Programmable liquid, gel and biohybrid compartments and methods of use

Abstract

Nano- to microscale liquid coacervate particles are provided. The liquid coacervate particles are produced by a process including stimulating a population of liquid droplets containing one or a mixture of components to induce a phase separation point of a first component, and maintaining stimulation at the phase separation point to form a coacervate domain of the first component within each of the droplets to form the liquid coacervate particles. The self-assembled nano, meso, micro and macro liquid coacervate particles and related coated substrates can have utility in drug delivery, bioanalytical systems, controlled cell culture, tissue engineering, biomanufacturing and drug discovery.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of PCT Patent Application No. PCT / US15 / 55836 filed Oct. 15, 2015, which claims the benefit of U.S. Provisional Application 62 / 064,057 filed Oct. 15, 2014, the disclosure of both of which is hereby incorporated by reference in its entirety.FEDERAL FUNDING LEGEND[0002]The invention was made with Government support under Federal Grant No. DMR-1121107 awarded by the National Science Foundation. The Government has certain rights in the invention.TECHNICAL FIELD[0003]The presently disclosed subject matter relates to programmable liquid, gel and biohybrid compartments and methods of use.BACKGROUND[0004]Multi-phase compartments are ubiquitous within biological cells, provide a powerful method for segregation of biomolecules, and are universal motifs in synthetic polymeric particle fabrication. Unfortunately, a broad platform for the in vitro programming of complex and hierarchical multi-phase structures has rem...

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Benefits of technology

[0005]In some embodiments, the presently disclosed subject matter is directed to a method for making nano- to microscale liquid coacervate particles, the method comprising: stimulating a population of droplets including a solution of one or a mixture of components, wherein the stimulation induces a phase separation point of a first component; and maintaining stimulation at the phase separation point to form a coacervate domain of the first component within each of the droplets, wherein liquid coacervate particles are formed. In some embodiments, the population of droplets are formed using one or a combination of mechanical agitation, sonication, or microfluidics. In some embodiments, the population of droplets are aqueous droplets. In some embodiments, the population of droplets are aqueous droplets formed by sonication of the solution in oil or microfluidics of the solution in oil. In some embodiments, the population of aqueous droplets are in the form of a water-in-oil emulsion. In some embodiments, the liquid coacervate particles are reversibly formed by cessation of stimulation followed by re-stimulation and re-maintaining stimulation. In some embodiments, the first component includes a polymer. In some embodiments, the polymer includes a polypeptide. In some embodiments, the polypeptide includes at least a portion of an elastin-like polypeptide (ELP). In some embodiments, the method further comprises stabilizing the coacervate domain of the first component within each of the droplets to form capsule structures, wherein the coacervate domain of the first component remains consolidated upon cessation of stimulation at the phase separation point of the first component. In some embodiments, stabilizing includes formation of cross-links by one or a combination of covalent coordination, ionic interaction, disulfide bonds, or hydrogen bonds. In some embodiments, the phase separation point is a phase separation temperature and the stimulus includes heating. In some embodiments, the one or a mixture of components includes: a polymer, a synthetic polymer, a hydrophilic polymer, a hydrophobic polymer, an amphiphilic polymer, an amphiphilic diblock polymer, a protein, a nucleic acid, an epoxy, or a polysaccharide, and combinations thereof. In some embodiments, the stimulating includes: addition or removal of one or more of the components, evaporation of the droplets, controlled diffusion of one or more of the components, electrostatic quenching of one or more of the components, inducing a reaction of one or more of the components, isomerization of one or more of the components, crosslinking of one or more of the component, or crystallization of one or more of the components, and combinations thereof. In some embodiments, a substrate is immersed within the population of aqueous droplets in the form of a water-in-oil emulsion, and a tunable degree of the coacervate domain of the first component is formed on a surface of the substrate based on a wetting property of the substrate. In some embodiments, a grafted molecule is present on the surface of the substrate, and the degree of formation of the coacervate domain of the first component on the surface of the substrate is controlled by one or both of a level of interaction of the first component with the molecule and the wetting property of the substrate. In some embodiments, the method further comprises stabilizing the coacervate domain on the surface of the substrate by one or a combination of mineralization or formation of cross-links by one or a combination of covalent coordination, ionic interaction, disulfide bonds, or hydrogen bonds, wherein the coacervate domain remains consolidated upon cessation of stimulation at the phase separation point. In some embodiments, the substrate includes one or more of a medical device, a stent, a vascular graft, a catheter, a biosensor, a drug reservoir, or a cell culture substrate. In some embodiments, the population of droplets are aqueous and the solution further includes one or a combination of a cell, a virus, or a nanoparticle having a coating of at least one component to cause recruitment of the coated cell, virus, or nanoparticle to the coacervate domain of the respective component within each of the droplets. In some embodiments, the first component has an attached bioactive agent, wherein the bioactive agent includes one or a combination of: a drug, a protein, a peptide, a peptide hormone, a ligand, a cell-signaling ligand, or an RGD cell binding domain, to cause recruitment of the drug, protein, peptide, peptide hormone, ligand, cell-signaling ligand, or RGD cell binding domain to the coacervate domain of the first component within each of the droplets. In some embodiments, the first component is a polypeptide and the bioactive agent is attached through an amino acid linkage or through a chemical linkage through a reactive peptide residue. In some embodiments, the polypeptide attached to the bioactive agent includes a protease cleavage site. In some embodiments, the solution includes one or more additional components each having an additional phase separation point, the method further comprising: stimulating the population of aqueous droplets, wherein stimulation induces a phase separation point of the additional component; maintaining stimulation at the additional phase separation point to form a coacervate domain of the additional component within each of the droplets; and optionally repeating the stimulating and maintaining for one or more additional components. In some embodiments, the first component and the additional components include polymers. In some embodiments, the polymers include polypeptides. In some embodiments, the polypeptides include at least a portion of an elastin-like polypeptide (ELP). In some embodiments, the first component and the additional component(s) have similar phase separation points and a blended alloy coacervate domain is formed. In some embodiments, the coacervate domain of the first component and the coacervate domain(s) of the additional component(s) form a multilayered coacervate domain, a blended alloy coacervate domain, or a combination thereof. In some embodiments, the first phase separation point and the additional phase separation point(s) are each a phase separation temperature, and the stimulus includes heating. In some embodiments, the solution includes at least the first component and a surfactant for controlling a size of the coacervate domain. In some embodiments, the surfactant includes an amphiphilic diblock polymer. In some embodiments, the first component is a hydrophobic ELP polymer and the amphiphilic diblock polymer is an ELP diblock polymer. In some embodiments, a ratio of the hydrophobic ELP polymer to the amphiphilic ELP diblock polymer ranges from about 1:1 to about 50:1, and the size of an outermost coacervate domain ranges from about 50 nm to about 20 μm. In some embodiments, the method further comprises stabilizing at least an outermost coacervate domain within each of the droplets to form capsule structures, wherein the outermost coacervate domain remains consolidated upon cessation of stimulation at the phase separation point for the outermost coacervate domain. In some embodiments, stabilizing includes formation of cross-links by one or a combination of covalent coordination, ionic interaction, disulfide bonds, or hydrogen bonds. In some embodiments, a substrate is immersed within the population of aqueous droplets in the form a water-in-oil emulsion, and a tunable degree of the coacervate of the first component and the additional component(s) is formed on a surface of the substrate based on a wetting property of the substrate. In some embodiments, a molecule is grafted on the surface of the substrate, and the degree of formation of the coacervate domain of the first component and the additional component(s) on the surface of the substrate is controlled by one or both a level of interaction of one or both of the first component and the additional component(s) with the molecule and the wetting property of the substrate. In some embodiments, the coacervate domain of the first component and the additional component(s) on the surface of the substrate is in the form of a single layer coacervate domain, a multilayered coacervate domain, a blended alloy coacervate domain, or combinations thereof. In some embodiments, the method further comprises stabilizing at least an outermost coacervate domain on the surface of the substrate by one or a combination of mineralization or formation of cross-links by one or a combination of covalent coordination, ionic interaction, disulfide bonds, or hydrogen bonds, wherein the outermost coacervate domain remains consolidated upon cessation of stimulation at the phase separation point for the outermost coacervate domain. In some embodiments, the substrate includes one or more of a medical device, a stent, a vascular graft, a catheter, a biosensor, a drug reservoir or a cell culture substrate. In some embodiments, the population of droplets are aqueous and the solution further includes one or a combination of a cell, a virus, or a nanoparticle having a coating of at least one component to cause recruitment of the coated cell, virus, or nanoparticle to the coacervate domain of the respective component within each of the droplets. In some embodiments, one or more of the components has an attached bioactive agent, wherein the bioactive agent includes one or a combination of: a drug, a protein, a peptide, a peptide hormone, a ligand, a cell-signaling ligand, or an RGD cell binding domain, to cause recruitment of the drug, protein, peptide, peptide hormone, ligand, cell-signaling ligand, or RGD cell binding domain to the coacervate domain of the component within each of the droplets. In some embodiments, at least two components have the attached bioactive agent. In some embodiments, the one or more components is a polypeptide and the bioactive agent is attached through an amino acid linkage or through a chemical linkage through a reactive peptide residue. In some embodiments, the one or more polypeptides attached to the bioactive agent includes a protease cleavage site.

[0006]In some embodiments, the presently disclosed subject matter is directed to a method for coating a substrate, the method comprising: stimulating a solution of one or a mixture of components, wherein a substrate is immersed within the solution, wherein the stimulation induces a phase separation point of a first component; maintaining stimulation at the phase separation point to form a degree of a coacervate domain of the first component on a surface of the substrate based on a wetting property of the substrate; and repeating the stimulating and maintaining for one or more additional components in the mixture to form a coacervate domain of the additional component. In some embodiments, the solution is aqueous. In some embodiments, the first component and the additional component(s) include polymers. In some embodiments, the polymers include polypeptides. In some embodiments, the polypeptides include at least a portion of an elastin-like polypeptide (ELP). In some embodiments, the first component and the additional component(s) have similar phase separation points and a blended alloy coacervate domain is formed on the surface of the substrate. In some embodiments, the coacervate domain of the first component and the coacervate domain(s) of the additional component(s) on the surface of the substrate is in the form of a single layer coacervate domain, a multilayered coacervate domain, a blended alloy coacervate domain, or combinations thereof. In some embodiments, a molecule is grafted on the surface of the substrate, and the degree of formation of the coacervate domain of the first component and the additional component(s) on the surface of the substrate is controlled by one or both a level of interaction of one or both of the first component and the additional component(s) with the molecule and the wetting property of the substrate. In some embodiments, the method further comprises stabilizing at least an outermost coacervate domain on the surface of the substrate by one or a combination of mineralization or formation of cross-links by one or a combination of covalent coordination, ionic interaction, disulfide bonds, or hydrogen bonds, wherein the outermost coacervate domain remains consolidated upon cessation of stimulation at the phase separation point for the outermost coacervate domain. In some embodiments, the substrate includes one or more of a medical device, a stent, a vascular graft, a catheter, a biosensor, a drug reservoir, or a cell culture substrate. In some embodiments, the solution further includes one or a combination of a cell, a virus, or a nanoparticle, and wherein the cell, virus, or nanoparticle includes a coating of at least one of the components to cause recruitment of the cell, virus, or nanoparticle to the coacervate domain of the respective component. In some embodiments, one or more of the components has an attached bioactive agent, wherein the bioactive agent includes one or a combination of: a drug, a protein, a peptide, a peptide hormone, a ligand, a cell-signaling ligand, or an RGD cell binding domain, to cause recruitment of the drug, protein, peptide, peptide hormone, ligand, cell-signaling ligand, or RGD cell binding domain to the coacervate domain of the respective component. In some embodiments, at least two of the components have the attached bioactive agent. In some embodiments, the one or more components is a polypeptide and the bioactive agent is attached through an amino acid linkage or through a chemical linkage through a reactive peptide residue. In some embodiments, the one or more polypeptides attached to the bioactive agent includes a protease cleavage site. In some embodiments, the first phase separation point and the additional phase separation point(s) are each a phase separation temperature, and the stimulus includes heating. In some embodiments, the one or a mixture of components includes: a polymer, a synthetic polymer, a hydrophilic polymer, a hydrophobic polymer, an amphiphilic polymer, an amphiphilic diblock polymer, a protein, a nucleic acid, an epoxy, or a polysaccharide, and combinations thereof. In some embodiments, the stimulating includes: addition or removal of one or more of the components, evaporation of the solution, controlled diffusion of one or more of the components, electrostatic quenching of one or more of the components, inducing a reaction of one or more of the components, crosslinking of one or more of the components, isomerization of one or more of the components, or crystallization of one or more of the components, and combinations thereof.

[0007]In some embodiments, the presently disclosed subject matter is directed to a coated substrate produced by a process comprising: stimulating a solution of one or a mixture of components, wherein a substrate is immersed within the solution, wherein the stimulation induces a phase separation point of a first component; maintaining stimulation at the phase separation point to form a degree of a coacervate domain of the first component on a surface of the substrate based on a wetting property of the substrate; and repeating the stimulating and maintaining for one or more additional components in the mixture to form a coacervate domain of the additional component. In some embodiments, the process further comprises stabilizing at least an outermost coacervate domain on the surface of the substrate by one or a combination of mineralization or formation of cross-links by one or a combination of covalent coordination, ionic interaction, disulfide bonds, or hydrogen bonds, wherein the outermost coacervate domain remains consolidated upon cessation of stimulation at the phase separation point for the outermost coacervate domain. In some embodiments, the substrate includes one or more of a medical device, a stent, a vascular graft, a catheter, a biosensor, a drug reservoir or a cell culture substrate.

[0008]In some embodiments, the presently disclosed subject matter is directed to a coated substrate produced by a process comprising: stimulating a population of aqueous droplets in the form of a water-in-oil emulsion, wherein the droplets include a solution of one or a mixture of components, wherein a substrate is immersed within the population of aqueous droplets, and wherein the stimulation induces a phase separation point of a first component; maintaining stimulation at the phase separation point to form a tunable degree of a coacervate domain of the first component on a surface of the substrate based on a wetting property of the substrate; and repeating the stimulating and maintaining for one or more additional components in the mixture to form a coacervate domain of the additional component. In some embodiments, the process further comprises stabilizing at least an outermost coacervate domain on the surface of the substrate by one or a combination of mineralization or formation of cross-links by one or a combination of covalent coordination, ionic interaction, disulfide bonds, or hydrogen bonds, wherein the outermost coacervate domain remains consolidated upon cessation of stimulation at the phase separation point for the outermost coacervate domain. In some embodiments, the substrate includes one or more of a medical device, a stent, a vascular graft, a catheter, a drug reservoir, a biosensor, or a cell culture substrate.

[0009]In some embodiments, the presently disclosed subject matter is directed to a nano- to microscale liquid coacervate particle composition produced by a process comprising: stimulating a population of droplets including a solution of one or a mixture of components, wherein the stimulation induces a phase separation point of a first component; maintaining stimulation at the phase separation point to form a coacervate domain of the first component within each of the droplets, wherein liquid coacervate particles are formed; and optionally repeating the stimulating and maintaining for the one or more additional components in the mixture to form a coacervate domain of the additional component within each of the droplets. In some embodiments, the process further comprises stabilizing at least an outermost coacervate domain within each of the droplets to form capsule structures, wherein the outermost coacervate domain remains consolidated upon cessation of stimulation at the phase separation point for the outermost coacervate domain. In some embodiments, stabilizing includes formation of cross-links by one or a combination of covalent coordination, ionic interaction, disulfide bonds, or hydrogen bonds. In some embodiments, the first component and the additional components include polymers. In some embodiments, the polymers include polypeptides. In some embodiments, the polypeptides include at least a portion of an elastin-like polypeptide (ELP). In some embodiments, the first component and the additional component(s) have similar phase separation points and a blended alloy coacervate domain is formed. In some embodiments, the coacervate domain of the first component and the coacervate domain(s) of the additional component(s) form a multilayered coacervate domain, a blended alloy coacervate domain, or a combination thereof. In some embodiments, the first phase separation point and the additional phase separation point(s) are each a phase separation temperature, and the stimulus includes heating. In some embodiments, the population of droplets are aqueous droplets. In some embodiments, the solution includes at least the first component and a surfactant for controlling a size of the coacervate domain. In some embodiments, the population of droplets are aqueous droplets and the surfactant includes an amphiphilic diblock polymer. In some embodiments, the first component is a hydrophobic ELP polymer and the amphiphilic diblock polymer is an ELP diblock polymer. In some embodiments, a ratio of the hydrophobic ELP polymer to the amphiphilic ELP diblock polymer ranges from about 1:1 to about 50:1, and a size of an outermost coacervate domain ranges from about 50 nm to about 20 μm. In some embodiments, the population of droplets are aqueous and the solution further includes one or a combination of a cell, a virus, or a nanoparticle having a coating of at least one component to cause recruitment of the coated cell, virus, or nanoparticle to the coacervate domain of the respective component within each of the droplets. In some embodiments, one or more of the components has an attached bioactive agent, wherein the bioactive agent includes one or a combination of: a drug, a protein, a peptide, a peptide hormone, a ligand, a cell-signaling ligand, or an RGD cell binding domain, to cause recruitment of the drug, protein, peptide, peptide hormone, ligand, cell-signaling ligand, or RGD cell binding domain to the coacervate domain of the respective component within each of the droplets. In some embodiments, at least two components have the attached bioactive agent. In some embodiments, the one or more components is a polypeptide and the bioactive agent is attached through an amino acid linkage or through a chemical linkage through a reactive peptide residue. In some embodiments, the one or more polypeptides attached to the bioactive agent includes a protease cleavage site.

Problems solved by technology

Unfortunately, a broad platform for the in vitro programming of complex and hierarchical multi-phase structures has remained elusive.

However, these approaches have been limited to varying extents by: the need for complex and specialized fluidic devices, low fabrication throughput, limitations in achievable particle size, and constraints on material components due to assembly requirements.

Thus, both current microfluidic and existing bulk techniques for fabrication of hierarchical liquid-liquid, gel and particle systems are severely lacking in scalability, size control, ease of fabrication, and morphological diversity.

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