U.S. patent application number 15/487543 was filed with the patent office on 2017-08-03 for programmable liquid, gel and biohybrid compartments and methods of use.
The applicant listed for this patent is Duke University. Invention is credited to Nick J. Carroll, Ashutosh Chilkoti, Gabriel P. Lopez, Joseph R. Simon.
Application Number | 20170216808 15/487543 |
Document ID | / |
Family ID | 55747376 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170216808 |
Kind Code |
A1 |
Lopez; Gabriel P. ; et
al. |
August 3, 2017 |
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.
Inventors: |
Lopez; Gabriel P.; (Durham,
NC) ; Simon; Joseph R.; (Durham, NC) ;
Carroll; Nick J.; (Durham, NC) ; Chilkoti;
Ashutosh; (Durham, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Duke University |
Durham |
NC |
US |
|
|
Family ID: |
55747376 |
Appl. No.: |
15/487543 |
Filed: |
April 14, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US15/55836 |
Oct 15, 2015 |
|
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15487543 |
|
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62064057 |
Oct 15, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 29/085 20130101;
A61L 31/16 20130101; A61K 9/146 20130101; C12M 23/20 20130101; B01J
2/00 20130101; B01J 2/06 20130101; B01J 13/06 20130101; C07K 19/00
20130101; A61K 38/39 20130101; A61L 27/34 20130101; A61L 2300/62
20130101; A61K 38/00 20130101; C07K 14/78 20130101; A61L 27/54
20130101; A61L 31/10 20130101 |
International
Class: |
B01J 13/06 20060101
B01J013/06; C12M 1/00 20060101 C12M001/00; A61L 31/10 20060101
A61L031/10; A61L 29/08 20060101 A61L029/08; A61K 9/14 20060101
A61K009/14; A61L 27/34 20060101 A61L027/34 |
Goverment Interests
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.
Claims
1. 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.
2. The method of claim 1, wherein the population of droplets are
formed using one or a combination of mechanical agitation,
sonication, or microfluidics.
3. The method of claim 1, wherein the population of droplets are
aqueous droplets formed by sonication of the solution in oil or
microfluidics of the solution in oil.
4. The method of claim 1, wherein the liquid coacervate particles
are reversibly formed by cessation of stimulation followed by
re-stimulation and re-maintaining stimulation.
5. The method of claim 1, wherein the first component includes a
polymer.
6. The method of claim 5, wherein the polymer includes a
polypeptide.
7. The method of claim 6, wherein the polypeptide includes at least
a portion of an elastin-like polypeptide (ELP).
8. The method of claim 1, wherein 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.
9. The method of claim 1, wherein 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.
10. 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.
11. The method of claim 10, wherein the solution is aqueous.
12. The method of claim 10, wherein the first component and the
additional component(s) include polymers.
13. The method of claim 12, wherein the polymers include
polypeptides.
14. The method of claim 13, wherein the polypeptides include at
least a portion of an elastin-like polypeptide (ELP).
15. The method of claim 10, wherein 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.
16. The method of claim 10, wherein 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.
17. 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.
18. The liquid coacervate particle of claim 17, wherein 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.
19. The liquid coacervate particle of claim 18, wherein stabilizing
includes formation of cross-links by one or a combination of
covalent coordination, ionic interaction, disulfide bonds, or
hydrogen bonds.
20. The liquid coacervate particle of claim 17, wherein the first
component and the additional components include polymers.
21. The liquid coacervate particle of claim 20, wherein the
polymers include polypeptides.
22. The liquid coacervate particle of claim 21, wherein the
polypeptides include at least a portion of an elastin-like
polypeptide (ELP).
23. The liquid coacervate particle of claim 17, wherein the first
component and the additional component(s) have similar phase
separation points and a blended alloy coacervate domain is
formed.
24. The liquid coacervate particle of claim 17, wherein the
population of droplets are aqueous droplets.
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.
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
remained elusive. Techniques such as layer-by-layer deposition,
molecular self-assembly, and microfluidic emulsion templating
enable a high degree of control over the layering of distinct
liquid or polymeric phases. 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.
SUMMARY OF THE DISCLOSURE
[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 .mu.m. 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 .mu.m. 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The foregoing aspects and other features of the disclosure
are explained in the following description, taken in connection
with the accompanying drawings, wherein:
[0011] FIG. 1A depicts droplets containing multiple fluorescently
labeled components according to at least one embodiment of the
present disclosure;
[0012] FIG. 1B depicts a schematic representing mixtures of up to
three components in a solution according to at least one embodiment
of the present disclosure;
[0013] FIG. 1C depicts nanoscale, mesoscale and microscale
coacervate domains evolved from droplets containing mixtures of
components according to at least one embodiment of the present
disclosure;
[0014] FIG. 2 depicts time-lapse darkfield microscope images of
thermally-induced spinodal decomposition of polymer within droplets
according to at least one embodiment of the present disclosure;
[0015] FIGS. 3A-3O depict spinodal decomposition of multiple ELP
components for formation of blended and multilayered microparticles
according to at least one embodiment of the present disclosure;
[0016] FIG. 4 depicts the formation of multi-liquid phase
coacervate domains and functionalized biomaterials according to at
least one embodiment of the present disclosure;
[0017] FIGS. 5A-5P depicts nano-, meso- and microparticles formed
by arrested coarsening according to at least one embodiment of the
present disclosure;
[0018] FIG. 6 depicts isothermal and nonisothermal paths from
one-phase region to two-phase region on a phase diagram via droplet
solvent evaporation according to at least one embodiment of the
present disclosure;
[0019] FIGS. 7A-7F depicts structures formed by the droplet
evaporation method according to at least one embodiment of the
present disclosure;
[0020] FIG. 8 depicts a schematic illustration of multilayer
polymer coacervate coatings according to at least one embodiment of
the present disclosure.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0021] For the purposes of promoting an understanding of the
principles of the present disclosure, reference will now be made to
preferred embodiments and specific language will be used to
describe the same. It will nevertheless be understood that no
limitation of the scope of the disclosure is thereby intended, such
alteration and further modifications of the disclosure as
illustrated herein, being contemplated as would normally occur to
one skilled in the art to which the disclosure relates.
[0022] Articles "a" and "an" are used herein to refer to one or to
more than one (i.e. at least one) of the grammatical object of the
article. By way of example, "an element" means at least one element
and can include more than one element.
[0023] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this disclosure belongs.
[0024] The presently disclosed invention provides the ability to
program the self-assembly of hierarchical liquid compartments
comprised of polymers from solutions and suspensions by encoding
polymer phase transition behavior and surface interactions at the
building block level. The ability to encode information needed for
programmable self-assembly into complex patterns is a hallmark of
biological materials and emulating these features allows for the
formation of a variety of practically useful materials. The
inventions of the present disclosure advantageously affords the
diversity to program phase separation events in liquid structures
spanning the nano- to micro-scale. The present disclosure is valid
for both synthetic and biological materials. The inventions are
demonstrated in the examples infra, which include stimulus-induced
self-assembly of phase-transitioned aqueous architectures comprised
of disordered proteins engineered via recombinant genetics. The
inventions disclosed herein enable exquisite control of polymeric
building blocks through the tailoring of structure, molecular
weight and composition at the individual amino acid (monomer)
level. The self-assembled nano, meso, micro and macro structures
may further find utility in drug delivery, bioanalytical systems,
controlled cell culture, tissue engineering, biomanufacturing and
drug discovery.
[0025] The presently disclosed invention provides the ability to
program the self-assembly of hierarchical liquid coacervate
particles. In one embodiment a method is provided for making nano-
to microscale liquid coacervate particles. The method includes
stimulating a population of droplets having a solution of one or a
mixture of components to induce a phase separation point of a first
component. By maintaining stimulation at the phase separation
point, a coacervate domain of the first component within each of
the droplets can be formed. Liquid coacervate particles are formed
through maintaining stimulation of the droplet containing one
component or a mixture of components.
[0026] The solution can include one or more additional components
each having an additional phase separation point. The method can
include further stimulating of the population of aqueous droplets
to induce a phase separation point of the additional components and
maintaining stimulation at the additional phase separation point to
form a coacervate domain of the additional component within each of
the droplets. Optionally, repeating the stimulating and the
maintaining for one or more additional components can be performed
to form additional coacervate domains within the droplets. Examples
of the liquid coacervate particles of the invention are shown in
FIGS. 1-6.
[0027] The formation of multiple all-aqueous protein-rich
compartments with distinct boundaries was directed by programming
the phase behavior of thermally-responsive elastin-like polypeptide
(ELP) components (FIG. 1B) within the confinement of water
microdroplets (FIG. 1A). This approach enables reversibly
fabrication of (1) single- and multi-modal population protein
compartments spanning the nano-, meso-, and micro-scale and (2)
multilayered micro-architectures (FIG. 1C) by controlling phase
behavior via simple modification of the ELP components. FIG. 1A
depicts droplets containing multiple fluorescently labeled ELP
polymers. FIG. 1B depicts a schematic representing mixtures of up
to three polymers in a solution. FIG. 1C depicts nanoscale,
mesoscale and microscale particles in the top portion, and
multilayered microscale particles in the bottom portion, evolved
from homogeneous droplets containing mixtures of ELP polymers by
controlled phase separation and wetting.
[0028] ELPs are a class of disordered, stimuli responsive protein
polymers based on the repetitive elastin-based motif
Val-Pro-Gly-Xaa-Gly (SEQ ID NO: 1), where Xaa is a tailorable guest
residue. The hallmark characteristic of these polymers is their
lower critical solution temperature (LCST) phase transition
behavior, where above the cloud point transition temperature (T sub
t) they phase separate to form polymer rich coacervate domains. For
this demonstration, the strategy centers on heating through the
solution coexistence boundary into the unstable region of the
miscibility gap to direct phase transition via spinodal
decomposition; here, spontaneous amplification of concentration
fluctuations results in rapid formation and coarsening of
protein-rich domains that are separated from a water-rich phase
(FIG. 2).
[0029] FIG. 2 depicts time-lapse darkfield microscope images of
thermally-induced spinodal decomposition of polymer within water
drops. Phase separated microdomains coarsen with time to form a
liquid-liquid structure comprised of a polymer-rich coacervate
(dark spherical center) surrounded by a water-rich layer. Within
the confinement of microdroplets, spinodal decomposition provides a
powerful approach for building compartmentalized liquid-liquid
structures through the modulation of polymeric component phase
behavior. It should be noted that other viable phase separation
mechanisms include: nucleation and growth (metastable region);
multi-component mixing-driven phase separation via component
addition or removal, solvent evaporation, and/or controlled
diffusion; electrostatic quenching; reaction-induced; crosslinking;
and crystallization.
[0030] In one embodiment, a nano- to microscale liquid coacervate
particle composition is provided that is produced by a process
including 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. In this
manner liquid coacervate particles are formed. Optionally, the
method includes 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.
[0031] The population of droplets can be formed using one or a
combination of mechanical agitation, sonication, or microfluidics.
The population of droplets can be formed by sonication of a
solution in oil or microfluidics of a solution in oil. The
population of droplets can be aqueous. The aqueous droplets can be
in the form of a water-in-oil emulsion.
[0032] The liquid coacervate particles can be reversibly formed by
cessation of stimulation followed by re-stimulation and
re-maintaining the stimulation. Such methods can be useful as
bioreactors or for temporarily sequestering reactants.
[0033] The programming of hierarchical aqueous polymeric layers
immiscible not only with the surrounding water solvent, but also
orthogonal to other co-existing polymeric phases is demonstrated.
The results reveal the phase tunability of polymer mixtures through
simple tailoring of the monomer (amino acid) sequence and molecular
weight. Five different homopolymers (labeled U1 through U5) were
used, each with distinct phase diagrams. FIGS. 3A-3F depict
spinodal decomposition of multiple ELP components for formation of
blended and multilayered microparticles. FIGS. 3A-3C depicts
temperature-controlled spectrophotometry of bulk ELP solutions. The
bulk phase transition temperatures of various homopolymer mixtures
are characterized using spectrophotometry (FIGS. 3A-3C). A mixture
of U3 and U4 shows two independent spinodal decomposition events,
as depicted in FIG. 3A. A mixture of U1, U2, and U3 shows three
independent spinodal decomposition events, as depicted in FIG. 3B.
A mixture of U2 and U5 shows a single spinodal decomposition event,
as depicted in FIG. 3C.
[0034] When the cloud point temperatures of different components
within a homopolymer mixture are sufficiently separated from one
another, observe multiple phase separation events were observed
(FIGS. 3A and 3B, solid curves) that correspond to the phase
separation events of their respective individual components (dashed
curves). Conversely, simultaneous phase separation (FIG. 3C, solid
curve) of two ELP homopolymers were observed in a mixture if the
transition temperature of their individual components is similar
(dashed curves). By carefully traversing these multicomponent phase
diagrams it was possible to controllably form either (1)
multilayered or (2) blended `alloy` coacervates via discretized
heating and selectively inducing various phase separation events as
predetermined in bulk.
[0035] FIGS. 3D-3O depict fluorescent microscope images of
hierarchically structured microparticles. Double-layered
coacervates made of U4 (lightest coacervate in far-right image) and
U3 (larger coacervate encapsulating U4 in far-right image) are
depicted in FIGS. 3D-3G. Triple-layered coacervates made of U1
(light coacervate of middle-left image), U2 (coacervate of
middle-right image encapsulating U1), and U3 (coacervate of
far-right image encapsulating U2) are depicted in FIGS. 3H-3K.
Alloy coacervates made of U2 and U5 are depicted in FIGS. 3L-3O.
The inset images of FIGS. 3D-3O depict fluorescent channel(s) of
ELPs that have and/or are phase separating. The double-layer (FIGS.
3D-3G) and triple-layer (FIGS. 3H-3K) protein coacervates were
assembled by exploiting the effects of micro-confinement on the
phase separation process. By encapsulating mixtures of homopolymers
inside droplets and subsequently heating in a step-wise fashion,
the unstable region of the phase diagram for each successive
increasingly hydrophilic protein component was targeted; the system
is held above each transition temperature until phase separation of
the target protein proceeds to completion. Subsequent phase
separation events in this manner result in the formation of n
coacervate layers, where n is the number of polymeric components
inside the droplets with dissimilar phase diagrams. Only one
polymer type populates each phase-transitioned layer.
[0036] The formation of wetting architectures is described by
component interfacial tensions (.gamma. sub i/j) and the spreading
parameter (S):
S=.gamma.sub(IP/Water)-(.gamma.sub(IP/MP)+.gamma.sub(MP/Water)).
A positive spreading parameter (S>0) results in the formation of
wetting layers, which is due to a high degree (.gtoreq.80% by
composition) of chemical homology between the protein polymers. By
contrast, a negative spreading parameter (S<0) will result in
Janus structures. Thus, interfacial energies and the accompanying
spreading behavior are a key module for structural design with the
method presented herein.
[0037] In contrast to distinct layered liquid phases, protein
coacervate `alloy` blends were formed when the phase transition is
initiated for a binary mixture comprised of two different polymers
with different chemical composition but with similar phase
transition temperatures, as evidenced by inspection of FIGS. 3L-3O.
Thus, both physical and chemical properties can be engineered into
each liquid phase layer by tailoring the system phase behavior at
the building block level.
The types and spatiotemporal distributions of signaling molecules
detected by target cells in extracellular regulation are the
subject of extensive investigations. Thus, there is a growing need
for creating diverse extracellular matrices (ECMs) from an array of
programmable biologically functional building blocks, with
versatile modular design. The method of manipulating polymer
assembly can allow (i) high-throughput encapsulation of cells,
cellular components (e.g., organelles) or viruses within
biocompatible (e.g., polypeptide) gel microarrays, (ii) capturing
cells, cellular components (e.g., organelles) or viruses within
tailorable layers of single population or `alloy` blends of gels,
each with distinct intrinsic physicochemical functionalization,
(iii) capturing cells, cellular components (e.g., organelles) or
viruses within capsule-like structures with modular core and shell
properties, (iv) spatial organization of immobilized cell-signaling
ligands and within the gel matrix, (v) tailoring of gel ECM
mechanical properties, (vi) integration of degradable (e.g.,
protease-cleavable) linkages, and (vii) combinations thereof (shown
schematically in FIG. 4). In FIG. 4, the dotted triangle represents
integrin ligands (recreate cell-cell and cell-ECM interactions),
the black triangle represents immobilized soluble biomolecules
(growth factors, cytokines), the white triangle represents
recapitulated cell-cell interactions (ephrins, cadherins, CAMs),
and X represents protease sensitive degradation sites. The
flexibility of these methods can be taken advantage of for forming
multi-liquid phase compartments to create functionalized
biomaterials for ECM assembly that have potential for direct
translation to regenerative medicine, cell-culture, cell-based
therapies, drug discovery, drug delivery, and tissue engineering
related markets.
[0038] The specific characteristics of phase transitioned
liquid-liquid phases depend strongly on molecular and surface
interactions. Validating this was possible by utilizing protein
diblock surfactants (called D1) to self-assemble populations of
uniform polymer-rich compartments with controllable size. FIGS.
5A-5P depict nano-, meso- and microparticles formed by arrested
coarsening. FIGS. 5M-5N depicts a schematic showing sequences of
the ELP diblock (FIG. 5M) and U1 uniblock (FIG. 5N). The ELP
diblocks consist of two adjoined blocks of dissimilar amino acid
repeats. The amphiphilic nature of these diblock polypeptides
enables the temperature-triggered self-assembly of nanoscale
micelles (D.apprxeq.56 nm) when D1 is the lone component. FIGS.
5A-5D depicts fluorescent microscope images of water drops
containing various ratios of darker-labeled ELP diblock and
lighter-labeled U1. The diblock arrests coarsening of
phase-separated U1 uniblock domains by acting as a
surface-stabilizing agent. In the case of a homopolymer/diblock
mixture, phase-separated polypeptide compartments that span the
nano-, meso-, and micro-scale (FIGS. 5M-5N) were fabricated by
simply modulating the stoichiometric ratio of the components. Upon
heating the mixture into the unstable region of the homopolymer
phase diagram, the homopolymer begins to phase separate via
spinodal decomposition. Coarsening of the polymeric-rich liquid
domains is arrested at a specific size by the stabilizing protein
amphiphiles; the protein compartment size is a function of diblock
quantity. This strategy provides access to the generally
less-accessible meso-scale and offers a mechanism for the
fabrication of particles spanning multiple length scales via
programmed phase separation.
[0039] The resultant protein compartments have a narrow size
distribution (FIGS. 5I-5L), presumably due to mass conservation and
molecular packing. FIGS. 5I-5L depict a schematic of the
polypeptide compartments formed by varying U1/diblock ratios. The
diameters of the resultant protein phase puncta are: (U1 to diblock
ratio in parentheses): 56.+-.3 nm (0:1), 625.+-.34 nm (1:1),
3.+-.0.6 .mu.m (5:1), and 17.+-.1 .mu.m (50:1). Furthermore, these
phase-transitioned domains do not experience appreciable
precipitation and are kinetically stable for up to 24 hours.
[0040] Biomaterial can be fabricated by covalently crosslinking the
coarsen-arrested protein microcompartments. Multi-modal populations
of protein-rich water compartments can be fabricated by
supplementing the aqueous precursor solution with a second
population of U2 homopolymer.
[0041] By simply changing the stoichiometric ratios of mixtures U1,
U2, and D1 two distinct, orthogonally phase-separated populations
of similar-sized microcompartments were created (FIG. 5O) and a
single coacervate compartment coexisting with numerous smaller
compartments (FIG. 5P); this type of multi-component protein
compartmentalization can be useful for recreating complex
non-membrane bound intracellular microenvironments through
controlled segregation of biomacromolecules. FIGS. 5O-5P depict
fluorescent microscope images of distinct uniblock polypeptide
domains. FIG. 5O consists of a bimodal population of U1 (top-left
inset) and U2 (top-middle inset) microcompartments with ratio of
total uniblock to diblock (top-right inset) of 5:1. FIG. 5P depicts
fully coarsened U1 coacervate (top-left inset) surrounded by U2
microcompartments (top-middle inset) with ratio of total uniblock
to diblock (top-right inset) of 50:1. Top panels of FIGS. 5O and 5P
show U1 (left), U2 (middle) and D1 (right) fluorescent channels;
bottom panels of FIGS. 5O and 5P are overlay images.
[0042] This technology can enable one-pot fabrication of complex
hierarchical biological and synthetic particle architectures
through preprogrammed phase separation and self-assembly. The
ability to evolve complex structures is in contrast to microfluidic
or layer-by-layer methods, where each layer or phase is added in
sequential steps; thus, massive scale up in production, size
control, and reduced fabrication costs is facilitated by the
technology. Additionally, phase separated low-surface energy water
compartments, crowded with biomolecules, mimics non-membrane bound
organelles within the cytoplasm and nucleoplasm of cells. Thus, the
system enables an avenue for a more thorough understanding of the
mechanisms of microdomain formation for the broad implementation
of: tissue engineering pathways through cell encapsulation, protein
microdomains as intra- and extracellular switches, protocells as
bioreactors, drug delivery vehicles and depots, and fundamental
studies of biological macromolecule assembly into condensed phases.
Within this paradigm, other materials include: hydrophilic and
hydrophobic liquid monomers; synthetic polymers; protein and
nucleic acid components; water/polymer and water/monomer mixtures;
epoxies; non-polymeric liquids; cells; organelles; viruses;
polysaccharides; polymer/monomer combinations; and mixtures
thereof.
[0043] FIG. 6 depicts isothermal (top arrow) and nonisothermal
(bottom arrow) paths from one-phase region to two-phase region on a
phase diagram via droplet solvent evaporation.
[0044] In another embodiment, methods are provided for coating a
substrate. The substrate can include one or more of a medical
device, a stent, a vascular graft, a catheter, a biosensor, a drug
reservoir, or a cell culture substrate. Examples of coating of
substrates are shown in FIGS. 7-8.
[0045] FIGS. 7A-7F depicts structures formed by the droplet
evaporation method, including non-wetting (FIG. 7A), partially
wetting (FIG. 7B) and fully wetting (FIG. 7C) phase-transitioned
coacervates on a substrate surface. Additional structures can
include multilayer compartments (FIG. 7D), and micelles or other
particles (FIG. 7E) and cells (FIG. 7F) encapsulated within the
resultant coacervates.
[0046] The approach is not limited to creating hierarchical liquid
compartments within emulsions using temperature-induced phase
separation. The platform is amenable to evolving the
phase-separated structures presented herein within surface-bound
drops via controlled evaporation. The strategy is to control
evaporation of solvent (e.g., water) from a multicomponent droplet
to move across the phase diagram from the one-phase region to the
two phase region, as shown schematically in FIG. 6. This
methodology obviates the requirement of an external continuous
fluid (e.g., oil) and provides modulated control of the components
via extent of solvent evaporation. The top arrow in FIG. 6
represents the ideal horizontal isothermal path across the phase
diagram into the two-phase region. Droplet cooling may occur due to
evaporation; the bottom arrow in FIG. 6 represents a pathway
whereby phase separation occurs via a non-isothermal evaporative
process. In both examples, coacervate formation is expected to
occur because the final state of the system falls within the
two-phase region of the phase diagram.
[0047] The surface chemistry and topography of the substrate is key
in determining the properties of the resultant phase-transitioned
coacervates. For example, if the coacervate does not wet the
surface preferentially to water, a spherical coacervate to form
within the drop is expected, as illustrated in FIG. 7A. Surface
coatings that can result in this non-wetting behavior include, but
are not limited to: PEG-silane, PEG-thiols on gold, POEGMA brushes,
and zwitterionic surfaces. In the case of a coacervate contact
angle between 0.degree. and 180.degree. with respect to water, the
coacervate can achieve formation of a partial sphere (FIG. 7B). If
the coacervate completely wets the surface, a uniform film may form
at the bottom of the drop upon coacervation (FIG. 7C); this film
could be attained by tethering polymers or oligomerss to the
surface expressing the same (or similar) physical properties to
those in the coacervate. The approach also applies to hierarchical
structures evolved within the drops. This fabrication strategy can
easily be extended to multilayer compartments (FIG. 7D),
coacervates encapsulating micelles and other nano- to
micro-particles (FIG. 7E), and cell, organelle or virus
encapsulation (FIG. 7F) within the formed coacervates.
[0048] Other examples whereby the concepts, methods and materials
described supra can be used include the formation of multilayer,
blend or micro- and nanostructured thin film coatings on surfaces
of solid materials by tailoring the phase behavior and surface
interactions of polymers. Of particular interest is the formation
of such complex coatings from peptide polymers and other bioactive
polymers, which may be tailored to have a number of desirable
biological properties for biomedical applications.
[0049] Formation of liquid coacervate phases in the form of thin
films (coatings) on a desired surface can be accomplished using any
of the means described above (temperature change, evaporation,
change in solvent quality (e.g. by addition of cosolutes) by
placing the surface (e.g. biomedical device) of interest to be
coated into a homogeneous solution of the polymer mixtures either
before or while phase separation (coacervation) is occurring. FIG.
8 depicts a schematic illustration of multilayer polymer coacervate
coatings. In this example, tailoring n constituent phase
transitions in multicomponent systems enables formation of n
immiscible wetting layers (n=3 above). In other examples, each
layer can consist of a single component, immiscible with other
components or can consist of blends, where multiple chemically
heterogeneous components are tuned to be miscible in the same
layer, and combinations thereof.
[0050] Morphology of the coating on the surface depends on the
sequence of selective coacervation (phase separation) of polymeric
components in solution onto the surface of interest, and the
wetting behavior of the phase separating polymer on that surface.
In analogy to the structures depicted in FIGS. 7A-7C, wetting can
determine the lateral morphology of the phase separating layers, so
that structures can range from uniformly spread multilayers to
poorly spread layers that display a complex topography.
[0051] Examples of the types of biomedical devices that can be
coated include stents, vascular grafts, catheters, biosensors, cell
culture substrata or any material or device for which a
biofunctional coating comprising the polymer layer is desired.
[0052] Examples of the types of bioactive function for these
coatings include, drug delivery, drug elution, cell encapsulation,
biosensing, diagnostics, theranostics, permselective membranes,
lubrication layers, and templates for biomineralization of other
forms of materials self-assembly.
[0053] After (or during) formation of the polymer layer by
selective coacervation and controlled wetting, it may be necessary
to stabilize them by crosslinking, mineralization or otherwise
encapsulating them. Crosslinking may be by covalent, coordination,
ionic, disulfide and hydrogen bonding for example.
[0054] One method for coating a substrate includes stimulating a
solution having one or a mixture of components, wherein the
substrate is immersed within the solution, and the stimulation
induces a phase separation point of a first component. The
stimulation is maintained at the phase separation point to form a
degree of a coacervate domain of the first component on a surface
of the substrate. The degree of coacervate domain formed on the
surface is based on the wetting property of the substrate. The
stimulating and maintaining is repeated for one or more additional
components in the mixture to form a coacervate domain of the
additional component(s).
[0055] Another method for coating a substrate includes stimulating
a population of aqueous droplets in the form of a water-in-oil
emulsion where the droplets include a solution of one or a mixture
of components. The substrate is immersed within the population of
aqueous droplets. Stimulation of the droplets induces a phase
separation point of a first component and maintaining stimulation
at the phase separation point forms a tunable degree of a
coacervate domain of the first component on a surface of the
substrate. The tunable degree of the coacervate domain that is
formed on the surface is based on the wetting property of the
substrate. The stimulating and maintaining is repeated for one or
more additional components in the mixture to form a coacervate
domain of the additional component(s).
[0056] The coated substrates, formed by the methods described
herein, can include a grafted molecule present on the surface of
the substrate. In this case, the degree of formation of the
coacervate domain of the first component, and/or additional
components, on the surface of the substrate can be controlled by
one or both of the level of interaction of the respective component
with the molecule and with the wetting property of the
substrate.
[0057] The coacervate domain of the first component and the
additional component(s) on the surface of the substrate can be in
the form of a single layer coacervate domain, a multilayered
coacervate domain, a blended alloy coacervate domain, or
combinations thereof.
[0058] In one embodiment, a coated substrate is provided that is
produced by a process including 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.
[0059] In one embodiment, a coated substrate is provided that is
produced by a process including 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.
[0060] With respect to the methods and processes described herein
for formation of nano- to microscale liquid coacervate particles
and coated substrates, the methods and processes of formation can
further include stabilizing at least an outermost coacervate domain
of the droplet or of the outermost coacervate domain on the surface
of the substrate. Stabilization can be effected 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. After stabilization of the
outermoset coacervate domain of the liquid droplet, this domain
remains consolidated upon cessation of stimulation at the phase
separation point for this coacervate domain, and capsule structures
are formed. After stabilization of the outermoset coacervate domain
on the surface of the substrate, this domain remains consolidated
upon cessation of stimulation at the phase separation point for
this coacervate domain. In addition to the outermost coacervate
domains, one or more of the additional coacervate domains can be
stabilized in the droplets and on the substrate surfaces. After
stabilization, the coacervate domain(s) can remain consolidated
upon cessation of stimulation at the respective phase separation
point of the respective component(s) of the domain.
[0061] The components of the present methods and processes can
include, for example, but are not limited to, 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. The component can include a polymer. The polymer can
include a polypeptide. The polypeptide can include at least a
portion of an ELP elastin-like polypeptide (ELP).
[0062] In some embodiments, any component, or mixture of components
can include an attached bioactive agent. The bioactive agent can
include 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 a component. In an embodiment
where a component is a polypeptide, the bioactive agent can be
attached through an amino acid linkage or through a chemical
linkage through a reactive peptide residue. The reactive peptide
residue can include, for example, but is not limited to lysine,
cysteine, and aspartic acid. The polypeptide attached to the
bioactive agent can include a protease cleavage site. In some
embodiments, the population of droplets are aqueous and the
solution 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.
[0063] The phase separation point of any component, or mixture
thereof, can be a phase separation temperature. For components or
mixtures having a phase separation temperature, the stimulus can
include heating. The phase separation point can also include other
phase separation characteristics, the characteristics relating to
the stimulus applied to affect a phase separation. For example, the
stimulation can include: addition or removal of one or more of the
components, evaporation of the droplets or 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, 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.
[0064] In some embodiments of the present disclosure, the solution
includes at least one surfactant for controlling a size of one or
more coacervate domains. The surfactant can include an amphiphilic
diblock polymer. For example, but not limited thereto, a component
can be a hydrophobic ELP polymer and the amphiphilic diblock
polymer can be an ELP diblock polymer. By changing the ratio of a
surfactant to a component, the resulting coacervate domain size can
be manipulated. In some embodiments, a ratio of the hydrophobic ELP
polymer to the amphiphilic ELP diblock polymer can range from about
1:1 to about 50:1, which can result in the size of an outermost
coacervate domain ranging from about 50 nm to about 20 .mu.m.
[0065] In at least one embodiment, two or more components can have
similar phase separation points. When stimulation induces a phase
separation point of the two or more components, a blended alloy
coacervate domain can be formed. For example, but not limited
thereto, where a first phase separation point of a first component
and an additional phase separation point of an additional
temperature are each similar phase separation temperatures, and the
stimulus includes heating, an alloy coacervate domain can be
formed. The coacervate domains of the various components can form a
multilayered coacervate domain, a blended alloy coacervate domain,
or a combination thereof.
[0066] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. These patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0067] One skilled in the art will readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The present examples along with the methods described
herein are presently representative of preferred embodiments, are
exemplary, and are not intended as limitations on the scope of the
invention. Changes therein and other uses will occur to those
skilled in the art which are encompassed within the spirit of the
invention as defined by the scope of the claims.
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