U.S. patent application number 12/666168 was filed with the patent office on 2010-10-07 for stimuli responsive nanofibers.
This patent application is currently assigned to Innovative Surface Technologies, Inc.. Invention is credited to Patrick Guire, Tahmina Naqvi, Jie Wen.
Application Number | 20100255581 12/666168 |
Document ID | / |
Family ID | 40186249 |
Filed Date | 2010-10-07 |
United States Patent
Application |
20100255581 |
Kind Code |
A1 |
Naqvi; Tahmina ; et
al. |
October 7, 2010 |
STIMULI RESPONSIVE NANOFIBERS
Abstract
A stimuli responsive nanofiber that includes a stimuli
responsive polymer, such as a thermally responsive polymer, and a
cross-linking agent having at least two latent reactive activatable
groups. The nanofiber may also include a biologically active
material or a functional polymer. The stimuli responsive nanofiber
can be used to modify the surface of a substrate. When the
nanofiber includes a thermally responsive polymer, the physical
properties of the surface can be controlled by controlling the
temperature of the system, thus controlling the ability of the
surface to bind to a biologically active material of interest.
Inventors: |
Naqvi; Tahmina; (Blaine,
MN) ; Wen; Jie; (Eden Prairie, MN) ; Guire;
Patrick; (Hopkins, MN) |
Correspondence
Address: |
Fulbright & Jaworski L.L.P.;Attn: MNIPDOCKET
Innovative Surface Technologies, Inc., 600 Congress Avenue, Suite 2400
Austin
TX
78701
US
|
Assignee: |
Innovative Surface Technologies,
Inc.
St. Paul
MN
|
Family ID: |
40186249 |
Appl. No.: |
12/666168 |
Filed: |
June 20, 2008 |
PCT Filed: |
June 20, 2008 |
PCT NO: |
PCT/US08/67708 |
371 Date: |
June 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60945801 |
Jun 22, 2007 |
|
|
|
Current U.S.
Class: |
435/396 |
Current CPC
Class: |
A61L 27/16 20130101;
D01F 6/22 20130101; A61L 29/085 20130101; A61L 27/34 20130101; A61L
27/16 20130101; A61L 29/14 20130101; A61L 31/16 20130101; A61L
29/085 20130101; A61L 27/34 20130101; A61L 31/048 20130101; C12M
23/20 20130101; A61L 29/041 20130101; A61L 27/50 20130101; D01F
1/10 20130101; C12N 5/0068 20130101; D06M 2400/01 20130101; A61L
31/10 20130101; C12N 2533/30 20130101; A61L 31/14 20130101; C12N
2539/00 20130101; C12M 25/14 20130101; D01D 5/0007 20130101; C12M
23/08 20130101; G01N 33/48 20130101; A61L 29/041 20130101; A61L
29/16 20130101; C12N 2537/10 20130101; D01F 6/625 20130101; A61L
2300/00 20130101; C08L 33/26 20130101; C08L 33/26 20130101; D06M
15/285 20130101; D01F 6/26 20130101; A61L 31/048 20130101; A61L
27/54 20130101; A61L 31/10 20130101; A61L 2400/12 20130101; C08L
33/26 20130101; C08L 33/26 20130101; C08L 33/26 20130101; C08L
33/26 20130101 |
Class at
Publication: |
435/396 |
International
Class: |
C12N 5/07 20100101
C12N005/07 |
Claims
1.-17. (canceled)
18. A cell culture article comprising a surface and a thermally
responsive polymer, wherein the thermally responsive polymer is
associated with the surface via a crosslinking agent having at
least two latent reactive groups.
19. The article according to claim 18 wherein the thermally
responsive polymer comprises a polymer which has a first physical
property at a first predetermined temperature range and a second
physical property at a second predetermined temperature range.
20. The article according to claim 18 wherein at least one of the
latent reactive groups comprises a photochemical reactive
group.
21. The article according to claim 20 wherein the photochemical
reactive group is an aryl ketone.
22. The article according to claim 21 wherein the aryl ketone is
selected from the group acetophenone, benzophenone, anthraquinone,
anthrone, acridone, xanthone, and thioxanthone.
23. The article according to claim 20 wherein all of the latent
reactive groups of the crosslinking agent are photochemical
reactive groups.
24. The article according to claim 23 wherein all of the
photochemical reactive groups are the same.
25. The article according to claim 18 wherein at least one of the
latent reactive groups is an aryl azide.
26. The article according to claim 18 wherein at least one of the
latent reactive groups comprises a thermally-reactive group.
27. The article according to claim 23 wherein the
thermally-reactive group includes a heat labile bond selected from
oxygen-oxygen bonds, nitrogen-oxygen bonds, and nitrogen-nitrogen
bonds.
28. The article according to claim 26 wherein all of the latent
reactive groups of the crosslinking agent comprise
thermally-reactive groups.
29. The article according to claim 28 wherein all of the
thermally-reactive groups are the same.
30. The article according to claim 18 wherein the crosslinking
agent is a monomeric material.
31. The article according to claim 18 wherein the crosslinking
agent is a compound having a formulae selected from (a)-(g):
L-(D-T-C(R.sup.1)(XP)CHR.sup.2GR.sup.3C(.dbd.O)R.sup.4).sub.m (a)
wherein L is a linking group; D is CR.sup.6R.sup.7; T is
(--CH.sub.2--).sub.x, (--CH.sub.2CH.sub.2--O--).sub.x,
(--CH.sub.2CH.sub.2CH.sub.2--O--).sub.x,
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--O--).sub.x or forms a bond;
R.sup.1 is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl,
aryloxyalkyl or aryloxyaryl group; X is O, S, or NR.sup.8R.sup.9; P
is a hydrogen atom or a protecting group, with the proviso that P
is absent when X is NR.sup.8R.sup.9; R.sup.2 is a hydrogen atom, an
alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; G is
O, S, SO, SO.sub.2, NR.sup.10, (CH.sub.2).sub.t--O-- or C.dbd.O;
R.sup.3 and R.sup.4 are each independently an alkyl, aryl,
arylalkyl, heteroaryl, or an heteroarylalkyl group or optionally,
R.sup.3 and R.sup.4 can be tethered together via
(--CH.sub.2--).sub.q,
(--CH.sub.2--).sub.rC.dbd.O(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS(--CH.sub.2--).sub.x,
(--CH.sub.2--).sub.rS.dbd.O(--CH.sub.2--).sub.s or
(--CH.sub.2--).sub.rS(O).sub.2(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rNR(--CH.sub.2--).sub.s; R.sup.10 is a hydrogen
atom or an alkyl, aryl or arylalkyl group; R.sup.6 and R.sup.7 are
each independently a hydrogen atom, an alkyl, aryl, arylalkyl,
heteroaryl or heteroarylalkyl group; R.sup.8 and R.sup.9 are each
independently a hydrogen atom, an alkyl, aryl, or arylalkyl group;
R is a hydrogen atom, an alkyl or an aryl group; q is an integer
from 1 to about 7; r is an integer from 0 to about 3; s is an
integer from 0 to about 3; m is an integer from 2 to about 10; t is
an integer from 1 to about 10; and x is an integer from 1 to about
500; L-(T-C(R.sup.1)(XP)CHR.sup.2GR.sup.3C(.dbd.O)R.sup.4).sub.m
(b) wherein L is a linking group; T is (--CH.sub.2--).sub.x,
(--CH.sub.2CH.sub.2--O--).sub.x,
(--CH.sub.2CH.sub.2CH.sub.2--O--).sub.x,
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--O--).sub.x or forms a bond;
R.sup.1 is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl,
aryloxyalkyl or aryloxyaryl group; X is O, S, or NR.sup.8R.sup.9; P
is a hydrogen atom or a protecting group, with the proviso that P
is absent when X is NR.sup.8R.sup.9; R.sup.2 is a hydrogen atom, an
alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; G is
O, S, SO, SO.sub.2, NR.sup.10, (CH.sub.2).sub.t--O-- or C.dbd.O;
R.sup.3 and R.sup.4 are each independently an alkyl, aryl,
arylalkyl, heteroaryl, or an heteroarylalkyl group, or optionally,
R.sup.3 and R.sup.4 can be tethered together via
(--CH.sub.2--).sub.q,
(--CH.sub.2--).sub.rC.dbd.O(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS.dbd.O(--CH.sub.2--).sub.s or
(--CH.sub.2--).sub.rS(O).sub.2(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.r--NR(--CH.sub.2--).sub.s; R.sup.10 is a
hydrogen atom or an alkyl, aryl or arylalkyl group; R.sup.8 and
R.sup.9 are each independently a hydrogen atom, an alkyl, aryl, or
arylalkyl group; R is a hydrogen atom, an alkyl or aryl group; q is
an integer from 1 to about 7; r is an integer from 0 to about 3; s
is an integer from 0 to about 3; m is an integer from 2 to about
10; t is an integer from 1 to about 10; and x is an integer from 1
to about 500; L-(GTZR.sup.3C(.dbd.O)R.sup.4).sub.m (c) wherein L is
a linking group; T is (--CH.sub.2--).sub.x,
(--CH.sub.2CH.sub.2--O--).sub.x,
(--CH.sub.2CH.sub.2CH.sub.2--O--).sub.x,
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--O--).sub.x or forms a bond; G
is O, S, SO, SO.sub.2, NR.sup.10, (CH.sub.2).sub.t--O-- or C.dbd.O;
Z is C.dbd.O, COO, or CONH when T is (--CH.sub.2--).sub.x; R.sup.3
and R.sup.4 are each independently an alkyl, aryl, arylalkyl,
heteroaryl, or an heteroarylalkyl group, or optionally, R.sup.3 and
R.sup.4 can be tethered together via (--CH.sub.2--).sub.q,
(--CH.sub.2--).sub.rC.dbd.O(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS.dbd.O(--CH.sub.2--).sub.s or
(--CH.sub.2--).sub.rS(O).sub.2(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rNR(--CH.sub.2--).sub.s; R is a hydrogen atom or
an alkyl or aryl group; q is an integer from 1 to about 7; r is an
integer from 0 to about 3; s is an integer from 0 to about 3; m is
an integer from 2 to about 10; t is an integer from 1 to about 10;
and x is an integer from 1 to about 500;
L-(TGQR.sup.3C(.dbd.O)R.sup.4).sub.m (d) wherein L is a linking
group; T is (--CH.sub.2--).sub.x, (--CH.sub.2CH.sub.2--O--).sub.x,
(--CH.sub.2CH.sub.2CH.sub.2--O--).sub.x,
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--O--).sub.x or forms a bond; G
is O, S, SO, SO.sub.2, NR.sup.10, (CH.sub.2).sub.t--O-- or C.dbd.O;
Q is (--CH.sub.2--).sub.p, (--CH.sub.2CH.sub.2--O--).sub.p,
(--CH.sub.2CH.sub.2CH.sub.2--O--).sub.p or
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--O--).sub.p; R.sup.3 and
R.sup.4 are each independently an alkyl, aryl, arylalkyl,
heteroaryl, or an heteroarylalkyl group, or optionally, R.sup.3 and
R.sup.4 can be tethered together via (--CH.sub.2--).sub.q,
(--CH.sub.2--).sub.rC.dbd.O(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS.dbd.O(--CH.sub.2--).sub.s or
(--CH.sub.2--).sub.rS(O).sub.2(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rNR(--CH.sub.2--).sub.s; R.sup.10 is a hydrogen
atom or an alkyl, aryl, or an arylalkyl group; R is a hydrogen atom
or an alkyl or aryl group; q is an integer from 1 to about 7; r is
an integer from 0 to about 3; s is an integer from 0 to about 3; m
is an integer from 2 to about 10; p is an integer from 1 to about
10; t is an integer from 1 to about 10; and x is an integer from 1
to about 500;
L-(--CH.sub.2--).sub.xxC(R.sup.1)(GR.sup.3C(.dbd.O)R.sup.4).sub.m
(e) wherein L is a linking group; R.sup.1 is a hydrogen atom, an
alkyl, alkyloxyalkyl, aryl, aryloxyalkyl, or aryloxyaryl group;
each G is G is O, S, SO, SO.sub.2, NR.sup.10, (CH.sub.2).sub.t--O--
or C.dbd.O; each R.sup.3 and R.sup.4 is independently an alkyl,
aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group, or
optionally, R.sup.3 and R.sup.4 can be tethered together via
(--CH.sub.2--).sub.q,
(--CH.sub.2--).sub.rC.dbd.O(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS.dbd.O(--CH.sub.2--).sub.s or
(--CH.sub.2--).sub.rS(O).sub.2(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rNR(--CH.sub.2--).sub.s; each R.sup.10 is a
hydrogen atom or an alkyl, aryl, or an arylalkyl group; each R is a
hydrogen atom or an alkyl or aryl group; each q is an integer from
1 to about 7; each r is an integer from 0 to about 3; each s is an
integer from 0 to about 3; m is an integer from 2 to about 10; each
t is an integer from 1 to about 10; and xx is an integer from 1 to
about 10;
L-(-C(R.sup.1)(XP)CHR.sup.2GR.sup.3C(.dbd.O)R.sup.4).sub.m (f)
wherein L is a linking group; R.sup.1 is a hydrogen atom, an alkyl,
alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; X is O, S,
or NR.sup.8R.sup.9; P is a hydrogen atom or a protecting group,
with the 27. that P is absent when X is NR.sup.8R.sup.9; R.sup.2 is
a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or
aryloxyaryl group; G is G is O, S, SO, SO.sub.2, NR.sup.10,
(CH.sub.2).sub.t--O-- or C.dbd.O; R.sup.3 and R.sup.4 are each
independently an alkyl, aryl, arylalkyl, heteroaryl, or an
heteroarylalkyl group, or optionally, R.sup.3 and R.sup.4 can be
tethered together via (--CH.sub.2--).sub.q,
(--CH.sub.2--).sub.rC.dbd.O(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS.dbd.O(--CH.sub.2--).sub.s or
(--CH.sub.2--).sub.rS(O).sub.2(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rNR(--CH.sub.2--).sub.s; R.sup.8 and R.sup.9 are
each independently a hydrogen atom, an alkyl, aryl, or arylalkyl
group; R.sup.10 is a hydrogen atom or an alkyl, aryl, or an
arylalkyl group; R is a hydrogen atom, an alkyl or an aryl group; q
is an integer from 1 to about 7; r is an integer from 0 to about 3;
s is an integer from 0 to about 3; m is an integer from 2 to about
10; and t is an integer from 1 to about 10; and
L-(GR.sup.3C(.dbd.O)R.sup.4).sub.m; (g) wherein L is a linking
group; G is O, S, SO, SO.sub.2, NR.sup.10, (CH.sub.2).sub.t--O-- or
C.dbd.O; R.sup.3 and R.sup.4 are each independently an alkyl, aryl,
arylalkyl, heteroaryl, or an heteroarylalkyl group, or optionally,
R.sup.3 and R.sup.4 can be tethered together via
(--CH.sub.2--).sub.q,
(--CH.sub.2--).sub.rC.dbd.O(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS.dbd.O(--CH.sub.2--).sub.s or
(--CH.sub.2--).sub.rS(O).sub.2(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rNR(--CH.sub.2--).sub.s; R is a hydrogen atom,
an alkyl or an aryl group; q is an integer from 1 to about 7; r is
an integer from 0 to about 3; s is an integer from 0 to about 3; m
is an integer from 2 to about 10; and t is an integer from 1 to
about 10.
32. The article according to claim 31 wherein the crosslinking
agent is a compound of formula: ##STR00010##
33. The article according to claim 18 wherein the cell culture
article comprises a nanofiber or nanofiber mesh.
34. The article according to claim 18 wherein the cell culture
article is selected from slides, multi-well plates, Petri dishes,
tissue culture plates, tissue culture flasks, and coverslips.
35. The article according to claim 33 wherein the nanofiber or
nanofiber mesh is provided on a surface of a slide, multi-well
plate, Petri dish, tissue culture plate, tissue culture flask or
coverslip.
36. The article according to claim 18 wherein the surface comprises
a polyolefin, polystyrene, poly(methyl)methacrylate,
polyacrylonitrile, poly(vinylacetate), poly(vinyl alcohol),
chlorine-containing polymer, polyoxymethylene, polycarbonate,
polyamide, polyimide, polyurethane, phenolic, amino-epoxy resin,
polyester, silicone, cellulose-based plastic, fluoropolymer or
rubber-like plastics.
37. The article according to claim 18 wherein the surface comprises
pyrolytic carbon, parylene coated surface, or a silylated surface
of glass, ceramic or metal.
38. The article according to claim 18 wherein the thermally
responsive polymer comprises poly(isopropylacrylamide), copolymers
of polyethylene glycol and poly(isopropylacrylamide), or mixtures
thereof.
39. A cell culture article comprising a surface and a stimuli
responsive polymer which undergoes a physical or chemical change
when exposed to external stimuli, wherein the stimuli responsive
polymer is associated with the surface via a crosslinking agent
having at least two latent reactive groups.
40. A kit for preparing a cell culture article, the kit comprising:
a stimuli responsive polymer suitable for cell culture, which
polymer undergoes a physical or chemical change when exposed to
external stimuli; and a crosslinking agent having at least two
latent reactive groups, the crosslinking agent being adapted for
associating the stimuli responsive polymer to a surface of a cell
culture article.
41. A method for preparing a cell culture article, the method
comprising steps of: (a) contacting a surface of the cell culture
article with a coating composition, the coating composition
comprising a stimuli responsive polymer which undergoes a physical
or chemical change when exposed to external stimuli, and a
crosslinking agent having at least two latent reactive groups; and
(b) treating the coating composition to activate latent reactive
groups of the crosslinking agent, thereby coupling the stimuli
responsive polymer to the surface of the cell culture article in a
manner in which at least some of the latent reactive groups remain
in an inactive state.
42. The method according to claim 41 further comprising a step of
coupling a biologically active material to the surface of the cell
culture device by activating remaining latent reactive groups of
step (b).
43. A method for preparing a cell culture article, the method
comprising steps of: (a) contacting a surface of the cell culture
article with a coating composition comprising a stimuli responsive
polymer which undergoes a physical or chemical change when exposed
to external stimuli, a crosslinking agent having at least two
latent reactive groups, and a biologically active material; and (b)
treating the coating composition to activate latent reactive groups
of the crosslinking agent to form a coating on the cell culture
device surface.
44. A method for preparing a cell culture article, the method
comprising steps of: (a) contacting a surface of the cell culture
article with a crosslinking agent having at least two latent
reactive groups; (b) treating the crosslinking agent to activate
less than all of the latent reactive groups of the crosslinking
agent, thereby coupling the crosslinking agent to the surface of
the cell culture and allowing at least some of the latent reactive
groups to remain in an inactive state; (c) contacting the surface
of the cell culture article with a stimuli responsive polymer which
undergoes a physical or chemical change when exposed to external
stimuli; (d) treating the surface of the cell culture article to
activate latent reactive groups which are present in an inactive
state subsequent to step (b), thereby coupling the stimuli
responsive polymer to the cell culture article surface.
45. A method for preparing a cell culture article, the method
comprising steps of: (a) preparing a composition comprising a
stimuli responsive polymer which undergoes a physical or chemical
change when exposed to external stimuli, and a crosslinking agent
having at least two latent reactive groups; (b) forming a cell
culture article from the composition of step (a); and (c) treating
the cell culture article formed in step (b) to activate latent
reactive groups of the crosslinking agent.
46. The method according to claim 45, wherein the step of forming a
cell culture article comprises forming a nanofiber.
47. A method for preparing a cell culture article, the method
comprising steps of: (a) preparing a composition comprising a
polymer and a crosslinking agent having at least two latent
reactive groups; (b) forming a cell culture article from the
composition of step (a); (c) contacting a surface of the cell
culture article with a stimuli responsive polymer which undergoes a
physical or chemical change when exposed to external stimuli; and
(d) treating the surface of the cell culture article to activate
latent reactive groups of the crosslinking agent, thereby coupling
the stimuli responsive polymer to the cell culture article
surface.
48. The method according to claim 47 wherein the polymer of step
(a) comprises a polyolefin, polystyrene, poly(methyl)methacrylate,
polyacrylonitrile, poly(vinylacetate), poly(vinyl alcohol),
chlorine-containing polymer, polyoxymethylene, polycarbonate,
polyamide, polyimide, polyurethane, phenolic, amino-epoxy resin,
polyester, silicone, cellulose-based plastic, fluoropolymer or
rubber-like plastic.
49. The article according to claim 47 wherein the polymer of step
(a) comprises pyrolytic carbon, or a silylated surface of glass,
ceramic or metal.
Description
TECHNICAL FIELD
[0001] The present invention generally relates to stimuli
responsive nanofibers and stimuli responsive nanofiber modified
surfaces. More particularly, the present invention is directed to
nanofibers including a thermally responsive polymer, a
multi-functional cross-linking agent, and optionally a biologically
active material or a functional polymer that is reactive with a
biologically active material. The stimuli responsive nanofibers can
be used to modify a surface of a substrate, such as a cell culture
device.
BACKGROUND
[0002] Nanofibers are being considered for a variety of
applications because of their unique properties including high
surface area, small fiber diameter, layer thinness, high
permeability, and low basis weight. More attention has been focused
on functionalized nanofibers having the capability of incorporating
active chemistry, especially in biomedical applications such as
wound dressing, biosensors and scaffolds for tissue
engineering.
[0003] Nanofibers may be fabricated by electrostatic spinning (also
referred to as electrospinning). The technique of electrospinning
of liquids and/or solutions capable of forming fibers, is well
known and has been described in a number of patents, such as, for
example, U.S. Pat. Nos. 4,043,331 and 5,522,879. The process of
electrospinning generally involves the introduction of a solution
or liquid into an electric field, so that the solution or liquid is
caused to produce fibers. These fibers are generally drawn to a
conductor at an attractive electrical potential for collection.
During the conversion of the solution or liquid into fibers, the
fibers harden and/or dry. This hardening and/or drying may be
caused by cooling of the liquid, i.e., where the liquid is normally
a solid at room temperature; by evaporation of a solvent, e.g., by
dehydration (physically induced hardening); or by a curing
mechanism (chemically induced hardening).
[0004] The process of electrostatic spinning has typically been
directed toward the use of the fibers to create a mat or other
non-woven material, as disclosed, for example, in U.S. Pat. No.
4,043,331. Nanofibers ranging from 50 nm to 5 .mu.m in diameter can
be electrospun into a nonwoven or an aligned nanofiber mesh. Due to
the small fiber diameters, electrospun textiles inherently possess
a very high surface area and a small pore size. These properties
make electrospun fabrics potential candidates for a number of
applications including: membranes, tissue scaffolding, and other
biomedical applications.
[0005] Nanofibers can be used to modify the surface of a substrate
to achieve a desired surface characteristic. Most nanofiber
surfaces have to be engineered to obtain the ability to immobilize
biomolecules. Surface modification of synthetic biomaterials, with
the intent to improve biocompatibility, has been extensively
studied, and many common techniques have been considered for
polymer nanofiber modification. For example, Sanders et al in
"Fibro-Porous Meshes Made from Polyurethane Micro-Fibers: Effects
of Surface Charge on Tissue Response" Biomaterials 26, 813-818
(2005) introduced different surface charges on electrospun
polyurethane (PU) fiber surfaces through plasma-induced surface
polymerization of negatively or positively charged monomers. The
surface charged PU fiber mesh was implanted in rat subcutaneous
dorsum for 5 weeks to evaluate tissue compatibility, and it was
found that negatively charged surfaces may facilitate vessel
ingrowth into the fibroporous mesh biomaterials. Ma et al. in
"Surface Engineering of Electrospun Polyethylene Terephthalate
(PET) Nanofibers Towards Development of a New Material for Blood
Vessel Engineering" Biomaterials 26, 2527-2536 (2005) conjugated
gelatin onto formaldehyde pretreated polyethylene terephthalate
(PET) nanofibers through a grafted polymethacrylic acid spacer and
found that the gelatin modification improved the spreading and
proliferation of endothelial cells (ECs) on the PET nanofibers, and
also preserved the EC's phenotype. Chua et al. in "Stable
Immobilization of Rat Hepatocyte Spheroids on Galactosylated
Nanofiber Scaffold" Biomaterials 26, 2537-2547 (2005) introduced
galactose ligand onto poly(e-caprolactone-co-ethyl ethylene
phosphate) (PCLEEP) nanofiber scaffold via covalent conjugation to
a poly(acrylic acid) spacer UV-grafted onto the fiber surface.
Hepatocyte attachment, ammonia metabolism, albumin secretion and
cytochrome P450 enzymatic activity were investigated on the 3-D
galactosylated PCLEEP nanofiber scaffold as well as the functional
2-D film substrate.
SUMMARY
[0006] The methods and techniques summarized above are costly,
complicated, or material specific. Thus, there is a need for a
surface modification approach that is more general and easy to use
and can be applied under mild conditions to a wide variety of
nanofibers.
[0007] According to one embodiment, the present invention is a
stimuli responsive nanofiber including a stimuli responsive
polymer. One example of a stimuli responsive nanofiber is a
thermally responsive nanofiber including a thermally responsive
polymer. In either of these embodiments, the stimuli responsive
nanofiber may include a cross-linking agent having at least two
latent reactive activatable groups. In use, photochemically,
electrochemically or thermally latent reactive groups will form
covalent bonds when subjected to a source of energy. Suitable
energy sources include radiation, electrical and thermal energy. In
some embodiments, the radiation energy is visible, ultraviolet,
infrared, x-ray or microwave electromagnetic radiation.
[0008] The cross-linking agent may have at least two latent
reactive activatable groups. These latent reactive groups may be
the same or may be different. For example, all of the latent
reactive groups may be photochemically reactive groups.
Alternatively, in other embodiments of the invention the
cross-linking agent may include both photochemically and thermally
reactive groups. Further, the cross-linking agent may be monomeric
or polymeric materials or may be a mixture of both monomeric and
polymeric materials.
[0009] According to a further embodiment of the present invention,
the thermally responsive polymer is poly(isopropylacrylamide) as
well as derivatives of poly(isopropylacrylamide) such as graft
copolymer derivatives with polyethylene glycol derivatives.
[0010] According to another embodiment, the present invention is a
method of treating a surface of a substrate including the steps of
combining a stimuli responsive polymer, such as a thermally
responsive polymer, and a cross-linking agent having at least two
latent reactive activatable groups; forming at least one nanofiber
from the combined mixture; contacting the surface with the
nanofiber; and forming a bond between the nanofiber and the
surface.
[0011] According to another embodiment, the present invention is a
surface coating for a surface of an article. The surface coating
includes a stimuli responsive nanofiber including a nanofiber
coated with a stimuli responsive polymer, such as a thermally
responsive polymer, and a cross-linking agent having at least two
latent reactive activatable groups. Optionally, the coated
nanofiber or the coated surface may include a biologically active
material or, alternatively, a functional polymer.
[0012] According to yet another embodiment, the present invention
is an article including a surface coating having a thermally
responsive nanofiber. According to a further embodiment, the
thermally responsive nanofiber includes a thermally responsive
polymer and a cross-linking agent having at least two latent
reactive activatable groups.
[0013] According to still yet another embodiment, the present
invention is a cell culture device including a surface coating
having a thermally responsive nanofiber. The thermally responsive
nanofiber includes a thermally responsive polymer, a cross-linking
agent having at least two latent reactive activatable groups, and a
biologically active material.
[0014] According to other embodiments of the present invention, the
stimuli responsive nanofiber may have a diameter ranging from 1 to
100 microns and still other embodiments may have a diameter ranging
from 1 nm to 1000 nm. The stimuli responsive nanofiber may have an
aspect ratio in a range of about at least 10 to at least 100.
[0015] According to yet a further embodiment of the present
invention, the thermally responsive nanofiber has first physical
property at a first predetermined temperature range and a second
physical property at a second predetermined temperature range. The
thermally responsive nanofiber is capable of transitioning from a
first physical property to a second physical property upon the
application or removal of heat to or from the system
[0016] Various modifications and additions can be made to the
exemplary embodiments discussed without departing from the scope of
the present invention. For example, while the embodiments described
above refer to particular features, the scope of this invention
also includes embodiments having different combinations of features
and embodiments that do not include all of the described features.
Accordingly, the scope of the present invention is intended to
embrace all such alternatives, modifications, and variations as
fall within the scope of the claims, together with all equivalents
thereof.
DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is an electronic image of a polycaprolactone
nanofiber described in Example 1.
[0018] FIG. 2 is an electronic image of a polyisopropylacrylamide
nanofiber described in Example 3.
[0019] FIGS. 3 and 4 illustrate protein absorption profiles of
polyisopropylacrylamide coated polystyrene and
polyisopropylacrylamide nanofibers described in Example 4.
[0020] FIGS. 5 and 6 are electronic images of cell lift up times
from various surfaces described in Example 5.
[0021] FIG. 7 is an electronic image of T47-D cells cultured on
nanofiber and flat surfaces.
[0022] FIG. 8 is an electronic image of replated BAEC cells.
DETAILED DESCRIPTION
[0023] Stimuli responsive or "smart" materials are materials that
have one or more properties that can be altered in a controlled
fashion by the application of external stimuli, such as stress,
temperature, moisture, pH, applied electric or magnetic fields,
ionic strength, or biomolecules such as glucose or antigens.
Representative stimuli responsive materials and polymers as well as
their physical characteristic are reported by Gil et al.,
"Stimuli-responsive polymers and their bioconjugates," Prog. Polym.
Sci., 29, 1173-1222 (2004) which is incorporated by reference
herein. A thermally responsive polymer is one example of these
materials. A thermally responsive material is a material in which a
physical property is altered in response to a change in temperature
in the surrounding environment or system. A thermally responsive
polymer may change from a hydrophilic state to hydrophobic state
when the temperature of the system or its surroundings rises above
a lower critical solution temperature (LCST). When in a hydrophilic
state, the polymer chains become swollen. Conversely, in a
hydrophobic state, the polymer chains collapse, and the polymer
becomes insoluble in water. In most cases, the process can be
reversible.
[0024] One embodiment of the present invention is directed to a
thermally responsive nanofiber. The thermally responsive nanofiber
can be used to modify a surface of a substrate to provide a
functionalized surface. More particularly, the thermally responsive
nanofiber can be used to provide a thermally responsive surface on
a substrate. The physical property of the thermally responsive
nanofiber modified surface of the substrate changes in response to
a change in temperature in the system. Biologically active
materials can be immobilized on the nanofiber modified surface by
reacting with the functional groups accessible or exposed on the
surface of the substrate. Typically, the biologically active
materials retain all or a portion of their bioactivity after having
been immobilized on the thermally responsive nanofiber modified
surface. The ability of biologically active materials to bond with
the surface of the substrate can be affected depending on the
physical state of the modified surface. Thus, by controlling the
temperature of the modified surface, the ability to bind to a
biological material can be controlled.
[0025] According to one embodiment of the present invention the
thermally responsive nanofiber includes a thermally responsive
polymer, a biologically active material, and a cross-linking agent
having at least two latent reactive activatable groups. The
thermally responsive nanofiber can be used to modify the surface of
a substrate by bonding the nanofiber to the surface by the
formation of a covalent bond between the surface of the substrate
and the nanofiber. At least one of the latent reactive activatable
groups undergoes activation when subjected to a suitable energy
source to form a covalent bond between the surface of the substrate
and the thermally responsive nanofiber. The remaining latent
reactive group(s) are left accessible or exposed on the surface of
the substrate. The biologically active material included in the
nanofiber or the accessible or exposed latent reactive groups on
the surface may be used for further surface modification of the
substrate.
[0026] A number of processing techniques such as drawing, template
synthesis, phase separation, self-assembly or electrospinning have
been used to prepare nanofibers.
[0027] For example, a thermally responsive nanofiber can be formed
by electrospinning a fiber-forming combination that includes a
thermally responsive polymer, a biologically active material, and a
cross-linking agent having at least two latent reactive activatable
groups. Electrospinning generally involves the introduction of a
polymer or other fiber-forming solution or liquid into an electric
field, so that the solution or liquid is caused to produce fibers.
When a strong electrostatic field is applied to a fiber-forming
combination held in a syringe with a capillary outlet, a pendant
droplet of the fiber-forming mixture from the capillary outlet is
deformed into a Taylor cone. When the voltage surpasses a threshold
value, the electric forces overcome the surface tension on the
droplet, and a charged jet of the solution or liquid is ejected
from the tip of the Taylor cone. The ejected jet then moves toward
a collecting metal screen that acts as a counterelectrode having a
lower electrical potential. The jet is split into small charged
fibers or fibrils and any solvent present evaporates leaving behind
a nonwoven fabric mat formed on the screen.
[0028] In one embodiment, electrostatically spun fibers can be
produced having very thin diameters. Parameters that influence the
diameter, consistency, and uniformity of the electrospun fibers
include the thermally responsive polymer, the molecular weight of
the polymer; the cross-linker concentration (loading) in the
fiber-forming mixture, the flow rate of the polymer solution, the
applied voltage, and the needle collector distance. According to
one embodiment of the present invention, a stimuli responsive
nanofiber has a diameter ranging from about 1 nm to about 100
.mu.m. In other embodiments, the stimuli responsive nanofiber has a
diameter in a range of about 1 nm to about 1000 nm. Further, the
nanofiber may have an aspect ratio in a range of about at least 10
to about at least 100. It will be appreciated that, because of the
very small diameter of the fibers, the fibers have a high surface
area per unit of mass. This high surface area to mass ratio permits
fiber-forming material solutions to be transformed from solvated
fiber-forming materials to solid nanofibers in fractions of a
second.
[0029] The stimuli responsive polymer used to form the nanofiber
may be selected from any stimuli responsive, fiber-forming material
that is compatible with the cross-linking agent. In one embodiment,
a selected thermally responsive polymer should be capable of
undergoing a rapid change from a first physical property to a
second physical property when the temperature of the system has
risen above a lower critical solution temperature. Exemplary
thermally responsive, fiber forming polymers include, but are not
limited to, poly(isopropylacrylamide) and mixtures and copolymers
thereof. Other thermally responsive polymers include random
copolymers of 2-(2-methoxyethoxy)ethyl methacrylate and
oligo(ethylene glycol) methacrylate.
[0030] According to one embodiment of the present invention, the
thermally responsive polymer is poly(isopropylacrylamide).
Poly(isopropylacrylamide) changes from a primarily hydrophobic
state to a primarily hydrophilic state upon reaching a lower
critical solution temperature of approximately 20 to 32.degree. C.
Poly-N-isopropylacrylamide (PIPAAm) has been one of the most
studied thermo-responsive polymer not only because it displays a
low critical solution temperature (LCST) of around 32.degree. C.,
close to body temperature, but also because its LCST is relatively
insensitive to environmental conditions. Slight variations of pH,
concentration or chemical environment affect the LCST by only a few
degrees. The main mechanism of PIPAAm's aqueous phase separation is
the thermally induced release of water molecules bound to polymer
isopropyl side groups, resulting in intra- and intermolecular
hydrophobic interactions between isopropyl groups above the
LCST.
[0031] The inclusion of cross-linking agents within the composition
forming the thermally responsive nanofiber, allows the thermally
responsive nanofiber to be compatible with a wide range of support
surfaces. The latent reactive cross-linking agents can be used
alone or in combination with other materials to provide a desired
surface characteristic.
[0032] Suitable cross-linking agents include either monomeric
(small molecule materials) or polymeric materials having at least
two latent reactive activatable groups that are capable of forming
covalent bonds with other materials when subjected to a source of
energy such as radiation, electrical or thermal energy. In general,
latent reactive activatable groups are chemical entities that
respond to specific applied external energy or stimuli to generate
active species with resultant covalent bonding to an adjacent
chemical structure. Latent reactive groups are those groups that
retain their covalent bonds under storage conditions but that form
covalent bonds with other molecules upon activation by an external
energy source. In some embodiments, latent reactive groups form
active species such as free radicals. These free radicals may
include nitrenes, carbine or excited states of ketones upon
absorption of externally applied electric, electromagnetic or
thermal energy. Various examples of known latent reactive groups
are reported in U.S. Pat. Nos. 4,973,493; 5,258,041; 5,563,056;
5,637,460; or 6,278,018.
[0033] Eight commercially available multifunctional
photocrosslinkers based on trichloromethyl triazine are available
either from Aldrich Chemicals, Produits Chimiques Auxiliaires et de
Syntheses, (Longjumeau, France), Shin-Nakamara Chemical, Midori
Chemicals Co., Ltd. or Panchim S.A. (France). The eight compounds
include 2,4,6-tris(trichloromethyl)-1,3,5 triazine,
2-(methyl)-4,6-bis(trichloromethyl)-1,3,5-triazine,
2-(4-methoxynaphthyl)-4,6-bis(trichloromethyl)-1,3,5-triazine,
2-(4-ethoxynaphthyl)-4,6-bis(trichloromethyl)-1,3,5-triazine,
4-(4-carboxylphenyl)-2,6-bis(trichloromethyl)-1,3,5-triazine,
2-(4-methoxyphenyl)-4,6-bis(trichloromethyl)-1,3,5-triazine,
2-(1-ethen-2-2'-furyl)-4,6-bis(trichloromethyl)-1,3,5-triazine and
2-(4-methoxystyryl)-4,6-bis(trichloromethyl)-1,3,5-triazine.
[0034] In some embodiments, the latent reactive groups are the
same, while in other embodiments the latent reactive groups may be
different. For example, the latent reactive groups may be two
different groups that are both activated by radiation. In other
embodiments one latent reactive group may by activated by radiation
while another latent reactive group may be activated by heat.
Suitable cross-linking agents include bi-, tri- and
multi-functional monomeric and polymeric materials.
[0035] Latent reactive groups that are reactive to thermal or heat
energy include a variety of reactive moieties and may include known
compounds that decompose thermally to form reactive species that
will then form covalent bonds. The covalent bonds allow the
cross-linking to bind to adjacent materials. Suitable
thermally-reactive groups typically have a pair of atoms having a
heat sensitive or labile bond. Heat labile bonds include
oxygen-oxygen bonds such as peroxide bonds, nitrogen-oxygen bonds,
and nitrogen-nitrogen bonds. Such bonds will react or decompose at
temperatures in a range of not more than 80-200.degree. C.
[0036] Both thermally generated carbenes and nitrenes undergo a
variety of chemical reactions, including carbon bond insertion,
migration, hydrogen abstraction, and dimerization. Examples of
carbene generators include diazirines and diazo-compounds. Examples
of nitrene generators include aryl azides, particularly
perfluorinated aryl azides, acyl azides, and triazolium ylides. In
addition, groups that upon heating form reactive triplet states,
such as dioxetanes, or radical anions and radical cations may also
be used to form the thermally-reactive group.
[0037] In one embodiment the thermally-reactive group of the
cross-linking agent includes a peroxide --(O--O)-- group.
Thermally-reactive peroxide-containing groups include, for example,
thermally-reactive diacyl peroxide groups, thermally-reactive
peroxydicarbonate groups, thermally-reactive dialkylperoxide
groups, thermally-reactive peroxyester groups, thermally-reactive
peroxyketal groups, and thermally-reactive dioxetane groups.
[0038] Dioxetanes are four-membered cyclic peroxides that react or
decompose at lower temperatures compared to standard peroxides due
to the ring strain of the molecules. The initial step in the
decomposition of dioxetanes is cleavage of the O--O bond, the
second step breaks the C--C bond creating one carbonyl in the
excited triplet state, and one in an excited singlet state. The
excited triplet state carbonyl can extract a hydrogen from an
adjacent material, forming two radical species, one on the adjacent
material and one on the carbon of the carbonyl with the oxygen and
will form a new covalent bond between the thermally reactive
dioxetane and the adjacent material.
[0039] Representative thermally reactive moieties are reported in
US 20060030669 and other representative thermal latent reactive
groups are reported in U.S. Pat. No. 5,258,041. Both of these
documents are hereby incorporated by reference.
[0040] Latent reactive groups that are reactive to electromagnetic
radiation, such as ultraviolet or visible radiation, are typically
referred to as photochemical reactive groups.
[0041] The use of latent reactive activatable species in the form
of latent reactive activatable aryl ketones is useful. Exemplary
latent reactive activatable aryl ketones include acetophenone,
benzophenone, anthraquinone, anthrone, anthrone-like heterocycles
(i.e., heterocyclic analogs of anthrone such as those having N, O,
or S in the 10-position), and their substituted (e.g., ring
substituted) derivatives. Examples of aryl ketones include
heterocyclic derivatives of anthrone, including acridone, xanthone,
and thioxanthone, and their ring substituted derivatives. In
particular, thioxanthone, and its derivatives, having excitation
energies greater than about 360 nm are useful.
[0042] The functional groups of such ketones are suitable since
they are readily capable of undergoing an
activation/inactivation/reactivation cycle. Benzophenone is an
exemplary photochemically reactive activatable group, since it is
capable of photochemical excitation with the initial formation of
an excited singlet state that undergoes intersystem crossing to the
triplet state. The excited triplet state can insert into
carbon-hydrogen bonds by abstraction of a hydrogen atom (from a
support surface, for example), thus creating a radical pair.
Subsequent collapse of the radical pair leads to formation of a new
carbon-carbon bond. If a reactive bond (e.g., carbon-hydrogen) is
not available for bonding, the ultraviolet light-induced excitation
of the benzophenone group is reversible and the molecule returns to
ground state energy level upon removal of the energy source.
Photochemically reactive activatable aryl ketones such as
benzophenone and acetophenone are of particular importance inasmuch
as these groups are subject to multiple reactivation in water and
hence provide increased coating efficiency.
[0043] In some embodiments of the invention, photochemically
reactive cross-linking agents may be derived from three different
types of molecular families. Some families include one or more
hydrophilic portions, i.e., a hydroxyl group (that may be
protected), amines, alkoxy groups, etc. Other families may include
hydrophobic and amphiphilic portions. In one embodiment, the family
has the formula:
L-((D-T-C(R.sup.1)(XP)CHR.sup.2GR.sup.3C(.dbd.O)R.sup.4)).sub.m.
[0044] L is a linking group. D is O, S, SO, SO.sub.2, NR.sup.5 or
CR.sup.6R.sup.7. T is (--CH.sub.2--).sub.x,
(--CH.sub.2CH.sub.2--O--).sub.x,
(--CH.sub.2CH.sub.2CH.sub.2--O--).sub.x or
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--O--).sub.x. R.sup.1 is a
hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or
aryloxyaryl group. X is O, S, or NR.sup.8R.sup.9. P is a hydrogen
atom or a protecting group, with the proviso that P is absent when
X is NR.sup.8R.sup.9. R.sup.2 is a hydrogen atom, an alkyl,
alkyloxyalkyl, aryl, aryloxylalkyl or aryloxyaryl group. G is O, S,
SO, SO.sub.2, NR.sup.10, (CH.sub.2).sub.t--O-- or C.dbd.O. R.sup.3
and R.sup.4 are each independently an alkyl, aryl, arylalkyl,
heteroaryl, or a heteroarylalkyl group or when R.sup.3 and R.sup.4
are tethered together via (--CH.sub.2--).sub.q,
(--CH.sub.2--).sub.rC.dbd.O(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS.dbd.O(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS(O).sub.2(--CH.sub.2--).sub.s, or
(--CH.sub.2--).sub.rNR(--CH.sub.2--).sub.s. R.sup.5 and R.sup.10
are each independently a hydrogen atom or an alkyl, aryl, or
arylalkyl group. R.sup.6 and R.sup.7 are each independently a
hydrogen atom, an alkyl, aryl, arylalkyl, heteroaryl or
heteroarylalkyl group. R.sup.8 and R.sup.9 are each independently a
hydrogen atom, an alkyl, aryl, or arylalkyl group, R is a hydrogen
atom, an alkyl group or an aryl group, q is an integer from 1 to
about 7, r is an integer from 0 to about 3, s is an integer from 0
to about 3, m is an integer from 2 to about 10, t is an integer
from 1 to about 10 and x is an integer from 1 to about 500.
[0045] In one embodiment, L is a branched or unbranched alkyl chain
having between about 2 and about 10 carbon atoms.
[0046] In another embodiment, D is an oxygen atom (O).
[0047] In still another embodiment, T is (--CH.sub.2--).sub.x or
(--CH.sub.2CH.sub.2--O--).sub.x and x is 1 or 2.
[0048] In still yet another embodiment, R.sup.1 is a hydrogen
atom.
[0049] In yet another embodiment, X is an oxygen atom, O, and P is
a hydrogen atom.
[0050] In another embodiment, R.sup.2 is a hydrogen atom.
[0051] In still another embodiment, G is an oxygen atom, O.
[0052] In still yet another embodiment, R.sup.3 and R.sup.4 are
each individually aryl groups, which can be further substituted,
and m is 3.
[0053] In one particular embodiment, L is
##STR00001##
D is O, T is (--CH.sub.2--).sub.x, R.sup.1 is a hydrogen atom, X is
O, P is a hydrogen atom, R.sup.2 is a hydrogen atom, G is O,
R.sup.3 and R.sup.4 are phenyl groups, m is 3 and x is 1.
[0054] In yet another particular embodiment, L is
(--CH.sub.2--).sub.y, D is O, T is (--CH.sub.2--).sub.z, R.sup.1 is
a hydrogen atom, X is O, P is a hydrogen atom, R.sup.2 is a
hydrogen atom, G is O, R.sup.3 and R.sup.4 are phenyl groups, m is
2, x is 1 and y is an integer from 2 to about 6, and in particular,
y is 2, 4 or 6.
[0055] In certain embodiments, x is an integer from about 1 to
about 500, more particularly from about 1 to about 400, from about
1 to about 250, from about 1 to about 200, from about 1 to about
150, from about 1 to about 100, from about 1 to about 50, from
about 1 to about 25 or from about 1 to about 10.
[0056] In another embodiment, the family has the formula:
L-((T-C(R.sup.1)(XP)CHR.sup.2GR.sup.3C(.dbd.O)R.sup.4)).sub.m,
and L, T, R.sup.1, X, P, R.sup.2, G, R.sup.3, R.sup.4, R.sup.8,
R.sup.9, R.sup.10, R, q, r, s, m, t and x are as defined above.
[0057] In one embodiment, L has a formula according to structure
(I):
##STR00002##
[0058] A and J are each independently a hydrogen atom, an alkyl
group, an aryl group, or together with B form a cyclic ring,
provided when A and J are each independently a hydrogen atom, an
alkyl group, or an aryl group then B is not present, B is
NR.sup.11, O, or (--CH.sub.2--).sub.z, provided when A, B and J
form a ring, then A and J are (--CH.sub.2--).sub.z or C.dbd.O,
R.sup.11 is a hydrogen atom, an alkyl group, an aryl group or
denotes a bond with T, each z independently is an integer from 0 to
3 and provided when either A or J is C.dbd.O, then B is NR.sup.11,
O, or (--CH.sub.2--).sub.z and z must be at least 1.
[0059] In another embodiment, T is --CH.sub.2--.
[0060] In another embodiment, the family has the formula:
L-((GTZR.sup.3C(.dbd.O)R.sup.4)).sub.m, and L, T, G, R.sup.3,
R.sup.4, R.sup.10, R, q, r, s, m, t and x are as defined above. Z
can be a C.dbd.O, COO or CONH when T is (--CH.sub.2--).sub.x.
[0061] In one embodiment, L has a formula according to structure
(I):
##STR00003##
and A, B, J, R.sup.11, and z are as defined above.
[0062] In another embodiment, L has a formula according to
structure (II):
##STR00004##
R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17 are each
independently a hydrogen atom, an alkyl or aryl group or denotes a
bond with T, provided at least two of R.sup.12, R.sup.13, R.sup.14,
R.sup.15, R.sup.16, R.sup.17 are bonded with T and each K,
independently is CH or N.
[0063] In another embodiment, the family has the formula:
L-((TGQR.sup.3C(.dbd.O)R.sup.4)).sub.m,
L, G, R.sup.3, R.sup.4, R.sup.10, R, q, r, s, m, t and x are as
defined above. T is (--CH.sub.2--).sub.x,
(--CH.sub.2CH.sub.2--O--).sub.x,
(--CH.sub.2CH.sub.2CH.sub.2--O--).sub.x,
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--O--).sub.x or forms a bond. Q
is (--CH.sub.2--).sub.p, (--CH.sub.2CH.sub.2--O--).sub.p,
(--CH.sub.2CH.sub.2CH.sub.2--O--).sub.p or
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--O--).sub.p and p is an integer
from 1 to about 10.
[0064] In one embodiment, L has a formula according to structure
(I):
##STR00005##
A, B, J, R.sup.11, and z are as defined above.
[0065] In another embodiment, L has a formula according to
structure (II):
##STR00006##
R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17 are each
independently a hydrogen atom, an alkyl or aryl group or denotes a
bond with T, provided at least two of R.sup.12, R.sup.13, R.sup.14,
R.sup.15, R.sup.16, R.sup.17 are bonded with T and each K,
independently is CH or N.
[0066] In still yet another embodiment, compounds of the present
invention provide that R.sup.3 and R.sup.4 are both phenyl groups
and are tethered together via a CO, a S or a CH.sub.2.
[0067] In yet another embodiment, compounds of the present
invention provide when R.sup.3 and R.sup.4 are phenyl groups, the
phenyl groups can each independently be substituted with at least
one alkyloxyalkyl group, such as
CH.sub.3O--(CH.sub.2CH.sub.2O--).sub.n--, or
CH.sub.3O(--CH.sub.2CH.sub.2CH.sub.2O--).sub.n-- a hydroxylated
alkoxy group, such as HO--CH.sub.2CH.sub.2O--,
HO(--CH.sub.2CH.sub.2O--).sub.n-- or
HO(--CH.sub.2CH.sub.2CH.sub.2O--).sub.n--, etc. wherein n is an
integer from 1 to about 10.
[0068] In another embodiment the family has the formula:
L-(((-CH.sub.2--).sub.xxC(R.sup.1)((G)R.sup.3C(.dbd.O)R.sup.4).sub.2).su-
b.m.
[0069] L, each R, R.sup.1, each G, each R.sup.3, each R.sup.4, each
R.sup.10, each q, each r, each s, each t and m are as defined above
and xx is an integer from 1 to about 10.
[0070] In one embodiment, L has a formula according to structure
(I):
##STR00007##
A, B, J, R.sup.11, and z are as defined above.
[0071] In another embodiment, A and B are both hydrogen atoms.
[0072] In still another embodiment, xx is 1.
[0073] In yet another embodiment, R.sup.1 is H.
[0074] In still yet another embodiment, G is
(--CH.sub.2--).sub.tO-- and t is 1.
[0075] In another embodiment, R.sup.3 and R.sup.4 are each
individually aryl groups.
[0076] In still yet another embodiment, xx is 1, R.sup.1 is H, each
G is (--CH.sub.2--).sub.tO--, t is 1 and each of R.sup.3 and
R.sup.4 are each individually aryl groups.
[0077] In another embodiment of the invention, the family has the
formula:
L-((-C(R.sup.1)(XP)CHR.sup.2GR.sup.3C(.dbd.O)R.sup.4).sub.m.
L, R, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.8, R.sup.9,
R.sup.10, X, P, G, q, r, s, t, and m are as defined above.
[0078] In one embodiment, L is
##STR00008##
and R.sup.20 and R.sup.21 are each individually a hydrogen atom, an
alkyl group or an aryl group.
[0079] In another embodiment, R.sup.1 is H.
[0080] In still another embodiment, wherein X is O.
[0081] In yet another embodiment, P is H.
[0082] In still yet another embodiment, R.sup.2 is H.
[0083] In another embodiment, G is (--CH.sub.2--).sub.tO-- and t is
1.
[0084] In still another embodiment, R.sup.3 and R.sup.4 are each
individually aryl groups.
[0085] In yet another embodiment, R.sup.1 is H, X is O, P is H,
R.sup.2 is H, G is (--CH.sub.2--).sub.tO--, t is 1, R.sup.3 and
R.sup.4 are each individually aryl groups and R.sup.20 and R.sup.21
are both methyl groups.
[0086] In yet another embodiment, the present invention provides a
family of compounds having the formula:
L-((GR.sup.3C(.dbd.O)R.sup.4)).sub.m,
L is a linking group; G is O, S, SO, SO.sub.2, NR.sup.10,
(CH.sub.2).sub.t--O-- or C.dbd.O; R.sup.3 and R.sup.4 are each
independently an alkyl, aryl, arylalkyl, heteroaryl, or an
heteroarylalkyl group or when R.sup.3 and R.sup.4 are tethered
together via (--CH.sub.2--).sub.q,
(--CH.sub.2--).sub.rC.dbd.O(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rS.dbd.O(--CH.sub.2--).sub.s or
(--CH.sub.2--).sub.rS(O).sub.2(--CH.sub.2--).sub.s,
(--CH.sub.2--).sub.rNR(--CH.sub.2--).sub.s; R.sup.10 is a hydrogen
atom or an alkyl, aryl, or an arylalkyl group; R is a hydrogen
atom, an alkyl or an aryl group; q is an integer from 1 to about 7;
r is an integer from 0 to about 3; s is an integer from 0 to about
3; m is an integer from 2 to about 10; and t is an integer from 1
to about 10.
[0087] In one embodiment, L is
##STR00009##
[0088] In another embodiment, G is C.dbd.O.
[0089] In still another embodiment, R.sup.3 and R.sup.4 are each
individually aryl groups.
[0090] In yet another embodiment, G is C.dbd.O and R.sup.3 and
R.sup.4 are each individually aryl groups.
[0091] "Alkyl" by itself or as part of another substituent refers
to a saturated or unsaturated branched, straight-chain or cyclic
monovalent hydrocarbon radical having the stated number of carbon
atoms (i.e., C.sub.1-C.sub.6 means one to six carbon atoms) that is
derived by the removal of one hydrogen atom from a single carbon
atom of a parent alkane, alkene or alkyne. Typical alkyl groups
include, but are not limited to, methyl; ethyls such as ethanyl,
ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl,
cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl,
cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl,
prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl,
2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,
but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,
but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl,
buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl,
cyclobuta-1,3-dien-1-yl, but-1-yn-1-yl, but-1-yn-3-yl,
but-3-yn-1-yl, etc.; and the like. Where specific levels of
saturation are intended, the nomenclature "alkanyl," "alkenyl"
and/or "alkynyl" is used, as defined below. "Lower alkyl" refers to
alkyl groups having from 1 to 6 carbon atoms.
[0092] "Alkanyl" by itself or as part of another substituent refers
to a saturated branched, straight-chain or cyclic alkyl derived by
the removal of one hydrogen atom from a single carbon atom of a
parent alkane. Typical alkanyl groups include, but are not limited
to, methanyl; ethanyl; propanyls such as propan-1-yl,
propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butanyls such as
butan-1-yl, butan-2-yl(sec-butyl), 2-methyl-propan-1-yl(isobutyl),
2-methyl-propan-2-yl(t-butyl), cyclobutan-1-yl, etc.; and the
like.
[0093] "Alkenyl" by itself or as part of another substituent refers
to an unsaturated branched, straight-chain or cyclic alkyl having
at least one carbon-carbon double bond derived by the removal of
one hydrogen atom from a single carbon atom of a parent alkene. The
group may be in either the cis or trans conformation about the
double bond(s). Typical alkenyl groups include, but are not limited
to, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl,
prop-2-en-1-yl, prop-2-en-2-yl, cycloprop-1-en-1-yl;
cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl,
2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,
buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl,
cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the
like.
[0094] "Alkyloxyalkyl" refers to a moiety having two alkyl groups
tethered together via an oxygen bond. Suitable alkyloxyalkyl groups
include polyoxyalkylenes, such as polyethyleneoxides,
polypropyleneoxides, etc. that are terminated with an alkyl group,
such as a methyl group. A general formula for such compounds can be
depicted as R'--(OR'').sub.n or (R'O).sub.nR'' wherein n is an
integer from 1 to about 10, and R' and R'' are alkyl or alkylene
groups.
[0095] "Alkynyl" by itself or as part of another substituent refers
to an unsaturated branched, straight-chain or cyclic alkyl having
at least one carbon-carbon triple bond derived by the removal of
one hydrogen atom from a single carbon atom of a parent alkyne.
Typical alkynyl groups include, but are not limited to, ethynyl;
propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls
such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the
like.
[0096] "Alkyldiyl" by itself or as part of another substituent
refers to a saturated or unsaturated, branched, straight-chain or
cyclic divalent hydrocarbon group having the stated number of
carbon atoms (i.e., C.sub.1-C.sub.6 means from one to six carbon
atoms) derived by the removal of one hydrogen atom from each of two
different carbon atoms of a parent alkane, alkene or alkyne, or by
the removal of two hydrogen atoms from a single carbon atom of a
parent alkane, alkene or alkyne. The two monovalent radical centers
or each valency of the divalent radical center can form bonds with
the same or different atoms. Typical alkyldiyl groups include, but
are not limited to, methandiyl; ethyldiyls such as ethan-1,1-diyl,
ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl; propyldiyls such as
propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl, propan-1,3-diyl,
cyclopropan-1,1-diyl, cyclopropan-1,2-diyl, prop-1-en-1,1-diyl,
prop-1-en-1,2-diyl, prop-2-en-1,2-diyl, prop-1-en-1,3-diyl,
cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,
cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such
as, butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl,
butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,
cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,
but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,
but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,
2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,
buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl,
buta-1,3-dien-1,4-diyl, cyclobut-1-en-1,2-diyl,
cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,
cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,
but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.;
and the like. Where specific levels of saturation are intended, the
nomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used.
Where it is specifically intended that the two valencies be on the
same carbon atom, the nomenclature "alkylidene" is used. A "lower
alkyldiyl" is an alkyldiyl group having from 1 to 6 carbon atoms.
In some embodiments the alkyldiyl groups are saturated acyclic
alkanyldiyl groups in which the radical centers are at the terminal
carbons, e.g., methandiyl(methano); ethan-1,2-diyl(ethano);
propan-1,3-diyl(propano); butan-1,4-diyl(butano); and the like
(also referred to as alkylenes, defined infra).
[0097] "Alkylene" by itself or as part of another substituent
refers to a straight-chain saturated or unsaturated alkyldiyl group
having two terminal monovalent radical centers derived by the
removal of one hydrogen atom from each of the two terminal carbon
atoms of straight-chain parent alkane, alkene or alkyne. The
location of a double bond or triple bond, if present, in a
particular alkylene is indicated in square brackets. Typical
alkylene groups include, but are not limited to, methylene
(methano); ethylenes such as ethano, etheno, ethyno; propylenes
such as propano, prop[1]eno, propa[1,2]dieno, prop[1]yno, etc.;
butylenes such as butano, but[1]eno, but[2]eno, buta[1,3]dieno,
but[1]yno, but[2]yno, buta[1,3]diyno, etc.; and the like. Where
specific levels of saturation are intended, the nomenclature
alkano, alkeno and/or alkyno is used. In some embodiments, the
alkylene group is (C.sub.1-C.sub.6) or (C.sub.1-C.sub.3) alkylene.
Other embodiments include straight-chain saturated alkano groups,
e.g., methano, ethano, propano, butano, and the like.
[0098] "Aryl" by itself or as part of another substituent refers to
a monovalent aromatic hydrocarbon group having the stated number of
carbon atoms (i.e., C.sub.5-C.sub.15 means from 5 to 15 carbon
atoms) derived by the removal of one hydrogen atom from a single
carbon atom of a parent aromatic ring system. Typical aryl groups
include, but are not limited to, groups derived from aceanthrylene,
acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,
chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,
hexylene, as-indacene, s-indacene, indane, indene, naphthalene,
octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene,
pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,
pleiadene, pyrene, pyranthrene, rubicene, triphenylene,
trinaphthalene, and the like, as well as the various hydro isomers
thereof. In some embodiments, the aryl group is (C.sub.5-C.sub.15)
aryl or, alternatively, (C.sub.5-C.sub.10) aryl. Other embodiments
include phenyl and naphthyl.
[0099] "Arylakl" by itself or as part of another substituent refers
to an acyclic alkyl radical in which one of the hydrogen atoms
bonded to a carbon atom, typically a terminal or sp.sup.3 carbon
atom, is replaced with an aryl group. Typical arylalkyl groups
include, but are not limited to, benzyl, 2-phenylethan-1-yl,
2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,
2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and
the like. Where specific alkyl moieties are intended, the
nomenclature arylalkanyl, arylalkenyl and/or arylalkynyl is used.
In some embodiments, the arylalkyl group is (C.sub.7-C.sub.30)
arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the
arylalkyl group is (C.sub.1-C.sub.10) and the aryl moiety is
(C.sub.5-C.sub.20) or, alternatively, an arylalkyl group is
(C.sub.7-C.sub.20) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl
moiety of the arylalkyl group is (C.sub.1-C.sub.8) and the aryl
moiety is (C.sub.6-C.sub.12).
[0100] "Aryloxyalkyl" refers to a moiety having an aryl group and
an alkyl group tethered together via an oxygen bond. Suitable
aryloxyalkyl groups include phenyloxyalkylenes, such as
methoxyphenyl or ethoxyphenyl.
[0101] "Cycloalkyl" by itself or as part of another substituent
refers to a cyclic version of an "alkyl" group. Typical cycloalkyl
groups include, but are not limited to, cyclopropyl; cyclobutyls
such as cyclobutanyl and cyclobutenyl; cyclopentyls such as
cyclopentanyl and cycloalkenyl; cyclohexyls such as cyclohexanyl
and cyclohexenyl; and the like.
[0102] "Cycloheteroalkyl" by itself or as part of another
substituent refers to a saturated or unsaturated cyclic alkyl
radical in which one or more carbon atoms (and any associated
hydrogen atoms) are independently replaced with the same or
different heteroatom. Typical heteroatoms to replace the carbon
atom(s) include, but are not limited to, N, P, O, S or Si. Where a
specific level of saturation is intended, the nomenclature
"cycloheteroalkanyl" or "cycloheteroalkenyl" is used. Typical
cycloheteroalkyl groups include, but are not limited to, groups
derived from epoxides, imidazolidine, morpholine, piperazine,
piperidine, pyrazolidine, pyrrolidine, quinuclidine, and the
like.
[0103] "Halogen" or "Halo" by themselves or as part of another
substituent, unless otherwise stated, refer to fluoro, chloro,
bromo and iodo.
[0104] "Haloalkyl" by itself or as part of another substituent
refers to an alkyl group in which one or more of the hydrogen atoms
are replaced with a halogen. Thus, the term "haloalkyl" is meant to
include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to
perhaloalkyls. For example, the expression "(C.sub.1-C.sub.2)
haloalkyl" includes fluoromethyl, difluoromethyl, trifluoromethyl,
1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl,
1,1,1-trifluoroethyl or perfluoroethyl.
[0105] "Heteroalkyl, Heteroalkynyl, Heteroalkenyl, Heteroalkynyl"
by itself or as part of another substituent refer to alkyl,
alkanyl, alkenyl and alkynyl radical, respectively, in which one or
more of the carbon atoms (and any associated hydrogen atoms) are
each independently replaced with the same or different heteroatomic
groups. Typical heteroatomic groups include, but are not limited
to, --O--, --S--, --O--O--, --S--S--, --O--S--, --NR'--,
.dbd.N--N.dbd., --N.dbd.N--, --N.dbd.N--NR'--, --PH--,
--P(O).sub.2--, --O--P(O).sub.2--, --S(O)--, --S(O).sub.2--,
--SnH.sub.2-- and the like, where R' is hydrogen, alkyl,
substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl or
substituted aryl.
[0106] "Heteroaryl" by itself or as part of another substituent,
refers to a monovalent heteroaromatic radical derived by the
removal of one hydrogen atom from a single atom of a parent
heteroaromatic ring system. Typical heteroaryl groups include, but
are not limited to, groups derived from acridine, arsindole,
carbazole, .beta.-carboline, benzoxazine, benzimidazole, chromane,
chromene, cinnoline, furan, imidazole, indazole, indole, indoline,
indolizine, isobenzofuran, isochromene, isoindole, isoindoline,
isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole,
oxazole, perimidine, phenanthridine, phenanthroline, phenazine,
phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,
pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine,
quinazoline, quinoline, quinolizine, quinoxaline, tetrazole,
thiadiazole, thiazole, thiophene, triazole, xanthene, and the like.
The heteroaryl group may be from 5-20 membered heteroaryl or,
alternatively, from 5-10 membered heteroaryl. In some embodiments,
the heteroaryl groups are those derived from thiophene, pyrrole,
benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole,
oxazole and pyrazine.
[0107] "Heteroarylalkyl" by itself or as part of another
substituent refers to an acyclic alkyl group in which one of the
hydrogen atoms bonded to a carbon atom, typically a terminal or
sp.sup.3 carbon atom, is replaced with a heteroaryl group. Where
specific alkyl moieties are intended, the nomenclature
heteroarylalkanyl, heteroarylalkenyl and/or heteroarylalkynyl is
used. In some embodiments, the heteroarylalkyl group is a 6-21
membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl
moiety of the heteroarylalkyl is (C.sub.1-C.sub.6) alkyl and the
heteroaryl moiety is a 5-15-membered heteroaryl. In other
embodiments, the heteroarylalkyl is a 6-13 membered
heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety is
(C.sub.1-C.sub.3) alkyl and the heteroaryl moiety is a 5-10
membered heteroaryl.
[0108] "Hydroxyalkyl" by itself or as part of another substituent
refers to an alkyl group in which one or more of the hydrogen atoms
are replaced with a hydroxyl substituent. Thus, the term
"hydroxyalkyl" is meant to include monohydroxyalkyls,
dihydroxyalkyls or trihydroxyalkyls.
[0109] "Parent Aromatic Ring System" refers to an unsaturated
cyclic or polycyclic ring system having a conjugated .pi. electron
system. Specifically included within the definition of "parent
aromatic ring system" are fused ring systems in which one or more
of the rings are aromatic and one or more of the rings are
saturated or unsaturated, such as, for example, fluorene, indane,
indene, phenalene, tetrahydronaphthalene, etc. Typical parent
aromatic ring systems include, but are not limited to,
aceanthrylene, acenaphthylene, acephenanthrylene, anthracene,
azulene, benzene, chrysene, coronene, fluoranthene, fluorene,
hexacene, hexaphene, hexylene, indacene, s-indacene, indane,
indene, naphthalene, octacene, octaphene, octalene, ovalene,
penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,
phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,
rubicene, tetrahydronaphthalene, triphenylene, trinaphthalene, and
the like, as well as the various hydro isomers thereof.
[0110] "Parent Heteroaromatic Ring System" refers to a parent
aromatic ring system in which one or more carbon atoms (and any
associated hydrogen atoms) are independently replaced with the same
or different heteroatom. Typical heteroatoms to replace the carbon
atoms include, but are not limited to, N, P, O, S, Si, etc.
Specifically included within the definition of "parent
heteroaromatic ring systems" are fused ring systems in which one or
more of the rings are aromatic and one or more of the rings are
saturated or unsaturated, such as, for example, arsindole,
benzodioxan, benzofuran, chromane, chromene, indole, indoline,
xanthene, etc. Typical parent heteroaromatic ring systems include,
but are not limited to, arsindole, carbazole, .beta.-carboline,
chromane, chromene, cinnoline, furan, imidazole, indazole, indole,
indoline, indolizine, isobenzofuran, isochromene, isoindole,
isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,
oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,
phenazine, phthalazine, pteridine, purine, pyran, pyrazine,
pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine,
quinazoline, quinoline, quinolizine, quinoxaline, tetrazole,
thiadiazole, thiazole, thiophene, triazole, xanthene, and the
like.
[0111] "Leaving group" is a group that is displaced during a
reaction by a nucleophilic reagent. Suitable leaving groups include
S(O).sub.2Me, --SMe or halo (e.g., F, Cl, Br, I).
[0112] "Linking group" is a group that serves as an intermediate
locus between two or more end groups. The nature of the linking
group can vary widely, and can include virtually any combination of
atoms or groups useful for spacing one molecular moiety from
another. For example, the linker may be an acyclic hydrocarbon
bridge (e.g., a saturated or unsaturated alkyleno such as methano,
ethano, etheno, propano, prop[1]eno, butano, but[1]eno, but[2]eno,
buta[1,3]dieno, and the like), a monocyclic or polycyclic
hydrocarbon bridge (e.g., [1,2]benzeno, [2,3]naphthaleno, and the
like), a simple acyclic heteroatomic or heteroalkyldiyl bridge
(e.g., --O--, --S--, --S--O--, --NH--, --PH--, --C(O)--,
--C(O)NH--, --S(O)--, --S(O).sub.2--, --S(O)NH--, --S(O).sub.2NH--,
--O--CH.sub.2--, --CH.sub.2--O--CH.sub.2--,
--O--CH.dbd.CH--CH.sub.2--, and the like), a monocyclic or
polycyclic heteroaryl bridge (e.g., [3,4]furano, pyridino,
thiopheno, piperidino, piperazino, pyrazidino, pyrrolidino, and the
like) or combinations of such bridges.
[0113] "Protecting group" is a group that is appended to, for
example, a hydroxyl oxygen in place of a labile hydrogen atom.
Suitable hydroxyl protecting group(s) include esters (acetate,
ethylacetate), ethers (methyl, ethyl), ethoxylated derivatives
(ethylene glycol, propylene glycol) and the like that can be
removed under either acidic or basic conditions so that the
protecting group is removed and replaced with a hydrogen atom.
Guidance for selecting appropriate protecting groups, as well as
synthetic strategies for their attachment and removal, may be
found, for example, in Greene & Wuts, Protective Groups in
Organic Synthesis, 3d Edition, John Wiley & Sons, Inc., New
York (1999) and the references cited therein (hereinafter "Greene
& Wuts").
[0114] Plastics or porous membranes such as polyolefins,
polystyrenes, poly(methyl)methacrylates, polyacrylonitriles,
poly(vinylacetates), poly (vinyl alcohols), chlorine-containing
polymers such as poly(vinyl)chloride, polyoxymethylenes,
polycarbonates, polyamides, polyimides, polyurethanes, phenolics,
amino-epoxy resins, polyesters, silicones, cellulose-based
plastics, fluoropolymers and rubber-like plastics can all be used
as supports, providing surfaces that can be modified as described
herein. In addition, supports such as those formed of pyrolytic
carbon, parylene coated surfaces, and silylated surfaces of glass,
ceramic, or metal are suitable for surface modification.
[0115] The method of the present invention may involve the
attachment of a biologically active material to a support surface.
For example, a thermally responsive nanofiber including a
cross-linking agent is provided having two or more latent reactive
activatable groups in the presence of a support surface. According
to an alternative embodiment, the nanofiber may also include a
biologically active material or a functional polymer that is
reactive with a biologically active material. At least one of the
latent reactive groups is activated and covalently bonded to the
surface. The remaining latent reactive groups are allowed to remain
in their inactive state and are later activated in order to bind a
biologically active material or a functional polymer in order to
attach the biologically active material to the surface of the
substrate.
[0116] A functional polymer is a polymer having one or more
functional groups that will react with a biologically active
material. Representative functional groups include carboxy, ester,
epoxy, hydroxyl, amido, amino, thio N-hydroxy succinimide,
isocyanate, anhydride, azide, aldehyde, cyanuryl chloride or
phosphine groups that will react with a biologically active
material
[0117] Alternatively, the biologically active material or
functional polymer provided in the thermally responsive nanofiber
composition may bind to a second biological material in order to
attach the second biological material to the surface of the
substrate through manipulation of the physical properties of the
support surface via, for example, the application or removal of
heat from the system.
[0118] The steps of the method can be performed in any suitable
order. For example, a thermally responsive nanofiber including a
thermally responsive polymer and cross-linking agent, as described
herein, may be physically absorbed in or adsorbed to a suitable
support surface by hydrophobic interactions. Upon photoactivation,
at least one of the photoactivatable groups (e.g., benzophenone
groups) undergoes covalent bond formation at the support surface.
With the absence of abstractable hydrogens in the proximity of the
remaining unbonded photoactivatable group(s), and removal of the
photoactivation source, the photoactivatable group returns from an
excited state to a ground state. These remaining photoactivatable
groups are then capable of being reactivated when a biologically
active material intended for immobilization is present, and when
the treated surface is exposed to another round of illumination.
This method can be described as a "two-step" approach, where the
thermally responsive nanofiber is applied in the first step to
create a latent reactive surface, and in the second step, the
biologically active material is added for attachment to the
activated surface.
[0119] Alternatively, the method, described as a "one-step" method,
provides that the thermally responsive nanofibers of the present
invention are mixed together with the biologically active material
to form a composition. The resultant composition is used to surface
modify materials in a single photoactivation step. In this case,
photoactivation triggers not only covalent bond formation of at
least one photoactivatable group with the surface of the substrate,
but also simultaneously triggers covalent bond formation with any
adjacent biologically active materials residing on the surface.
[0120] In an alternative embodiment, the thermally responsive
nanofiber is formed from a combination or mixture including a
thermally responsive polymer, a cross-linking agent having at least
two latent activatable groups, and a biologically active material.
At least one of the latent reactive groups undergoes covalent bond
formation at the support surface to bond the nanofiber to the
surface of the substrate. The remaining latent reactive group(s)
can undergo photoactivation to react with a second biologically
active material. Alternatively, the biologically active material
incorporated into the nanofiber can itself react with a second
biologically active material to provide for further
functionalization of the substrate.
[0121] In another alternative method, the thermally responsive
nanofibers of the present invention are used to pretreat a
substrate surface prior to the application and bonding of molecules
that have themselves been functionalized with latent reactive
groups. This method is useful in situations where a particularly
difficult substrate requires maximal coating durability. In this
manner, the number of covalent bonds formed between the substrate
surface and the target molecule derivatized with latent reactive
groups can typically be increased, as compared to surface
modification with a desired latent reactive group-containing target
molecule alone.
[0122] After the surface of a substrate has been coated or treated
with the thermally responsive nanofibers of the present invention,
the thermally responsive surface can then be fine tuned by the
application or removal of heat to the system to selectively bind
and release a biological material of interest. Heat can be applied
to the system to transition the thermally responsive nanofiber
bound to the surface from a hydrophilic state to a hydrophobic
state. In a hydrophobic state at a temperature higher than LCST,
the polymer chains collapse and the surface becomes hydrophobic. In
this state the thermally responsive nanofiber surface may attract
or repel select target molecules. Alternatively, heat can also be
removed from the system by cooling the substrate below the LCST.
Once cooled, the thermally responsive nanofiber may revert back to
its initial hydrophilic state, once again showing an altered
affinity for a particular target molecule.
[0123] Suitable biologically active or target molecules for use in
the present invention encompass a diverse group of materials or
substances. These materials may be used in either an underivatized
form or previously derivatized. Moreover, target molecules can be
immobilized singly or in combination with other types of target
molecules.
[0124] Target molecules can be immobilized to the surface after
(e.g., sequentially) the surface has been primed with the thermally
responsive nanofibers of the present invention. Alternatively,
target molecules are immobilized during (e.g., simultaneously with)
attachment of the thermally responsive nanofibers to the surface of
the substrate.
[0125] Typically, target molecules are selected so as to confer
particular desired properties to the surface and/or to the device
or article bearing the surface. According to one embodiment of the
present invention, the target molecule or material is a
biologically active material. Biologically active materials which
may be immobilized on the surface of the nanofiber modified
substrate, or alternatively, provided as a part of the nanofiber
composition, generally include, but are not limited to, the
following: enzymes, proteins, carbohydrates, nucleic acids, and
mixtures thereof. Further examples of suitable target molecules,
including biological materials, and the surface properties they are
typically used to provide, is represented by the following
nonlimiting list.
TABLE-US-00001 TARGET MOLECULE FUNCTIONAL ACTIVITY Synthetic
Polymers Sulfonic acid-substituted Lubricity, negatively charged
surface, polyacrylamide hydrophilicity Polyacrylamide Lubricity,
protein repulsion, hydrophilicity Polyethylene glycol Lubricity,
cell and protein repulsion, hydrophilicity Polyethyleneimine
Positively charged surface Polylactic acid Bioerodible surface
Polyvinyl alcohol Lubricity, hydrophilicity Polyvinyl pyrrolidone
Lubricity, hydrophilicity Quaternary amine-substituted Lubricity,
positively charged surface polyacrylamide Silicone Lubricity,
hydrophobicity Conductive polymeric Electric conductivity
materials, e.g., polyvinylpyridine, polyacetylene, polypyrrole)
Carbohydrates Alginic acid Lubricity, hydrophilicity Cellulose
Lubricity, hydrophilicity, bio-degradable glucose source Chitosan
Positively charged surface, hydrophilicity, hemostatsis Glycogen
Hydrophilicity, biodegradable glucose source Heparin
Antithrombogenicity, hydrophilicity, cell and growth factor
attachment, protein affinity Hyaluronic acid Lubricity, negatively
charged surface Pectin Lubricity, hydrophilicity Mono-,
di-saccharides Hydrophilicity Dextran sulfate Chromatography media,
hydrophilicity Proteins Antibodies Antigen binding, immunoassay
Antithrombotic agents (e.g. Antithrombogenic surface antithrombin
III) Albumin Nonthrombogenic surface Attachment proteins/peptides
Cell attachment (e.g. collagen) Enzymes Catalytic surface
Extracellular matrix Cell attachment and growth proteins/peptides
Growth factors, Cell growth proteins/peptides Hirudin
Antithrombogenic surface Thrombolytic proteins (e.g., Thrombolytic
activity streptokinase, plasmin, urokinase) Lipids Fatty acids
Hydrophobicity, biocompatibility Mono-, di- and triglycerides
Hydrophobicity, lubricity, bio-degradable fatty acid source
Phospholipids Hydrophobicity, lubricity, bio-degradable fatty acid
source Prostaglandins/leukotrienes Nonthrombogenic
surface/immobilized messenger Nucleic Acids DNA Substrate for
nucleases/affinity binding, genomic assay RNA Substrate for
nucleases/affinity binding, genomic assay Nucleosides, nucleotides
Source of purines, pyrimidines, enzyme cofactor
Drugs/Vitamins/Cofactors Enzyme cofactors Immobilized enzyme Heme
compounds Globin bindings/surface oxygenation Drugs Drug activity
Nonpolymeric Materials Dyes (e.g., azo dyestuffs) Coloring agent
Fluorescent compounds Fuorescence (e.g., fluorescein)
[0126] The thermally responsive nanofibers of the present invention
can be used in a wide variety of applications including: filters,
scaffolds for tissue engineering, protective clothing,
reinforcement of composite materials, and sensor technologies.
[0127] Medical articles that can be fabricated from or coated or
treated with the thermally responsive nanofibers of the present
invention can include, but are not limited to, the following:
catheters including urinary catheters and vascular catheters (e.g.,
peripheral and central vascular catheters), wound drainage tubes,
arterial grafts, soft tissue patches, gloves, shunts, stents,
tracheal catheters, wound dressings, sutures, guide wires and
prosthetic devices (e.g., heart valves and LVADs). Vascular
catheters which can be prepared according to the present invention
include, but are not limited to, single and multiple lumen central
venous catheters, peripherally inserted central venous catheters,
emergency infusion catheters, percutaneous sheath introducer
systems, thermodilution catheters, including the hubs and ports of
such vascular catheters, leads to electronic devices such as
pacemakers, defibrillators, artificial hearts, and implanted
biosensors.
[0128] Additional articles that can be fabricated from or have a
surface that can be coated or treated with the thermally responsive
nanofibers of the present invention can include, but are not
limited to, the following: slides, multi-well plates, Petri dishes,
tissue culture slides, tissue culture plates, tissue culture
flasks, cell culture devices, or column supports and/or
chromatography media.
[0129] In another embodiment, the thermally responsive nanofibers
of the present invention can be applied to a microscope slide or
"chip" for biomolecule immobilization.
[0130] In yet another embodiment, the thermally responsive
nanofibers of the present invention can be applied to a surface of
a cell culture device to provide a thermally responsive surface
[0131] Various types of mammalian cells have been seeded on tissue
culture polystyrene (TCPS) coated with poly-isopropylacrylamide
(PIPAAm). The cells adhered, proliferated and differentiated in the
same manner as uncoated TCPS. With the cells on bare TCPS,
digestive trypsin treatment is carried out to dissolve the
extracellular matrix and to chelate and remove Ca ions to release
the cells, which in the process lose their cell surface receptors,
gap junctions and underlying extracellular matrix. Another
alternative for cell release is the use of cell scrapers, the
mechanical use of which generates irregularly shaped tissue
fragments. With thermo-responsive polymer coated dishes the cells
are detached in a non invasive fashion only by reducing the culture
temperature from 20.degree.-32.degree. C. at a temperature at which
the polymer hydrates. In contrast to enzymatic digestion, both
adhesive proteins and cell-cell junctions between the confluent
cells are preserved, enabling generation of a three dimensional
functional tissue that lacks any scaffold.
[0132] Cell sheet engineering is a unique technique that has arisen
from the use of thermo-responsive polymer as a cell culture
substrate. At 37.degree. C., PIPAAm becomes hydrophobic, promoting
protein adsorption and thereby cell adhesion. By lowering the
temperature to 20.degree.-32.degree. C., cells can be released from
the underlying substrate. The change from hydrophobic to
hydrophilic character over this transition results in the release
of proteins and adherent cells from the culture substrate. Through
this technique, cell-cell contacts, gap junctions and surface
receptors are maintained as well as the underlying extracellular
matrix (ECM). The intact ECM serves as glue to layer cell sheets to
form homogenous tissue grafts for example highly pulsatile cardiac
tissue grafts or heterogeneous tissue grafts by layering sheets
from various different cell types, for example endothelial cells
and hepatocytes. The cell sheets thus generated have been highly
applicable to animal transplant studies. Transplant experiments
have been done to compare the response of dissociated cells versus
cell sheet injections. Dissociated cardiomyocytes equivalent to
four cell sheets were injected into left subcutaneous dorsal tissue
and four cell sheet layers obtained from low temperature lift off
mediated by thermo-responsive polymer, were transplanted into the
right subcutaneous tissue. The isolated cells formed a lump under
the skin while the sheet transplanted site remained smooth. One
week after the transplant, the respective sites were opened and
cross sectional views of the right side indicated a flat square
cardiac graft with no visible necrosis and connexin 43 (a gap
junction marker) staining revealed the presence of numerous gap
junctions.
[0133] The left side showed cell dense graft surface zones with
central cell-void areas and only a few depositions were seen when
stained for connexin 43. The grafting of PIPAAm on tissue culture
polystyrene and its success with the culture and harvest of various
cell types has led to the development of commercially available
tissue culture polystyrene dishes by Cell Seed Inc. (Tokyo, Japan).
The use of these surfaces completely abandons the use of trypsin
when collecting cells as detachment is achieved by lowering the
culture temperature. This eliminates the use of laborious
pipetting, saving on both labor, time and cell/tissue damage.
[0134] The culture surfaces can also be functionalized by
co-polymerization of PIPAAm with its carboxylate derivatized
analog, 2-carboxyisopropylacrylamide (CIPAAm). Insulin was
immobilized on culture surfaces by standard amide bond formation
with the CIPAAm carboxylate group. The surfaces with immobilized
insulin showed an increase in proliferation of bovine carotid
artery endothelial (BAEC) cells even without the addition of serum.
Similarly, the carboxyl groups on CIPPAm sequences can be used to
immobilize cell adhesive sequences such as RGDS which promotes BAEC
cell adhesion and proliferation without the addition of fetal
bovine serum in the culture medium. Thus, the culture of cells and
their low temperature lift off obviates the need of using serum
which has both cost and safety (prions and bovine spongiform
encephalopathy) concerns regarding its use. These surfaces would be
useful for serum free culture of cells and cell sheets which can
then be used in various tissue engineering and transplant
applications.
[0135] The spontaneous cell sheet generation from PIPAAm-grafted
TCPS is a relatively slow process, occurring gradually from the
sheet periphery toward the interior. Thus, significant incubation
time is required to lift up the intact, viable cell sheet
completely. Rapid recovery of cell sheets is considered important
to maintain biological function and viability of recovered cell
sheets, as well as for practical assembly of tissue structures. The
rate limiting step to cell recovery is the hydration of
hydrophobized PIPAAm segments interacting with the cell sheet,
incorporation of a highly water permeable substrate to interface is
desirable between cell sheets and the thermo-responsive surfaces.
Several approaches have been tried in this regard to make the
detachment of cell sheets a faster process. It has been shown that
placing hydrophilically-modified PVDF membranes on confluent
Madin-Darby canine kidney (MDCK) cells incubated at 20.degree. C.
for one hour helps in the easy lift of cells. Another set of
experiments has utilized porous membranes (PET) grafted with
PIPAAm. As mentioned earlier, on PIPAAm-grafted TCPS dishes, water
required to hydrate PIPAAm at a lower temperature can readily
penetrate the culture matrix from only the periphery of each cell
to the interface between the cell and grafted PIPAAm chains. On
porous membranes, water hydration of PIPAAm is supplied through
pores underneath adherent cells, as well as from the periphery of
each cell. Ready, rapid access of bulk water to PIPAAm grafts
through pores beneath attached cells should accelerate single cell
and cell sheet detachment. The pore size of a membrane is an
important factor in determining the cell adhesion and growth. In
general, cells do not grow on surfaces which have a pore size
greater than their pseudopodium. On membranes with pore size
greater than 5 .mu.m the fibroblast adhesion and suppression was
found to be greatly reduced.
[0136] Nanofibers produced via the process of electrospinning may
have unprecedented porosity (>70%), a high surface to volume
ratio, and a wide range of pore diameter distribution and high
interconnectivity, all physical properties ideal for promoting cell
attachment and growth. Furthermore, the nanotopography of
electrospun nanofibers closely resembles the nanofibrillar and
nanoporous 3D geometry of the ECM and basement membrane. The higher
surface area allows for a higher percentage of cellular attachment
as well as for multiple focal adhesion points on different fibers
due to nano-sized fiber diameters. Because the diameters of
nanofibers are orders of magnitude smaller than the size of the
cells, cells are able to organize, spread or attach to adsorbed
proteins at multiple focal points.
[0137] Electrospun nanofibers are capable of supporting a wide
variety of cell types. Human umbilical cord endothelial cells
attached and proliferated better when seeded onto 50:50 poly
(L-lactic acid-co-.epsilon.-caprolactone) (PLCL) fibers with a
diameter of 300 nm compared to 7 .mu.m microfibers. Cells attached
to microfibers were round in shape and non-proliferative, whereas
on nanofibers, the cells were nicely spread out and anchored on
multiple fibers. Elias and co-workers have reported osteoblast
adhesion, proliferation, alkaline phosphatase activity and ECM
secretion on carbon nanofibers increased with decreasing fiber
diameter in the range of 60-200 nm. Nanogrooved surfaces can induce
contact guidance of human corneal epithelial cells, causing them to
elongate and align their cytoskeleton along the topological
features. Highly porous PLLA scaffolds with nanoscale pores created
using a liquid-liquid phase separation have been used for the
culture of neural stem cells and were shown to have a positive
effect on neurite outgrowth. Recent studies show that the growth of
NIH 3T3 fibroblasts and normal rat kidney cells on polyamide
nanofibrillar surfaces resulted in changes in morphology, actin
organization, focal adhesion assembly, fibronectin secretion and
rates of cell proliferation that are more representative of
fibroblast phenotype in vivo. Breast epithelial cells on the same
surface underwent morphogenesis to form multicellular spheroids
unlike the same cells cultured on glass. It has also been shown
that the commercially available polyamide nanofibers provide a
better substrate for cell attachment for weakly adherent cell
lines, for example PC12, a neuronal cell line. Polyamide
electrospun nanofibers have also been shown to support the
attachment and proliferation of mouse embryonic stem cells (ES-D3).
These cells differentiated into neurons, oligodendrocytes and
astrocytes based upon the culture media selected. Fetal bovine
chondrocytes seeded on nanofibers poly (.epsilon.-caprolactone)
(PCL) scaffolds were able to maintain the chondrocytic phenotype
during three weeks of culture, specifically upregulating collagen
type JIB expression, indicative of mature chondrocyte phenotype.
These studies demonstrate that nanofiber scaffolds are not only
cytocompatible but can also be used to stimulate and encourage cell
proliferation and phenotypic behavior.
[0138] To induce specific biological responses from the attached
cells, the nanofibers may also be functionalized using bioactive
molecules. Functionalization is typically carried out by either
conjugating the molecules to the surface of the nanofibers or by
incorporating the bioactive molecules in the spinning solution.
Polyacrylic acid (PAA) grafted onto poly
(.epsilon.-caprolactone-coethyl ethylene phosphate) (PCLEEP) allows
for the conjugation of galactose ligand, which mediates hepatocytes
attachment. Hepatocytes cultured on these PCLEEP functionalized
nanofiber scaffolds formed 20-100 .mu.m spheroid aggregates that
engulfed the nanofibers. To underscore the importance of culture
substrate, others have shown that aminated nanofiber meshes
supported a higher degree of cell adhesion and proliferation of
hematopoietic stem/progenitor cells compared to aminated films.
Similarly conjugation of bone morphogenetic protein-2 (BMP-2) on
chitosan nanofibers resulted in better proliferation, alkaline
phosphatase activity and calcium deposition of osteoblastic
cells.
[0139] In sum, the nanoscale nature of the electrospun polymeric
nanofibers mimics the natural ECM. ECM-like properties of the
nanofibers can be used to stimulate and encourage cell
proliferation and differentiation. Moreover, the cells are able to
maintain their in vivo like morphology and function. Thus, the
combination of fiber composition, morphology, alignment and the
capacity to incorporate bioactive molecules or growth factors helps
recreate the functions of native ECM.
[0140] The invention will be further described with reference to
the following non-limiting examples. It will be apparent to those
skilled in the art that many changes can be made in the embodiments
described without departing from the scope of the present
invention. Thus the scope of the present invention should not be
limited to the embodiments described in this application, but only
by embodiments described by the language of the claims and the
equivalents of those embodiments. Unless otherwise indicated, all
percentages are by weight.
EXAMPLES
Example 1
Electrospinning of PCL and PS Nanofibers
[0141] Poly (.epsilon.-caprolactone) (PCL), with an average
molecular weight of 80 kDa and Polystyrene (350,000 Da) were
purchased from Aldrich chemicals (Milwaukee, Wis.). 0.14 g/ml
solutions were prepared by dissolving 14 g of PCL or PS in 100 ml
of organic solvent mixture (1:1) composed of tetrahydrofuran
(Fisher Scientific) and N,N-dimethylformamide (Alfa Aesar, Ward
Hill, Mass.) and mixing it well by shaking the mixture for 24 h at
room temperature. The polymer solution was placed in a plastic
syringe fitted with a 27G blunt needle (Strategic Applications,
Inc., Libertyville, Ill.). A syringe pump (KD Scientific, USA) was
used to feed the polymer solution into the needle tip. Nanofiber
meshes were fabricated by electrospinning using a high voltage
power supply (Gamma High Voltage Research, USA). The nanofibers
were collected onto grounded aluminum foil located at a fixed
distance from the needle tip. The meshes were then removed, placed
in a vacuum chamber for at least 48 h to remove organic solvent
residue and then stored in a dessicator. The nanofibers were
evaluated with a microscope (Olympus BX 60).
[0142] Parameters that significantly influence the diameter,
consistency and uniformity of the electrospun PCL and PS fibers
were polymer concentration, applied voltage, solution feeding rate
and needle-collector distance. These parameters were optimized
until unbeaded and uniform fibers were spun continuously without
needle clogging. Three polymer concentrations (0.10 g/ml, 0.12
g/ml, and 0.14 g/ml), two voltages (17 kv, 20 kv) and three
needle-collector distances (8 cm, 12 cm, 15 cm) were investigated
to obtain non-defect nanofibers. The optimized conditions are shown
in Table 1.
TABLE-US-00002 TABLE 1 Electrospinning Parameters Polymer
concentration 0.14 g/ml Applied voltage 20 kv Flow rate 0.02 ml/min
Needle-collector distance 12 cm
[0143] FIG. 1. shows the typical SEM image of PCL nanofibers. The
average fiber diameter of nanofibers is, 453.+-.146 nm. Highly
porous structure was observed in the formulation tested. The
porosity measured by a liquid displacement method was 0.90
Example 2
Coating of Nanofiber Meshes with (PIPAAm)
[0144] Various coating approaches were employed to obtain a thin
coating of the thermo-responsive polymer on different culture
substrates. The substrates included TCPS, Thermanox coverslips
(Nunc), commercially available nanofiber meshes (Surmodics Inc.,
Corning Inc.) and in-house PCL nanofibers. The Thermanox coverslips
and nanofiber inserts were dip coated in an IPA (isopropyl alcohol)
solution of 20 mg/ml PIPAAm (polyisopropylacrylamide Aldrich
chemicals, Mw+20-25 KDa, WI) and 0.8 mg/ml TriLite
(tris[2-hydroxy-3-(4-benzoylphenoxy)propl]isocyanurate. The pieces
were dip coated by immersing in the coating solution for 10 seconds
and then extracted at a speed of 0.5 cm/sec. The meshes were air
dried and then UV illuminated (300-400 nm, Harland Medical UVM400,
MN) for 5 minutes. Various dipping speeds, concentrations, number
of dips and immerse times were tried. The efficacy of the coated
surfaces was tested by the attachment and detachment behavior of
the BAEC cells (Lonza Biosciences, NJ, USA) at 37.degree. C. and
20.degree. C. respectively. Although the above mentioned conditions
worked well for cell attachment and detachment, this dipping method
could not coat the tissue culture formatted surfaces (for example,
multi well dishes or 100 mm dishes). Therefore, another approach
was tried where the multi well dishes, nanofiber inserts,
commercially available nanofiber 96 well and 100 mm dishes were
first coated with 0.8 mg/ml solution of TriLite. The TriLite
solution was immediately withdrawn and UV illuminated for 30
seconds. PIPAAm solution (20 mg/ml in IPA) was then added to the
wells, immediately withdrawn and UV illuminated for 2.0 minutes.
The treated surfaces were rinsed with IPA and tissue culture grade
sterile water before plating the cells.
Example 3
Smart Polymer Nanofibers
[0145] Four formulations containing 1.0 wt % TriLite were prepared
to synthesize smart polymer photoreactive nanofibers. These
formulations were PS in (DMF/THF), PCL in (DMF/THF), PIPAAm in
(IPA/DMF), PIPAAm-co-PEG (1%) in water. The nanofibers were
fabricated by the electrospinning process of Example 2. The
parameters such as, polymer concentration, solvent ratio, applied
voltage and needle-collector distance, were optimized until
unbeaded and uniform fibers with an average diameter under 500 nm
can be spun continuously without needle clogging. The optimized
conditions are shown in Table 2. After drying, all the nanofibers
except PS and PCL were illuminated for 5 minutes under a UV lamp
(Harland Medical UVM400, Eden Prairie, Minn.). The nanofibers were
evaluated under a microscope. PEG-PIPAAm was synthesized by free
radical copolymerization of N-isopropylacrylamide (Aldrich) with
poly (ethyleneglycol) methyl ether methacrylate (Mw 2,000, Aldrich)
in water using ammonium persulfate (Sigma) as initiator and
N,N,N',N'-tetramethylethylenediamine (Aldrich) as a catalyst.
TABLE-US-00003 TABLE 2 Feeding Needle- Polymer Applied Rate
Collector Polymer Solvent Concentration Voltage ml/min Distance PS
THF/DMF 14% 20 kv .02 12 cm PCL THF/DMF 14% 20 kv .02 12 cm PIPAAm
IPA/DMF 25% 16 kv 0.1 6 cm PEG- water 5% 16 kv 0.2 6 cm PIPAAm
Example 4
Surface Characterization and Screening of PIPAAm Coated
Nanofibers
[0146] As the coated nanofiber meshes were completed they were
examined for surface topography, protein adsorption, and contact
angle. Initially they were screened in house for uniformity by
microscopic examination (looking for changes in pore size, and
obvious delamination or uncoated areas) and for contact angles.
Comparison between bulk PIPAAm, the coated and uncoated nanofiber
mesh, and the hydrophobic polystyrene core will provide evidence
for surface changes. Microscopic examination of the coated
nanofibers showed no obvious delamination or changes in the
morphology compared to the uncoated nanofibers.
[0147] Coatings which passed initial screening were assessed for
protein adsorption. PIPAAm surfaces at 37.degree. C. should adsorb
considerably more protein than at 25.degree. C. because of the
phase transition. Coated and uncoated TCPS coverslips, incubated in
1.times.PBS buffer at 37.degree. C. and 4.degree. C. for two hours,
then quickly removed and placed in a solution of 1 mg/ml BSA for 6
hours at 37.degree. C. and 4.degree. C. This time period should be
enough for protein adsorption to occur. Following the BSA
incubation, the pieces were rinsed three times with 1.times.PBS and
placed in HRP-labeled anti-BSA antibody (Sigma) for 30 minutes,
followed by a standard rinse and HRP colorimetric assay. Pieces
were considered coated with the thermo-responsive polymer if the
difference in protein adsorption between 37.degree. C. and
25.degree. C. incubated coated pieces exceeds one standard
deviation and differs significantly from that of uncoated
pieces.
[0148] Alternatively, we also adsorbed a cell adhesive protein,
Fibronectin (FN). Bovine plasma FN (Biomedical Technology Inc, MA)
was adsorbed onto the nanofibrillar surfaces by incubation of 10
.mu.g/ml FN in PBS solution at 37.degree. C. and 25.degree. C. for
6 hours. The coated pieces were then vigorously washed with PBS for
five times. They were blocked with 0.1% bovine serum albumin (BSA)
in PBS for one hour and reacted with 2.0 mg/ml rabbit polyclonal
anti bovine FN antibody (Biogenesis, Inc, UK) at a 1:200 dilution
(final concentration, 10 .mu.g/ml) for 2 hours at 37.degree. C. and
25.degree. C. respectively. Following five washes with PBS,
containing 0.1% BSA, they were incubated for an additional one hour
with anti rabbit IgG-HRP antibody (Chemicon International, CA) with
a 1:1000 dilution (final concentration 15 ug/ml) and incubated with
HRP substrate for 10 minutes. The color development was quenched
with I.0N H2S04 and absorbance measurements were taken at 450 nm
with a spectrophotometer (Spectramax M2).
[0149] A ten fold difference in protein adsorption was seen on
PIPAAm coated surfaces incubated at 37.degree. C. and 25.degree. C.
respectively. Surfaces that showed a difference in protein
adsorption at 37.degree. C. and 25.degree. C. were further
evaluated for their cell attachment and detachment profile by
plating different cell lines.
Example 5
Cultured Bovine and Human Cells
[0150] Bovine Aortic Endothelial Cells (BAEC) and T47-D cells were
pre-cultured in 75 cm.sup.2 flasks in DMEM-F12+10% FBS. The cells
were trypsinized and plated on PIPAAm coated nanofibrillar and TCPS
surfaces. The cells were also plated on commercially available
PIPAAm coated TCPS surfaces (Cell Seed Inc). Both cell lines were
plated at a density of 100,000 cells/well in a 6-well PIPAAm coated
dishes. Bare TCPS and Cell Seed surfaces were used as control
surfaces and similar numbers of cells were plated on them. The
cells were cultured for a period of 48 hours in a humidified
atmosphere with 5% C02 at 37.degree. C. Both the cell lines
attached well to the coated surfaces which indicate that the
coating is thin enough for the cells to attach. Forty eight hours
later, the cells were moved to room temperature. The BAEC cells
plated on PIPAAm coated nanofibers, TCPS and Cell seed surfaces
started to lift up in about 15-20 minutes. Approximately, after
about 35 minutes complete cell sheets lifted up (FIG. 4). The
results were more dramatic with T47-D cells. After 25 minutes
incubation at room temperature, the cells begun to sheet off from
the PIPAAm/TriLite coated nanofibrillar and TCPS surfaces while the
cells plated on Cell Seed surfaces failed to lift up even after 120
minutes of incubation at room temperature. It was observed that on
Cell Seed surfaces, there was no cell detachment while 50-70% of
the cells lifted up from PIPAAm/TriLite coated surfaces in about
half the time (FIG. 5).
Example 6
Cultured Human Epithelial Cells
[0151] It has been shown that cells growing on nanofibrillar
surfaces form more in vivo like morphologies. These surfaces are
also permissive for epithelial cells to undergo morphogenesis. We
have shown that our coating on smart polymer surfaces does not
interfere with the nanofibrillar properties of the matrix and cells
still undergo morphogenesis or form more in vivo like structures in
addition to being detached by mere temperature reduction.
[0152] For morphogenesis studies, T47-D breast epithelial cells
were cultured on nanofibrillar and flat surfaces coated with
PIPAAm/TriLite. The controls were bare nanofibers and TCPS. The
cells were cultured in DMEM+10% fetal bovine serum (FBS) in an
atmosphere of 5% CO.sub.2, 95% air at 37.degree. C. This particular
cell line has been selected as it has shown to demonstrate tubular
and spheroidal structures under conditions that promote three
dimensional interactions with collagen or matrigel. After, 10 days
in culture, cells were fixed with 4% paraformaldehyde and incubated
with Phalloidin Alexa Fluor 594 (1:500, Molecular Probes, OR) for
30 minutes at room temperature. The cells were rinsed three times
with PBS and observed under an inverted fluorescent microscope
(Zeiss Axiovert 200M). Phalloidin binds to filamentous actin
(F-actin) and provides visualization of cytoskeletal organization
of the cells. After 5 days in culture, a mixed population of
spheroids and tubular cells was observed on nanofibers. By day 8,
multicellular spheroids were dominant although some tubules still
persisted. In contrast, the growth of T47-D cells on flat surfaces
showed a monolayer with a group of stress fibers. We have shown
that our coating on the nanofiber surface with the
thermo-responsive polymer does not affect the nanofibrillar
topology and hence morphogenesis of T47-D cells or more in vivo
like cells can be obtained on these surfaces by mere reduction of
temperature. To show that detached cells recover quickly on fresh
surfaces and still retain their morphology after temperature
reduction, the second set of cells was grown to confluency for
about 5-10 days at 37.degree. C. The cells were then moved to room
temperature for about 15-40 minutes. The detached cell sheet was
gently removed with the help of a 10.0 ml pipette to a fresh tissue
culture surface. The cells were allowed to settle down and were
then fixed with 4% paraformaldehyde after 30 minutes incubation at
37.degree. C. Replated cells were stained with phalloidin F-actin
to show that the advantage of growing cells on thermo-responsive
nanofibrillar surfaces as opposed to flat thermo-responsive
surfaces is the ability to achieve and retain in vivo like
morphology (D).
[0153] FIG. 7A shows the T47-D cells cultured on PIPAAm/TriLite
coated nanofibers for a period of 10 days and stained with
Phalloidin F-actin. Note the presence of multicellular spheroids
and the peripheral organization of actin filaments. A magnified
image (400 .mu.m) of the spheroid of FIG. 7B shows the lumen
extending through the spheroid. T47-D cells cultured on
PIPAAm/TriLite coated TCPS for period of 10 days were also fixed
and stained for phalloidin F-actin. Note the spread out morphology
and organization of stress fibers in the cells of FIG. 7C. T47-D
cells lifted up through temperature reduction were replated on
fresh nanofibers. FIG. 7D shows that replated T47-D cells maintain
their tubular and spheroidal morphology and peripheral organization
of actin (200 .mu.m).
[0154] Replated cell sheets were also analyzed for conexxin 43
expression which is considered to be a major component of gap
junctional channel. BAEC cells were plated at a density of 50,000
cells/22 mm well. The cells were cultured until confluency and then
lifted up by moving the dish to 20.degree. C. for 15 minutes. The
sheets were transferred onto fresh nanofibrillar surfaces with the
help of a 10 ml pipette and the curled up edges were uncurled by
adding a drop of medium onto the sheet. The sheet was then
transferred to the 5% CO2 humidified incubator at 37.degree. C. and
were allowed to attach. Thirty minutes later the cells were fixed
with 4% paraformaldehyde and stained with 1:1000 dilution of anti
connexin 43 (Sigma). Staining for Connexin 43 showed diffused
expression of connexin 43 through out the entire sheet suggesting
the presence of intact gap junctions (FIG. 8).
* * * * *