U.S. patent application number 14/201965 was filed with the patent office on 2014-07-17 for nanofibers containing latent reactive groups.
This patent application is currently assigned to Innovative Surface Technologies, Inc.. The applicant listed for this patent is Innovative Surface Technologies, Inc.. Invention is credited to Patrick E. Guire, Jie Wen.
Application Number | 20140199468 14/201965 |
Document ID | / |
Family ID | 40186251 |
Filed Date | 2014-07-17 |
United States Patent
Application |
20140199468 |
Kind Code |
A1 |
Wen; Jie ; et al. |
July 17, 2014 |
NANOFIBERS CONTAINING LATENT REACTIVE GROUPS
Abstract
A nanofiber is formed by combining one or more natural or
synthetic polymeric materials and one or more than one
cross-linking agents having at least two latent reactive
activatable groups. The latent reactive activatable nanofiber may
be used to modify the surface of a substrate by activating at least
one of the latent reactive activatable groups to bond the nanofiber
to the surface by the formation of a covalent bond between the
surface of the substrate and the latent reactive activatable group.
Some of the remaining latent reactive activatable group(s) are left
accessible on the surface of the substrate, and may be used for
further surface modification of the substrate. Biologically active
materials may be immobilized on the nanofiber modified surface by
reacting with the latent reactive groups that are accessible on the
surface of the substrate.
Inventors: |
Wen; Jie; (St. Johns,
FL) ; Guire; Patrick E.; (Hopkins, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Innovative Surface Technologies, Inc. |
St. Paul |
MN |
US |
|
|
Assignee: |
Innovative Surface Technologies,
Inc.
St. Paul
MN
|
Family ID: |
40186251 |
Appl. No.: |
14/201965 |
Filed: |
March 10, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12666173 |
Sep 29, 2010 |
8709809 |
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PCT/US08/67739 |
Jun 20, 2008 |
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14201965 |
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60945807 |
Jun 22, 2007 |
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Current U.S.
Class: |
427/2.11 ;
264/10; 264/165; 264/495; 427/180; 427/2.25; 427/2.26; 427/2.3;
427/2.31; 427/513 |
Current CPC
Class: |
B29C 35/08 20130101;
D01F 6/625 20130101; D06M 15/273 20130101; A61L 31/14 20130101;
D06M 15/285 20130101; D01D 5/003 20130101; A61L 2400/12 20130101;
D01F 1/10 20130101; A61L 27/50 20130101; A61L 29/14 20130101; D06M
2400/01 20130101; D06M 15/263 20130101; B05D 3/067 20130101; C12M
23/20 20130101; D01D 5/0007 20130101 |
Class at
Publication: |
427/2.11 ;
264/10; 264/495; 264/165; 427/2.25; 427/2.26; 427/2.3; 427/2.31;
427/180; 427/513 |
International
Class: |
D01D 5/00 20060101
D01D005/00; B05D 3/06 20060101 B05D003/06; B29C 35/08 20060101
B29C035/08 |
Claims
1.-56. (canceled)
57. A method of producing a latent reactive nanofiber comprising
steps of: (a) preparing a composition comprising a cross-linking
agent having at least two latent photochemically reactive groups,
and a fiber forming material; and (b) forming a nanofiber from the
composition of (a).
58. The method according to claim 57 wherein the step of forming a
nanofiber comprises electrospinning the composition of (a).
59. The method according to claim 57 further comprising a step of
treating the formed nanofiber to activate at least some of the
latent photochemically reactive groups of the cross-linking agent,
thereby forming a crosslinked nanofiber.
60. The method according to claim 57 wherein the cross-linking
agent is a tri-functional monomeric or polymeric material.
61. The method according to claim 57 wherein the cross-linking
agent has a formula:
L-(T-C(R.sup.1)(XP)CHR.sup.2GR.sup.3C(.dbd.O)R.sup.4).sub.m wherein
L is a linking group; T is (--CH.sub.2--).sub.x,
(--CH.sub.2CH.sub.2--O--).sub.x,
(--CH.sub.2--CH.sub.2--CH.sub.2--O--).sub.x, or
(--CH.sub.2--CH.sub.2--CH.sub.2--CH.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, 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,
(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.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.
62. The method according to claim 57 wherein the cross-linking
agent is tris[2-hydroxy-3-(4-benzoylphenoxy)propyl]isocyanurate
having formula: ##STR00010##
63. The method according to claim 57 wherein the fiber forming
material is a synthetic or natural polymer.
64. The method according to claim 63 wherein the fiber forming
material is a biodegradable polymer selected from polyesters,
polyamides, polyurethanes, polyorthoesters, polycaprolactone,
polyiminocarbonates, aliphatic carbonates, polyphosphazenes,
polyanhydrides, and copolymers of these.
65. The method according to claim 63 wherein the fiber forming
material comprises a polymer having peptide, nucleotide or
saccharide monomeric units.
66. The method according to claim 63 wherein the fiber forming
material is a thermally responsive polymeric material.
67. The method according to claim 66 wherein the thermally
responsive polymeric material comprises poly(N-isopropylacrylamide)
or polyethylene glycol-poly(N-isopropylacrylamide).
68. The method according to claim 63 wherein the fiber forming
material comprises two or more polymeric materials.
69. The method according to claim 57 wherein the nanofiber further
comprises a biologically active material or a functional
polymer.
70. A method of coating a surface of a substrate comprising steps
of: (a) providing a latent reactive nanofiber according to claim
57; and (b) contacting the surface with the formed nanofiber.
71. The method according to claim 70 further comprising a step of
treating the surface to activate latent photochemically reactive
groups of the cross-linking agent, thereby coupling the nanofiber
to the surface via the cross-linking agent.
72. The method according to claim 70 wherein the substrate
comprises plastic, pyrolytic carbon, glass, ceramic or metal.
73. The method according to claim 70 wherein the substrate
comprises a catheter, wound drainage tube, arterial graft, soft
tissue patch, glove, shunt, stent, wound dressing, sutures, guide
wire or prosthetic device.
74. The method according to claim 70 wherein the substrate
comprises a slide, microliter well, microliter plate, Petri dish,
tissue culture slide, tissue culture plate, tissue culture flask,
cell culture plate, column support, chromatography media,
microscope slide or chip.
Description
TECHNICAL FIELD
[0001] The present invention generally relates to nanofibers and
nanofiber modified surfaces. More particularly, the present
invention is directed to nanofibers including one or more
multi-functional cross-linking agents each having at least two
latent reactive activatable groups. The nanofibers containing
latent reactive activatable cross-linking agents may be used to
modify a surface of a substrate.
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 liquid
into an electric field, so that the 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 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. Recently, efforts have focused on using
electrospinning techniques to produce nonwoven membranes of
nanofibers.
[0005] Nanofibers can be used to modify the surface of a substrate.
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 teraphthalate
(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 of the present invention, a
nanofiber includes one or more natural or synthetic polymeric
materials and one or more cross-linking agents each having at least
two latent reactive activatable groups. In use, photochemically or
thermally latent reactive groups will form covalent bonds when
subjected to a source of energy. Suitable energy sources include
radiation and thermally 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 various embodiments of the present invention,
the polymeric material of the nanofiber may be hydrophilic,
hydrophobic, amphophilic or thermally responsive, depending on the
desired application. According to yet a further embodiment of the
present invention, the nanofiber also may be either biodegradable
or non-biodegradable polymers. In still further embodiments the
nanofiber may include a biologically active material.
[0010] The nanofiber typically has a diameter ranging from 1 nm to
100 microns and may have a diameter ranging from 1 nm to 1000 nm.
The nanofiber may have an aspect ratio in a range of about at least
10 to at least 100.
[0011] According to another embodiment of the present invention, a
latent reactive activatable nanofiber is produced by combining one
or more polymeric materials with one or more cross-linking agents
each having at least two latent reactive activatable groups and
forming at least one nanofiber from the combination. The nanofiber
may be formed by electrospinning the combination containing the
polymeric materials and the cross-linking agent. According to yet a
further embodiment of the present invention, the combination may
also include biologically active materials or be further combined
with a functional polymer that will subsequently react with
biologically active materials. Functional polymers include any
suitable polymer having one or more functional groups that will
react with a biologically active material. Representative
functional groups for these polymers include carboxy, ester, epoxy,
hydroxyl, amido, amino, thio, N-hydroxy succinimide, isocyanate,
anhydride, azide, aldehyde, cyanuryl chloride or phosphine
groups.
[0012] According to yet another embodiment, the present invention
provides method of coating a surface of a substrate. According to
one embodiment of the present invention, the method includes
combining one or more polymeric materials and one or more
cross-linking agents each having at least two latent reactive
activatable groups, forming at least one nanofiber from the
combination, contacting the surface of the substrate with the
nanofiber; and forming a bond between the nanofiber and the
surface. According to a further embodiment of the present
invention, the method includes activating at least one of the
latent reactive activatable groups with a source of energy to bond
the nanofiber to a biologically active material. According to an
alternative embodiment of the present invention, the method
includes simultaneously activating a first latent reactive
activatable group to bond the nanofiber to the surface and a second
latent reactive activatable group to bond the nanofiber to a
biologically active material.
[0013] According to still another embodiment, the present invention
provides an article having a surface coating including a plurality
of nanofibers including one or more natural or synthetic polymeric
materials and one or more cross-linking agents each having at least
two latent reactive activatable groups. In some embodiments, a
biologically active material is bonded to the nanofibers.
[0014] According to yet still another embodiment, the present
invention is a cell culture plate including a surface coating
having at least one nanofiber including one or more polymeric
materials and one or more cross-linking agents each having at least
two latent reactive activatable groups.
[0015] While multiple embodiments are disclosed, still other
embodiments of the present invention will become apparent to those
skilled in the art from the following detailed description, which
illustrates and describes exemplary embodiments of the invention.
Accordingly, the detailed description is to be regarded as
illustrative in nature and not restrictive.
DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1A-1D are electronic images of polycaprolactone
nanofibers prepared by the process described in Example 1.
[0017] FIGS. 2-4 illustrate functional group densities for
nanofibers containing carboxy and amine groups that are described
in Example 7.
[0018] FIG. 5 illustrates protein immobilization levels for
nanofibers described in Example 10.
[0019] FIG. 6 illustrates horse radish peroxidase activity for
nanofibers described in Example 11.
[0020] FIG. 7 graphs enzymatic degradation of nanofibers described
in Example 12.
[0021] FIGS. 8A-8D are electronic images of enzymatically degraded
nanofibers that are described in Example 12.
DETAILED DESCRIPTION
[0022] The present invention is directed toward a latent reactive
activatable nanofiber. The latent reactive activatable nanofiber
can be used to modify a surface of a substrate to provide a
functionalized surface. Biologically active materials may be
immobilized on the nanofiber modified surface by reacting with the
latent reactive groups exposed on the surface of the substrate.
Typically, the biologically active materials retain at least some
of their bioactivity after having been immobilized on the nanofiber
modified surface.
[0023] According to one embodiment of the present invention the
nanofiber includes one or more natural or synthetic polymeric
materials and cross-linking agents having at least two latent
reactive activatable groups. According to a further embodiment of
the present invention, the nanofiber may be biodegradable or
non-biodegradable and may also include a biologically active
material. The latent reactive activatable nanofiber can be used to
modify the surface of a substrate by activating at least one of the
latent reactive activatable groups to bond the nanofiber to the
surface by the formation of a covalent bond between the surface of
the substrate and the latent reactive activatable group. The
remaining latent reactive activatable group(s) are left accessible
on the surface of the substrate, and may be used for further
surface modification of the substrate.
[0024] A number of processing techniques such as drawing, template
synthesis, phase separation, self-assembly or electrospinning have
been used to prepare nanofibers. In one embodiment, the nanofiber
can be formed by electrospinning a fiber-forming combination that
includes one or more polymeric materials and cross-linking agents
having at least two latent reactive activatable groups. According
to an alternative embodiment of the present invention, the
fiber-forming combination may also include biologically active
materials. Electrospinning generally involves the introduction of
one or more polymeric materials or other fiber-forming solutions or
liquid into an electric field, so that the solution or liquid
produces nanofibers. 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 combination 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
counter electrode having a lower electrical potential. The jet is
split into small charged fibers or fibrils and any solvent present
evaporates leaving behind a nonwoven mat formed on the screen.
[0025] Electrostatically spun fibers can be produced having very
thin diameters. Parameters that influence the diameter,
consistency, and uniformity of the electrospun fibers include the
polymeric material and cross-linker concentration (loading) in the
fiber-forming combination, the applied voltage, and needle
collector distance. According to one embodiment of the present
invention, a nanofiber has a diameter ranging from about 1 nm to
about 100 .mu.m. In other embodiments, the 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 at least about 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 solutions or liquids to be transformed from liquid or
solvated fiber-forming materials to solid nanofibers in fractions
of a second.
[0026] The polymeric material used to form the nanofiber may be
selected from any fiber forming material which is compatible with
the cross-linking agents. Depending upon the intended application,
the fiber-forming polymeric material may be hydrophilic,
hydrophobic or amphiphilic. Additionally, the fiber-forming
polymeric material may be a thermally responsive polymeric
material.
[0027] Synthetic or natural, biodegradable or non-biodegradable
polymers may form the nanofiber. A "synthetic polymer" refers to a
polymer that is synthetically prepared and that includes
non-naturally occurring monomeric units. For example, a synthetic
polymer can include non-natural monomeric units such as acrylate or
acrylamide units. Synthetic polymers are typically formed by
traditional polymerization reactions, such as addition,
condensation, or free-radical polymerizations. Synthetic polymers
can also include those having natural monomeric units, such as
naturally-occurring peptide, nucleotide, and saccharide monomeric
units in combination with non-natural monomeric units (for example
synthetic peptide, nucleotide, and saccharide derivatives). These
types of synthetic polymers can be produced by standard synthetic
techniques, such as by solid phase synthesis, or recombinantly,
when allowed.
[0028] A "natural polymer" refers to a polymer that is either
naturally, recombinantly, or synthetically prepared and that
consists of naturally occurring monomeric units in the polymeric
backbone. In some cases, the natural polymer may be modified,
processed, derivitized, or otherwise treated to change the chemical
and/or physical properties of the natural polymer. In these
instances, the term "natural polymer" will be modified to reflect
the change to the natural polymer (for example, a "derivitized
natural polymer", or a "deglycosylated natural polymer").
[0029] Nanofiber materials, for example, may include both addition
polymer and condensation polymer materials such as polyolefin,
polyacetal, polyamide, polyester, cellulose ether and ester,
polyalkylene sulfide, polyarylene oxide, polysulfone, modified
polysulfone polymers and mixtures thereof. Exemplary materials
within these generic classed include polyethylene,
poly(.epsilon.-caprolactone), poly(lactate), poly(glycolate),
polypropylene, poly(vinylchloride), polymethylmethacrylate (and
other acrylic resins), polystyrene, and copolymers thereof
(including ABA type block copolymers), poly(vinylidene fluoride),
poly(vinylidene chloride), polyvinyl alcohol in various degrees of
hydrolysis (87% to 99.5) in crosslinked and non-crosslinked forms.
Exemplary addition polymers tend to be glassy (a Tg greater than
room temperature). This is the case for polyvinylchloride and
polymethylmethacrylate, polystyrene polymer compositions, or alloys
or low in crystallinity for polyvinylidene fluoride and polyvinyl
alcohol materials.
[0030] In some embodiments of the invention the nanofiber material
is a polyamide condensation polymer. In more specific embodiments,
the polyamide condensation polymer is a nylon polymer. The term
"nylon" is a generic name for all long chain synthetic polyamides.
Typically, nylon nomenclature includes a series of numbers such as
in nylon-6,6 which indicates that the starting materials are a
C.sub.6 diamine and a C.sub.6 diacid (the first digit indicating a
C.sub.6 diamine and the second digit indicating a C.sub.6
dicarboxylic acid compound). Another nylon can be made by the poly
condensation of epsilon caprolactam in the presence of a small
amount of water. This reaction forms a nylon-6 (made from a cyclic
lactam--also known as epsilon-aminocaproic acid) that is a linear
polyamide. Further, nylon copolymers are also contemplated.
Copolymers can be made by combining various diamine compounds,
various diacid compounds and various cyclic lactam structures in a
reaction mixture and then forming the nylon with randomly
positioned monomeric materials in a polyamide structure. For
example, a nylon 6,6-6,10 material is a nylon manufactured from
hexamethylene diamine and a C.sub.6 and a C.sub.10 blend of
diacids. A nylon 6-6,6-6,10 is a nylon manufactured by
copolymerization of epsilon aminocaproic acid, hexamethylene
diamine and a blend of a C.sub.6 and a C.sub.10 diacid
material.
[0031] Block copolymers can also be used as nanofiber materials. In
preparing a composition for the preparation of nanofibers, a
solvent system can be chosen such that both blocks are soluble in
the solvent. One example is an ABA (styrene-EP-styrene) or AB
(styrene-EP) polymer in methylene chloride solvent. Examples of
such block copolymers are a Kraton.TM.-type of AB and ABA block
polymers including styrene/butadiene and styrene/hydrogenated
butadiene(ethylene propylene), a Pebax.TM.-type of
epsilon-caprolactam/ethylene oxide and a Sympatex.TM.-type of
polyester/ethylene oxide and polyurethanes of ethylene oxide and
isocyanates.
[0032] Addition polymers such as polyvinylidene fluoride,
syndiotactic polystyrene, copolymers of vinylidene fluoride and
hexafluoropropylene, polyvinyl alcohol, polyvinyl acetate,
amorphous addition polymers, such as poly(acrylonitrile) and its
copolymers with acrylic acid and methacrylates, polystyrene,
poly(vinyl chloride) and its various copolymers, poly(methyl
methacrylate) and its various copolymers, can be solution spun with
relative ease because they are soluble at low pressures and
temperatures. Highly crystalline polymer like polyethylene and
polypropylene generally require higher temperature and high
pressure solvents if they are to be solution spun.
[0033] Nanofibers can also be formed from polymeric compositions
comprising two or more polymeric materials in polymer admixture,
alloy format, or in a crosslinked chemically bonded structure. Two
related polymer materials can be blended to provide the nanofiber
with beneficial properties. For example, a high molecular weight
polyvinylchloride can be blended with a low molecular weight
polyvinylchloride. Similarly, a high molecular weight nylon
material can be blended with a low molecular weight nylon material.
Further, differing species of a general polymeric genus can be
blended. For example, a high molecular weight styrene material can
be blended with a low molecular weight, high impact polystyrene. A
Nylon-6 material can be blended with a nylon copolymer such as a
Nylon-6; 6,6; 6,10 copolymer. Further, a polyvinyl alcohol having a
low degree of hydrolysis such as a 87% hydrolyzed polyvinyl alcohol
can be blended with a fully or super hydrolyzed polyvinyl alcohol
having a degree of hydrolysis between 98 and 99.9% and higher. All
of these materials in admixture can be crosslinked using
appropriate crosslinking mechanisms. Nylons can be crosslinked
using crosslinking agents that are reactive with the nitrogen atom
in the amide linkage. Polyvinyl alcohol materials can be
crosslinked using hydroxyl reactive materials such as
monoaldehydes, such as formaldehyde, ureas, melamine-formaldehyde
resin and its analogues, boric acids, and other inorganic
compounds, dialdehydes, diacids, urethanes, epoxies, and other
known crosslinking agents. Crosslinking reagent reacts and forms
covalent bonds between polymer chains to substantially improve
molecular weight, chemical resistance, overall strength and
resistance to mechanical degradation.
[0034] Biodegradable polymers can also be used in the preparation
of an article associated with the nanofibrillar structure. Examples
of classes of synthetic polymers that have been studied as
biodegradable materials include polyesters, polyamides,
polyurethanes, polyorthoesters, polycaprolactone (PCL),
polyiminocarbonates, aliphatic carbonates, polyphosphazenes,
polyanhydrides, and copolymers thereof. Specific examples of
biodegradable materials that can be used in connection with, for
example, implantable medical devices include polylactide,
polygylcolide, polydioxanone, poly(lactide-co-glycolide),
poly(glycolide-co-polydioxanone), polyanhydrides,
poly(glycolide-co-trimethylene carbonate), and
poly(glycolide-co-caprolactone). Blends of these polymers with
other biodegradable polymers can also be used.
[0035] In some embodiments, the nanofibers are non-biodegradable
polymers. Non-biodegradable refers to polymers that are generally
not able to be non-enzymatically, hydrolytically or enzymatically
degraded. For example, the non-biodegradable polymer is resistant
to degradation that may be caused by proteases. Non-biodegradable
polymers may include either natural or synthetic polymers.
[0036] The inclusion of cross-linking agents within the composition
forming the nanofiber, allows the nanofiber to be compatible with a
wide range of support surfaces. The cross-linking agents can be
used alone or in combination with other materials to provide a
desired surface characteristic.
[0037] 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, electrochemical or
thermal energy. Various examples of known or commercially available
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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] Representative thermally reactive moieties are reported in
US 20060030669 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.
[0045] Latent reactive groups that are reactive to electromagnetic
radiation, such as ultraviolet or visible radiation, are typically
referred to as photochemical reactive groups.
[0046] 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.
[0047] The functional groups of such ketones are suitable because
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.
[0048] 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 or amphophilic portion. 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,
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.xC.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.tNR(--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 am 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.
[0049] In one embodiment, L is a branched or unbranched alkyl chain
having between about 2 and about 10 carbon atoms.
[0050] In another embodiment, D is an oxygen atom (O).
[0051] 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.
[0052] In still yet another embodiment, R.sup.1 is a hydrogen
atom.
[0053] In yet another embodiment, X is an oxygen atom, O, and P is
a hydrogen atom.
[0054] In another embodiment, R.sup.2 is a hydrogen atom.
[0055] In still another embodiment, G is an oxygen atom, O.
[0056] 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.
[0057] 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.
[0058] In yet another particular embodiment, L is
(--CH.sub.2--).sub.y, 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
2, x is 1 and y is an integer from 2 to about 6, and in particular,
y is 2, 4 or 6.
[0059] 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.
[0060] 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.16, R, q, r, s, m, t and x are as defined above.
[0061] In one embodiment, L has a formula according to structure
(I):
##STR00002##
[0062] 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.
[0063] In another embodiment, T is --CH.sub.2--.
[0064] 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.
[0065] 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.
[0066] In another embodiment, L has a formula according to
structure (II):
##STR00004##
[0067] 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.
[0068] 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.
[0069] In one embodiment, L has a formula according to structure
(I):
##STR00005##
A, B, J, R.sup.11, and z are as defined above.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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).s-
ub.m.
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.
[0074] In one embodiment, L has a formula according to structure
(I):
##STR00007##
A, B, J, R.sup.11, and z are as defined above.
[0075] In another embodiment, A and B are both hydrogen atoms.
[0076] In still another embodiment, xx is 1.
[0077] In yet another embodiment, R.sup.1 is H.
[0078] In still yet another embodiment, G is
(--CF.sub.2--).sub.tO-- and t is 1.
[0079] In another embodiment, R.sup.1 and R.sup.4 are each
individually aryl groups.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] In another embodiment, R.sup.1 is H.
[0084] In still another embodiment, X is O.
[0085] In yet another embodiment, P is H.
[0086] In still yet another embodiment, R.sup.2 is H.
[0087] In another embodiment, G is (--CH.sub.2--).sub.tO-- and t is
1.
[0088] In still another embodiment, R.sup.3 and R.sup.4 are each
individually aryl groups.
[0089] 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.
[0090] 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, G, R, R.sup.3, R.sup.4, R.sup.10, q, r, s, m and t are as
defined above.
[0091] In one embodiment, L is
##STR00009##
[0092] In another embodiment, G is C.dbd.O.
[0093] In still another embodiment, R.sup.3 and R.sup.4 are each
individually aryl groups.
[0094] In yet another embodiment, G is C.dbd.O and R.sup.3 and
R.sup.4 are each individually aryl groups.
[0095] In yet another embodiment, the present invention provides a
family of compounds having the formula:
-((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.tO-- 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.
[0096] "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.
[0097] "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.
[0098] "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-1yl; 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-l, cyclobut-1-en-1-yl,
cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the
like.
[0099] "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.n--R'' wherein n is an
integer from 1 to about 10, and R' and R'' are alkyl or alkylene
groups.
[0100] "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.
[0101] "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).
[0102] "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.1C.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.
[0103] "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,
hexalene, as-indacene, s-indacene, indane, indene, naphthalene,
octacene, octaphene, octalene, ovalene, penta-2,4-dine, 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.
[0104] "Arylalkyl" 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.
Preferably, an arylalkyl group is (C.sub.7C.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.6-C.sub.20),
more preferably, 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).
[0105] "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, ethoxyphenyl, etc.
[0106] "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.
[0107] "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 limed to, N, P, O, S, Si, etc. 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.
[0108] "Halogen" or "Halo" by themselves or as part of another
substituent, unless otherwise stated, refer to fluoro, chloro,
bromo and iodo.
[0109] "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, perfluoroethyl, etc.
[0110] "Heteroalkyl, Heteroalkanyl, 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).sub.2--, --S(O).sub.2--,
and the like, where R' is hydrogen, alkyl, substituted alkyl,
cycloalkyl, substituted cycloalkyl, aryl or substituted aryl.
[0111] "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, quinolizene, quinoxaline, tetrazole,
thiadiazole, thiazole, thiophene, triazole, xanthene, and the like.
Preferably, the heteroaryl group is from 5-20 membered heteroaryl,
more preferably from 5-10 membered heteroaryl. Suitable heteroaryl
groups are those derived from thiophene, pyrrole, benzothiophene,
benzofuran, indole, pyridine, quinoline, imidazole, oxazole and
pyrazine.
[0112] "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, heteroarylakenyl 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.
[0113] "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, trihydroxyalkyls, etc.
[0114] "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, hexalene, 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.
[0115] "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.
[0116] "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).
[0117] "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.
[0118] "Protecting groups" 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").
[0119] There are a variety of substrate materials that may be used
in the present invention. Plastics such as polyolefins,
polystyrenes, poly(methyl)methacrylates, polyacrylonitriles,
poly(vinylacetates), poly (vinyl alcohols), chlorine-containing
polymeric material such as poly(vinyl) chloride, polyoxymethylenes,
polycarbonates, polyamides, polyimides, polyurethanes, phenolics,
amino-epoxy resins, polyesters, silicones, cellulose-based
plastics, and rubber-like plastics may 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.
[0120] The method of the present invention may involve the
attachment or bonding of a biologically active material to a
support surface. For example, a nanofiber including a cross-linking
agent is provided having two or more latent reactive activatable
groups in the presence of a support surface. At least one of the
latent reactive activatable groups is activated and covalently
bonded to the surface. The remaining latent reactive activatable
groups are allowed to revert to their inactive state and are later
reactivated in order to later bind a biologically active material
in order to attach the biologically active-material to the surface
of the substrate.
[0121] The steps of the method may be performed in any suitable
order. For example, a nanofiber including a cross-linking agent, as
described herein, can be physically absorbed or adsorbed to a
suitable support surface by the hydrophobic interactions. Upon
activation by a source of energy, at least one of the latent
reactive activatable 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
latent reactive activatable group(s), and removal of the source of
energy, the latent reactive activatable group returns from an
excited state to a ground state. These remaining latent reactive
activatable 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 latent reactive activatable nanofiber is
applied in the first step to create a latent reactive activatable
surface, and in the second step, the biologically active material
is added for attachment to the activated surface.
[0122] Alternatively, the method, described as a "one-step" method,
provides that the latent reactive activatable nanofibers of the
present invention are combined or mixed together with the
biologically active material to form a composition. The resultant
composition is used to surface modify materials in a single step of
activation by a source of energy. In this case, activation by a
source of energy triggers not only covalent bond formation of at
least one latent reactive activatable group with the surface of the
substrate, but also simultaneously triggers covalent bond formation
with any adjacent biologically active materials residing on the
surface.
[0123] In an alternative embodiment, the nanofiber is formed from a
combination or mixture including a polymeric material, a
cross-linking agent having at least two latent reactive activatable
groups, and a biologically active material. At least one of the
latent reactive activatable groups undergoes covalent bond
formation at the support surface to bond the nanofiber to the
surface of the substrate. The remaining latent reactive activatable
group(s) can undergo activation by a source of energy 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.
[0124] In another alternative method, latent reactive activatable
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.
[0125] Suitable biologically active or other target molecules for
use in the present invention for attachment to a support surface,
encompass a diverse group of substances. Target molecules can 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.
[0126] Target molecules can be immobilized to the surface either
after (e.g., sequentially) the surface has been primed with the
latent reactive activatable nanofibers of the present invention.
Alternatively, target molecules are immobilized during (e.g.,
simultaneously with) attachment of the latent reactive activatable
nanofibers to the surface of the substrate.
[0127] 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 biologically active 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
Polymeric Materials 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 materials, Electric conductivity 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 proteins/ Cell attachment and growth peptides
Growth factors, proteins/ Cell growth 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 Fluorescence (e.g., fluorescein)
[0128] Target molecules can also be functional polymers. Functional
polymers are defined as polymers with functional groups which can
be used for further chemical reactions. The functional groups
include but are not limited to carboxyl, amine, thiol, epoxy, NHS,
aldehyde, azide, phosphine, or hydroxyl.
[0129] The latent reactive activatable 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.
[0130] Medical articles that can be fabricated from or coated or
treated with the latent reactive activatable 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.
[0131] Additional articles that can be fabricated from or have a
surface that can be coated or treated with the latent reactive
activatable nanofibers of the present invention can include, but
are not limited to, the following: slides, microtiter wells,
microtiter plates, Petri dishes, tissue culture slides, tissue
culture plates, tissue culture flasks, cell culture plates, or
column supports and/or chromatography media.
[0132] In another embodiment, the latent reactive activatable
nanofibers of the present invention can be applied to a microscope
slide or "chip" for biomolecule immobilization.
[0133] In yet another embodiment the latent reactive activatable
nanofibers of the present invention can be applied to a surface of
a cell culture plate.
[0134] 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 Photoreactive Nanofibers
[0135] Poly (.epsilon.-caprolactone) (PCL), with an average
molecular weight of 80 kDa was purchased from Aldrich Chemicals
(Milwaukee, Wis.). 0.14 g/ml PCL solution was prepared by
dissolving 14 g of PCL in 100 ml of organic solvent mixture (1:1)
composed of tetrahydrofuran and N,N-dimethylformamide and mixing it
well by vortexing the mixture for 24 h at room temperature. Polymer
solutions with 1%, 5%, and 10% weight percent of photocrosslinker
content (such as TriLite, tris
[2-hydroxy-3-(4-benzoylphenoxy)propyl]isocyanurate) were made by
adding different amounts of crosslinker in the PCL solution. The
polymer solution was placed in a plastic syringe fitted with a 27G
needle. A syringe pump (KD Scientific, USA) was used to feed the
polymer solution into the needle tip. A high voltage power supply
(Gamma High Voltage Research, USA) was used to charge the needle
tip. The nanofibers were collected onto grounded aluminum foil
target located at a certain distance from the needle tip. The fiber
meshes were then removed, placed in a vacuum chamber for at least
48 h to remove organic solvent residue, and then stored in a
desiccator. The nanofibers were evaluated under microscope. Other
photoreactive nanofibers were also prepared by electrospinning
TriLite containing polymer solutions. The polymers include nylon
6/6 (Aldrich), polystyrene (Mw 170,000, Aldrich),
poly(N-isopropylacrylamide) (PIPAAm, Mw 20,000-25,000, Aldrich),
and PEG-PIPAAm, PEG-PIPAAm was synthesized by free radical
copolymerization of N-isopropylacrylamide (Aldrich) with
poly(ethylene glycol) methyl either methacrylate (Mw 2,000,
Aldrich) in water using ammonium persulfate (Aldrich) as initiator
and N,N,N',N'-tetramethylethylenediamine (Aldrich) as catalyst. A
photoreactive polymer PVB-BP was synthesized by the reaction of
poly(vinyl butyral) (Mw 70,000-100,000, Polysciences) with
benzophenone acid chloride which was prepared by the reaction of
4-benzoylbenzoic acid (Aldrich) and oxalyl chloride (Aldrich).
Photoreactive PVB-BP nanofibers were prepared by electrospinning
PVB-BP solution without TriLite. The electrospinning conditions are
summarized in Table 1.
TABLE-US-00002 TABLE 1 Electrospinning Parameters Polymer Col-
concen- Applied Feeding lection tration Voltage Rate Distance
Polymer Solvent (% w/w) (kv) (ml/min) (cm) PCL THF/DMF 14 20 0.3 12
Nylon 6/6 trifluoro- 20 17 0.1 10 ethanol Polystyrene THF/DMF 14 20
0.2 12 PIPAAm IPA/DMF 25 16 0.2 6 PEG-PIPAAm water 5 12 0.2 6
PVB-BP THF/DMF 25 17 0.1 13
[0136] The morphology of all the nanofibers was investigated using
a Hitachi S-3500N SEM. The fiber samples were mounted on an
aluminum stub using carbon tape and gold sputter-coated before
viewing. The average diameter of the nanofibers was determined
based on the measurements of at least 20 fibers. FIG. 1 shows the
typical SEM images of nanofibers with different photocrosslinker
concentration. The average fiber diameters of 0%, 1%, 5%, and 10%
nanofibers are 208.+-.146 nm, 212.+-.80 nm, 453.+-.146 nm,
315.+-.160 nm, respectively. Highly porous structure was observed
in all four formulations of FIG. 1.
Example 2
Acid Derivatized Nanofibers by Polymer Deposition
[0137] Poly(acrylic acid) (PAA) was used to provide carboxylic
acids on the nanofiber surface. FAA sodium salt with an average
molecular weight of 5 kDa was purchased from Aldrich Chemicals. A
certain amount of photoreactive PCL nanofiber mesh was immersed in
20 ml 50-100 mg/ml PAA aqueous solution in a quartz round dish
(Quartz Scientific, Inc., Fairport Harbor, Ohio). Mild agitation
was applied to remove the air bubbles trapped in the nanofibers. UV
irradiation was then applied to the mixture in a UVP CL-1000
Ultraviolet Cross linker (40 watt, 254 nm, distance from light
source is 12.7 cm). The nanofiber mesh was flipped over and UV
illumination applied again. The coated nanofiber meshes were washed
with deionized water for 24 hours and then dried under vacuum to
constant weight.
Example 3
Amine Derivatized Nanofibers by Polymer Deposition
[0138] Poly(dimethyl acrylamide-co-aminopropyl methacrylamide)
(DMA:APMA. 80/20) was used to provide amino groups on the surface.
The copolymer with an average molecular weight of 5 kDa was
synthesized by free-radical copolymerization of DMA and APMA
hydrochloride. A certain amount of photoreactive PCL nanofiber mesh
was immersed in 20 ml 50 mg/ml PDMA/APMA aqueous solution in a
quarts round dish (Quartz Scientific, Inc., Fairport Harbor, Ohio).
Mild agitation was applied to remove the air bubbles trapped in the
nanofibers. UV irradiation was then applied to the mixture in a UVP
CL-1000 Ultraviolet Crosslinker (40 watt, 254 nm, distance from
light source is 12.7 cm). The nanofiber mesh was flipped over and
UV illumination applied again. The coated nanofiber meshes were
washed with deionized water for 24 hours and then dried under
vacuum to constant weight.
Example 4
Epoxy Derivatized Nanofibers by Polymer Deposition
[0139] Poly(glycidyl methacrylate) (Mw 25,000 Polysciences) was
used to provide epoxy groups on the surface. A certain amount of
photoreactive PCL nanofiber mesh was immersed in 10 ml 50 mg/ml
Poly(glycidyl methacrylate) water/DMSO solution in a quartz round
dish (Quartz Scientific, Inc., Fairport Harbor, Ohio). Mild
agitation was applied to remove the air bubbles trapped in the
nanofibers. UV irradiation was then applied to the mixture in a UVP
CL-1000 Ultraviolet Crosslinker (40 watt, 254 nm, distance from
light source is 12.7 cm). The nanofiber mesh was flipped over and
UV illumination applied again. The coated nanofiber meshes were
washed with deionized water for 24 hours and then dried under
vacuum to constant weight.
Example 5
Acid Derivatized Nanofibers by Self-Assembly Monolayer (SAM)
[0140] SAM acid was used to provide carboxylic acids on the
nanofiber surface. SAM acid was synthesized by ISurTec, Inc. A
certain amount of photoreactive PCL nanofiber mesh was immersed in
1.0 mg/ml aqueous solution of SAM acid in a quartz round dish
(Quartz Scientific, Inc., Fairport Harbor, Ohio). Mild agitation
was applied to remove the air bubbles trapped in the nanofibers. UV
irradiation was then applied to the mixture in a UVP CL-1000
Ultraviolet Crosslinker (40 watt, 254 nm, distance from light
source is 12.7 cm). The nanofiber mesh was flipped over and UV
illumination applied again. The coated nanofiber meshes were washed
with deionized water for 24 hours and then dried under vacuum to
constant weight.
Example 6
Acid or Amine Derivatized Nanofibers by Graft Polymerization
[0141] Preweighed PCL nanofiber meshes were immersed into 20 ml of
50 mg/ml acrylic acid (Aldrich) or 3-aminopropyl methacrylamide
(APMA.HCl, Polysciences) aqueous solution in an amber glass bottle.
The mixture was bubbled with argon for 2 hrs and transferred to a
quartz round dish (Quartz Scientific, Inc., Fairport Harbor, Ohio),
followed by 2 min of UV irradiation (Harland Medical UVM400, Minn.,
distance from light source was 8 inches) on each side of the fiber
mesh. Thereafter, samples were rinsed with distilled water three
times, washed with water overnight and lyophilized.
Example 7
Functionality Characterization
[0142] Functional groups (i.e. carboxy and amino) on the nanofibers
were measured by reversible ionic dye binding. Calibrations were
done with the respective dyes in the solvents used for elution. The
flurescent/UV/vis measurements were performed on a SpectraMax M2
Multi-detection Reader from Molecular Devices.
Carboxy Groups
[0143] PCL nanofiber samples were shaken overnight in 10 ml of 10
mg/l thionin (Aldrich Chemicals) in ethanol at room temperature,
rinsed three times with ethanol for 30 s each, and then immersed in
10 ml of a solution of 0.01 N HCl in a 1:1 mixture of ethanol and
water. After shaking for 1.5 h, fluorescence of the solution was
recorded at 620 nm (excitation 485 nm).
Amino Groups
[0144] PCL nanofiber samples were shaken overnight in a solution of
50 mmol/L Orange II (Aldrich Chemicals) in water (pH 3, HCl) at
room temperature. The samples were washed three times with water
(pH 3) and immersed in 10 ml of water (pH 12, NaOH). After shaking
for 1.5 min, the UV/Vis absorption of the solution was recorded at
479 nm.
[0145] The functional groups on the nanofiber surface were
determined based on 1:1 complexation between functional groups and
dye molecules.
[0146] The functional group density was reported as nmol of
functional groups per mg of nanofibers (FIGS. 2-4). FIG. 2 shows
that PAA deposition on 1%, 5%, and 10% nanofibers yielded carboxy
group densities of 282, 203 and 572 nmol/mg, respectively.
Theoretically, nanofibers with higher crosslinker content should
give higher functional density, given the diameters remain the
same. However, the functional group density on 5% nanofibers was
slightly lower than that of 1% nanofibers, it should be noted that
the same mass of nanofibers with bigger diameter would possess
smaller surface area. Therefore, even though 5% nanofibers had more
crosslinker in total weight, it might have less accessible
photogroups on the fiber surface, leading to a lower density of PAA
on the surface. Using the bulk density of PCL (1.12 g/ml) and the
diameter of the nanofibers determined by SEM, the density of nmol
functional group per mg nanofiber can be converted to number of
functional group per nm.sup.2 fiber surface. Recalculated
functional group densities were 10, 16, and 30 groups/nm.sup.2 for
1%, 5% and 10% nanofibers (Table 2), which are all above 0.1 group
nm.sup.2, the minimum density level we expected. As shown in FIG.
3, the amine density on surfaces created by (80:20) DMA:APMA
deposition was lower than carboxy density generated by PAA
deposition, which was partially due to 20% amination on DMA:APMA
versus 100% carboxylation on PAA. Graft polymerization of APMA to
photoreactive nanofibers gave low amine densities (2 nmol/mg, 8
nmol/mg and 7 nmol/mg), indicating poor grafting efficiency, which
was probably due to the presence of impurities in the monomer APMA.
FIG. 4 shows that all three functionalization methods could
generate a high density of carboxy groups on 1% nanofibers with the
order of carboxy density from high to low being PAA>AA
graft>acid-SAM.
TABLE-US-00003 TABLE 2 Carboxy Group Densities and Photogroup
Content Carboxy Density Carboxy Density Diameter (nmol/mg
(group/nm.sup.2 (nm) nanofibers) fiber surface) 1% Nanofiber 212
282 10 5% Nanofiber 453 203 16 10% Nanofiber 315 572 30
Example 8
Porosity Measurement
[0147] The porosity of the nanofiber meshes was determined by a
liquid displacement method. The mesh sample was immersed in a
graduated cylinder containing V.sub.1 volume of isopropanol (IPA).
A bath sonication is applied to force IPA to enter the pores and
get rid of the air bubbles. After 10 min, the volume is recorded as
V.sub.2. The wetted mesh sample was removed from the cylinder and
the residual IPA volume is V.sub.3. (V.sub.1-V.sub.2) was the
volume of IPA held in the fibers, which represents the volume of
porous space in the fibers, whereas (V.sub.2-V.sub.3) was the total
volume of filter and porous space. Thus the porosity of the filter
was obtained as (V.sub.1-V.sub.3)/(V.sub.2-V.sub.3).
TABLE-US-00004 TABLE 3 Porosity of Nanofiber with and without PAA
Coating No PAA coating PAA coated 0% TriLite 89.9% 1% TriLite .sup.
87% 89% 5% TriLite 87.5% 95.8%.sup. 10% TriLite .sup. 90% 92%
Example 9
Biomolecule Immobilization
[0148] Horse Radish Peroxidase (HRP, PeroxidaseType XII, Sigma) was
immobilized on PCL nanofibers through an EDC/NHS coupling method.
Carboxy-functionalized nanofiber meshes were immersed in a fresh
solution containing 10 mg/ml EDC and 5 mg/ml NHS, in water,
adjusted to pH 4.5. After incubation on a shaker (100 rpm) at
4.degree. C. for 30 min, the activated samples were removed, rinsed
quickly with ice cold water and immediately immersed in protein
solution (5.0 ug/ml, PBS, pH 7.4). After gentle agitation at room
temperature for 2 hours, the nanofibers were removed and rinsed
with PBS, then washed extensively with PBS-0.1% Triton overnight.
The protein immobilized nanofiber was rinsed and analyzed for
protein and activity assays.
Example 10
Bicinchoninic Acid (BCA) Protein Assay
[0149] The protein loading on the nanofibers including the ones for
nonspecific protein adsorption was determined by standard BCA
assay. Preweighed protein conjugated nanofibers were dissolved in 2
ml of 1.0 N NaOH containing 2% SDS overnight at 37.degree. C. The
solution was then neutralized with 1N HCl and 1 ml of the solution
was added to 250 .mu.l 6.1 N TCA solution. After 10 min incubation
at 4.degree. C., the sample was centrifuged at 14 k rpm for 5 min
to form a protein pellet. The pellet was washed with 200 .mu.l cold
acetone twice by centrifugation and dried on a heat block at
95.degree. C. for 5 min. The protein pellet was dissolved in 40
.mu.l of 5% SDS solution in 0.1 N NaOH and 960 .mu.l of distilled
water, then used for protein assay using a BCA assay kit (Pierce,
Rockford, Ill.). Protein loading level was determined as the weight
percentage of immobilized protein per dry weight of nanofibers.
[0150] FIG. 5 shows the protein immobilization levels on 1%
nanofibers through different surface modifications. BSA was used to
construct the calibration curve. PAA modified nanofibers showed the
highest protein immobilization (1.7 .mu.g/mg), followed by AA
grafted nanofibers (1.4 .mu.g/mg) and acid-SAM coated nanofibers
(0.7 .mu.g/mg). The order correlates the order of carboxy density
on 1% nanofibers.
Example 11
Bioactivity of Immobilized Protein
[0151] The bioactivity of immobilized HRP was determined using a
TMB substrate solution. Color development was initiated after 2 ml
substrate solution (KPL) was added to HRP conjugated nanofibers.
After 10 min, sulfuric acid was added to stop the color development
and absorbance at 450 nm was measured. A standard curve of HRP was
used to calculate the bioactivity of immobilized HRP.
[0152] HRP activity was measured by HRP-catalyzed TMB oxidation. As
shown in FIG. 6, HRP conjugated on PAA modified nanofibers showed
highest activity while lower activity was found on acid-SAM coated
and AA grafted nanofibers. Given that the protein level on AA
grafted nanofibers was almost twice as much as that of acid-SAM
coated nanofibers, the similar activity indicates acid-SAM might be
a better spacer candidate for protein conjugation. The activity
difference between PAA deposition and AA grafting suggests the
orientation of PAA chains on the nanofibers could play an important
role in protein activity.
Example 12
Degradation of Photocrosslinked Nanofibers
[0153] Degradation was studied in two degradation buffers: 1) PBS,
pH 7.4; 2) PBS with 50 U/ml Lipase from P. cepacia. The samples for
the degradation study were prepared as follows. After
electrospinning, the fibers were removed from the aluminum
collector by floating them in water to loosen them from the
collector and then lyophilized. The fiber meshes were then
crosslinked under UV irradiation (UVP CL-1000 Ultraviolet
Crosslinker, 40 watt, 254 nm, distance from light source is 5
inches) for 15 min. 40.about.50 mg of nanofiber was placed into a
15 ml centrifuge tube and 10 ml degradation buffer was added. The
tubes were placed on a shaker in a 37.degree. C. incubator. The
samples were withdrawn at predetermined time points, washed three
times with distilled water by centrifugation and dried to constant
weight under vacuum. The experiment was carried out in triplicate.
Degradation was calculated as:
% Weight loss=(M.sub.2-M.sub.1)/M.sub.1.times.100%
where M.sub.2 and M.sub.1 are the mass of nanofibers after and
before degradation.
[0154] The one important feature of degradable polymers as
biomaterials is that they disappear in the body after they have
fulfilled their functions and no second surgery is needed to remove
them. Different applications require different degradation rates.
It is important to understand the degradation behavior of a
material and hopefully control it. The degradation is influenced
not only by the polymer physicochemical properties such as
molecular weight, crystallinity, chain orientation, and other
morphological variables, but also by the environmental conditions.
Two conditions were investigated in the degradation study:
hydrolysis and enzymatic degradation. It is well known that, as a
bulk material, the degradation of PCL is very slow due to its high
hydrophobicity and high degree of crystallinity. Once PCL is
fabricated into nanofibers, it may degrade faster because of a
significant increase of surface area. On the other band,
degradation rates may slow down due to crosslinking of PCL by the
benzophenone groups. The degradation of PCL nanofibers with four
different crosslinker loadings (0%, 1%, 5%, 10% wt/wt) was
conducted in phosphate buffered saline PBS (pH 7.4) and PBS
containing 50 units/ml Lipase. The results showed that after 23
weeks in PBS, 10.66% weight loss was found for PCL nanofibers with
0% crosslinker, whereas no signs of degradation (less than 4%)
showed on nanofibers crosslinked with 1%, 5% and 10% crosslinker.
However in the presence of Lipase, the nanofibers degraded much
faster with 93.6%, 41.0%, 8.6% and 3.7% weight loss for nanofibers
with 0%, 1%, 5% and 10% crosslinker after 24 hrs (FIG. 7). It is
concluded that photocrosslinking greatly affects the degradation of
nanofibers. The degradation rate slowed down with the increased
crosslinker content. It is possible to tune the degradation of
nanofibers by changing the photocrosslinker content, which has
great promise especially when one material is needed for different
applications that require different degradation rates. SEM images
showed that after 5 hrs, significant degradation was observed in 0%
and 1% nanofibers with fiber surfaces becoming rough, while 5% and
10% nanofibers mostly remained intact with fiber surfaces remaining
smooth (FIG. 8).
Example 13
Immobilization of Lysozyme to Photoreactive PCL Nanofibers Using
Direct UV Illumination
[0155] Sixteen nanofiber pieces were cut from larger nanofiber
sheets that were electrospun by ISurTec. The nanofiber sheets were
prepared using four different TriLite (TL) loadings. The TriLite
loadings were: 0%, 1%, 5% and 10%. Eight of the sixteen pieces were
prepared for use in a BCA protein assay, while the other eight
pieces were prepared for an activity assay. Each of the nanofiber
pieces were weighed prior to incubation with lysozyme.
[0156] A lysozyme solution was prepared using lysozyme from chicken
egg white (Amresco, Solon, Ohio) The lysozyme was prepared at 50
mg/ml in dH.sub.20. The nanofibers were incubated in the lysozyme
solution for one hour at room temperature with shaking.
[0157] After the one hour incubation in the lysozyme solution, the
nanofibers were removed from the lysozyme solution and placed on a
piece of Teflon for the UV illumination. The fibers were
illuminated for a total of two minutes (30 seconds per side
.times.2).
[0158] After UV illumination, the nanofibers were placed into new
scintillation vials and washed overnight with two ml of PBS/0.1%
Triton (Sigma-Aldrich, Milwaukee, Wis.) to remove any unbound
lysozyme. The nanofibers were washed at room temperature on the
shaker.
[0159] Following the overnight wash in PBS/0.1% Triton, each of the
nanofiber pieces were rinsed with dH.sub.2O and placed into new
scintillation vials. The nanofiber pieces tor the activity assay
were used immediately for the assay.
[0160] Two ml of a 1N NaOH/2% SDS (Sigma-Aldrich, Milwaukee, Wis.)
solution was added to the nanofibers for the BCA protein assay to
dissolve them. The nanofibers were incubated with the NaOH/SDS
solution overnight at 37.degree. C.
Example 14
Lysozyme Activity
[0161] A. Immobilized Lysozyme Activity Assay:
[0162] An EnzChek.RTM. Lysozyme Assay Kit (Molecular Probes,
Euguene, Oreg.) was used to determine the activity level of the
immobilized lysozyme on the NFs. All of the reagents used for the
assay were prepared according to the kit instructions.
[0163] A standard curve was prepared in a 96 well plate according
to the kit instructions. 1.5 ml of substrate solution (prepared
with kit reagents according to the kit instructions) was added to
each of the scintillation vials containing the nanofiber pieces.
The standards and nanofiber pieces were incubated with the
substrate solution for one hour and 50 minutes at 37.degree. C.
(protected from light).
[0164] After the incubation with the substrate solution, 100 .mu.l
of the supernatant from each nanofiber sample was loaded in
triplicate to the 96 well plate containing the standards and
fluorescence was measured at 518 nm.
[0165] B. BCA Protein Assay:
[0166] 1) Precipitate Lysozyme Using Trichloroacetic Acid (TCA)
[0167] Trichloroacetic acid (Sigma-Aldrich, Milwaukee, Wis.) was
used to precipitate the lysozyme from the solutions containing the
dissolved nanofibers.
[0168] The solutions containing the dissolved nanofibers were
adjusted to pH 2 using 1N HCL and then placed into eppendorf tubes.
TCA was then added to the solutions (1 volume:4 volumes) and the
tubes were placed on ice for 10 minutes.
[0169] After the 10 minute incubation on ice, the tubes were spun
in the microfuge at 14,000 rpm for 5 minutes. The supernatant was
removed, leaving the protein pellet intact.
[0170] Two hundred .mu.l of cold acetone was then added to each
tube to wash the pellet. The tubes were spun again at 14,000 rpm
for 5 minutes and the supernatant was removed. This acetone wash
was repeated twice for a total of three acetone washes.
[0171] After the final acetone wash, the protein pellets were dried
for 10 minutes in a heat block to remove any residual acetone.
[0172] 2) Prepare Protein Samples for BCA Assay
[0173] After drying the protein pellets, forty .mu.l of a 0.2N
NaOH/5% SDS solution was added to each tube to dissolve the
pellets. 960 .mu.l of dH.sub.2O was then added to each tube to
bring the total volume to 1 ml. The protein solutions were
transferred to glass test tubes for the assay.
[0174] 3) Prepare Lysozyme Standard Curve
[0175] A standard curve was prepared using lysozyme (Amresco,
Solon, Ohio) in dH.sub.2O. Ten standards were prepared in glass
test tubes ranging in concentration from 10 .mu.g/ml to 0.0195
.mu.g/ml (1 ml total volume per standard.)
[0176] 4) Incubate Standards and Experimental Samples With BCA
Working Reagent
[0177] A QuantiPro.TM. BCA Assay Kit (Sigma-Aldrich, Milwaukee,
Wis.) was used for the assay. One ml of BCA working reagent
(prepared according to kit instructions) was added to each of the
standards and experimental samples (2 ml total volume per tube).
The standards and samples were then incubated at 37.degree. C. for
three hours. Two hundred .mu.l of the standard and experimental
solutions was loaded in triplicate to a 96 well plate and
absorbance was measured at 562 nm.
[0178] The results confirmed that a significant amount of lysozyme
was conjugated onto PCL nanofibers by direct UV illumination,
however, the immobilized lysozyme showed limited activity,
indicating the loss of activity during direct UV conjugation.
[0179] 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.
* * * * *