U.S. patent application number 11/539345 was filed with the patent office on 2007-04-12 for latent reactive blood compatible agents.
Invention is credited to Richard A. Amos, Aron B. Anderson, Peter H. Duquette, Terrence P. Everson, Patrick E. Guire.
Application Number | 20070082022 11/539345 |
Document ID | / |
Family ID | 26760475 |
Filed Date | 2007-04-12 |
United States Patent
Application |
20070082022 |
Kind Code |
A1 |
Guire; Patrick E. ; et
al. |
April 12, 2007 |
LATENT REACTIVE BLOOD COMPATIBLE AGENTS
Abstract
A reagent and related method for use in passivating a
biomaterial surface, the reagent including a latent reactive group
and a bifunctional aliphatic acid (e.g., fatty acid), in
combination with a spacer group linking the latent reactive group
to the aliphatic acid in a manner that preserves the desired
function of each group. Once bound to the surface, via the latent
reactive group, the reagent presents the aliphatic acid to the
physiological environment, in vivo, in a manner (e.g.,
concentration and orientation) sufficient to hold and orient
albumin.
Inventors: |
Guire; Patrick E.; (Eden
Praire, MN) ; Anderson; Aron B.; (Minnetonka, MN)
; Amos; Richard A.; (St. Anthony, MN) ; Everson;
Terrence P.; (Eagan, MN) ; Duquette; Peter H.;
(Edina, MN) |
Correspondence
Address: |
INTELLECTUAL PROPERTY GROUP;FREDRIKSON & BYRON, P.A.
200 SOUTH SIXTH STREET
SUITE 4000
MINNEAPOLIS
MN
55402
US
|
Family ID: |
26760475 |
Appl. No.: |
11/539345 |
Filed: |
October 6, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11212930 |
Aug 26, 2005 |
7144573 |
|
|
11539345 |
Oct 6, 2006 |
|
|
|
10422160 |
Apr 24, 2003 |
7071235 |
|
|
11212930 |
Aug 26, 2005 |
|
|
|
10207944 |
Jul 29, 2002 |
6555587 |
|
|
10422160 |
Apr 24, 2003 |
|
|
|
09177318 |
Oct 22, 1998 |
6465525 |
|
|
10207944 |
Jul 29, 2002 |
|
|
|
60078383 |
Mar 18, 1998 |
|
|
|
Current U.S.
Class: |
424/423 ;
424/78.08; 427/2.26 |
Current CPC
Class: |
A61K 31/12 20130101;
A61K 47/6957 20170801; A61L 27/227 20130101; A61L 33/0011 20130101;
A61L 29/048 20130101; A61L 31/10 20130101; A61K 31/74 20130101;
A61L 31/047 20130101; A61L 27/507 20130101; A61L 33/0029 20130101;
A61L 33/0082 20130101; A61L 27/34 20130101; A61L 29/085
20130101 |
Class at
Publication: |
424/423 ;
424/078.08; 427/002.26 |
International
Class: |
A61K 31/74 20060101
A61K031/74; A61F 2/02 20060101 A61F002/02 |
Claims
1-56. (canceled)
57. A passivating biomaterial comprising a surface prepared by (a)
providing a surface derivatized with a nucleophilic species, (b)
reacting the surface with a reactive molecule under conditions
suitable to react the reactive molecule with the nucleophilic
species in order to form a bifunctional aliphatic acid attached to
the surface by a covalent linkage.
58. A biomaterial according to claim 57 wherein the biomaterial is
selected from the group consisting of polyolefins, polystyrenes,
poly(methyl)methacrylates, polyacrylonitriles, poly(vinylacetates),
poly (vinyl alcohols), chlorine-containing polymers such as
poly(vinyl)chloride, polyoxymethylenes, polycarbonates, polyamides,
polyimides, polyurethanes, phenolics, amino-epoxy resins,
polyesters, silicones, cellulose-based plastics, and rubber-like
plastics.
59. A passivated biomaterial surface, comprising a biomaterial
surface having covalently attached thereto a reagent according to
claim 57, the surface being positioned in vivo under conditions
suitable to permit albumin molecules to be attracted and bound
thereto in order to passivate the surface.
60. A medical article fabricated from a passivating biomaterial
according to claim 59.
61. A medical article according to claim 60, wherein the article
comprises a blood-contacting medical device for in vivo
application.
62. A passivated biomaterial surface comprising the surface of
claim 59 having a proteinaceous material bound thereto.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application filed Apr. 24, 2003 and assigned Ser. No. 10/422,160,
which is a divisional of U.S. patent application filed Jul. 29,
2002 and assigned Ser. No. 10/207,944, which is a divisional of
U.S. patent application filed Oct. 22, 1998 and assigned Ser. No.
09/177,318, which claims the benefit of provisional U.S. patent
application filed Mar. 18, 1998 and assigned Ser. No. 60/078,383,
the entire disclosure of which is incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates to reagents and methods for
rendering a surface biocompatible, and in particular to reagents
and methods for "passivating" the surface of an implantable medical
device in order to render it hemocompatible. In another aspect, the
invention relates to biomedical devices, per se, and in particular
those having biocompatible, including hemocompatible,
tissue-contacting surfaces.
BACKGROUND OF THE INVENTION
[0003] Manufacturers of implantable medical devices have long
attempted to understand, and in turn improve, the performance of
materials used in blood-contacting applications (Leonard, E. F., et
al. Ann. N.Y. Acad. Sci. 516, New York, Acad. Sci., New York,
1987). The biological response of the body, as well as problems
with infection, have hindered the application of implantable,
disposable, and extracorporeal devices. Anticoagulant drugs, such
as heparin and coumadin, can improve the use of such devices,
although anticoagulants have their own corresponding risks and
drawbacks. For these reasons, development of materials having
greater compatibility with blood has been pursued aggressively
(Sevastianov, V. I., CRC Crit. Rev. Biocomp. 4:109, 1988).
[0004] Two general strategies that have been used to develop
improved blood-contacting materials include modifying the chemistry
of the bulk material itself, and/or modifying the interfacial
properties of the material. With regard to the latter approach,
several classes of materials have been covalently bonded onto
blood-contacting surfaces with the goal of improving blood
compatibility. These include anticoagulants, such as heparin and
hirudin; hydrogels; polyethylene oxide (PEO); albumin binding
agents; cell membrane components; prostaglandins; and sulfonated
polymers. These approaches have met with varying degrees of success
in terms of reducing protein adsorption, platelet adhesion and
activation, and thrombus formation. Unfortunately, no approach has
yet been shown to be universally applicable for improving
blood-biomaterial interactions.
[0005] As mentioned above, albumin binding agents have been
considered for use on biomaterials. Biomaterials having a high
surface concentration of albumin have been shown to be less likely
to initiate the fibrin cascade and platelet attachment than those
having a high concentration of other serum proteins, such as
fibrinogen, fibronectin, or immunoglobulins. On many polymeric
materials, however, fibrinogen is often the predominant protein
adsorbed from protein mixtures or plasma. For these reasons,
investigators have attempted to immobilize albumin onto materials
or to design biomaterial surfaces that will enhance binding of
endogenous albumin from blood, thus mitigating the adsorption of
fibrinogen and consequent thrombogenic phenomena.
[0006] In this respect, a number of different approaches have been
employed to date. These approaches include passive adsorption or
covalent immobilization of albumin to the surface, and the
development of surfaces designed to selectively bind endogenous
albumin from circulating blood, the latter using alkyl
chain-modified materials and other means.
[0007] Munro, et al., U.S. Pat. No. 4,530,974, discloses a method
of adsorbing albumin to a water-insoluble polymer such as
polyurethane by covalently binding to the surface a nonionic
hydrophobic aliphatic chain to which serum albumin will selectively
bind.
[0008] Frautschi et al., U.S. Pat. No. 5,017,670 and U.S. Pat. No.
5,098,977, teach methods for covalent attachment of aliphatic
extensions of 12 to 22 carbon atoms to water-insoluble polymers
containing aromatic rings and ring structures with adjacent
secondary hydroxyls for increased albumin binding.
[0009] Eaton, U.S. Pat. No. 5,073,171, describes a biocompatible
prosthetic device incorporating an amount of an albumin binding dye
effective to form a coating of endogeneous albumin on the device
when the device is in contact with a physiological fluid containing
albumin.
[0010] While some or all of these various strategies can be used to
enhance the binding of endogenous albumin to blood-contacting
material surfaces, and in turn to reduce fibrinogen binding, these
approaches are each limited in one or more respects. Alkyl
chain-modified surfaces have been shown to increase albumin binding
and decrease fibrinogen binding, but these effects were fairly
limited, for instance, to a short term time frame (generally less
than one hour). In addition, various other surface modification
methods discussed above are useful for only a narrow range of
substrate materials.
[0011] On another subject, the assignee of this application has
developed the ability to attach bioactive groups to a surface by
covalently bonding those groups, directly or indirectly, 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 photoreactive groups 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 5,512,329 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.
[0012] It would be highly desirable to be able to attach albumin to
a biomaterial surface in a manner that is suitably stable for
extended use, particularly in a manner that permits the albumin to
be replenished over time and in the course of use.
SUMMARY OF THE INVENTION
[0013] The present invention provides a novel reagent for use in
passivating a biomaterial surface, the reagent comprising a latent
reactive group and a bifunctional aliphatic acid, in combination
with a spacer group linking the latent reactive group to the
aliphatic acid in a manner that preserves the desired function of
each group. The reagent can be used to passivate a surface by
activating the latent reactive group in the presence of the surface
in order to covalently bond the reagent to the surface. Once bound
to the surface, the reagent presents the aliphatic acid to the
physiological environment, in vivo, in a manner (e.g.,
concentration and orientation) sufficient to hold and orient
albumin. Preferably, over time, the reagent surface is able to
replenish itself by replacing albumin molecules that have become
unbound or deteriorated with new albumin molecules. Albumin (e.g.,
human serum albumin (HSA)), is defined as any naturally occurring
proteinaceous moiety containing a fatty acid binding site.
[0014] In a preferred embodiment, the reagent is of the general
formula (X).sub.m--Y-(Z).sub.n where X is a latent reactive (e.g.,
photoreactive) group, Y is a spacer radical, and Z is a
bifunctional aliphatic acid, as each are described herein. The
values of m and n are .gtoreq.1 and while m can equal n, it is not
necessary. The aliphatic acid is `bifunctional` in that it provides
both an aliphatic region and an anionic (e.g., carboxylic acid)
region. Once attached to a surface, these portions cooperate in the
process of attracting and binding of albumin in order to passivate
the surface.
[0015] In the preferred embodiment where both m and n=1, the
reagent is termed a heterobifunctional reagent. The aliphatic acid
is preferably attached to the latent reactive group by means of a
divalent spacer group in a manner that does not detrimentally
affect the function of either the aliphatic or anionic portions.
Higher-valent spacer groups can also be selected which permit the
attachment of multiple aliphatic acid and latent reactive groups,
again in a manner which does not detrimentally affect the functions
of the respective groups. In this case m does not necessarily equal
n and both are .gtoreq.1.
[0016] In a further embodiment, the spacer group can be a
multivalent polymer having multiple sites along the backbone which
permit covalent attachment of the aliphatic acid and latent
reactive groups. These groups can be attached to a preformed
reactive polymer using conventional chemical coupling techniques or
may be incorporated during the polymerization process by use of
appropriately substituted monomers. In this embodiment, m does not
necessarily equal n and typically both are larger than one.
[0017] The invention further provides a method for preparing a
passivating reagent, as well as a method of using the reagent to
passivate the surface of a synthetic or natural biomaterial. In yet
a further embodiment, the invention provides a surface coated with
a passivating reagent of this invention, and in turn, an article
fabricated from a material providing a surface coated or coatable
with such a reagent. In yet a further embodiment, the invention
provides a passivated biomaterial surface having reagent attached
thereto and albumin attracted and attached to the bound
reagent.
DETAILED DESCRIPTION
[0018] The present invention permits the binding of albumin to a
surface to be enhanced by the use of a surface modification
reagent. The reagent includes a bifunctional aliphatic acid capable
of being attached to a surface in an amount and orientation that
improves the ability of the surface to attract and bind albumin.
While not intending to be bound by theory, it appears that a
surface bearing a reagent of this invention exhibits improved
albumin binding by virtue of both hydrophobic interactions (of the
alkyl chain) and ionic interactions (of the anionic moiety) with
albumin. It is expected that the hydrophobic interactions serve to
hold and orient the free albumin molecule, while the ionic
interactions serve to maintain the albumin molecule in position by
the addition of attractive ionic forces. In a particularly
preferred embodiment, the bifunctional aliphatic acid is attached
to either alkane, oxyalkane, or hydrophobic polymeric backbones to
allow both aliphatic and ionic regions of the bifunctional acid
analog to spacially orient away from the biomaterial surface to
induce better binding with native albumin. The reagent, in turn,
permits albumin binding surfaces to be created using a variety of
medical device materials, and in particular, for use in
blood-contacting medical devices.
Bifunctional Aliphatic Acid
[0019] The bifunctional aliphatic acid of the present invention
("Z" group) includes both an aliphatic portion and an anionic
portion. The word "aliphatic", as used herein, refers to a
substantially linear portion, e.g., a hydrocarbon backbone, capable
of forming hydrophobic interactions with albumin. The word
"anionic", in turn, refers to a charged portion capable of forming
further ionic interactions with the albumin molecule. By the use of
a reagent of this invention, these portions can be covalently
attached to a surface in a manner that retains their desired
function, in order to attract and bind native albumin from blood
and other bodily fluids.
[0020] In a preferred embodiment, the invention includes
photoactivatible molecules having fatty acid functional groups,
including polymers having multiple photoactivatible and fatty acid
functional groups, as well as heterobifunctional molecules.
Photoactivatible polyacrylamide copolymers containing multiple
pendant fatty acid analogs and multiple pendant photogroups have
been synthesized from acrylamide, a benzophenone-substituted
acrylamide, and N-substituted acrylamide monomers containing the
fatty acid analog. Photoactivatible polyvinylpyrrolidones have also
been prepared in a similar fashion. Polyacrylamide or
polyvinylpyrrolidone copolymers with a single end-point photogroup
and multiple pendant fatty acid analogs have also been synthesized.
Finally, photoactivatible, heterobifunctional molecules having a
benzophenone on one end and a fatty acid group on the other end
optionally separated by a spacer have been made, wherein that
spacer can be a hydrophobic alkyl chain or a more hydrophilic
polyethyleneglycol (PEG) chain.
Spacer Group
[0021] Suitable spacers ("Y" groups) for use in preparing
heterobifunctional reagents of the present invention include any
di- or higher-functional spacers capable of covalently attaching a
latent reactive group to an aliphatic acid in a manner that permits
them both to be used for their intended purpose. Although the
spacer may itself provide a desired chemical and/or physical
function, preferably the spacer is non-interfering, in that it does
not detrimentally affect the use of the aliphatic and ionic
portions for their intended purposes. In the case of the polymeric
reagents of the invention, the spacer group serves to attach the
aliphatic acid to the backbone of the polymer.
[0022] The spacer may be either aliphatic or polymeric and contain
various heteroatoms such as O, N, and S in place of carbon.
Constituent atoms of the spacers need not be aligned linearly. For
example, aromatic rings, which lack abstractable hydrogen atoms (as
defined below), can be included as part of the spacer design in
those reagents where the latent reactive group functions by
initiating covalent bond formation via hydrogen atom abstraction.
In its precursor form (i.e., prior to attachment of a photoreactive
group and aliphatic acid), a spacer can be terminated with any
suitable functionalities, such as hydroxyl, amino, carboxyl, and
sulfhydryl groups, which are suitable for use in attaching a
photoreactive group and the aliphatic acid by a suitable chemical
reaction, e.g., conventional coupling chemistry.
[0023] Alternatively, the spacer can be formed in the course of
combining a precursor containing (or capable of attaching) the
photoreactive group with another containing (or capable of
attaching) the aliphatic acid. For example, the aliphatic acid
could be reacted with an aliphatic diamine to give an aliphatic
amine derivative of the bifunctional aliphatic acid and which could
be coupled with a carboxylic acid containing the photogroup. To
those skilled in the art, it would be obvious that the photogroup
could be attached to any appropriate thermochemical group which
would react with any appropriate nucleophile containing O, N or
S.
[0024] Examples of suitable spacer groups include, but are not
limited to, the groups consisting of substituted or unsubstituted
alkylene, oxyalkylene, cycloalkylene, arylene, oxyarylene, or
aralkylene group, and having amides, ethers, and carbonates as
linking functional groups to the photoactivatible group, and the
bifunctional aliphatic fatty acid.
[0025] The spacer of the invention can also comprise a polymer
which serves as a 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, 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.
[0026] 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
polyvinylpyrrolidone 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 polyolthoesters.
[0027] The polymeric backbone is chosen to provide a backbone
capable of bearing one or more photoreactive groups, and one or
more fatty acid functional groups. The polymeric backbone is also
selected to provide a spacer between the surface and the various
photoreactive groups and fatty acid functional groups. In this
manner, the reagent can be bonded to a surface or to an adjacent
reagent molecule, to provide the fatty acid functional 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.
Photoreactive Group
[0028] In a preferred embodiment one or more photoreactive groups
are provided by the X groups attached to the central Y spacer
radical. Upon exposure to a suitable light source, each of the
photoreactive groups are subject to activation. The term
"photoreactive group", as used herein, refers to a chemical group
that responds to an applied external energy source in order to
undergo active specie generation, resulting in covalent bonding to
an adjacent chemical structure (e.g., an aliphatic carbon-hydrogen
bond).
[0029] Preferred X groups 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 groups 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.
[0030] Photoreactive aryl ketones are 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 group, since
it is capable of photochemical excitation with the initial
formation of an excited singlet state that undergoes intersystem
crossing to the triplet state. The excited triplet state can insert
into carbon-hydrogen bonds by abstraction of a hydrogen atom (for
example, from a support surface or target molecule in the solution
and in bonding proximity to the agent), 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. Hence, photoreactive aryl ketones are
particularly preferred.
[0031] The azides constitute a preferred class of latent reactive
groups 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 ethyl azidoforinate, 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 groups
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 (--CO--CN.sub.2--CO--O--) such as t-butyl alpha
diazoacetoacetate. Other photoreactive groups include aliphatic azo
compounds such as azobisisobutyronitrile, diazirines (--CHN.sub.2)
such as 3-trifluoromethyl-3-phenyldiazirine and ketenes
(--CH.dbd.C.dbd.O) such as ketene and diphenylketene.
[0032] Upon activation of the photoreactive groups, 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. TABLE-US-00001
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
Preparation of Reagents
[0033] Reagents of the present invention can be prepared by any
suitable means, depending upon the selection of either a
heterobifunctional reagent or a polymeric reagent. In the case of
the heterobifunctional reagents, the fatty acid residue is provided
by a fatty acid possessing a chemically reactive group on the alkyl
chain which permits covalent coupling of the remainder of the
heterobifunctional molecule to the fatty acid with preservation of
the carboxylic acid functionality. Preferably, the site of the
reactive group is in close proximity to the carboxylic acid group
so as to minimize effects on the binding activity of the
hydrophobic alkyl chain. Most preferably, the fatty acid residue
can be provided by a compound such n-tetradecylsuccinic anhydride
(TDSA). Reaction of such a molecule with a second molecule
possessing a nucleophilic species such as a primary amine results
in opening of the anhydride ring to give a fatty acid with an amide
linkage to the remainder of the molecule. This reaction generates a
pair of regioisomers depending upon the direction of the anhydride
ring opening. The second molecule in this reaction can be provided
by a spacer group, with or without a photoactivatible group, which
possesses a group capable of reaction with the fatty acid compound.
Most preferably, this spacer group possesses an amine which is
highly reactive with an anhydride species. The spacer group is
typically a bifunctional molecule which can have the
photoactivatible group attached prior to reaction with the fatty
acid derivative or the reverse order of reaction can be used. The
bifunctional spacer can be either heterobifunctional or
homobifunctional, with the former requiring a differential
reactivity in the first and second reaction steps and the latter
requiring an efficient method of separating the monofunctionalized
spacer following the first reaction. Optionally, no spacer is
required and a photoactivatible group possessing functionality
capable of reaction with the fatty acid derivative can be used. The
above examples are nonlimiting and the methods of accomplishing
these coupling reactions are apparent to those skilled in the
art.
[0034] Polymeric reagents of the invention can be prepared by
derivatization of preformed polymers possessing reactive groups
along the backbone of the polymer capable of reaction with the
photoactivatible groups and the fatty acid derivatives. For
example, polyacrylamide, polyvinylpyrrolidone, or siloxanes
functionalized with amine groups along the backbone, with or
without a spacer group, can be reacted with 4-benzoylbenzoyl
chloride (BBA-Cl) and TDSA to provide the photoactivatible and
fatty acid ligands respectively. Alternatively, the
photoactivatible and fatty acid groups can be prepared in the form
of polymerizable monomers which can then be copolymerized with
themselves and other monomers to provide polymers of the invention.
In a further embodiment of the invention, the photoactivatible
group can be introduced in the form of a chain transfer agent along
with the fatty acid monomer and other comonomers so as to provide a
polymer having the photoactivatible group at the end of the polymer
chain. For example, a chain transfer agent possessing two
derivatized benzophenones as the photoactivatible groups and a
mercaptan as the chain transfer agent can be used to copolymerize a
fatty acid monomer and acrylamide or N-vinylpyrrolidone monomers to
provide polymers of the invention. Alternatively, this polymer
could be prepared with reactive groups along the backbone, followed
by reaction with a fatty acid derivative.
Surfaces and Methods of Attachment.
[0035] The reagent of the present invention can be used to modify
any suitable surface. Where the latent reactive group is a
photoreactive group of the preferred type, it is particularly
preferred that the surface provide abstractable hydrogen atoms
suitable for covalent bonding with the activated group.
[0036] Plastics such as polyolefins, polystyrenes,
poly(methyl)methacrylates, polyacrylonitriles, poly(vinylacetates),
poly (vinyl alcohols), chlorine-containing polymers such as
poly(vinyl) chloride, polyoxymethylenes, polycarbonates,
polyamides, polyimides, polyurethanes, phenolics, amino-epoxy
resins, polyesters, silicones, cellulose-based plastics, and
rubber-like plastics can all be used as supports, providing
surfaces that can be modified as described herein. See generally,
"Plastics", pp. 462-464, in Concise Encyclopedia of Polymer Science
and Engineering, Kroschwitz, ed., John Wiley and Sons, 1990, the
disclosure of which is incorporated herein by reference. In
addition, supports such as those formed of pyrolytic carbon and
silylated surfaces of glass, ceramic, or metal are suitable for
surface modification.
[0037] Any suitable technique can be used for reagent binding to a
surface, and such techniques can be selected and optimized for each
material, process, or device. The reagent can be successfully
applied to clean material surfaces as listed above by spray, dip,
or brush coating of a solution of the fatty acid binding reagent.
The surface may be air-dried prior to illumination or the surface
can be illuminated while submerged in the coating solution. 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.
[0038] The "two-step" method would involve a first step of
photocoupling a hydrocarbon backbone to the surface, followed by a
second step of attaching (e.g., thermochemically) one or more fatty
acid derivatives to the immobilized backbone. For example, this two
step approach could involve covalently attaching a photoreactive
hydrocarbon backbone containing nucleophiles which could be used to
thermochemically couple fatty acid derivatives to the surface, or
directly attaching thermochemical groups (e.g. amines) to the
surface, followed by thermochemical attachment of one or more fatty
acid derivatives.
[0039] Alternatively, chemically reactive groups can be introduced
on the surface by a variety of non-photochemical methods, followed
by chemical coupling of the fatty acid group to the modified
surface. For example, amine groups can be introduced on a surface
by plasma treatment with a mixture of methane and ammonia and the
resulting amines can then be reached with TDSA to chemically couple
the fatty acid derivative to the surface through an amide linkage.
When desired, other approaches can be used for surface modification
using the reagent of the present invention. This approach is
particularly useful in those situations in which a support is
difficult to modify using conventional chemistry, or for situations
that require exceptional durability and stability of the target
molecule on the surface.
EXAMPLES
[0040] The invention will be further described with reference to
the following non-limiting Examples, which incorporate the
following table of formulas. It will be apparent to those skilled
in the art that many changes can be made in the embodiments
described without departing from the scope of the present
invention. Thus the scope of the present invention should not be
limited to the embodiments described in this application, but only
by embodiments described by the language of the claims and the
equivalents of those embodiments. Unless otherwise indicated, all
percentages are by weight. TABLE-US-00002 Compound/ Formula Example
Notation ##STR1## 1/1 4-Benzoylbenzoyl chloride ##STR2## 2/2
4-Bromoethyl- benzophenone ##STR3## 3/3 Poly(ethylene
glycol).sub.200Mono-4- benzoylbenzyl Ether ##STR4## 4/4
Poly(ethylene glycol).sub.200Mono-4- benzoylbenzyl Ether
Monomethanesulfonate ##STR5## 5/5 Monoaminopoly (ethylene
glycol).sub.200 Mono-4- benzoylbenzyl Ether ##STR6## 6/6 Mono-2-
(carboxymethyl) hexadecanamidopoly (ethylene glycol).sub.200Mono-4-
benzoylbenyl Ether ##STR7## 7/6 Mono-3- carboxyhepta-
decanamidopoly (ethylene glycol).sub.200Mono-4- benzoylbenzyl Ether
##STR8## 8/7 Mono-2- (carboxymethyl) hexadecanamidotetra (ethylene
glycol) Mono-4- benzoylbenzyl Ether ##STR9## 9/7 Mono-3-
carboxyhepta- decanamidotetra (ethylene glycol) Mono-4-
benzoylbenzyl Ether ##STR10## 10/8 N-[2-(4- Benzoylbenzyloxy)
ethyl]-2- (carboxymethyl) hexadecanamide ##STR11## 11/8 N-[2-(4-
Benzoylbenzyloxy) ethyl]-3- carboxylhepta- decanamide ##STR12##
12/9 N-[12- (Benzoylbenzyloxy) dodecyl]-2- (carboxymethyl)
hexadecanamide ##STR13## 13/9 N-[12- (Benzoylbenzyloxy)
dodecyl]-3-carboxy- heptadecanamide ##STR14## 14/10 N-[3-(4-
Benzoylbenzamido) propyl]-2- (carboxymethyl) hexadecanamide
##STR15## 15/10 N-[3-(4- Benzoylbenzamido) propyl]-3- carboxyhepta-
decamide ##STR16## 16/11 N-(3-Benzoylphenyl)- 2-(carboxymethyl)
hexadecanamide ##STR17## 17/11 N-(3-Benzoylphenyl)- 3-carboxyhepta-
decanamide ##STR18## 18/12 N-(4-Benzoylphenyl)- 2-(carboxymethyl)
hexadecanamide ##STR19## 19/12 N-(4-Benzoylphenyl)- 3-carboxyhepta-
decanamide ##STR20## 20/13 Monohexadecanamido- poly(ethylene
glycol).sub.200Mono-4- benzoylbenzyl Ether ##STR21## 21/14 Mono-3-
Carboxypropana- midpoly (ethylene glycol).sub.200Mono-4-
benzoylbenzyl Ether ##STR22## 22/15 Hexadecyl 4- benzoylbenzil
ether ##STR23## 23/16 Poly(ethylene glycol).sub.200Monohexadecyl
Mono-4- benzoylbenzyl Ether ##STR24## 24/17 Poly(ethylene
glycol).sub.200Mono-15- carboxypentadecyl Mono-4- benzoylbenzyl
Ether ##STR25## 25/18 Mono-15- carboxypenta- decanamidopoly
(ethylene glycol).sub.200 Mono-4- benzoylbenzyl Ether ##STR26##
26/19 N-[3- Methacrylamido) propyl]-2- (carboxymethyl)hexade-
canamide ##STR27## 27/19 N-[3- Methacrylamido) propyl]-3-
carboxyhepta- decanamide ##STR28## 28/20 N-[3-(4- Benzoylbenzamido)
propyl]methacrylamide ##STR29## 29/21 N-(2-Mercaptoethyl)-
3,5-bis(4- benzoylbenzyloxy) benzamide ##STR30## 30/22
Photoreactive Endpoint Copolymer of Acrylamide and Fatty Acid
Monomers ##STR31## 31/23 Photoreactive Random Copolymer of
Acrylamide and Fatty Acid Monomers ##STR32## 32A-C/24 Photoreactive
Endpoint Copolymer of N-Vinylpyrrolidone and Fatty Acid Monomers
##STR33## 33A-D/25 Photoreactive Random Copolymer of
N-Vinylpyrrolidone and Fatty Acid Monomers ##STR34## 34/26
Photoreactive Siloxane Copolymer Containing Fatty Acids Ligands
Example 1
Preparation of 4-Benzoylbenzoyl Chloride (BBA-Cl) (Compound 1)
[0041] 4-Benzoylbenzoic acid (BBA), 1.0 kg (4.42 moles), was added
to a dry 5 liter Morton flask equipped with reflux condenser and
overhead stirrer, followed by the addition of 645 ml (8.84 moles)
of thionyl chloride and 725 ml of toluene. Dimethylformamide (DMF),
3.5 ml, was then added and the mixture was heated at reflux for 4
hours. After cooling, the solvents were removed under reduced
pressure and the residual thionyl chloride was removed by three
evaporations using 3.times.500 ml of toluene. The product was
recrystallized from toluene/hexane (1/4) to give 988 g (91% yield)
after drying in a vacuum oven. Product melting point was
92-94.degree. C. Nuclear magnetic resonance (NMR) analysis at 80
MHz was consistent with the desired product. The final compound was
stored for use in the preparation of photoactivatable compounds, as
described for instance in Examples 10 and 20.
Example 2
Preparation of 4-Bromomethylbenzophenone (BMBP) (Compound 2)
[0042] 4-Methylbenzophenone, 750 g (3.82 moles), was added to a 5
liter Morton flask equipped with an overhead stirrer and dissolved
in 2850 ml of benzene. The solution was then heated to reflux,
followed by the dropwise addition of 610 g (3.82 moles) of bromine
in 330 ml of benzene. The addition rate was approximately 1.5
ml/min and the flask was illuminated with a 90 watt (90 joule/sec)
halogen spotlight to initiate the reaction. A timer was used with
the lamp to provide a 10% duty cycle (on 5 seconds, off 40
seconds), followed in one hour by a 20% duty cycle (on 10 seconds,
off 40 seconds). At the end of the addition, the product was
analyzed by gas chromatography and was found to contain 71% of the
desired 4-bromomethylbenzophenone, 8% of the dibromo product, and
20% unreacted 4-methylbenzophenone. After cooling, the reaction
mixture was washed with 10 g of sodium bisulfite in 100 ml of
water, followed by washing with 3.times.200 ml of water. The
product was dried over sodium sulfate and recrystallized twice from
toluene/hexane (1/3 by volume (v/v)). After drying under vacuum,
635 g of BMBP were isolated, providing a yield of 60% and having a
melting point of 112-114.degree. C. Analysis on an NMR spectrometer
was consistent with the desired product. The final compound was
stored for use in the preparation of photoactivatable compounds, as
described for instance in Examples 3, 7, 8, 9, 15, and 21.
Example 3
Preparation of Poly(ethylene glycol).sub.200 Mono-4-benzoylbenzyl
Ether (Compound 3)
[0043] The poly(ethylene glycol).sub.200 (PEG), 72.72 g (0.363
mol), was azeotroped with 200 ml of toluene for two hours to remove
moisture, followed by the removal of excess toluene under vacuum.
The PEG residue was then dissolved in 400 ml of anhydrous
tetrahydrofuran (THF) while stirring under argon at 4.degree. C.
Sodium hydride, 2.90 g of a 60% mixture in mineral oil (72.5 mmol),
was added in portions and the mixture was stirred 1 hour at room
temperature. BMBP, 20.0 g (72.7 mmol), prepared according to the
general method described in Example 2, was added as a solution in
100 ml of THF over a 2 hour period and the mixture was stirred 16
hours at room temperature under argon. The reaction was quenched
with aqueous ammonium chloride (36 g in 200 ml of water) and the
organic solvent was removed under vacuum. The residue was dissolved
in brine, extracted with chloroform, and the resulting organic
extracts were dried over sodium sulfate. The product was isolated
as a viscous oil by adding the chloroform solution to diethyl
ether, resulting in precipitation of 27.64 g of the desired
product. The product was used without additional purification.
Analysis on an NMR spectrometer was consistent with the desired
product.
Example 4
Preparation of Poly(ethylene glycol.sub.200 Mono-4-benzoylbenzyl
Ether Monomethanesulfonate (Compound 4)
[0044] Compound 3, 3.0 g (7.61 mmol), prepared according to the
general method described in Example 3, was dissolved in 25 ml of
methylene chloride, followed by the addition of 1.5 g (14.8 mmol)
of triethylamine (TEA). The mixture was cooled on an ice bath under
argon and 1.3 g (11.3 mmol) of methanesulfonyl chloride (MsCl) were
added dropwise over a 10 minute period. The reaction temperature
was allowed to rise to ambient temperature overnight. The
precipitated salts were removed by filtration and the solvent was
removed under vacuum. The residue was dissolved in toluene and
filtered to remove solids, followed by evaporation under vacuum to
give 3.01 g of product. No further purification of the product was
done at this point. Analysis on an NMR spectrometer was consistent
with the desired product.
Example 5
Preparation of Monoaminopoly(ethylene glycol).sub.200
Mono-4-benzoylbenzyl Ether (Compound 5)
[0045] Compound 4, 17.97 g (38.07 mmol), prepared according to the
general method described in Example 4, was dissolved in 100 ml of
anhydrous THF in a thick-walled tube, followed by the addition of
100 ml of concentrated ammonium hydroxide. The tube was sealed and
the two phase mixture was stirred vigorously at 65.degree. C. for
16 hours. The solvent was removed under vacuum and the resulting
residue was dissolved in chloroform. The product was loaded on a
silica gel flash chromatography column and eluted with
chloroform/acetone/acetic acid (60/40/1 v/v) until all of the less
polar impurities were removed. The product was then eluted with
chloroform/methanol/acetic acid/water (85/15/1/1 v/v). The
fractions which were UV, ninhydrin, and Dragendorff positive were
pooled and the solvent was removed under vacuum to give 8.63 g of
product. Analysis on an NMR spectrometer was consistent with the
desired product.
Example 6
Preparation of Mono-2-(carboxymethyl)hexadecanamidopoly(ethylene
glycol).sub.200 Mono-4-benzoylbenzyl Ether (Compound 6) and
Mono-3-carboxyheptadecanamidopoly(ethylene glycol.sub.200
Mono-4-benzoylbenzyl Ether (Compound 7)
[0046] Compound 5, 3.03 g (7.71 mmol), prepared according to the
general method described in Example 5, and TEA, 2.24 g (22.1 mmol),
were dissolved in 30 ml of methylene chloride, followed by the
addition of 2.40 g (8.10 mmol) of TDSA as the solid. The reaction
mixture was stirred 18 hours at room temperature under argon. The
solvents were removed under vacuum and the resulting oil was
purified by silica gel flash chromatography using a solvent
gradient: 500 ml of ether/hexane (75/25 v/v); 500 ml of
ether/hexane/acetic acid (75/25/1 v/v); chloroform/acetone/acetic
acid (60/40/1 v/v); and chloroform/methanol/acetic acid/water
(85/15/1/1 v/v). The fractions were pooled to give two separate UV
and Dragendorff positive materials representing the regioisomers
resulting from ring opening of the anhydride ring. Evaporation of
solvent gave 1.35 g of product in one fraction and 0.893 g in the
second. Analysis on an NMR spectrometer was consistent with the
desired products.
Example 7
Preparation of Mono-2-(carboxymethyl)hexadecanamidotetra(ethylene
glycol)Mono-4-benzoylbenzyl Ether (Compound 8) and
Mono-3-carboxyheptadecanamidotetra(ethylene glycol)
Mono-4-benzoylbenzyl Ether (Compound 9)
[0047] The tetraethylene glycol (TEG), 7.063 g (36.4 mmol), was
azeotroped with 200 ml of toluene for two hours to remove moisture,
followed by the removal of excess toluene under vacuum. The TEG
residue was then dissolved in 70 ml of anhydrous THF while stirring
under argon on an ice bath. Sodium hydride, 1.45 g of a 60% mixture
in mineral oil (36.3 mmol), was added and the mixture was stirred 1
hour at room temperature. BMBP, 5.0 g (18.2 mmol), prepared
according to the general method described in Example 2, was added
and the mixture was stirred 16 hours at room temperature under
argon. The reaction was quenched with aqueous ammonium chloride (9
g in 40 ml of water) and the organic solvent was removed under
vacuum. The residue was dissolved in saturated brine, extracted
with chloroform, and the resulting organic extracts were dried over
sodium sulfate. The product was isolated as a viscous oil by adding
the chloroform solution to diethyl ether. The crude product, 7.6 g,
was used without additional purification.
[0048] The entire product from above was dissolved in 200 ml of
methylene chloride, followed by the addition of 3.96 g (39.1 mmol)
of TEA. The mixture was cooled to 4.degree. C. under argon and 3.35
g (29.2 mmol) of MsCl were added. After 6 hours, an additional 1 ml
each of TEA and MsCl were added and the reaction was left to stir
for 16 hours to insure complete reaction. The precipitated salts
were removed by filtration and the solvent was removed under
vacuum. The residue was dissolved in toluene and filtered to remove
solids, followed by evaporation under vacuum. No further
purification of the product was done at this point.
[0049] The entire mesylate product from above was dissolved in 50
ml of THF in a thick-walled glass tube, followed by the addition of
50 ml of concentrated ammonium hydroxide. The tube was sealed and
the two phase mixture was stirred vigorously at 65.degree. C. for
16 hours. The solvent was removed under vacuum and the resulting
residue was dissolved in 20 ml of chloroform. After drying over
sodium sulfate, the product was precipitated by addition of the
chloroform solution to diethyl ether resulting in approximately 4.5
g of a brown viscous oil. A portion of the product, approximately 1
g, was purified by silica gel flash chromatography using a solvent
gradient of ether/hexane/acetic acid (75/25/1 v/v), followed by
chloroform/acetone/acetic acid (60/40/1 v/v), and
chloroform/ethanol/water/acetic acid (85/15/1/1 v/v). A total of
220 mg of purified product were isolated. Analysis on an NMR
spectrometer was consistent with the desired product.
[0050] The amine product from above, 0.220 g (0.568 mmol), and TEA,
63 mg (0.623 mmol), were dissolved in 20 ml of methylene chloride,
followed by the addition of 0.185 g (0.625 mmol) of TDSA. The
reaction mixture was stirred 48 hours at room temperature under
argon. The solvents were removed under vacuum and the resulting oil
was purified by silica gel flash chromatography using an
chloroform/methanol/water/acetic acid (85/15/1/1 v/v). The
appropriate fractions were pooled, evaporated, redissolved in
chloroform, and dried over sodium sulfate. Evaporation of solvent
gave 234 mg of a waxy solid as a mixture of regioisomers resulting
from opening of the anhydride ring. Analysis on an NMR spectrometer
was consistent with the desired products.
Example 8
Preparation of
N-[2-(4-Benzoylbenzyloxy)ethyl]-2-(carboxymethyl)hexadecanamide
(Compound 10) and
N-[2-(4-Benzoylbenzyloxy)ethyl]-3-carboxylheptadecanamide (Compound
11)
[0051] Anhydrous ethanolamine, 1.00 g (16.4 mmol), was dissolved in
5 ml of anhydrous THF with stirring under argon. Sodium hydride,
0.655 g (16.4 mmol) of a 60% mineral oil dispersion, was added as a
solid followed by an additional 5 ml of anhydrous THF. The
resulting mixture was stirred at room for 45 minutes at which time
no more hydrogen evolution was observed. The BMBP, 4.50 g (16.4
mmol), prepared according to the general method described in
Example 2, was added as a solution in 25 ml of THF over a 30 minute
period. The reaction was allowed to stir overnight at room
temperature. The reaction was quenched with water and the product
was extracted with chloroform. The organic extract was washed with
0.1 N HCl and the aqueous solution was washed one time with
chloroform. The aqueous was then evaporated under vacuum, dissolved
in 10% methanol in chloroform (v/v) and dried over sodium sulfate.
Evaporation of solvent gave 2.62 g of a pale yellow solid which was
used without additional purification.
[0052] The above amine, 0.625 g (2.14 mmol), and TDSA, 0.467 g
(1.57 mmol), were dissolved in 10 ml of methylene chloride,
followed by the addition of 660 .mu.l (4.74 mmol) of TEA. The
resulting solution was stirred at room temperature for 16 hours to
complete the reaction. The product was diluted with water and
treated with 5% HCl, followed by separation of the organic layer
and drying over sodium sulfate. The solvent was removed under
vacuum and the product was purified using silica gel flash
chromatography with a solvent gradient of chloroform followed by
2.5% and 5% (v/v) methanol in chloroform. The appropriate fractions
were pooled to give 357 mg of product as a pair of regioisomers
resulting from the opening of the anhydride ring. Analysis on an
NMR spectrometer was consistent with the desired products.
Example 9
Preparation of
N-[12-(Benzoylbenzyloxy)dodecyl]-2-(carboxymethyl)hexadecanamide
(Compound 12) and
N-[12-(Benzoylbenzyloxy)dodecyl]-3-carboxyheptadecanamide (Compound
13)
[0053] 1,12-Dodecanediol, 5.0 g (24.7 mmol), was dissolved in 50 ml
of anhydrous THF in a dry flask under nitrogen. The sodium hydride,
0.494 g of a 60% dispersion in mineral oil (12.4 mmol), was added
in portions over a five minute period. The resulting mixture was
stirred at room temperature for one hour. BMBP, 3.40 g (12.4 mmol),
prepared according to the general method described in Example 2,
was added as a solid along with sodium iodide (0.185 g, 1.23 mmol)
and tetra-n-butylammonium bromide (0.398 g, 1.23 mmol). The mixture
was stirred at a gentle reflux for 24 hours. The reaction was then
cooled, quenched with water, acidified with 5% HCl, and extracted
with chloroform. The organic extracts were dried over sodium
sulfate and the solvent was removed under vacuum. The product was
purified on a silica gel flash chromatography column using
chloroform to elute non-polar impurities, followed by elution of
the product with chloroform/ethyl acetate (80/20 v/v). Pooling of
appropriate fractions and evaporation of solvent gave 3.42 g of
product, a 70% yield. Analysis on an NMR spectrometer was
consistent with the desired product.
[0054] The above alcohol, 1.30 g (3.28 mmol), was dissolved in 13
ml of anhydrous methylene chloride, followed by 0.829 g (8.19 mmol)
of TEA and cooling on an ice bath under argon. MsCl, 0.563 g (4.91
mmol), was added dropwise over a five minute period, followed by
stirring at room temperature for 16 hours. The reaction was diluted
with water, acidified with 5% HCl, and extracted with chloroform.
The organic extracts were dried over sodium sulfate and evaporated
to give 1.56 g of a yellow oil. This product was used without
further purification. Analysis on an NMR spectrometer was
consistent with the desired product.
[0055] The above mesylate, 1.56 g (3.28 mmol), was dissolved in 25
ml of THF in a thick-walled tube, followed by the addition of 25 ml
of ammonium hydroxide. The tube was sealed and the mixture was
stirred vigorously for 72 hours at 80.degree. C. The mixture was
treated with 200 ml of water and the product was extracted with
chloroform. The organic extracts were dried over sodium sulfate and
the product was purified on a silica gel flash chromatography
column. The column was eluted with chloroform and
chloroform/methanol (95/5 v/v) until the less polar impurities were
removed, followed by elution of the desired product using
chloroform/methanol/ammonium hydroxide (70/25/5 v/v). Pooling of
the ninhydrin and UV active fractions and evaporation of solvent
gave 0.526 g of product, a 40% yield. Analysis on an NMR
spectrometer was consistent with the desired product.
[0056] The above amine, 0.440 g (1.11 mmol), was dissolved in 7 ml
of methylene chloride, followed by 0.329 g (1.11 mmol) of TDSA and
0.337 g (3.33 mmol) of TEA. The resulting mixture was stirred at
room temperature for 36 hours. The reaction was then diluted with
water, acidified with 5% HCl, and extracted with chloroform. The
organic extracts were dried over sodium sulfate and the residue
after evaporation was purified on silica gel flash chromatography.
A solvent gradient of chloroform, 2.5% methanol in chloroform
(v/v), and 5% methanol in chloroform (v/v) was used to elute the
product. A total of 378 mg of product were isolated as a partially
resolved pair of regioisomers resulting from opening of the
anhydride ring. Analysis on an NMR spectrometer was consistent with
the desired products.
Example 10
Preparation of
N-[3-(4-Benzoylbenzamido)propyl]-2-(carboxymethyl)hexadecanamide
(Compound 14) and
N-[3-(4-Benzoylbenzamido)propyl]-3-carboxyheptadecanamide (Compound
15)
[0057] 1,3-Diaminopropane, 1.910 kg (25.77 mol), was placed in a 12
liter Morton flask and diluted with 1000 ml of methylene chloride.
After cooling to below 10.degree. C. on an ice bath, a solution of
1.005 kg (5.175 mol) of t-butyl phenyl carbonate in 250 ml of
methylene chloride was added slowly to the diamine while keeping
the temperature below 15.degree. C. at all times. Once the addition
was complete, the mixture was warmed to room temperature for 2
hours to complete the reaction. The reaction was further diluted
with 900 ml of methylene chloride, followed by the addition of 500
g of ice and a slow addition of 2500 ml of 2.2 N NaOH. The organic
layer was separated and the basic aqueous solution was extracted
with 3.times.1250 ml of methylene chloride, keeping each extract
separate. Each of these separate extracts was successively washed
with 1250 ml of 0.6 N NaOH, beginning with the first extract and
proceeding to the last. This wash procedure was repeated and the
organic extracts were combined and dried over sodium sulfate.
Evaporation of solvent yielded 825 g of product for a 92% yield.
This product was used without any further purification. Analysis on
an NMR spectrometer was consistent with the desired product.
[0058] The above amine, 0.774 g (4.44 mmol), was diluted with 20 ml
of anhydrous methylene chloride, followed by the addition of 1.24 g
(12.3 mmol) of TEA and a dropwise addition of 10 ml of anhydrous
methylene chloride containing of 1.0 g (4.09 mmol) of BBA-CL,
prepared according to the general method described in Example 1,
After stirring 1.5 hours at room temperature, the reaction was
diluted with water and acidified with 1 N HCl. The product was
extracted with chloroform and the organic extracts were dried over
sodium sulfate. Silica gel flash chromatography using
chloroform/methanol (90/10 v/v) gave 1.68 g of product, slightly
greater than theoretical because of solvent residues. Mass spectral
analysis confirmed the desired product.
[0059] The above product, 1.5 g (3.95 mmol), was dissolved in 10 ml
of trifluoroacetic acid under a nitrogen atmosphere. After stirring
3 hours at room temperature to remove the t-butyloxycarbonyl
(t-BOC) protecting group, the solvent was removed under reduced
pressure and the product was purified using silica gel flash
chromatography. After removal of the less polar impurities with
chloroform/methanol (90/10 v/v), the eluting solvent was switched
to chloroform/methanol/ammonium hydroxide (70/25/5 v/v) for
isolation of the desired product. Pooling of the appropriate
fractions and evaporation of solvent gave 1.77 g of product.
Analysis on an NMR spectrometer was consistent with the desired
product.
[0060] A portion of above amine product, 0.500 g (1.77 mmol), was
dissolved in 10 ml of anhydrous methylene chloride under an argon
atmosphere. TEA, 0.197 g (1.95 mmol), was added, followed by 0.577
g (1.95 mmol) of TDSA. The reaction was stirred for four hours at
room temperature. The mixture was diluted with water, extracted
with methylene chloride, and the organic extracts were dried over
sodium sulfate. After vacuum removal of solvents, the product was
purified by silica gel flash chromatography using a
chloroform/methanol/acetic acid/water (85/15/1/1 v/v) system. A
repeat chromatography using a 0.fwdarw.5% methanol in chloroform
(v/v) system gave a more pure product. A total of 0.259 g of
product (25% yield) were isolated as a pair of regioisomers
resulting from opening of the anhydride ring. Analysis on an NMR
spectrometer was consistent with the desired products.
Example 11
Preparation of N-(3-Benzoylphenyl)-2-(carboxymethyl)hexadecanamide
(Compound 16) and N-(3-Benzoylphenyl)-3-carboxyheptadecanamide
(Compound 17)
[0061] The 3-aminobenzophenone, 0.500 g (2.53 mmol), was dissolved
in 5.0 ml of dry DMF along with 0.512 g (5.06 mmol) of TEA and
0.030 g (0.25 mmol) of 4-dimethylaminopyridine. While stirring
under argon, 0.826 g (2.79 mmol) of TDSA were added and the
resulting solution was stirred at 45.degree. C. overnight. The
reaction was diluted with water and the desired product was
extracted with chloroform. After drying over sodium sulfate, the
solvent was removed and the product was purified on silica gel
flash chromatography. The less polar impurities were eluted with
chloroform and the product was eluted with a 2.5.fwdarw.5.0%
methanol in chloroform (v/v) gradient. A total of 1.048 g of
product were isolated with a partial resolution of the two
regioisomers resulting from opening of the anhydride ring system.
Analysis on an NMR spectrometer was consistent with the desired
products.
Example 12
Preparation of N-(4-Benzoylphenyl)-2-(carboxymethyl)hexadecanamide
(Compound 18) and N-(4-Benzoylphenyl)-3-carboxyheptadecanamide
(Compound 19)
[0062] The 4-aminobenzophenone, 0.500 g (2.53 mmol), was dissolved
in 7.0 ml of dry DMF along with 0.512 g (5.06 mmol) of TEA and
0.030 g (0.25 mmol) of 4-dimethylaminopyridine. While stirring
under argon, 0.826 g (2.79 mmol) of TDSA were added and the
resulting solution was stirred at 55.degree. C. for 80 hours. At
this time, thin layer chromatography (TLC) revealed partial
conversion to a less polar UV active product. The reaction was
diluted with water and the desired product was extracted with
chloroform. After drying over sodium sulfate, the solvent was
removed and the product was purified on silica gel flash
chromatography. The less polar impurities were eluted with
chloroform and the product was eluted with a 2.5.fwdarw.5.0%
methanol in chloroform (v/v) gradient. A total of 0.753 g of
product were isolated with a partial resolution of the two
regioisomers resulting from opening of the anhydride ring system.
Analysis on an NMR spectrometer was consistent with the desired
products.
Example 13
Preparation of Monohexadecanamidopoly(ethylene glycol).sub.200
Mono-4-benzoylbenzyl Ether (Compound 20)
[0063] Compound 5, 0.914 g (2.32 mmol), prepared according to the
general method described in Example 5, was dissolved in 10 ml of
anhydrous chloroform with stirring under argon. TEA, 0.516 g (5.10
mmol), was added followed by the slow dropwise addition of 0.701 g
(2.55 mmol) of palmitoyl chloride. The resulting mixture was
stirred at room temperature overnight. The reaction was diluted
with water and the product was extracted with chloroform. After
drying over sodium sulfate, the solvent was removed under vacuum
and the product was purified by silica gel chromatography. A
chloroform/methanol (95/5) solvent was used to elute the product,
yielding 382 mg of a viscous oil. Analysis on an NMR spectrometer
was consistent with the desired product.
Example 14
Preparation of Mono-3-Carboxypropanamidopoly(ethylene
glycol).sub.200 Mono-4-benzoylbenzyl Ether (Compound 21)
[0064] Compound 5, 0.500 g (1.27 mmol), prepared according to the
general method described in Example 5, was dissolved in 5 ml of
anhydrous chloroform along with 0.14 g (1.40 mmol) of succinic
anhydride. After solution was complete, 0.141 g (1.39 mmol) of TEA
were added with stirring under argon. The resulting mixture was
stirred at room temperature for 24 hours. The solvent was then
removed under vacuum and the product was purified on a silica gel
flash chromatography column using a chloroform solvent, followed by
a chloroform/methanol (95/5 to 90/10 v/v) solvent gradient. Pooling
of appropriate fractions and evaporation of solvent gave 447 mg of
a viscous oil. Analysis on an NMR spectrometer was consistent with
the desired product.
Example 15
Preparation of Hexadecyl 4-Benzoylbenzyl Ether (Compound 22)
[0065] 1-Hexadecanol, 5.0 g (20.6 mmol), was dissolved in 10 ml of
anhydrous THF with warming, followed by slow addition of 0.840 g
(21.0 mmol) of a 60% dispersion of NaH in mineral oil. Once the
hydrogen evolution was complete, 6.35 g (23.1 mmol) of BMBP,
prepared according to the general method described in Example 2,
were added. The reaction mixture was stirred at 50.degree. C. under
argon for one hour and then at room temperature for 16 hours. After
this time the reaction was quenched with water and the product was
extracted with chloroform. After drying over sodium sulfate, the
solvent was removed under vacuum and the residue was purified by
silica gel flash chromatography using a hexane/ether (90/10)
solvent. Appropriate fractions were pooled and evaporated to give
8.01 g of a waxy solid, an 88.9% yield. Analysis on an NMR
spectrometer was consistent with the desired product.
Example 16
Preparation of Poly(ethylene glycol).sub.200 Monohexadecyl
Mono-4-benzoylbenzyl Ether (Compound 23)
[0066] Compound 3, 1.00 g (2.54 mmol), prepared according to the
general method described in Example 3, was dissolved in 10 ml of
anhydrous THF under an argon atmosphere. Sodium hydride, 0.112 g
(2.80 mmol) of a 60% dispersion in mineral oil, was added in
portions while stirring on an ice bath. The mixture was allowed to
stir 20 minutes at room temperature, followed by the addition of
0.776 g (2.54 mmol) of 1-bromohexadecane. The mixture was stirred
overnight at room temperature. The reaction was quenched with water
and the product was extracted with chloroform. After drying over
sodium sulfate and removal of solvent, the product was purified by
silica gel flash chromatography using a chloroform/methanol/acetic
acid/water (85/15/1/1 v/v) solvent as eluent. The appropriate
fractions were pooled to give 1.357 g of product, an 86% yield.
Analysis on an NMR spectrometer was consistent with the desired
product.
Example 17
Preparation of Poly(ethylene glycol).sub.200
Mono-15-carboxypentadecyl Mono-4-benzoylbenzyl Ether (Compound
24)
[0067] 10-Hydroxyhexadecanoic acid, 0.785 g (2.88 mmol), was
dissolved in 20 ml of anhydrous DMF in a dry flask under argon.
Sodium hydride, 0.260 g (6.5 mmol) of a 60% dispersion in mineral
oil, was then added and the resulting slurry was stirred at
60.degree. C. for four hours. After this time, Compound 4, 1.24 g
(2.62 mmol), prepared according to the general method described in
Example 4, was added as a solution in 7 ml of DMF. The resulting
slurry was stirred at room temperature for 72 hours. After this
time, the reaction was quenched with water and the product was
extracted with chloroform. After drying over sodium sulfate, the
product was purified on a silica gel flash chromatography column.
The column was eluted with chloroform/methanol (95/5 v/v) until the
less polar impurities were removed, followed by elution of the
product with chloroform/methanol/acetic acid/water (90/10/1/1 v/v).
The appropriate fractions were pooled and evaporated to yield 1.24
g of product, a 74% yield. Analysis on an NMR spectrometer was
consistent with the desired product.
Example 18
Preparation of Mono-15-carboxypentadecanamidopoly(ethylene
glycol).sub.200 Mono-4-benzoylbenzyl Ether (Compound 25)
[0068] Hexadecanedioic acid, 0.500 g (1.75 mmol), was dissolved in
5 ml of anhydrous DMF with stirring under an argon atmosphere.
N-Hydroxysuccinimide, 0.442 g (3.84 mmol) and
dicyclohexylcarbodiimide, 1.44 g (6.98 mmol), were added and the
mixture was stirred for six hours at room temperature. The
resulting solid was removed by filtration and the filter cake was
washed with 1 ml of DMF. The solution was then reacted with 0.747 g
(1.90 mmol) of Compound 5, prepared according to the general method
described in Example 5, dissolved in 5 ml of DMF and 0.389 g (3.84
mmol) of TEA. After stirring two hours at room temperature, TLC
showed complete consumption of the starting amine. The product was
purified on a silica gel flash chromatography column by eluting
less polar impurities using chloroform and elution of the desired
product using a chloroform/methanol/acetic acid/water (85/15/1/1
v/v) solvent. The appropriate fractions were pooled and evaporated
to give 1.356 g of product. Analysis on an NMR spectrometer was
consistent with the desired product.
Example 19
Preparation of
N-[3-Methacrylamido)propyl]-2-(carboxymethyl)hexadecanamide
(Compound 26) and
N-[3-Methacrylamido)propyl]-3-carboxyheptadecanamide (Compound
27)
[0069] N-(3-Aminopropyl)methacrylamide hydrochloride (APMA-HCl),
6.064 (33.9 mmol), was dissolved in anhydrous methylene chloride
along with 10.24 g (101 mmol) of TEA. TDSA, 10.0 g (33.7 mmol), was
immediately added and the mixture was stirred 48 hours at room
temperature with moisture protection from a drying tube. After this
time, the reaction was acidified with 1 N HCl, extracted with
chloroform, and dried over sodium sulfate. The product was purified
on a silica gel chromatography column using a
chloroform/methanol/acetic acid/water (85/15/1/1 v/v) solvent. The
appropriate fractions were pooled, 100 ppm of phenothiazine were
added, and the solvent was removed under reduced pressure to give
16.0 g of product as a pair of regioisomers resulting from opening
of the anhydride ring. Analysis on an NMR spectrometer was
consistent with the desired products.
Example 20
Preparation of N-[3-(4-Benzoylbenzamido)propyl]methacrylamide
(BBA-APMA) (Compound 28)
[0070] APMA-HCl, 120.0 g (0.672 mol), was suspended in 800 ml of
chloroform along with 25 mg of phenothiazine. The solution was
cooled to below 10.degree. C., followed by the addition of 172.5 g
(0.705 mol) of BBA-Cl, prepared according to the general method
described in Example 1. A solution of 150.3 g (1.49 moles) of TEA
in 50 ml of chloroform was prepared and the solution was added
dropwise to the above suspension over a 1-1.5 hour time period
while stirring on an ice bath. After completion of the addition,
the ice bath was removed and the solution was stirred for 2.5 hours
to complete the reaction. The mixture was then washed with 600 ml
of 0.3 N HCl followed by 2.times.300 ml of 0.07 N HCl. The
chloroform solution was then dried over sodium sulfate and the
product was recrystallized twice using a toluene/chloroform (4/1
v/v) mixture. Phenothiazine, 25 mg, was added prior to the second
recrystallization to prevent premature polymerization. The yield
was 212 g (90% yield) with a melting point of 147-151.degree. C.
Analysis on an NMR spectrometer was consistent with the desired
product.
Example 21
Preparation of
N-(2-Mercaptoethyl)-3,5-bis(4-benzoylbenzyloxy)benzamide (Compound
29)
[0071] A photoactivatable chain transfer reagent was prepared in
the following manner, and used in the manner described in Examples
22 and 24. 3,5-Dihydroxybenzoic acid, 46.2 g (0.30 moles), was
weighed into a 250 ml flask equipped with a Soxhlet extractor and
condenser. Methanol, 48.6 ml, and concentrated sulfuric acid, 0.8
ml, were added to the flask and 48 g of 3A molecular sieves were
placed in the Soxhlet extractor. The extractor was diluted with
methanol and the mixture was heated at reflux overnight. Gas
chromatographic analysis on the resulting product showed a 98%
conversion to the desired methyl ester. The solvent was removed
under reduced pressure to give approximately 59 g of crude product.
This product was used in the following step without further
purification. A small sample was purified for NMR analysis,
resulting in a spectrum consistent with the desired product.
[0072] The entire methyl ester product from above was placed in a 2
liter flask with overhead stirrer and condenser, followed by the
addition of 173.25 g (0.63 mol) of BMBP, prepared according to the
general method described in Example 2, 207 g (1.50 mol) of
potassium carbonate, and 1200 ml of acetone. The resulting mixture
was then refluxed overnight to give complete reaction as indicated
by TLC. The solids were removed by filtration and the acetone was
evaporated under reduced pressure to give 49 g of crude product.
The solids were diluted with 1 liter of water and extracted with
3.times.1 liter of chloroform. The extracts were combined with the
acetone soluble fraction and dried over sodium sulfate, yielding
177 g of crude product. The product was recrystallized from
acetonitrile to give 150.2 g of a white solid, a 90% yield for the
first two steps. Melting point of the product was 131.5.degree. C.
(DSC) and analysis on an NMR spectrometer was consistent with the
desired product.
[0073] The methyl 3,5-bis(4-benzoylbenzyloxy)benzoate, 60.05 g
(0.108 mol), was placed in a 2 liter flask, followed by the
addition of 120 ml of water, 480 ml of methanol, and 6.48 g (0.162
mol) of sodium hydroxide. The mixture was heated at reflux for
three hours to complete hydrolysis of the ester. After cooling, the
methanol was removed under reduced pressure and the sodium salt of
the acid was dissolved in 2400 ml of warm water. The acid was
precipitated using concentrated hydrochloric acid, filtered, washed
with water, and dried in a vacuum oven to give 58.2 g of a white
solid (99% yield). Melting point on the product was 188.3.degree.
C. (DSC) and analysis on an NMR spectrometer was consistent with
the desired product.
[0074] The 3,5-bis(4-benzoylbenzyloxy)benzoic acid, 20.0 g (36.86
mmol), was added to a 250 ml flask, followed by 36 ml of toluene,
5.4 ml (74.0 mmol) of thionyl chloride, and 28 .mu.l of DMF. The
mixture was refluxed for four hours to form the acid chloride.
After cooling, the solvent and excess thionyl chloride were removed
under reduced pressure. Residual thionyl chloride was removed by
four additional evaporations using 20 ml of chloroform each. The
crude material was recrystallized from toluene to give 18.45 g of
product, an 89% yield. Melting point of product was 126.9.degree.
C. (DSC) and analysis on an NMR spectrometer was consistent with
the desired product.
[0075] The 2-aminoethanethiol hydrochloride, 4.19 g (36.7 mmol),
was added to a 250 ml flask equipped with an overhead stirrer,
followed by 15 ml of chloroform and 10.64 ml (76.5 mmol) of TEA.
After cooling the amine solution on an ice bath, a solution of
3,5-bis(4-benzoylbenzyloxy)benzoyl chloride, 18.4 g (32.8 mmol), in
50 ml of chloroform was added dropwise over a 50 minute period.
Cooling on ice was continued 30 minutes, followed by warming to
room temperature for two hours. The product was diluted with 150 ml
of chloroform and washed with 5.times.250 ml of 0.1 N hydrochloric
acid. The product was dried over sodium sulfate and recrystallized
twice from toluene/hexane (15/1 v/v) to give 13.3 g of product, a
67% yield. Melting point on the product was 115.9.degree. C. (DSC)
and analysis on an NMR spectrometer was consistent with the desired
product.
Example 22
Preparation of a Photoreactive Endpoint Copolymer of Acrylamide and
Fatty Acid Monomers (Compound 30)
[0076] Acrylamide, 0.640 g (9.00 mmol), was dissolved in 9 ml of
THF, followed by the addition of 0.299 g (0.68 mmol) of Compounds
26 and 27, prepared according to the general method described in
Example 19, 0.060 g (0.10 mmol) of Compound 29, prepared according
to the general method described in Example 21, 9 .mu.l (0.060 mmol)
of N,N,N',N'-tetramethylethylenediamine (TEMED), and 0.049 g (0.30
mmol) of 2,2'-azobisisobutyronitrile (AIBN). The solution was
sparged two minutes with helium, two minutes with argon, and was
then sealed and heated overnight at 55.degree. C. The resulting
suspension was diluted with 5 ml of additional THF and added to
diethyl ether, followed by filtration to isolate the solid. After
drying in a vacuum oven, 0.966 g of a white solid were isolated.
Analysis of the polymer revealed 0.073 mmol of BBA per gram of
polymer.
Example 23
Preparation of a Photoreactive Random Copolymer of Acrylamide and
Fatty Acid Monomers (Compound 31)
[0077] Acrylamide, 0.657 g (9.24 mmol), was dissolved in 9 ml of
THF, followed by the addition of 0.307 g (0.70 mmol) of Compounds
26 and 27, prepared according to the general method described in
Example 19, 0.036 g (0.10 mmol) of Compound 28, prepared according
to the general method described in Example 20, 9 .mu.l (0.060 mmol)
of TEMED, and 0.026 g (0.16 mmol) of AIBN. The solution was sparged
two minutes with helium, two minutes with argon, and was then
sealed and heated overnight at 55.degree. C. The resulting
suspension was diluted with 5 ml of additional THF and added to
diethyl ether, followed by filtration to isolate the solid. After
drying in a vacuum oven, 0.997 g of a white solid were isolated.
Analysis of the polymer revealed 0.086 mmol of BBA per gram of
polymer.
Example 24
Preparation of a Photoreactive Endpoint Copolymer of
N-Vinylpyrrolidone and Fatty Acid Monomers (Compounds 32A-C)
[0078] N-Vinylpyrrolidone, 0.915 g (8.23 mmol), was dissolved in 3
ml of THF, followed by the addition of 0.271 g (0.618 mmol) of
Compounds 26 and 27, prepared according to the general method
described in Example 19, 0.070 g (0.116 mmol) of Compound 29,
prepared according to the general method described in Example 21, 1
.mu.l (0.01 mmol) of TEMED, and 0.057 g (0.347 mmol) of AIBN. This
composition was designed to make TDSA 7 mole % of the monomers in
the reaction mixture. The solution was sparged two minutes with
helium, two minutes with argon, and was then sealed and heated
overnight at 55.degree. C. The polymer was precipitated by the
addition of diethyl ether, followed by isolation with filtration.
After drying in a vacuum oven, 1.10 g of a white solid were
isolated. Analysis of Compound 32A revealed 0.109 mmol of BBA per
gram of polymer.
[0079] The above procedure was followed using the following
quantities of reagents in 4 ml of THF: N-vinylpyrrolidone, 0.433 g
(3.90 mmol); Compounds 26 and 27, 0.507 g (1.16 mmol) Compound 29,
0.060 g (0.10 mmol); TEMED, 3 .mu.l (0.02 mmol); and AIBN, 0.049 g
(0.298 mmol). This composition was designed to make TDSA 23 mole %
of the monomers in the reaction mixture. After isolation following
the above procedure, 0.808 g of a white solid were isolated.
Analysis of Compound 32B revealed 0.083 mmol of BBA per gram of
polymer.
[0080] The above procedure was followed using the following
quantities of reagents in 3 ml of THF: N-vinylpyrrolidone, 0.181 g
(1.63 mmol); Compounds 26 and 27, 0.759 g (1.73 mmol); Compound 29,
0.060 g (0.10 mmol); TEMED, 1 .mu.l (0.01 mmol); and AIBN, 0.049 g
(0.298 mmol). This composition was designed to make TDSA 50 mole %
of the monomers in the reaction mixture. After isolation following
the above procedure, 0.705 g of a white solid were isolated.
Analysis of Compound 32C revealed 0.102 mmol of BBA per gram of
polymer.
Example 25
Preparation of a Photoreactive Random Copolymer of
N-Vinylpyrrolidone and Fatty Acid Monomers (Compounds 33A-D)
[0081] N-Vinylpyrrolidone, 0.749 g (6.74 mmol), was dissolved in
8.8 ml of THF, followed by the addition of 0.224 g (0.511 mmol) of
Compounds 26 and 27, prepared according to the general method
described in Example 19, 0.027 g (0.077 mmol) of Compound 28,
prepared according to the general method described in Example 20, 1
.mu.l (0.01 mmol) of TEMED, and 0.019 g (0.116 mmol) AIBN. This
composition was designed to make TDSA 7 mole % of the monomers in
the reaction mixture. The solution was sparged two minutes with
helium, two minutes with argon, and was then sealed and heated
overnight at 55.degree. C. The polymer was precipitated by the
addition of diethyl ether, followed by isolation with filtration.
After drying in a vacuum oven, 0.353 g of a white solid were
isolated. Analysis of the Compound 33A revealed 0.112 mmol of BBA
per gram of polymer.
[0082] The above procedure was followed using the following
quantities of reagents in 3 ml of THF: N-vinylpyrrolidone, 0.362 g
(3.26 mmol); Compounds 26 and 27, 0.621 g (1.42 mmol); Compound 28,
0.017 g (0.049 mmol); TEMED, 1 .mu.l (0.01 mmol); and AIBN, 0.012 g
(0.073 mmol). This composition was designed to make TDSA 30 mole %
of the monomers in the reaction mixture. After isolation following
the above procedure, 0.770 g of a white solid were isolated.
Analysis of Compound 33B revealed 0.052 mmol of BBA per gram of
polymer.
[0083] The above procedure was followed using the following
quantities of reagents in 3 ml of THF: N-vinylpyrrolidone, 0.196 g
(1.76 mmol); Compounds 26 and 27, 0.791 g (1.80 mmol); Compound 28,
0.013 g (0.037 mmol); TEMED, 1 .mu.l (0.01 mmol); and AIBN, 0.009 g
(0.055 mmol). This composition was designed to make TDSA 50 mole %
of the monomers in the reaction mixture. After isolation following
the above procedure, 0.708 g of a white solid were isolated.
Analysis of Compound 33C revealed 0.048 mmol of BBA per gram of
polymer.
[0084] The above procedure was followed using the following
quantities of reagents in 7 ml of THF: N-vinylpyrrolidone, 0.188 g
(1.69 mmol); Compounds 26 and 27, 1.792 g (4.09 mmol); Compound 28,
0.020 g (0.057 mmol); TEMED, 1 .mu.l (0.01 mmol); and AIBN, 0.014 g
(0.085 mmol). This composition was designed to make TDSA 70 mole %
of the monomers in the reaction mixture. After isolation following
the above procedure, 0.879 g of a white solid were isolated.
Analysis of Compound 33D revealed 0.058 mmol of BBA per gram of
polymer.
Example 26
Preparation of a Photoreactive Siloxane Copolymer Containing Fatty
Acid Ligands (Compound 34)
[0085] An aminopropylmethylsiloxane-dimethylsiloxane copolymer,
5.00 g of a 6-7 mole % amine monomer content, was dissolved in 50
ml of dry methylene chloride, followed by the addition of 0.79 g
(7.81 mmol) of TEA. BBA-Cl, 0.19 g (0.78 mmol), prepared according
to the general method described in Example 1, was then added and
the mixture was stirred 3 hours at room temperature. TDSA, 0.924 g
(3.12 mmol), was then added and the solution was stirred 24 hours
at room temperature. The reaction was then diluted with water and
the pH was adjusted to approximately 6 using 0.1 N HCl. The organic
layer was removed and dried over sodium sulfate. The solvent was
removed under reduced pressure and the resulting oil was diluted
with hexane. The precipitate was removed by filtration and
evaporation of the solvent gave 4.75 g of a viscous oil. Analysis
of the polymer revealed 0.013 mmol of BBA per gram of polymer.
Example 27
Fatty Acid Immobilization on an Amine Derivatized Surface
[0086] A polymer surface is derivatized by plasma treatment using a
3/1 mixture of methane and ammonia gases (v/v). (See, e.g., the
general method described in U.S. Pat. No. 5,643,580). A mixture of
methane (490 SCCM) and ammonia (161 SCCM) are introduced into the
plasma chamber along with the polymer part to be coated. The gases
are maintained at a pressure of 0.2-0.3 torr and a 300-500 watt
glow discharge is established within the chamber. The sample is
treated for a total of 3-5 minutes under these conditions.
Formation of an amine derivatized surface is verified by surface
analysis using Electron Spectroscopy for Chemical Analysis (ESCA)
and Time of Flight Secondary Ion Mass Spectrometry (TOF-SIMS).
[0087] TDSA is dissolved at a concentration of 30 mg/ml in a
solvent compatible with both the polymer substrate and the
anhydride. TEA, 1.5 equivalents relative to the anhydride, are
added to the solution and the final mixture is allowed to incubate
with the amine derivatized surface for 24 hours at room temperature
to permit maximal coupling of the fatty acid to the surface. The
final surface is then washed with fresh solvent to remove all
unreacted materials and the final wash is a dilute acid wash to
remove any remaining TEA.
Example 28
Surface Modification of Selected Substrates with Reagents
[0088] Three polymers commonly-used as biomaterials were
surface-modified with novel compounds described above. The polymer
substrates included polyethylene (PE), polyvinylchloride (PVC), and
polyurethane (PU). These polymers were obtained as flat sheets and
used as 1.times.1 cm squares, 1 cm circular disks or obtained in
cylindrical form (tubes or rods) and used as short segments. The
shape and size of the part was chosen based on the particular assay
to be conducted with the coated substrates.
[0089] Coating solutions were prepared by dissolving the reagents
at concentrations ranging from 1-15 mg/ml in neat isopropanol (IPA)
or deionized water/IPA solutions. The reagents were applied to the
polymer substrates using dip coating methods. Parts were suspended
vertically, immersed in the solution at 2 cm/sec, allowed to dwell
for five seconds, and then withdrawn at a rate of 0.1 cm/sec. After
removal of the substrate from the coating solution, it was air
dried until the solvent was no longer visible, often within about 1
minute. The substrate with the coating was then suspended midway
between two opposed Dymax UV curing lamps, each outfitted with a
Heraeus Q402Z4 bulb. At the distance of placement of the lamps, the
parts received approximately 1.5 mW/cm.sup.2 in the wavelength
range 330-340 nm. The substrate was rotated at 3 rpm during the two
minutes of illumination to ensure that the surface was evenly
bathed in light. After illumination, the parts were removed from
the lamp chamber and washed in IPA, using two sequential 30-minute
washes in fresh solvent. The coated samples were then stored in the
dark at ambient temperature until used.
Example 29
Surface Analysis of Polymer Substrates Modified with Compounds 8,
9, 18, 19, 32 and 33
[0090] Three different techniques (staining, ESCA, and TOF-SIMS)
were used to evaluate the surfaces of modified substrates to
confirm the presence and uniformity of the compounds.
[0091] PE and PVC flat materials were modified with
heterobifunctional reagents (Compounds 8, 9, 18, 19) and polymeric
reagents (Compounds 32 and 33, having varying monomer
compositions). Reagents were prepared in IPA at 1.0 mg/ml and
applied using the methods described in Example 28.
[0092] First, the coated materials were stained with Toluidine Blue
0, a positively-charged, visible-wavelength dye. Samples were
immersed in a solution of the dye (0.02% w/v in water) for 30
seconds, removed from solution, and rinsed with DI water. This
staining protocol was useful for identifying qualitatively the
presence of each of the reagents on the material surface. The
results of the dye binding suggested that the surface modification
procedures were successful in immobilizing the reagents on the
substrate surfaces. There was some variability in the darkness of
the stain, both from different reagents on the same material and
for the same reagents on different materials. The staining was
grossly uniform to the naked eye over the surfaces of the material,
suggesting that the reagent was not pooling or segregating when
applied to the surface and that the coverage of the surface was
relatively uniform.
[0093] ESCA was used to analyze quantitatively the surface chemical
composition of the modified substrates. PE and PVC modified with
heterobifunctional reagents (Compounds 8, 9, 18, 19) and polymeric
reagents (Compounds 32 and 33, having varying molar compositions)
were analyzed with a Perkin Elmer Model 5400 ESCA system using
monochromatic Al X-rays with analysis at a 65 degree takeoff angle.
Survey spectra were collected to calculate the atomic
concentrations in the surface.
[0094] The results of the ESCA measurements (Tables 1 and 2) on the
surface modified materials were useful for indicating the presence
and chemical composition of the coatings. For the PVC substrate,
the atomic concentration of the chlorine atom (Cl) was used to
determine whether the coating masked the substrate material. By
comparing the amounts of Cl detected on the surface of the PVC
after modification, it was clear that the Cl was greatly reduced on
the surface-modified substrates. Together with the results of the
dye binding described above, this suggested that the reagents
covered the surface completely, but were thin enough to detect the
underlying substrate. For the PE substrate, which in the uncoated
state should have an atomic concentration of 100% carbon (as ESCA
cannot detect H atoms), the modified and unmodified samples could
simply be compared using the carbon concentration. On all of the
modified samples the carbon concentration was reduced by about 20%.
It was also evident that nitrogen was present on the surfaces of
the modified PE and PVC, but not on the uncoated surfaces. This was
indicative of the nitrogen in each of the reagents. Finally, the
similarity in the atomic concentrations of C, O, and N on the
surfaces of PE and PVC samples modified with each compound supports
the presence and completeness of the coating. TABLE-US-00003 TABLE
1 Atomic Concentration summary for PE samples (atomic %). Sample
[C] [O] [N] [Cl] [Si] [Na] Uncoated 100 -- -- -- -- -- Compound 33D
83.4 10.5 6.2 -- -- -- Compound 33C 80.9 11.3 7.8 -- -- -- Compound
33B 80.7 11.1 8.3 -- -- -- Compound 32C 80.3 12.4 7.3 -- -- --
Compound 32B 79.3 12.1 8.1 -- 0.5 -- Compounds 18, 19 83.8 13.9 2.3
-- -- -- Compounds 8, 9 81.4 16.8 1.6 -- -- 0.2
[0095] TABLE-US-00004 TABLE 2 Atomic Concentration Summary for PVC
samples (atomic %). Sample [C] [O] [N] [Cl] [Si] [Na] Uncoated 74.2
7.5 -- 17.6 -- 0.5 Compound 33D 80.4 11.5 6.2 1.9 -- -- Compound
33C 78.8 12.1 8.8 0.4 -- -- Compound 33B 77.8 11.6 9.1 1.3 -- 0.1
Compound 32C 79.9 12.4 6.8 0.8 -- -- Compound 32B 77.6 12.2 9.1 0.4
0.7 -- Compounds 18, 19 83.4 12.8 2.4 1.4 -- -- Compounds 8, 9 79.7
17.4 1.4 1.1 -- 0.3
[0096] TOF-SIMS was conducted to ensure that the coatings were
located on the outermost surface of the substrates. TOF-SIMS is
sensitive to the chemical structure within the outer 10 .ANG. of a
surface. TOF-SIMS was performed by Physical Electronics (Eden
Prairie, Minn.) using a Physical Electronics model number 7200
instrument. Positive- and negative-ion spectra were recorded for
each of the surfaces. In addition, scans of the surface were used
to determine the uniformity of chemical fragments which were
indicative of the coatings (independent of the substrate
chemistry). The surfaces (substrates and coatings) analyzed by
TOF-SIMS were the same as those analyzed by ESCA, described above.
For the coated substrates, the TOF-SIMS spectra were substantially
different from the spectra for the uncoated PE or PVC material. For
example, there were many chemical fragments containing nitrogen,
which is not present in either of the base materials. There were
many high molecular weight fragments in the positive ion spectra
(between 200 and 600 mass/charge units) associated with the
heterobifunctional reagents (Compounds 8, 9 and 18, 19). The
polymer-based reagents (Compounds 32, 33) had regular repeating
fragment fingerprints indicative of the polymer backbone. Also
confirming that the reagents were present on the surfaces of the
materials, was that the fragment patterns for each compound were
similar on the two different substrates. In addition, the scans of
the surface to detect the presence of peaks uniquely associated
with the coating reagents indicated that the reagents were
relatively uniformly distributed over the surface of the substrate,
further confirming the results of the Toluidine Blue O staining
tests described previously.
Example 30
Human Serum Albumin (HSA) Adsorption from Buffer and Platelet Poor
Plasma
[0097] Adsorption of human serum albumin (HSA) from single protein
buffer solution and from diluted human platelet poor plasma (PPP)
onto the polymer materials was quantified using radiolabeled
protein. Fatty acid-free HSA (Sigma Chemical, St. Louis Mo.) was
radiolabeled with .sup.3H using sodium borohydride techniques
(Means and Feeney, Biochemistry 7, 2192 (1968)). Buffer solutions
of HSA were prepared by dissolving unlabeled HSA to a concentration
of 0.1 mg/ml in Tris-saline (TN) buffer solution (50 mM Tris, 150
mM NaCl, pH 7.5). The resulting solution was then spiked with an
aliquot of the .sup.3H-HSA such that the specific activity was
approximately 1000 dpm/.mu.g HSA for the total solution. Plasma
solutions were prepared using a commercially-available PPP (George
King Biomedical; Overland Park, Kans.) prepared from blood
anticoagulated with sodium citrate (3.8%). Just prior to an
adsorption experiment, the PPP was diluted 4:1 with phosphate
buffered saline (10 mM phosphate, 150 mM NaCl, pH 7.4; PBS) and
then spiked with the radiolabeled HSA such that the specific
activity was approximately 6000 dpm/.mu.g of HSA in the diluted
plasma.
[0098] Adsorption experiments were conducted identically for both
the buffer and PPP solutions containing .sup.3H-HSA. Circular disks
(1 cm) of the surface-modified PE and PVC were placed in 20 ml
scintillation vials; uncoated disks of the same materials were used
as controls. The pieces were hydrated in 2 ml of TN overnight at
room temperature. On the day of the experiment, .sup.3H-HSA
solutions (buffer or PPP) were prepared as described above. The
hydration buffer was aspirated from the polymer samples and 1.0 ml
of the radiolabeled HSA solution was added to the vial. The vials
were gently agitated on an orbital shaker for 2 hours at room
temperature. The HSA solution was aspirated and 4 ml of TNT
solution (50 mM Tris, 150 mM NaCl, 0.05% Tween20, pH 7.5) were
added to each vial; the vials were shaken for 15 minutes at room
temperature. The TNT wash step was repeated two times and the disks
were transferred to clean, dry scintillation vials. Two ml of THF
were added to each vial and the samples were strongly agitated on
an orbital shaker overnight. To each vial, 10 ml of Hionic Fluor
were added and thoroughly mixed by vortexing. The vials were
counted using a liquid scintillation counter (Packard 1900 CA). The
surface concentration of HSA was calculated from these data using
the specific activity of the HSA adsorption solution and the
surface area of the disks.
[0099] PE and PVC were modified with heterobifunctional and
polymeric compounds using the same procedures as described in
Example 28. The results of the binding of .sup.3H-HSA out of TN
buffer solution onto the modified and uncoated PE and PVC materials
are shown in Table 3. TABLE-US-00005 TABLE 3 Adsorption of HSA from
TN buffer onto modified PE and PVC surfaces Surface concentration
of HSA (.mu.g/cm.sup.2) Surface PE PVC Uncoated 0.069 .+-. 0.001
0.066 .+-. 0.000 Compound 32B 0.068 .+-. 0.001 0.051 .+-. 0.001
Compound 32C 0.050 .+-. 0.001 0.036 .+-. 0.001 Compound 33B 0.071
.+-. 0.002 0.066 .+-. 0.001 Compound 33C 0.054 .+-. 0.000 0.045
.+-. 0.006 Compound 33D 0.136 .+-. 0.000 0.036 .+-. 0.005 Compounds
18, 19 0.128 .+-. 0.005 0.098 .+-. 0.006 Compounds 10, 11 0.167
.+-. 0.006 0.168 .+-. 0.007 Compounds 14, 15 0.191 .+-. 0.001 0.200
.+-. 0.010 Compound 8, 9 0.191 .+-. 0.010 0.159 .+-. 0.007
[0100] The results of HSA binding from buffer solution indicated
that many of the polymeric reagents bound HSA at similar levels to
uncoated surfaces, whereas the heterobifunctional compounds
enhanced binding by 2- to 3-fold over uncoated.
Example 31
HSA Binding from Plasma to PE Modified with Compounds 8, 9, 18, 19,
30, 32, and 33
[0101] PE flat substrates were modified with Compounds 8, 9, 18,
19, 30, 32, and 33. Compounds 8, 9, 18, 19, 32, and 33 were
prepared in IPA at concentration of 1 mg/ml and Compound 30 was
prepared in IPA/water (80/20 v/v), and substrates were coated
following the procedure as described in Example 28. HSA binding
from PPP was measured as described in Example 30; the specific
activity was 2,003 dpm/.mu.g. TABLE-US-00006 TABLE 4 HSA binding
from PPP onto PE Surface concentration Surface (PE)
(.mu.g/cm.sup.2) Uncoated 0.008 .+-. 0.001 Compound 32B 0.064 .+-.
0.012 Compound 33A 0.010 .+-. 0.000 Compound 33B 0.168 .+-. 0.002
Compound 30 0.015 .+-. 0.000 Compounds 18, 19 0.017 .+-. 0.002
Compounds 8, 9 0.012 .+-. 0.001
Example 32
HSA Binding from Plasma to PVC Modified with Compounds 8, 9, 32,
and 33
[0102] PVC flat substrates were modified with Compounds 8, 9, 32,
and 33. The compounds were prepared in IPA at concentration of 1
mg/ml, and were applied to the substrates following the procedure
as described in Example 28. HSA binding from PPP was measured as
described in Example 30; in this experiment the specific activity
was 3,150 dpm/.mu.g HSA. TABLE-US-00007 TABLE 5 HSA binding from
PPP onto PVC Surface concentration Surface (.mu.g/cm.sup.2)
Uncoated 0.0183 .+-. 0.0005 Compound 32C 0.0460 .+-. 0.0040
Compound 32B 0.0420 .+-. 0.0010 Compound 33C 0.1720 .+-. 0.0120
Compound 33B 0.0830 .+-. 0.0020 Compounds 8, 9 0.0296 .+-.
0.0010
Example 33
HSA Binding from Plasma to PE Modified with Compounds 14, 15
[0103] PE flat substrates were modified with Compounds 14, 15. The
compounds were prepared in IPA at concentrations ranging from 1-10
mg/ml and applied as one coat or three coats, otherwise following
the procedure as described in Example 28. HSA binding from PPP was
measured as described in Example 30; specific activity of HSA was
5,636 dpm/.mu.g in experiment #1 and #2. The results are shown in
Table 6. TABLE-US-00008 TABLE 6 HSA binding from PPP onto PE
modified with Compounds 14, 15 Surface concentration of HSA
(.mu.g/cm.sup.2) Surface Experiment #1 Experiment #2 Uncoated PE
0.14 .+-. 0.004 0.12 .+-. 0.005 1 mg/ml (3 coats) 0.16 .+-. 0.008
n.d.* 2.5 mg/ml (3 coats) 0.28 .+-. 0.011 n.d. 5 mg/ml (1 coat)
n.d. 0.48 .+-. 0.014 5 mg/ml (3 coats) 0.48 .+-. 0.016 1.22 .+-.
0.046 7.5 mg/ml (1 coat) n.d. 0.67 .+-. 0.024 7.5 mg/ml (3 coats)
0.91 .+-. 0.018 1.02 .+-. 0.053 10 mg/ml (1 coat) n.d. 0.52 .+-.
0.012 10 mg/ml (3 coats) 0.70 .+-. 0.037 1.18 .+-. 0.111 *n.d. is
not determined
[0104] The results of this experiment indicate that increasing the
concentration of applied reagent yields surfaces which show
increased binding of HSA from PPP. In addition, increasing the
number of coats of reagent applied to the surface yields increased
binding of HSA from PPP.
Example 34
HSA Binding from Plasma to PE Modified with Compounds 10, 11
[0105] PE flat substrates were modified with Compounds 10, 11. The
compounds were prepared in IPA at concentrations ranging from 1-15
mg/ml and applied in three coats, otherwise following the procedure
as described in Example 28. HSA binding from PPP was measured as
described in Example 30; specific activity of the HSA in plasma was
5,977 dpm/1 g in Experiment #1 and 6,636 dpm/.mu.g in Experiment
#2. TABLE-US-00009 TABLE 7 HSA binding from PPP onto PE modified
with Compounds 10, 11 Surface concentration of HSA (.mu.g/cm.sup.2)
Surface Experiment #1 Experiment #2 Uncoated PE 0.19 .+-. 0.024
0.22 .+-. 0.017 1 mg/ml 0.43 .+-. 0.026 n.d. 2.5 mg/ml 0.25 .+-.
0.016 n.d. 5 mg/ml 0.64 .+-. 0.030 n.d. 7.5 mg/ml 0.76 .+-. 0.084
n.d. 10 mg/ml 1.04 .+-. 0.076 1.26 .+-. 0.092 12.5 mg/ml n.d. 0.91
.+-. 0.047 15 mg/ml n.d. 1.02 .+-. 0.052
[0106] The results of this experiment indicate that increasing the
concentration of applied reagent yields increased HSA binding,
although it appears as though the HSA binding reaches a plateau
where further increases in the reagent applied to the surface
provide no additional benefit. This may indicate that the surface
has become saturated with reagent.
Example 35
HSA Binding to PE Modified with Compounds 8, 9
[0107] PE flat substrates were modified with Compounds 8, 9. The
compounds were prepared in IPA at concentrations ranging from 1-10
mg/ml and applied as one coat or three coats, otherwise following
the procedure as described in Example 28. HSA binding from PPP was
measured as described in Example 30; specific activity in plasma
was 6,045 dpm/.mu.g HSA. TABLE-US-00010 TABLE 8 HSA binding from
PPP onto PE modified with Compounds 8, 9 Surface concentration of
HSA (.mu.g/cm.sup.2) Surface One coat Three coats Uncoated PE 0.196
.+-. 0.034 n.a. 1 mg/ml 0.194 .+-. 0.021 0.309 .+-. 0.039 2.5 mg/ml
0.3403 .+-. 0.034 0.642 .+-. 0.069 5 mg/ml 0.627 .+-. 0.067 0.692
.+-. 0.024 7.5 mg/ml 1.043 .+-. 0.083 0.873 .+-. 0.063 10 mg/ml
1.071 .+-. 0.197 1.067 .+-. 0.013
[0108] These coatings on PE and PVC enhanced HSA binding from
buffer and plasma by as much as 10-fold. With some reagents (10,
11, 14, 15, and 8, 9), increasing concentration of coating solution
produced surfaces with increasing capacity to bind HSA. This
plateau occurred near 7.5 mg/ml for reagent 14, 15. For Compounds
8, 9, 10, 11, this plateau occurred near 10 mg/ml.
Example 36
Fibrinogen (Fgn) Adsorption from PPP onto Modified Substrates
[0109] PE and PVC substrates were modified with Compounds 8, 9, 18,
19, 32, and 33. The compounds were prepared in IPA at a
concentration of 1.0 mg/ml and applied as a single coat, otherwise
following the procedure as described in Example 28.
[0110] Adsorption of Fgn from human plasma (PPP) onto the control
and surface-modified materials was quantified by using .sup.3H-Fgn.
Fgn was radiolabeled with .sup.3H using sodium borohydride
techniques (Means and Feeney, Biochemistry 7, 2192 (1968)) and
stored frozen at -80.degree. C. until used. Plasma solutions of Fgn
for adsorption experiments were prepared using PPP (George King
Biomedical; Overland Park, Kans.). On the day of the adsorption
experiment, PPP was diluted 4:1 with TN buffer. The diluted PPP was
then spiked with an aliquot of the stock .sup.3H-Fgn solution to
give a working solution with specific activity 1,816 dpm/.mu.g
Fgn.
[0111] Polymer samples (1 cm circular disks) were placed in 20 ml
scintillation vials and hydrated overnight in 2.0 ml of TN at room
temperature prior to protein adsorption. On the day of the
experiment, the buffer solution was aspirated and 1.0 ml of the
diluted PPP containing the .sup.3H-Fgn was added to completely
cover the polymer sample. The substrates were incubated in the
.sup.3H-Fgn solution for 2 hours at 23.degree. C. The PPP solution
was aspirated and the substrates washed three times with TNT (15
minutes each time). Disks were placed in clean scintillation vials,
dissolved with THF, and counted for radioactivity as described in
Example 30 for the HSA adsorption experiments. Surface
concentrations of Fgn were calculated using the specific activity
of the Fgn in the solution and the surface area of the polymer
samples. The experimental results of the fibrinogen absorption
experiments are shown in Table 10. TABLE-US-00011 TABLE 10 Fgn
adsorption to PE and PVC modified with Compounds 8, 9, 18, 19, 32,
and 33 Surface concentration of Fgn (.mu.g/cm.sup.2) Surface
treatment PE PVC Uncoated 0.231 .+-. 0.152 0.269 .+-. 0.060
Compound 33C 0.148 .+-. 0.044 0.092 .+-. 0.003 Compound 33B 0.160
.+-. 0.013 0.112 .+-. 0.013 Compound 32C 0.129 .+-. 0.016 0.180
.+-. 0.005 Compound 32B 0.167 .+-. 0.016 0.249 .+-. 0.018 Compounds
18, 19 0.131 .+-. 0.051 0.193 .+-. 0.029 Compounds 8, 9 0.194 .+-.
0.032 0.222 .+-. 0.062
[0112] With these reagents, Fgn binding to modified surfaces was
equal to or less than adsorption to uncoated surfaces. It is
possible that the enhanced binding of HSA was responsible for
reduced binding of Fgn. Surfaces that reduce the binding of Fgn are
generally less likely to induce subsequent unfavorable responses
from blood, such as fibrin formation and platelet adhesion.
Example 37
Binding of Anti-HSA Antibodies to Modified PE Exposed to HSA
[0113] PE substrates were modified with Compounds 8, 9, 18, 19, 30,
31, 32, and 33. The compounds were prepared in IPA at a
concentration of 1.0 mg/ml and applied as a single coat, otherwise
following the procedure as described in Example 28.
[0114] The binding of polyclonal anti-HSA antibodies was conducted
using an ELISA technique to determine whether bound albumin
maintained native structure in the absorbed state. Sheep anti-(HSA)
antibodies conjugated to horseradish peroxidase (HRP) were obtained
from Biodesign (Kennebunk, Me.). Polymer samples were hydrated with
TN for 2 hours, and the protein solution was prepared with an HSA
concentration of 1.0 mg/ml in TN. 1 ml of the protein solution was
added to the samples and incubated for 2 hours at room temperature.
After the adsorption, the solution was aspirated and the samples
rinsed with TNT buffer. 1 ml of 1% BSA was added as a blocking step
and incubated for one hour. The samples were washed twice with TNT
for 30 min. each. After the wash, the samples were briefly rinsed
with TN and incubated with the sheep-Ab-HRP in TN (diluted 1:2000),
at room temperature for 1 hour with gentle agitation. The samples
were washed 4 times with 3 mls TNT per vial by vortex. The pieces
were transferred to test tubes and 1 ml TMB/peroxide solution was
added. The color was allowed to develop for 15 minutes. The
absorbance of the solutions was read at 655 nm using a
spectrophotometer. The absorbance is directly proportional to the
surface concentration of HRP and, therefore, also proportional to
the surface concentration of anti-HSA antibody bound to the
substrate surfaces. TABLE-US-00012 TABLE 11 Results of anti-albumin
antibody binding to HSA exposed surface Surface Bound Ab
(A.sub.655) Uncoated 0.134 .+-. 0.005 Compound 32A 0.354 .+-. 0.030
Compound 32B 0.335 .+-. 0.022 Compound 32C 0.338 .+-. 0.017
Compound 33A 0.311 .+-. 0.026 Compound 33B 0.385 .+-. 0.034
Compound 33C 0.352 .+-. 0.020 Compound 30 0.332 .+-. 0.016 Compound
31 0.289 .+-. 0.025 Compounds 8, 9 0.456 .+-. 0.016 Compounds 18,
19 0.488 .+-. 0.020
[0115] The results of anti-HSA antibody binding to HSA previously
absorbed from buffer to the uncoated and surface-modified materials
indicated that there was little difference among the reagents
tested. All surfaces bound high concentrations of antibody, about 3
to 4-fold higher than uncoated surfaces.
Example 38
Platelet Attachment and Activation from Platelet Rich Plasma (PRP)
on Modified PE and PVC
[0116] The surface-modified materials were incubated with platelet
rich plasma (PRP) and then examined with a scanning electron
microscope (SEM) to determine the influence of surface chemistry on
platelet attachment and activation. Blood was collected fresh from
human volunteers into 3.8% sodium citrate using 9:1 ratio of blood
to anticoagulant. The blood was centrifuged at 1200 rpm for 15 min.
to separate PRP from blood. The PRP was collected and kept at room
temperature until used (less than 1 hour). The test samples (1 inch
squares) were placed in a 6-well plate, 1 sample per well. To
quantify the platelets in the plasma, a sample of the PRP was taken
and diluted 1:100 with 1% ammonium oxalate. A capillary tube was
used to transfer a small amount of solution to a hemacytometer, and
the sample was incubated in a covered petri dish for 30 minutes for
the platelets to settle. The platelets were counted under a phase
contrast microscope and determined to be between
1.4-4.4.times.10.sup.14 platelets/ml. The PRP solution was added
onto the top of the samples until the entire surface was covered,
and the samples were incubated one hour at room temperature with no
agitation. After incubation, the PRP was removed carefully by
aspiration and 3 mls of Tyrode's buffer (138 mM NaCl, 2.9 mM KCl,
12 mM sodium bicarbonate, pH 7.4) was gently added to each well.
The plates were agitated slightly on an orbital shaker for 15 min.;
the solution was changed and the wash repeated. The wash solution
was aspirated and 2.0 ml of Karnovsky's fixative (25 mls
formaldehyde+5 mls 25% glutaraldehyde+20 mls of a solution of 23%
NaH.sub.2PO.sub.4--H.sub.2O+77% NaHPO.sub.4 anhydrous) were added
to each well. The plate was wrapped with parafilm and incubated
overnight with slight agitation. The fixative was aspirated and the
samples were washed three times each with pure water, 15 minutes
for each wash. The samples were then dehydrated with an ethanol
series of 25, 50, 75, and 100%, for 15 minutes each. The samples
were kept at 4.degree. C. in 100% ethanol until mounted (up to 4
days). Samples were mounted and coated with Pd/Au and observed
using a JEOL 840 scanning electron microscope. Photos were taken of
different areas on the sample surface at several magnifications to
give a representative view of each sample. The platelets were
counted and judged for degree of activation using morphological
descriptions based on Goodman et al Scanning Electron
Microscopy/1984/I, 279-290 (1984).
[0117] The SEM results for two representative platelet attachment
experiments are shown in Tables 12 and 13. From the SEM
photographs, surface densities of bound platelets were estimated.
The lowest platelet densities were found on the Compound 33C
polymer consistently on both substrates. The Compound 32C polymer
also had low platelet densities consistently. The predominant
platelet morphologies are summarized in Table 13. Platelets that
were rounded or dendritic were interpreted to be less activated;
whereas the platelets that were spreading or fully spread and
showed substantial aggregation were interpreted to be more
extensively activated. For PE, the uncoated substrate had the
highest platelet densities as well as the most fully spread
platelet morphology. For PVC, the uncoated surface was poor but not
the worst surface. TABLE-US-00013 TABLE 12 Platelet Densities on
modified surfaces (platelets/cm.sup.2 .times. 10.sup.-6). Reagent
PE PVC Uncoated 980 .+-. 50 650 .+-. 0 Compound 32C 420 .+-. 0 400
.+-. 30 Compound 33C 220 .+-. 5 200 .+-. 30 Compounds 18, 19 900
.+-. 30 1000 .+-. 200 Compounds 8, 9 400 .+-. 40 400 .+-. 0
[0118] TABLE-US-00014 TABLE 13 Morphology of platelets attached to
modified surfaces. Surface PE PVC Compound Few aggregates,
platelets N/A 32B mostly round or dendritic Compound Some
aggregates, Few aggregates, platelets 32C platelets mostly round or
mostly round or dendritic dendritic Compound Few aggregates,
platelets No aggregates, platelets 33C mostly round or dendritic
mostly round Compound Few aggregates, platelets Some aggregates,
platelets 33D mostly round or dendritic dendritic or spread
dendritic Compounds Many aggregates, most Many aggregates, most 18,
19 platelets spread or fully platelets spreading or fully spread
spread Compounds Many aggregates, most Few aggregates, most 8, 9
platelets spread or fully platelets are spreading spread Uncoated
Many aggregates, most Few aggregates, platelets are platelets are
fully spread spread dendritic or fully spread
[0119] The polymeric reagents performed the best at reducing
platelet attachment and activation on both substrates. The
heterobifunctional reagents 8 and 9 performed similarly to the
polymeric reagent 32C. The heterobifunctional reagents 18 and 19
were similar to or worse than the uncoated surface, depending on
the substrate.
Example 39
Acute Dog Jugular Vein Implants with Catheters Modified with
Compounds 6, 7
[0120] Surfaces modified with Compounds 6, 7 were tested using an
acute, dog, jugular vein implant model. Surface-modified and
control samples were implanted for one hour in the external jugular
veins of 15-25 kg mixed-breed dogs. Attachment of
.sup.111In-labeled, autologous platelets was monitored spatially
and quantitatively in real time using gamma camera imaging.
[0121] In each experiment, the dog was anesthetized with
pentobarbital and secured in a supine position. No anticoagulant
was given to the animals prior to or during the experiments. Ninety
ml of blood was drawn into citrate/dextrose (9:1 v/v) and the
platelets were isolated and labeled with .sup.111In-oxine. The
labeled platelets were reinfused into the dog and allowed to
circulate for 20 minutes. In quick succession, one rod modified
with a fatty acid derivative and one uncoated control rod were
implanted bilaterally in the left and right external jugular veins.
By using an uncoated control rod in each experiment, any
variability in the response of individual animals to the implanted
materials was accounted for. Immediately after insertion of the
rods, the neck region of the dog was monitored continuously for one
hour with a Picker 4/15 digital gamma camera to follow in real time
the attachment of platelets onto the rods. The gamma camera allowed
both digital quantification and spatial resolution of the
radioactive counts. The data collected with the camera was
transferred to a dedicated microcomputer to calculate the relative
platelet adhesion rates on the coated and control materials. After
the one hour scan, the animal was heparinized systemically, to stop
any additional thrombogenesis, and euthanized with an intravenous
injection of KCl. Each jugular vein was exposed and opened
longitudinally to reveal the rod in place in the vein. After the
rods were photographed, they were removed and the thrombus was
stripped, lyophilized and weighed. TABLE-US-00015 TABLE 14
Comparison of platelet attachment on PU modified with Compounds 6,
7. Platelet attachment Surface rate versus control Uncoated PU 1.00
.+-. 0.43 Modified with Compounds 6, 7 0.39 .+-. 0.35
[0122] The coated PU surface performed significantly better than
the uncoated surface, reducing platelet adhesion in this acute test
of blood compatibility.
Example 40
Five-Month Sheep Mitral Valve Implants Using Modified Silicone
Rubber Heart Valves
[0123] Silicone rubber (SR) heart valves are modified with reagents
14, 15. The reagent is prepared at 5 mg/ml in IPA and applied,
using procedures as described in Example 28, in three coats to the
surface of the SR portions of a polymeric, tri-leaflet valve. The
valves are sterilized using ethylene oxide and implanted in the
mitral position in juvenile sheep using procedures described
previously Irwin, E. D., et al, J. Invest. Sur. 6, 133-141 (1993).
Three valves treated with the reagents are implanted. Valves are
left in place for approximately 150 days. At the end of the implant
period, the sheep are sacrificed and the hearts are explanted. The
valve, including the surrounding heart tissue is removed and placed
in buffered formalin. The valves are examined visually and
photographed.
[0124] The appearance of the explanted valve leaflets should be
improved by the coating. The coated valves should have minimal
thrombus on the surface of the leaflets, whereas the uncoated SR
valves should have substantial thrombus covering much of the
surface of the leaflets. Furthermore, the thrombus present on the
surface may be significantly mineralized, a further detrimental
outcome that would potentially shorten the usable lifetime of the
valve.
* * * * *