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| United States Patent |
5,556,632
|
|
Kohler
, et al.
|
September 17, 1996
|
Phospholipid modified non-thombogenic surface
Abstract
A biocompatible material comprising a substrate and a phospholipid moiety
covalently attached thereto in an amount and orientation effective to
provide an improved nonthrombogenic surface relative to the substrate
without the phospholipid moiety covalently attached thereto is provided.
In particularly preferred embodiments, the biocompatible material has the
following structure:
##STR1##
wherein: R.sup.1 is a (C.sub.1 -C.sub.30)alkyl group; R.sup.2 is a
(C.sub.1 -C.sub.30)alkylene group; m is 1-4; n is 1-4; and L is a divalent
linking group covalently bonded to the substrate and to the phospholipid
moiety.
| Inventors:
|
Kohler; Anja S. (Stillwater, MN);
Mooradian; Daniel L. (Eagan, MN);
Furcht; Leo T. (Minneapolis, MN)
|
| Assignee:
|
Regents of the University of Minnesota (Minneapolis, MN)
|
| Appl. No.:
|
406124 |
| Filed:
|
March 17, 1995 |
| Intern'l Class: |
A61F 002/02 |
| Field of Search: |
424/423
523/112,113
514/822
|
References Cited
U.S. Patent Documents
| 4480041 | Oct., 1984 | Myles et al. | 436/508.
|
| 4927879 | May., 1990 | Pidgeon | 525/54.
|
| 4931498 | Jun., 1990 | Pidgeon | 525/54.
|
| 4973493 | Nov., 1990 | Guire | 427/2.
|
| 4979959 | Dec., 1990 | Guire | 623/66.
|
| 5256641 | Oct., 1993 | Yatvin et al. | 514/2.
|
| Foreign Patent Documents |
| WO92/06719 | Apr., 1992 | WO.
| |
Other References
J. D. Andrade, et al., "Surfaces and Blood Compatability, Current
Hypotheses" Trans. Am. Soc. Artif. Intern. Organs, 75-84 (1987).
L. I. Barsukov, et al., "Affinity Chromatography of the Phosphatidylcholine
Exchange Protein from Bovine Liver," Biochimica et Biophysica Acta, 513,
198-204 (1978).
L. D. Bergelson (ed.), Lipid Biochemical Preparations, 267-269 (1980).
Ewan J. Campbell, et al., "Biocompatible Surfaces Using
Methacryloylphosphorylcholine Laurylmethacrylate Copolymer," ASAIO
Journal, M853-M857 (1994).
James A. Hayward, et al., "Biomembrane surfaces as models for polymer
design: the potential for haemocompatibility," Biomaterials, 5, 135-141
(May 1984).
Kazuhiko Ishihara, et al., "Reduced thrombogenicity of polymers having
phospholipid polar groups,"Journal of Biomedical Materials Research, 24,
1069-1077 (1990).
Kazuhiko Ishihara, et al., "Hemocompatibility of human whole blood on
polymers with a phospholipid polar group and its mechanism, " Journal of
Biomedical Materials Research, 26, 1543-1552 (1992).
Robert J. Markovich, et al., "Silica Subsurface Amine Effect on the
Chemical Stability and Chromatographic Properties of End-Capped
Immobilized Artificial Membrane Surfaces, " Anal. Chem. , 63, 1851-1860
(1991).
Charles Pidgeon et al., "Immobilized Artificial Membrane Chromatography:
Supports Composed of Membrane Lipids," Analytical Biochemistry, 176, 36-47
(1989).
Charles Pidgeon et al., "Immobilized Artificial Membrane Chromatography:
Rapid Purification of Functional Membrane Proteins," Analytical
Biochemistry, 194, 163-173 (1991).
Tomoko Ueda, et al., "Protein adsorption on biomedical polymers with a
phosphorylcholine moiety adsorbed with phospholipid," J. Biomater. Sci.
Polymer Edn., 3, 185-194 (1991).
J. Yu, et al., "Polymeric biomaterials: influence of phosphorylcholine
polar groups on protein adsorption and complement activation," The
International Journal of Artificial Organs, 17, 499-504 (1994).
|
Primary Examiner: Azpuru; Carlos
Attorney, Agent or Firm: Mueting, Raasch, Gebhardt & Schwappach, P.A.
Goverment Interests
STATEMENT OF GOVERNMENT RIGHTS
The present invention was made with the support of the United States
Government via grants from the National Institute of Health (Grant Nos.
R37-CA2463 and R01-EY09065. The government has certain rights in the
invention.
Claims
What is claimed is:
1. A biocompatible material comprising a substrate and a phospholipid
moiety covalently attached thereto in an amount and orientation effective
to provide an improved nonthrombogenic surface relative to the substrate
without the phospholipid moiety covalently attached thereto.
2. The biocompatible material of claim 1 wherein the phospholipid moiety
contains a choline moiety and a phosphate polar group.
3. The biocompatible material of claim 2 having the following structure:
##STR8##
wherein: (a) R.sup.1 is a (C.sub.1 -C.sub.30)alkyl group;
(b) R.sup.2 is a (C.sub.1 -C.sub.30)alkylene group;
(c) m is 1-4;
(d) n is 1-4; and
(e) L is a divalent linking group covalently bonded to the substrate and to
the phospholipid moiety.
4. The biocompatible material of claim 3 wherein:
(a) R.sup.1 is a (C.sub.12 -C.sub.20)alkyl group;
(b) R.sup.2 is a (C.sub.4 -C.sub.10)alkylene group;
(c) m is 1; and
(d) n is 1.
5. The biocompatible material of claim 3 wherein L is an amide-containing
linking group.
6. The biocompatible material of claim 5 wherein L is
--C(O)--NH--(CH.sub.2).sub.p -- wherein p is 0-20.
7. The biocompatible material of claim 6 wherein L is
--C(O)--NH--(CH.sub.2).sub.p -- wherein p is 1-6.
8. The biocompatible material of claim 1 wherein the substrate is an
organic polymer.
9. The biocompatible material of claim 8 wherein the organic polymer is
selected from the group consisting of polypropylene and
polytetrafluoroethylene.
10. A medical device comprising the biocompatible material of claim 1.
11. A medical device comprising the biocompatible material of claim 3.
12. A method for enhancing the biocompatibility of a substrate comprising:
(a) covalently attaching a phospholipid compound to the substrate in an
amount and orientation effective to provide an improved nonthrombogenic
surface relative to the substrate without the phospholipid moiety
covalently attached thereto; and
(b) contacting the substrate with blood.
13. The method of claim 12 wherein the phospholipid compound has the
following structure:
##STR9##
wherein: (a) R.sup.3 is a (C.sub.1 -C.sub.30)alkyl group having 0-4 double
bonds;
(b) R.sup.4 is a (C.sub.1 -C.sub.30)alkyl group having 0-4 double bonds;
(c) m is 1-4;
(d) n is 1-4; and
(e) either R.sup.3 or R.sup.4 has at least one double bond.
14. The method of claim 13 wherein the substrate is an aminated substrate
and the phospholipid compound is covalently attached to the aminated
substrate by:
(a) oxidizing the at least one double bond in R.sup.3 or R.sup.4 of the
phospholipid compound to form a carboxylated phospholipid; and
(b) combining the carboxylated phospholipid with the aminated substrate and
a carboxyl-reactive crosslinker to form an amide linkage and covalently
attach the phospholipid to the substrate.
15. The method of claim 14 wherein the carboxyl-reactive crosslinker is
selected from the group consisting of
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride,
N,N'-dicyclohexylcarbodiimide, 4-(p-azidosalicylamido)butylamine, and
diisopropylcarbodiimide.
16. The method of claim 12 wherein the substrate is an organic polymer.
17. The method of claim 16 wherein the organic polymer is selected from the
group consisting of polypropylene and polytetrafluoroethylene.
18. The method of claim 16 wherein the substrate having improved
biocompatibility is contained in a medical device.
19. A biocompatible material comprising a substrate and a phospholipid
moiety covalently attached thereto in an amount and orientation effective
to provide an improved nonthrombogenic surface relative to the substrate
without the phospholipid moiety covalently attached thereto, having the
following structure:
##STR10##
wherein: (a) R.sup.1 is a (C.sub.1 -C.sub.20)alkyl group;
(b) R.sup.2 is a (C.sub.4 -C.sub.10)alkylene group;
(c) m is 1;
(d) n is 1; and
(e) L is --C(O)--NH--(CH.sub.2).sub.p -- wherein p is 1-6.
Description
FIELD OF THE INVENTION
The present invention relates generally to biocompatible materials. More
specifically, the present invention relates to biocompatible materials
having a covalently bound phospholipid moiety. The materials produced in
accordance with the invention have various medical applications, such as
in medical devices, surgical equipment, vascular grafts, etc.
BACKGROUND OF THE INVENTION
The implantation of vascular grafts and medical devices such as artificial
organs, artificial heart valves, artificial joints, synthetic and
intraocular lenses, electrodes, catheters, and various other prosthetic
devices into or on the body is a rapidly developing area of medicine.
However, this has been hampered by the lack of suitable synthetic
materials that are stable when contacted with physiological fluids,
particularly blood.
Adverse reactions between materials and blood components are predominant
factors limiting the use of synthetic materials that come into contact
with physiological fluids. For example, catheters, vascular grafts, and
the like, tend to serve as a nidus, or focus, for the formation of thrombi
(blood clots). Initial contact of such materials with blood results in
deposition of plasma proteins, such as albumin, fibrinogen,
immunoglobulin, coagulation factors, and complement components. The
adsorption of fibrinogen onto the surface of the material causes platelet
adhesion, activation, and aggregation. Other cell adhesive proteins, such
as fibronectin, vitronectin, and von Willebrand factor (vWF) also promote
platelet adhesion. As a result, the continual use of anticoagulants in
conjunction with the introduction of such materials to the body is often
necessary.
Furthermore, complement activation occurs when materials are introduced
into blood. Adsorption of large amounts of IgG, IgM, and C3b onto surfaces
causes activation. Subsequently, complexes may be formed which contribute
to undesirable immune responses, such as proteolysis, cell lysis,
opsonization, anaphylaxis, and chemotaxis. As a result, these responses
render such materials incompatible with the living body.
A number of approaches have been suggested to improve the biocompatibility
of medical devices. One approach has been to modify the surface of the
material to prevent undesirable protein adhesion by providing the material
with a low polarity surface, a negatively charged surface, or a surface
coated with biological materials, such as enzymes, endothelial cells, and
proteins. Another approach has been to bind anticoagulants to the surface
of biologically inert materials to impart antithrombogenic characteristics
to the materials. Still another approach used in the art has been the
copolymerization of various phospholipids which are used as coating
materials for various substrates. Partial polymeric backbone coatings have
also been used in a similar fashion. However, many of these methods can
result in a leaching or "stripping off" of the coating.
Additionally, quaternary amines have been bound to polymer surfaces,
followed by the binding of heparin thereto. Conversely, heparin has been
complexed with a quaternary amine prior to coating the complex onto a
polymeric surface. Both of these methods have the disadvantage of being
nonpermanent or leachable systems, i.e., the heparin would gradually be
lost from the polymer material into the surrounding medium. Furthermore,
coated systems generally have limited viability due to the instability of
the anticoagulant.
Thus, a need exists for a biocompatible material for use in medical devices
that retains antithrombogenic properties, i.e., reduced platelet adhesion
and activation, for an extended period of time.
SUMMARY OF THE INVENTION
The invention provides a biocompatible material comprising a substrate and
a phospholipid moiety covalently attached thereto in an amount and
orientation effective to provide an improved nonthrombogenic surface
relative to the substrate without the phospholipid moiety covalently
attached thereto. Preferably, the phospholipid moiety contains a choline
moiety and a phosphate polar group. More preferably, the phospholipid
moiety has the following structure:
##STR2##
wherein: R.sup.1 is a (C.sub.1 -C.sub.30)alkyl group; R.sup.2 is a
(C.sub.1 -C.sub.30)alkylene group; m is 1-4; n is 1-4; and L is a divalent
linking group, preferably containing an amide functionality, covalently
bonded to the substrate and to the phospholipid moiety.
The present invention is also directed to biocompatible medical devices
comprising substrates which have been modified with a covalently bound
phospholipid moiety, such as a phosphatidylcholine moiety.
The materials modified in accordance with the present invention are
prepared by covalently attaching a phospholipid moiety to the substrate
material by initially derivatizing at least one fatty acid chain of a
phospholipid of the following formula:
##STR3##
wherein: R.sup.3 is a (C.sub.1 -C.sub.30 )alkyl group having 0-4 double
bonds; R.sup.4 is a (C.sub.1 -C.sub.30)alkyl group having 0-4 double
bonds; m is 1-4; n is 1-4; and either R.sup.3 or R.sup.4 has at least one
double bond. The derivatized phospholipid, preferably a carboxylated
phospholipid, is then reacted with a modified substrate, preferably an
aminated substrate, to form a covalent linkage. Once modified, the
resulting product exhibits improved biocompatibility.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph illustrating the number of adherent platelets to various
material substrates, the materials shown being unmodified surface
materials as compared to those modified in accordance with the invention.
The substrates are glass, aminated glass (N-glass), and
phosphatidylcholine-grafted glass (PC-glass).
FIG. 2 illustrates comparative spread platelet area for the various
materials, both unmodified and modified according to the invention. The
substrates are glass, aminated glass (N-glass), and
phosphatidylcholine-grafted glass (PC-glass).
FIG. 3 is a graph illustrating the number of platelets adherent to
unmodified polypropylene samples as compared to modified polypropylene in
accordance with the coupling process of the invention. The substrates are
polypropylene (PP), aminated polypropylene (PP-N), phosphatidylcholine
adsorbed onto polypropylene (PP-Ads-PC) and phosphatidycholine grafted
onto polypropylene (PP-N-PC).
FIG. 4A is a photograph of adherent platelets on polypropylene-adsorbed
phosphatidylcholine (PP-Ads-PC).
FIG. 4B is a photograph of adherent platelets on phosphatidylcholine
covalently linked to polypropylene (PP-N-PC).
FIG. 4C is a photograph of adherent platelets on polypropylene (PP).
FIG. 4D is a photograph of adherent platelets on aminated polypropylene
(PP-N).
FIG. 5 is a graph illustrating the number of platelets adherent to
unmodified polytetrafluoroethylene (PTFE), aminated PTFE and PTFE modified
with phosphatidylcholine.
FIG. 6 is a graph illustrating albumin (BSA) adsorption on various
materials.
FIG. 7 is a graph containing fibrinogen adsorption assay data for various
materials, indicating the amount of adsorbed fibrinogen.
FIG. 8 shows comparative data from a Heparinized Blood Complement
Activation Assay using various materials.
DETAILED DESCRIPTION OF THE INVENTION
The biocompatibility of materials used in medical devices or other
implantable materials or materials that are not necessarily implanted but
that come into contact with blood is improved by covalently attaching a
phospholipid moiety, e.g., a phosphatidylcholine moiety. By covalently
attaching a phospholipid moiety to a substrate, the extent and severity of
adverse reactions between the substrate and blood is reduced. The term
"biocompatible" as used herein means that the material shows reduced
platelet adhesion or spreading upon interaction with blood when compared
to the material without the phospholipid.
Blood compatibility is much more complex than the compatibility of a
material with other bodily fluids or tissues. This is because of the
complex mixture of red cells, white cells, platelets, inorganic ions, and
plasma proteins such as albumins, fibrinogens, and globulins in blood.
Blood forms a clot or thrombus when injury occurs or when it is contacted
by a foreign substance. Almost all materials set off this clot-forming
process, and generally soon thereafter become coated with an irreversible
clot of varying size. Such clots could have an adverse effect on the
utility of such materials. Thus, particularly preferred biocompatible
materials of the present invention are particularly advantageous because
they do not cause any significant coagulation or reaction of natural blood
components as would occur in vivo, such as blood platelet adhesion and
activation.
The biocompatible materials of the present invention include a substrate
and a phospholipid moiety covalently attached thereto in an amount and
orientation effective to provide an improved nonthrombogenic surface
relative to the substrate without the phospholipid moiety covalently
attached thereto. By this it is meant that for a substrate to which there
is a phospholipid moiety covalently attached, there is a reduction in the
number of platelets attached to the substrate surface per unit area
relative to the same substrate without the phospholipid moiety covalently
attached thereto. Preferably, there is at least a 50% reduction in the
number of adherent platelets per unit area of the substrate surface upon
covalently attaching the phospholipid moiety. More preferably, there is at
least an 80% reduction in the number of adherent platelets per unit area
of substrate surface when a phospholipid is covalently attached to the
substrate. Even more preferable is a 90% reduction in the number of
adherent platelets per unit area of substrate surface when a phospholipid
is covalently attached to the substrate. Most preferably, the substrate
surface is substantially nonthrombogenic, i.e., it causes little or no
platelet adhesion to occur.
In particularly preferred embodiments, the biocompatible materials of the
present invention cause little or no platelet activation, in addition to
low platelet adhesion, as determined by platelet spread. That is, for
substrates to which platelets do adhere, the platelets generally remain
rounded and exhibit little or no spreading. Thus, there is little
thrombosis that can occur. The biocompatible materials of the present
invention are particularly advantageous because they do not cause
significant thrombosis, even though protein deposition occurs. That is,
although fibrinogen adsorbs to the surface of the substrates to which a
phospholipid is covalently attached, the fibrinogen is not present in an
orientation that causes adverse platelet adhesion and activation.
Substrates to which the phospholipid can be attached in accordance with the
invention include any synthetic or natural material that is insoluble in
physiological fluids. It can be a metal such as titanium or stainless
steel, glasses such as soda glass and silica glass, inorganic materials,
and organic polymers. Preferably, it is an organic polymer that has
demonstrated its relative biocompatibility for use in various medical
devices. Examples of polymeric substrates useful for the invention are
synthetic polymers such as polyurethanes, polycarbonates, silicone
elastomers, polypropylene, polyethylene, polyvinyl chlorides, polyesters,
nylons, polyvinyl pyrrolidones, polymethacrylates such as
polymethylmethacrylate (PMMA), n-Butyl cyanoacrylate, polyvinyl alcohols,
cellulosics, polyvinylidene fluoride (PVDF), polytetrafluoroethylene,
polytetrafluoroethylene(polyester), ethylene tetrafluoroethylene copolymer
(ETFE), acrylonitrile butadiene ethylene, polyamide, polyimide, styrene
acrylonitrile, and the like. As long as the substrate can be modified, as
by amination for example, for covalent attachment of the phospholipid, it
can be used as a substrate.
Among the polyurethanes suitable for use in the present invention are the
elastomeric, segmented polyether polyurethanes derived from repeating
units of polytetramethylene ether glycol and a mixture of methylene
diisocyanate and a diol (or diamine) coupler. Examples of commercially
available polyurethanes include, for example, those with the designation
TYGOTHANE from Norton Chemical Co., Akron, Ohio, RENATHANE from Renal
Systems, Inc., Minneapolis, Minn., and TECOFLEX from Thermedic, Inc.,
Woburn, Mass. Among the silicone elastomers suitable for use in the
present invention are the medical grade elastomers such as the silicone
elastomer commercially available under the designation SILASTIC from Dow
Corning Corp., Midland, Mich. Among the polycarbonates suitable for use in
the present invention are the bisphenol A polycarbonates such as the one
commercially available under the designation LEXAN from General Electric,
Pittsfield, Mass.
Preferably, the phospholipid moiety attached to the biocompatible materials
of the present invention contains a choline moiety and phosphate polar
group and variable lengths of fatty acid chains. That is, the
phospholipids are derivatives of phosphatidylcholine. Particularly
preferred such biocompatible materials are represented by the following
structure:
##STR4##
wherein: R.sup.1 is a (C.sub.1 -C.sub.30 )alkyl group; R.sup.2 is a
(C.sub.1 -C.sub.30 )alkylene group; m is 1-4; n is 1-4; and L is a
divalent linking group covalently bonded to the substrate and to the
phospholipid moiety. Most preferably, R.sup.1 is a (C.sub.12
-C.sub.20)alkyl group; R.sup.2 is a (C.sub.4 -C.sub.10) group; m is 1; and
n is 1.
It should be understood that this representation does not mean that there
is only one phospholipid moiety attached to the substrate. Rather, the
phospholipid moiety attached to the substrate through the linking group L
is present on the substrate in an amount effective to improve the
nonthrombogenic characteristics of the substrate surface. Furthermore, it
should be understood that the biocompatible materials of the present
invention can include one or more types of phospholipid moieties
covalently attached to the same substrate. That is, more than one type of
phospholipid moiety can be covalently attached to any one substrate
surface.
The phospholipid moieties are attached to the substrates in a manner such
that the polar head groups, i.e., the phosphate end of the molecules, are
exposed at the substrate surface. This is accomplished by covalently
attaching at least one of the fatty acid chains of the phospholipid
molecules to the substrate. Preferably, only one of the fatty acid chains
of each phospholipid molecule is covalently attached to the substrate. In
this way, there is one fatty acid chain free per phospholipid moiety to
provide advantageous surface properties. This can be accomplished by any
number of conventional methods known in the art using suitable
crosslinking agents.
The covalent linkage occurs through a divalent linking group that is
covalently bonded to the substrate and covalently bonded to the
phospholipid moiety. This can be accomplished through a variety of
functionalities depending on the functionality of the phospholipid and the
substrate at the attachment sites of each. As long as a covalent linkage
is accomplished, there is no criticality to the type of divalent linking
group used. Typically, divalent linking groups include amide
functionalities. Other linking groups may also be used, such as esters,
acyl halides, alkyl halides, sulfones, sulfoxides, sulfide linkages and
the like. A preferred amide-containing linking group is
--C(O)--NH--(CH.sub.2).sub.p --wherein p is 0-20, preferably p is 1-6.
This linkage can occur by incorporating a crosslinking agent can be used
as between the substrate and the phospholipid, or the crosslinking agent
can be used as a template to activate the substrate and phospholipid
functionalities to interact.
For example, phosphatidylcholine can be attached to a silica substrate
through an amide linkage as shown in the following structure:
##STR5##
This covalent attachment can be accomplished by aminating the substrate,
oxidizing the phospholipid, and combining the two in the presence of a
carboxyl-reactive crosslinker, such as a carbodiimide, to form the amide
linkage.
In particularly preferred embodiments, the present invention provides a
method for improving the nonthrombogenic characteristics of a substrate by
oxidizing a phospholipid compound having the following structure:
##STR6##
wherein: R.sup.3 is a (C.sub.1 -C.sub.30 )alkyl group having 0-4 double
bonds; R.sup.4 is a (C.sub.1 -C.sub.30)alkyl group having 0-4 double
bonds; m is 1-4; n is 1-4; and either R.sup.3 or R.sup.4 has at least one
double bond. The oxidation is carried out under conditions that
selectively oxidize the at least one double bond in R.sup.3 or R.sup.4 of
the phospholipid compound to form a carboxylated phospholipid. The
resultant carboxylated phospholipid is then combined with an aminated
substrate and a carboxyl-reactive crosslinker to form an amide linkage and
covalently attach the phospholipid to the substrate. Suitable
carboxyl-reactive crosslinkers are selected from the group consisting of
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride,N,N'-dicyclohe
xylcarbodiimide, 4-(p-azidosalicylamido)-butylamine, and
diisopropylcarbodiimide.
In a particularly preferred embodiment, carboxylated phophatidylcholine is
prepared by dissolving egg lecithin in a strong acid, e.g., an aqueous
acetic acid solution, and adding an oxidizing agent capable of oxidizing a
carbon-carbon double bond. The double bond is thereby converted to a
carboxyl group (--COOH) as shown below:
##STR7##
This carboxylated phosphatidylcholine is capable of being acted upon to
form a covalent linkage with an appropriately modified substrate, e.g., an
aminated substrate.
A substrate can be aminated by a variety of techniques depending on the
substrate material. The coupling of the carboxylated phosphatidylcholine
to the aminated substrate can be effectuated through the use of
carbodiimide crosslinking agents, such as EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), DIPCDI (diisopropyl
carbodiimide), and the like. This type of crosslinking agent acts as a
template to activate the carboxyl groups of the phospholipid moieties and
the amine groups of the aminated substrate, thereby forming an amide
linkage.
The above process illustrates the type of procedure by which various
biocompatible materials may be coated with phosphatidylcholine in such a
manner that the resulting material has reduced platelet adhesion and
activation. The resulting biocompatible materials, when prepared in
accordance with the invention, have demonstrable suppressed platelet
adherence and reduced thrombogenicity. Suppressed platelet adherence and
activation on the materials modified according to the invention may be
demonstrated by standard procedures known in the art. For example, one
technique is to expose treated materials to blood for a period of time
sufficient for the blood proteins to react with the material, and
subsequently rinsing with PBS and placing in a saline solution containing
a fixing material, such as formaldehyde. Microscopy may be used to observe
the surface clarity of the material in order to assess the adherence of
blood components.
Medical devices in which the biocompatible material of the present
invention can be incorporated include, but are not limited to, surgical
implants, prostheses, and any artificial part or device which replaces or
augments a part of a living body or comes into contact with bodily fluids,
particularly blood. Various medical devices and equipment usable in
accordance with the invention are known in the art. Examples of devices
include catheters, suture material, tubing, and fiber membranes. Examples
of catheters include central venous catheters, thoracic drain catheters,
angioplasty balloon catheters. Examples of tubing include tubing used in
extracorporeal circuitry, such as whole blood oxygenators. Examples of
membranes include polycarbonate membranes, haemodialysis membranes,
membranes used in diagnostic or biosensor devices. Also included are
devices used in diagnosis, as well as polyester yarn suture material such
as polyethylene ribbon, and polypropylene hollow fiber membranes.
Further illustrations of medical devices include the following:
autotransfusion devices, blood filters, blood pumps, blood temperature
monitors, bone growth stimulators, breathing circuit connectors, bulldog
clamps, cannulae, grafts, implantible pumps, impotence and incontinence
implants, intra-occular lenses, leads, lead adapters, lead connectors,
nasal buttons, orbital implants, cardiac insulation pads, cardiac jackets,
clips, covers, dialators, dialyzers, disposable temperature probes, domes,
drainage products, drapes, ear wicks, electrodes, embolic devices,
esophageal stethoscopes, fracture fixation devices, gloves, guide wires,
hemofiltration devices, hubs, intra-arterial blood gas sensors,
intracardiac suction devices, intrauterine pressure devices, nasal spetal
splints, nasal tampons, needles, ophthalmic devices, oxygenators, PAP
brushes, periodontal fiber adhesives, pessary, retention cuffs, sheeting,
staples, stomach ports, surgical instruments, transducer protectors,
ureteral stents, vaginal contraceptives, valves, vessel loops, water and
saline bubbles, achtabular cups, annuloplasty ring, aortic/coronary
locators, artificial pancreas, batteries, bone cement, breast implants,
cardiac materials, such as fabrics, felts, mesh, patches, cement spacers,
cochlear implant, defibrillators, generators, orthopedic implants,
pacemakers, pateIlar buttons, penile implant, pledgets, plugs, ports,
prosthetic heart valves, sheeting, shunts, umbilical tape, valved
conduits, and vascular access devices.
Reasonable modifications and variations are possible from the foregoing
disclosure without departing from either the spirit or scope of the
present invention as defined by the claims. Objects and advantages of this
invention will now be illustrated by the following examples, but the
particular materials and amounts thereof recited in these examples, as
well as other conditions and details, should not be construed to unduly
limit this invention.
EXAMPLES
Example 1
Preparation of Phosphatidylcholine Starting Material
Initially, 500 mg of egg lecithin was dissolved in 50 ml of 90% acetic acid
in a 500-ml Erlenmeyer flask. A 120-ml solution of an oxidizing reagent of
0.024 M KMnO.sub.4 and 0.04 M NaIO.sub.4 was prepared by combining 0.455 g
of KMnO.sub.4 in 60 ml of distilled H.sub.2 O and 0.513 g of NaIO.sub.4 in
60 ml of distilled H.sub.2 O. The oxidizing reagent was then added
dropwise to the egg lecithin solution and gently stirred until the
solution remained purple in color upon further addition of the oxidizing
reagent (approximately 100 ml was added). The resulting solution was
allowed to mix for 30 minutes. Subsequently, the solution was decolorized
using 15 ml of 20% sodium bisulfite. The pH was then adjusted to 2.0 with
3M HCl. Extraction was performed with a chloroform/methanol 2:1 solution
by adding 360 ml of the chloroform/methanol to a 500-ml Erlenmeyer flask.
The resulting volume was then split into three polypropylene centrifuge
bottles, each of a 250 ml capacity, and centrifuged at 3,000 rpm for 10
minutes. The top aqueous layer was removed, and the remainder divided
between two bottles. The remaining phase was washed using 50 ml distilled
H.sub.2 O per bottle and a very small amount of NaCl so as to improve
phase separation. The preparation was once again centrifuged at 3,000 rpm
for 10 minutes. The upper phase was removed and discarded. A majority of
the chloroform was removed from the resulting product phase using a rotary
evaporator under vacuum. The temperature did not exceed 35.degree. C.
Thin layer chromatography (TLC) analysis was used to verify the purity and
identity of the carboxylated phosphatidylcholine. Initially, a small
sample of starting egg lecithin and carboxylated phosphatidylcholine were
dissolved in 2:1 chloroform/methanol solution. The sample was spotted onto
Silica Gel GF glass plates (available from Analtech, Inc.), dried, and
placed in a KOPLAN jar containing a developing system. The developing
system used included chloroform/methanol/concentrated aqueous ammonia
(65:35:8 by volume). The solvent front was allowed to migrate to within
one inch of the top of the plate, removed, and then air-dried. Finally,
the plate was placed in a jar containing iodine crystals and visualized.
Retardation factor, R.sub.f, was measured using the distance of solvent
migration. The carboxylated lecithin had an R.sub.f value of 0.15, as
compared to starting egg lecithin, which had an R.sub.f value of 0.040.
The R.sub.f value was 0.15 for modified phosphatidylcholine (PC) and 0.40
for unmodified phosphatidylcholine. The product was stored in 50%
dioxane/water solution for a 1.5% solution at -20.degree. C.
Example 2
Preparation of Aminopropylsilane Substrate
Glass cover slips were immersed in 3N HCl for 30 minutes. They were then
rinsed with water and placed in 0.1 M NaOH for 10 minutes. Subsequently,
the slips were blotted dry and placed in a 50-ml PYREX dish. A 30 ml
solution of a 20% aminopropylsilanc (APS) was added to the dish and the
mixture remained for 30 minutes. APS was removed and the mixture was
rinsed and subsequently placed in an oven at 110.degree. C. under vacuum
for 2 hours. The silica cover slips were aminated, possessing free amino
groups on the surface.
Example 3
Preparation of Phosphatidylcholine-Grafied Material
First, the pH of the phosphatidylcholine starting material made according
to Example 1 was adjusted to 5.0 using a dioxane solution and 0.1 M NaOH.
1-Ethyl-3-(3-dimcthylaminopropyl)carbodiimide (EDC, 340 mg) was added to a
round-bottom flask containing the dioxane/phosphatidylcholine solution.
The mixture was then added to a compartmentalized PYREX dish into four
wells, containing the aminated cover slips prepared according to Example
2. The pH was then adjusted to 5.0. The dishes were then shaken on an
orbital shaker at 100 rpm for 24 hours. The solvent was then removed, and
the product was respectively rinsed with dioxanc, methanol, NaCl and water
in subsequent washings. The samples were allowed to dry and placed into
separate vials.
Example 4
Analysis of Phosphatidylcholine-Modified Material
Verification of the modified material was performed using X-Ray
Photoelectron Spectroscopy (XPS) analysis to identify phosphorous content
of the grafted material. Either XPS or ESCA surface analysis techniques
may be used to determine elemental and chemical composition of the top 2
to 20 layers of a solid sample. XPS has the capability of nondestructive
depth-profiling in order to calculate phospholipid layer thickness.
Materials were analyzed by XPS using the Perkin-Elmer Model 555 at a
45.degree. incident angle. The surface area of the beam was 1 mm .times.3
mm with an average penetration depth of 50 Angsttoms. Binding energy was
measured by eV value. Corresponding eV positions were obtained for each
sample, which was used to calculate the relative percent of each element
found in the substrate.
The following Table summarizes the data which demonstrates both amination
of surfaces using positive elemental analysis for nitrogen, and
demonstrates grafting of phosphatidylcholine onto substrates using
positive elemental analysis of phosphorous.
TABLE I
______________________________________
XPS Atomic Concentration Table
Substrate % N % P % C % Si % O
______________________________________
Glass 1.2 0 17 24 56
Si wafer 1.1 0 14 40 45
Quartz 1.5 0 24 25 49
NH.sub.2 -Glass
3.9 0 25 21 47
NH.sub.2 -Si wafer
3.3 0 27 32 36
NH.sub.2 -Quartz
4.1 0 28 23 43
Phosphatidylcholine-Glass
3.6 0.73 60.5 12.1 22.9
(side 1)
Phosphatidylcholine-Glass
4.2 0.68 66.0 8.1 20.9
(side 2)
______________________________________
As can be seen from the data in Table I, the phosphatidylcholine is
covalently bound to the surface material. The carbon content increased
dramatically upon grafting the phosphatidylcholine due to the presence of
lengthy fatty acid chains. Oxygen and silica contents decreased because
the bulk of oxygen and silica were not subject to the incident X-rays due
to the phospholipid occupancy of the top 50 Angstroms. The nitrogen
content increased with amination of the surface. The presence of
phosphorous in the phosphatidylcholine-grafted surface indicated
successful grafting of phosphatidylcholine onto the surface material.
Example 5
Atomic Force Microscopy Analysis of Modified Surface Materials
Atomic Force Microscopy (AFM) analysis was used to demonstrate the
different surface morphologies for different samples of unmodified and
materials modified according to the invention. The topography of the
synthesized materials was analyzed using NANOSCOPE III (available from
Digital Instruments).
Roughness analysis was obtained from these samples: glass, hydrated glass,
aminated glass, hydrated and aminated glass, phosphatidylcholine-grafted
glass, hydrated phosphatidylcholine-grafted glass. The samples were
analyzed in air and water, from which surface plots were constructed.
Surface plots displayed the selected image with color-coded height
information in a three-dimensional oblique perspective, from which
roughness parameters were calculated. RMS value is the standard deviation
of the difference between the highest and lowest points within a given
area (Z values). The following Table shows the roughness values for the
same samples as listed above.
TABLE II
______________________________________
Roughness Values from AFM Analysis
Material RMS @ 1.0 .mu..sup.2
RMS @ 4.0 .mu..sup.2
______________________________________
Glass 0.628 0.938
Hydrated glass 0.210 0.244
Aminated glass 0.652 1.1175
Hydrated aminated glass
0.325 0.673
Phosphatidylcholine-glass
6.555 7.241
Hydrated phosphatidylcholine-
1.601 1.664
glass
______________________________________
As can be seen from the above data, phosphatidylcholine-grafted glass is
the "roughest" surface when nonhydrated. Upon hydration, the RMS value of
phosphatidylcholine-grafted glass decreased four-fold. In comparison, the
hydrated glass RMS value decreased three-fold, and the aminated glass
surface RMS value decreased two-fold. Therefore, upon exposure to water,
the phosphatidylcholinegrafted glass surface exhibited a substantial
decrease in surface roughness.
Example 6
Surface Characteristics using Contact Angle Analysis
Contact angle analysis demonstrates the nature of surface materials, which
is used to illustrate and compare the physical properties of modified and
unmodified surface materials. Advancing and receding contact angles were
recorded for glass, aminated glass, and phosphatidylcholine-glass using an
NRL C.A. GONIOMETER (Rame-hart, Inc., Model#100-00115) with 0.08 ml
droplet volume. The resulting data is shown below in the Table III.
TABLE III
______________________________________
Contact Angle Data
Advancing Receding Contact
Material Contact Angle
Angle
______________________________________
Glass
Average: 52.7 .+-. 3.0
32.0 .+-. 1.7
(n = 9) (n = 6)
Aminated Glass
Average: 70.6 .+-. 0.76
34.8 .+-. 1.4
(n = 12) (n = 8)
Phosphatidylcholine-Glass
XPS P/C ratio: 0.009
Average: 67.0 .+-. 5.7
13.5 .+-. 2.0
(n = 9) (n = 6)
Phosphatidylcholine-Glass
XPS P/C ratio: 0.018
Average: 58.1 .+-. 3.2
8.8 .+-. 4.2
(n = 9) (n = 6)
______________________________________
As can be seen from the data in the above table, the droplet from from the
glass and aminated glass samples was smooth and uniform. However, the
contact angle measurements of the phosphatidylcholine-grafted glass sample
exhibited a stick/slip phenomenon, which indicates nonuniformity at a
microscopic level.
The significant differences between the receding contact angles of the
samples suggest that the phosphatidylcholine-grafted glass has a unique
interaction with water. The low receding contact angle of 8.8.degree.
indicates a strong affinity of the phosphatidylcholine-grafted glass for
water. Aminated glass and unmodified glass surfaces had receding contact
angles of 32 and 35 respectively, indicating interactions with water
identical to each other. Hence, the phosphatidylcholine-grafted glass
surface has physical and chemical properties which are distinct from the
other materials.
Example 7
Demonstration of Suppressed Platelet Adherence and Thrombogenicity
The differences in cell behavior toward materials modified according to the
invention were examined by measuring platelet adherence to the modified
surfaces of materials as compared to unmodified surfaces when exposed to
blood. Whole blood was drawn from human donors, and nine parts whole blood
was mixed with one part citrate citric dextrose (CCD) containing 3.50 ml
of 0.1 M citric acid, 46.50 ml of 0.1 citrate, and 1.25 g of dextrose at
pH 6.5. The resulting solution was centrifuged at 800 rpm in a SORVALL
table-top centrifuge for 20 minutes at room temperature. Subsequently, the
platelet-rich plasma (PRP) was removed from the red and white blood cell
portions. The samples (i.e., underivatized glass, aminated glass, and
phosphatidylcholine-glass) were placed in a PYREX dish. Added to each of
the samples was 160 microliters of the platelet-rich plasma using
EPPENDORF micropipets. The solutions stood for approximately 20 minutes.
The plasma was then removed and the samples were rinsed with phosphate
buffered saline (PBS). The samples were fixed using 3.7% formaldehyde for
15 minutes and rinsed with PBS. The samples were stained with Coomassie
Brilliant Blue (CBB) for 90 minutes, destained, rinsed, and mounted on a
slide.
Materials modified in accordance with the invention, including glass, Si
wafer, and quartz, were exposed to whole blood for 20 minutes. The blood
was subsequently removed from the exposed materials. The materials were
then rinsed twice with PBS and placed in a saline solution containing
formaldehyde as a fixing agent for the biological components remaining on
the materials. The materials were then examined for surface clarity using
standard microscopy techniques.
Platelet number, spread area, and averages thereof were calculated by
collecting several images of materials exposed to the platelet adhesion
assay. Several images were taken of each sample which had been exposed to
the platelet adhesion assay using OPTIMUS Imaging Software (obtained from
BioScan, Edmonds, Wash.). The number of platelets was then calculated for
the different materials.
FIG. 1 is a graph illustrating comparative platelet adhesion (number of
platelets per unit area) to various material substrates. In contrast to
the unmodified material and the aminated material, the
phosphatidylcholine-modified glass surfaces (XPS P/C ratio was 0.018)
exhibited minimal platelet adhesion. FIG. 2 and Table IV shows the
comparative platelet spread results for unmodified and modified materials
in accordance with the invention. The data demonstrates the minimal
platelet adhesion and platelet spread of phosphatidylcholine-grafied
materials.
TABLE IV
______________________________________
Platelet Spread Data
Adherent
Material Platelets Spread Area (.mu..sup.2)
______________________________________
Glass 61.6 .+-. 11.37
31.8 .+-. 6.5
Aminated Glass 73.9 .+-. 11.4
20.82 .+-. 3.90
Phosphatidylcholine-Glass
0 0
______________________________________
As can be seen from the data in the above table and FIGS. 1 and 2,
phosphatidylcholine-grafted glass had negligible platelet adhesion and
virtually no platelet spreading.
Example 8
Other Materials Grafted with Phosphatidylcholine Polypropylene
The process of preparing the phosphatidylcholine-linked composition was
carried out in accordance with the invention, but instead the surface
material used was polypropylene (PP). Unmodified polypropylene and
aminated polypropylene (having 0.6-0.8 nmol/cm.sup.2 NH.sub.2 groups) was
obtained from Neomecs, Inc., St. Louis Park, Minn. Polypropylene with
adsorbed phosphatidylcholine was obtained by subjecting unmodified
polypropylene to the coupling procedure described in Example 3 (without
the amine modified surface, the procedure simply resulted in adsorbed,
rather than covalently linked, phosphatidylcholine). Aminated
polypropylene with covalently linked phosphatidylcholine was obtained in
the manner described in Example 3.
The resulting products were comparatively tested using standard microscopy
techniques. When compared to unmodified polypropylene surfaces or
polypropylene surfaces modified according to different methods, the
comparative test results demonstrated that other substances, such as
polypropylene, is also receptive to the process of the invention and
likewise suppresses platelet adhesion. See FIG. 3 and Table V below.
TABLE V
______________________________________
Platelet Adhesion Data
Adherent Spread Area
Material Platelets (microns)
______________________________________
polypropylene with adsorbed
81 .+-. 13
25.6 .+-. 3.6
phosphatidylcholine (PP-Ads-PC)
phosphatidycholine linked to aminated
10 .+-. 6 26.4 .+-. 7.1
polypropylene (PP-N-PC)
polypropylene (PP) 63 .+-. 9 30.0 .+-. 5.2
aminated polypropylene (PP-N)
138 .+-. 7
13.8 .+-. 4.4
______________________________________
As can be seen from the platelet adhesion data in the above table and FIG.
3, the phosphatidylcholine-polypropylene showed suppressed platelet
adhesion. FIGS. 4A through 4D are photographs of the above surfaces which
depict surface characteristics consistent with the above data (FIG.
4A=PP-Ads-PC; FIG. 4B=PP-N-PC; FIG. 4C=PP; FIG. 4D=PP-N).
Polytetrafluoroethylene
The same experiment as above was performed, but instead the substrate
material used was polytetrafluoroethylene. Unmodified
polytetrafluoroethylene and aminated polytetrafluoroethylene (having 0.8
nmol/cm.sup.2 NH.sub.2 groups) was obtained from Neomecs, Inc., St. Louis
Park, Minn. Aminated polytetrafluoroethylene with covalently linked
phosphatidylcholine was obtained in the manner described in Example 3.
The graph illustrating platelet adhesion on polytetrafluoroethylene is
shown in FIG. 5. As can be seen from FIG. 5, polytetrafluoroethylene
likewise demonstrated reduced platelet adhesion onto the surface when
prepared in accordance with the invention, as compared to both aminated
polytetrafluoroethylene and unmodified polytetrafluoroethylene alone.
These results show that phosphatidylcholine can be grafted onto various
substrate materials and are capable of obtaining the desired
biocompatibility characteristics of the invention.
Example 9
Albumin Adsorption and Fibrinogen Adsorption
Albumin and fibrinogen are plasma proteins that adsorb to material surfaces
and influence blood compatibility. The adsorption of albumin and
fibrinogen on glass, aminated glass, and phosphatidylcholine-grafted glass
was therefore measured.
Albumin Adsorption
Radio-labeled albumin, .sup.125 I-BSA (available from Sigma), was used in
concentrations 0.009, 0.15, 0.9, and 1.5 mg BSA/ml, with a 45:1 ratio of
BSA/.sup.125 I-BSA. Replicate samples of underivatized glass, aminated
glass and PC-glass samples were placed in FALCON 24-well plates, to each
sample was added 250 microliters of heated BSA solution. The plates were
incubated at 37.degree. C. for 2 hours. Subsequently, the samples were
aspirated and rinsed with four subsequent washings of PBS. The samples
were then removed from the plate and placed in individual scintillation
vials. To each sample was added 5 ml scintillation cocktail and the
samples were counted using a BECKMAN LS 6500 Scintillation Counter. After
24 hours, the cocktail was removed from the vials and transferred to
separate scintillation vials for counting. The original samples were
exposed to 5 ml of 1% SDS solution for 24 hours. After incubation, the SDS
was removed from the samples and counted. To the remaining samples was
added 5 ml of scintillation cocktail and these samples were then counted.
FIG. 6 is a graph illustrating albumin (BSA) adsorption after 2 hours of
incubation on glass (G), aminated glass (N), and phosphatidylcholine
covalently linked to glass (P) at several albumin concentrations including
1.5 mg/ml, 0.9 mg/ml, 0.09 mg/ml, and 0.005 mg/ml. As can be seen from
FIG. 6, there is no preferential adsorption of albumin to
phosphatidylcholine-grafted glass surfaces.
Fibrinogen Adsorption
.sup.125 I-Fibrinogen (human fibrinogen available from ICN, Irvine, Calif.)
adsorption to the test surfaces was measured from 1.0% human plasma in
Tris buffer at a pH of 7.4. The .sup.125 I-Fibrinogen used had a specific
activity of 4.15 mCi/mg and a concentration of 0.851 mCi/ml. To 70
microliters of platelet poor plasma was added 24 microliters of .sup.125
I-fibrinogen and subsequently diluted using 7 ml Tris buffer. Triplicate
samples of glass, aminated glass, and PC-glass were placed in FALCON
24-well plates, and 300 microliters of the fibrinogen solution was added
to each sample. The samples were then incubated at room temperature for 2
hours and 20 minutes. The fibrinogen solution was removed and then samples
were washed three times with Tris buffer. The samples were then placed
into glass scintillation vials and 5 ml of scintillation cocktail was
added to each vial. The samples were then counted with a BECKMAN LS 6500
Scintillation Counter. FIG. 7 is a graph illustrating fibrinogen
adsorption assay results.
Example 10
Complement Activation Assay
Complement activation assays are useful in determining the compatibility of
various substances with the immune system. The degree to which complement
enzymes are activated by contact with a foreign material can be measured,
thereby indicating the corresponding response of the immune system to that
material. Blood from human donors was drawn into sterile
heparin-containing tubes. Heparinized blood was tested on several samples,
including glass, aminated glass and PC-glass. Controls were run with no
materials present. The samples were incubated in 50-ml polypropylene tubes
at 37.degree. C. for 10 to 30 minutes. Subsequently, EDTA was added to
individual centrifuging tubes and aliquots were frozen at -70.degree. C.
Complement activation was determined utilizing the AMERSHAM standard human
complement C3a activation radioimmunoassay kit. Samples of glass, aminated
glass, and phospholipid-grafted glass were measured for C3a activation in
heparinized blood. Complement protein adsorption on the samples was
evaluated using radiolabeled antibodies specific for C3a des Arg. As can
be seen from FIG. 8, there was no elevation in complement activation on
the phophatidylcholine-modified glass as compared to the level of
activation seen when no material was present. On the other hand, the
aminated glass demonstrated significant complement activation.
The complete disclosures of all patents, patent applications, and
publications are incorporated herein by reference as if each were
individually incorporated by reference. The invention has been described
with reference to various specific and preferred embodiments and
techniques. However, it should be understood that many variations and
modifications may be made while remaining within the spirit and scope of
the invention.
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