U.S. patent application number 10/357131 was filed with the patent office on 2003-09-25 for latent reactive polymers with biologically active moieties.
Invention is credited to Amos, Richard A., Clapper, David L., Everson, Terrence P., Hu, Sheau-Ping, Swanson, Melvin J..
Application Number | 20030181423 10/357131 |
Document ID | / |
Family ID | 25438061 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030181423 |
Kind Code |
A1 |
Clapper, David L. ; et
al. |
September 25, 2003 |
Latent reactive polymers with biologically active moieties
Abstract
A polybifunctional reagent having a polymeric backbone, one or
more pendent photoreactive moieties, and two or more pendent
bioactive groups. The reagent can be activated to form a bulk
material or can be brought into contact with the surface of a
previously formed biomaterial and activated to form a coating. The
pendent bioactive groups function by promoting the attachment of
specific molecules or cells to the bulk material or coated surface.
Bioactive groups can include proteins, peptides, carbohydrates,
nucleic acids and other molecules that are capable of binding
noncovalently to specific and complimentary portions of molecules
or cells.
Inventors: |
Clapper, David L.;
(Shorewood, MN) ; Swanson, Melvin J.; (Carver,
MN) ; Hu, Sheau-Ping; (Falcon Heights, MN) ;
Amos, Richard A.; (St. Anthony, MN) ; Everson,
Terrence P.; (Eagan, MN) |
Correspondence
Address: |
INTELLECTUAL PROPERTY GROUP
FREDRIKSON & BYRON, P.A.
4000 PILLSBURY CENTER
200 SOUTH SIXTH STREET
MINNEAPOLIS
MN
55402
US
|
Family ID: |
25438061 |
Appl. No.: |
10/357131 |
Filed: |
February 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10357131 |
Feb 3, 2003 |
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09591564 |
Jun 9, 2000 |
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6514734 |
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09591564 |
Jun 9, 2000 |
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08916913 |
Aug 15, 1997 |
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6121027 |
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Current U.S.
Class: |
514/100 ;
435/180 |
Current CPC
Class: |
A61L 31/04 20130101;
C12Q 1/56 20130101; G01N 33/6854 20130101; G01N 2333/815 20130101;
A61L 33/0011 20130101; C12N 11/087 20200101; G01N 2333/78 20130101;
A61L 29/04 20130101; G01N 2333/974 20130101; C08J 7/12 20130101;
G01N 33/6845 20130101; G01N 33/54366 20130101; Y10S 530/816
20130101; A61L 31/10 20130101; C12Q 1/6834 20130101; G01N 33/68
20130101; G01N 2333/8128 20130101; A61L 27/14 20130101; Y10S
530/815 20130101; A61L 33/0047 20130101; A61L 29/085 20130101; A61L
27/34 20130101; G01N 33/54353 20130101 |
Class at
Publication: |
514/100 ;
435/180 |
International
Class: |
A61K 031/665 |
Claims
What is claimed is:
1. A polybifunctional reagent comprising a plurality of molecules
each comprising a polymeric backbone bearing (a) one or more
pendent photoreactive moieties capable of being activated by
exposure to a suitable energy source, and (b) two or more pendent
bioactive groups capable of specific, noncovalent interactions with
complimentary groups, the reagent being capable, upon activation of
the photoreactive moieties, of forming a bulk material or surface
coating in order to promote the attraction of such complimentary
groups.
2. A polybifunctional reagent according to claim 1, wherein the
photoreactive moieties can be activated to form intermolecular
covalent bonds between the reagent molecule and a biomaterial
surface in order to form a coating thereon.
3. A polybifunctional reagent according to claim 1, wherein the
photoreactive moieties can be activated to form intermolecular
covalent bonds between adjacent reagent molecules in order to form
a bulk material.
4. A polybifunctional reagent according to claim 1, wherein the
bioactive groups participate in a specific binding reaction with
complementary molecules or cell receptors.
5. A polybifunctional reagent according to claim 4, wherein the
bioactive groups are each, independently, selected from the group
consisting of proteins, peptides, amino acids, carbohydrates, and
nucleic acids, each being capable of binding noncovalently to
specific and complimentary portions of molecules or cells.
6. A polybifunctional reagent according to claim 1, wherein the
bioactive groups each have a known or identifiable complementary
binding partner and are each, independently, selected from the
group consisting of antithrombotic agents, cell attachment factors,
receptors, ligands, growth factors, antibiotics, enzymes and
nucleic acids.
7. A polybifunctional reagent according to claim 6 wherein the
bioactive groups comprise antithrombotic agents selected from the
group consisting of heparin, hirudin, lysine, prostaglandins,
streptokinase, urokinase, and plasminogen activator.
8. A polybifunctional reagent according to claim 6 wherein the
bioactive groups comprise cell attachment factors selected from the
group consisting of surface adhesion molecules and cell-cell
adhesion molecules.
9. A polybifunctional reagent according to claim 8 wherein the
bioactive groups comprise surface adhesion molecules selected from
the group consisting of laminin, fibronectin, collagen,
vitronectin, tenascin, fibrinogen, thrombospondin, osteopontin, von
Willibrand Factor, and bone sialoprotein and active domains
thereof.
10. A polybifunctional reagent according to claim 8 wherein the
bioactive groups comprise cell-cell adhesion molecules selected
from the group consisting of N-cadherin and P-cadherin and active
domains thereof.
11. A polybifunctional reagent according to claim 6 wherein the
bioactive groups comprise growth factors selected from the group
consisting of fibroblastic growth factors, epidermal growth factor,
platelet-derived growth factors, transforming growth factors,
vascular endothelial growth factor, bone morphogenic proteins and
other bone growth factors, and neural growth factors.
12. A polybifunctional reagent according to claim 6 wherein the
bioactive groups comprise a ligand or receptor selected from the
group consisting of antibodies, antigens, avidin, streptavidin,
biotin, heparin, type IV collagen, protein A, and protein G.
13. A polybifunctional reagent according to claim 6 wherein the
bioactive groups comprise an antibiotic selected from the group
consisting of antibiotic peptides.
14. A polybifunctional reagent according to claim 6 wherein the
bioactive groups comprise enzymes.
15. A polybifunctional reagent according to claim 6 wherein the
bioactive groups comprise nucleic acid sequences capable of
selectively binding complimentary nucleic acid sequences.
16. A polybifunctional reagent according to claim 1 wherein the
polymeric backbone comprises a synthetic polymer selected from the
group consisting of addition type polymers, such as the vinyl
polymers.
17. A polybifunctional reagent according to claim 1 wherein the
photogroups each comprise a photoactivatable ketone.
18. A polybifunctional reagent comprising a synthetic polymeric
backbone bearing (a) one or more pendent photoreactive moieties in
the form of photoactivatable ketones, and (b) two or more pendent
bioactive groups selected from the group consisting of proteins,
peptides, amino acids, carbohydrates, nucleic acids and other
molecules that are capable of binding noncovalently to specific and
complimentary portions of molecules or cells.
19. A method of coating a biomaterial surface, the method
comprising the steps of (a) providing a polybifunctional reagent
comprising a polymeric backbone bearing (i) one or more pendent
photoreactive moieties capable of being activated by exposure to a
suitable energy source, and (ii) two or more pendent bioactive
groups capable of specific, noncovalent interactions with
complimentary groups, (b) contacting the surface with the reagent,
and (c) activating the photoreactive moieties in order to crosslink
the reagent molecules to themselves and/or to the surface.
20. A method according to claim 19 wherein the reagent is coated on
the surface by spraying, dipping or brushing.
21. A method for forming a bulk biomaterial, the method comprising
the steps of (a) providing a polybifunctional reagent comprising a
polymeric backbone bearing (i) one or more pendent photoreactive
moieties capable of being activated by exposure to a suitable
energy source, and (ii) two or more pendent bioactive groups
capable of specific, noncovalent interactions with complimentary
groups, and (b) activating the reagent to form a bulk
biomaterial.
22. A coated biomaterial surface, comprising the bound residues of
an activated polybifunctional reagent that initially comprised a
polymeric backbone bearing (a) one or more pendent photoreactive
moieties capable of being activated by exposure to a suitable
energy source, and (b) two or more pendent bioactive groups capable
of specific, noncovalent interactions with complimentary
groups.
23. A bulk material comprising the bound residues of an activated
polybifunctional reagent that initially comprised a polymeric
backbone bearing (a) one or more pendent photoreactive moieties
capable of being activated by exposure to a suitable energy source,
and (b) two or more pendent bioactive groups capable of specific,
noncovalent interactions with complimentary groups.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. Ser. No.
09/591,564, filed Jun. 9, 2000, which is a continuation of U.S.
Ser. No. 08/916,913, filed Aug. 15, 1997, the entire disclosure of
which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] In one aspect, this invention relates to reagents that can
be used to modify biomaterial surfaces or to fabricate new
biomaterials. In another aspect, the invention relates to
biomaterials having surfaces that have been prepared or modified to
provide desired bioactive function.
BACKGROUND OF THE INVENTION
[0003] Biomaterials have long been used to fabricate biomedical
devices for use in both in vitro and in vivo applications. A
variety of biomaterials can be used for the fabrication of such
devices, including ceramics, metals, polymers, and combinations
thereof. Historically, such biomaterials were considered suitable
for use in fabricating biomedical devices if they provided a
suitable combination of such basic properties as inertness, low
toxicity, and the ability to be fabricated into desired devices.
(Hanker, J. S. and B. L. Giammara, Science 242:885-892, 1988).
[0004] As the result of more recent advances, devices can now be
provided with surfaces having various desirable characteristics,
e.g., in order to better interface with surrounding tissue or
solutions. For instance, approaches have been developed to promote
the attachment of specific cells or molecules to device surfaces. A
device surface, for instance, can be provided with a bioactive
group that is capable of attracting and/or attaching to various
molecules or cells. Examples of such bioactive groups include
antigens for binding to antibodies, ligands for binding to cell
surface receptors, and enzyme substrates for binding to
enzymes.
[0005] Such bioactive groups have been provided on the surfaces of
biomaterials in a variety of ways. In one approach, biomaterials
can be fabricated from molecules that themselves present the
desired bioactive groups on the surfaces of devices after
fabrication. However, desirable bioactive groups are typically
hydrophilic and cannot be incorporated into most metals or
hydrophobic polymeric biomaterials at effective concentrations
without disrupting the structural integrity of such
biomaterials.
[0006] An alternative approach involves adding bioactive groups to
the surfaces of biomaterials, e.g., after they have been fabricated
into medical devices. Such bioactive groups can occasionally be
added by adsorption. However, groups that have been added by
adsorption cannot typically be retained on surfaces at high levels
or for long periods of time.
[0007] The retention of such bioactive groups on a surface can be
improved by covalent bonding of those groups to the surface. For
instance, U.S. Pat. Nos. 4,722,906, 4,979,959, 4,973,493 and
5,263,992 relate to devices having biocompatible agents covalently
bound via a photoreactive group and a chemical linking moiety to
the biomaterial surface. U.S. Pat. Nos. 5,258,041 and 5,217,492
relate to the attachment of biomolecules to a surface through the
use of long chain chemical spacers. U.S. Pat. Nos. 5,002,582 and
5,263,992 relate to the preparation and use of polymeric surfaces,
wherein polymeric agents providing desirable properties are
covalently bound via a photoreactive moiety to the surface. In
particular, the polymers themselves exhibit the desired
characteristics, and in the preferred embodiment, are substantially
free of other (e.g., bioactive) groups.
[0008] Others have used photochemistry to modify the surfaces of
biomedical devices, e.g., to coat vascular grafts. (See, e.g.,
Kito, H. et. al., ASAIO Journal 39:M506-M511, 1993. See also
Clapper, D. L., et. al., Trans. Soc. Biomat. 16:42, 1993).
[0009] Cholakis and Sefton synthesized a polymer having a polyvinyl
alcohol (PVA) backbone and heparin bioactive groups. The polymer
was coupled to polyethylene tubing via nonlatent reactive
chemistry, and the resultant surface was evaluated for
thromboresistance in a series of in vitro and in vivo assays. For
whatever reason, the heparin in the polymer prepared by Cholakis
and Sefton did not provide effective activity. (Cholakis, C. H. and
M. V. Sefton, J. Biomed. Mater. Res. 23:399-415, 1989. See also
Cholakis, C. H., et. al., J. Biomed. Mater. Res. 23:417-441,
1989).
[0010] Finally, Kinoshita et. al. disclose the use of reactive
chemistry to generate polyacrylic acid backbones on porous
polyethylene, with collagen molecules being subsequently coupled to
carboxyl moieties on the polyacrylic acid backbones. (See
Kinoshita, Y., et. al., Biomaterials 14:209-215, 1993).
[0011] Generally, the resultant coating in the above-captioned
situations is provided in the form of bioactive groups covalently
coupled to biomaterial surfaces by means of short linear spacers.
This approach works well with large molecular weight bioactive
groups, such as collagen and fibronectin, where the use of short
spacers is desired and the size of the bioactive group is quite
large compared to that of the spacer itself.
[0012] The approaches described above, however, with the possible
exception of Kinoshita et al., are not optimal for coating small
molecular weight bioactive groups. Kinoshita does appear to coat
small molecular weight molecules, although it describes a laborious
multistep process that can detrimentally affect both yield and
reproducibility.
[0013] Small molecular weight bioactive groups are typically
provided in the form of either small regions derived from much
larger molecules (e.g., cell attachment peptides derived from
fibronectin) or as small molecules that normally diffuse freely to
produce their effects (e.g., antibiotics or growth factors). It
appears that short spacers can unduly limit the freedom of movement
of such small bioactive groups, and in turn, impair their activity
when immobilized. What are clearly needed are methods and
compositions for providing improved concentrations of bioactive
groups, and particularly small molecular weight groups, to a
biomaterial surface in a manner that permits improved freedom of
movement of the bioactive groups.
SUMMARY OF THE INVENTION
[0014] The present invention addresses the needs described above by
providing a "polybifunctional" reagent comprising a polymeric
backbone bearing one or more pendent photoreactive moieties and one
or more, and preferably two or more, pendent bioactive groups. The
reagent preferably includes a high molecular weight polymer
backbone, preferably linear, having attached thereto an optimal
density of both bioactive groups and photoreactive moieties. The
reagent permits useful densities of bioactive groups to be coupled
to a biomaterial surface, via one or more photoreactive groups. The
backbone, in turn, provides a spacer function of sufficient length
to provide the bioactive groups with greater freedom of movement
than that which could otherwise be achieved, e.g., by the use of
individual spacers (as described above).
[0015] As an added advantage, the present reagent permits the
formation of inter- and intra-molecular covalent bonds within
and/or between polymer backbones and the biomaterial surface,
thereby providing an optimal and controllable combination of such
properties as coating density, freedom of movement, tenacity and
stability.
[0016] In addition to its use in modifying a biomaterial surface, a
reagent of the invention provides other benefits as well. The
photoreactive moieties allow individual polymer molecules to couple
efficiently (e.g., crosslink) with adjacent polymer molecules. This
crosslinking characteristic allows the polymers to generate thick
coatings upon biomaterial surfaces and/or to generate independent
films and bulk materials, either in vitro or in vivo.
[0017] The present invention also discloses a method for
synthesizing a polybifunctional reagent and for providing a coated
surface, such as the surface of a biomaterial, or biomedical device
fabricated from such a biomaterial. The coated surface, having
molecules of the polybifunctional reagent attached thereto in order
to provide the device with desirable properties or attributes.
[0018] The photoreactive moieties can be activated in order to
attach the polybifunctional reagent to a surface providing
abstractable hydrogen atoms in such a manner that the pendent
bioactive group(s) retain their desired bioactive function.
Preferably, the reagent is attached to the surface in a "one step"
method, that is, by applying a reagent to the surface and there
activating one or more of its photoreactive groups in order to form
a coating. In contrast, a "two step" method would involve a first
step of immobilizing a polymeric backbone via photochemical means,
and a second step of attaching (e.g., thermochemically) one or more
bioactive groups to the immobilized backbone.
[0019] Preferred polybifunctional reagents of the invention can be
used to coat the surfaces of existing biomaterials and/or to
generate new biomaterials, e.g., by the formation of bulk
materials. In either case, they can improve the surface properties
of a biomedical device by providing covalently bound bioactive
groups at the device surface. Preferred bioactive groups, in turn,
act by either noncovalently binding to, or acting upon, specific
complimentary portions of molecules or cells that come into contact
with such groups.
[0020] In one preferred embodiment, a polybifunctional reagent of
the invention is synthesized having a polymeric backbone, one or
more photoreactive moieties, and two or more bioactive groups. The
polymeric molecule of the invention is brought into contact with
the surface of a previously formed biomaterial or into contact with
another polymeric molecule of the invention. The photoreactive
moieties are energized via an external stimulation to form, by
means of active specie generation, a covalent bond between the
reagent molecule and either the biomaterial surface or another
reagent molecule. For instance, a biomaterial can be wetted in a
solution containing a suitable reagent (typically for 0.1-5
minutes) and then exposed to light (typically for 0.1-2 minutes) to
achieve covalent coupling.
[0021] Preferred bioactive groups function by promoting the
attachment of specific molecules or cells to the surface. Preferred
bioactive groups include, but are not limited to, proteins,
peptides, carbohydrates, nucleic acids and other molecules that are
capable of binding noncovalently to specific and complimentary
portions of molecules or cells. Examples of such specific binding
include cell surface receptors binding to ligands, antigens binding
to antibodies, and enzyme substrates binding to enzymes.
Preferably, the polymeric backbone comprises a synthetic polymeric
backbone selected from the group consisting of addition type
polymers, such as the vinyl polymers. More preferably, the
photogroups each comprise a reversibly photoactivatable ketone.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0022] As used herein, the following terms and words will have the
following ascribed meanings:
[0023] "biomaterial" will refer to a material that is substantially
insoluble in aqueous systems and that provides one or more surfaces
for contact with fluids containing biological molecules, e.g., in
vivo or in vitro aqueous systems containing tissues, cells, or
biomolecules;
[0024] "device" will refer to a functional object fabricated from a
biomaterial;
[0025] "coating", when used as a noun, will refer to one or more
polymer layers on a biomaterial surface, and in particular, to one
or more layers immobilized on a biomaterial surface by the
activation of a polybifunctional reagent of the present
invention;
[0026] "polybifunctional reagent", when used in the context of the
presently claimed reagent, will refer to a molecule comprising a
polymer backbone, to which are covalently bonded one or more
photoreactive moieties and two or more bioactive groups;
[0027] "a photoreactive moiety" will refer to a chemical group that
responds to a specific applied external energy source in order to
undergo active specie generation, resulting in covalent bonding to
an adjacent molecule or biomaterial surface;
[0028] "bioactive group" will refer to a molecule having a desired
specific biological activity, such as a binding or enzymatic
(catalytic) activity;
[0029] "polymer backbone" will refer to a natural polymer or a
synthetic polymer, e.g., resulting from addition or condensation
polymerization;
[0030] Preferred reagents of the invention comprise a synthetic
polymer which serves as a backbone, one or more pendent
photoreactive moieties which can be activated to provide covalent
bonding to surfaces or adjacent polymer molecules, and two or more
pendent low molecular weight biologically active moieties
(bioactive groups).
[0031] Backbone. The polymer backbone can be either synthetic or
naturally occurring, and is preferably a synthetic polymer selected
from the group consisting of oligomers, homopolymers, and
copolymers resulting from addition or condensation polymerization.
Naturally occurring polymers, such as polysaccharides and
polypeptides, can be used as well. Preferred backbones are
biologically inert, in that they do not provide a biological
function that is inconsistent with, or detrimental to, their use in
the manner described.
[0032] Such polymer backbones can include acrylics such as those
polymerized from hydroxyethyl acrylate, hydroxyethyl methacrylate,
glyceryl acrylate, glyceryl methacrylate, acrylic acid, methacrylic
acid, acrylamide and methacrylamide; vinyls such as polyvinyl
pyrrolidone and polyvinyl alcohol; nylons such as polycaprolactam;
derivatives of polylauryl lactam, polyhexamethylene adipamide and
polyhexamethylene dodecanediamide, and polyurethanes; polyethers
such as polyethylene oxide, polypropylene oxide, and polybutylene
oxide; and biodegradable polymers such as polylactic acid,
polyglycolic acid, polydioxanone, polyanhydrides, and
polyorthoesters.
[0033] The polymeric backbone is chosen to provide a backbone
capable of beating one or more photoreactive moieties and two or
more bioactive groups. The polymeric backbone is also selected to
provide a spacer between the surface and the various photoreactive
moieties and bioactive groups. In this manner, the reagent can be
bonded to a surface or to an adjacent reagent molecule, to provide
the bioactive groups with sufficient freedom of movement to
demonstrate optimal activity. The polymer backbones are preferably
water soluble, with polyacrylamide and polyvinylpyrrolidone being
particularly preferred polymers.
[0034] Photoreactive moieties. Polybifunctional reagents of the
invention carry one or more pendent latent reactive (preferably
photoreactive) moieties covalently bonded to the polymer backbone.
Photoreactive moieties are defined herein, and preferred moieties
are sufficiently stable to be stored under conditions in which they
retain such properties. See, e.g., U.S. Pat. No. 5,002,582, the
disclosure of which is incorporated herein by reference. Latent
reactive moieties can be chosen that are responsive to various
portions of the electromagnetic spectrum, with those responsive to
ultraviolet and visible portions of the spectrum (referred to
herein as "photoreactive") being particularly preferred.
[0035] Photoreactive moieties respond to specific applied external
stimuli to undergo active specie generation with resultant covalent
boding to an adjacent chemical structure, e.g., as provided by the
same or a different molecule. Photoreactive moieties are those
groups of atoms in a molecule that retain their covalent bonds
unchanged under conditions of storage but that, upon activation by
an external energy source, form covalent bonds with other
molecules.
[0036] The photoreactive moieties generate active species such as
free radicals and particularly nitrenes, carbenes, and excited
states of ketones upon absorption of external electric,
electromagnetic or kinetic (thermal) energy. Photoreactive moieties
may be chosen to be responsive to various portions of the
electromagnetic spectrum, and photoreactive moieties that are
responsive to e.g., ultraviolet and visible portions of the
spectrum are preferred and are referred to herein occasionally as
"photochemical" moiety.
[0037] Photoreactive aryl ketones are particularly preferred, such
as acetophenone, benzophenone, anthraquinone, anthrone, and
anthrone-like heterocycles (i.e., heterocyclic analogues of
anthrone such as those having N, O, or S in the 10- position), or
their substituted (e.g., ring substituted) derivatives. The
functional groups of such ketones are preferred since they are
readily capable of undergoing the
activation/inactivation/reactivation cycle described herein.
Benzophenone is a particularly preferred photoreactive moiety,
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.
Photoactivatable 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. Hence, photoreactive aryl ketones are
particularly preferred.
[0038] The azides constitute a preferred class of latent reactive
moieties and include arylazides (C.sub.6R.sub.5N.sub.3) such as
phenyl azide and particularly 4-fluoro-3-nitrophenyl azide, acyl
azides (--CO--N.sub.3) such as benzoyl azide and p-methylbenzoyl
azide, azido formates (--O--CO--N.sub.3) such as ethyl
azidoformate, phenyl azidoformate, sulfonyl azides
(--SO.sub.2--N.sub.3) such as benzenesulfonyl azide, and phosphoryl
azides (RO).sub.2PON.sub.3 such as diphenyl phosphoryl azide and
diethyl phosphoryl azide. Diazo compounds constitute another class
of photoreactive moieties and include diazoalkanes (--CHN.sub.2)
such as diazomethane and diphenyldiazomethane, diazoketones
(--CO--CHN.sub.2) such as diazoacetophenone and
1-trifluoromethyl-1-diazo-2-pentanone, diazoacetates
(--O--CO--CHN.sub.2) such as t-butyl diazoacetate and phenyl
diazoacetate, and beta-keto-alpha-diazoacetates
(--CO--CN.sub.2--CO--O--) such as t-butyl alpha diazoacetoacetate.
Other photoreactive moieties include the aliphatic azo compounds
such as azobisisobutyronitrile, the diazirines (--CHN.sub.2) such
as 3-trifluoromethyl-3-phenyldiazirine, the ketenes
(--CH.dbd.C.dbd.O) such as ketene and diphenylketene.
[0039] Upon activation of the photoreactive moieties, the coating
adhesion molecules are covalently bound to each other and/or to the
material surface by covalent bonds through residues of the
photoreactive groups. Exemplary photoreactive groups, and their
residues upon activation, are shown as follows.
1 Photoreactive Group Residue Functionality aryl azides amine
R--NH--R' acyl azides amide R--CO--NH--R' azidoformates carbamate
R--O--CO--NH--R' sulfonyl azides sulfonamide R--SO.sub.2--NH--R'
phosphoryl azides phosphoramide (RO).sub.2PO--NH--R' diazoalkanes
new C--C bond diazoketones new C--C bond and ketone diazoacetates
new C--C bond and ester beta-keto-alpha-diazoacetates new C--C bond
and beta-ketoester aliphatic azo new C--C bond diazirines new C--C
bond ketenes new C--C bond photoactivated ketones new C--C bond and
alcohol
[0040] Bioactive Groups. Low molecular weight bioactive groups of
the present invention are typically those that are intended to
enhance or alter the function or performance of a particular
biomedical device in a physiological environment. In a particularly
preferred embodiment, the bioactive group is selected from the
group consisting of cell attachment factors, growth factors,
antithrombotic factors, binding receptors, ligands, enzymes,
antibiotics, and nucleic acids. A reagent molecule of this
invention includes at least one pendent bioactive group. The use of
two or more pendent bioactive groups is presently preferred,
however, since the presence of several such groups per reagent
molecule tends to facilitate the use of such reagents.
[0041] Desirable cell attachment factors include attachment
peptides (defined below), as well as large proteins or
glycoproteins (typically 100-1000 kilodaltons in size) which in
their native state can be firmly bound to a substrate or to an
adjacent cell, bind to a specific cell surface receptor, and
mechanically attach a cell to the substrate or to an adjacent cell.
Naturally occurring attachment factors are primarily large
molecular weight proteins, with molecular weights above 100,000
daltons.
[0042] Attachment factors bind to specific cell surface receptors,
and mechanically attach cells to the substrate (referred to as
"substrate adhesion molecules" herein) or to adjacent cells
(referred to as "cell-cell adhesion molecules" herein) [Alberts, B.
et. al., Molecular Biology of the Cell 2nd ed., Garland Publ.,
Inc., New York (1989)]. In addition to promoting cell attachment,
each type of attachment factor can promote other cell responses,
including cell migration and differentiation. Suitable attachment
factors for the present invention include substrate adhesion
molecules such as the proteins laminin, fibronectin, collagens,
vitronectin, tenascin, fibrinogen, thrombospondin, osteopontin, von
Willibrand Factor, and bone sialoprotein. Other suitable attachment
factors include cell-cell adhesion molecules ("cadherins") such as
N-cadherin and P-cadherin.
[0043] Attachment factors useful in this invention typically
comprise amino acid sequences or functional analogues thereof that
possess the biological activity of a specific domain of a native
attachment factor, with the attachment peptide typically being
about 3 to about 20 amino acids in length. Native cell attachment
factors typically have one or more domains that bind to cell
surface receptors and produce the cell attachment, migration, and
differentiation activities of the parent molecules. These domains
consist of specific amino acid sequences, several of which have
been synthesized and reported to promote the attachment, spreading
and/or proliferation of cells. These domains and functional
analogues of these domains are termed "attachment peptides".
[0044] Examples of attachment peptides from fibronectin include,
but are not limited to, RGD (arg-gly-asp) [Kleinman, H. K, et.al.,
Vitamins and Hormones 47:161-186, 1993], REDV (arg-glu-asp-val)
[Hubbell, J. A., et. al., Ann. N.Y. Acad. Sci. 665:253-258, 1992],
and C/H-V (WQPPRARI or trp-gln-pro-pro-arg-ala-arg-ile) [Mooradian,
D. L., et. al., Invest. Ophth. & Vis. Sci. 34:153-164,
1993].
[0045] Examples of attachment peptides from laminin include, but
are not limited to, YIGSR (tyr-ile-gly-ser-arg) and SIKVAV
(ser-ile-lys-val-ala-val) [Kleinman, H. K, et. al., Vitamins and
Hormones 47:161-186, 1993] and F-9 (RYVVLPRPVCFEKGMNYTVR or
arg-tyr-val-val-leu-pro-arg-pro-val-cys-phe-glu-lys-gly-met-asn-tyr-thr-v-
al-arg) [Charonis, A. S., et. al., J. Cell Biol. 107:1253-1260,
1988].
[0046] Examples of attachment peptides from type IV collagen
include, but are not limited to, HEP-III (GEFYFDLRLKGDK or
gly-glu-phe-tyr-phe-asp-leu- -arg-leu-lys-gly-asp-lys) [Koliakos,
G. G, et. al., J. Biol. Chem. 264:2313-2323, 1989]. Desirably,
attachment peptides used in this invention have between about 3 and
about 30 amino acid residues in their amino acid sequences.
Preferably, attachment peptides have not more than about 15 amino
acid residues in their amino acid sequences.
[0047] Other desirable bioactive groups present in the invention
include growth factors, such as fibroblastic growth factors,
epidermal growth factor, platelet-derived growth factors,
transforming growth factors, vascular endothelial growth factor,
bone morphogenic proteins and other bone growth factors, neural
growth factors, and the like.
[0048] Yet other desirable bioactive groups present in the
invention include antithrombotic agents that inhibit thrombus
formation or accumulation on blood contacting devices. Desirable
antithrombotic agents include heparin and hirudin (which inhibit
clotting cascade proteins such as thrombin) as well as lysine.
Other desirable antithrombotic agents include prostaglandins such
as PGI.sub.2, PGE.sub.1, and PGD.sub.2, which inhibit platelet
adhesion and activation. Still other desirable antithrombotic
agents include fibrinolytic enzymes such as streptokinase,
urokinase, and plasminogen activator, which degrade fibrin clots.
Another desirable bioactive group consists of lysine, which binds
specifically to plasminogen, which in turn degrades fibrin
clots.
[0049] Other desirable bioactive groups present in the invention
include binding receptors, such as antibodies and antigens.
Antibodies present on a biomaterial surface can bind to and remove
specific antigens from aqueous media that comes into contact with
the immobilized antibodies. Similarly, antigens present on a
biomaterial surface can bind to and remove specific antibodies from
aqueous media that comes into contact with the immobilized
antigens.
[0050] Other desirable bioactive groups consist of receptors and
their corresponding ligands. For example, avidin and streptavidin
bind specifically to biotin, with avidin and streptavidin being
receptors and biotin being a ligand. Similarly, fibroblastic growth
factors and vascular endothelial growth factor bind with high
affinity to heparin, and transforming growth factor beta and
certain bone morphogenic proteins bind to type IV collagen. Also
included are immunoglobulin specific binding proteins derived from
bacterial sources, such as protein A and protein G, and synthetic
analogues thereof.
[0051] Yet other desirable bioactive groups present in the
invention include enzymes that can bind to and catalyze specific
changes in substrate molecules present in aqueous media that comes
into contact with the immobilized enzymes. Other desirable
bioactive groups consist of nucleic acid sequences (e.g., DNA, RNA,
and cDNA), which selectively bind complimentary nucleic acid
sequences. Surfaces coated with specific nucleic acid sequences are
used in diagnostic assays to identify the presence of complimentary
nucleic acid sequences in test samples.
[0052] Still other desirable bioactive groups present in the
invention include antibiotics that inhibit microbial growth on
biomaterial surfaces. Certain desirable antibiotics may inhibit
microbial growth by binding to specific components on bacteria. A
particularly desirable class of antibiotics are the antibiotic
peptides which seem to inhibit microbial growth by altering the
permeability of the plasma membrane via mechanisms which, at least
in part, may not involve specific complimentary ligand-receptor
binding [Zazloff, M., Curr. Opinion Immunol. 4:3-7, 1992].
[0053] Biomaterials. Preferred biomaterials include those formed of
synthetic polymers, including oligomers, homopolymers, and
copolymers resulting from either addition or condensation
polymerizations. Examples of suitable addition polymers include,
but are not limited to, acrylics such as those polymerized from
methyl acrylate, methyl methacrylate, hydroxyethyl methacrylate,
hydroxyethyl acrylate, acrylic acid, methacrylic acid, glyceryl
acrylate, glyceryl methacrylate, methacrylamide, and acrylamide;
vinyls such as ethylene, propylene, styrene, vinyl chloride, vinyl
acetate, vinyl pyrrolidone, and vinylidene difluoride. Examples of
condensation polymers include, but are not limited to, nylons such
as polycaprolactam, polylauryl lactam, polyhexamethylene adipamide,
and polyhexamethylene dodecanediamide, and also polyurethanes,
polycarbonates, polyamides, polysulfones, poly(ethylene
terephthalate), polylactic acid, polyglycolic acid,
polydimethylsiloxanes, and polyetheretherketone.
[0054] Certain natural materials are also suitable biomaterials,
including human tissue such as bone, cartilage, skin and teeth; and
other organic materials such as wood, cellulose, compressed carbon,
and rubber.
[0055] Other suitable biomaterials are composed of substances that
do not possess abstractable hydrogens to which the photogroups can
form covalent bonds. One such class of biomaterials can be made
suitable for coating via photochemistry by applying a suitable
primer coating which bonds to the biomaterial surface and provides
a suitable substrate for binding by the photogroups. A subset of
this group includes metals and ceramics which have oxide groups on
their surfaces and are made suitable for coupling via
photochemistry by adding a primer coating that binds to the oxide
groups and provides abstractable hydrogens. The metals include, but
are not limited to, titanium, stainless steel, and cobalt chromium.
The ceramics include, but are not limited to, silicon nitride,
silicon carbide, zirconia, and alumina, as well as glass, silica,
and sapphire. One suitable class of primers for metals and ceramics
consists of organosilane reagents, which bond to the oxide surface
and provide hydrocarbon groups (Brzoska, J. B., et. al., Langmuir
10:4367-4373, 1994). The investigators have also discovered that
--SiH groups are suitable alternatives for bonding of
photogroups.
[0056] A second class of biomaterials that require an organic
primer are the noble metals, which include gold, silver, copper,
and platinum. Functional groups with high affinity to noble metals
include --CN, --SH, and --NH.sub.2, and organic reagents with these
functional groups are used to apply organic monolayers onto such
metals (Grabar, K. C., et. al., Anal. Chem. 67:735-743, 1995).
[0057] Another class of biomaterials that do not possess
abstractable hydrogens are fibrous or porous. The invention
polymers form covalently crosslinked polymer networks that fill the
pores or form films around individual fibers and are therefore
physically entrapped. Expanded polytetrafluoroethylene is such a
biomaterial.
[0058] Biomaterials can be used to fabricate a number of devices
capable of being coated with bioactive groups using a
polybifunctional reagent of the present invention. Implant devices
are one general class of suitable devices, and include, but are not
limited to, vascular devices such as grafts, stents, catheters,
valves, artificial hearts, and heart assist devices; orthopedic
devices such as joint implants, fracture repair devices, and
artificial tendons; dental devices such as dental implants and
fracture repair devices; ophthalmic devices such as lenses and
glaucoma drain shunts; and other catheters, synthetic prostheses
and artificial organs. Other suitable biomedical devices include
dialysis tubing and membranes, blood oxygenator tubing and
membranes, blood bags, sutures, membranes, cell culture devices,
chromatographic support materials, biosensors, and the like.
[0059] Preparation of Reagents. Those skilled in the art, given the
present teaching, will appreciate the manner in which reagents of
the present invention can be prepared using conventional techniques
and materials. In one preferred method, a polymer backbone is
prepared by the copolymerization of a base monomer, such as
acrylamide or N-vinylpyrrolidone, with monomers having pendent
photoreactive and/or thermochemically reactive groups. The polymers
prepared by this copolymerization are then derivatized with the
bioactive molecule by reaction through the thermochemically
reactive groups. An example of such a coupling is the reaction
between an N-oxysuccinimide (NOS) ester on the polymeric backbone
with an amine group on the bioactive molecule.
[0060] An alternative preferred method involves the preparation of
monomers that contain the desired bioactive group as well as a
polymerizable function, such as a vinyl group. Such monomers can
then be copolymerized with monomers containing photoreactive groups
and with a base monomer such as acrylamide or
N-vinylpyrrolidone.
[0061] A preferred procedure used to synthesize latent reactive
peptide polymers involves the synthesis of N-substituted
methacrylamide monomers containing each peptide (peptide monomer)
and a methacrylamide monomer containing a substituted benzophenone
(4-benzoylbenzoic acid, BBA). The peptide monomers were prepared by
reacting the sulfhydryl moiety of each peptide with the maleimide
moiety of N-[3-(6-maleimidylhexanamido)propyl]- methacrylamide (Mal
MAm). Then, each peptide monomer was copolymerized with acrylamide
and the monomer containing BBA (BBA-APMA) to produce the final
latent reactive peptide polymer.
[0062] Various parameters can be controlled to provide reagents
having a desired ratio (whether on a molar or weight basis) of
polymeric backbone, photoreactive moeities and bioactive groups.
For instance, the backbone itself will typically provide between
about 40 and about 400 carbon atoms per photoreactive group, and
preferably between about 60 and about 300 carbon atoms.
[0063] With respect to the bioactive group, the length of the
backbone can vary depending on such factors as the size of the
bioactive group and the desired coating density. For instance, for
relatively small bioactive groups (MW less than 3000) the polymeric
backbone will typically be in the range of about 5 to about 200
carbon atoms per bioactive group, and preferably between about 10
and about 100. For larger bioactive groups, such as those having a
molecular weight between about 3000 and about 50,000, the preferred
backbone provides, on the average, between about 10 and about 5000
carbon atoms between bioactive group, and preferably between about
50 and 1000 carbon atoms. In each case, those skilled in the art,
given the present description, will be able to determine the
conditions suitable to provide an optimal combination of bioactive
group density and freedom of movement.
[0064] Coating method. Reagents of the present invention can be
coated onto biomaterial surfaces using techniques (e.g., dipping,
spraying, brushing) within the skill of those in the relevant art.
In a preferred embodiment, the polybifunctional reagent is first
synthesized and then brought into contact (i.e., sufficient
proximity to permit binding) with a previously formed biomaterial.
The photoreactive group is energized via an external stimulation
(e.g., exposure to a suitable light source) to form, via free
active specie generation, a covalent bond between the reagent and
either another polybifunctional reagent molecule or the biomaterial
surface. This coating method is herein termed the "one step coating
method", since photoreactive coupling chemistry attaches an
invention polymer to a biomaterial surface, and no subsequent steps
are required to add the bioactive group. The external stimulation
that is employed desirably is electromagnetic radiation, and
preferably is radiation in the ultraviolet, visible or infrared
regions of the electromagnetic spectrum.
[0065] Photoactivatible polymers of the invention can also be used
to immobilize biomoieties in patterns on the surfaces of
biomaterials, for example using techniques previously described for
generating patterns of coating with features of 50-350 mm in size.
(See, Matsuda, T. and T. Sugawara, J. Biomed. Mater. Res.
29:749-756 (1995)). For example, hydrophilic patterns that inhibit
the attachment and growth of endothelial cells can be generated by:
1) synthesizing latent reactive hydrophilic polymers, 2) adding the
latent reactive polymers to tissue culture polystyrene plates, 3)
illuminating the polymers through a pattern photomask, and 4)
removing nonimmobilized polymers by washing with an appropriate
solvent.
[0066] Such an approach can be employed with polymers of the
present invention in order to immobilize patterns of specific
biomoieties. For example, microarrays of specific binding molecules
(e.g., antibodies, antigens/haptens, nucleic acid probes, etc.) can
be immobilized on optical, electrochemical or semiconductor sensor
surfaces to provide simultaneous multianalyte assay capabilities or
multiple sensitivity range assays for single analytes. Patterned
immobilization also provides a useful tool for developing a
"laboratory on a chip," in which sequential processing/reaction
steps occur along a fluid movement path in a multistep microvolume
assay system. Patterning of cell attachment factors, for instance,
those that promote the attachment of neural cells to electrodes,
will permit the development of: 1) new generations of
ultrasensitive biosensors and 2) artificial limbs that are directly
controlled by the patient's nervous system.
[0067] Reagents of the invention can be covalently coupled to
previously formed biomaterials to serve as surface coatings. The
present reagent molecules can also be covalently coupled to
adjacent molecules, in order to form films or bulk biomaterials.
The surface coatings, films, and bulk biomaterials resulting from
coupling via photoreactive moieties provide useful densities of
bioactive groups on the surface of the resultant biomaterials.
[0068] Use of devices. Bioactive polymers of the present invention
are used to modify the surfaces of existing biomaterials or to
generate new biomaterials. Biomedical devices that contain the
resultant biomaterials are used for a variety of in vitro and in
vivo applications. For example, biomedical devices possessing cell
attachment groups or growth factors as biomoieties promote the
attachment and/or growth of cells on in vitro cell culture devices
and improve tissue integration with implant devices such as
vascular grafts, orthopedic implants, dental implants, cornea
lenses, and breast implants. Biomedical devices possessing
antithrombotic factors as biomoieties prevent thrombosis on the
surfaces of blood contacting devices, such as catheters, heart
valves, vascular stents, vascular grafts, stent grafts, artificial
hearts, and blood oxygenators.
[0069] Biomedical devices such as resins or membranes possessing
receptors or ligands as biomoieties can be used for affinity
purification of a broad range of biomolecules. For example, heparin
(which is also an antithrombotic moiety) is used to specifically
bind and purify several clotting factors, protease inhibitors,
lipoproteins, growth factors, lipolytic enzymes, extracellular
matrix proteins and viral coat proteins. Staphylococcal Protein A
specifically binds immunoglobulins and has proven to be very useful
for purification of antibodies. Streptavidin is a protein that
binds specifically to biotin with extremely high affinity.
Streptavidin and biotin are a very useful pair of reagents as a
secondary binding pair in diagnostic assays. Many times signal
amplification, enhanced sensitivity and faster test performance can
be achieved by using immobilized streptavidin.
[0070] Biomedical devices having surface-coated antibodies or
antigens can be used in diagnostic tests that depend on the
specificity of binding for sensitive detection of the complimentary
antigen or antibody. The antibodies or antigens can be immobilized
onto membranes, plastic tubes, microplate wells or solid state
biosensor devices. Immobilized antibodies are also important for
purification of a variety of biopharmaceutical agents. Proteins
produced in bacteria or fungi by genetic engineering techniques can
be purified by affinity purification with immobilized antibodies.
Blood fractions, such as clotting factor VIII (antihemophiliac
factor) are also purified by immobilized antibodies.
[0071] Biomedical devices having surfaces coated with nucleic acid
sequences can be used to selectively bind complimentary nucleic
acid sequences. Such devices are used in diagnostic assays to
identify the presence of complimentary nucleic acid sequences in
test samples. Devices having surface-coated enzymes as biomoieties
can be used for a broad range of enzyme reactors, to catalyze
either synthetic processes (e.g., making chiral pharmaceuticals) or
degradative/conversion processes (e.g., degrading starch and
converting glucose to fructose for making high fructose corn
syrup).
[0072] Coated antimicrobial agents can be used to inhibit bacterial
growth on the surfaces of devices. Such antimicrobial surfaces can
reduce the rate of infections associated with implant devices,
including several types of catheters (intravascular, peritoneal,
hemodialysis, hydrocephalus, and urological), arteriovenous shunts,
heart valves, vascular grafts, tracheotomy tubes, orthopedic and
penile implants. Several in vitro devices can also benefit from
such surfaces, e.g., by inhibiting biofilm formation. These include
contact lens cases, dental unit water lines, plumbing used in food
and pharmaceutical industries, food packaging, table tops and other
surfaces used for food handling, and air filters.
EXAMPLES
[0073] The invention will be further described with reference to
the following nonlimiting 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. Unless otherwise indicated, all percentages are by
weight.
Example 1
Peptide Polymers
[0074] A. Synthesis of 4-Benzoylbenzoyl Chloride (BBA-Cl)
[0075] 4-Benzoylbenzoic acid (BBA), 200.0 g (0.884 moles), was
added to a dry 2 liter round bottom flask, followed by the addition
of 273 ml of thionyl chloride. Dimethylformamide (DMF), 684 ul, was
then added and the mixture was refluxed for 3-4 hours. After
cooling, the excess thionyl chloride was removed on a rotary
evaporator at water aspirator pressure. Any remaining thionyl
chloride was removed by repeated evaporation with 3.times.100 ml of
toluene. The final product was then recrystallized from 5:1 hexane:
toluene with typical yields of BBA-Cl at >90% and a melting
point of 92-94.degree. C.
[0076] B. Synthesis of
N-[3-(4-Benzoylbenzamido)propyl]methacrylamide (BBA-APMA)
[0077] N-(3-Aminopropyl)methacrylamide hydrochloride (APMA-HCl, 120
g, 0.672 moles), from Eastman Kodak Co., Rochester, N.Y.) were
added to a dry 2 liter, three-neck round bottom flask equipped with
an overhead stirrer. Phenothiazine, 23-25 mg, was added as an
inhibitor, followed by 800 ml of chloroform. The suspension was
cooled below 10.degree. C. on an ice bath and 172.5 g (0.705 moles)
of BBA-Cl were added as a solid. Triethylamine, 207 ml (1.485
moles), in 50 ml of chloroform was then added dropwise over a 1-1.5
hour time period. The ice bath was removed and stirring at ambient
temperature was continued for 2.5 hours. The product was then
washed with 600 ml of 0.3 N HCl and 2.times.300 ml of 0.07 N HCl.
After drying over sodium sulfate, the chloroform was removed under
reduced pressure and the product was recrystallized twice from 4:1
toluene: chloroform using 23-25 mg of phenothiazine in each
recrystallization to prevent polymerization. Typical yields of
BBA-APMA were 90% with a melting point of 147-1510.degree. C.
[0078] C. Synthesis of
N-[3-(6-Maleimidohexanamido)propyl]methacrylamide (Mal-MAm)
[0079] 6-Maleimidohexanoic acid was prepared by dissolving
6-aminohexanoic acid (100.0 g, 0.762 moles) in 300 ml of acetic
acid in a three-neck, 3 liter flask equipped with an overhead
stirrer and drying tube. Maleic anhydride, 78.5 g (0.801 moles),
was dissolved in 200 ml of acetic acid and added to the
6-aminohexanoic acid solution. The mixture was stirred one hour
while heating on a boiling water bath, resulting in the formation
of a white solid. After cooling overnight at room temperature, the
solid was collected by filtration and rinsed with 2.times.50 ml of
hexane. Typical yield of the (Z)-4-oxo-5-aza-2-undecendioic acid
was 90-95% with a melting point of 160-165.degree. C.
[0080] (Z)-4-Oxo-5-aza-2-undecendioic acid, 150.0 g (0.654 moles),
acetic anhydride, 68 ml (73.5 g, 0.721 moles), and phenothiazine,
500 mg, were added to a 2 liter three-neck round bottom flask
equipped with an overhead stirrer. Triethylamine (TEA), 91 ml
(0.653 moles), and 600 ml of tetrahydrofuran (THF) were added and
the mixture was heated to reflux while stirring. After a total of 4
hours of reflux, the dark mixture was cooled to <60.degree. C.
and poured into a solution of 250 ml of 12 N HCl in 3 liters of
water. The mixture was stirred 3 hours at room temperature and then
was filtered through a filtration pad (Celite 545, J. T. Baker,
Jackson, Tenn.) to remove solids. The filtrate was extracted with
4.times.500 ml of chloroform and the combined extracts were dried
over sodium sulfate. After adding 15 mg of phenothiazine to prevent
polymerization, the solvent was removed under reduced pressure. The
6-maleimidohexanoic acid was recrystallized from 2:1 hexane:
chloroform to give typical yields of 55-60% with a melting point of
81-85.degree. C.
[0081] The N-oxysuccinimide ester (NOS) of 6-maleimidohexanoic acid
was prepared by dissolving 1.0 g (4.73 mmole) of
6-maleimidohexanoic acid and 0.572 g (4.97 mmole) of
N-hydroxysuccinimide (NHS) in 10 ml of dry dioxane, followed by the
addition of 1.074 g (5.21 mmole) of 1,3-dicyclohexylcarbodiimide
(DCC). The reaction mixture was allowed to stir overnight at room
temperature. The 1,3-dicyclohexylurea byproduct was removed by
filtration and the filter cake was rinsed with 3.times.10 ml of
dioxane. Phenothiazine (0.2 mg) was added and the solution was
evaporated under reduced pressure. The resulting solid was
extracted with hexane to remove any excess DCC and this product was
used without any additional purification.
[0082] The N-succimimidyl 6-maleimidohexanoate, 414 mg (1.34
mmole), and N-(3-aminopropyl)methacrylamide hydrochloride, 200 mg
(1.12 mmole), were diluted with 10 ml of chloroform, followed by
the addition of 153 .mu.l (1.10 mmole) of TEA over a 1 hour period
at room temperature. The mixture was allowed to stir overnight at
room temperature. The product was isolated by evaporation and
purified by silica gel flash chromatography using a 99:1, followed
by a 97:3 chloroform : methanol gradient. Pooling of fractions,
addition of 10 mg p-methoxyphenol, and evaporation of solvent gave
261 mg of product. Mass spectral analysis of a sample gave
M.sup.+=335 (10.7%) and NMR showed maleimide (6.6 ppm) and allylic
methyl (2.0 ppm) proton peaks.
[0083] D. Synthesis of Peptide Monomers
[0084] Five peptides were used as biomoieties. Each peptide moiety
was synthesized by standard solid-phase synthesis methods and is
identified below by its common name, a representative literature
citation, the parent protein from which it was identified, and the
specific sequence used (indicated by standard single letter
notation for identifying amino acids).
2 Common name Literature citation Parent protein Sequence used RGD
Kleinman, et al.sup.1 fibronectin CKKGRGDSPAF C/H-V Mooradian, et
al.sup.2 fibronectin CKKWQPPRARI C/H-II McCarthy, et al.sup.3
fibronectin CKNNQKSEPLIGRKKT F-9 Charonis, et al.sup.4 laminin
RYVVLPRPVCFEKK HEP-III Koliakos, et al.sup.5 type IV collagen
CKGEFYFDLRLKGDK .sup.1Kleinman, H. K, et. al., Vitamins and
Hormones 47:161-186 (1993). .sup.2Mooradian, D. L., et. al.,
Invest. Ophth. & Vis. Sci. 34:153-164 (1993). .sup.3McCarthy,
J. B., et. al., Biochem. 27:1380-1388 (1988). .sup.4Charonis, A.
S., et. al., J. Cell Biol. 107:1253-1260 (1988). .sup.5Koliakos, G.
G, et. al., J. Biol. Chem. 264:2313-2323 (1989).
[0085] For each peptide sequence, the portion of the sequence that
is not underlined represents the native sequence that is present in
the parent protein. The portion of the sequence that is underlined
represents amino acids that were added to provide specific
functional groups. The lysine residues (K) were added to provide
primary amines (epsilon amino groups) that were used for
radiolabelling via reductive methylation. Cysteine residues (C)
were added to provide sulfhydryl groups that were used for coupling
each peptide to maleimide groups present on monomers that were
subsequently polymerized to produce the peptide polymers. C/H-II
contained sufficient lysine residues in its native sequence and did
not require the addition of additional lysine residues; similarly
F-9 contained a cysteine residue as part of its native sequence and
did not require the addition of an additional cysteine residue.
[0086] An appropriate quantity of Mal-MAm was removed from a stock
solution of Mal-MAm in chloroform and was placed in a reaction
vial, dried under nitrogen stream, and redissolved in
dimethylsulfoxide (DMSO). An equal molar amount of each peptide was
dissolved in degassed 50 mM acetate buffer (pH 5), added to the
reaction vial, and the mixture was stirred for 60-90 minutes at
room temperature.
3 Peptide Mal-MAm DMSO peptide acetate buffer reaction time type
(.mu.mole) (ml) (.mu.mole) (ml) (min.) RGD 53.4 2 53.4 10 90 F-9
40.4 1 40.4 7 60 C/H-V 8.6 0.2 8.6 1.3 90 C/H-II 38 2 38 10 90
HEP-III 6.4 0.3 6.4 2.7 90
[0087] E. Synthesis of Photoreactive Polyacrylamides Using Peptide
Monomers (Peptide Polymers)
[0088] BBA-APMA was dissolved at a concentration of 10 mg/ml in
DMSO, and acrylamide was dissolved at a concentration of 100 mg/ml
in water. The peptide monomers were not purified after being
synthesized and remained dissolved in solutions of acetate buffer
containing DMSO as described above. The appropriate molar amounts
of BBA-APMA monomer and acrylamide were then added to each reaction
vial. Each mixture was degassed by water aspiration for 15 minutes.
Ammonium persulfate (10% stock solution in water) and
N,N,N',N'-tetramethylethylenediamine (TEMED) were added (in the
amounts indicated below) to catalyze the polymerizations. Each
mixture was degassed again and incubated overnight at room
temperature in a sealed dessicator. Each resultant peptide
copolymer was dialyzed against water (using Spectra/Por 50,000 MWCO
dialysis tubing from Spectrum Medical Industries, Houston, Tex.) at
4.degree. C. to remove unincorporated reactants and then
lyophilized.
[0089] The following table indicates the amount of each reactant
that was added for each copolymerization.
4 RGD F-9 C/H-V C/H-II HEP-III Peptide monomer (.mu.mol) 53.4 40.4
8.6 38.0 6.4 BBA-APMA monomer (.mu.mol) 21.4 16.2 3.44 15.2 2.56
Acrylamide (.mu.mol) 873 986 168 986 176 10% ammonium persulfate
130 93 40 130 33 (.mu.l) TEMED (.mu.l) 26 19 8 26 6.6
[0090] The recovered amounts of each peptide polymer after
lyophilization were 72 mg of RGD, 135 mg of F-9, 100 mg of C/H-II;
15.2 mg of C/H-V, and 17.8 mg of HEP-III
[0091] F. Coupling of Peptide Polymers to Biomaterials
[0092] Three biomaterials were used: polystyrene (PS), polyurethane
(PU), and silicone rubber (SR). Breakaway 96-well plate size
polystyrene strips (Immulon I Removawell Strips, from Dynatech
Laboratories, Inc., Chantilly, Va.) were used to determine the
immobilized levels of each peptide polymer in radiolabelling
experiments. Both 24- and 48-well PS culture plates (that were
nonsterile and nonplasma treated) were obtained from Coming Costar
Corp. (Cambridge, Mass.) and used for conducting the cell growth
bioactivity assays. Flat PU sheets (Pellethane 55-D) and flat SR
sheets were each obtained from Specialty Silicone Fabricators, Inc.
(Paso Robles, Calif.) and punched to produce discs with diameters
of 6, 10, and 15 mm diameters that respectively fit inside wells of
96, 48, and 24 well culture plates. The discs were used in both the
radiolabelling assays and bioactivity assays.
[0093] Two different protocols were used to apply the peptides
(peptide polymers or peptide reagent controls) to biomaterials,
with the major difference being whether or not the peptides were
dried onto the biomaterials before being illuminated to activate
the latent reactive groups. With the dry immobilization protocol,
the peptides were diluted in 50% (v/v) isopropanol (IPA) in water,
added to biomaterials as indicated below, and dried before
illumination. With the wet immobilization protocol, the peptides
were diluted in water, added to biomaterials as indicated below,
and not allowed to dry before illumination.
[0094] With each immobilization protocol, the final added
concentration of peptide moieties was 50 .mu.g/ml, and the
following volumes were added per well of each type of culture
plate: 50 .mu.l/well of 96-well plates, 100 .mu.l/well of 48-well
plates, 200 .mu.l/well of 24-well plates. As was indicated above,
discs of PU and SR were placed in the bottoms of the plates for the
coating and evaluation procedures.
[0095] The samples were illuminated with a Dymax lamp (model no.
PC-2, Dymax Corporation, Torrington, Conn.) which contained a
Heraeus bulb (W. C. Heraeus GmbH, Hanau, Federal Republic of
Germany) to activate the photogroups present in each polymer, and
produce covalent bonding to the biomaterial. The illumination
duration was for 1-2 minutes at an intensity of 1-2 mW/cm.sup.2 in
the wavelength range of 330-340 nm. Adsorption controls were also
generated with peptide polymers and peptide reagent controls that
were not illuminated.
[0096] Following either photoimmobilization or adsorption, the
peptide polymers and peptide reagent controls, respectively, were
extensively washed on an orbital shaker (.about.150-200 rpm) to
remove peptides that were not tenaciously bound to the substrate.
The wash steps included: 1) an overnight wash with three changes in
phosphate buffered saline (PBS), pH 7.3, containing I % Tween 20
detergent, 2) a 30 minute wash/sterilization step in 70% (vol/vol)
ethanol in water, and 3) four washes in sterile PBS.
[0097] G. Quantitation of Immobilized Levels of Peptides on
Biomaterials
[0098] Two peptide polymers (RGD polymer and F-9 polymer) and their
respective peptide reagent controls were radiolabelled with tritium
by reductive methylation and used to determine the level of each
peptide that was immobilized onto each biomaterial. The peptide
reagent controls were not incorporated into polymers and consisted
of RGD (sequence GRGDSPKKC) and F-9. The four tritium labeled
peptides were respectively called, [.sup.3H]-RGD polymer,
[.sup.3H]-F-9 polymer, [.sup.3H]-RGD reagent control, and
[.sup.3H]-F-9 reagent control. Each tritium-labeled peptide was
coated onto each biomaterial (PS breakaway strips, 6 mm discs of
PU, or 6 mm discs of SR) using the dry immobilization protocol and
the wash procedure described herein.
[0099] After the wash procedure, the PS break away strips were
broken into individual wells, placed in scintillation vials (1
well/vial), dissolved in THF and counted in Aquasol-2 Fluor (DuPont
NEN.RTM., Boston, Mass.) to determine the dpm's/sample. The PU
discs were swelled in THF and counted in Aquasol-2. The SR discs
were dissolved in Soluene-350 Tissue Solubilizer and counted in
Hionic Fluor (each from Packard Instrument Co., Meriden, Conn.).
After the biomaterials were counted by liquid scintillation
spectrometry, the final loading densities of each peptide
(ng/cm.sup.2) were calculated from the known specific activities
(dpm/ng) of each tritiated reagent. A summary of the loading
density results is given in the table below. Each value is the
average of three or more determinations. Immobilized refers to
illuminated samples. Adsorbed refers to nonilluminated samples.
ND=not determined.
5 Peptide Peptide Peptide Polymer Polymer Reagent Immobilized
Adsorbed Control Adsorbed Peptide Biomaterial (ng/cm.sup.2)
(ng/cm.sup.2) (ng/cm.sup.2) F-9 PS 2018 321 57 SR 1565 178 75 PU
139 90 31 RGD PS 2575 ND 66 SR 1375 156 65 PU 562 109 23
[0100] In all cases, the immobilized peptide polymers (i.e., the
polymer coatings which had been illuminated) exhibited the greatest
loading densities. The loading densities of the peptide polymers
were also biomaterial dependent, with the greatest retained levels
of peptide polymers being on PS and SR, and the lowest retained
levels being on PU. In each case, the retained levels of
photoimmobilized peptide polymers were on the order of 1.5- to
9-fold greater than the adsorbed peptide polymers and on the order
of 4.5- to 39-fold greater than the adsorbed peptide reagent
controls.
[0101] H. Cell Attachment Activity of Immobilized Peptides
[0102] Calf pulmonary artery endothelial (CPAE) cells were
purchased from ATCC (American Type Culture Collection, Rockville,
Md.) and cultured as indicated by ATCC. The attachment assays were
conducted in 48-well PS culture plates. When PU and SR were
evaluated, discs of each material (with or without coatings) were
placed in the bottoms of the culture plate wells. When PS was
evaluated, the bottoms of the wells were coated. For each assay,
uncoated and peptide coated biomaterials (PS, PU, and SR) were
seeded at 50,000 cells per well in serum free media containing 2
mg/ml of bovine serum albumin (BSA fraction V, from Sigma Chemical
Company, St. Louis, Mo.). The cells were allowed to attach to each
biomaterial for two hours. Then the unattached cells were removed
by aspiration, and the wells were rinsed twice with Hank's Balanced
Salt Solution (Celox Corp., Hopkins, Minn.). Finally, the attached
cells were quantitated by the addition of culture media containing
a metabolic dye, MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
which is converted from a yellow tetrazolium salt into an
insoluble, purple formazan in the presence of viable cells. After a
two hour incubation in culture media containing MTT, the media was
removed, and the formazan dye that had been deposited inside the
viable cells was solubilized (with acidic isopropanol) and read on
a spectrophotometer at 570 nm. The absorbance of the formazan is
directly proportional to the number of attached, viable cells
present per well.
[0103] The following table summarizes results of cell attachment
assays comparing immobilized peptide polymers and adsorbed peptide
reagent controls. In each case, the relative numbers of cells that
attached to the peptide-coated biomaterials (as determined by MTT
dye) were divided by the number of cells that attached to the
uncoated (UC) biomaterials to obtain a relative cell attachment
score. Each value represents the average of 1 to 4 different
experiments, with each experiment being conducted with 3 or 4
replicates. The peptides were immobilized onto PS using the wet
immobilization protocol described herein and the peptides were
immobilized onto SR and PU via the dry immobilization protocol.
Immobil. Peptide Polymer refers to illuminated peptide polymer.
Adsorbed Peptide Reagent refers to nonilluminated peptide reagent.
ND=not determined.
6 Polystyrene Silicone Rubber Polyurethane Ad- Ad- Ad- Immobil.
sorbed Immobil. sorbed Immobil. sorbed Peptide Peptide Peptide
Peptide Peptide Peptide Peptide Polymer Reagent Polymer Reagent
Polymer Reagent RGD 10 1.4 6.2 1.1 4.3 ND F-9 5.7 0.8 0.9 ND 1.9 ND
C/H-V ND ND 1.3 0.4 1.4 ND
[0104] These results show that the photoimmobilized peptide
polymers enhanced cell attachment to all three biomaterials. The
greatest improvements were observed with RGD polymer
photoimmobilized onto all three biomaterials and F-9 polymer
photoimmobilized onto PS (4.3 to 10 fold improvements), with lesser
improvements observed with F-9 polymer on PU and C/H-V polymer on
SR and PU (1.3 to 1.9 fold improvements). In each case where
adsorbed peptide reagent controls were compared to photoimmobilized
peptide polymers, cell attachment was greater on the peptide
polymers.
[0105] I. Cell Growth Activity of Immobilized Peptides
[0106] The growth assays were conducted in 24-well PS culture
plates. When PU and SR were evaluated, discs of each material (with
or without coatings) were placed in the bottoms of the culture
plate wells. When PS was evaluated, the bottoms of the wells were
coated. For each assay, uncoated and peptide coated biomaterials
(PS, PU, and SR) were seeded with CPAE cells at 1500 cells per well
and allowed to proliferate in vitro for 4 to 7 days. Then, the
media was aspirated and the cell growth was quantitated using MTT
dye.
[0107] The following table summarizes results that compared cell
growth on uncoated substrates, adsorbed peptides, and peptide
polymers. As with the attachment assays, the relative numbers of
cells growing on each peptide coated biomaterial was divided by the
number of cells growing on the uncoated (UC) biomaterials to obtain
a relative cell growth score. Each value represents the average of
1 to 4 different experiments, with each experiment being conducted
with 3 or 4 replicates. The dry immobilization protocol described
herein was used to immobilize all peptides evaluated. Immobil.
Peptide Polymer refers to illuminated peptide polymer. Adsorbed
Peptide Reagent refers to non illuminated peptide reagent. ND=not
determined.
7 Polystyrene Silicone Rubber Polyurethane Ad- Ad- Ad- Immobil.
sorbed Immobil. sorbed Immobil. sorbed Peptide Peptide Peptide
Peptide Peptide Peptide Peptide Polymer Reagent Polymer Reagent
Polymer Reagent RGD 17.5 1.2 1.7 1.1 9.4 1.7 F-9 14.8 1.0 2.4 1.7
7.7 1.7 C/H-V 11.3 1.0 1.0 2.0 3.9 0.7
[0108] In addition to the peptides shown in the table above, C/H-II
and HEP-III were also evaluated for growth on PS. With these two
peptides, growth on adsorbed peptides was 1.0 and 1.1 times that
observed on uncoated PS, and growth on peptide polymers was 10.5
and 13 times that observed on uncoated PS. These two peptides
(polymers and reagent controls) were immobilized via the wet
immobilization protocol. The results with C/H-II and HEP-III are
the averages of 1 and 2 experiments, respectively, with each
experiment being conducted with 4 replicates.
[0109] These growth assays show that all five peptide polymers
photoimmobilized onto PS promoted growth that was 10.5 to 17.5 fold
greater than growth on uncoated PS. Also, the three peptide
polymers enhanced cell growth on PU by 3.9 to 9.4 fold. Only slight
improvements in cell growth were observed on SR.
Example 2
Hirudin Polymer
[0110] A. Synthesis of N-Succinimidyl
6-(4-Benzoylbenzamido)hexanoate (BBA-EAC-NOS)
[0111] BBA-Cl (30.00 g, 0.123 moles), prepared as described in
Example 1, was dissolved in 450 ml of toluene. An amount (16.1 g,
0.123 moles) of 6-aminohexanoic acid (which will be alternatively
referred to herein as .epsilon.-aminocaproic acid, or as its
abbreviated form EAC) was dissolved in 375 ml of 1 N NaOH and this
solution was added to the solution of the acid chloride. The
mixture was stirred vigorously to generate an emulsion for 45
minutes at room temperature. The product was then acidified with 1
N HCl and extracted with 3.times.450 ml of ethyl acetate. The
combined extracts were dried over magnesium sulfate, filtered, and
evaporated under reduced pressure. The
6-(4-benzoylbenzamido)hexanoic acid was recrystallized from
toluene:ethyl acetate to give 36.65 g of product, m.p.
106-109.degree. C.
[0112] The 6-(4-benzoylbenzamido)hexanoic acid (25 g, 73.7 mmoles),
was added to a dry flask and dissolved in 500 ml of dry
1,4-dioxane. NHS (15.5 g, 0.135 moles) was added and the flask was
cooled on an ice bath under a dry N.sub.2 atmosphere. DCC (27.81 g,
0.135 moles), in 15 ml of 1,4-dioxane was then added and the
mixture was stirred overnight. After filtration to remove the
1,3-dicyclohexylurea, the solvent was removed under reduced
pressure and the product was recrystallized twice from ethanol to
give 23.65 g of a white solid, m.p. 121-123.degree. C.
[0113] B. Synthesis of a Photoreactive Polyacrylamide Containing
Hirudin Ligands (Hirudin Polymer)
[0114] Methacryloyl-EAC-BBA was prepared by reacting 112 mg (0.629
mmole) of APMA-HCl, 358 mg (0.818 mmole) BBA-EAC-NOS and 80 mg (104
.mu.l, 0.692 mmole) TEMED in 22 ml DMSO. The mixture was stirred
for 4.5 hours. Then the NOS polymer was prepared by adding to this
mixture 4.20 gm (59.1 mmole) acrylamide, 532 mg (3.15 mmole)
N-acryloxysuccinimide (Eastman Kodak, Rochester, N.Y.) and 64 mg
(0.39 mmole) of 2,2'-azobisisobutyronit- rile (AIBN). The mixture
was sparged with helium and incubated at 50.degree. C. overnight. A
portion of the resulting polymer solution (10 ml) was diluted with
10 ml DMSO and slowly added to vigorously stirred acetone (200 ml)
to precipitate the polymer. The polymer was collected, washed with
acetone to remove impurities, and dried under vacuum. A total of
1.47 g was recovered.
[0115] Recombinant hirudin with a purity of greater than 90% and an
activity of 16,500.+-.2000 ATU/mg was obtained from Transgene
Laboratories (Strasbourg, France). (See below for definition of
ATU.) Hirudin is an antithrombotic agent that acts by binding to
and inhibiting the proteolytic activity of thrombin. Hirudin (10.5
mg, 1.52 .mu.mole) was dissolved in 1 ml of 0.1 M carbonate buffer.
The NOS polymer (prepared as described above) was dissolved in DMSO
at 4 mg/ml. Then 2 mg of the NOS polymer was added to the hirudin
solution and mixed overnight. Half of the reaction mixture was
dialyzed in a Spectra/Por 50,000 MWCO tubing against water,
followed by lyophilization. A total of 5.9 mg of hirudin polymer
was recovered, which contained 5.0 mg hirudin and 0.2 mole BBA per
mole of hirudin. The hirudin was quantitated with a BCA protein
assay kit (from Pierce Chemical Company, Rockford, Ill.) and the
BBA content was determined spectrophotometrically.
[0116] A control polymer ("ethanolamine polymer") was prepared by
adding ethanolamine instead of hirudin to the NOS polymer. The
resultant ethanolamine polymer is uncharged and was used as a
control in experiments that compared the binding of thrombin to a
similar polymer that contained ethanolamine instead of hirudin.
[0117] C. Assay for Activities of Hirudin and Hirudin Polymer In
Solution
[0118] The specific activities of hirudin and hirudin polymer were
determined by standard protocols provided by Transgene and are
expressed as antithrombin units (ATU's) per mg of hirudin. One ATU
is the amount of hirudin that is required to inhibit the
proteolytic activity of one NIH unit of thrombin. For these assays,
a known amount of bovine thrombin (175-350 NIH units/mg., from
Sigma Chemical Co., St. Louis, Mo.) was preincubated with a series
of dilutions of hirudin or hirudin polymer, and the remaining
thrombin activity was determined with a chromogenic substrate,
Chromozym TH (from Boehringer Mannheim Corp, Indianapolis, Ind.).
The specific activities of hinidin assayed before and after
incorporation into the hirudin polymer were 11,710 and 10,204
ATU/mg, respectively. Therefore incorporation of hirudin into the
polymer produced only a 13% decrease in its activity.
[0119] D. Coupling of Hirudin Polymer and Ethanolamine Polymer to
Biomaterials.
[0120] Flat sheets of three biomaterials were used: 1) polyethylene
(PE, primary reference material from the National Institutes of
Health, Bethesda, Md.), 2) SR (medical grade SILASTIC.RTM. from Dow
Corning Corporation, Midland, Mich.), and 3) PU (Tecoflex.RTM. from
ThermoCardiosystems, Woburn, Mass.). Samples of each biomaterial
were cut into either 6 mm diameter disks or 1.times.1 cm squares.
To remove surface contaminants prior to coatings, the PU and PE
samples were washed by brief immersion in IPA and SR was extracted
1 hour with hexane and dried overnight. In addition, the SR samples
were treated with an argon plasma (3 min., 250 watts, 250 mtorr)
just prior to application of the hirudin polymer or ethanolamine
polymer.
[0121] The hirudin polymer was diluted to 1-25 .mu.g/ml in 75:25
(v/v) water:IPA and added to one side of each biomaterial sample
that was washed or extracted as described above. The added hirudin
polymer solutions were allowed to dry and were then illuminated
with a Dymax lamp. To remove loosely adherent hirudin polymer that
remained after the coating procedures, each sample was washed 3
times for 15 minutes and then overnight in a 1% solution to Tween
20 in PBS. Then the Tween 20 was removed by rinsing the samples in
deionized water.
[0122] Three types of control samples were also prepared: 1)
uncoated controls to which hirudin polymer was not added, 2)
samples to which the hirudin polymer was added but not illuminated,
and 3) SR samples that were coated with the ethanolamine polymer.
For the latter control, the ethanolamine polymer (prepared as
described herein) was diluted in deionized water to 1 or 5
.mu.g/200 .mu.l. Then, 200 .mu.l aliquots of each stock solution
were added to one side of 1 cm.sup.2 samples of SR, allowed to dry,
photoactivated, and washed in Tween 20/PBS and deionized water as
described herein for the hirudin polymer.
[0123] E. Quantitation of Hirudin Loading on Biomaterials.
[0124] Hirudin was radiolabeled via reductive methylation,
incorporated into a hirudin polymer as described herein, and used
to quantitate the amount of hirudin polymer that was immobilized
onto each of three biomaterials as described herein. To count the
retained tritium, the samples were dissolved in THF, diluted into
Aquasol, and counted in a Packard 1900CA liquid scintillation
counter. The results presented in the table below show that the
amount of retained hirudin polymer was proportional to the amount
added, and the amount retained after photoactivation is 2.3 to 66
times that retained without photoactivation. Each result is the
average of 4 determinations. N.A.=not assayed.
8 Added hirudin Retained hirudin polymer polymer without
photoactivation after photoactivation Biomaterial .mu.g/cm.sup.2
.mu.g/cm.sup.2 .mu.g/cm.sup.2 PE 1.0 0.0003 0.020 PE 5.0 N.A. 0.10
PE 25 N.A. 0.15 PU 1.0 0.027 0.062 PU 5.0 N.A. 0.114 PU 25 N.A.
0.312 SR 1.0 N.A. 0.019 SR 5.0 N.A. 0.043
[0125] F. Assay for Activity of Hirudin Polymer Coatings on
Biomaterials.
[0126] Hirudin polymer that had not been labeled with tritium was
coated onto each biomaterial as described herein. The activity of
the immobilized hirudin polymer was then assayed by quantitating
the binding of added tritium labeled thrombin (.sup.3H-Thr). The
.sup.3H-Thr was prepared by labeling human thrombin (4000 NIH
units/mg protein, from Sigma Chemical Co.) via reductive
methylation. Each coated sample was incubated in a solution of 2
.mu.g/ml of .sup.3H-Thr in a Tris buffer (0.05 M Tris-HCl, 0.1 M
NaCl, 0.1% PEG 3350, pH 8.5) for one hour and rinsed with the same
buffer containing no thrombin to remove unbound thrombin. The
samples were then dissolved THF, diluted in Aquasol and
counted.
[0127] The results presented in the table below show that the
amount of thrombin that was retained by hirudin-coated biomaterials
was proportional to the amount of immobilized hirudin. Each result
is the average of 4 determinations. SR+EP.sup.1 and SR+EP.sup.5
indicate SR samples that were coated with the ethanolamine polymer
added at 1.0 and 5 .mu.g/cm.sup.2, respectively. N.A. equals not
assayed. Control experiments were conducted in which thrombin was
added to uncoated biomaterials, and the amounts of thrombin that
bound to uncoated PE, PU, and SR were 0.01, 0.012, and 0.006
.mu.g/cm ,respectively. Comparisons of thrombin binding to uncoated
PE and PU versus the same biomaterials coated with 25
.mu.g/cm.sup.2 of hirudin polymer show that the latter promoted 200
and 23 fold greater thrombin binding, respectively. Finally, the
results with SR coated with the ethanolamine polymer show that the
hirudin moiety is essential for binding thrombin.
9 Added hirudin polymer Bound thrombin Biomaterial (.mu.g/cm.sup.2)
(.mu.g/cm.sup.2) PE 0 N.A. PE 1.0 0.020 PE 5.0 0.014 PE 25 0.200 PU
0 N.A. PU 1.0 0.028 PU 5.0 0.137 PU 25 0.280 SR 0 N.A. SR 1.0 0.004
SR 5.0 0.047 SR + EP.sup.1 0 0.004 SR + EP.sup.5 0 0.005
Example 3
Heparin Polymer
[0128] A. Synthesis of N-Succinimidyl 6-Maleimidohexanoate.
[0129] 6-Maleimidohexanoic acid, 20.0 g (94.7 mmol) was dissolved
in 100 ml of chloroform, followed by the addition of 60.1 g (0.473
mol) of oxalyl chloride. The resulting solution was then stirred
for 2 hours at room temperature. The excess oxalyl chloride was
removed under reduced pressure and the resulting acid chloride was
azeotroped with 4.times.25 ml of chloroform to remove the excess
oxalyl chloride. The acid chloride product was dissolved in 100 ml
of chloroform, followed by the addition of 12.0 g (0.104 moles) of
NHS and a slow addition of 11.48 g (0.113 mol) of TEA. The mixture
was stirred at room temperature overnight. After washing the
reaction mixture with 4.times.100 ml of water, the chloroform
solution was dried over sodium sulfate. Removal of solvent gave
24.0 g of product for an 82% yield. Analysis on an NMR spectrometer
was consistent with the desired product and it was used without
further purification.
[0130] B. Synthesis of a Photoreactive Polyacrylamide Containing
Hydrazide Ligands (Hydrazide Polymer).
[0131] Acrylamide, 8.339 g (0.117 mol), was dissolved in 112 ml of
THF, followed by 0.241 g (1.50 mmol) of AIBN, 0.112 ml (0.74 mmol)
of TEMED, 1.284 g (3.70 mmol) of BBA-APMA (prepared as described
herein), and 0.377 g (1.2 mmol) of N-succinimidyl
6-maleimidohexanoate (prepared as described herein). The solution
was deoxygenated with a helium sparge for 4 minutes, followed by an
argon sparge for 4 minutes. The sealed vessel was then heated
overnight at 55.degree. C. to complete the polymerization. The
precipitated polymer was isolated by filtration and was washed by
stirring for 30 minutes with 100 ml of THF. The final product was
recovered by filtration and dried in a vacuum oven to provide 9.64
g of solid, a 96% yield.
[0132] The above polymer (1.0 g) was dissolved in 50 ml of 0.05 M
phosphate buffer at pH 8 and this solution was added to a second
solution containing 0.696 g (5.89 mmol) of oxalic dihydrazide in 50
ml of 0.05 M phosphate buffer at pH 8. The combined solutions were
stirred overnight at room temperature. The product was put into
dialysis against deionized water using 6000-8000 molecular weight
cutoff dialysis tubing. After six changes of water over two days,
the polymer was isolated by lyophilization to give 850 mg of
product. An analysis for hydrazide groups on this polymer gave a
value of 0.0656 mmol of NH.sub.2/g of polymer, 55% of theory.
[0133] C. Synthesis of a Photoreactive Polyacrylamide Containing
Heparin Ligands (Heparin Polymer).
[0134] It is known that a controlled periodate oxidation of the
uronic acid residues present in heparin will generate aldehyde
functional groups, while retaining reasonable heparin activity.
Unbleached heparin with an activity of 152 units/mg (from Celsus
Laboratories, Cincinnati, Ohio) was dissolved at 250 mg/ml in 0.1 M
acetate buffer (pH 5.5) and oxidized with sodium periodate at 20
mg/ml for 30 minutes to generate free aldehyde groups. Remaining
periodate was inactivated by the addition of excess ethylene
glycol. Then, the ethylene glycol and small molecular weight
reaction products were removed by dialyzing overnight at 4.degree.
against 0.1 M acetate buffer (pH 5.5) using Spectra/Por 6,000
molecular weight cutoff dialysis tubing (from Spectrum Medical
Industries). The oxidized heparin retained 95 units/mg of
activity.
[0135] The concentration of oxidized heparin was adjusted to 15
mg/ml in 0.1 M acetate buffer (pH 5.5) and was reacted overnight
with an equal volume of 20 mg/ml photopolyhydrazide in water at
room temperature. The heparin polymer was used to coat biomaterials
without further purification.
[0136] D. Immobilization of Heparin Polymer onto Regenerated
Cellulose Membrane.
[0137] The heparin polymer was synthesized as described herein and
immobilized onto regenerated cellulose (RC) membranes having a pore
size of 0.45 mm. One inch diameter RC membranes were incubated with
heparin polymer for 15 minutes, air dried and then illuminated for
45 seconds on each side. The discs were washed first in 10.times.
PBS and then in PBS to remove unbound heparin polymer.
[0138] E. Evaluation of Thrombin Inhibition by Heparin Coated
Membranes.
[0139] The antithrombotic activity of heparin is due to its
inhibition of thrombin, which is a protease that is known to
participate in the clotting cascade. Heparin inhibits thrombin
activity by first binding to antithrombin III (ATIII). Then the
heparin/ATIII complex binds to and inactivates thrombin, after
which the heparin is released and can bind to another ATIII. The
assay for inhibition of thrombin by immobilized heparin was
conducted by measuring the cleavage of a chromogenic peptide
substrate by thrombin and used previously described methods.
[0140] Each assay was conducted in 1 ml of PBS which contained 0.85
mg BSA (Sigma Chemical Co.), 10 mU human thrombin (Sigma Chemical
Co.), 100 mU/ml ATIII (Baxter Biotech, Chicago, Ill.), and 0.17
.mu.mole of the chromogenic thrombin substrate S-2238 (Kabi
Pharmacia, Franklin, Ohio). To this assay solution was added either
uncoated or heparin coated membranes (to evaluate heparin activity
on the membranes) or standard concentrations of heparin (to
generate standard curves of heparin content versus absorbance). The
amounts of heparin that were added ranged from 2.5 to 25 mU. The
color generated, measured as absorbance at 405 nm, by thrombin
mediated cleavage of the S-2238 was read using a spectrophotometer
after 2 hours of incubation at 37.degree. C. The absorbance was
directly related to the activity of the thrombin and, thus,
inversely related to the amount of activation of ATIII induced by
the heparin in solution or immobilized on the surface of the
substrate. Activity of surface bound heparin was calculated by
comparing the absorbance values generated with the membranes to the
absorbance values generated with known amounts of added
heparin.
[0141] This assay was then used to evaluate the heparin activity
present on the coated and uncoated RC membranes. The coated
membrane had heparin activity of 255 mU/sq.cm. whereas the uncoated
had <0.1 mU/sq. cm.
Example 4
Lysine Polymers
[0142] A. Synthesis of N-.alpha.-[6-(maleimido)hexanoyl]lysine.
[0143] 6-Maleimidohexanoic acid, 2.24 g (10.6 mmol) (prepared as
described in Example 1) was dissolved in 10.76 g (84.8 mmol) of
oxalyl chloride and stirred as a neat solution for 4 hours at room
temperature. The excess oxalyl chloride was then removed under
reduced pressure and the resulting acid chloride was dissolved in
25 ml of methylene chloride. This solution was added with stirring
to a solution of 3.60 g (10.6 mmol) N-.epsilon.-t-BOC lysine
t-butyl ester hydrochloride (Bachem Calif.) in 25 ml of methylene
chloride and 3.21 g (31.7 mmol) of TEA. The resulting mixture was
stirred overnight under nitrogen. After this time, the mixture was
treated with water and the organic layer was separated and dried
over sodium sulfate. The solvent was removed and the product was
purified on a silica gel flash chromatography column using a 0-5%
methanol in chloroform solvent gradient. Pooling of the desired
fractions and evaporation of solvent gave 5.20 g of product (98%
yield). Analysis on an NMR spectrometer was consistent with the
desired product.
[0144] The protected amino acid derivative, 0.566 g (1.14 mmol) was
dissolved in 5 ml of trifluoroacetic acid with stirring. After
stirring four hours at room temperature, the solvent was removed
under reduced pressure. The resulting oil was tritruated with ether
to remove residual trifluoroacetic acid to give 373 mg of product
for a 98% yield. Analysis on an NMR spectrometer was consistent
with the desired product.
[0145] B. Synthesis of a Photoreactive Polyacrylamide Containing
.epsilon.-Amino Lysine Ligands (Lysine Polymer).
[0146] Acrylamide (0.22 g, 3.10 mmol), BBA-APMA (0.014 g, 0.039
mmol), and N-.alpha.-[6-(maleimido)hexanoyl]lysine (0.266 g, 0.784
mmol; prepared as described herein) were dissolved in 7.3 ml of dry
DMSO. To initiate the polymerization, 8 mg (0.047 mmol) of AIBN and
4.0 .mu.l of TEMED were added, followed by sparging with nitrogen
to remove all oxygen. The mixture was then heated at 55.degree. C.
for 16 hours followed by evaporation of the DMSO under reduced
pressure. The product was dissolved in DI water and dialyzed three
days using 6-8K molecular weight cut off (MWCO) tubing against DI
water. The resulting solution was lyophilized to give 160 mg of
product.
[0147] C. Generation of Lysine Polymer Coatings on Polyurethane
(PU).
[0148] PU sheets were cut into 1.times.1 cm pieces, washed with IPA
and air dried. To improve wetting of the lysine polymer solution on
PU, the PU pieces were treated with argon plasma at 250 watts, 0.25
torr, for 1 minute. The PU pieces were then immersed in an aqueous
solution of lysine polymer (prepared as described herein, 1 mg/ml)
for 5 minutes, air dried, and illuminated for 30 seconds. The
samples were then washed overnight (in three changes of phosphate
buffered saline, pH 7.4, which contained 1% Tween 20) to remove
unbound lysine polymer. The coated PU pieces were stored in PBS
containing 0.02% sodium azide until evaluated.
[0149] D. Quantitation of Lysine Polymer Coatings.
[0150] The lysine polymer (prepared as described above) was
radiolabelled via reductive methylation and used to quantitate the
levels immobilized onto polyurethane. The tritiated lysine polymer
was coated onto PU pieces with or without illumination to determine
the density of lysine polymer that was immobilized. After the wash
procedure, samples were dissolved in Soluene-350 and counted in
Hionic fluor (each from Packard Instrument Co., Meriden, Conn.).
The table below shows the immobilized levels (.+-.SEM) expressed in
terms of .mu.g/cm.sup.2 and nmole/cm.sup.2 of lysine moiety. Each
level is the average of 4 replicates. The results show that 1.51
.mu.g/cm.sup.2 is immobilized after illumination, which is more
than sufficient to produce a monolayer coating and is 3.8 times as
much polymer as was retained with the adsorbed control.
10 Lysine polymer level Lysine moiety level Treatment
(.mu.g/cm.sup.2) (nmole/cm.sup.2) Adsorbed 0.40 .+-. 0.03 0.359
.+-. 0.027 Illuminated 1.51 .+-. 0.23 1.36 .+-. 0.21
[0151] E. Evaluation of Plasminogen Binding by Lysine Coated
Polyurethane.
[0152] Others have described the covalent coupling of lysine to
silane derivatized glass and the resultant lysine derivatized glass
was reported to promote plasminogen binding, with the bound lysine
exhibiting significant proteolytic activity. Such a surface is
expected to demonstrate improved resistance to thrombus formation
when placed in contact with blood. The coating chemistry used in
the previous study utilized a short spacer and was limited to glass
as a surface, whereas the photoreactive lysine polymer can be
applied at high densities to a large range of biomaterials.
[0153] The lysine moiety in the lysine polymer is coupled via the
.alpha.-amino group to the polymer backbone, and the
.epsilon.-amino group is free to bind plasminogen. Therefore PU
that is coated with the lysine polymer is expected to inhibit
thrombus formation by reversibly binding plasminogen from blood,
with the bound plasmin demonstrating proteolytic activity that
cleaves fibrin and prevents fibrin clot formation on the coated
surface.
Example 5
Prostaglandin Polymers
[0154] A. Synthesis of a Photoreactive Polyacrylamide Containing
Primary Amine Ligands (Amine Polymer)
[0155] A solution of acrylamide (7.46 g, 105.1 mmoles), APMA-HCl
(2.14 g, 11.9 mmoles), and BBA-APMA (0.837 g, 2.39 mmoles) is
prepared in 170 ml of DMSO. To this solution is added AIBN (0.246
g, 1.50 mmoles) and TEMED (0.131 g, 1.13 mmoles). The solution is
then deoxygenated by sparging with helium gas for a period of 10
minutes and is sealed and placed in a 55.degree. C. oven for 18
hours to complete the polymerization. The polymer solution is
diluted with water and dialyzed against deionized water using
12,000-14,000 MWCO dialysis tubing to remove solvent, unreacted
monomers, and low molecular weight oligomers. The final product is
isolated by lyophilization, and the photogroup load is determined
by UV absorbance at 265 nm. The amine content of the polymer is
determined using a trinitrobenzenesulfonate (TNBS) method. The
photogroup and amine load can be changed by adjusting the quantity
of monomers used in the polymerization.
[0156] B. Synthesis of a Photoreactive Polyacrylamide Containing
Prostaglandin E.sub.1 Ligands (Prostaglandin E.sub.1 Polymer).
[0157] A solution of prostaglandin E.sub.1 (Sigma Chemical Co.) (30
mg, 0.0846 mmole) in 5 ml of dry 1,4-dioxane is prepared, and NHS
(10.7 mg, 0.0931 mmole) and DCC (26.2 mg, 0.127 mmole) are added to
the solution. The mixture is stirred overnight at room temperature
with formation of the 1,3-dicyclohexylurea (DCU) byproduct. The
solid is removed by filtration, and the filter cake is rinsed with
1,4-dioxane. The solvent is removed under reduced pressure, and the
resulting product is stored under dry conditions and used without
further purification.
[0158] The amine polymer (synthesized as described above) is
dissolved in DMSO at a concentration of 10 mg/ml, followed by the
addition of 1.5 equivalents of the NOS-derivatized prostaglandin
E.sub.1 relative to the amine content of the amine polymer
solution. Five equivalents of triethylamine are added to help
catalyze the reaction. After an overnight reaction, the polymer
solution is placed in dialysis against deionized water using
12,000-14,000 MWCO dialysis tubing to remove excess low molecular
weight reactants. The product is isolated by lyophilization.
[0159] C. Synthesis of a Photoreactive Polyacrylamide Containing
Carbacyclin Ligands (Carbacyclin Polymer).
[0160] A solution of carbacyclin (Sigma Chemical Co.) (5 mg, 0.0143
mmole) in 2 ml of dry 1,4-dioxane is prepared, and NHS (1.8 mg,
0.0157 mmole) and DCC (4.4 mg, 0.0215 mmole) are added to the
solution. The mixture is stirred overnight at room temperature with
formation of the DCU byproduct. The solid is removed by filtration,
and the filter cake is rinsed with 1,4-dioxane. The solvent is
removed under reduced pressure, and the resulting product is stored
under dry conditions and used without further purification.
[0161] The amine polymer (synthesized as described above) is
dissolved in DMSO at a concentration of 10 mg/ml, followed by the
addition of 1.5 equivalents of the NOS-derivatized carbacyclin
relative to the amine content of the amine polymer solution. Five
equivalents of TEA are added to help catalyze the reaction. After
an overnight reaction, the polymer solution is placed in dialysis
against deionized water using 12,000-14,000 MWCO dialysis tubing to
remove excess low molecular weight reactants. The product is
isolated by lyophilization.
[0162] D. Prostaglandin Polymer Coatings
[0163] Each prostaglandin polymer (synthesized as described above)
is diluted to 5 mg/ml in 50% (v/v) IPA in water and added to
biomaterial samples (polyurethane, silicone rubber and
polyethylene). The volume of prostaglandin containing polymer
solution that is added to each polymer is just sufficient to cover
the surface of each biomaterial (about 100 ml/cm.sup.2). The
polymer solution is allowed to dry onto each sample, after which
each sample is illuminated for 1-2 minutes.
[0164] Both prostaglandin E.sub.1 and carbacyclin (which is a
stable analog of prostaglandin I.sub.2; PGI.sub.2) are known to
inhibit platelet activation and thrombus formation. Therefore the
prostaglandin coatings generated are expected to inhibit platelet
activation and thrombus formation on biomaterials.
Example 6
Protein A Polymer
[0165] A. Synthesis of N-Succinimidyl 6-Methacrylamidohexanoate
(MAm-EAC-NOS)
[0166] The .epsilon.-aminocaproic acid (EAC), 2.00 g (15.25 mmol),
was added to a dry round bottom flask, followed by the addition of
2.58 g (16.73 mmol) of methacrylic anhydride. The resulting mixture
was stirred at room temperature for two hours, followed by
trituration with hexane. The hexane was decanted and the product
was triturated two additional times to give 3.03 g of the acylated
product (yield >99%). Without further purification, the product
was dissolved in 50 ml of chloroform, followed by the addition of
1.922 g (16.7 mmol) of NHS and 6.26 g (30.3 mmol) of DCC. The
mixture was stirred overnight at room temperature with protection
from moisture. The resulting solid was removed by filtration and
the filter cake was rinsed with chloroform. The solvent was removed
under reduced pressure with 5 ppm of the monomethyl ether of
hydroquinone (MEHQ) to prevent polymerization. The residue, 4.50 g,
was redissolved in 45 ml of dry THF and the solution was used
without further purification.
[0167] B. Synthesis of a Photoreactive Polyacrylamide Containing
Protein A Ligands (Protein A Polymer).
[0168] To prepare the latent reactive NOS polymer, acrylamide (1.0
gm, 14.1 mmole) was dissolved in 15 ml of dry THF. To that solution
was added 44 mg (0.149 mmole) of MAm-EAC-NOS (synthesized as
described herein) and 158 mg (0.45 mmole) of BBA-APMA (synthesized
as described in Example 1). For the initiator, 500 mg (3.04 mmole)
of AIBN was added, followed by the addition of 50 .mu.l of TEMED.
The solution was bubbled with nitrogen and incubated at 55.degree.
C. for 18 hours to allow polymerization. The insoluble polymer was
collected by filtration, and then dissolved in dry DMSO. The
polymer was precipitated by being added dropwise to stirred
ethanol, and was then collected by filtration and dried for storage
until used. The product yield was 0.906 gm.
[0169] To couple protein A to the latent reactive NOS polymer,
recombinant staphylococcal protein A (from Calbiochem-Novabiochem
Corp., San Diego, Calif.) was dissolved at 10 mg/ml in 0.1 M
carbonate buffer, pH 9. The latent reactive NOS polymer was
dissolved at 100 mg/ml in 50 mM phosphate buffer, pH 6.8. Then 200
.mu.l (20 mg) of the NOS polymer was added to 1 ml (10 mg) of the
protein A solution, and the mixture was incubated overnight at
4.degree. C. Evaluation by standard sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) revealed that more
than 80% of the added protein A was incorporated into the resultant
protein A polymer. The protein A polymer solution was used to coat
biomaterials without further purification.
[0170] C. Generation of Protein A Polymer Coatings on
Biomaterials.
[0171] The protein A polymer was then photocoupled to two types of
membranes, polysulfone (PSO) membranes with a pore size of 0.2 mm
(HT-200 membranes from Gelman Sciences, Ann Arbor, Mich.) and
regenerated cellulose (RC) membranes with a pore size of 0.45 mm
(no. SM18606, from Sartorius, Edgewood, N.Y.). Each membrane was in
the form of a disc that was 1 inch in diameter. Prior to addition
of the protein A polymer, each membrane was washed first in 1:1
(v/v) isopropanol:0.1 N HCl and then in water.
[0172] The protein A polymer (estimated concentration of 8.67
mg/ml) was then added to each type of membrane (1 ml of polymer per
4-6 discs) and incubated overnight at 4.degree. C. Then the discs
were removed from the polymer solution, air dried, and illuminated
for 1 minute on each side in a controlled temperature chamber (at
10.degree. C.). Illumination was produced with a Dymax lamp as
described herein.
[0173] The membranes were then washed to remove unbound protein A
polymer. The wash was achieved by placing the coated disks in
membrane holders (MAC-25 holder, from Amicon, Beverly, Mass.), with
2-4 membranes being placed in each holder. The membranes were then
sequentially washed with: 1) 10 ml of 0.1 M glycine in 2% acetic
acid, 2) 10 ml of 10.times. PBS, and 3) 30 ml of PBS. The washed
membranes were then stored in PBS containing 0.05% sodium azide
until used.
[0174] D. Evaluation of Activity of Protein A Polymer Coatings on
Biomaterials.
[0175] Protein A is a bacterial protein that binds specifically to
the F.sub.c region of immunoglobulin G (IgG) molecules. The
activity of the protein A coating on each type of membrane was
evaluated by assaying for binding by added IgG. Uncoated membranes
were used as controls. For this assay, 2 ml of rabbit serum was
diluted 1:5 in PBS and perfused through the coated membranes at 2-3
m/min. The membranes were then washed with PBS to remove unbound
IgG. The bound IgG was then eluted with 0.1 M glycine in 2% acetic
acid. The amount of eluted IgG was determined by measuring the
absorbance of the eluant at 280 nm and using an extinction
coefficient (.epsilon..sub.280) of 1.4 ml/cm-mg to calculate the mg
of eluted IgG. Also the IgG that eluted from each membrane type was
evaluated for purity by reduced SDS PAGE analysis. With the
biomaterials coated with protein A polymer, the eluted protein was
greater than 90% light and heavy chains of IgG. In contrast, the
major protein that eluted from uncoated controls was albumin.
[0176] Three discs of each type (PSO or RC) were placed in a MAC-25
holder and evaluated using this procedure. The table below shows
the average amounts of IgG that eluted from each membrane type;
with each value being the average of 10 determinations (10 cycles)
for 3 PSO disks and the average of 3 determinations (3 cycles) for
3 RC disks.
11 IgG eluted) Fold greater IgG on Membrane type Coating (mg/disc)
protein A coating Polysulfone uncoated control 0.020 Polysulfone
protein A polymer 0.680 34 Regenerated uncoated control 0.006
cellulose Regenerated protein A polymer 0.383 64 cellulose
[0177] These results show that each type of coated membrane binds
34-64 fold more IgG than does its respective uncoated control.
Also, protein A in the polymer has a stable conformation and is
tenaciously bound, since there is no decrease in IgG elution after
10 cycles of serum addition and IgG elution.
Example 7
IgG Polymers
[0178] A. Synthesis of a Photoreactive Polyacrylamide Containing
N-Oxysuccinimide Ligands (NOS Polymer).
[0179] Acrylamide, 3.897 g (0.0548 mol), was dissolved in 53 ml of
THF, followed by 0.115 g (0.70 mmol) of AIBN, 0.053 ml of TEMED,
0.204 g (0.58 mmol) of BBA-APMA (prepared as described in Example
1), and 0.899 g (2.9 mmol) of N-succinimidyl 6-maleimidohexanoate
(prepared as described in Example 3). The solution was deoxygenated
with a helium sparge for 4 minutes, followed by an argon sparge for
4 minutes. The sealed vessel was then heated overnight at
55.degree. C. to complete the polymerization. The precipitated
polymer was isolated by filtration and was washed by stirring for
30 minutes with 100 ml of THF. The final product was recovered by
filtration and dried in a vacuum oven to provide 4.688 g of solid,
a 94% yield.
[0180] B. Synthesis and Immobilization of a Photoreactive
Polyacrylamide Containing IgG Ligands.
[0181] IgG molecules are a class of antibody molecules that bind to
specific antigens. Tritiated rabbit anti-glucose oxidase IgG was
used so that the tritium label could be used to quantitate the
immobilized level of IgG and glucose oxidase binding could be
evaluated to assay IgG activity. NOS polymer (50 mg) (prepared as
described herein) was added to 100 mg of [.sup.3H] IgG (in 100 ml
of 0.1 M sodium carbonate at pH 9) and allowed to react overnight
at 4.degree. C. The IgG polymer was used without further
purification.
[0182] Polyester membrane (Accuwick from Pall Corporation) was cut
into 6 mm discs, and 4 .mu.l aliquots of the IgG polymer were
spotted on each of 19 discs. Three discs were left as controls that
were neither illuminated or washed. Eight discs were illuminated
for 1 minute and 8 discs were left nonilluminated. The latter 8
illuminated and 8 nonilluminated discs were washed with 25 mM
bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (BIS-TRIS) at pH
7.2 containing 1% lactose, 1% BSA and 0.1% Brij 35.
[0183] To quantitate the immobilized levels of IgG polymer, the 3
control (uncoated, unwashed) discs and 3 each of the illuminated
and nonilluminated conditions were dissolved in Soluene (0.5 ml)
and counted in 5 mls of Hionic Fluor. The results are reported in
the table below. A comparison of the illuminated (washed) to the
control (uncoated, unwashed) shows that 75% of the added IgG
polymer was retained after illumination. In contrast, with the
nonilluminated samples, only 12.5% of the added IgG polymer was
retained.
12 IgG Amount on discs (ug) IgG Activity (A.sub.655) Treatment n
mean SEM n mean SEM Control (uncoated, 3 2.08 0.03 5 not NA
unwashed) assayed Nonilluminated 3 0.26 0.01 5 0.669 0.021 (washed)
Illuminated (washed) 3 1.56 0.03 5 1.079 0.059
[0184] To quantitate the activity of the immobilized IgG, the
remaining 5 illuminated and 5 nonilluminated discs were incubated
with glucose oxidase at 0.1 mg/ml in PBS for 1 hour and washed 5
times with TNT (0.05 M Tris(hydroxymethyl)aminomethane, 0.15 M
NaCl, 0.05% Tween-20). Each disc was then transferred to wells in a
96 well microtiter plate and assayed by adding 200 .mu.l of
3,3',5,5'-tetramethylbenzidine (TMB) chromogen mixture (100 .mu.l
of TMB reagent from Kirkegaard & Perry Laboratories, Inc., 100
.mu.l 0.2 M sodium phosphate pH 5.5, 10 mg of glucose and 4 .mu.g
of horseradish peroxidase) and allowing the color to develop for 20
minutes. Aliquots (100 .mu.l) were then transferred to a separate
microtiter plate and the absorbance was read at 655 nm. A
comparison of the illuminated to nonilluminated samples shows that
61% greater activity was expressed by the illuminated samples.
Example 8
Streptavidin Polymer
[0185] A. Synthesis of a Photoreactive Polyacrylamide Containing
Streptavidin Ligands
[0186] Streptavidin (from InFerGene Company, Benicia, Calif.) was
coupled to the NOS polymer (prepared as described in Example 6).
Streptavidin (15 mg) was dissolved in 1.5 ml of 0.1 M carbonate
buffer (pH 9.0). The NOS polymer was prepared and dissolved in 5 mM
acetate buffer (pH 5) to a final concentration of 100 mg/ml. The
NOS polymer solution (0.3 ml) was added to the streptavidin
solution (1.5 ml), and the mixture was stirred overnight at
4.degree. C. The resulting streptavidin polymer was used without
further purification or characterization.
[0187] B. Generation of Streptavidin Polymer Coatings on
Surfaces
[0188] Solid glass rods (3 mm diameter.times.3 cm length) were
washed by sonication in 1:1 (v/v) acetone in 0.1N HCl for 30
minutes, rinsed in water, acetone, dried at 100.degree. C. for 1
hr., cooled, and stored desiccated until used.
Bis(trimethoxysilylethyl)benzene (from United Chemical
Technologies, Inc., Bristol, Pa.) was diluted to 10% (v/v) in
acetone. The rods were dipped in the silane reagent for 30 seconds,
air dried, dipped in water for 30 seconds, removed and cured at
100.degree. C. for 15 minutes, and rinsed with acetone.
[0189] The organosilane primed glass rods were dipped into a
solution of streptavidin polymer for 30 seconds. The glass rods
were removed from the solution, allowed to air dry, and illuminated
for 30 seconds with a Dymax lamp. Adsorption controls were prepared
via the same protocol, except that they were not illuminated. Both
type of coated rods were washed with PBS containing 0.05% Tween 20
to remove nonadherent streptavidin polymer.
[0190] C. Evaluation of Immobilized Streptavidin Polymer
[0191] Streptavidin is a receptor that binds strongly to biotin as
its ligand. The activity of streptavidin polymer coating was
evaluated by quantifying the binding of added biotin derivatized
horseradish peroxidase (biotin-HPP, obtained from Pierce Chemical
Company, Rockford, Ill.). The glass rods were incubated for one
hour in a 9 .mu.g/ml solution of biotin-HIRP. The binding of
underivatized FFPP (added at 9 .mu.g/ml) was evaluated as a control
for nonspecific binding of HRP to the glass rods. The rods were
then washed with PBS containing 0.05% Tween 20 to remove nonbound
HRP, and the relative activity of bound HRP was evaluated with a
TMB peroxidase substrate system (Kirkegaard and Perry Laboratory,
Inc., Gaithersburg, Md.). IRP catalyzes the oxidation of TMB and
produces a color that is quantitated spectrophotometrically at 405
nm. Each result is the average of 3 determinations.
13 Absorbance of Absorbance of Coating on glass Biotin-HRP HRP
Uncoated control 0.067 0.085 Adsorbed streptavidin polymer 0.285
0.065 Covalent streptavidin polymer 1.005 0.056
[0192] The results show the expected trends, with the greatest
peroxidase activity being observed on rods that were coated with
photoimmobilized streptavidin polymer (covalent streptavidin
polymer) and to which had been added biotin-HRP. The adsorbed
(nonilluminated) streptavidin polymer produced 3.5 fold less
peroxidase activity, and the remaining variants which lacked
streptavidin and/or biotin exhibited little peroxidase
activity.
Example 9
Biotin Polymer
[0193] The photoreactive amine polymer (80 mg) (synthesized as
described in Example 5) is dissolved in 2 ml of DMSO. To the
polymer solution is added 40 mg of biotinamidocaproic acid
3-sulfo-N-hydroxysuccinimide ester (Sigma Chemical Co.) and 0.05 ml
triethylamine. The solution is mixed for two hours at room
temperature, then dialyzed against deionized water to remove any
biotin that is not coupled to the polymer.
[0194] A solution of the biotin polymer (1.0 mg/ml in deionized
water) is applied to wells of a polystyrene microtitration plate
and incubated for one hour, after which the plate is illuminated
for 1-2 minutes. The plate is then washed with deionized water to
remove unbound biotin polymer.
[0195] Biotin is a ligand that binds to streptavidin as its
receptor. Polystyrene microtitration plates are coated with biotin
polymer and evaluated for activity by assaying the binding of
streptavidin. A solution of straptavidin is added to the plates
that are coated with biotin polymer and unbound streptavidin is
removed by washing with deionized water. The retained streptavidin
is quantitated by adding biotin-HRP and evaluating HRP
activity.
Example 10
Magainin Polymer
[0196] A. Synthesis of Magainin Peptide Monomer
[0197] Magainin-2 was used in this example and was custom
synthesized for BSI by Bachem, Inc. (Torrance, Calif.) with a
cysteine being added to the carboxyl terminus of the peptide. The
resulting sequence of Magainin consisted of
GIGKFLHSAKKFGKAFVGETMNSC. As was described in Example 1, the
underlined C (C) denotes a nonnative amino acid that was added to
allow coupling via the sulfhydryl group.
[0198] Magainin (2.36 Emote) was dissolved in 0.5 ml of degassed
water. To this solution was added 2.36 .mu.mole of Mal-MAm
(dissolved in 20 .mu.l chloroform) and 0.5 ml ethanol. The reaction
was stirred for 90 minutes at room temperature, after which the
solution was dried under nitrogen and resuspended in 1 ml water.
The recovered magainin monomer solution was determined to have 5.5
mg/mil of magainin moiety, as determined by the MicroBCA assay (kit
from Pierce Chemical Company, Rockford, Ill.).
[0199] B. Synthesis of Photoreactive Polyacrylamide Containing
Magainin Ligand (Magainin Polymers)
[0200] BBA-APMA was dissolved at a concentration of 10 mg/ml in
DMSO, and acrylamide was dissolved at a concentration of 100 mg/ml
in water. The magainin monomer (0.48 .mu.mole in 220 .mu.l water)
was not purified after being synthesized (as described above). The
appropriate molar amounts of BBA-APMA (0.25 .mu.mol in 44 .mu.l of
THF) and acrylamide (6.9 .mu.mol in 120 .mu.l of water) were then
added to the reaction vial. An additional 300 .mu.l of THF was
added, and the mixture was degassed by water aspiration for 15
minutes. Ammonium persulfate (6.8 .mu.l of 10% stock solution in
water) and TEMED (1.5 .mu.l) were added to catalyze the
polymerization. The mixture was degassed again and incubated
overnight at room temperature in a sealed dessicator. The resultant
magainin polymer was dialyzed against water (using Spectra/Por
50,000 MWCO dialysis tubing; from Spectrum, Houston, Tex.) at
4.degree. C. to remove unincorporated reactants and then
lyophilized. Of the 1.2 mg of magainin peptide that was used to
synthesize the methacryloyl magainin, 0.35 mg was present in the
solubilized magainin polymer.
[0201] C. Evaluation of Immobilized Magainin Polymer
[0202] Magainin is a cationic peptide antibiotic that was
originally isolated from the skin of Xenopus laevis. It is active
against a broad spectrum of pathogens and acts at the surface of
the pathogens. The activity of the magainin polymer was evaluated
by a standard solution assay, which determined the minimum
inhibitory concentration (MIC) of magainin polymer that was
required to prevent the growth of bacteria. The MIC of magainin
polymer was 50 .mu.g/ml for both Escherichia coli (ATCC No. 25922)
and for Staphylococcus epidermidis (ATCC No. 12228), whereas native
monomeric magainin (not incorporated into either a magainin monomer
or the magainin polymer) had an MIC of 6.25-12.5 .mu.g/ml for E.
coli and 25 .mu.g/ml for S. epidermidis.
[0203] The magainin polymer is diluted to 250 .mu.g/ml in 50% (v/v)
IPA in water and added to biomaterial samples (PU, SR and PE). The
volume of magainin polymer solution that is added to each polymer
is just sufficient to cover the surface of each biomaterial (about
100 .mu.l/cm.sup.2). The polymer solution is allowed to dry onto
each sample, after which each sample is illuminated for 1-2
minutes. The coated samples are washed in 0.1 N HCl followed by
PBS.
[0204] The antimicrobial activity of immobilized magainin polymer
is evaluated with a centrifugation assay. Sheets of biomaterials
are cut into disks of 1.5 cm diameter, coated with magainin
polymer, and placed in individual wells of 24-well culture plates.
Bacteria (E. coli and S. epidermidis) are suspended at 200-400
colony forming units per ml (cfu/ml) in PBS. One ml aliquots of
bacterial suspensions are placed in wells that contain
magainin-coated biomaterials, and the plates are centrifuged at
3500 xg at 4.degree. C. for 20 minutes to sediment the bacteria on
the coated biomaterial disks. The disks are then placed in tryptic
soy agar (TSA) bacterial culture plates and overlaid with a thin
layer of TSA. After overnight incubation at 37.degree. C., the
colonies of bacteria growing on the disks are counted. The magainin
polymer coatings are expected to inhibit bacterial growth and
support the growth of fewer colonies of bacteria than uncoated
controls.
Example 11
.beta.-Galactosidase Polymer
[0205] A. Synthesis of a Photoreactive Polyacrylamide Containing
.beta.-Galactosidase (.beta.-Galactosidase Polymer)
[0206] A mixture containing 50 mg/ml of the NOS polymer (prepared
as described in Example 6) and 6.4 mg/ml .beta.-galactosidase (from
Boehringer Mannheim, Indianapolis, Ind.) in 0.1 M sodium carbonate,
pH 9, was prepared. The mixture was allowed to react at room
temperature for 1 hour and stored at 40.degree. C. overnight. The
resultant .beta.-galactosidase polymer solution was used without
purification for the generation of crosslinked films.
[0207] B. Generation of Crosslinked Films.
[0208] Films were cast by placing 40 ml aliquots of the
.beta.-galactosidase polymer solution (synthesized as described
herein) on a Teflon block and allowing each aliquot to dry. The
resulting films were illuminated for 0.5 or 4 minutes as described
above.
[0209] C. Assay for Integrity of the Films.
[0210] The integrity of the films was assayed by determining
whether they would retain their shape after being placed in a
solution of PBS. Films illuminated for 0.5 min. dissolved upon
exposure to saline; whereas films illuminated for 4 minutes
retained their shape. These results are consistent with
light-activation of the BBA groups producing covalent crosslinking
of invention polymer molecules.
[0211] The crosslinked .beta.-galactosidase polymer was washed 3
times with 1 ml of PBS to remove nonincorporated enzyme. The last
wash (0.2 ml) and the recovered film were each analyzed for enzyme
activity using o-nitrophenol-.beta.-D-galactopyranoside (o-NPG)
(from Pierce, Rockford, Ill.) at 1 mg/ml in water using the
protocol described in the "Worthington Enzyme Manual" (Worthington
Biochemical Corp., Freehold, N.J., 1977). The last wash showed no
.beta.-galactosidase activity while the film gave the yellow
nitrophenol product. This result demonstrated that the
.beta.-galactosidase moiety was active after the invention polymer
was crosslinked to form an insoluble biomaterial.
Example 12
DNA Polymer
[0212] A model oligodeoxynucleotide (oligoDNA) probe with a
sequence from exon 1 of the H-2K.sup.b gene of the major
histocompatibility complex was synthesized and used as a capture
probe. The sequence of the oligoDNA capture probe was
5'-GTCTGAGTCGGAGCCAGGGCGGCCGCCAACAGCAGGAGCA-3' and was synthesized
with an aliphatic C12 spacer at the 5' end that terminated with a
primary amine. The oligoDNA capture probe (80 .mu.g, or 6 nmole)
was coupled via the terminal amino group on the C12 spacer to 160
.mu.g NOS polymer described herein in 50 mM phosphate buffer (pH
8.5, 1 mM EDTA, 0.24 ml final volume) at room temperature for 2.5
hours. The resultant DNA polymer was utilized without further
purification or characterization.
[0213] The DNA-polymer was applied to microplate wells
(polypropylene plates from Coming Costar Corporation, Cambridge,
Mass.) at 10 pmole (in 0.1 ml solution) per well and incubated for
10-30 minutes. The plates containing DNA polymer solutions were
illuminated for 1.5 minutes with a Dymax lamp as described herein
except that a filter was used that removes light of wavelengths
shorter than 300 nm. A control consisted of oligoDNA capture probe
(10 pmole in 0.1 ml 50 mM phosphate buffer, pH 8.5, 1 mM EDTA) that
was added to wells, allowed to adsorb for 2.5 hr., and not
illuminated. The plates were washed with phosphate buffered saline
containing 0.05% Tween 20 in PBS to remove unbound DNA-polymer or
control oligoDNA capture probe.
[0214] A detection probe with a sequence complementary to the
capture probe described herein was synthesized with a biotin at the
5' end and used to evaluate the activity of the immobilized DNA
polymer. The sequence of the detection probe was
5'-CCGTGCACGCTGCTCCTGCTGTTGGCGGCCGCCC- TGGCTCCGACTCAGAC-3'. A
control detection probe consisting of a noncomplementary sequence
from exon 2 of the H-2K.sup.b gene was also synthesized with a
biotin moiety at the 5' end.
[0215] The binding of each detection probe was assayed by
subsequently adding a conjugate of streptavidin and horseradish
peroxidase (SA-HRP, available from Pierce, Rockford, Ill.) and
measuring the activity of the bound HRP. For this assay, the coated
plates were blocked with hybridization buffer (0.75 M NaCl, 0.075 M
citrate, pH 7.0, 0.1% lauroylsarcosine, 1% casein, and 0.02% sodium
dodecyl sulfate) at 55.degree. C. for 30 minutes. Complementary and
noncomplementary detection probes were added at 50 fmole per 0.1 ml
of hybridization buffer per well and incubated for one hour at
55.degree. C. The plates were then washed with 0.3 M NaCl, 0.03 M
citrate, pH 7.0 containing 0.1% SDS for 5 minutes at 55.degree. C.
SA-HRP was added at 0.5 .mu.g/ml and incubated for 30 minutes at
37.degree. C. The plates were then washed with 0.05% Tween 20 in
PBS, followed by addition of peroxidase substrate (TMB Microwell
Peroxidase substrate system from Kirkegarrd and Perry Laboratories,
Gaithersburg, Md.) and measurement of absorbance at 655 nm on a
microplate reader (model 3550, Bio-Rad Labs, Cambridge, Mass.).
Since the polypropylene plates were opaque, the reacted substrate
solutions were transferred to polystyrene plates to read the
absorbance.
[0216] Hybridization Signals (A.sub.655) from Polypropylene
Microwells Coated with Photoimmobilized DNA-polymer or Adsorbed
OligoDNA Capture Probe (n=3).
14 Complementary Noncomplementary detection probe detection probe
Adsorbed oligoDNA capture 0.037 .+-. 0.005 0.033 .+-. 0.001 probe
Photoimmobilized DNA 1.170 .+-. 0.079 0.068 .+-. 0.010 polymer
[0217] The results in the above table provide the hybridization
signals from polypropylene microwells coated with photoimmobilized
DNA-polymer or adsorbed oligoDNA capture probe (n=3). These results
demonstrate that the photoimmobilized DNA polymer binds 32-fold
more complementary detection probe than does the adsorbed control,
and neither coating binds the noncomplementary probe.
* * * * *