dramatically tune the electrical and mechanical properties of the tissue. The tissue can become electrically conductive by incorporating ion channels during the emulsification process. The addition of ion channels allows for the creation of biomimetic membranes that replicate and amplify the properties of the individual channel (selectivity, pore size exclusion). The system allows the incorporation of virtually any natural or artificial channel since the channels self-arrange in the monolayer / bilayer due to the hydrophobic effect. Some of the channels that have been incorporated through this method are gramicidin (self-inserting peptide), Outer Membrane Protein (OmpF), and Ammonium Transporter Protein (AmtB) (integral membrane protein), as well as artificial channels.

[0104] The mechanical properties of the tissue can be tuned with the addition of a hydrogel during the emulsification process. For example, adding a 1% agarose to the aqueous phase during emulsification results in tissues with a higher Young Modulus.

[0105] These networks can mimic a natural tissue, and they can be used in a wide range of applications such as bio-inks, electrically-driven separation membranes, sensors, and bioreactors, among others.

[0106] The networks can be used as bio-inks. Briefly, the gel was assembled inside the cartridge of a commercial bioprinter (Cellink BioX). Then the material was extruded using a pneumatic printhead, which allowed to create patterned shapes (previously drawn in CAD software) enabling the layered fabrication of three-dimensional (3D) biomimetic structures. The network has also been used to fabricate biomimetic membranes for electrically driven separations when ion channels with specific properties are incorporated into the network. Again, the ion channels include peptides, integral membrane proteins, and artificial channels. To fabricate the membrane the tissue was placed in a glass / or plastic container separated by two hydrogel layers to each side. The hydrogels were contacted with an aqueous media. The assembly follows the order: aqueous solution / hydrogel / tissue / hydrogel / aqueous solution. The application of voltage between the two aqueous solutions drives the transport of charged molecules or ions, that can cross the membrane through ion channels from one aqueous chamber to the other one. The selectivity of the membrane is controlled by the width of the tissue in addition to the selectivity of the channels used since each layer of droplets acts like a membrane.

[0107] For example, two layers of droplets containing a channel that is 90% selectivity for a specific molecule, will act like two 90%-selectivity membranes in series, giving an overall 99% selectivity provided that all the droplets contain the selective channel. This unique feature allows fine-tuning of the selectivity of the membrane even when using low selectivity channels. These membranes have potential applications in water treatment, nutrient recovery, mining industry separations, and biomedical devices.

[0108] In the process described herein, thousands to billions of droplets are simultaneously produced by emulsification. In this process, the droplet formation and the monolayer coating of the droplets occur simultaneously since the adsorption of surfactants into the water droplets lowers the surface tension of water allowing the formation of more droplets. The mixing energy supplied to the systems reduces the time for monolayer formation (which is mostly controlled by diffusion in the other approaches) enabling the completion of the process in a few minutes regardless of the volume of the tissue that is being produced. Additionally, the stochastic nature of this droplet formation process allows for a normal distribution of droplet sizes, which improves the jamming of the system during the force application (e.g., centrifugation) step. The droplets are forced into contact by centrifugation which is faster than gravity-based approaches.

[0109] As a result, the process described herein can produce a network composed of millions of droplets in as little as 5 minutes, while printing a similar-sized tissue could take up to two hours with the present technologies. Further, this process is scalable to an industrial level (potentially millions of square meters of membranes for example), which will allow users to access the full potential of droplet interface bilayer networks.

[0110] Additionally, the application of these networks in a membrane for water treatment and resource recovery can solve major problems in the industry of commercial biomimetic membranes also related to scalability. Scaling up the production of biomimetic membranes can be challenging. Achieving large-scale production while maintaining consistent quality and performance can be costly and technically demanding. This new process of membrane fabrication offers 1) simplicity and scalability of membrane fabrication, 2) self-healing membranes that can reform once damaged, 3) high levels of selectivity, and 4) ability to be fabricated with a wide range of materials. In this matter, the fabrication of membranes that are made with polymers instead of lipids solves a problem since so far literature has only reported the fabrication of droplet-based tissues with lipids, which can be unstable and unsuitable for the fabrication of water treatment and mining industry membranes due to the aggressive conditions of temperature and pH of these processes.

[0111] Example 2: 3D-printable bilayer-stabilized jammed emulsions as scalable biological tissue mimics

[0112] The invention refers to a process to make artificial tissues using the droplet interface bilayer technique, as well as applications of the resulting tissues. The process consists of forming a water in-oil emulsion using bilayer-forming amphiphilic molecules as emulsifiers. Lipids and polymers have been used as emulsifiers. The list includes (but is not limited to) amphiphilic molecules such as l,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1, 2-di oleoyl -sn- glycero-3-phosphocholine (DOPC), polybutadiene polyethylene oxide (PB-PEO), polyisoprene polyethylene oxide (PI-PEO) and other block copolymers. Emulsification allows for the formation of aqueous droplets enclosed by a monolayer. After the droplets are formed, the solution is centrifuged to create a tightly packed jammed system, and the excess oil is discarded. The contact of the droplets creates a gel-like substance that mimics natural tissues since each droplet within the gel is enclosed by a bilayer, thus creating a network of communicating water compartments. This bilayer is known as a droplet interface bilayer (DIB). The droplet size can be controlled through the tuning of the surfactant concentration, and the mixing energy supplied during emulsification. The process enables the formation of small tissues (volumes in the pL scale) up to industrial-scale tissues (volumes in the liter scale). The resulting material has outstanding properties compared to the initial building blocks. The material exhibits viscoelasticity, self-healing behavior, and it is nonconductive despite its high water content (over 99%). Electrically speaking the material behaves as an RC -circuit.

[0113] Slight modifications of the process can be applied to dramatically tune the electrical and mechanical properties of the tissue. The tissue can become electrically conductive by incorporating ion channels during the emulsification process. The addition of ion channels allows for the creation of biomimetic membranes that replicate and amplify the properties of the individual channel (selectivity, pore size exclusion). The system allows the incorporation of virtually any natural or artificial channel since the channels self-arrange in the monolayer / bilayer due to the hydrophobic effect. Some of the channels that have been incorporated through this method are gramicidin (self inserting peptide), Outer Membrane Protein (OmpF), and Ammonium Transporter Protein (AmtB) (integral membrane protein), as well as artificial channels such as LAP5nlO (recently disclosed as a highly selective lithium channel). The mechanical properties of the tissue can be tuned with the addition of a hydrogel during the emulsification process. For example, adding a 1% agarose to the aqueous phase during emulsification results in tissues with a higher Young Modulus.

[0114] A purpose of this invention is to mimic a natural tissue, and it can be used in a wide range of applications such as bio-inks, electrically-driven separation membranes, sensors, and bioreactors, among others. Two of these applications are demonstrated herein. The gels have been used as bio-inks. Briefly, the gel was assembled inside the cartridge of a commercial bioprinter (Cellink BioX). Then the material was extruded using a pneumatic printhead, which allowed to create patterned shapes (previously drawn in CAD software) enabling the layered fabrication of three-dimensional (3D) biomimetic structures. The tissue can also be used to fabricate biomimetic membranes for electrically driven separations when ion channels with specific properties are incorporated into the network. Again, the ion channels include peptides, integral membrane proteins, and artificial channels. To fabricate the membrane the tissue was placed in a glass / or plastic container separated by two hydrogel layers to each side. The hydrogels were contacted with an aqueous media. The assembly follows the order: aqueous solution / hydrogel / tissue / hydrogel / aqueous solution. The application of voltage between the two aqueous solutions drives the transport of charged molecules or ions, that can cross the membrane through ion channels from one aqueous chamber to the other one. The selectivity of the membrane is controlled by the width of the tissue in addition to the selectivity of the channels used since each layer of droplets acts like a membrane. For example, two layers of droplets containing a channel that is 90% selectivity for a specific molecule, will act like two 90%- selectivity membranes in series, giving an overall 99% selectivity provided that all the droplets contain the selective channel. This unique feature allows fine-tuning of the selectivity of the membrane even when using low selectivity channels. These membranes have potential applications in water treatment, nutrient recovery, mining industry separations, and biomedical devices.

[0115] Present technologies include the fabrication of droplet interface bilayer tissues through 3D printing with a piezoelectric droplet generator, where each droplet is individually produced and deposited in an oil medium containing amphipathic molecules. Similar works in literature reported the creation of a droplet network by using a microfluidic droplet generator where monodisperse arranges of droplets are assembled one droplet at a time. In both approaches the processes of droplet and monolayer formation are sequential. The droplets coated by a monolayer are assembled into networks of droplets by releasing the droplets in a stagnant hydrophobic medium. The droplets fall by gravity forming layers of droplets that need to be precisely located next to each other to obtain a highly packed network. Once printed the networks are not reconfigurable.

[0116] The invention is unique since presents a process where thousands to billions of droplets are simultaneously produced by emulsification. In this process, the droplet formation and the monolayer coating of the droplets occur simultaneously since the adsorption of surfactants into the water droplets lowers the surface tension of water allowing the formation of more droplets. The mixing energy supplied to the systems reduces the time for monolayer formation (which is mostly controlled by diffusion in the other approaches) enabling the completion of the process in a few minutes regardless of the volume of the tissue that is being produced. Additionally, the stochastic nature of this droplet formation process allows for a normal distribution of droplet sizes, which is crucial for the jamming of the system during the centrifugation step. The droplets are forced into contact by centrifugation which is faster than gravity-based approaches. As a result, the process render a tissue composed of millions of droplets in as little as 5 minutes, while printing a similar-sized tissue could take up to two hours with the present technologies. Moreover, this invention presents a novel approach toward the production of a material that can be utilized as a raw material for other processes (bioink, membrane fabrication). This separates this invention from present technologies which yield assemblies of tissues with limited manipulation capabilities after printing. Finally, this is the first time that a membrane for electrically driven separations made with droplet interface bilayer tissues has been hypothesized and demonstrated.

[0117] This invention solves the scalability of artificial tissues since current technologies only allow the assembly of tissues in the mm scale. The process presented in this invention is scalable to an industrial level (potentially millions of square meters of membranes for example), which will allow unlocking the full potential of droplet interface bilayer networks.

[0118] Additionally, the application of these tissues in a membrane for water treatment / resource recovery will solve major problems in the industry of commercial biomimetic membranes also related to scalability. Scaling up the production of biomimetic membranes can be challenging. Achieving large-scale production while maintaining consistent quality and performance can be costly and technically demanding. This new process of membrane fabrication offers 1) simplicity and scalability of membrane fabrication, 2) self-healing membranes that can reform once damaged, 3) high levels of selectivity, and 4) ability to be fabricated with a wide range of materials. In this matter, the fabrication of membranes that are made with polymers instead of lipids solves a problem since so far literature has only reported the fabrication of droplet-based tissues with lipids, which can be unstable and unsuitable for the fabrication of water treatment and mining industry membranes due to the aggressive conditions of temperature and pH of these processes.

[0119] The present work allows tissues to be produced in a few minutes, and the amount produced yields cm-sized tissues. Emulsification is a well-known process in the industry (for example, used for latex, paintings, food industry), which offers the ability to scale up the invention. The process of emulsification offers the possibility of using the hydrophobic effect to incorporate proteins that usually do not self-insert in bilayers as well the option of using artificial channels. Further, the present process demonstrates enhanced stability due to the fact that the building blocks can be lipids but also polymers that can tolerate better aggressive conditions in temperature and pH, and that are the gold standard in industrial membrane fabrication. The process yields a material that can have multiple uses, acting as a raw material for other fabrication processes.

[0120] There are several potential applications for these tissues and the membranes fabricated with these tissues including a platform for drug screening and testing, a controlled system to investigate cellular signaling pathways, drug delivery, fabrication of sensors for toxins and environmental pollutants, development of artificial organs, creation of soft robots, use in biocomputing, among others.

[0121] The present example introduces a rapid and scalable method to create jammed emulsions that are bilayer-stabilized which can mimic the features of compartmentalization found in biological tissues and can be functionalized with membrane proteins to confer specific properties to the bilayer network. The method is highly adaptable and can be used with a wide range of amphiphiles. Three different ion channels are used in this work to showcase features such as tunable conductance, selective transport, and memristance. In addition, the rheological properties of the material make it suitable for 3D printing. JIBEs demonstrate excellent stability in aqueous media, which allows for constructing structures that can interact with their environment. The incorporation of other membrane proteins or artificial ion channels will enable novel applications in fields such as separations, tissue engineering, drug delivery, and soft robotics, unlocking the full potential of JIBE.

[0122] Specifically, the present work provides Jammed Interconnected by Bilayer Emulsions (JIBEs) as a new class of functional biomimetic tissue-like materials because of their ability to emulate biological tissue mechanical and electrical properties and to incorporate biological and bioinspired channels. JIBEs were fabricated using a simple and scalable emulsificationcentrifugation process. This fabrication approach produces a network of polydisperse aqueous compartments interconnected by bilayers and the structures formed integrate elements of highly concentrated emulsions (HCEs) and droplet interface bilayers (DIBs) (Bayley, H. et al., 2008, Molecular BioSystems, 4, 1191; Foudazi, R. et al., 2015, Advances in Colloid and Interface Science, 220, 78).

[0123] The creation of stable gel-like materials through jammed emulsions has been demonstrated in previous works with non-bilayer-forming surfactants, primarily for oil-in-water emulsions (Letteri, R. A. et al., 2017, Advanced Materials, 29, 1702921; Sridharan, S. et al., 2021, Advanced Functional Materials, 31, 2101749). Significantly less work has been done for aqueous droplets. Current approaches to stabilize high internal phase water-in-oil emulsions rely on attractive Pickering emulsions, where nanoparticle-stabilized droplets are crosslinked using an amino-functionalized polymer (Wu, B. et al., 2021, Advanced Materials, 33, 2102392). From the biomimetic perspective and for a number of possible biomimetic applications, the fabrication of aqueous gels is preferred since they resemble the environment found in cells. Additionally, most molecules involved in cell communication and signaling are water-soluble, thus functional tissue-like networks could be constructed using water-in-oil HCEs.

[0124] Networks of aqueous lipid-coated droplets suspended in a hydrophobic medium have been stabilized by forming lipid bilayers between them, known as droplet interface bilayers (DIBs, Fig. 1 A) (Bayley, H. et al., 2008, Molecular BioSystems, 4, 1191). This technique was originally described as a method to create an artificial model membrane (Funakoshi, K. et al., 2006, Analytical Chemistry, 78, 8169). DIB networks have been created by self-assembly of droplets dispensed manually, by microfluidic devices, or using a piezoelectric instrument (Holden, M. A. et al., 2007, Journal of the American Chemical Society, 129, 8650; Hwang, W. L. et al., 2007, Journal of the American Chemical Society, 129, 11854; Stanley, C. E. et al., 2010, Chemical Communications, 46, 7592; Villar, G. et al., 2013, Science, 340, 48). The fabrication of such constructs using the droplet-by-droplet method provides advantages of precision and control but is lengthy and poorly scalable, typically taking many hours to form a micro-meter-sized network. Additionally, the handling of these structures is extremely limited, its printability is constricted to an oil phase, and its fabrication has been demonstrated only with DPhPC lipids. Thus, the development of materials around this concept has been challenging.

[0125] The present work introduces JIBEs as a class of tissue-like fabricated materials through a rapid & scalable process (Fig. IF). The process yields a water-compatible 3D-printable hydrogel (Fig. 1C, Fig. ID). This method allows for the rapid production of milliliter volumes of a prototissue in under 15 minutes, generating a network of millions of picoliter water compartments separated by bilayers. It is demonstrated that this innovative class of materials can be synthesized from a range of amphiphilic-bilayer forming molecules and can be further functionalized with self-inserting peptides or integral membrane proteins to create conductive ion pathways within the material. In addition to characterizing this material's electrical and rheological properties, the present example showcases its printability using a commercial extrusion bioprinter. Two applications of this material are presented herein: first, as an exceptional biomimetic membrane for electrically driven ion separations, exhibiting high selectivity for ammonium ions; and second, as an alamethicin-based memristor with potential for applications in neuromorphic computing (Fig. IE).

[0126] Fast and Scalable Biomimetic Tissue Fabrication

[0127] The process developed for assembling JIBEs is illustrated in Fig. 2A. This new approach to tissue-like material preparation involves three main steps. First, a low-shear mixing process (such as simple vortexing or the use of a homogenizer) using water, oil, and a bilayer-forming amphiphile is employed to produce a water-in-oil emulsion. As a result, the water phase is dispersed into droplets within the oil phase with each droplet coated by a lipid or block copolymer monolayer. The droplets are polydisperse in size and their dimensions depend on emulsification parameters such as the mixing speed and the selection of oil and amphiphiles. In this work, successful JIBEs were formed from a range of lipids and block copolymers, including diphytanoyl phosphatidylcholine (DPhPC), dipalmitoylphosphatidylcholine (DOPC), soy phosphatidylcholine (PC), polybutadiene-polyethylene oxide (PB-PEO), and polyimidepolyethylene oxide (PI-PEO), demonstrating the adaptability of the method to a wide range of bilayer forming amphiphiles. The volumetric percentage of water to oil varied depending on the formulation but initially was between 44 - 50%. The oil selection also depended on the surfactant. Mixtures of hexadecane and silicone oil AR20 resulted in the effective emulsification of lipids such as DPhPC and DOPC, while squalane and hexadecane mixtures were more successful for the block copolymers. When the emulsification was complete (usually in less than 10 min) the emulsion was white in color and fluid in nature (Fig. 2F). During this step, the collision of droplets resulted in the spontaneous formation of DIBs for lipids and polymer as shown in Fig. 2B and Fig. 2D. However, the presence of oil hindered the formation of a continuous bilayer network. In the second step, the droplets were centrifuged at a centrifugal force >8000g for a time of less than 5 min which jammed the droplets and reduced the entrapped oil from an initial value of 56-50% to under 5%. This process created a continuous network of bilayers between droplets as assessed by their electrical properties such as capacitance (Fig. 2H). Because of jamming, the initially spherical droplets adopted a polyhedral shape since the packing density exceeded the theoretical maximum for random close packing of spheres (Fig. 2B through Fig. 2E) (Cameron, N. R , 1996, Biopolymers Liquid Crystalline Polymers Phase Emulsion, 163). Finally, in the last step, the excess oil was removed by decantation, obtaining JIBEs with a solid gel-like appearance and shape retention. The complete process can be carried out in less than 15 minutes and can be scaled to produce large volumes, thus enabling scalable and rapid fabrication.

[0128] Material Properties of JIBEs

[0129] JIBEs have outstanding properties compared to its constituent materials and comparable soft materials while having advantages of modularity, functionality, and printability. Comprising a network of bilayer-separated aqueous compartments, they exhibit properties of bilayers such as selective water permeability, self-healing, flexibility and elasticity, membrane potential, and the ability to incorporate membrane proteins and biomimetic ion channels (Allen-Benton, M. et al., 2019, Experimental Biology and Medicine, 244, 709; Huang, Y. et al., 2022, Advances in Colloid and Interface Science, 304, 102666; Rawicz, W. et al., 2000, Biophysical Journal, 79, 328; Shen, Y-X. et al., 2014, Journal of Membrane Science, 454, 359). Simultaneously, they possess characteristics found in HCEs and other jammed systems (such as granular hydrogels), which confers their rheological properties and 3D printability (Cheng, W. et al., 2020, VIEW, I, 20200060). All these properties will be discussed in detail in the following sections.

[0130] Electrical Properties and Tunability Through Ion Channels

[0131] Even though JIBEs have a water content of over 95%, they are electrically non- conductive due to the presence of a bilayer stabilizing the network. At the same time, they exhibit the characteristic capacitive behavior observed in bilayers (Gross, L. C. M. et al., 2011, Langmuir, 27, 14335), as is evident from Fig. 2H. DPhPC and PBis-PEOs JIBEs exhibit a square-wave current response when subject to a triangular voltage waveform. These properties have also been noted at the two-droplet DIB scale (Taylor, G. J. et al., 2015, Soft Matter, 11, 7592). Thus, the JIBEs simply behave as a larger resistor-capacitor (RC) circuit since it is an ensemble of small RC circuits (Hwang. W. L. et al., 2007, Journal of the American Chemical Society, 129, 11854). It is important to note that the capacitive response arises after the centrifugation step, which is consistent with the almost complete exclusion of oil from the network. This insulating behavior is analogous to the barrier properties found in biological tissues such as the epidermis, particularly the stratum comeum (Yamamoto, Y., 1976, Medical and biological engineering, 14, 151). When measured under the same conditions the capacitance for DPhPC is significantly higher than for polymers, which has two origins. At the molecular level, PB-PEO forms a thicker bilayer than lipids, which results in lower capacitance as this parameter decreases with the distance of the charged plates (Salipante, P. F. et al., 2012, Soft Matter, 8, 3810; Vreeker, E. et al., 2024, bioRxiv, 2024.2005.2016.594548). Additionally, at the macroscopic level, the PB-PEO has smaller droplets which could result in more RC circuits in series. In this configuration, the reciprocal of the total capacitance is the sum of the reciprocals of the individual capacitances.

[0132] The conductance of JIBEs can be easily modulated by the incorporation of ion channels in the bilayers. As a demonstration, Outer Membrane Protein F (OmpF) was incorporated into JIBEs made with DPhPC lipids. OmpF is a beta-barrel porin found in gram-negative bacteria. The protein inserts into the lipid bilayer forming an elliptical channel with dimensions of 0.8nm x 1.08 nm which causes high ion conductance, and it has previously been inserted into DIBs and other bilayer based membrane architectures (Lee, S., 2022, Small, 18, 2200007; Tu, Y.-M. et al., 2020, Nature Materials, 19, 347). At a mass lipid-to-protein ratio (LPR) of 200, a remarkable contrast between the controls and the protein-communicated networks was observed, as presented in Fig. 21. OmpF-functionalized JIBEs showed over 4000 times higher Na+transport than non-functionalized JIBEs reflecting around 1000 OmpF-functional pathways within the network, considering the single-channel conductance of 0.74 nS (Lee, S., 2022, Small, 18, 2200007). This example demonstrates the simplicity of modifying the macroscale properties of the JIBEs by incorporating integral membrane proteins to resemble naturally occurring conductive tissues. Additionally, in the future, the incorporation of other integral membrane proteins with special features such as temperature sensing, mechanosensitive properties, lightactivation, and others, can unlock the full potential of JIBEs as functional materials. The present work demonstrates how this material could be used, with gramicidin A and alamethicin ion channels, for the development of membranes for electrically driven ion separations and to fabricate biomimetic iontronic memristors.

[0133] Selective Permeability

[0134] Lipid bilayers allow the passage of small uncharged molecules, such as water, while rejecting the passage of ions (Paula, S. et al., 1996, Biophysical Journal, 70, 339). This is also the case for DTBs, where two droplet studies have shown water permeation rates that correlate with the nature of the amphiphile and temperature in a range of 40 - 80 pm / s (Milianta, P. J. et al., 2015, Langmuir, 31, 12187; Thiam, A. R. et al., 2012, Langmuir, 28, 6291). PB-PEO polymersomes are reported to have water permeabilities ten times lower than lipid vesicles (Discher, B. M. et al., 1999, Science, 284, 1143). The selective permeability feature of bilayer has been employed to trigger osmotically-driven folding in droplet networks of small synthetic tissues in previous studies (Villar, G. et al., 2013, Science, 340, 48).

[0135] To test the selective permeability and actuation capabilities of macroscopic mm scale JIBEs two filaments of PB-PEO with different osmolyte concentrations were conjointly 3D- printed using two-printheads in a commercial bioprinter, as shown in Fig. 2G. As ions are nearly impermeable within the bilayer network, to equilibrate the system, water is transported from the filament with lower osmotic concentration to the one with higher concentration. As one side of the structure shrank and the other swelled, the entire structure folded in the direction of water transport over 24 hours resulting in an arch shape. Similar results were observed for osmotically unbalanced 3D printed-filaments of JIBEs made with highly economical soy-PC lipids. Moreover, this behavior is in agreement with numerical simulations, where droplets are modeled as point masses with an associated radius.

[0136] This demonstration also highlights the capabilities of JIBEs to create modular systems that can interact with each other due to their self-healing nature similar to what has been demonstrated for droplet-by-droplet 3D-printed DIB networks (Alcinesio, A. et al., 2022, Advanced Functional Materials, 32, 2107773).

[0137] Rheological Properties and Self-Healing

[0138] The rheology of JIBEs made with DPhPC or PBis-PEOe at a concentration of 5 mg / mL (to the water phase) was studied using a protocol described in the literature for the characterization of granular hydrogels (Qazi, T. H. et al., 2022, ACS Biomaterials Science & Engineering, 8, 1427). The scalability of this approach allowed macroscale characterization of these unique materials in a way that was not possible with tissues created using the droplet-by- droplet approach. The mechanical properties were measured at 25 °C using a parallel-plate geometry. The results are displayed in Fig. 3. Both lipid and polymer JIBEs demonstrated viscoelasticity, non-zero yield stress, shear-thinning behavior, and self-healing properties. JTBEs tissue materials are viscoelastic, exhibiting solid-like and liquid-like behavior under oscillatory conditions. Fig. 3A and Fig. 3E depict the studies of the material at a fixed strain of 1% and variable frequency. The elastic behavior of JIBEs can be observed at low frequencies indicated by an independent storage modulus, a characteristic of attractive emulsions (Datta, S. S. et al., 2011, Physical Review E, 84, 041404). For both materials, the storage is greater than the loss modulus indicative of a solid-like behavior at lower frequencies. The storage modulus is 46 Pa and 371 Pa for DPhPC and PBis-PEOe. respectively. One of the reasons behind higher storage in the polymer JIBEs could be due to the smaller droplet size, as smaller droplets have conducted to stiffer materials, for example, in jammed granular hydrogels and attractive Pickering emulsions (Qazi, T. H. et al., 2022, ACS Biomaterials Science & Engineering, 8, 1427; Wu, B. et al., 2021, Advanced Materials, 33, 2102362). Additionally, mechanical studies in planar-bilayers have demonstrated that polymer leads to more application favorable mechanical properties as assessed by higher rupture voltage compared to lipids (Vreeker, E. et al., 2024, bioRxiv, 2024.2005.2016.594548). Previously, DIB-based materials have been modeled as a purely elastic network of picoliter-size droplets and the predicted Young modulus of the material was estimated between ~ 100 to 200 Pa (Villar, G. et al., 2013, Science, 340, 48). Moreover, many biological tissues have a viscoelastic nature with an elastic modulus starting at 100 Pa (Levental, I. et al., 2007, Soft Matter, 3, 299).

[0139] Fig. 3B and Fig. present strain-dependence of the storage and loss modulus when the frequency is set at 0.5 rad / s. For DPhPC and PBis-PEOe, a peak in the loss modulus indicates the yielding behavior of the material. The occurrence of non-zero yield stress confirms jammed packing since this parameter represents the divisor line between unjammed and jammed states (Foudazi, R. et al., 2015, Advances in Colloid and Interface Science, 220, 78). The yield point lies within the same order of magnitude for lipids and polymer JIBEs with a yielding point occurring at 65% and 87%, respectively.

[0140] JIBEs behave as non-Newtonian fluids, as shown in Fig. 3C and Fig. 3G. The linear region of the shear rate-viscosity plot can be fitted to a power-law model, to determine coefficients K and n, where K represents the viscosity at a shear rate of 1 s ' and n is the flow behavior index. For DPhPC K=18.2 and n=0.340 (R2=99.1%) and PBI5-PEO6K=78.2 and n=0.176 (R2=99.8%). n<l is consistent with a shear-thinning fluid, as the viscosity decreases as the shear rate increases. These parameters are desirable for extrusion 3D printing as they are similar to other printable inks (Paxton, N. et al., 2017, Biofabrication, 9, 044107).

[0141] Finally, the self-healing nature of JIBEs was assessed by performing repeated time sweeps on the material at alternating high and low strain, as shown in Fig. 3D and Fig. 3H. The material recovery was studied at an oscillation strain of 0.5% where it behaves as a solid, and at 300% which is past the yielding point. The JIBEs’ self-healing character was observed by the storage modulus retrieval after the material had been yielded two times. Although the first yield produces a higher loss in the storage, after the second yield the material recovers 80% and >100% of the modulus for DPhPC and PBis-PECE respectively. These self-healing properties arise from the reversible nature of the DIB formation between the droplets (Bayley, H. et al., 2008, Molecular Biosystems, 4, 1191).

[0142] 3D-Printability

[0143] The rheological properties, including non-zero yield stress, shear thinning, and recovery after being subjected to a high shear rate make JIBEs a suitable material for 3D printing (Paxton, N. et al., 2017, Biofabrication, 9, 044107). Furthermore, the membrane-bound cell-like compartments of JIBEs as well as their capabilities of hosting integral membrane proteins represent a novel class of bio-ink when compared to the portfolio of materials currently used in bioprinting (Gungor-Ozkerim, P. S. et al., 2018, Biomaterials Science, 6, 915). The present work demonstrates and quantifies the feasibility of JIBEs as a bioink for extrusion-based 3D printing in air and aqueous solutions.

[0144] The printability of JIBEs was tested using a commercially available extrusion-based pneumatic bioprinter. Because of the accessibility and affordability of the material, the majority of the prints were made with PB-PEO polymers (Fig. 4) and soy-PC lipids. DPhPC lipids are also 3D-printable. JIBEs were successfully printed in air and into osmotically balanced aqueous solutions (Fig. 4A and Fig. 4C). The continuity, stability, and shape retention of printed filaments were analyzed. The material forms continuous, unbroken filaments during extrusion for a range of pressures and raster speeds (Fig. 41 and Fig. 4P). As expected, filament dimensions are directly proportional to the applied pressure and indirectly proportional to printing speed. When exposed to air for an extended period of time, printed filaments experience a degree of lateral spreading followed by evaporation of aqueous compartments because of the water permeability of bilayers (Fig. 4J), but when contained within an osmotically balanced aqueous environment, the prints remain stable for days to months without collapsing (Fig. 4Q).

[0145] Printed JIBEs demonstrated good stacking capabilities, enabling the construction of 3D structures with well-defined geometries. 3D prints with a height of up to 1 cm were selfsupported without the need for engineering inter-particle interactions which is usually a challenge for jammed water-in-oil emulsions (Wu. B. et al., 2021, Advanced Materials, 33, 2102362). Fig. 4B shows the 3D CAD model of a star that was used to test the stackability of the material. Again, the stability of the prints was tested in air and aqueous solutions. In the air, JIBE prints with PBis-PECE were not able to self-support the 1 cm tall structure. Nevertheless, by increasing the length of the hydrophobic tail with two extra PB units the mechanical stability was greatly improved rendering a print that accurately represents the CAD design as shown in Fig. 4D through Fig. 4G. In some cases, these structures are not stable beyond a day since the material dries in air. In contrast, printing directly in a buffered solution allowed for the printing ofPB 15-PEO6 as shown in Fig. 4K through Fig. 4N. The filament dimensions, including wall thickness, height, and overall diameter of printed stars, were comparable to those of a commonly used hydrogel bioink, Pluronic F127. Moreover, the ease with which JIBEs stabilize in water is remarkable. Architectures based on DIBs are constrained to a bulk oil phase and its stabilization on the aqueous phase has been achieved at small scales by prior encapsulation of the oil in water or hydrogel shells (Baxani, D. K. et al., 2016, Angewandte Chemie International Edition, 55, 14240; Villar, G. et al., 2011, Nature Nanotechnology, 6, 803). By contrast, JIBEs deposition in water results in an immediate bilayer formation at the interface which allow communication with its external environment as demonstrated in a subsequent section.

[0146] Overall, printed JIBEs demonstrated adequate shape-holding capabilities, fdament formation, and stability. In the future, 3D-printing capabilities can be further enhanced by postprinting crosslinking of the oil phase and / or by the incorporation of a hydrogel matrix inside individual droplets.

[0147] JIBEs as new membrane platform for electrically driven ion separations

[0148] JIBEs are presented herein as a new approach to fabricating selective biomimetic membranes since their unique architecture offers a middle ground between protein insertion, defect sealability, and membrane stability thus overcoming challenges faced by current approaches based on spherical vesicles and 2D (planar) nanosheets (Shen, Y.-X. et al., 2018, Nature Communications, 9, 2294; Tu, Y.-M. et al., 2020, Nature Materials, 19, 347; Zhao, Y. et al., 2022, Environmental Science & Technology, 56, 5179). As demonstrated with the OmpF proteins, JIBEs can be functionalized with membrane proteins to create conductive ion paths within the network. When using selective ion channels, this quality can be leveraged for electrically driven separations.

[0149] To demonstrate the potential of JIBEs in ion separations, the gramicidin A (gA) channel was used, which is well known for allowing the passage of monovalent ions while rejecting divalent ions (Heitz, F., 1983, Biophysical Chemistry, 18, 153). This peptide also exhibits a naturally occurring selectivity between monovalent cations which has been reported in black- lipid membrane studies as NH4+>K+>Na+(Andersen, O. S., 1983, Biophysical Journal, 41, 147; Myers, V. B., 1972, Biochimica et Biophysica Acta (BBA) - Biomembranes, 274, 313). To showcase the progression from a multi-channel single membrane up to a tissue-like scale, studies of the transport in a two-droplet DIB system were carried out as illustrated in Fig. 5A and Fig. 5B. The results agreed with the trends reported in the literature (Fig. 5C)

[0150] JIBEs-gA doped membranes were assembled as the intermediate layer separating two aqueous compartments. The JIBEs layer was supported on both sides by 2.5% agarose hydrogels. These hydrogels helped in forming droplet hydrogel bilayers at the interface between JIBEs membrane and the agarose (Leptihn, S. et al., 2013, Nature Protocols, 8, 1048). Thus, agarose offers support and connectivity to the aqueous phase while being a negligible source of resistance to ion transport. A schematic of the system is depicted in Fig. 5E, along with pictures of the actual setup (Fig. 5D). For single salt experiments, the transport was measured by the equivalent current recorded with a patch-clamp amplifier. As presented in Fig. 5F, DPhPC JIBEs membranes with 2 pM of gA demonstrated than two-droplet DIBs when performing single salt experiments. The tissue-like membrane exhibited a selectivity of K+ / Na+=5.5 and NH4+ / Na+=16.8. By comparison, the controls (without the peptide) presented almost negligible transport, and only due to the lipid's natural permeability to monovalent ions, showcasing a defect-free network since even minor flaws in the structure would cause massive non-specific transport. Additionally, mixed ion experiments were performed to further confirm the selective nature of the transport. In this case, JIBE-membranes doped with 150 pM of gA showed a selectivity between monovalent ions (K+ / Na+=2.9) and divalent ions (K+ / Ca2+=13.3) in a mixed salt environment that included sodium, potassium, and calcium ions after being subjected to a potential of 1000 mV for 48 h.

[0151] Although the natural selectivity of gramicidin is minor compared to other naturally occurring channels such as the potassium channel (Doyle, D. A. et al., 1998, Science, 280, 69), this demonstration highlights the high potential of JIBE-membranes, particularly because of the scal ability of the process, the simplicity of protein incorporation into the network, stability in water, and their self-healing nature that leads to a defect-free construct. In the future the incorporation of highly selective channels along with the reduction in the thickness of the membrane layer will facilitate the production of biomimetic membranes, with transport properties that rely solely on the characteristics of the embedded channels.

[0152] JIBEs as biomimetic memristors

[0153] Neuromorphic computing systems aim to mimic the complexity and plasticity of the brain. One of the key circuit elements to achieve this end is the memristor (Markovic, D. et al., 2020, Nature Reviews Physics, 2, 499), a component that varies its electrical resistance based on its previous history of current flow (Chua, L., 1971, IEEE Transactions on Circuit Theory, 18, 507). Commercial memristors rely on metal-oxide-semiconductor circuitry that has not reached the efficiency of their biological counterparts. Biomolecular memristors based on DIBs doped with memri stive-like ion channels offer a promising alternative to solid-state memristors (Makhoul-Mansour, M. M. et al., 2023, Journal of Composite Materials, 57, 659; Maraj, J. J. et al., 2023, Advanced Intelligent Systems, 5, 2300049; Maraj, J. J. et al., 2021, ACS Applied Electronic Materials, 3, 4448). However, so far two-droplet DIBs that have been demonstrated are highly unstable and difficult to manipulate due to their liquid construction (Venkatesan, G. A., 2016, Lab on a Chip, 16, 2116). JIBEs offer an advantage over the DIB architecture because of their capabilities to incorporate ion channels and their easiness of fabrication and manipulation.

[0154] A biomolecular memristor was assembled using alamethicin-doped JIBEs. Alamethicin is a peptide that has been traditionally used as a model for a voltage-gated channel since it reversibly inserts in the lipid membrane when the voltage surpasses a threshold (Fig. 6A). Previously, the insertion voltage for alamethicin in DPhPC DIBs was determined at 72.4 ±8.9 V at a scan rate of 100 mV / s and at room temperature (Najem, J. S. et al., 2018, ACS Nano, 12, 4702). JTBEs-doped with 6 pM Alamethicin were assembled on a 2 mm-thick PDMS support with a surface area of 0.25 cm2, with Ag / AgCl electrodes on opposite sides of the diagonal as shown in Fig. 6B. As a note, since JIBEs are a complex bilayer network, an ensemble of parallel and series circuits, usually takes some time (from 10 min to an hour) to begin observing ionic transport when the network is stimulated with a triangular waveform of + / - 150 mV at 10 mV / s. Fig. 6C shows the characteristic response of the system when no channels were added, in which case the current observed was negligible. Fig. 6D shows the characteristic response of a JIBE with Alamethicin, depicting the signature pinched I-V curve that identifies a memristor (Chua, L., 2014, Semiconductor Science and Technology, 29, 104001). The present work highlights the potential of JIBEs to emulate complex neuronal networks since a sample of 50 pL contains the order of 106droplets, offering the possibility of more closely emulating biosynapses as well as developing memristive devices that can have low-switching power and thus high energy efficiency.

[0155] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWe claim:

1. A method of making a synthetic material comprising a plurality of aqueous compartments and a bilayer network comprising amphiphilic molecules, the method comprising the steps of: providing an emulsion comprising a plurality of aqueous compartments encompassed by amphiphilic molecules dispersed within a hydrophobic liquid; applying a force to the emulsion; and removing the hydrophobic liquid to isolate the synthetic material.

2. The method of claim 1, wherein the plurality of aqueous compartments has a normal distribution of size.

3. The method of claim 1, wherein at least one aqueous compartment within the plurality of aqueous compartments is polyhedral.

4. The method of claim 1, wherein the amphiphilic molecule is a lipid.

5. The method of claim 1, wherein the amphiphilic molecule is DPhPC.

6. The method of claim 1, wherein the amphiphilic molecule is a polymer.

7. The method of claim 1, wherein the amphiphilic molecule is a polymer selected from polybutadiene (PB), polyisoprene (PI), polyethylene (PE), polypropylene (PP), polystyrene (PS), polyacrylonitrile (PAN), poly(ethylene terephthalate) (PET), poly(tetrafluoroethylene), polypropylene carbonate), and copolymers thereof.

8. The method of claim 1, wherein the amphiphilic molecule is a polymer selected from polyethylene oxide (PEO), polyvinylpyrrolidone (PVP), poly(acrylic acid) (PAA),poly(vinyl alcohol) (PVA), poly(acrylamide) (PAAm), poly(N,N-dimethylacrylamide) (PDMA), and copolymers thereof.

9. The method of claim 1, wherein the amphiphilic molecule is a block copolymer of at least one hydrophilic polymer and at least one hydrophobic polymer.

10. The method of claim 1, wherein the amphiphilic molecule is a block copolymer of polybutadiene polyethylene oxide (PB-PEO), a block copolymer of polyisoprene polyethylene oxide (PI-PEO), or a combination thereof.

11. The method of claim 1, wherein the hydrophobic liquid is selected from the group consisting of a lubricant, a long-chain hydrocarbon, a vegetable oil, a fatty acid ester, and combinations thereof.

12. The method of claim 1, wherein the hydrophobic liquid comprises squalane, hexadecane, silicone oil, or a combination thereof.

13. The method of claim 1, wherein the step of applying a force compacts the plurality of aqueous compartments.

14. The method of claim 1, wherein the step of applying a force to the emulsion comprises centrifuging the emulsion.

15. The method of claim 1, wherein the ratio of the volume of the plurality of aqueous compartments to the volume of the hydrophobic liquid in the emulsion is about 1:1.

16. The method of claim 1, wherein the step of removing the hydrophobic liquid comprises the step of decanting off the hydrophobic liquid above the synthetic material.

17. The method of claim 1, wherein the emulsion further comprises a channelforming molecule, a membrane protein, an artificial channel, an ionophore, or any combination thereof.

18. The method of claim 1, further comprising the step of extruding the synthetic material onto a surface open to air.

19. The method of claim 1, further comprising the step of extruding the synthetic material into an aqueous solution.

20. The method of claim 1, further comprising the step of extruding the synthetic material into water.

21. A synthetic material comprising a plurality of aqueous compartments and a bilayer network comprising amphiphilic molecules, wherein each aqueous compartment is entirely encompassed by amphiphilic molecules.

22. The synthetic material of claim 21, wherein the plurality of aqueous compartments has a normal distribution of size.

23. The synthetic material of claim 21, wherein at least one aqueous compartment within the plurality of aqueous compartments is polyhedral.

24. The synthetic material of claim 21, wherein the amphiphilic molecule is a lipid.

25. The synthetic material of claim 21, wherein the amphiphilic molecule is DPhPC.

26. The synthetic material of claim 21, wherein the amphiphilic molecule is a polymer.

27. The synthetic material of claim 21 , wherein the amphiphilic molecule is a polymer selected from polybutadiene (PB), polyisoprene (PI), polyethylene (PE), polypropylene (PP), polystyrene (PS), polyacrylonitrile (PAN), polyethylene terephthalate) (PET), poly(tetrafluoroethylene), polypropylene carbonate), and copolymers thereof.

28. The synthetic material of claim 21, wherein the bilayer network comprises a polymer selected from polyethylene oxide (PEO), polyvinylpyrrolidone (PVP), poly(acrylic acid) (PAA), poly(vinyl alcohol) (PVA), poly(acrylamide) (PAAm), poly(N,N- dimethylacrylamide) (PDMA), and copolymers thereof.

29. The synthetic material of claim 21, wherein the amphiphilic molecule is a block copolymer of at least one hydrophilic polymer and at least one hydrophobic polymer.

30. The synthetic material of claim 21, wherein the amphiphilic molecule is a block copolymer of polybutadiene polyethylene oxide (PB-PEO), a block copolymer of polyisoprene polyethylene oxide (PI-PEO), or a combination thereof.

31. The synthetic material of claim 21, wherein the synthetic material has a storage modulus of at least 40 Pa.

32. The synthetic material of claim 21, wherein the synthetic material is a hydrogel.

33. The synthetic material of claim 21, wherein the synthetic material is self-healing.

34. The synthetic material of claim 21, wherein the bilayer network further comprises a channel-forming molecule, a membrane protein, an artificial channel, an ionophore, or any combination thereof.

35. The synthetic material of claim 21, wherein the bilayer network further comprises a molecule selected from a porin, a pore-forming toxin, an aquaporin, alamethicin, gramicidin A (gA), outer membrane protein F (OmpF), outer membrane protein X (OmpX), hemolysin toxin(aHL), ferrichrome outer membrane transporter (FhuA), AquaporinO, aquaporin Z (AqpZ), magainin, cecrophin, melittin, maculatin, or combinations thereof.

36. The synthetic material of claim 21, wherein the bilayer network is water permeable.

37. The synthetic material of claim 21, wherein the aqueous compartments further comprise a drug, a therapeutic agent, an ion, or any combination thereof.

38. A bioink comprising the synthetic material of any of claims 21-37.

39. A soft robotic component comprising the synthetic material of any of claims 21- 37.

40. A permeable membrane comprising the synthetic material of any of claims 21-37.

41. A biomimetic memristor comprising the synthetic material of any of claims 21-37.

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