U.S. patent application number 13/604465 was filed with the patent office on 2013-03-14 for copolymer-stabilized emulsions.
This patent application is currently assigned to The Regents of the University of California. The applicant listed for this patent is Connie B. Chang, Timothy J. Deming, Sara M. Graves, Jarrod A. Hanson, Thomas G. Mason. Invention is credited to Connie B. Chang, Timothy J. Deming, Sara M. Graves, Jarrod A. Hanson, Thomas G. Mason.
Application Number | 20130064759 13/604465 |
Document ID | / |
Family ID | 40378464 |
Filed Date | 2013-03-14 |
United States Patent
Application |
20130064759 |
Kind Code |
A1 |
Mason; Thomas G. ; et
al. |
March 14, 2013 |
COPOLYMER-STABILIZED EMULSIONS
Abstract
An emulsion includes a substantially continuous liquid medium,
and a plurality of droplet structures dispersed within the
substantially continuous liquid medium. Each droplet structure of
the plurality of droplet structures includes an outer droplet of a
first liquid having an outer surface; an inner droplet of a second
liquid within the first droplet, the second liquid being immiscible
in the first liquid, wherein the inner and outer droplets have a
boundary surface region therebetween; an outer layer of block
copolymers disposed on the outer surface of the outer droplet; and
an inner layer of block copolymers disposed on the boundary surface
region between the outer and the inner droplets.
Inventors: |
Mason; Thomas G.; (Los
Angeles, CA) ; Deming; Timothy J.; (Los Angeles,
CA) ; Hanson; Jarrod A.; (Los Angeles, CA) ;
Chang; Connie B.; (Los Angeles, CA) ; Graves; Sara
M.; (Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mason; Thomas G.
Deming; Timothy J.
Hanson; Jarrod A.
Chang; Connie B.
Graves; Sara M. |
Los Angeles
Los Angeles
Los Angeles
Los Angeles
Los Angeles |
CA
CA
CA
CA
CA |
US
US
US
US
US |
|
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
40378464 |
Appl. No.: |
13/604465 |
Filed: |
September 5, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12391914 |
Feb 24, 2009 |
8283308 |
|
|
13604465 |
|
|
|
|
PCT/US2008/009882 |
Aug 20, 2008 |
|
|
|
12391914 |
|
|
|
|
60935605 |
Aug 21, 2007 |
|
|
|
Current U.S.
Class: |
424/1.11 ;
424/400; 424/9.3; 424/9.4; 424/9.5; 977/773; 977/774; 977/795;
977/906; 977/927; 977/928; 977/929; 977/930 |
Current CPC
Class: |
Y10S 977/929 20130101;
Y10S 977/93 20130101; Y02A 50/401 20180101; Y10S 977/928 20130101;
B82Y 5/00 20130101; Y10S 977/927 20130101; Y10T 428/2984 20150115;
A61K 47/20 20130101; B01F 17/005 20130101; Y10S 977/795 20130101;
Y10S 977/906 20130101; A61K 9/113 20130101; A61K 47/34 20130101;
Y10S 977/773 20130101; Y02A 50/30 20180101; A61K 47/183
20130101 |
Class at
Publication: |
424/1.11 ;
424/400; 424/9.3; 424/9.4; 424/9.5; 977/773; 977/795; 977/906;
977/927; 977/928; 977/929; 977/930; 977/774 |
International
Class: |
A61K 51/00 20060101
A61K051/00; A61K 9/113 20060101 A61K009/113; A01N 25/04 20060101
A01N025/04 |
Goverment Interests
[0002] This invention was made using U.S. Government support under
Grant No. CHE-0415275, awarded by the National Science Foundation.
The U.S. Government has certain rights in this invention.
Claims
1. An emulsion, comprising: a substantially continuous liquid
medium; and a plurality of droplet structures dispersed within said
substantially continuous liquid medium, wherein each droplet
structure of said plurality of droplet structures comprises: an
outer droplet of a first liquid having an outer surface; an inner
droplet of a second liquid having an inner surface within said
first droplet, said second liquid being immiscible in said first
liquid, wherein said inner and outer droplets have a film of said
first liquid in a surface boundary region therebetween; an outer
layer of block copolymers disposed on said outer surface of said
outer droplet; and an inner layer of block copolymers disposed on
said inner surface of said inner droplets in proximity to said
boundary surface region between said outer and said inner droplets,
wherein said block copolymers comprise a hydrophilic polymer block
and a hydrophobic polymer block that act in combination to
stabilize said droplet structure, and wherein said first liquid is
immiscible in said substantially continuous liquid medium.
2. An emulsion according to claim 1, wherein said droplet structure
has a maximum dimension corresponding to an undeformed droplet
diameter that is less than about 1000 nm and greater than about 10
nm.
3. An emulsion according to claim 1, wherein said droplet structure
has a maximum dimension corresponding to an undeformed droplet
diameter that is less than about 250 nm and greater than about 50
nm.
4. An emulsion according to claim 1, wherein block copolymers of
said inner layer of block copolymers are of a substantially same
molecular form as block copolymers of said outer layer of block
copolymers.
5. An emulsion according to claim 1, wherein said hydrophilic block
has a molecular weight in the range from about 200 Da to about
3,000,000 Da and said hydrophobic block has a molecular weight in
the range from about 200 Da to about 3,000,000 Da.
6. An emulsion according to claim 1, wherein a dimensionless ratio,
defined by the average radius of said inner droplet divided by the
average radius of said outer droplet, is less than about 0.9 and
greater than about 0.05.
7. An emulsion according to claim 1, wherein said inner layer of
block copolymers is a layer of di-block copolymers formed from
polymerization of two distinguishably different monomer types and
said outer layer of block copolymers is a layer of di-block
copolymers formed from polymerization of two said monomer
types.
8. An emulsion according to claim 1, wherein said hydrophilic
polymer block is a polypeptide block comprising predominantly
hydrophilic amino acids and said hydrophobic polymer block is a
polypeptide block comprising predominantly hydrophobic amino
acids.
9. An emulsion according to claim 1, wherein said hydrophilic
polymer block is a polypeptide block comprising a plurality of
types of hydrophilic amino acids and said hydrophobic polymer block
is a polypeptide block comprising a plurality of types of
hydrophobic amino acids.
10. An emulsion according to claim 8, wherein said hydrophilic
amino acids are selected from the group of hydrophilic amino acids
consisting of L-argenine, L-asparagine, L-aspartic acid,
L-cysteine, L-glutamic acid, L-glutamine, L-histidine, L-lysine,
L-serine, L-threonine, L-tyrosine, D-argenine, D-asparagine,
D-aspartic acid, D-cysteine, D-glutamic acid, D-glutamine,
D-histidine, D-lysine, D-serine, D-threonine, D-tyrosine,
DL-argenine, DL-asparagine, DL-aspartic acid, DL-cysteine,
DL-glutamic acid, DL-glutamine, DL-histidine, DL-lysine, DL-serine,
DL-threonine, DL-tyrosine, and any combination thereof.
11. An emulsion according to claim 8, wherein said hydrophobic
amino acids are selected from the group of hydrophobic amino acids
consisting of racemic-alanine, racemic-glycine, racemic-isoleucine,
racemic-leucine, racemic-methionine, racemic-phenylaniline,
racemic-proline, racemic-tryptophan, racemic-valine, and any
combination thereof.
12. An emulsion according to claim 10, wherein said hydrophobic
amino acids are selected from the group of hydrophobic amino acids
consisting of racemic-alanine, racemic-glycine, racemic-isoleucine,
racemic-leucine, racemic-methionine, racemic-phenylaniline,
racemic-proline, racemic-tryptophan, racemic-valine and any
combination thereof.
13. An emulsion according to claim 8, wherein said hydrophilic
amino acids and said hydrophobic amino acids are selected from the
group of amino acids consisting of alanine, arginine, asparagine,
aspartic acid, cysteine, glutamic acid, glutamine, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
proline, serine, threonine, tryptophan, tyrosine, valine, and any
combination thereof.
14. An emulsion according to claim 8, wherein said hydrophilic
amino acids are L-lysine and said hydrophobic amino acids are
racemic-leucine.
15. An emulsion according to claim 1, wherein said block copolymers
are bock copolypeptides having a structure satisfying the formula
K.sub.xrL.sub.y, K representing L-lysine and rL representing
racemic-leucine, wherein x is an integer in the range 10 to 200 and
y is an integer in the range 3 to 30.
16. An emulsion according to claim 1, wherein said second liquid of
said inner droplet is hydrophilic and further comprises, at least
one of blended or dispersed therein, at least one of
single-stranded DNA, double-stranded DNA, RNAs, oligonucleotides,
peptides, proteins, salts, viruses, vitamins, serums, lysates, ATP,
GTP, molecular motors, hydrophilic drug molecules, cells, vesicles,
nanodroplets, nanoparticles, fullerenes, single-walled carbon
nanotubes, multi-walled carbon nanotubes, cytoplasm, ribosomes,
enzymes, glucose, hemoglobin, golgi, dendrimers, surfactants,
lipids, albumin, anions, cations, buffers, sugars, saccharides,
quantum dots, clay nanoparticles, metal nanoclusters, metal
nanoparticles, magnetically responsive iron oxide nanoparticles,
organic nanospheres, organic nanoparticles, inorganic nanospheres,
inorganic nanoparticles, fluorescent dyes, transfection agents,
antiseptic materials, antimicrobial materials, materials that
absorb electromagnetic radiation, isotopically specific materials,
molecules containing radioactive isotopes, imaging-contrast
enhancement agents, agents that disrupt cellular functions, agents
that enhance cellular functions, agents that disrupt cellular
substructures, agents that modify cellular substructures, agents
that affect cellular metabolic pathways, agents that trigger
cellular apoptosis and combinations thereof.
17. An emulsion according to claim 1, wherein said first liquid of
said outer droplet is hydrophobic and further comprises, at least
one of blended or dispersed therein, at least one of fats, lipids,
waxes, natural oils, synthetic oils, silicone oils, volatile oils
essential oils, fragrances, cholesterol, steroids, hydrophobic drug
molecules, polymers, block copolymers, poly-acids, poly-bases,
polypeptides, block copolypeptides, micelles, quantum dots,
nanoparticles, nanoclusters, carbon nanotubes, fullerenes,
ferrofluids, thermotropic liquid crystals, lyotropic liquid
crystals, fluorinated liquids, brominated liquids, plant-derived
materials, animal-derived materials, bacterially-derived materials,
and combinations thereof.
18. An emulsion according to claim 16, wherein said first liquid of
said outer droplet is hydrophobic and further comprises, at least
one of blended or dispersed therein, at least one of fats, lipids,
waxes, natural oils, synthetic oils, silicone oils, volatile oils,
essential oils, fragrances, cholesterol, steroids, hydrophobic drug
molecules, polymers, block copolymers, polypeptides, block
copolypeptides, poly-acids, poly-bases, micelles, quantum dots,
nanoparticles, nanoclusters, carbon nanotubes, fullerenes,
ferrofluids, thermotropic liquid crystals, lyotropic liquid
crystals, fluorinated liquids, brominated liquids, plant-derived
materials, animal-derived materials, bacterially-derived materials,
and combinations thereof.
19. A droplet structure, comprising: an outer droplet of a first
liquid having an outer surface; an inner droplet of a second liquid
having an inner surface within said first droplet, said second
liquid being immiscible in said first liquid, wherein said inner
and outer droplets have a film of said first liquid in a boundary
surface region therebetween; an outer layer of block copolymers
disposed on said outer surface of said outer droplet; and an inner
layer of block copolymers disposed on said inner surface of said
inner droplet, wherein said block copolymers comprise a hydrophilic
polymer block and a hydrophobic polymer block that act in
combination to stabilize said outer surface of said outer droplet
from coalescing with said inner surface of said inner droplet and
to stabilize said droplet structure from coalescing with other
droplet structures.
20. A droplet structure according to claim 19, wherein said droplet
structure has a maximum dimension given by an undeformed droplet
diameter that is less than about 1000 nm and greater than about 10
nm.
21. A droplet structure according to claim 19, wherein said droplet
structure has a maximum dimension given by an undeformed droplet
diameter that is less than about 250 nm and greater than about 50
nm.
22. A droplet structure according to claim 19, wherein block
copolymers of said inner layer of block copolymers are of a
substantially same molecular form as block copolymers of said outer
layer of block copolymers.
23. A droplet structure according to claim 19, wherein said
hydrophilic block has a molecular weight in the range from about
200 Da to about 3,000,000 Da and said hydrophobic block has a
molecular weight in the range from about 200 Da to about 3,000,000
Da.
24. A droplet structure according to claim 19, wherein a ratio
given by a radius of said outer droplet divided by a radius of said
inner droplet is less than about 0.9 and greater than about
0.05.
25. A droplet structure according to claim 19, wherein said inner
layer of block copolymers is a layer of di-block copolymers formed
from polymerization of two distinguishably different monomer types
and said outer layer of block copolymers is a layer of di-block
copolymers formed from polymerization of two said monomer
types.
26. A droplet structure according to claim 19, wherein said
hydrophilic polymer block is a polypeptide block comprising
predominantly hydrophilic amino acids and said hydrophobic polymer
block is a polypeptide block comprising predominantly hydrophobic
amino acids.
27. A droplet structure according to claim 26, wherein at least one
of said polypeptide blocks comprises a surface moiety to provide
surface functionalization.
28. A droplet structure according to claim 19, wherein said
hydrophilic polymer block is a polypeptide block comprising
predominantly a plurality of types of hydrophilic amino acids and
said hydrophobic polymer block is a polypeptide block comprising
predominantly a plurality of types of hydrophobic amino acids.
29. A droplet structure according to claim 26, wherein said
hydrophilic amino acids are selected from the group of hydrophilic
amino acids consisting of L-argenine, L-asparagine, L-aspartic
acid, L-cysteine, L-glutamic acid, L-glutamine, L-histidine,
L-lysine, L-serine, L-threonine, L-tyrosine, D-argenine,
D-asparagine, D-aspartic acid, D-cysteine, D-glutamic acid,
D-glutamine, D-histidine, D-lysine, D-serine, D-threonine,
D-tyrosine, DL-argenine, DL-asparagine, DL-aspartic acid,
DL-cysteine, DL-glutamic acid, DL-glutamine, DL-histidine,
DL-lysine, DL-serine, DL-threonine, DL-tyrosine, and any
combination thereof.
30. A droplet structure according to claim 26, wherein said
hydrophobic amino acids are selected from the group of hydrophobic
amino acids consisting of racemic-alanine, racemic-glycine,
racemic-isoleucine, racemic-leucine, racemic-methionine,
racemic-phenylaniline, racemic-proline, racemic-tryptophan,
racemic-valine, and any combination thereof.
31. A droplet structure according to claim 29, wherein said
hydrophobic amino acids are selected from the group of hydrophobic
amino acids consisting of racemic-alanine, racemic-glycine,
racemic-isoleucine, racemic-leucine, racemic-methionine,
racemic-phenylaniline, racemic-proline, racemic-tryptophan,
racemic-valine, and any combination thereof.
32. A droplet structure according to claim 26, wherein said
hydrophilic amino acids and said hydrophobic amino acids are
selected from the group of amino acids consisting of alanine,
arginine, asparagine, aspartic acid, cysteine, glutamic acid,
glutamine, glycine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, proline, serine, threonine, tryptophan,
tyrosine, valine, and any combination thereof.
33. A droplet structure according to claim 26, wherein said
hydrophilic amino acids are L-lysine and said hydrophobic amino
acids are racemic-leucine.
34. A droplet structure according to claim 19, wherein said block
copolymers are bock copolypeptides having a structure satisfying
the formula K.sub.xrL.sub.y, K representing L-lysine and rL
representing racemic-leucine, wherein x is an integer in the range
from about 10 to about 200 and y is an integer in the range from
about 3 to about 30.
35. A droplet structure according to claim 19, wherein said second
liquid of said inner droplet is hydrophilic and further comprises,
at least one of blended or dispersed therein, at least one of
single-stranded DNA, double-stranded DNA, RNAs, oligonucleotides,
peptides, proteins, salts, viruses, vitamins, serums, lysates, ATP,
GTP, molecular motors, hydrophilic drug molecules, cells, vesicles,
nanodroplets, nanoparticles, fullerenes, single-walled carbon
nanotubes, multi-walled carbon nanotubes, cytoplasm, ribosomes,
enzymes, glucose, hemoglobin, golgi, dendrimers, surfactants,
lipids, albumins, anions, cations, buffers, sugars, saccharides,
quantum dots, clay nanoparticles, metal nanoclusters, metal
nanoparticles, magnetically responsive iron oxide nanoparticles,
organic nanospheres, organic nanoparticles, inorganic nanospheres,
inorganic nanoparticles, fluorescent dyes, transfection agents,
antiseptic materials, antimicrobial materials, materials that
absorb electromagnetic radiation, isotopically specific materials,
molecules containing radioactive isotopes, imaging-contrast
enhancement agents, agents that enhance magnetic resonance imaging,
agents that enhance x-ray imaging, agents that enhance neutron
imaging, agents that enhance positron-emission tomography, agents
that enhance light scattering, agents that disrupt cellular
functions, agents that enhance cellular functions, agents that
disrupt cellular substructures, agents that modify cellular
substructures, agents that affect cellular metabolic pathways,
agents that trigger cellular apoptosis and combinations
thereof.
36. A droplet structure according to claim 19, wherein said first
liquid of said outer droplet is hydrophobic and further comprises,
at least one of blended or dispersed therein, at least one of fats,
lipids, waxes, natural oils, synthetic oils, silicone oils,
volatile oils, essential oils, fragrances, cholesterol, steroids,
hydrophobic drug molecules, polymers, block polymers, poly-acids,
poly-bases, polypeptides, block polypeptides, micelles, quantum
dots, nanoparticles, nanoclusters, carbon nanotubes, fullerenes,
ferrofluids, thermotropic liquid crystals, lyotropic liquid
crystals, fluorinated liquids, brominated liquids, plant-derived
materials, animal-derived materials, bacterially-derived materials,
and combinations thereof.
37. A droplet structure according to claim 35, wherein said first
liquid of said outer droplet is hydrophobic and further comprises,
at least one of blended or dispersed therein, at least one of fats,
lipids, waxes, natural oils, synthetic oils, synthetic oils,
volatile oils, essential oils, fragrances, cholesterol, steroids,
hydrophobic drug molecules, polymers, block polymers, poly-acids,
poly-bases, polypeptides, block polypeptides, micelles, quantum
dots, nanoparticles, nanoclusters, carbon nanotubes, fullerenes,
ferrofluids, thermotropic liquid crystals, lyotropic liquid
crystals, fluorinated liquids, brominated liquids, plant-derived
materials, animal-derived materials, bacterially-derived materials,
and combinations thereof.
38. A nano-droplet structure, comprising: an outer droplet of a
first liquid having an outer surface; an inner droplet of a second
liquid having an inner surface arranged within said first droplet,
said second liquid being immiscible in said first liquid, wherein
said inner and outer droplets have a film of said first liquid in a
boundary surface region therebetween; an outer layer of block
copolypeptides disposed on said outer surface of said outer
droplet; and an inner layer of block copolypeptides disposed on
said inner surface proximate to said boundary surface region
between said outer and said inner droplets, wherein said block
copolypeptides have a structure satisfying the formula
K.sub.xrL.sub.y, K representing L-lysine and rL representing
racemic-leucine, wherein x is an integer in the range from about 10
to about 200 and y is an integer in the range from about 3 to about
30, and wherein said nano-droplet structure has a maximum dimension
given by an undeformed outer droplet diameter that is less than
about 300 nm and greater than about 10 nm.
39. A nano-droplet structure according to claim 38, wherein said
second liquid of said inner droplet is hydrophilic and further
comprises, at least one of blended or dispersed therein, at least
one of single-stranded DNA, double-stranded DNA, RNAs,
oligonucleotides, peptides, proteins, salts, viruses, vitamins,
serums, lysates, ATP, GTP, molecular motors, hydrophilic drug
molecules, cells, vesicles, nanodroplets, nanoparticles,
fullerenes, single-walled carbon nanotubes, multi-walled carbon
nanotubes, cytoplasm, ribosomes, enzymes, glucose, hemoglobin,
golgi, dendrimers, surfactants, lipids, albumins, anions, cations,
buffers, sugars, saccharides, quantum dots, clay nanoparticles,
metal nanoclusters, metal nanoparticles, magnetically responsive
iron oxide nanoparticles, organic nanospheres, organic
nanoparticles, inorganic nanospheres, inorganic nanoparticles,
fluorescent dyes, transfection agents, antiseptic materials,
antimicrobial materials, materials that absorb electromagnetic
radiation, isotopically specific materials, molecules containing
radioactive isotopes, imaging-contrast enhancement agents, agents
that enhance magnetic resonance imaging, agents that enhance x-ray
imaging, agents that enhance neutron imaging, agents that enhance
positron-emission tomography, agents that enhance light scattering,
agents that disrupt cellular functions, agents that enhance
cellular functions, agents that disrupt cellular substructures,
agents that modify cellular substructures, agents that affect
cellular metabolic pathways, agents that trigger cellular apoptosis
and combinations thereof.
40. A nano-droplet structure according to claim 38, wherein said
first liquid of said outer droplet is hydrophobic and further
comprises, at least one of blended or dispersed therein, at least
one of fats, lipids, waxes, natural oils, synthetic oils, silicone
oils, volatile oils, essential oils, fragrances, cholesterol,
steroids, hydrophobic drug molecules, polymers, block copolymers,
polypeptides, block polypeptides, poly-acids, poly-bases, micelles,
quantum dots, nanoparticles, nanoclusters, carbon nanotubes,
fullerenes, ferrofluids, thermotropic liquid crystals, lyotropic
liquid crystals, fluorinated liquids, brominated liquids,
plant-derived materials, animal-derived materials,
bacterially-derived materials, and combinations thereof.
41. A droplet structure according to claim 39, wherein said first
liquid of said outer droplet is hydrophobic and further comprises,
at least one of blended or dispersed therein, at least one of fats,
lipids, waxes, natural oils, synthetic oils, silicone oils,
volatile oils, essential oils, fragrances, cholesterol, steroids,
hydrophobic drug molecules, polymers, block polymers, polypeptides,
block polypeptides, poly-acids, poly-bases, micelles, quantum dots,
nanoparticles, nanoclusters, carbon nanotubes, fullerenes,
ferrofluids, thermotropic liquid crystals, lyotropic liquid
crystals, fluorinated liquids, brominated liquids, plant-derived
materials, animal-derived materials, bacterially-derived materials,
and combinations thereof.
42. An emulsion, comprising: a liquid medium; and a plurality of
nano-droplets dispersed within said liquid medium, wherein each of
said plurality of nano-droplets comprises an inner droplet of a
first liquid surrounded by a second liquid, said first liquid being
immiscible in said second liquid and said second liquid being
immiscible in said liquid medium, and wherein said plurality of
nano-droplets have an ensemble average undeformed outer droplet
diameter of at least about 10 nm and less than about 300 nm.
43. An emulsion according to claim 42, wherein said liquid medium
is a same material as said first liquid.
44. An emulsion according to claim 42, further comprising block
copolymers adsorbed onto at least one of a surface of said
plurality of nano-droplets and a surface of said inner
droplets.
45. An emulsion according to claim 44, wherein said block
copolymers each comprises a hydrophilic polymer block and a
hydrophobic polymer block that act in combination to stabilize at
least one of said plurality of nano-droplets or said inner
droplets.
46. An emulsion according to claim 42, further comprising first
block copolymers adsorbed onto a surface of said plurality of
nano-droplets and second block copolymers adsorbed onto a surface
of said inner droplets.
47. An emulsion according to claim 46, wherein said first and
second block copolymers each comprises a hydrophilic polymer block
and a hydrophobic polymer block that act in combination to
stabilize at least one of said plurality of nano-droplets or said
inner droplets.
48. An emulsion according to claim 47, wherein said first and
second block copolymers are of a substantially same molecular
structure.
49. An emulsion, comprising: a substantially continuous liquid
medium; and a plurality of droplet structures dispersed within said
substantially continuous liquid medium, wherein each droplet
structure of said plurality of droplet structures comprises: a
droplet of a liquid having an outer surface; and a layer of block
copolymers disposed on said outer surface of said droplet, wherein
said block copolymers comprise a hydrophilic polymer block and a
hydrophobic polymer block that act in combination to stabilize said
droplet structure, and wherein said liquid of said plurality of
droplet structures is immiscible in said substantially continuous
liquid medium.
50. An emulsion according to claim 49, wherein said hydrophilic
polymer block is a polypeptide block comprising predominantly
hydrophilic amino acids and said hydrophobic polymer block is a
polypeptide block comprising predominantly hydrophobic amino
acids.
51. An emulsion according to claim 49, wherein said hydrophilic
polymer block is a polypeptide block comprising a plurality of
types of predominantly hydrophilic amino acids and said hydrophobic
polymer block is a polypeptide block comprising a plurality of
types of predominantly hydrophobic amino acids.
52. An emulsion according to claim 50, wherein said hydrophilic
amino acids and said hydrophobic amino acids are selected from the
group of amino acids consisting of alanine, arginine, asparagine,
aspartic acid, cysteine, glutamic acid, glutamine, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
proline, serine, threonine, tryptophan, tyrosine, valine, and any
combination thereof.
53. An emulsion according to claim 52, wherein said each droplet
structure has a maximum dimension corresponding to an undeformed
droplet diameter that is less than about 300 nm and greater than
about 10 nm.
54. A droplet structure, comprising: a droplet of a liquid having
an outer surface; and a layer of block copolymers disposed on said
outer surface of said droplet, wherein said block copolymers
comprise a hydrophilic polymer block and a hydrophobic polymer
block that act in combination to stabilize said droplet
structure.
55. A droplet structure according to claim 54, wherein said
hydrophilic polymer block is a polypeptide block comprising
predominantly hydrophilic amino acids and said hydrophobic polymer
block is a polypeptide block comprising predominantly hydrophobic
amino acids.
56. A droplet structure according to claim 54, wherein said
hydrophilic polymer block is a polypeptide block comprising
predominantly a plurality of types of hydrophilic amino acids and
said hydrophobic polymer block is a polypeptide block comprising
predominantly a plurality of types of hydrophobic amino acids.
57. A droplet structure according to claim 55, wherein said
hydrophilic amino acids and said hydrophobic amino acids are
selected from the group of amino acids consisting of alanine,
arginine, asparagine, aspartic acid, cysteine, glutamic acid,
glutamine, glycine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, proline, serine, threonine, tryptophan,
tyrosine, valine, and any combination thereof.
58. A method of producing an emulsion, comprising: providing a
first liquid and a second liquid, said first liquid being
immiscible in said second liquid; adding a selected quantity of
block copolymers to at least one of said first and second liquids;
and emulsifying said first liquid in said second liquid to produce
a plurality of droplets of said first liquid dispersed in said
second liquid, wherein said block copolymers stabilize said
plurality of droplets from coalescing.
59. A method of producing an emulsion according to claim 58,
wherein each droplet of said plurality of droplets of said first
liquid comprises an inner droplet therein of said second liquid
that is produced substantially simultaneously with the production
of said plurality of droplets during said emulsifying.
60. A method of producing an emulsion according to claim 58,
wherein each droplet of said plurality of droplets of said first
liquid comprises a plurality of inner droplets therein of said
second liquid that are produced substantially simultaneously with
the production of said plurality of droplets during said
emulsifying.
61. A method of producing an emulsion according to claim 58,
further comprising selecting said quantity of block copolymers to
add to provide said plurality of droplets to have a preselected
ensemble average diameter.
62. A method of producing an emulsion according to claim 58,
further comprising selecting a molecular composition of said block
copolymers during said adding to provide said plurality of droplets
having a preselected ensemble average diameter.
63. A method of producing an emulsion, comprising: at least one of
adding a surfactant to at least one of a first liquid and a second
liquid, or adding surfactant precursors to at least one of said
first liquid and said second liquid; emulsifying said first liquid
in said second liquid to form a plurality of droplets of said first
liquid immersed in said second liquid to provide a simple emulsion,
said first liquid being immiscible in said second liquid; adding at
least one of said surfactant or said surfactant precursors to a
third liquid; and emulsifying said simple emulsion in said third
liquid to form a plurality of droplets of said simple emulsion to
provide a double emulsion, said second liquid being immiscible in
said third liquid, wherein said plurality of droplets of said
double emulsion each comprises at least one droplet of said first
liquid therein.
64. An emulsion produced according to the method of any one of
claims 58-63.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application No. 60/935,605 filed Aug. 21, 2007, the entire contents
of which are hereby incorporated by reference.
BACKGROUND
[0003] 1. Field of Invention
[0004] The present invention relates to droplet structures,
emulsions of droplet structures and methods of producing the
droplet structures and emulsions; and more particularly to droplet
structures, emulsions of droplet structures and methods of
producing the droplet structures that are stabilized with block
copolymers.
[0005] 2. Discussion of Related Art
[0006] Simple emulsions are dispersions of droplets of one liquid
in another immiscible liquid; the droplets are typically formed by
applied shear and stabilized against subsequent coalescence by a
surfactant that provides an interfacial repulsion (J. Bibette, F.
Leal-Calderon, and P. Poulin, Rep. Prog. Phys. 62, 969 (1999)).
(All references cited anywhere in any section of this specification
are incorporated herein by reference.) Two of the most common types
are `direct` oil-in-water (O/W) emulsions and `inverse`
water-in-oil (W/O) emulsions. Surfactants are amphiphilic molecules
that can take many different forms: ionic (e.g. anionic, cationic,
zwitterionic), non-ionic (e.g. ethoxylated alkane chains), and
polymeric (e.g. simple, diblock, and triblock polymers). Because
they are amphiphilic, surfactants tend to preferentially adsorb
onto oil-water interfaces. The relative solubility of the
surfactant in the oil and the water, the concentration of the
surfactant, and the degree of interfacial repulsion that the
surfactant provides once it has adsorbed onto the interfaces are
important factors in determining the stability and longevity of
emulsions that are formed by an applied shear flow or other sources
of non-thermal external stresses that can cause bigger droplet
structures to be ruptured into smaller droplet structures.
[0007] Beyond simple emulsions, higher levels of topological
complexity exist. For instance, a W/O emulsion can be sheared into
an aqueous continuous liquid, thereby creating a dispersion of oil
droplets which themselves contain smaller water droplets. Through
judicious choice of the surfactants, both the `inner` water
droplets inside the oil droplets, as well as the larger oil
droplets themselves, can remain stable over long periods of time.
This type of emulsion is called a water-in-oil-in-water (W/O/W)
emulsion. Emulsion systems that have this level of topological
complexity are generically called `double emulsions` because,
starting from the continuous liquid phase, two oil-water
interfacial layers must be penetrated to reach the center of the
smallest droplet structure. Indeed, through successive controlled
emulsification steps, it is possible to fabricate triple emulsions
and even higher topologically ordered multiple emulsions that
contain many interfacial layers that must be penetrated in order to
reach the center of the smallest droplet structure in the system. A
W/O/W double emulsion may have outer oil droplets that each contain
only one inner water droplet. However, it is also possible for oil
droplets in a W/O/W double emulsion to contain many inner water
droplets. Sometimes, this is mistakenly referred to as a "multiple
emulsion". Instead, more properly, it should be referred to as a
W/O/W double emulsion that has outer oil droplets that generally
each contain a plurality of multiple inner droplets. Two average
droplet volume fractions can be used to characterize a double
emulsion roughly: the average `inner volume fraction` of water
droplets inside the oil droplets, and the average `outer volume
fraction` of the W/O droplets that exist in the continuous aqueous
solution. Generally, there is a full distribution of radii
corresponding to inner water droplets and also a different
distribution of radii corresponding to outer water droplets. It can
be desirable for these distributions to exhibit monomodal peaks
that are fairly sharp, so the droplet sizes are more highly
controlled, or `uniform`. Another structural aspect that
characterizes double emulsions is the probability distribution of
the number of inner droplets per outer droplet. Although we focus
primarily on creating W/O/W double emulsions (i.e. water-borne
double droplets) herein, it is equally possible to create
oil-in-water-in-oil (O/W/O) double emulsions that do not have an
aqueous continuous phase. For oil-in-water single emulsions and for
water-in-oil-in-water double emulsions, .phi. is typically used to
designate the oil volume fraction: the volume of oil contained
within the emulsion system divided by the total volume of the
emulsion system.
[0008] In recent years, two primary pathways, structured
microfluidic and sequential emulsification, have provided highly
uniform W/O/W double emulsions that typically have average outer
droplet diameters greater than about one micron. The first pathway
is through relatively low-throughput microfluidic methods. In one
implementation of this pathway, a W/O/W emulsion is created using a
first cross-channel flow junction to produce water droplets in oil
and then using a second cross-channel flow junction to rupture the
W/O droplets into a continuous aqueous phase (S. Okushima et al.,
Langmuir 20, 9905 (2004)). Alternatively, porous glass
emulsification and membrane emulsification methods, rather than
micromachined fluidic channels, can be used to provide highly
uniform W/O emulsions at higher throughput. This implementation
permits quite robust incorporation of many inner droplets into
double emulsions. A second implementation of microfluidic rupturing
is by structuring the flow of an innermost water jet, an
intermediate oil jet, and an outermost water jet using microfluidic
channels, such that the capillary instability of the inner and
outer interfaces occurs simultaneously (A. S. Utada et al., Science
308, 537 (2005)). This method is good for encapsulating objects in
the innermost aqueous jet into a W/O/W double emulsion containing a
single inner droplet. However, it is significantly more difficult
to coordinate the flows so that double emulsions containing a
specific number of multiple inner droplets are formed at the
desired internal volume fraction. In both of these microfluidic
approaches, appropriate surfactants must be present in the liquid
phases in order to preserve the stability of the emulsion after
formation.
[0009] The second pathway is the more traditional form of
sequential emulsification without the use of micromachined
channels. In sequential emulsification, a W/O emulsion is first
created, and then this simple inverse emulsion is, in turn,
emulsified into an aqueous surfactant solution using shear (W.
Yafei, Z. Tao, and H. Gang, Langmuir 22, 67 (2006)). If desired,
both the water and the oil droplets in this W/O/W double emulsion
can be size-fractionated to make them monodisperse. Without
fractionation, the traditional method can be very high-throughput
and can produce many liters per hour. If a high level of
monodispersity is desired, then the fractionation necessarily slows
down the process. In a variation on this method, a high-throughput
approach for making the oil droplets quasi-monodisperse by shearing
a premixed double emulsion in a thin gap (C. Goubault et al.,
Langmuir 17, 5184 (2001)) uses a method previously developed for
making monodisperse simple emulsions (T. G. Mason, and J. Bibette,
Phys. Rev. Lett. 77, 3481 (1996); T. G. Mason, and J. Bibette,
Langmuir 13, 4600 (1997)). As for double emulsions produced using
microfluidic methods, the choice of surfactants for sequential
emulsification is also important in order to obtain the desired
properties of stability and release.
[0010] Similar to small molecule surfactants and lipids, synthetic
block copolymers are able to self-assemble into ordered
nanostructures via microphase separation of the polymeric
components (A. J. Link, M. L. Mock, and D. A. Tirrell, Curr Opin
Biotech 14, 603 (2003)). However, the ability of block copolymers
to assemble into hierarchically structured materials or distinct
tertiary structures, similar to those found in biological systems
(e.g. proteins), has been limited by the random coiled nature of
most common polymers as well as the limited functionality of the
polymer domains. Incorporation of elements that encourage
hydrogen-bonding (G. A. Silva et al., Science 303, 1352 (2004)),
amphiphilicity (D. E. Discher, and A. Eisenberg, Science 297, 967
(2002)), crystallization (G. D. Fasman, Prediction of protein
structure and the principles of protein conformation (Plenum Press,
New York, 1989), pp. xiii), and liquid crystal formation (D. J.
Pochan et al., Macromolecules 35, 5358 (2002)) would all serve to
influence structural evolution (J. Rodriguez-Hernandez, and S.
Lecommandoux, J Am Chem Soc 127, 2026 (2005)). Increasing the
complexity of copolymer sequences (di- to tri- to tetra-blocks,
etc.) would also enhance the potential for hierarchical assembly
(I. W. Hamley, Soft Matter 1, 36 (2005)). The main limitation in
utilizing these strategies is that the synthetic chemistry
necessary for preparation of functional, multicomponent block
copolymers is a major hindrance due to incompatibilities of
different monomers with a given polymerization method (A. J. Link,
M. L. Mock, and D. A. Tirrell, Curr Opin Biotech 14, 603 (2003)).
Furthermore, since most common synthetic polymers lack the
intricate complexity found in biopolymers (e.g. secondary
structure, complex functionality and stereochemistry), they may
never be able to faithfully mimic the behavior of self-assembled
biological macromolecules. For these reasons, prior to
investigating emulsion systems, we have studied the self-assembly
of block copolypeptides as synthetic materials that possess the
ability to aggregate into specifically defined, functional
nanostructures, including vesicles and hydrogels. These
non-emulsion materials typically form through interactions between
the copolypeptide molecules resulting in "bottom-up" self-assembly.
However, the use of synthetic constituents (i.e. non-amino acid
monomers) to form synthetic polymer blocks and the use of higher
tri-block and multi-block polymer structures are not excluded from
some of the general concepts of the current invention.
[0011] In work preceding the invention described herein, we focused
our efforts on studying the roles of chain length and block
composition on the assembly of small, charged diblock copolypeptide
amphiphiles, where we utilized the structure directing properties
of a rod-like .alpha.-helical segment in the hydrophobic domain.
Specifically, we prepared and studied the aqueous self-assembly of
a series of poly(L-lysine)-b-poly(L-leucine) block copolypeptides,
K.sub.xL.sub.y, where x ranged from 20 to 80, and y ranged from 10
to 30 residues, as well as the poly(L-glutamatic
acid)-b-poly(L-leucine) block copolypeptide, E.sub.60L.sub.20 (E.
P. Holowka, D. J. Pochan, and T. J. Deming, J Am Chem Soc 127,
12423 (2005)). The poly(L-lysine-HBr) and
poly(L-glutamate-Na.sup.+) segments are highly charged
polyelectrolytes at neutral pH and dissolve readily in water. In
earlier work, we found that samples with high K to L molar ratios
(e.g. K.sub.180L.sub.20) could be dissolved directly into deionized
water, yielding transparent hydrogels composed of twisted fibrils
(A. P. Nowak et al., Nature 417, 424 (2002)). We reasoned that use
of shortened charged segments would relax repulsive polyelectrolyte
interactions and allow formation of charged polypeptide membranes.
In our first series of copolymers, the size of the oligoleucine
domain was held constant at 20 residues, and the oligolysine domain
was varied from 20 to 80 residues. Samples were processed by
suspending dry polymer in THF/water (1:1) followed by dialysis.
Analysis of these assemblies using DIC optical microscopy revealed
the presence of large, sheet-like membranes for K.sub.20L.sub.20,
and thin fibrils for K.sub.40L.sub.20. The K.sub.60L.sub.20 sample
was most promising, as only large vesicular assemblies were
observed by differential interference contrast (DIC) microscopy (E.
P. Holowka, D. J. Pochan, and T. J. Deming, J Am Chem Soc 127,
12423 (2005)).
[0012] The K.sub.60L.sub.20 polypeptide vesicles obtained directly
from dialysis are polydisperse and range in diameter from ca. 5
.mu.m down to 0.8 .mu.m as determined using DIC and DLS (FIG. 1).
For applications such as drug delivery via blood circulation, a
vesicle diameter of ca. 50 nm to about 100 nm, even up to about 200
nm, is desired. We found that aqueous suspensions of
K.sub.60L.sub.20 vesicles could be extruded through nuclear
track-etched polycarbonate (PC) membranes with little loss of
polypeptide material. After two passes through a filter, reductions
in vesicle diameter to values in close agreement to filter pore
size were observed. These results showed that the charged
polypeptide vesicles are readily extruded, allowing good control
over vesicle diameter in the tens to hundreds of nanometers range
(FIG. 1). Dynamic light scattering (DLS) size analysis revealed
that the extruded vesicles were also less polydisperse than before
extrusion and contained no micellar contaminants. The vesicular
morphology was also confirmed through TEM imaging of the sub-micron
K.sub.60L.sub.20 suspensions. The extruded vesicles were monitored
for 6 weeks using DLS and were found to be stable since the average
diameters did not change for most samples. The vesicles were also
found to have high thermal stability. An aqueous suspension of 1
.mu.m vesicles was held at 80.degree. C. for 30 minutes, after
which no vesicle disruption could be detected (E. P. Holowka, D. J.
Pochan, and T. J. Deming, J Am Chem Soc 127, 12423 (2005)). Only
after heating to 100.degree. C. for 30 minutes were the vesicles
disrupted, yielding large flat membrane sheets.
[0013] Stability of these highly charged polypeptide vesicles in
ionic media is important for use in most applications ranging from
personal care products to drug delivery. Although the
K.sub.60L.sub.20 vesicles are unstable and cluster at high salt
concentrations (>0.5 M), they are stable in 100 mM PBS buffer as
well as serum-free DMEM cell culture media (E. P. Holowka, D. J.
Pochan, and T. J. Deming, J Am Chem Soc 127, 12423 (2005)).
Addition of serum, which contains anionic proteins, results in
vesicle disruption, most likely due to polyion complexation between
the serum proteins and the oppositely charged polylysine chains.
Accordingly, we found that the negatively charged polypeptide
vesicles prepared using E.sub.60L.sub.20 are stable in DMEM with
10% fetal bovine serum. Based on these results, we believe these
charged polypeptide vesicles show potential as encapsulants for
water-soluble solutes as an alternative to liposomes. Another
feature of these charged polypeptide vesicles is the potential for
facile functionalization of the hydrophilic polypeptide chains at
the vesicle surface through either chemical conjugation to amine or
carboxylate residues, or by careful choice of charged residues. For
example, we recently reported the preparation of arginine-leucine
(i.e. R.sub.60L.sub.20) vesicles that are able to readily enter
cells due to the many guanidinium groups of the arginine segments
(E. P. Holowka et al., Nat Mater 6, 52 (2007)). In this case, the
arginine residues played a dual role, where they were both
structure directing in vesicle formation, as well as functional for
cell binding and entry. The key attributes of block copolypeptides
that are advantageous for the design of biomimetic membranes with
multifunctional properties are the ability to place structural and
functional elements in precise locations within polymer chains. In
embodiments of this invention, the copolypeptides populating the
interfaces of droplets can also make use of such multifunctional
properties, including controlling the morphology and topology of
the droplet structures and how they interact with cells and other
target materials in applications.
[0014] Due to their compartmentalized internal structure, W/O/W
double emulsions can provide advantages over simple oil-in-water
(O/W) emulsions for encapsulation, such as the ability to carry
simultaneously both polar cargos (such as water-soluble molecules
or water dispersable colloids in the inner water droplet) and
nonpolar cargos (such as oil-soluble molecules or oil dispersable
colloids in the outer oil droplet), deliver combination therapies
of oil-soluble and water-soluble drug molecules to a very specific
localized region (e.g. through targeting moieties on molecules that
decorate the outer an inner surfaces of the droplets), as well as
improved control over temporal release of therapeutic molecules
(Pays, K. et al. Double emulsions: how does release occur?Journal
of Controlled Release 79, 193-205 (2002); Davis, S. S. &
Walker, I. M. Multiple Emulsions as Targetable Delivery Systems.
Methods in Enzymology 149, 51-64 (1987); Okochi, H. & Nakano,
M. Preparation and evaluation of W/O/W type emulsions containing
vancomycin. Advanced Drug Delivery Reviews 45, 5-26 (2000)). The
preparation of double emulsions typically requires mixtures of
surfactants for stability, and the formation of double
nanoemulsions, where both inner and outer droplets are sub-100 nm,
has never before been achieved (Garti, N. Double emulsions--Scope,
limitations and new achievements. Colloids and Sutfaces
A-Physicochemical and Engineering Aspects 123, 233-246 (1997);
Loscertales, I. G. et al. Micro/nano encapsutation via electrified
coaxial liquid jets. Science 295, 1695-1698 (2002); Utada, A. S. et
al. Monodisperse double emulsions generated from a microcapillary
device. Science 308, 537-541 (2005)).
[0015] While offering certain advantages over ordinary O/W
emulsions, stable W/O/W emulsions generally do not form
spontaneously using a single surfactant and standard emulsification
methods according to conventional methods (Garti, N. Double
emulsions--Scope, limitations and new achievements. Colloids and
Surfaces A-Physicochemical and Engineering Aspects 123, 233-246
(1997); Morais, J. M., Santos, O. D. H., Nunes, J. R. L., Zanatta,
C. F., Rocha-Filho, P. A. W/O/W Multiple emulsions obtained by
one-step emulsification method and evaluation of the involved
variables. Journal of Dispersion Science and Technology 29, 63-69
(2008)). Microfluidics can be used to make double emulsions that
are microns in size and highly uniform (Loscertales, I. G. et al.
Micro/nano encapsutation via electrified coaxial liquid jets.
Science 295, 1695-1698 (2002); Utada, A. S. et al. Monodisperse
double emulsions generated from a microcapillary device. Science
308, 537-541 (2005)), yet the throughput can be low compared to
commercial processes for making polydisperse single emulsions
(Mason, T. G., Wilking, J. N., Meleson, K., Chang, C. B. &
Graves, S. M. Nanoemulsions: formation, structure, and physical
properties. Journal of Physics-Condensed Matter 18, R635-R666
(2006)). Typical methods for making W/O/W emulsions involve a
two-step process of first forming an `inverse` water-in-oil (W/O)
emulsion, followed by emulsification of this mixture in water using
a combination of surfactants (Ficheux, M. F., Bonakdar, L.,
Leal-Calderon, F. & Bibette, J. Some stability criteria for
double emulsions. Langmuir 14, 2702-2706 (1998); Wang, Y. F., Tao,
Z. & Gang, H. Structural evolution of polymer-stabilized double
emulsions. Langmuir 22, 67-73 (2006); Garti, N. Double
emulsions--Scope, limitations and new achievements. Colloids and
Surfaces A-Physicochemical and Engineering Aspects 123, 233-246
(1997); Goubault, C. et al. Shear rupturing of complex fluids:
Application to the preparation of quasi-monodisperse
water-in-oil-in-water double emulsions. Langmuir 17, 5184-5188
(2001); Okushima, S., Nisisako, T., Torii, T. & Higuchi, T.
Controlled production of monodisperse double emulsions by two-step
droplet breakup in microfluidic devices. Langmuir 20, 9905-9908
(2004)). This process allows control of both inner and outer
droplet volumes if the emulsions in both stages are monodisperse,
yet this process has not been used to form stable nanoscale
droplets (i.e. having both inner and outer droplet diameters that
are nanoscale). Moreover, this approach requires a difficult search
for surfactant combinations that can co-exist without destabilizing
either inner or outer droplet interfaces (Ficheux, M. F., Bonakdar,
L., Leal-Calderon, F. & Bibette, J. Some stability criteria for
double emulsions. Langmuir 14, 2702-2706 (1998)). Consequently,
there is a need for improving stability against evolution of the
droplet sizes (e.g. through coalescence and/or coarsening) and
reducing droplet sizes in the development of double emulsions for
applications (Benichou, A., Aserin, A., Garti, N. Double emulsions
stabilized with hybrids of natural polymers for entrapment and slow
release of active matters. Advances in Colloid and Interface
Science 108-109, 29-41 (2004)).
SUMMARY
[0016] An emulsion according to an embodiment of the current
invention includes a substantially continuous liquid medium, and a
plurality of droplet structures dispersed within said substantially
continuous liquid medium. Each droplet structure of the plurality
of droplet structures according to this embodiment of the current
invention includes an outer droplet of a first liquid having an
outer surface; an inner droplet of a second liquid within the first
droplet, the second liquid being immiscible in the first liquid,
wherein the inner and outer droplets have a boundary surface region
therebetween; an outer layer of block copolymers disposed on the
outer surface of the outer droplet; and an inner layer of block
copolymers disposed on the boundary surface region between the
outer and the inner droplets. The block copolymers include a
hydrophilic polymer block and a hydrophobic polymer block that act
in combination to stabilize the droplet structure, and the first
liquid is immiscible in the substantially continuous liquid
medium.
[0017] An emulsion according to an embodiment of the current
invention includes a liquid medium and a plurality of nano-droplets
dispersed within the liquid medium. Each of the plurality of
nano-droplets includes an inner droplet of a first liquid
surrounded by a second liquid, the first liquid being immiscible in
the second liquid and the second liquid being immiscible in the
liquid medium. The plurality of nano-droplets have an ensemble
average diameter of at least about 10 nm and less than about 200
nm.
[0018] An emulsion according to an embodiment of the current
invention includes a substantially continuous liquid medium and a
plurality of droplet structures dispersed within the substantially
continuous liquid medium. Each droplet structure of the plurality
of droplet structures includes a droplet of a liquid having an
outer surface, and a layer of block copolymers disposed on the
outer surface of the droplet. The block copolymers comprise a
hydrophilic polymer block and a hydrophobic polymer block that act
in combination to stabilize the droplet structure, and the liquid
of the plurality of droplet structures is immiscible in the
substantially continuous liquid medium.
[0019] A method of producing an emulsion according to an embodiment
of the current invention includes providing a first liquid and a
second liquid, the first liquid being immiscible in the second
liquid; adding a selected quantity of block copolymers to at least
one of the first and second liquids; and emulsifying the first
liquid in the second liquid to produce a plurality of droplets of
the first liquid dispersed in the second liquid. The block
copolymers stabilize said plurality of droplets from
coalescing.
[0020] A method of producing an emulsion according to an embodiment
of the current invention includes at least one of adding a
surfactant to at least one of a first liquid and a second liquid,
or adding surfactant precursors to at least one of the first liquid
and the second liquid; emulsifying the first liquid in the second
liquid to form a plurality of droplets of the first liquid immersed
in the second liquid to provide a simple emulsion, the first liquid
being immiscible in the second liquid; adding at least one of the
same surfactant or the same surfactant precursors to a third
liquid; and emulsifying the simple emulsion in the third liquid to
form a plurality of droplets of the simple emulsion to provide a
double emulsion, the second liquid being immiscible in the third
liquid. The plurality of droplets of the double emulsion each
comprises at least one droplet of the first liquid therein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Additional features of this invention are provided in the
following detailed description of various embodiments of the
invention with reference to the drawings. Furthermore, the
above-discussed and other attendant advantages of the present
invention will become better understood by reference to the
detailed description when taken in conjunction with the
accompanying drawings, in which:
[0022] FIGS. 1A and 1B show optical micrographs using differential
interference contrast (DIC) of 1% (w/v) suspensions of polypeptide
vesicles extruded through 1.0 .mu.m polycarbonate (PC) membranes
(Bars=5 .mu.m). (A)=K.sub.60L.sub.20 and (B)=E.sub.60L.sub.20. FIG.
1C shows TEM image of a uranyl acetate negatively stained 0.1%
(w/v) K.sub.60L.sub.20 vesicle suspension filtered through a 0.1
.mu.m PC membrane (Bar=350 nm). FIG. 1D shows the average diameter
of 1% (w/v) aqueous suspensions of vesicles of K.sub.60L.sub.20
(filled circles) and E.sub.60L.sub.20 (open diamonds) as a function
of PC membrane pore size. Vesicle diameters were determined using
dynamic light scattering (DLS).
[0023] FIGS. 2A and 2B show schematic representative block
copolypeptides used for emulsification, K.sub.xrL.sub.y and
K.sub.xL.sub.y, respectively. FIG. 2C is a schematic illustration
of emulsification processes according to some embodiments of the
current invention using K.sub.xrL.sub.y block copolypeptides to
generate water-in-oil-in-water double emulsions. FIG. 2D is a
schematic illustration of emulsification processes according to
some embodiments of the current invention using K.sub.xL.sub.y
block copolypeptides to generate single oil-in-water emulsions. For
FIGS. 2C and 2D, step (i) indicates ordinary emulsification such as
can be produced using a rotary mixer, to produce microscale
droplets, and step (ii) indicates more extreme emulsification, such
as can be produced using a microfluidic homogenizer, to produce
nanoscale droplets. A detailed section of the interfaces of a
resulting droplet structures, showing the copolypeptides at the
interfaces, for double and direct emulsions are also illustrated
schematically at the right side of FIGS. 2C and 2D,
respectively.
[0024] FIGS. 3A-3D show cryogenic transmission electron microscopy
(Cryo-TEM) images and dynamic light scattering (DLS) data for
K.sub.xrL.sub.y block copolypeptide double emulsions prepared using
a microfluidic homogenizer (Microfluidics Microfluidizer.RTM. 110S
equipped with 75 .mu.m channel dimension interaction chamber). All
emulsions were prepared under the following conditions: number of
discrete passes through microfluidic homogenizer N=6, input air
pressure to microfluidic homogenizer p=130 psi, copolypeptide
concentration in water C=1 mM and total oil volume fraction
.phi.=0.20. The oil is polydimethylsiloxane (PDMS) silicone oil
having 10 cSt viscosity. Droplet radii from DLS data were
determined using cumulant analysis. Cryo-TEM images of
K.sub.xrL.sub.y emulsions (Bars=100 nm), (A)=K.sub.40rL.sub.5,
(B)=K.sub.40rL.sub.10, (C)=K.sub.40rL.sub.20, and
(D)=K.sub.40rL.sub.30. FIG. 3E shows the measured effective droplet
radii (near the outer droplet radii) of double emulsions by DLS (in
nanometers) as a function of the length of the K and rL blocks in
the copolypeptide. FIG. 3F is a histogram (i.e. probability
distribution) of the ratio of inner to outer droplet radii for
K.sub.40rL.sub.10 measured from Cryo-TEM images. FIG. 3G shows
average droplet radius, <a> (nm) as a function of block
copolymer concentration C in (mM) for K.sub.40rL.sub.10. This
concentration corresponds to the aqueous phases in which this
copolypeptide is soluble. FIG. 3H shows average droplet radius,
<a> (nm) as determined by DLS, as a function of
racemic-leucine (rL) block length at a fixed K.sub.40 block
length.
[0025] FIGS. 4A-4D show images detailing methods to tune the sizes
of W/O/W double emulsion droplets. FIG. 4A shows laser confocal
scanning microscopy (LCSM) images of 0.1 mM FITC labeled
K.sub.40rL.sub.10 emulsion (total oil volume fraction .phi.=0.20,
10 cSt PDMS silicone oil) prepared using an ultrasonic tip
homogenizer (Bar=5 .mu.m). FIGS. 4B-4D are emulsions prepared using
a microfluidic homogenizer (75 .mu.m interaction chamber). (B)
Cryo-TEM image of a K.sub.40rL.sub.10 emulsion: N=6, p=130 psi, C=1
mM and .phi.=0.20 (Bar=100 nm). (C) Cryo-TEM image of the plug
isolated by ultracentrifugation of a K.sub.40rL.sub.20 emulsion:
N=6, p=130 psi, C=1.5 mM and .phi.=0.20, (Bar=100 nm). (D) Remnant
suspension of smaller double nanoenmulsions obtained by
ultracentrifugation and separation of a K.sub.40rL.sub.20 emulsion:
N=6, p=130 psi, C=1.5 mM and .phi.=0.20, (Bar=100 nm). FIG. 4E
shows average droplet radius, <a> of K.sub.40rL.sub.20
emulsions, determined by DLS as a function of copolypeptide
concentration: N=6, p=130 psi, C=0.1 to 1.5 mM and .phi.=0.20. FIG.
4F shows average droplet radius, <a>, of K.sub.40rL.sub.y
determined by DLS as a function of racemic-leucine (rL) block
length, y, for: N=6, p=130 psi, C=1 mM and .phi.=0.20.
[0026] FIG. 5A shows an LCSM image of a FITC labeled 0.1 mM
K.sub.40rL.sub.10 double emulsion (.phi.=0.20, 10 cSt PDMS silicone
oil) prepared using a ultrasonic tip homogenizer (10 see, Bar=5
.mu.m). The FITC-labeled copolypeptide fluoresces green. FIG. 5B
shows multi-color fluorescence micrograph overlay of a FITC labeled
C=0.1 mM K.sub.40rL.sub.10 double emulsion prepared using a
ultrasonic homogenizer (10 sec) with 0.01 M pyrene (fluoresces
blue) in the outer droplets of 10 cSt silicone oil (.phi.=0.20),
and nanoscale InGaP/ZnS quantum dots (fluoresce red) in the inner
aqueous droplets (Bar=5 .mu.m).
[0027] FIGS. 6A-6B show LCSM images of FITC labeled 0.1 mM
copolypeptide double emulsions (.phi.=0.20, 10 cSt PDMS silicone
oil) prepared using an ultrasonic tip homogenizer (10 sec).
(A)=K.sub.60L.sub.20, (B)=K.sub.40rL.sub.10. FIG. 6C shows a
cryo-TEM image of a 1.0 mM E.sub.40rL.sub.10 emulsion prepared
using a microfluidic homogenizer: N=6, p=130 psi, C=1 mM and
.phi.=0.20 (Bar=250 nm). FIG. 6D shows Cryo-TEM image of a 1.0 mM
K.sub.40rL.sub.10 prepared using a microfluidic homogenizer: N=6,
p=130 psi, C=1 mM and .phi.=0.20 (Bar=250 nm). FIG. 6E shows a
photograph of phase separation of silicone oil and water due to
non-emulsification of the oil with the water-soluble K.sub.60
homopolymer polypeptide after similar attempts to make emulsions
and/or double emulsions using similar external excitation at
similar polypeptide concentrations and total oil volume fractions
as in the other panels.
[0028] FIGS. 7A-7C show CTEM images for K.sub.x(rac-L).sub.y
stabilized double emulsions prepared using a microfluidic
homogenizer under the following conditions: number of passes N=6,
homogenizer inlet air pressure p=130 psi, block copolypeptide
concentration C=1.0 mM, and total oil volume fraction .phi.=0.20
(PDMS silicone oil 10 cSt). (Bars=200 nm):
(a)=K.sub.40(rac-L).sub.5, (b)=K.sub.40(rac-L).sub.10, and
(c)=K.sub.40(rac-L).sub.30. FIG. 7D shows a histogram displaying
the observed probability distribution (in %) as a function of the
ratio of inner radius a.sub.i to outer radius a.sub.o (i.e. I/O
ratio) determined by measuring a.sub.i and a.sub.o from at least 50
double emulsion droplets observed in a cryo-TEM image of a
K.sub.40(rac-L).sub.10 emulsion.
[0029] FIGS. 8A-8D show cryo-TEM (CTEM) images of various block
copolypeptides used to stabilize double emulsions. CTEM images of
(FIG. 8A) R.sub.40(rac-L).sub.10 (R=L-arginine hydrobromide used in
the polymerization) and (FIG. 8B) E.sub.40(rac-L).sub.10
(E=L-glutamic acid sodium salt used in the polymerization)
stabilized double emulsions prepared using a microfluidic
homogenizer under the following conditions: N=6, homogenizer inlet
air pressure p=130 psi, block copolypeptide concentration C=1.0 mM,
and oil volume fraction .phi.=0.20 (PDMS silicone oil 10 cSt). CTEM
images of (FIG. 8C) K.sub.60(rac-V).sub.20 (V=valine) and (FIG. 8D)
K.sub.60(rac-A).sub.20 (A=alanine) stabilized double emulsions
created using an ultrasonic homogenizer for 1 minute with, block
copolypeptide concentration C=1.0 mM, and oil volume fraction
.phi.=0.20 (PDMS silicone oil 10 cSt). All scale bars=200 nm.
[0030] FIGS. 9A and 9B show comparison of emulsification properties
of copolypeptides. (FIG. 9A) Photograph of emulsions containing
toluene as the oil phase using K.sub.60L.sub.20 and
K.sub.40(rac-L).sub.20 surfactants created using an ultrasonic
homogenizer for 1 minute with block copolypeptide concentrations
C=0.1 mM, and oil volume fractions .phi.=0.20. The image was taken
3 hours after emulsification, where the K.sub.60L.sub.20 sample
showed noticeable phase separation (oil layer at top). (FIG. 9B)
Photograph of attempted emulsification of PDMS silicone oil and
water using the homopolypeptide K.sub.60 as a surfactant. This
sample rapidly and completely phase separated, indicating that the
homopolymer polypeptide K.sub.60 did not provide adequate
stabilization of oil-water interfaces and any droplets that were
transiently produced during the excitation rapidly coalesced after
that excitation was ceased.
[0031] FIGS. 10A-10C are plots of dynamic light scattering (DLS)
data showing how double emulsion droplet sizes are affected by
different experimental parameters. All samples were prepared using
a microfluidic homogenizer (75 .mu.m interaction chamber) under the
following conditions: number of passes N=6, homogenizer inlet air
pressure p=130 psi. Diameters were determined using cumulant
analysis of the (DLS) correlation function and are estimates of
average outer droplet diameters of the W/O/W double emulsions.
(FIG. 10A) Plot of measured average diameter of double droplet
structures vs. K.sub.40(rac-L).sub.20 block copolypeptide
concentration C. (FIG. 10B) Plot of measured average diameter vs.
oil volume fraction .phi.. (FIG. 10C) Plot of average diameter of
double droplet structures vs. hydrophobic (rac-L) length obtained
by varying x in different samples of K.sub.40(rac-L).sub.x.
[0032] FIGS. 11A-11D are fluorescence microscopy and CTEM images
showing influence of silicone oil capped with acetamide groups
(PBA) on hydrogen bonding in the oil phase of emulsions. (FIG. 11A)
Fluorescence microscopy image of W/O/W double emulsions stabilized
using fluorescently dyed FITC-K.sub.60(rac-L).sub.20 containing
multiple inner water droplets (note that the L-block is racemic):
C=0.1 mM, PBA oil volume fraction .phi.=0.20 created using an
ultrasonic tip homogenizer for 10 seconds. (FIG. 11B) Fluorescence
microscopy image of single O/W emulsions stabilized with
FITC-K.sub.60L.sub.20 (note that the L block is not racemic): C=0.1
mM, PBA oil volume fraction .phi.=0.20 created using an ultrasonic
tip homogenizer for 10 seconds. (FIG. 11C) CTEM image of nanoscale
W/O/W double emulsion droplets with multiple inner water droplets
prepared with PBA as the oil phase. (FIG. 11D) CTEM image of
nanoscale double emulsion droplets using 300 cSt PDMS (identical
viscosity to PBA) as a control oil phase where single inner aqueous
droplets are dominant. Emulsion samples for (11C) and (11D) were
prepared with K.sub.60(rac-L).sub.20 using a microfluidic
homogenizer (75 .mu.m interaction chamber) under the following
conditions: number of passes N=6, homogenizer inlet air pressure
p=130 psi, block copolypeptide concentration C=1.0 mM, and oil
volume fraction .phi.=0.20. Scale bars: (11A) and (11B)=5 .mu.m;
(11C) and (11D)=100 nm. PBA=bis[3-(acetamido)-propyl]terminated
polydimethylsiloxane (number-weighted molecular weight
M.sub.n=2,500, and viscosity 300 cSt).
[0033] FIGS. 12A-12C show cryogenic transmission electron
microscopy (CTEM) images for copolypeptide-stabilized single and
double emulsions prepared using a microfluidic homogenizer, showing
how the racemic nature of the L-block can influence the type of
emulsion generated. Vitrified water gives a lighter background and
silicone oil, which has a greater density of higher atomic number
atoms, appears darker and provides contrast. Emulsions were
prepared under the following conditions: N=6, p=130 psi, C=1.0 mM,
and oil volume fraction .phi.=0.20 (PDMS silicone oil 10 cSt).
(FIG. 12A) CTEM image of a K.sub.40(rac-L).sub.20 stabilized W/O/W
double emulsion. (FIG. 12B) CTEM image of a K.sub.60L.sub.20
stabilized single O/W emulsion. (FIG. 12C) CTEM image of
size-fractionated droplets isolated from a K.sub.40(rac-L).sub.20
stabilized double emulsion by low speed centrifugation followed by
ultracentrifugation (using a Beckman ultracentrifuge with SW28
swinging bucket rotor and typical speeds from about 3,000 to about
25,000 RPM). All bars=200 nm.
[0034] FIGS. 13A and 13B show fluorescence micrographs of double
emulsions containing polar and nonpolar cargos. Samples prepared
using an ultrasonic tip homogenizer (10 sec at 35% power) with
.phi.=0.2 and C=0.1 mM. The oil phase fluoresces blue due to
entrapped pyrene (0.01 M), and an internal aqueous phase, if
present, fluoresces red due to encapsulation of InGaP quantum dots
(at concentration of 2 .mu.M). The polypeptides fluoresce green due
to labelling with fluorescein (FITC). Before imaging, the droplets
were dialyzed against and subsequently diluted with pure water to
remove most of the quantum dots and therefore red fluorescence from
the external continuous aqueous phase. (FIG. 13A) FITC-labeled
K.sub.40(rac-L).sub.10 stabilized water-in-oil-in-water double
emulsion loaded with both pyrene (blue fluorescence) in the outer
oil droplets and quantum dots in the inner water droplets (red
fluorescence). (FIG. 13B) FITC-labeled K.sub.60L.sub.20 stabilized
oil-in-water emulsion loaded with pyrene (blue fluorescence) in the
oil droplets. Because K.sub.60L.sub.20 forms a direct emulsion, no
red fluorescence is seen within the droplets, confirming the
absence of inner water droplets for this particular composition.
Both scale bars are .mu.m.
[0035] FIG. 14 shows circular dichroism spectra of block
copolypeptide solutions (1.0 mg/mL) in ultrapure water. The minima
at 208 and 222 nm in the (rac-K).sub.60L.sub.20 sample are
characteristic of the .alpha.-helical conformation.
.diamond-solid.=(rac-K).sub.60L.sub.20, and
c=(rac-K).sub.40(rac-L).sub.20.
DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS
[0036] Emulsions are dispersions of droplets of one liquid phase
material in another immiscible liquid phase material that can be
formed, typically through flow-induced rupturing of bigger droplets
into smaller ones. A surfactant, which consists of amphiphilic
molecules which are surface-active, which is soluble in at least
one liquid phase, and which prefers adsorbing on the interfaces
between the two immiscible liquids, is usually added in order to
prevent subsequent droplet coalescence (i.e. fusion) and to keep
the size distribution of the droplets from changing over time.
Simple emulsions are generally classified as oil-in-water (i.e. O/W
or "direct") and water-in-oil (i.e. W/O or "inverse"), and these
different morphologies can be obtained by using an appropriate
surfactant that provides adequate stability and can sometimes be
influenced through the order of addition of the components while
shearing.
[0037] The following prefixes: racemic-, r, r-, and rac-, and
similar common prefix abbreviations, are used interchangeably to
refer to racemic forms of amino acids, oligopeptides, and
polypeptide blocks throughout this specification. Likewise,
abbreviations cryo-TEM and CTEM are used to refer to cryogenic
transmission electron microscopy. The variables p and P are used
interchangeably to refer to the same input gas pressure to the
microfluidic homogenizer, and .phi. and q are used interchangeably
to refer to the total oil volume fraction.
[0038] Oil-in-water emulsions comprised of microscale droplets are
common products and have been made for centuries. A simple example
is mayonnaise, typically made from egg yolk, which contains both
stabilizing amphiphilic lipid and protein molecules, and olive oil
that is added in a thin stream while beating the mixture with a
whisk or spoon. Some of the mechanical shear energy is stored in
the additional droplet interfacial area that is created as the
droplets are ruptured down to a smaller size. Typical mechanical
devices can produce shear rates that can achieve droplet rupturing
down to droplet diameters that are typically around three hundred
nanometers, but it is very difficult to achieve a reduction of the
peak in the size distribution below this limit. Historically,
sub-micron emulsions are known as "mini-emulsions", and these have
been created using microfluidic and ultrasonic means for the past
twenty years. The term emulsifying used herein is intended to have
a broad meaning that can include the process of exciting two
immiscible liquids (each of which can include additional components
mixed, blended, and/or suspended therein) which are placed in
proximity and/or contact, and introducing some form of non-thermal
energy to excite and rupture interfacial boundaries between the two
liquids in order to form discrete droplets of one immiscible liquid
substantially surrounded by the other immiscible liquid. While
emulsifying, bigger droplets are typically broken down into smaller
droplets (e.g. through interfacial "capillary" instabilities that
develop when larger droplets become significantly deformed),
thereby forming additional interfacial area. Moreover, while
emulsifying, single emulsions, double emulsions, higher-ordered
multiple emulsions, and combinations thereof can be formed. The
aforementioned methods of emulsifying provide extremely high shear
or flow rates that can stretch and rupture even very small
droplets. Indeed, there are reports in the literature of the use of
ultrasonic dispersers or microfluidic homogenizers that have
obtained droplets down into the nanoscale domain: the average
droplet sizes are below 100 nm. There is some ambiguity in whether
"size" refers to radius or diameter, but this factor of two is a
very minor issue, considering the wide range of droplet sizes that
can exist from the micellar scale of 2-3 nm all the way up to
droplets having macroscopic dimensions.
[0039] Here we show that W/O/W double emulsions can be prepared in
a simple process and stabilized over many months using
single-component, synthetic amphiphilic diblock copolypeptide
surfactants according to some embodiments of the current invention.
These surfactants even stabilize droplets subjected to extreme
flow, leading to direct, mass-production of robust W/O/W double
emulsions that have nanoscale inner droplets and also nanoscale
outer droplets, and are therefore are amenable to nanostructured
encapsulation applications in foods, cosmetics, and drug
delivery.
[0040] Since amphiphilic diblock polypeptides could function also
as surfactants on oil-water interfaces, as well as have properties
of self-assembly that reflect their propensity to form vesicles, we
have examined the possibility of generating stable double emulsions
using minimal shear and a single interfacial agent that is not
biased against complex droplet topologies according to some
embodiments of the current invention.
[0041] We find that it is possible to make direct emulsions and
nanoemulsions, as well as double emulsions and double
nanoemulsions, stabilized by amphiphilic diblock copolypeptides
according to some embodiments of the current invention. Results
according to some embodiments of the current invention actually
indicate that, for a wide range of molecular weights of the
hydrophilic blocks and hydrophobic blocks, the preferred morphology
after applying the shear is the double emulsion. Double emulsions
can provide a drug delivery vehicle, for example, that can package
both water-soluble drugs and oil-soluble drugs. Moreover, the
copolypeptide that stabilizes the double emulsion droplets can also
be engineered and tailored to provide desirable biochemical
interactions, such as biological cell targeting, cellular and
subcellular membrane disruption, and enzymatic funtionalities,
which can enhance the delivery and performance of drug molecules
that may be incorporated into the droplet structure.
[0042] A method of producing emulsions, emulsions produced and
droplet structures within the emulsions according to some
embodiments of the current invention are illustrated schematically
in FIGS. 2C and 2D. The method according to an embodiment of the
current invention includes providing a first liquid and a second
liquid, the first liquid being immiscible in the second liquid,
adding a selected quantity of block copolymers to at least one of
the first and second liquids, and emulsifying the first liquid in
the second liquid to produce a plurality of droplets of the first
liquid dispersed in the second liquid. The block copolymers act to
stabilize the plurality of droplets against coarsening or other
structural evolution that could potentially occur through
coalescence or other destabilizing mechanisms. The block copolymers
can be, but are not limited to, block copolypeptides. Furthermore,
the block copolypeptides can be formed from natural occurring
and/or synthetic monomers. The first and second liquids can be an
oil (e.g. non-polar) and water (e.g. polar), for example, or vice
versa, according to some embodiments of the current invention.
However, the invention is not limited to just oil and water as the
only pair of immiscible liquids. Other types of polar and non-polar
liquid-like materials that are immiscible (e.g. potentially even
fluorinated-oils and hydrocarbon-oils that are immiscible) may be
used according to other embodiments of the current invention.
Therefore, when the terms hydrophobic and hydrophilic are used in
this specification and the claims, these terms are more broadly
intended to refer to relative differences in molecular interactions
between different types of immiscible liquids or immiscible
liquid-like materials.
[0043] An emulsion according to an embodiment of the current
invention can include a substantially continuous liquid medium, and
a plurality of droplet structures dispersed within said
substantially continuous liquid medium. FIGS. 2C and 2D show two
examples of emulsions: double (2C) and direct (2D). However, higher
order emulsion can be included according to other embodiments of
the current invention. A droplet structure according to an
embodiment of the current invention can include an outer droplet of
a first liquid having an outer surface, an inner droplet of a
second liquid within the first droplet, the second liquid being
immiscible in the first liquid, wherein the inner and outer
droplets have a boundary surface region therebetween. The droplet
structure can also include an outer layer of block copolymers
disposed on the outer surface of the outer droplet, and an inner
layer of block copolymers disposed on the boundary surface region
between the outer and the inner droplets. The term "disposed on" is
intended to be a general term which can include, but is not limited
to, adsorption onto the surface. For example, the block copolymers
may have a portion extending into a portion of the droplet and
another portion extending out of the droplet at the surface region,
as is illustrated schematically in the droplets on the right hand
side of FIGS. 2C and 2D. The term "layer" is intended to be a broad
term that includes case in which the block copolymers are loosely
arranged around the surface region of the droplet, which can
include cases in which the layer is permeable as well as cases in
which the layer does not form a completely enclosed surface. The
block copolymers can include a hydrophilic polymer block and a
hydrophobic polymer block that act in combination to stabilize the
droplet structure. The block copolymer layers shown are
schematically representative and may not be to scale. The first
liquid is immiscible in the substantially continuous liquid medium
according to this embodiment of the current invention.
[0044] For most compositions that form stable double emulsions, the
block copolymer disposed on the interfaces provides a repulsive
potential energy of interaction that is significantly stronger than
the "thermal energy" corresponding to equilibrium thermal
fluctuations, k.sub.BT, where k.sub.B is Boltzmann's constant and T
is the temperature, between the interfaces of inner droplets and
the interfaces of the outer droplets that contain them according to
some embodiments of the current invention. In addition, for most
compositions that form stable double emulsions, the block copolymer
also provides a repulsive potential energy of interaction between
the interfaces of outer droplets that encounter other outer
droplets that is significantly stronger than thermal energy. For
some compositions that form stable double emulsions, the block
copolymer can provide a repulsive potential energy of interaction
between the interfaces of multiple inner droplets within an outer
droplet that is significantly greater than thermal energy. The
aforementioned repulsive potential energy of interaction
notwithstanding, it is possible for an attractive potential energy
of interaction between liquid interfaces to also exist. This
attractive potential energy of interaction can lead to aggregation
of outer droplets, inner droplets, or a combination thereof in a
manner that would not cause coalescence, would not cause film
rupturing, and would not destroy the integrity of droplet
structures in single, double, and multiple emulsions. Any such
attractive potential of interaction may lead to the formation of a
secondary minimum in the interaction potential between droplet
interfaces, yet this does not necessarily imply that the droplet
structures would be destabilized. An example of such an attractive
potential energy of interaction is a depletion attraction that may
arise between outer droplets as a result of excess copolymer
content that may be present in the continuous liquid phase.
[0045] For W/O/W double emulsions, although the interfacial
stability of an oil film between the surface of the inner water-oil
interface and the surface of the outer oil-water interface
conferred by adsorbed amphiphilic molecules at the interfaces can
be important for ensuring long-term stability of the droplets
against evolution of the inner and outer droplet sizes, additional
potential factors could influence the long-term stability of the
droplets. One potential factor is the potential presence or absence
of an osmotic pressure and/or osmotic pressures due to either
hydrophilic contents and/or hydrophobic contents of materials
loaded into the liquid phases within the double emulsion droplets.
For instance, in some methods of producing double emulsions, it is
possible for an inner water droplet to contain excess
copolypeptide, some of which resides in the water phase of the
inner droplet, not just at the water-oil interface at the surface
of the inner water droplet. This excess polymer could create an
osmotic pressure. Sometimes having such an osmotic pressure created
by a water-soluble material and/or water-dispersed material could
be desirable for stabilizing droplets against longer-term
coarsening processes such as Ostwald ripening. Another potential
factor is the potential presence of a soluble material in the
continuous water phase outside all of the double droplets that
could potentially create an osmotic pressure. Yet another potential
factor is the potential presence of an oil-soluble material and/or
oil-dispersed material in the outer oil droplets that could
potentially create an osmotic pressure. Relative differences in
these potential osmotic pressures and the relative solubilities of
the oil, water, and other materials in each of the respective other
materials can also potentially have an influence on the migration
of materials that could potentially change the sizes of inner and
outer droplets. The long-term observations we have made for some
double emulsion compositions indicate that significant stability of
both inner droplet and outer droplet sizes can be achieved in
certain embodiments of the current invention.
[0046] Concurrent with or subsequent to emulsification of double
emulsion droplets, alteration of the liquid material in the inner
droplets could be created and used to solidify or otherwise
introduce elastic structures in the inner droplet material.
Concurrent with or subsequent to emulsification of double emulsion
droplets, alteration of the liquid material in the outer droplets
could be created and used to solidify or otherwise introduce
elastic structures in the outer droplet material. Concurrent with
or subsequent to emulsification of double emulsion droplets,
alteration of the continuous liquid material outside the double
droplets could be created and used to solidify or otherwise
introduce elastic emulsion structures in the continuous liquid
material. Concurrent with or subsequent to emulsification of double
emulsion droplets, alteration of the liquid material of inner
droplets, of the liquid material of outer droplets, or of the
liquid material of the continuous phase, or a combination thereof,
could be created and used to solidify, create structural changes,
and/or otherwise introduce elasticity in the double droplet
structures. Said alteration could consist of structural changes
and/or solidification induced by phase changes (e.g. induced by
temperature changes), gelation, crosslinking, polymerization,
photopolymerization, chemical reactions, increase in volume
fraction of soluble and/or dispersed species (e.g. through
transport of lower molecular weight materials in the inner droplet
and/or outer droplet), jamming of dispersed species, glassilication
of dispersed species (e.g. by inducing an attraction between
dispersed species), and self-assembly. Likewise, concurrent with or
subsequent to emulsification of double emulsion droplets,
structural changes that influence the elasticity of a layer of
amphiphilic molecules adsorbed at the interfaces of the inner
and/or outer droplets could also be altered and controlled. Such
alteration might be achieved by selecting amphiphilic molecules
that can potentially crosslink to create an elastic layer at the
interface of inner droplets and/or outer droplets. Such
crosslinking might be induced by electromagnetic radiation, heat,
chemical reactions, or a combination thereof.
[0047] There are many classes of drug molecules, and the
classification can be made in different ways by emphasizing
different criteria. Some drug molecules are hydrophobic, some drug
molecules are hydrophilic, and some drug molecules even possess a
significant degree of amphiphilic nature. By referring to drug
molecules, we include all types of molecules that can be used to
interact with and affect the viability and function of biological
structures and biological entities, including but not limited to
biomolecules, sub-cellular structures, biomembranes, cytoplasm,
nucleus, extracellular matrix, organelles, cells, synapses,
tissues, organs, and organisms.
[0048] Drugs, such as drug molecules or formulations of a plurality
of drug molecules, that could be introduced into the liquid phases
of emulsions, double emulsions, and multiple emulsions include but
are not limited to: anti-perspirant drugs, anti-itch drugs,
anti-infection drugs, anti-inflammatory drugs, anti-arthritis
drugs, anti-bursitis drugs, anti-acne drugs, anti-pain drugs,
anti-headache drugs, anti-migraine drugs, anti-influenza drugs,
anti-depression drugs, anti-diabetes drugs, anti-viral drugs,
anti-venin drugs, anti-fungal drugs, anti-(methicillin resistant
staphylococcus aureus) drugs, anti-biotic drugs, anti-bacterial,
anti-microbial, anti-hunger drugs, anti-malnutrition drugs,
anti-(acquired immunodeficiency syndrome) drugs, anti-(human
immunodeficiency virus) drugs, anti-herpes drugs, anti-hepatitis
drugs, anti-spirochete drugs, anti-(Lyme disease) drugs,
anti-cholesterol drugs, anti-dandruff drugs, anti-(hair loss)
drugs, anti-dermatitis drugs, anti-swelling drugs, anti-addiction
drugs, anti-dementia drugs, anti-(Alzheimer's disease) drugs,
anti-(Parkinson's disease) drugs, anti-prion drugs, anti-(urinary
tract infection) drugs, anti-schizophrenia drugs, anti-hemorrhoid
drugs, anti-worm drugs, anti-cancer drugs, anti-seizure drugs,
anti-epileptic drugs, anti-manic drugs, anti-anxiety drugs,
anti-histamine drugs, anti-coagulant drugs, anti-septic drugs,
anti-bacterial drugs, anti-tuberculosis drugs, anti-insomnia drugs,
anti-fibromyalgia drugs, anti-incontinence drugs, anti-dermatitis
drugs, anti-angiogenesis drugs, anti-allergy drugs, anti-(hay
fever) drugs, anti-asthma drugs, anti-(high blood pressure) drugs,
anti-(blood clotting) drugs, anti-(motion sickness) drugs,
anti-(weight gain) drugs, anti-(weight loss) drugs, anti-obesity
drugs, anti-flatulence drugs, anti-burp drugs, anti-constipation
drugs, anti-malaria drugs, anti-wart drugs, anti-(skin burn) drugs,
anti-(skin sunburn) drugs, anti-(skin wrinkle) drugs, anti-hives
drugs, anti-conjunctivitis drugs, anti-(skin boil) drugs,
anti-(cold sore) drugs, anti-psychotic drugs, anti-(skin cancer)
drugs, anti-eczema drugs, anti-anemia drugs, anti-jaundice drugs,
anti-encephalitis drugs, anti-dementia drugs, anti-(premenstrual
pain) drugs, anti-chlamydia drugs, anti-protozoan drugs,
anti-thrombosis drugs, anti-toothache drugs, anti-earache drugs,
anti-tuberculosis drugs, anti-bronchitis drugs, anti-pneumonia
drugs, anti-polio drugs, anti-tetanus drugs, anti-(venereal
disease) drugs, anti-(attention deficit disorder) drugs, anti-(lip
chapping) drugs, anti-osteoporosis drugs, anti-(heart disease)
drugs, anti-(heart attack) drugs, anti-(heart failure), anti-stroke
drugs, anti-arrhythmia drugs, anti-(peripheral artery disease)
agents, anti-platelet agents, anti-anginal drugs, anti-ageing
drugs, anti-(memory loss) drugs, anti-hypertension drugs,
anti-psoriasis drugs, anti-anorexia drugs, anti-diarrhea drugs,
anti-gout drugs, anti-hypothyroid drugs, anti-(organ transplant
rejection) drugs, anti-parasite drugs, anti-(erectile dysfunction)
drugs, anti-vaginitis drugs, anti-(hot flash) drugs, insect and
spider repellants, anesthesia agents, hormones, enzymes, catalysts,
inhibitors, promoters, moisturizers, vitality enhancers, skin
regeneration agents, skin re-growth agents, hair growth agents,
hair re-growth agents, attention enhancers, muscular strength
enhancers, male potency enhancers, female fertility enhancers,
birth control agents, decongestants, anesthetic agents, ocular
treatment agents, smoking cessation enabling agents, nicotine
substitution agents, penicillin-related drugs,
cephalosporin-related drugs, sulfa-related drugs, mycin-related
drugs, endocrine drugs, cardiovascular drugs, pulmonary drugs,
central nervous system drugs, gastrointestinal drugs, muscle
relaxant drugs, sedative drugs, tranquilizers, hypnotic drugs,
analgesic drugs, general anesthetic drugs, vaccines,
menopause-related drugs, and diuretic drugs.
[0049] Imaging enhancement agents that could be introduced into the
liquid phases of emulsions, double emulsions, and multiple
emulsions include but are not limited to: magnetic resonance
imaging (MRI) enhancement agents, x-ray computerized tomography
(CT) enhancement agents, positron emission tomography (PET)
enhancement agents, ultrasound imaging enhancement agents, and
optical imaging enhancement agents.
[0050] By non-thermal energy, we mean all forms of energy that are
not related to equilibrium fluctuations of the constituents of an
emulsion system, regardless of whether the emulsion system is a
single emulsion or a double emulsion. For instance,
out-of-equilibrium imbalances in the local concentrations of
constituent materials in an emulsion system could lead to entropic
driving stresses that are strong enough to cause droplets of one
liquid material to form in another immiscible liquid material
without the direct application of external viscous flows. This kind
of "spontaneous emulsification" results from local differences in
the chemical potential of constituents within the emulsion system
that can potentially be strong enough to drive the formation of
droplets. Consequently, we regard the restructuring processes
giving rise to "spontaneous emulsification" as a form of
non-thermal energy, even if there may be some debate about this
classification in the prior art. Moreover, "spontaneous
emulsification" and other non-equilibrium imbalances in the
chemical potential of the species in an emulsion system could also
be used to drive the formation of co-polypeptide stabilized double
emulsions. Thus, we include entropic driving stresses that lead to
spontaneous emulsification and other forms of non-equilibrium
transport processes (such as heat- and convection-generating
chemical reactions) in what we intend as forms of non-thermal
energy.
[0051] FIG. 2C shows an example of producing double emulsions
according to some embodiments of the current invention. FIG. 2D
shows an example of producing single emulsions according to some
embodiments of the current invention. The invention is not limited
to only direct and double emulsions and is not limited to double
emulsions in which droplets have only a single inner droplet.
Triple and higher order emulsions are intended to be included
within the scope of the current invention. In addition, double
emulsions that have one, two or more than two droplets within the
larger droplets are included within the scope of the current
invention. In the examples of FIGS. 2C and 2D, the double and
direct emulsions resulting after step i can be further processed
with a microfluidic homogenizer (Microfluidizer.RTM. 110S with 75
.mu.m microchannel interaction chamber) to reduce the droplet sizes
(e.g. see after step ii in FIGS. 2C and 2D). Various additional
processing after the initial emulsification can also be performed
within the scope of the current invention.
[0052] The amphiphilic block copolypeptides, K.sub.xrL.sub.y, where
rL (or, equivalently rac-L) signifies a racemic oligoleucine
domain, were synthesized using transition metal mediated
polymerization of .alpha.-amino acid N-carboxy anhydrides (T. J.
Deming, Macromolecules 32, 4500 (1999)). The block copolypeptide is
composed of a random coil, positively charged poly L-lysine block
bound to a racemic oligoleucine block that lacks a stable secondary
structure (FIG. 2A). In order to form an emulsion according to an
embodiment of the current invention, we began by dissolving a block
copolypeptide in water at a desired concentration (C), followed by
the addition of oil to give a particular final oil volume fraction
.phi., (FIG. 2C). For copolypeptides typically having racemic
hydrophobic blocks, application of shear using a handheld rotary
shearing wand (IKA Ultra-Turrax T8 with the S8N-8G dispersing
element) formed a microscale premixed emulsion composed of
polydisperse (W/O/W) double emulsion droplets ranging in size from
1 to 20 .mu.m (e.g. FIG. 2C after step i). (Note that the double
emulsion at this stage was obtained according to this embodiment of
the invention without a two-stage emulsification or microfluidic
emulsifier as has been described in some conventional processes in
the prior art.) This premixed emulsion was then fed into a
high-pressure microfluidic homogenizer (e.g. Microfluidizer.RTM.
Model 110S) typically having an inlet gas pressure p=130 psi
(corresponding to liquid pressures in the interaction chamber that
are roughly 240 times this inlet gas pressure), which sheared the
large droplets into sub-micron and nanoscale droplets (e.g. FIG. 2C
after step ii). Optionally, in order to obtain increasingly more
monodisperse droplets, the resulting sub-micron and nanoscale
emulsion can be re-introduced into the microfluidic homogenizer for
N multiple passes, where the integer N is the pass number. This
method allows for a straightforward way to produce bulk quantities
of sub-micron and nanoscale double emulsion droplets (FIG. 2C).
Although for this method of emulsification, we typically perform
the emulsifying by using N discrete passes through the microlluidic
homogenizer, there are alternative methods of continuous
recirculation of the emulsion through the interaction chamber that
would also be suitable for performing the emulsifying (i.e. through
continuous recirculation emulsification). The use of multiple
passes and/or recirculation can have desirable consequences of
reducing the overall diameters of the droplet structures and also
reducing the polydispersity of the droplet size distributions. In a
similar manner, by altering the hydrophobic block of the
copolypeptide to be non-racemic by controlling and tailoring the
polymer synthesis and then following the same physical
emulsification process, direct oil-in-water emulsions that are
coated with copolypeptide can also be formed (FIG. 2D).
[0053] Cryogenic transmission electron microscopy (cryo-TEM or
CTEM) can be used to observe and image unperturbed droplet
structures in both double and direct emulsions that have been
rapidly vitrified in ice without having to introduce staining
agents. The images show that double emulsions are indeed formed for
a variety of K.sub.xrL.sub.Y polypeptide surfactants at 1.0 mM
(FIGS. 3A-D). Also, in the cryo-TEM images of K.sub.40rL.sub.20 and
K.sub.40rL.sub.30 in particular, there exists a large population of
droplets having diameters of about 100 nm and smaller (FIGS. 3C and
3D, respectively). This is surprising given the relatively low
concentration of block copolypeptide in solution. In addition, the
emulsions produced according to this embodiment of the current
invention show some interesting trends concerning the structure of
the inner droplet. For many liquid and copolypeptide compositions
passed through the microfluidic homogenizer, only one inner aqueous
droplet is formed per oil droplet. The efficiency of this process
can be very high (>95%). From these images (FIGS. 3A-D), there
is a relatively consistent ratio of inner to outer droplet radius
for all double droplets in these samples. A histogram detailing the
probability of observing a dimensionless ratio (i.e. "I/O ratio")
given by: the radius of the inner droplet a.sub.i divided by the
radius of the outer droplet a.sub.o containing it, is shown in FIG.
3F. The histogram shows a consistent average value of about
<a.sub.i/a.sub.o>.apprxeq.0.5 (i.e. 50%) corresponding to a
monomodal peak for a K.sub.40rL.sub.10 emulsion (FIG. 3F). Indeed,
although there are variations in a.sub.i and a.sub.o from droplet
to droplet, this average ratio of
<a.sub.i/a.sub.o>.apprxeq.0.5 also was observed for some
other compositions of double emulsions that have been imaged. In
addition, dynamic light scattering (DLS) results of the
hydrodynamic radii for emulsions made using a variety of block
copolypeptides confirm that sub-micron droplets are formed for a
large range of block copolypeptide compositions (FIGS. 3E, 3G, and
3H).
[0054] Control of the droplet size can be an important issue for
drug delivery applications. There are three main means of
controlling the size of our double emulsions according to some
embodiments of the current invention. One method relies on
manipulation of the emulsification conditions through the energy of
the non-thermal excitation (e.g. applied shear and extensional flow
stresses), the flow properties of the liquids (e.g. viscosity or
viscoelasticity), and the interfacial tension between the liquids.
A second method involves performing size separations after
emulsification, such as centrifugation, filtering, and sorting of
droplets in preformed emulsions. A third method varies the
composition and concentration of the block copolypeptides through
synthesis. Indirectly, this third method also provides a means of
varying important physical properties, such as solubility of the
copolypeptides in the liquids, the interfacial tension between the
liquids in the presence of adsorbed copolypeptide, the viscosity of
the liquid solutions containing co-polypeptide, the structural
morphology of each of the blocks of the copolypeptide that may
confer interfacial stability that preserves droplet stability and
inhibits interfacial coalescence. Although we use a chemical
process to synthesize the copolypeptides, control over the
production of copolypeptides could be achieved through other means,
such as genetic expression in bioreactors containing genetically
modified bio-organisms. We have the ability to not only produce
small double emulsion droplets (with submicron outer droplets and
even smaller inner droplets), as discussed previously, but also
larger double emulsion droplets (>1 .mu.m). Micron scale
emulsion droplets could be made using low flow rates obtained from
a handheld homogenizer or somewhat higher flow rates via ultrasonic
homogenization (e.g. using an ultrasonic tip homogenizer). When
ultrasonic homogenization was used to emulsify a 0.1 mM FITC
labeled K.sub.40rL.sub.10 copolypeptide emulsion, laser scanning
confocal microscopy (LCSM) demonstrated that we could form larger
double emulsion droplets in the size range from 1 .mu.m to .mu.m
(FIG. 4A). In addition, we can take this solution of larger double
emulsion droplets and further emulsify it into smaller double
emulsion droplets by passing it through a microfluidic homogenizer
(FIG. 4B).
[0055] For certain applications, further size separation may be
desired. For these applications, we could also use centrifugation
to fractionate the emulsion and isolate emulsions of a desired size
range. To accomplish such separation, a 1.5 mM K.sub.40rL.sub.20
emulsion (.phi.=0.2) that had been passed through the microfluidic
homogenizer for six passes (N=6) was placed in a desktop centrifuge
set to a low speed of 3,500 rpm (revolutions per minute) for 4
hours. Due to the differences in mass densities of the droplet
structures with respect to the continuous liquid phase, the larger
droplets rose to the top as a plug more rapidly and could be
separated out easily from the extremely dilute suspension of
smaller droplets below (i.e. the remnants). We were able to
separate out larger droplets that had diameters greater than 300
nm. The remnant suspension had droplet diameters less than 300 nm,
therefore, a higher speed centrifuge was needed to further
fractionate the sizes of the droplets. The remnant suspension was
placed in an ultracentrifuge and centrifuged for 24 hrs at 20,000
rpm. The cryo-TEM images of these layers showed that the droplet
sizes can be segregated to a very narrow size range (FIGS. 4C and
4D). A plug formed on top of the centrifuged sample and images
showed that the diameter of the outer droplets ranged from about 30
nm to about 200 nm (FIG. 4C), and the remnant suspension had outer
droplet diameters ranging from about 10 nm to about 30 nm (FIG.
4D). This fractionation procedure demonstrates that isolation of
emulsion droplets of a desired size between 10 nm and 10 .mu.m is
quite feasible. Through this centrifugation procedure, we have also
demonstrated that it is possible to raise the volume fraction of
oil droplets in O/W emulsions and also oil droplets containing
inner water droplets in W/O/W double emulsions without
destabilizing either inner or outer droplet interfaces.
[0056] Another means for controlling double emulsion droplet size
was through variation of the block copolypeptides. A simple way to
do this was by changing polypeptide concentration. Dynamic light
scattering (DLS) results showed that as the K.sub.40rL.sub.20
copolypeptide concentration was increased from 0.1 mM to 1.5 mM the
average droplet radius decreased from about 400 nm at 0.1 mM to
about 160 nm at 1.5 mM. Another way to decrease the size of the
emulsion droplet was to increase the length of the oligoleucine
segment in a copolymer. As the oligoleucine length was increased
from K.sub.40rL.sub.5 to K.sub.40rL.sub.30, while holding the
lysine length the same (K.sub.40), the size of the outer oil
droplets decreased on average from about 470 nm to about 320
nm.
[0057] Vesicles are composed of lamellar membranes that separate an
inner aqueous compartment from an outer continuous liquid, where
the inner liquid can serve as a container for hydrophilic cargos.
In a similar fashion, double emulsions encapsulate an inner aqueous
droplet using a relatively thick oil film that exists between
layers of amphiphilic molecules that are present at two distinct
oil-water interfaces. One advantage in this system can be that the
thicker oil film (which we also refer to as an `oil layer`) located
between the interfaces of the inner and outer droplets can act as a
reservoir for a hydrophobic cargo. To verify this idea, we
incorporated both water-soluble and oil soluble fluorescent markers
into our copolypeptide stabilized emulsions. The water-soluble
fluorescent markers were InGaP/ZnS quantum dots with an emission
wavelength at 630 nm (red), and the hydrophobic fluorescent marker
was pyrene due to its high solubility in silicone oil and its blue
fluorescence. In addition, by using a green-fluorescently labeled
FITC functionalized K.sub.40rL.sub.10 copolymer to stabilize the
emulsion, we could simultaneously image localization of both
hydrophilic and hydrophobic markers as well as the copolypeptide.
Fluorescence LSCM imaging of a 0.1 mM FITC labeled
K.sub.40rL.sub.10 emulsion without pyrene or quantum dots showed
large double emulsion droplets in the range from about 1 .mu.m to
about 5 .mu.m diameters (FIG. 5A). A triple labeled emulsion was
made by emulsifing 0.1 mM FITC labeled K.sub.40rL.sub.10 with 0.01
M pyrene in 10 cSt silicone oil (.phi.=0.2) in the presence of the
InGaP/ZnS quantum dots. The 3 different fluorescent dyes were
imaged using fluorescence overlay microscopy. The overlayed
fluorescence image shows the segregation of the hydrophilic quantum
dots (red) into the inner aqueous liquid, the hydrophobic pyrene
(blue) into the oil liquid, and the FITC labeled polypeptide
(green) stabilizing the outer interface (FIG. 5B). The labeling of
the inner droplet interfaces cannot be seen likely due to quenching
of the fluorescence of the FITC labeled polypeptide by the quantum
dots contained in the inner droplets. Supporting this hypothesis,
FITC fluorescence around the inner droplet can be seen in the LCSM
image of the FITC-K.sub.40rL.sub.10 emulsion without quantum dots
(FIG. 5A).
[0058] Utilizing our synthetic methods, we can alter the
compositions and conformations of our copolypeptides, and also
incorporate other amino acids into the polypeptide chains. To
demonstrate the effects of changing chain conformations, we altered
the hydrophobic domain from a randomly copolymerized racemic
oligoleucine segment, as in K.sub.40rL.sub.10, to an
enantiomerically pure oligoleucine segment that adopts a stable
.alpha.-helical structure, as in K.sub.60L.sub.20. LCSM images of
the emulsions produced from ultrasonication of both of these
FITC-labeled polypeptides at 0.1 mM showed that both samples formed
similar double emulsions (FIGS. 6A and 6B). It is also important to
note that double emulsion formation is not exclusive to block
copolypeptides containing poly L-lysine as the hydrophilic block,
but can also form with negatively charged poly L-glutamate
hydrophilic segments, for example. A 1.0 mM E.sub.40rL.sub.10
copolypeptide emulsion was prepared using a microfluidic
homogenizer. The cryo-TEM images of this sample showed that double
emulsions were formed similar to those with the block copolypeptide
K.sub.40rL.sub.10 (FIGS. 6C-6D). In addition, emulsification was
attempted using the homopolymer K.sub.60, containing no hydrophobic
domain, and no emulsion was formed after ultrasonication (FIG. 6E).
Although K.sub.60 has good solubility in water, one would not
expect it to have strong amphiphilic properties, since it lacks a
hydrophobic block. We also allowed aqueous solutions of
K.sub.xrL.sub.y copolypeptides to remain in contact with oil
layers, to see if spontaneous formation of double emulsions occurs
without the application of shear. Spontaneous formation was not
seen after a one-week incubation period.
[0059] Additional Features and Variations:
[0060] Copolypeptide-Stabilized Emulsions
[0061] There are many potential compositional variations that can
be used with the basic process that we have found to make both
simple and double emulsions comprised of droplets having
microscale, sub-microscale, and nanoscale radii. The basic elements
of the process according to some embodiments of the current
invention are: a first liquid (e.g. water), a different second
immiscible liquid (e.g. oil), and amphiphilic co-polypeptides that
have significant solubility in at least one or possibly even both
of the two liquids (e.g. soluble in water). The co-polypeptide is
added into at least one of the liquids in which it is soluble, and
non-thermal energy is supplied to the system of liquids and
copolypeptides to disturb interfaces between the two liquids,
resulting in irreversible net growth of the interfacial surface
area through the formation of droplets and the creation of a
metastable emulsion. This applied non-thermal energy can be
supplied in many different forms, including a mechanical shear
flow, through ultrasonic waves, electromagnetic fields and waves,
gravity, concentration gradients, or through a pressure drop that
causes extensional flow. As interfaces between the liquids are
extended by the non-thermal energy that is introduced to cause
emulsification, the interfaces can become unstable to capillary
instabilities, causing larger droplets or films to break down into
smaller droplets. Depending upon the type of liquids and the
composition and structure of the copolypeptide used to stabilize
the interfaces, these smaller droplets may or may not contain inner
droplets of the other liquid phase (e.g. form W/O/W double
emulsions).
[0062] To load the inner droplets of W/O/W double emulsions with
desired cargo, prior to emulsification, the continuous liquid phase
can contain many different kinds of desired dissolved and dispersed
cargo elements prior to the emulsification. In the most common case
that we have investigated, the continuous liquid phase is water. In
this case, the following kinds of cargo could be loaded into the
aqueous inner droplets (and also the continuous aqueous phase):
single-stranded DNA, double-stranded DNA, single-stranded RNA,
double-stranded RNA, mRNA, tRNA, rRNA, miRNA, siRNA, piRNA,
rasiRNA, tasiRNA, hcRNA, scnRNA, RNA polymerases, nucleotides,
oligonucleotides, transposons, peptides, oligo-peptides,
poly-peptides, proteins, microtubules, actin filaments,
intermediate filaments, bundling proteins, crosslinking proteins,
transfection agents, salts, anions, cations, acids, bases, buffers,
viruses, vitamins, serums, lysates, ATP and GTP (e.g. molecular
energy sources), molecular motors, hydrophilic drug molecules,
cells, vesicles, nanodroplets, nanoemulsions, fullercnes, single
and multi-walled carbon nanotubes, cytoplasm, ribosomes, enzymes,
glucose, golgi, dendrimers, surflactants, lipids, lipoproteins,
oligonucleotide-pepeptide copolymers, globulins, albumins, human
serum albumin, bovine serum albumin, sugars, emulsans, saccharides,
oligo-saccharides, poly-saccharides, biocompatible polymers,
biodegradable polymers, quantum dots, clay nanoparticles, metal
nanoclusters and nanoparticles, magnetically responsive iron oxide
nanoparticles, organic and inorganic nanospheres and nanoparticles,
isotopically substituted hydrophilic molecules, imaging enhancement
agents, and fluorescent dyes. Mixtures of these components in the
continuous phase can also be made, provided that they remain stably
dispersed. For W/O/W double emulsions stabilized by amphiphilic
co-polypeptides, a wide range of hydrophilic materials and
water-dispersed materials that are smaller than the final droplet
size (or can be compressed into a volume that is less than the
inner water droplet volume) and that prefers to be in the aqueous
phase can potentially be incorporated into the inner water
droplets.
[0063] In the case of W/O/W double emulsions, the composition of
dispersed materials in the inner water droplets is determined by
the composition of the aqueous liquid prior to the application of
non-thermal energy that disturbs the interfaces between the liquids
(i.e. emulsification). After the emulsification, the inner water
droplets will contain the same components that are in the outer
continuous water portion. After the emulsification is over, the
outer water portion can be separated from the droplets and
retained, since it may have valuable components in it. Following
this separation, the double emulsion can be re-dispersed in a
different continuous aqueous liquid containing block copolypeptide
and possibly also another surfactant that would be suitable for
maintaining the stability of the droplets over long periods of time
in a desired product. In this manner, the composition of the
continuous aqueous liquid and that of the inner water droplets
(i.e. inside the oil droplets) can be set differently: the inner
water droplets can contain the desired drug molecules and particles
at the desired concentration, whereas the continuous aqueous liquid
does not have to contain them.
[0064] The second immiscible liquid (e.g. oil) can also contain a
wide array of different molecular, polymer, and particulate
materials. Assuming that the second liquid is hydrophobic (e.g.
oil), then the following could be incorporated into the dispersed
droplet liquid: fats, lipids, waxes, natural oils, essential oils,
fragrances, cholesterol, steroids, hydrophobic drug molecules,
hydrophobic polymers, hydrophobic polypeptides, poly-(lactic acid),
poly-(lactic-co-glycolic acid), poly-(lactic-glycolic acid),
biocompatible polymers, biodegradable polymers, micelles, quantum
dots, nanoparticles, nanoclusters, carbon nanotubes, fullerenes,
ferrofluids, imaging enhancement agents, fluorescent dyes, and
liquid crystals. In the case of a W/O/W double emulsion, the oil
could contain oil-soluble drug molecules and indicators that would
surround the inner water droplet and potentially facilitate and/or
improve the desired function of the contents of the water droplet
in a cooperative manner.
[0065] The liquid portions of the emulsions and/or droplets may be
changed to solid or liquid crystalline portions after the emulsion
is formed. If a polymerizable oil is used (e.g. ultraviolet
crosslinkable silicone oil), then the oil can be made into a rigid
crosslinked polymer by illuminating the emulsion or double emulsion
with ultraviolet light. Alternatively, if the oil is paraffinic,
then cooling the emulsion or double emulsion, once formed, below
the solidification temperature of the paraffin would enable the
liquid oil to become solidified.
[0066] The ability to form stable emulsions and double emulsions is
not limited to 10 cSt PDMS silicone oil. We have found that other
silicone oils having kinematic viscosities in the range from about
0.65 cSt to 1,000 cSt at room temperature, corresponding to
viscosities in the range from about 1 cP to about 1,000 cP also
form stable emulsions and stable double emulsions. Other
embodiments of the current invention could extend this range of
viscosities from about 0.1 cP to more than 10,000 cP. Since heating
liquids generally lowers their viscosities, emulsification at hot
temperatures could conceivably be used to obtain desired emulsion
compositions (e.g. higher viscosity oils) and structures (e.g.
reduced droplet sizes). Also, natural oils can be emulsified with
our copolypeptides, including soybean oil and methyl oleate.
Organic solvents, such as toluene, dichlorobenzene, and dodecane,
have also been emulsified in a continuous phase of water using
block copolypeptides.
[0067] We are able to produce stable droplets in O/W emulsions and
stable double droplets in W/O/W double emulsions using oil volume
fractions 0 ranging from the extremely dilute limit (e.g.
10.sup.-5) to the concentrated regime above 0.9. Typically, for
forming an O/W emulsion or for forming a W/O/W double emulsion, the
emulsification is carried out for .phi.<0.5, and more usually
O/W emulsions and W/O/W double emulsions are made at
.phi..apprxeq.0.1 to .phi.=0.2. Using a higher .phi. can increase
the throughput of the droplet production in the emulsification
process, so this can be desirable, yet the average dimensions of
the droplet structures can also depend on .phi., too. The inner
droplet radii and volume fractions can also be varied over a wide
range. We are also able to make normal direct emulsions and double
emulsions over a wide range of radii from the microscale to the
nanoscale.
[0068] Many different kinds of equipment and devices can be used to
supply the energy that disturbs the interfaces in order to create
the emulsion or double emulsion: colloid mills, mixers, stirrers,
homogenizers, ultrasonic dispersers, magnetic dispersers,
electromagnetic dielectrophoretic excitation, microfluidic devices,
and porous membrane extrusion. Our studies indicate that, for
co-polypeptide stabilization, a variety of different methods can be
used to provide non-thermal energetic excitations that disturb the
interfaces, and, provided the energy is sufficient to significantly
disturb the interfaces, the same emulsion morphology results. We
have shown that W/O/W double emulsions can be produced with the
same composition including co-polypeptide using stirrers,
ultrasonic dispersers, and microfluidic homogenizers.
[0069] The following describes in more detail experimental
procedures used for the above-noted examples.
[0070] General Methods and Materials
[0071] Tetrahydrofuran (THF) was dried by passage through a column
packed with alumina under nitrogen prior to use (A. B. Pangborn et
al., Organometallics 15, 1518 (1996)). Molecular weights were
obtained by tandem gel permeation chromatography/light scattering
(GPC/LS) performed at 60.degree. C. on a SSI pump equipped with a
Wyatt DAWN EOS light scattering detector and Wyatt Optilab DSP.
Separations were effected by 10.sup.5, 10.sup.4, and 10.sup.3 .ANG.
Phenomenex 5 .mu.m columns using 0.1 M LiBr in DMF as eluent and
polypeptide concentration of approximately 5 mg/mL. Infrared
spectra were recorded on a Perkin Elmer RXI FTIR Spectrophotometer
calibrated using polystyrene film. .sup.1H NMR spectra were
recorded on a Bruker AVANCE 400 MHz spectrometer. Deionized (DI)
water was purified using a Purelab Option 560 reverse osmosis
purifier. Millipore water was obtained from a Millipore Milli-Q
Biocel A10 purification unit. Silicone oil (poly-(dimethylsiloxane)
or PDMS) is supplied by Gelest, Inc. with viscosities ranging from
1 cSt to 1,000 cSt (corresponding to different average molecular
weights of the PDMS).
[0072] Block Copolypeptide Synthesis--General
[0073] The .alpha.-amino acid-N-carboxyanhydride NCA monomers were
synthesized using previously published literature protocols (H. R.
Kricheldorf, .alpha.-Aminoacid-N-Carboxyanhydrides and Related
Materials (Springer-Verlag, NY, 1987)). All of the block
copolypeptides were polymerized using the (PMe.sub.3).sub.4Co
initiator (H. F. Klein, and H. H. Karsch, Chem. Ber. 108, 944
(1975)). The resulting polypeptides were characterized using GPC,
.sup.1H NMR and IR spectroscopy (T. J. Deming, Macromolecules 32,
4500 (1999)). The compositions of the copolymers were determined by
analysis of the integration values of the .sup.1H NMR spectra
recorded in D.sub.2O. All compositions were found to be within 5%
of predicted values. From measured polymer chain length
distributions, the polydispersity index (Mw/Mn) ranged from 1.1 to
1.3.
[0074]
Poly(N.sub..epsilon.CBZ-L-lysine).sub.40-b-poly(rac-leucine).sub.20
[0075] In the drybox, L-Lysine NCA (10.00 g, 33 mmol) was dissolved
in THF (200 mL) and placed in a 500 mL flat bottom flask that could
be sealed with a plastic stopper. An aliquot of (PMe.sub.3).sub.4Co
(16 mL of a 48 mg/mL solution in THF) was then added via syringe to
the flask. A stir bar was added and the flask sealed and let stir
for 45 minutes. An aliquot (50 .mu.L) was removed from the
polymerization for GPC analysis (Mn=11,000, Mw/Mn=1.24). L-Leucine
NCA (1.3 g, 8.2 mmol) and D-Leucine NCA (1.3 g, 8.2 mmol) were
dissolved in THF (52 mL) and then added to the polymerization
mixture. After stirring for another 16 h, the solution was removed
from the drybox and the THF removed under reduced pressure. FTIR
analysis showed complete consumption of monomer and was similar to
previously reported results (V. Breedveld ec al., Macromolecules
37, 3943 (2004)).
[0076] Poly(L-Lysine.HBr).sub.40-b-poly(rac-Leucine).sub.20,
K.sub.40rL.sub.10
[0077] The
poly(N.sub..epsilon.CBZ-L-lysine).sub.40-b-poly(rac-leucine).su-
b.20 from above was dissolved in trifluoroacetic acid (TFA) (350
mL), transferred to a 1 L flat bottom flask and placed into an ice
bath. HBr (33% in acetic acid) was then added (40 mL, 131 mmol) and
the reaction stirred for 2 hrs. Deprotected polymer was isolated by
addition of diethyl ether to the reaction mixture, followed by
centrifugation. The isolated polymer was then dissolved in DI water
and dialyzed (6,000-8,000 MWCO membrane) against tetrasodium EDTA
(3 mmol, 4 days), 0.1 M HCl (2 days), DI water (1 day), 0.1 M LiBr
(2 days), DI water (2 days), changing each solution 3 times/day.
The dialyzed polymer was isolated by freeze-drying to give the
product as a dry white powder (4.8 g, 70.2%). FTIR and .sup.1H-NMR
were performed and shown to be similar to previous results (V.
Breedveld et al., Macromolecules 37, 3943 (2004)).
[0078] FITC Functionalized K.sub.40rL.sub.10
[0079] The K.sub.40rL.sub.10 copolymer was prepared as described
above. GPC analysis of the first segment (poly CBZ-lysine):
Mn=10,500, Mw/Mn=1.20. The deprotected copolymer (150 mg,
1.3.times.10.sup.-2 mmol) was dissolved in water and placed in a
125 mL flat bottom flask. NaHCO.sub.3 (162 mg, 19 mmol) was added
to the solution. Fluorescein isothiocyanate (FITC) (5 mg,
1.3.times.10.sup.-2 mmol) was dissolved in dry DMSO (1 mL) and
added to the polymer solution. A stir bar was added and the
reaction mixture was stirred overnight. The polymer solution was
dialyzed (6,000-8,000 MWCO membrane) for 3 days against DI water,
changing water 3 times/day. The dialyzed polymer was isolated by
freeze-drying to yield a yellow-orange polymer containing
approximately 1 fluorescein unit per polymer chain (130 mg,
87%).
[0080] Emulsification of Silicone Oil Using K.sub.40rL.sub.10
[0081] The freeze-dried K.sub.40rL.sub.10 copolypeptide was first
dissolved in de-ionized water at the desired concentration. The
range of block copolypeptide concentrations, C, varied from
1.0.times.10.sup.-4 mM to 1.7 mM. Silicone oil (10 cSt, Gelest
PDMS) was added to give the desired volume fraction .phi. of oil to
the continuous phase (0.01.ltoreq..phi..ltoreq.0.8). A premix
emulsion was prepared by applying shear using either a handheld
homogenizer (IKA Ultra-Turrax T8 with the S8N-8G dispersing
element) or a handheld ultrasonic homogenizer (Cole-Palmer 4710
Series Model ASI at an output of 35-40%). This premix emulsion was
then passed through a M-110S Microfluidizer.RTM. Processor with a
75 .mu.m stainless steel/ceramic interaction chamber and an input
air pressure p=130 psi. The emulsion was collected at the product
outlet of the microfluidic homogenizer, and then passed through the
microfluidic homogenizer five more passes (N=6 total), which
decreased the average droplet radius <a> and increased the
monodispersity of the sample. We have formed double emulsions of
copolypeptide, water, and various oils other than cSt and 100 cSt
silicone oil, including soybean oil and methyl oleate. Cryo-TEM has
also confirmed the formation of double emulsions using the
following copolypeptide compositions: K.sub.20rL.sub.10,
K.sub.40rL.sub.5, K.sub.40rL.sub.10, K.sub.40rL.sub.20,
K.sub.40rL.sub.30, K.sub.40rL.sub.20, and E.sub.40rL.sub.10
(E=Glutamic Acid).
[0082] Fractionation of Emulsions
[0083] A 1.5 mM K.sub.40rL.sub.20 emulsion (prepared as above) was
centrifuged in a 15 mL plastic centrifuge tube for 24 h at 3,500
rpm using an IEC HN-S tabletop centrifuge. A 0.5 mm plug formed,
and was separated from the remnants. A plug was formed at the top
of the tube (droplet sizes >300 nm, by cryo-TEM), due the
different densities of silicone oil and water (0.973 g/mL for 10
cSt PDMS silicone oil vs. 1.0 g/mL for water). The plug was
isolated from the rest of the sample, designated as primary
remnants, and these primary remnants were further fractionated at
20,000 rpm for 4 hrs using a Beckman L8-55 ultracentrifuge. A plug
formed on the top of the suspension (droplet sizes ranging from 30
nm to 200 nm, by cryo-TEM) along with droplets remaining in
suspension as secondary remnants (droplet sizes ranging from 10 nm
to 30 nm, by cryo-TEM).
[0084] Dynamic Light Scattering (DLS)
[0085] The diameters of emulsion droplets were estimated by dynamic
light scattering (DLS) with a Photocor-FC board and software. The
samples were diluted to obtain an intensity reading of between
1.times.10.sup.5 and 6.times.10.sup.5. Each measurement was
performed at a scattering angle of 90.degree. for 500 seconds, with
linear channel spacing and an adjustable baseline. The fitting
procedure used was cumulant analysis with an adjustable baseline to
fit the data and calculate average droplet radii.
[0086] Loading of Three Fluorescent Probes into Different
Components of K.sub.40rL.sub.10 Emulsions
[0087] To label the hydrophobic liquid, we dissolved pyrene in the
silicone oil component at a concentration of 0.01 M, quantum dots
(Evident Technologies, Type T2-MP 650 nm Macoun Red InGaP/ZnS,
amine-functionalized) were dispersed in the water component at a
concentration of 2 .mu.M. To prepare the emulsion,
FITC-K.sub.40rL.sub.10 (150 .mu.L of an 0.1 mM solution) was
combined with InGaP quantum dots (50 .mu.L of an 8 .mu.M solution)
and pyrene in 10 cst silicon oil (50 .mu.L of a 0.01 M pyrene
solution). The mixture was emulsified using an ultrasonic tip
homogenizer (output of 35%) for 10 seconds.
[0088] Laser Scanning Confocal Microscopy (LSCM)
[0089] A 0.1 mM FITC-K.sub.40rL.sub.10 emulsion (.phi.=0.2, 10 cSt
silicone oil) was prepared by combining 800 .mu.L of a 0.1 mM
FITC-K.sub.40rL.sub.10 polypeptide solution and 200 .mu.L of 10 cSt
PDMS silicone oil, followed by emulsification for 10 s using a
handheld ultrasonic homogenizer (output of 35%). Prior to imaging,
an aliquot of the 0.1 mM FITC-K.sub.40rL.sub.10 emulsion suspension
was diluted by a factor of 10 with de-ionized (DI) water. One drop
of the emulsion was placed on a glass slide, followed by placement
of a cover slip. The samples were imaged using a Leica-SP MP
confocal and multiphoton inverted microscope equipped with a 488 nm
(blue) argon laser (JDS Uniphase) and a 561 nm (green) diode laser
(DPSS: Melles Griot) and a two-photon laser setup consisting of a
Spectra-Physics Millenia X 532 nm (green) diode pump laser and a
Tsunami Ti:sapphire picosecond-pulsed infrared laser tuned at 768
nm for ultraviolet excitation.
[0090] Fluorescence Microscopy
[0091] Prior to fluorescence imaging, emulsion suspensions were
diluted tenfold with DI water. A drop of emulsion was then placed
onto a glass slide and covered using a glass cover slip. The
samples were imaged using a Zeiss 200 fluorescence microscope.
[0092] Cryogenic TEM Imaging
[0093] Each emulsion sample was diluted tenfold with DI water prior
to imaging. An aliquot of each sample (5 .mu.L) was then placed on
a carbon grid. The grid was loaded into a Vitrobot (FEI) automated
vitrification device for automated sample blotting and
vitrification in liquid ethane. The grid was stored under liquid
nitrogen and then placed, using a cold stage, in a Phillips Tecnai
F20 electron microscope with an accelerating voltage of 120 kV.
Images were obtained on a Teitz SCX slow-scan CCD detector coupled
to the Leginon software package.
Additional Examples
[0094] The block copolypeptide surfactants we designed according to
some embodiments of the current invention have the general
structure poly(L-lysine.HBr).sub.x-b-poly(racemic-leucine).sub.y,
K.sub.x(rac-L).sub.y, where x ranged from 20 to 100, and y ranged
from 5 to 30 residues (FIG. 1A). The hydrophilic poly(L-lysine.HBr)
segments are highly charged at neutral pH, provide good water
solubility (Katchalski, E. & Sela, M. Synthesis and chemical
properties of poly-alpha-amino acids. Advances in Protein Chemistry
13, 243-492 (1958)), and possess abundant amine groups for chemical
functionalization (Niederhafner, P., Sebestik, J. & Je{hacek
over (z)}ek, J. Peptide dendrimers. Journal of Peptide Science 11,
757-788 (2005)). Unlike hydrophobic segments of other polymeric
amphiphiles, poly(L-leucine) segments adopt rod-like
.alpha.-helical conformations that give rise to strong interchain
associations and poor solubility in common organic solvents (Nowak,
A. P. et al. Rapidly recovering hydrogel scaffolds from
self-assembling diblock copolypeptide amphiphiles. Nature 417,
424-428 (2002)). We have shown that block copolymers of the
structure K.sub.xL.sub.y (e.g. K.sub.60L.sub.20) associate strongly
in water to form membranes via packing of the hydrophobic segments
(Holowka, E. P., Pochan, D. J. & Deming, T. J. Charged
polypeptide vesicles with controllable diameter. Journal of the
American Chemical Society 127, 12423-12428 (2005)). Consequently,
we have focused on poly(rac-leucine) since its disordered chain
conformation improves solubility (Table 1) (Kricheldorf, H. R.
& Mang, T. C-13-NMR Sequence-Analysis, 20. Stereospecificity of
the Polymerization of D,L-Leu-NCA and D,L-Val-NCA. Makromolekulare
Chemie-Macromolecular Chemistry and Physics 182, 3077-3098 (1981);
Breitenbach, J. W., Allinger, K. & Koref, A. Viskositatsstudien
an Losungen von DL-Phenylalanin-Polypeptiden. Monatsh. Chem. 86,
269 (1955)) and helps promote surface activity (Table 1), while its
peptidic nature allows for additional mechanical stabilization of
droplet interfaces via interchain H-bonding in the oil phase (Lapp,
C. & Marchal, J. Preparation De La Poly-D,L-Phenylalanine En
Helice Par Polymerisation De La D,L-Benzyl-4 Oxazolidine Dione-2-5.
Journal De Chimie Physique Et De Physico-Chimie Biologique 60,
756-766 (1963)).
[0095] Diblock copolypeptides were screened for emulsification
activity by adding PDMS silicone oil to aqueous
K.sub.x(rac-L).sub.y solutions (Table 1, FIGS. 2A-2B, 11A). The
resulting mixtures were sheared using a handheld rotary homogenizer
and then passed six times through a high-pressure microfluidic
homogenizer (FIG. 2C). All K.sub.x(rac-L).sub.y samples gave stable
W/O/W nanoemulsions that did not ripen or phase separate for over 9
months. Only copolypeptides with low hydrophobic content, e.g.
K.sub.40(rac-L).sub.5, gave emulsions that slowly phase separated
after 1 year. Other methods of mixing, including ultrasonic mixing,
also provided stable emulsions, but with droplets having diameters
of up to several microns. Use of hydrophobic segments longer than
thirty residues greatly diminished aqueous solubility (Table 1),
where K.sub.40(rac-L).sub.30 could only be dissolved up to 1 mM. As
controls, 0.1 mM suspensions of K.sub.60L.sub.20 and K.sub.60 were
also used as surfactants, where K.sub.60L.sub.20 did form stable
emulsions and K.sub.60 failed to emulsify oil and water mixtures
(FIGS. 9A-9B). These results indicated that K.sub.x(rac-L).sub.y
surfactants give stable emulsions over a broad range of
compositions and concentrations.
[0096] To probe droplet structure, block copolypeptide stabilized
emulsions were imaged using optical microscopy and cryogenic
transmission electron microscopy (CTEM). All samples with
K.sub.x(rac-L).sub.y were found to contain oil droplets, each
containing predominately a single internal aqueous droplet with
consistent inner to outer volume ratios (FIGS. 7A-8D). Contrasting
these results, the emulsions formed using K.sub.60L.sub.20
contained only simple oil droplets, revealing that the
racemic-leucine segments play a key role in stabilizing the double
emulsion structure in this embodiment of the current invention. As
copolypeptide hydrophobic content was decreased, droplet sizes
increased (Table 1, FIG. 10C), suggesting that copolymer
composition influences interfacial mean curvature. Average droplet
diameters were also found to increase when the concentration of
K.sub.40(rac-L).sub.20 was decreased (FIG. 10A). Likewise,
decreasing the oil volume fraction yielded smaller emulsion
droplets (FIG. 10B). Emulsions always formed such that water
remained the continuous liquid and did not invert up to oil volume
fractions approaching 50%. In addition to PDMS, other immiscible
liquids such as dodecane, soybean oil, and methyl oleate gave
emulsions using 1 mM K.sub.40(rac-L).sub.20 in water. The
versatility of various embodiments of the current invention was
shown by formation of stable emulsions using R.sub.40(rac-L).sub.10
or E.sub.40(rac-L).sub.10, containing guanidinium or carboxylate
functionality of L-arginine (R) and L-glutamate (E), respectively
(FIGS. 8A-8B).
[0097] Formation of nanoscale emulsion droplets is necessary for
many applications, such as drug delivery where the outer droplet
diameter generally needs to be less than 200 nm, and preferably
between 50 nm and 100 nm (Kataoka, K., Kwon, G. S., Yokoyama, M.,
Okano, T. & Sakurai, Y. Block-Copolymer Micelles as Vehicles
for Drug Delivery. Journal of Controlled Release 24, 119-132
(1993)). Although many conventional methods are available for
preparation of double emulsions, none allow preparation of outer
droplets in this size range (Garti, N. Double emulsions--Scope,
limitations and new achievements. Colloids and Surfaces
A-Physicochemical and Engineering Aspects 123, 233-246 (1997);
Loscertales, I. G. et al. Micro/nano encapsutation via electrified
coaxial liquid jets. Science 295, 1695-1698 (2002); Utada, A. S. et
al. Monodisperse double emulsions generated from a microcapillary
device. Science 308, 537-541 (2005); Benichou, A., Aserin, A.,
Garti, N. Double emulsions stabilized with hybrids of natural
polymers for entrapment and slow release of active matters.
Advances in Colloid and Interface Science 108-109, 29-41 (2004)).
Ultrasonic homogenization was used to prepare a
K.sub.40(rac-L).sub.20 emulsion yielding a polydisperse sample with
the smallest double emulsion droplets observed by CTEM being ca.
400 nm in diameter. These droplets were further reduced in size by
passage six times through a microfluidic homogenizer, yielding
droplet diameters ranging from ca. ten to a few hundred nanometers.
The stability of these double emulsions against both external and
internal coalescence allowed the use of centrifugation to
fractionate droplets into a desired size range. Centrifugation of
the sample in FIG. 12A gave a buoyant fraction containing droplets
hundreds of nanometers in diameter. The smaller droplets in the
remaining suspension were further separated by ultracentrifugation
(Mason, T. G., Wilking, J. N., Meleson, K., Chang, C. B. &
Graves, S. M. Nanoemulsions: formation, structure, and physical
properties. Journal of Physics-Condensed Matter 18, R635-R666
(2006)), yielding a fraction with droplets ranging from ca. 10 nm
to 100 nm in diameter (FIG. 12C). This fractionation procedure
shows that isolation of stable double emulsion droplets in the
nanoscale range is quite feasible, and that they are remarkably
stable to applied external stresses, such as shear stresses,
extensional stresses, and osmotic compressional stresses.
[0098] To demonstrate their encapsulating ability, both
water-soluble and oil soluble fluorescent markers were loaded into
copolypeptide stabilized double emulsions. A dispersion of
InGaP/ZnS quantum dots was mixed with fluorescein labeled
FITC-K.sub.40(rac-L).sub.10 prior to emulsification with PDMS
silicone oil containing pyrene. Using fluorescence microscopy, both
markers and the labeled polypeptide were imaged in the double
emulsion droplets (FIG. 5B). The images also showed the
compartmentalization of hydrophilic quantum dots (red) into the
inner aqueous phase, hydrophobic pyrene (blue) into the oil phase,
and the labeled polypeptide (green) stabilizing the outer
interface. Polypeptide at the inner interface was not observed
likely due to quenching of the fluorescein label by the quantum
dots. In samples prepared with K.sub.60L.sub.20 surfactant, only
simple oil droplets with no internal aqueous compartment were
observed (FIG. 13B). These cargos were observed to remain
encapsulated within the droplets for at least 3 months, showing
unprecedented enhanced stability of the inner aqueous compartment
compared to most double emulsion systems (Davis, S. S. &
Walker, I. M. Multiple Emulsions as Targetable Delivery Systems.
Methods in Enzymology 149, 51-64 (1987); Garti, N. Double
emulsions--Scope, limitations and new achievements. Colloids and
Surfaces A-Physicochemical and Engineering Aspects 123, 233-246
(1997); Benichou, A., Aserin, A., Garti, N. Double emulsions
stabilized with hybrids of natural polymers for entrapment and slow
release of active matters. Advances in Colloid and Interface
Science 108-109, 29-41 (2004)).
[0099] These K.sub.x(rac-L).sub.y surfactants were designed with
high hydrophilic contents (HC), namely the ratio of hydrophilic to
hydrophobic residues, which favor stabilization of O/W emulsions
where the oil is on the concave side of the curved interface of a
nanoscale droplet. Conversely, the inner water-oil interface of a
W/O/W double emulsion is best stabilized by a surfactant with a low
HC since the oil is on the convex side of the interface. The
opposite signs of these mean interfacial curvatures (Strey, R.
Microemulsion microstructure and interfacial curvature. Colloid and
Polymer Science 272, 1005-1019 (1994)) explain why single component
surfactants generally do not stabilize double emulsion droplets
and, consequently, combinations of surfactants are required
(Ficheux, M. F., Bonakdar, L., Leal-Calderon, F. & Bibette, J.
Some stability criteria for double emulsions. Langmuir 14,
2702-2706 (1998)). This also explains the formation of only O/W
emulsions prepared with K.sub.60L.sub.20, since the rod-like
oligoleucine segments are poorly solvated by the oil and tend to
aggregate in the oil phase (Nowak, A. P. et al. Rapidly recovering
hydrogel scaffolds from self-assembling diblock copolypeptide
amphiphiles. Nature 417, 424-428 (2002)). Based on these
observations, it appears that stabilizing an inner aqueous droplet
in a W/O/W double emulsion is significantly more likely when the
hydrophobic polypeptide segments disperse readily in the oil and
thereby prevent steric crowding of the large hydrophilic segments
in the aqueous phase.
[0100] The racemic-leucine segments in K.sub.x(rac-L).sub.y provide
a combination of features that stabilize double emulsion droplets.
The conformational flexibility of these segments improves oil
solubility, since it has been shown that poly(rac-leucine) is
soluble in organic solvents such as CH.sub.2Cl.sub.2 and
(CH.sub.3).sub.2SO whereas poly(L-leucine) is not (Kricheldorf, H.
R. & Mang, T. C-13-NMR Sequence-Analysis, 20. Stereospecificity
of the Polymerization of D,L-Leu-NCA and D,L-Val-NCA.
Makromolekulare Chemie-Macromolecular Chemistry and Physics 182,
3077-3098 (1981); Breitenbach, J. W., Allinger, K. & Koref, A.
Viskositatsstudien an Losungen von DL-Phenylalanin-Polypeptiden.
Monatsh. Chem. 86, 269 (1955)). This allows K.sub.x(rac-L).sub.y
chains to better stabilize an inner droplet oil-water interface as
the hydrophobic segments can disperse more readily in the oil.
Despite its improved solubility, in an oil solvent, nearly all
residues of poly(rac-leucine) will also be engaged in both
intramolecular and intermolecular H-bonds. Studies on racemic
polymers of both leucine and phenylalanine have demonstrated that
they associate in organic solvents via H-bonding (Lapp, C. &
Marchal, J. Preparation De La Poly-D,L-Phenylalanine En Helice Par
Polymerisation De La D,L-Benzyl-4 Oxazolidine Dione-2-5. Journal De
Chimie Physique Et De Physico-Chimie Biologique 60, 756-766
(1963)). At the interface of an inner aqueous droplet with oil, the
high HC of our polymers favors a low packing density of rac-leucine
segments in the oil phase that would allow few interchain H-bonds
and give a weakly stabilized interface (FIG. 2C). However, the
opposite curvature of the outer droplet oil-water interface allows
dense packing of the racemic-leucine segments in the oil phase,
favoring interchain H-bonding. Consequently, even though inner
aqueous droplets are likely unstable, they are prevented from
merging with the outer droplets, and forming simple emulsions,
since the outer interfaces are expected to be reinforced by H-bond
crosslinking. To test this concept, emulsions were prepared
containing a silicone oil capped with acetamide groups capable of
H-bonding to rac-leucine segments. Emulsification with
K.sub.60(rac-L).sub.20 gave W/O/W nanoemulsions containing multiple
internal droplets (FIGS. 11A-11D), supporting the hypothesis that
rac-leucine segments can stabilize droplets through H-bonding
interactions in the oil phase, thus inhibiting internal droplet
coalescence.
[0101] Our use of racemic, disordered hydrophobic polypeptide
segments that interact via H-bonding is a novel means for
stabilizing W/O/W double emulsions. This approach differs greatly
from protein and peptide stabilized emulsions where double
emulsions do not form without use of additional surfactants, and an
ordered amphiphilic helix is the most common source of surface
activity (Enser, M., Bloomberg, G. B., Brock, C., Clark, D. C. De
novo design and structure-activity relationships of peptide
emulsifiers and foaming agents. International Journal of Biological
Macromolecules 12, 118-124 (1990); Dickinson, E. Structure and
composition of adsorbed protein layers and the relationship to
emulsion stability. Journal of the Chemical Society Faraday
Transactions 88, 2973-2983 (1992); Saito, M., Ogasawara, M.,
Chikuni, K., Shimizu, M. Synthesis of a peptide emulsifier with an
amphiphilic structure. Bioscience, Biotechnology and Biochemistry
59, 388-392 (1995); Dalgleish, D. G. Conformations and structures
of milk proteins adsorbed to oil-water interfaces. Food Research
International 29, 541-547 (1996); Chang, C. B., Knobler, C. M.,
Gelbart, W. M., Mason, T. G. Curvature Dependence of Viral Protein
Structures on Encapsidated Nanoemulsion Droplets. ACS Nano 2
281-286 (2008)). Our strategy also can be applied to other
copolypeptides, since samples containing rac-valine and rac-alanine
hydrophobic segments also gave stable double nanoemulsions (FIGS.
8C, 8D). Use of block copolypeptide surfactants can overcome key
limitations of W/O/W double emulsions by allowing the unprecedented
straightforward preparation of nanoscale droplets, which also
exhibit high stability and can be used to simultaneously
encapsulate both oil-soluble and water-soluble cargos. (The term
cargo is used to refer to any material that one can add to the
liquid contained within any of the droplets, whether these droplets
are inner droplets or outer droplets of double emulsions or simple
droplets of direct emulsions.)
Methods Summary
[0102] K.sub.40(rac-L).sub.20 copolypeptide was first dissolved at
the desired concentration (e.g. 0.01 mM <C<1.5 mM) in
ultrapure water. PDMS silicone oil (10 cSt) was added to give the
desired volume fraction .phi. of oil to the continuous phase
(0.05<.phi.<0.50). A microscale emulsion (i.e. "premix"
emulsion) was prepared by either mixing for 1 minute using a
handheld homogenizer (IKA Ultra-Turrax T8 with the S8N-8G
dispersing element) or by mixing for 10 seconds using a handheld
ultrasonic tip homogenizer (Cole-Parmer 4710 Series Model ASI at an
output of 35-40%). This emulsion was then passed through a M-1100S
Microfluidizer.RTM.Processor with a 75 .mu.m stainless
steel/ceramic interaction chamber and an input air pressure p=130
psi. The emulsion was collected at the product outlet, and then
passed through the microfluidic homogenizer repeatedly for a total
of six passes (N=6), which decreased the average droplet radius
<a> (e.g. of the single droplets in a simple emulsion and of
the outer and inner droplets in a double emulsion) and increased
the monodispersity of the droplets in the emulsions. A similar
protocol was used for emulsions generated using other block
copolypeptide surfactants (Table 1, FIGS. 7A-7C). The ratio given
by the inner droplet radius divided by the outer droplet radius
(labeled as "I/O ratio") was relatively uniform for different
hydrophobic chain lengths at approximately 0.5 (Table 1, FIG. 7D).
Other amphiphilic block copolypeptides, where either the lysine or
leucine domains were substituted with different hydrophilic or
hydrophobic residues, respectively, were also found to form double
emulsions (FIGS. 8A-8D). The emulsification capability of different
polypeptide surfactants was also qualitatively evaluated using
toluene, which forms less stable emulsions, and with a control
homopolypeptide, K.sub.60 (FIGS. 9A, 9B), which does not yield
stable emulsions or stable double emulsions.
Supplementary Methods
[0103] Materials.
[0104] Tetrahydrofuran (THF) was dried by passage through a column
packed with alumina under nitrogen prior to use (Nowak, A. P. et
al. Rapidly recovering hydrogel scaffolds from self-assembling
diblock copolypeptide amphiphiles. Nature 417, 424-428 (2002)).
Molecular weights were obtained by tandem gel permeation
chromatography/light scattering (GPC/LS) performed at 60.degree. C.
on a SSI pump equipped with a Wyatt DAWN EOS light scattering
detector and Wyatt Optilab DSP. Separations were effected by
10.sup.5, 10.sup.4, and 10.sup.3 .ANG. Phenomenex 5 .mu.m columns
using 0.1 M LiBr in DMF as eluent and polypeptide concentrations of
approximately 5 mg/mL. Infrared spectra were recorded on a Perkin
Elmer RXI FTIR Spectrophotometer calibrated using polystyrene film.
.sup.1H NMR spectra were recorded on a Bruker AVANCE 400 MHz
spectrometer. Deionized (DI) water was purified using a Purelab
Option 560 reverse osmosis purifier. Ultrapure (18 M.OMEGA.) water
was obtained from a Millipore Milli-Q Biocel A10 purification unit.
Silicone oil (10 cSt, polydimethylsiloxane, PDMS) was supplied by
Gelest, Inc.
[0105] Block Copolypeptide Synthesis.
[0106] The .alpha.-amino acid-N-carboxyanhydride NCA monomers were
synthesized using previously published literature protocols (Id.).
The resulting polypeptides were characterized using GPC, .sup.1H
NMR and IR spectroscopy (Id.). The compositions of the copolymers
were determined by analysis of the integration values of the
.sup.1H NMR spectra recorded in D.sub.2O. All compositions were
found to be within 5% of predicted values. Polymer chain length
distributions (Mw/Mn) ranged from 1.1 to 1.3. K.sub.60L.sub.20 was
synthesized using a published procedure (Holowka, E. P., Pochan, D.
J. & Deming, T. J. Charged polypeptide vesicles with
controllable diameter. Journal of the American Chemical Society
127, 12423-12428 (2005)). Chain conformations of the hydrophobic
poly(leucine) segments were confirmed using circular dichroism
spectroscopy (FIG. 14), where the contributions from the
poly(lysine) segments were removed using poly(racemic-lysine)
segments as previously described (Nowak, A. P. et al. Rapidly
recovering hydrogel scaffolds from self-assembling diblock
copolypeptide amphiphiles. Nature 417, 424-428 (2002)).
[0107]
Poly(N.sub..epsilon.-CBZ-L-lysine).sub.40-b-poly(rac-leucine).sub.2-
0. In a nitrogen filled glove box, CBZ-L-Lysine NCA (10 g, 33 mmol)
was dissolved in THF (200 mL) and placed in a 500 mL flat bottom
flask that could be sealed with a plastic stopper. An aliquot of
(PMe.sub.3).sub.4Co (16 mL of a 48 mg/mL solution in THF) was then
added via syringe to the flask. A stir bar was added, then the
flask was sealed and allowed to stir for 45 minutes. An aliquot (50
.mu.L) was removed from the polymerization solution for GPC
analysis (Mn=11,000, Mw/Mn=1.24). L-Leucine NCA (1.3 g, 8.2 mmol)
and D-Leucine NCA (1.3 g, 8.2 mmol) were dissolved in THF (50 mL)
and then added to the polymerization mixture. After stirring for
another 16 h, FTIR analysis showed complete consumption of monomer,
similar to previously reported results (Id.).
[0108] Poly(L-lysine*HBr).sub.40-b-poly(rac-leucine).sub.20,
K.sub.40(rac-L).sub.20. The
poly(N.sub..epsilon.-CBZ-L-lysine).sub.40-b-poly(rac-leucine).sub.20
solution from above was removed from the drybox and the THF removed
under reduced pressure. The block copolypeptide was then dissolved
in trifluoroacetic acid (TFA) (350 mL), transferred to a 1 L flat
bottom flask, which was placed into an ice bath. HBr (33% in acetic
acid) was then added (40 mL, 131 mmol) and the reaction stirred for
2 hrs. Deprotected polymer was isolated by addition of diethyl
ether (400 mL) to the reaction mixture, followed by centrifugation.
The isolated polymer was then dissolved in DI water and dialyzed
(using a 6,000 to 8,000 MWCO membrane) in a 4 L container against
aqueous tetrasodium EDTA (3 mmol, 2 days), aqueous HCl (100 mmol, 2
days), DI water (1 day), aqueous LiBr (100 mmol, 2 days), and
finally DI water (2 days), changing each solution 3 times/day. The
dialyzed polymer was isolated by freeze-drying to give the product
as a dry white powder (4.8 g, 70%). FTIR and .sup.1H-NMR were
performed on the block copolypeptide and were found to be similar
to previous results (Id.).
[0109] FITC functionalized K.sub.40(rac-L).sub.10.
[0110] The K.sub.40(rac-L).sub.10 copolymer was prepared in a
manner similar to K.sub.40(rac-L).sub.20. GPC analysis of the first
segment (poly CBZ-L-lysine) gave: Mn=10,500, Mw/Mn=1.20. The
deprotected copolymer (150 mg, 1.3.times.10.sup.-2 mmol) was
dissolved in water and placed in a 125 mL flat bottom flask.
NaHCO.sub.3 (160 mg, 19 mmol) was then added to the solution.
Fluorescein isothiocyanate (FITC) (5.0 mg, 1.3.times.10.sup.-2
mmol) dissolved in dry DMSO (1 mL) was added to the polymer
solution. A stir bar was added and the reaction mixture was stirred
overnight. The polymer solution was dialyzed (using a 6,000 to
8,000 MWCO membrane) for 3 days against DI water, changing the
water 3 times/day. The dialyzed polymer was isolated by
freeze-drying to yield a yellow-orange polymer containing
approximately 1 fluorescein unit per polymer chain (130 mg, 87%).
The FITC functionalized K.sub.60L.sub.20 copolymer was prepared
using a similar procedure.
[0111] Loading of Fluorescent Probes into Different Phases of
FITC-K.sub.40(rac-L).sub.10 Stabilized Double Emulsions.
[0112] To label the hydrophobic phase, pyrene was dissolved in
silicone oil at a concentration of 0.01 M. To label the aqueous
phase, water soluble quantum dots (Evident Technologies, Type T2-MP
650 nm Macoun Red InGaP/ZnS, amine-functionalized) were dispersed
in the aqueous phase at a concentration of 2 .mu.M. To prepare the
emulsion, solutions of FITC-labeled K.sub.40(rac-L).sub.10 (150
.mu.L of a C=0.1 mM solution) and InGaP quantum dots (50 .mu.L of
an 8 .mu.M solution) were mixed with pyrene in 10 cSt silicone oil
(50 .mu.L of a 0.01 M pyrene solution). The mixture was emulsified
using an ultrasonic tip homogenizer (output of 35%) for 10 s. The
same procedure was followed for the FITC-K.sub.60L.sub.20 block
copolypeptide surfactant. Prior to imaging, the non-encapsulated
quantum dots were removed by dialysis against deionized water.
[0113] The invention has been described in detail with respect to
various embodiments, and it will now be apparent from the foregoing
to those skilled in the art that changes and modifications may be
made without departing from the invention in its broader aspects,
and the invention, therefore, as defined in the claims is intended
to cover all such changes and modifications as fall within the true
spirit of the invention.
[0114] For example, it can be desirable to make W/O/W double
emulsions at higher oil volume fractions .phi.. Double emulsions
can be routinely formed through emulsification at .phi. ranging
from dilute volume fractions .phi.<<1 up to .phi..apprxeq.0.4
of the primary dispersed phase of the outer droplet, and, through
simple and appropriate modifications of the procedures, up to about
.phi..apprxeq.0.6 in some embodiments. It is conceivable that
certain particular embodiments that extend this known regime could
achieve .phi.>0.6, up to about .phi..apprxeq.0.9. After
emulsifying at a particular 0, droplet structures (including simple
single droplets and also double droplets) can be subsequently
concentrated to higher .phi. by applying osmotic stresses through
methods including evaporation, dialysis, centrifugation,
ultracentrifugation, filtration, and microfluidic concentration.
The maximum volume fraction to which the emulsion can be
concentrated and still remain stable can depend on many factors,
including the droplet sizes and how the copolymers stabilize the
elastic interfaces. For certain embodiments, the concentration can
be achieved up to .phi..apprxeq.0.95. For nanoscale droplets,
reaching volume fractions of up to about .phi..apprxeq.0.8 through
concentration processes subsequent to the emulsification process is
more typical.
[0115] After making a double emulsion, we can typically use methods
of size fractionation such as centrifugation, ultracentrifugation,
outer-droplet size-dependent depletion attractions, etc. to
separate smaller outer droplets from larger ones according to some
embodiments of the current invention. This may also potentially be
used to separate out inner droplet volumes since the buoyancy of a
droplet depends on its density, which is determined both by inner
and outer droplet volumes.
[0116] The "boundary surface region" referred to herein includes
the following. Practitioners in this art typically say that there
is a "film" of the primary dispersed phase (i.e. oil) between the
secondary dispersed phase (i.e. inner droplet of water) and the
continuous phase (i.e. water solution). In a stable double
emulsion, there is a disjoining pressure of this film that can
resist thermal driving stresses, chemical driving stresses, and
mild external agitation (e.g. physical shear stresses), and
therefore the film is called "stable". Stability of the film is
equivalent to the resistance to coalescence of the two oil-water
interfaces that have mean curvatures of opposite signs (by commonly
accepted conventions). Stability of the outer droplets from
coarsening through coalescence is also generally desirable for
having a useful product that remains shelf-stable. In this case,
there is also at least a short-range repulsion that creates a
repulsive disjoining pressure in the water film of continuous phase
that separates the oil-water interfaces of two outer droplets that
may closely approach.
[0117] Also, other types of materials can be used as stabilizers or
surface modifiers, according to additional embodiments of the
current invention, which could potentially be incorporated into the
block copolymers that stabilize interfaces of double and multiple
emulsions. Some potential copolymers include: lipo-polypeptides,
glyco-polypeptides, and polynucleicacid-polypeptides (i.e.
polypeptide-polynucleotide copolymers). For instance, a charged
oligonucleotide or short polynucleotide (e.g. single-stranded DNA,
double-stranded DNA, RNA, etc) could be substituted for the
hydrophilic block and attached to a racemic hydrophobic block (e.g.
rac-L) to confer the desired solubility and interfacial stability
properties.
[0118] Another embodiment can include PEG-modified block
copolymers: poly-(ethylene glycol)-poly-(peptide) specifically for
use in making double emulsions and for decorating the surfaces of
stable double emulsions (even if such PEGylated molecules might not
create much additional interfacial stability). PEG and PEG
derivatives are known to provide good resistant coatings for drug
delivery vehicles, so it is anticipated that PEG-modified double
emulsions could remain longer in circulation in the
bloodstream.
[0119] It can be reasonably expected that the release of cargo,
such as drug molecules, contained in double emulsions and double
nanoemulsions can be triggered by a change in the pH, ionic
strength, temperature, chemical environment, or a combination
thereof. Such a change could affect the conformation, density, and
interactions between copolypeptides that reside at oil-water
interfaces, thereby altering the stability and creating conditions
suitable for release. Likewise, because of their liquid nature, it
can be expected that, according to some embodiments of the current
invention, double emulsions can exhibit excellent clearance
properties when introduced into an organism, including humans. This
clearance property refers to mechanisms by which the organism can
clear (i.e. digest, excrete, or otherwise get rid of) the droplet
materials and associated stabilizing materials.
[0120] It can be reasonably expected that the following natural
amino acids can be polymerized to become a portion of the molecular
composition of the copolymer that stabilizes droplet structures,
including but not limited to single droplet, double droplet, and
multiple droplet structures. This stabilization would encompass
nanoscale and larger droplet structures. These amino acids may come
in a variety of forms, including but not limited to chiral,
enantiomeric, and other molecular specifications, such as H-, L-,
Z- D-, LD-, and rac-forms. The categorization as `natural` is
somewhat arbitrary, but a good guide for `natural` amino acids can
be deduced from the lists of products in the catalogs of large
biochemical and chemical suppliers such as Sigma-Aldrich.RTM.. For
instance, in their catalog, a wide variety of synthetic precursors
are available for the following amino acids: Alanine, Arginine,
Asparagine, Aspartic Acid, Cysteine/Cystine, Glutamic Acid,
Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine,
Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan,
Tyrosine, Valine.
[0121] Although many of the examples described above demonstrate
that using fully racemic amino-acids for the hydrophobic block in
copolypeptides can promote the formation and stabilization of
double emulsion structures, general aspects of this invention are
not limited to only these examples. For example, it is possible to
design and synthesize copolypeptides having hydrophobic blocks that
contain a portion of non-racemic amino acids (i.e. either
subsections of several D-amino acids in a row and/or subsections of
several L-amino acids in a row within a hydrophobic block that
contains some racemic nature) and that these copolypeptides could
still confer a desirable property of promoting the formation and
stabilization of double emulsions according to some embodiments of
the current invention. Likewise, although we have shown examples
that using non-racemic amino acids in the hydrophobic block in
copolypeptides can tend to promote the formation and stabilization
of single emulsions, it is possible to design and synthesize
copolypeptides having hydrophobic blocks that contain a portion of
racemic amino acids and that these copolypeptides could still
confer a desirable property of promoting the formation and
stabilization of single emulsions according to some embodiments of
the current invention.
[0122] Other natural amino-acid-related structures can be
polymerized to form a portion of the molecular composition of the
copolymer that stabilizes double emulsions and double nanoemulsion
structures. These include the following: Amino Alcohols, Amino
Aldehydes, Amino Lactones, and n-Methyl Amino Acids.
[0123] Examples of unnatural amino acids and amino acid derivatives
that can be part of the copolymer that stabilizes single, double,
and multiple emulsions are: Alanine Derivatives, Alicyclic Amino
Acids, Arginine Derivatives, Aromatic Amino Acids, Asparagine
Derivatives, Aspartic Acid Derivatives, Beta-Amino Acids, Cysteine
Derivatives, DAB (2,4-Diaminobutyric Acid), DAP
(2,3-Diaminopropionic Acid), Glutamic Acid Derivatives, Glutamine
Derivatives, Glycine Derivatives, Histidine Derivatives, Homo-Amino
Acids, Isoleucine Derivatives, Leucine Derivatives, Linear Core
Amino Acids, Lysine Derivatives, n-Methyl Amino Acids, Norleucine,
Norvaline, Ornithine, Penicillamine, Phenylalanine Derivatives,
Phenylglycine Derivatives, Proline Derivatives, Pyroglutamine
Derivatives, Serine Derivatives, Threonine Derivatives, Tryptophan
Derivatives, Tyrosine Derivatives, Valine Derivatives, as well as
more than 100 `Other` derivative types of molecular compositions
and structures listed in common catalogues of biochemical and
chemical suppliers. For instance, there are more than a thousand
types of unnatural amino acid derivatives listed as products of
Sigma-Aldrich.RTM. in August, 2008. This number is likely to grow
and will provide alternative other molecular structures that can be
incorporated into copolypeptides used to stabilize emulsions,
double emulsions, and multiple emulsions. Another source of
potential molecular constituents that could be used to fabricate
complex amphiphilic copolymers suitable for stabilizing single
emulsions or double emulsions is BACHEM Americas Inc.'s 2008
"Building Blocks" and "Peptides and Biochemicals" catalogs
(www.bachem.com), which describes many kinds of amino acid
derivatives, special amino acids, resin-linked amino acids, and
other linkers and reagents.
[0124] The copolymers that stabilize droplets can have molecular
compositions and structures that include reactive groups (e.g.
polymerizable groups, pH-sensitive groups, photo-reactive groups,
and photo-polymerizable groups) which can be activated through
chemical or physical changes to provide linking and/or coupling
functionality between copolymer molecules on the same interface,
copolymer molecules on adjacent inner and outer interfaces within
the same double or multiple emulsion structure, between copolymer
molecules on the interfaces of adjacent inner droplets, and between
copolymer molecules on the interfaces of adjacent outer
droplets.
[0125] The copolymers that stabilize droplets structures can have
enzymatic and catalytic functionality. These include the following:
Enzymes, Analytical Enzymes, Cofactors, Collagenases, Enzyme
Inhibitors, Enzyme-Mediated Synthesis, Stabilizers, Enzyme
Substrates, Lectins, Molecular Biology Enzymes, Kinases,
Phosphatases, and Proteolytic Enzymes and Substrates. Other
desirable functional molecular components of the copolymers can
also be chosen and incorporated into copolymers, such as amine
protectors, guanidine protectors, and guanidinylation.
[0126] Useful synthetic structures that can be polymerized to form
a portion of the molecular composition of the copolymer stabilizes
droplet structures, including but not limited to single droplet,
double droplet, and multiple droplet structures, whether microscale
or nanoscale droplet structures. These synthetic structures
include, but are not limited to, the following: Poly-(ethylene
glycol) (PEG), Functionalized Oligoethylene Glycols, Monofunctional
PEGs, Homobifunctional PEGs, Heterobifunctional PEGs, PEGylated
oligonucleotides, and PEGylated peptides.
[0127] In general, synthetic derivative molecules that mimic at
least some aspects of the composition, structure, and function can
be reasonably anticipated to provide stabilization to double
emulsions similar to what we describe herein. Therefore, it can be
reasonably anticipated that new unnatural amino-acid-like molecules
developed in the future could also be used to stabilize double and
multiple emulsion structures.
TABLE-US-00001 TABLE 1 Block copolypeptide surfactants used to
prepare emulsions. All emulsions were prepared using a microfluidic
homogenizer under the following conditions: number of passes N = 6,
homogenizer inlet air pressure p = 130 psi, block copolypeptide
concentration C = 1.0 mM, Water Block Droplet Inner/Outer
Solubility Interfacial Copolypeptide M.sub.n .times. (10.sup.-3)*
Diameter (nm) Diameter Ratio CAC (M) Limit (mM) Tension
(dyne/cm).sup..dagger. K.sub.20(rac-L).sub.10 5.5 380 0.52 1.5
.times. 10.sup.-5 N/A N/A K.sub.40(rac-L).sub.5 11.0 430 0.48 1.1
.times. 10.sup.-4 11.5 N/A K.sub.40(rac-L).sub.10 10.5 200 0.47 2.0
.times. 10.sup.-5 8.5 N/A K.sub.40(rac-L).sub.20 11.0 120 0.57 9.7
.times. 10.sup.-7 3.0 25.3 K.sub.40(rac-L).sub.20** 11.0 60 0.45
N/A N/A N/A K.sub.40(rac-L).sub.30 11.1 60 0.52 3.6 .times.
10.sup.-7 1.0 N/A K.sub.60(rac-L).sub.20 16.2 N/A N/A 3.6 .times.
10.sup.-6 4.5 N/A K.sub.100(rac-L).sub.10 27.1 N/A N/A 3.6 .times.
10.sup.-5 N/A N/A K.sub.60(rac-A).sub.20 16.3 N/A N/A 4.1 .times.
10.sup.-5 N/A N/A K.sub.60(rac-V).sub.20 15.8 N/A N/A 4.9 .times.
10.sup.-6 N/A N/A K.sub.60L.sub.20.sup.# 16.2 130 N/A 7.1 .times.
10.sup.-7 2.5 33.4 R.sub.40(rac-L).sub.10 10.7 220 0.51 2.4 .times.
10.sup.-5 N/A N/A E.sub.40(rac-L).sub.10 9.1 210 0.52 2.4 .times.
10.sup.-5 N/A N/A *= number average molecular masses determined
using GPC-LS. **= This sample was fractionated from larger droplets
by centrifugation followed by ultracentrifugation. .sup.#= This
sample formed a simple WO emulsion. .sup..dagger.= Oil/water
interfacial tension data of 10 cSt PDMS in contact with: 10 mM
aqueous sodium dodecyl sulphate (SDS) solution = 12.4 dyne/cm; in
contact with deionized water = 40.7 dyne/cm. N/A = experiment not
performed
and oil volume fraction .phi.=0.20. Diameters (of the outer
droplets) and inner/outer diameter ratios were determined by
averaging measurements of at least 50 droplets from CTEM images.
Critical aggregation concentration (CAC) values were determined
using pyrene fluorescence at 20.degree. C. Water solubility limits
were measured by diluting 15 mM stock solutions of each polypeptide
until optically clear solutions were created. The block copolymers
had negligible solubility in PDMS. Oil/water interfacial tension
data were measured using the Du Nouy ring method using 10 cSt PDMS
and block copolypeptide solutions (0.1 mM, pull rate=0.01 mm/s,
25.degree. C.).
* * * * *