U.S. patent application number 11/296810 was filed with the patent office on 2006-05-11 for method for attachment of biomolecules to medical device surfaces.
This patent application is currently assigned to Medtronic, Inc.. Invention is credited to James R. Keogh, Paul V. Trescony.
Application Number | 20060099326 11/296810 |
Document ID | / |
Family ID | 46253931 |
Filed Date | 2006-05-11 |
United States Patent
Application |
20060099326 |
Kind Code |
A1 |
Keogh; James R. ; et
al. |
May 11, 2006 |
Method for attachment of biomolecules to medical device
surfaces
Abstract
A method for making a medical device having at least one
biomolecule immobilized on a substrate surface is provided. One
method of the present invention includes immobilizing a biomolecule
comprising an unsubstituted amide moiety on a biomaterial surface.
Another method of the present invention includes immobilizing a
biomolecule on a biomaterial surface comprising an unsubstituted
amide moiety. Still another method of the present invention may be
employed to crosslink biomolecules comprising unsubstituted amide
moieties immobilized on medical device surfaces. Additionally, one
method of the present invention may be employed to crosslink
biomolecules comprising unsubstituted amide moieties in solution,
thereby forming a crosslinked biomaterial or a crosslinked medical
device coating.
Inventors: |
Keogh; James R.; (Maplewood,
MN) ; Trescony; Paul V.; (Champlin, MN) |
Correspondence
Address: |
James R. Keogh;Medtronic, Inc.
MS LC340
710 Medtronic Parkway
Minneapolis
MN
55432
US
|
Assignee: |
Medtronic, Inc.
|
Family ID: |
46253931 |
Appl. No.: |
11/296810 |
Filed: |
December 7, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10620180 |
Jul 15, 2003 |
|
|
|
11296810 |
Dec 7, 2005 |
|
|
|
09257543 |
Feb 24, 1999 |
6617142 |
|
|
10620180 |
Jul 15, 2003 |
|
|
|
09001994 |
Dec 31, 1997 |
5945319 |
|
|
09257543 |
Feb 24, 1999 |
|
|
|
08635187 |
Apr 25, 1996 |
5821343 |
|
|
09001994 |
Dec 31, 1997 |
|
|
|
08984922 |
Dec 4, 1997 |
5891506 |
|
|
09257543 |
|
|
|
|
08694535 |
Aug 9, 1996 |
5728420 |
|
|
08984922 |
Dec 4, 1997 |
|
|
|
09012056 |
Jan 22, 1998 |
6033719 |
|
|
09257543 |
|
|
|
|
09001994 |
Dec 31, 1997 |
5945319 |
|
|
09012056 |
Jan 22, 1998 |
|
|
|
08984922 |
Dec 4, 1997 |
5891506 |
|
|
09012056 |
Jan 22, 1998 |
|
|
|
09010906 |
Jan 22, 1998 |
5928916 |
|
|
09257543 |
|
|
|
|
09001994 |
Dec 31, 1997 |
5945319 |
|
|
09010906 |
Jan 22, 1998 |
|
|
|
08984922 |
Dec 4, 1997 |
5891506 |
|
|
09010906 |
Jan 22, 1998 |
|
|
|
Current U.S.
Class: |
427/2.26 |
Current CPC
Class: |
A61L 27/3645 20130101;
A61L 27/54 20130101; Y10S 530/816 20130101; A61L 27/3625 20130101;
A61L 27/22 20130101; A61L 27/24 20130101; C08L 89/00 20130101; Y10S
530/815 20130101; A61L 33/0029 20130101; A61L 29/085 20130101; Y10S
530/81 20130101; A61L 27/28 20130101; A61L 33/128 20130101; G01N
33/543 20130101; A61K 38/43 20130101; A61L 27/3641 20130101; A61L
31/16 20130101; A61L 27/34 20130101; A61L 31/08 20130101; A61L
33/18 20130101; Y10S 530/811 20130101; C12N 11/02 20130101; A61L
33/0011 20130101; A61L 33/0047 20130101; A61L 31/10 20130101; C12N
11/14 20130101; A61L 2300/606 20130101; A61L 33/0082 20130101; A61L
33/12 20130101; A61L 27/34 20130101; C08L 89/00 20130101; A61L
31/10 20130101; C08L 89/00 20130101; A61L 27/24 20130101; C08L
89/00 20130101; A61L 29/085 20130101; C08L 89/00 20130101; A61L
33/0029 20130101; C08L 89/00 20130101 |
Class at
Publication: |
427/002.26 |
International
Class: |
A61K 6/083 20060101
A61K006/083; B05D 3/00 20060101 B05D003/00 |
Claims
1-34. (canceled)
35. A method of forming a coated tissue heart valve, the coating
imparting improved cell binding characteristics to the surface of
the heart valve, the method comprising the steps of: (a) providing
the heart valve; (b) providing a biomolecule, the biomolecule
comprising an arginine, glycine and aspartic acid amino acid
sequence; and (c) combining the biomolecule with the surface of the
tissue heart valve to form a chemical bond, the chemical bond
immobilizing the biomolecule on the surface, the immobilized
biomolecule forming the coating.
36. The method of claim 35 wherein the biomolecule is a naturally
occurring biomolecule.
37. The method of claim 35 wherein the biomolecule is a chemically
synthesized biomolecule.
38. The method of claim 35 wherein the biomolecule is a
peptide.
39. The method of claim 38 wherein the peptide is a cell attachment
peptide.
40. The method of claim 35 wherein the biomolecule is a
protein.
41. The method of claim 40 wherein the protein is a cell attachment
protein.
42. The method of claim 40 wherein the protein is fibrinogen.
43. The method of claim 40 wherein the protein is fibronectin.
44. The method of claim 40 wherein the protein is vitronectin.
45. The method of claim 40 wherein the protein is collagen.
46. The method of claim 35 wherein the heart valve is implantable
in a heart.
47. The method of claim 35 further comprising the step of combining
at least one reducing agent selected from the group consisting of
sodium borohydride, sodium cyanoborohydride and amine borane.
48. The method of claim 35 further comprising the step of combining
a chemical moiety of the biomolecule with a chemical moiety of the
surface to form the chemical bond.
49. The method of claim 48 wherein the chemical moiety of the
biomolecule is an aldehyde moiety.
50. The method of claim 49 wherein the aldehyde moiety is formed by
combining a periodate with a 2-aminoalcohol moiety.
51. The method of claim 50 wherein the periodate comprises at least
one of a periodic acid, a sodium periodate, an alkali metal
periodate, and a potassium periodate.
52. The method of claim 49 wherein the aldehyde moiety is formed by
combining a periodate with a 1,2-dihydroxy moiety.
53. The method of claim 52 wherein the periodate comprises at least
one of a periodic acid, a sodium periodate, an alkali metal
periodate, and a potassium periodate.
54. The method of claim 48 wherein the chemical moiety of the
biomolecule is an epoxide moiety.
55. The method of claim 48 wherein the chemical moiety of the
biomolecule is an isocyanate moiety.
56. The method of claim 48 wherein the chemical moiety of the
biomolecule is a 1,2-dicarbonyl moiety.
57. The method of claim 48 wherein the chemical moiety of the
biomolecule is a phosphate moiety.
58. The method of claim 48 wherein the chemical moiety of the
biomolecule is a sulphate moiety.
59. The method of claim 48 wherein the chemical moiety of the
biomolecule is a carboxylate moiety.
60. The method of claim 48 wherein the chemical moiety of the
surface is a guanidino moiety.
61. The method of claim 60 wherein the guanidino moiety is formed
by combining an amine moiety with a guanidino forming agent.
62. The method of claim 61 wherein the guanidino forming agent is
selected from the group consisting of S-ethylthiouronium bromide,
S-ethylthiouronium chloride, O-methylisourea, O-methylisouronium
sulfate, O-methylisourea hydrogen sulfate, S-methylisothiourea,
2-methyl-1-nitroisourea, aminoiminomethanesulfonic acid, cyanamide,
cyanoguanide, dicyandiamide, 3,5-dimethyl-1-guanylpyrazole nitrate
and 3,5-dimethylpyrazole.
63. The method of claim 61 further comprising the step of combining
a stabilizing agent.
64. The method of claim 63 wherein the stabilizing agent is a
borate ion.
65. The method of claim 61 wherein the amine moiety is formed by
combining an unsubstituted amide moiety with an amine forming
agent.
66. The method of claim 65 wherein the amine forming agent is
selected from the group consisting of bromine, bromide, bromite,
hypobromite, chlorine, chloride, chlorite, hypochlorite, lead
tetraacetate, benzyltrimethylammonium tribromide,
[bis(trifluoroacetoxy)iodo]benzene, hydroxy(tosyloxy)iodobenzene
and iodosylbenzene.
67. The method of claim 48 wherein the chemical moiety of the
surface is a primary amine moiety.
68. The method of claim 67 wherein the primary amine moiety is
formed by combining an unsubstituted amide moiety with an amine
forming agent.
69. The method of claim 68 wherein the amine forming agent is
selected from the group consisting of bromine, bromide, bromite,
hypobromite, chlorine, chloride, chlorite, hypochlorite, lead
tetraacetate, benzyltrimethylammonium tribromide,
[bis(trifluoroacetoxy)iodo]benzene, hydroxy(tosyloxy)iodobenzene
and iodosylbenzene.
70. The method of claim 48 wherein the chemical moiety of the
surface is an aldehyde moiety.
71. The method of claim 70 wherein the aldehyde moiety is formed by
combining a periodate with a 2-aminoalcohol moiety.
72. The method of claim 71 wherein the periodate comprises at least
one of a periodic acid, a sodium periodate, an alkali metal
periodate, and a potassium periodate.
73. The method of claim 70 wherein the aldehyde moiety is formed by
combining a periodate with a 1,2-dihydroxy moiety.
74. The method of claim 73 wherein the periodate comprises at least
one of a periodic acid, a sodium periodate, an alkali metal
periodate, and a potassium periodate.
75. The method of claim 48 wherein the chemical moiety of the
surface is an epoxide moiety.
76. The method of claim 48 wherein the chemical moiety of the
surface is an isocyanate moiety.
77. The method of claim 48 wherein the chemical moiety of the
surface is a 1,2-dicarbonyl moiety.
78. The method of claim 48 wherein the chemical moiety of the
surface is a phosphate moiety.
79. The method of claim 48 wherein the chemical moiety of the
surface is a sulphate moiety.
80. The method of claim 48 wherein the chemical moiety of the
surface is a carboxylate moiety.
81. The method of claim 48 wherein the chemical moiety of the
biomolecule is a guanidino moiety.
82. The method of claim 81 wherein the guanidino moiety is formed
by combining an amine moiety with a guanidino forming agent.
83. The method of claim 82 wherein the guanidino forming agent is
selected from the group consisting of S-ethylthiouronium bromide,
S-ethylthiouronium chloride, O-methylisourea, O-methylisouronium
sulfate, O-methylisourea hydrogen sulfate, S-methylisothiourea,
2-methyl-1-nitroisourea, aminoiminomethanesulfonic acid, cyanamide,
cyanoguanide, dicyandiamide, 3,5-dimethyl-1-guanylpyrazole nitrate
and 3,5-dimethylpyrazole.
84. The method of claim 82 further comprising the step of combining
a stabilizing agent.
85. The method of claim 84 wherein the stabilizing agent is a
borate ion.
86. The method of claim 82 wherein the amine moiety is formed by
combining an unsubstituted amide moiety with an amine forming
agent.
87. The method of claim 86 wherein the amine forming agent is
selected from the group consisting of bromine, bromide, bromite,
hypobromite, chlorine, chloride, chlorite, hypochlorite, lead
tetraacetate, benzyltrimethylammonium tribromide,
[bis(trifluoroacetoxy)iodo]benzene, hydroxy(tosyloxy)iodobenzene
and iodosylbenzene.
88. The method of claim 48 wherein the chemical moiety of the
biomolecule is a primary amine moiety.
89. The method of claim 88 wherein the primary amine moiety is
formed by combining an unsubstituted amide moiety with an amine
forming agent.
90. The method of claim 89 wherein the amine forming agent is
selected from the group consisting of bromine, bromide, bromite,
hypobromite, chlorine, chloride, chlorite, hypochlorite, lead
tetraacetate, benzyltrimethylammonium tribromide,
[bis(trifluoroacetoxy)iodo]benzene, hydroxy(tosyloxy)iodobenzene
and iodosylbenzene.
91. A method of making a tissue heart valve comprising: (a)
providing the heart valve; (b) providing a biomolecule, the
biomolecule comprising an arginine, glycine and aspartic acid amino
acid sequence; and (c) reacting a chemical moiety of the
biomolecule with a chemical moiety of the tissue heart valve to
form a chemical bond, the chemical bond immobilizing the
biomolecule on the surface of the tissue heart valve.
92. The method of claim 91 wherein the biomolecule is a naturally
occurring biomolecule.
93. The method of claim 91 wherein the biomolecule is a chemically
synthesized biomolecule.
94. The method of claim 91 wherein the biomolecule is a
peptide.
95. The method of claim 94 wherein the peptide is a cell attachment
peptide.
96. The method of claim 91 wherein the biomolecule is a
protein.
97. The method of claim 96 wherein the protein is a cell attachment
protein.
98. The method of claim 96 wherein the protein is fibrinogen.
98. The method of claim 96 wherein the protein is fibronectin.
99. The method of claim 96 wherein the protein is vitronectin.
100. The method of claim 96 wherein the protein is collagen.
101. The method of claim 91 wherein the heart valve is implantable
in a heart.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/001,994 for "Oxidative Method for
Attachment of Biomolecules to Medical Device Surfaces" to Keogh
filed Dec. 31, 1997, which is a continuation-in-part of U.S. patent
application Ser. No. 08/635,187 for "Oxidative Method of Attachment
of Biomolecules to Surfaces of Medical Devices" to Keogh filed Apr.
25, 1996.
[0002] This application is further a continuation-in-part of U.S.
patent application Ser. No. 08/984,922 for "Oxidative Method for
Attachment of Glycoproteins or Glycopeptides to Surfaces of Medical
Devices" to Keogh filed Dec. 4, 1997, which is a
continuation-in-part of U.S. patent application Ser. No. 08/694,535
for "Oxidative Method of Attachment of Glycoproteins to Surfaces of
Medical Devices" to Keogh filed Aug. 9, 1996, now U.S. Pat. No.
5,728,420 issued Mar. 17, 1998.
[0003] This application is also a continuation-in-part of U.S.
patent application Ser. No. 09/012,056 for "A Method for Covalent
Attachment of Biomolecules to Surfaces of Medical Devices" to Keogh
filed Jan. 22, 1998 which is a continuation-in-part of U.S. patent
application Ser. No. 09/001,994 for "Oxidative Method for
Attachment of Biomolecules to Medical Device Surfaces" to Keogh
filed Dec. 31, 1997, and is also a continuation-in-part of U.S.
patent application Ser. No. 08/984,922 for "Oxidative Method for
Attachment of Glycoproteins or Glycopeptides to Surfaces of Medical
Devices" to Keogh filed Dec. 4, 1997.
[0004] This application is additionally a continuation-in-part of
U.S. patent application Ser. No. 09/010,906 for "A Method for Ionic
Attachment of Biomolecules to Surfaces of Medical Devices" to Keogh
filed Jan. 22, 1998, which is a continuation-in-part of U.S. patent
application Ser. No. 09/001,994 for "Oxidative Method for
Attachment of Biomolecules to Medical Device Surfaces" to Keogh
filed Dec. 31, 1997, and is also a continuation-in-part of U.S.
patent application Ser. No. 08/984,922 for "Oxidative Method for
Attachment of Glycoproteins or Glycopeptides to Surfaces of Medical
Devices" to Keogh filed Dec. 4, 1997. All the foregoing patent
applications and patent are hereby incorporated by reference
herein, each in its respective entirety.
BACKGROUND OF THE INVENTION
[0005] For many years, a number of medical devices (e.g.,
pacemakers, vascular grafts, stents, heart valves, etc.) that
contact bodily tissue or fluids of living persons or animals have
been developed, manufactured, and used clinically. A major problem
with such articles is that their surfaces tend to adsorb a layer of
proteins from tissues and fluids such as tears, urine, lymph fluid,
blood, blood products, and other fluids and solids derived from
blood. The composition and organization of this adsorbed protein
layer is thought to influence, if not control, further biological
reactions. Adverse biological reactions such as thrombosis and
inflammation can diminish the useful lifetime of many devices.
[0006] Implantable medical devices also tend to serve as foci for
infection of the body by a number of bacterial species. These
device-associated infections are promoted by the tendency of these
organisms to adhere to and colonize the surface of the device.
Consequently, it has been of great interest to physicians and the
medical industry to develop surfaces that are less prone in
promoting the adverse biological reactions that typically accompany
the implantation of a medical device.
[0007] One approach for minimizing undesirable biological reactions
associated with medical devices is to attach various biomolecules
to their surfaces for the attachment and growth of a cell layer
which the body will accept. Biomolecules such as growth factors,
cell attachment proteins, and cell attachment peptides have been
used for this purpose. In addition, biomolecules such as
antithrombogenics, antiplatelets, anti-inflammatories,
antimicrobials, and the like have also been used to minimize
adverse biomaterial-associated reactions.
[0008] A number of approaches have been suggested to attach such
biomolecules. These approaches typically require the use of
coupling agents such as glutaraldehyde, cyanogen bromide,
p-benzoquinone, succinic anhydrides, carbodiimides, diisocyanates,
ethyl chloroformate, dipyridyl disulphide, epichlorohydrin, azides,
among others, which serve as attachment vehicles for coupling of
biomolecules to substrate surfaces. For example, covalent
attachment of biomolecules using water soluble carbodiimides is
described by Hoffman et al., "Covalent Binding of Biomolecules to
Radiation-Grafted Hydrogels on Inert Polymer Surfaces," Trans. Am.
Soc. Artif. Intern. Organs, 18, 10-18 (1972); and Ito et al.,
"Materials for Enhancing Cell Adhesion by Immobilization of
Cell-Adhesive Peptide," J. Biomed. Mat. Res., 25, 1325-1337
(1991).
[0009] One type of biomolecule which is commonly coupled to
biomaterial surfaces with coupling molecules is protein. Proteins
are polypeptides made up of amino acid residues. A protein
comprising two or more polypeptide chains is called an oligomeric
protein. In general, established coupling procedures couple
proteins to substrate surfaces through a protein's lysine amino
acid residues which comprise terminal amine moieties. However, not
all biomolecules, including some proteins and peptides, comprise
terminal amine moieties. In addition, a number of established
coupling procedures couple biomolecules which comprise reactive
moieties capable of forming bonds with amine moieties to substrate
surfaces which comprise terminal amine moieties.
[0010] Thus, what is needed are methods for creating terminal amine
moieties within biomolecules which lack terminal amine moieties.
These newly formed terminal amine moieties can then be used to
attach these modified biomolecules to a medical device substrate
surface which comprises chemical moieties capable of forming bonds
with amine moieties. In addition, methods are needed for creating
terminal amine moieties on medical device substrate surfaces which
lack terminal amine moieties. These newly formed terminal amine
moieties can then be used to attach biomolecules which comprise
chemical moieties capable of forming bonds with amine moieties.
[0011] In some cases, covalently coupling of a biomolecule to a
biomaterial surface is not desirable. Therefore, there also exists
a need for methods which may ionically couple a biomolecule to a
biomaterial surface. In fact, ionic coupling techniques have an
advantage of not altering the chemical composition of an attached
biomolecule, thereby reducing the possibility of destroying the
biological properties of an attached biomolecule. Ionic coupling of
biomolecules also has an advantage of releasing the biomolecule
under appropriate conditions. One example of the ionic attachment
of a biomolecule to a surface is set forth in U.S. Pat. No.
4,442,133 to Greco et al. In this case, a tridodecyl methylammonium
chloride (TDMAC) coating is used to ionically bind an antibiotic
agent.
[0012] Another type of biomolecule which is often coupled to
biomaterial surfaces is heparin. Heparin, an anionic biomolecule,
is of great interest to a number of investigators for the
development of non-thrombogenic blood-contact biomaterial surfaces.
Heparin, a negatively charged glycosaminoglycan, inhibits blood
coagulation primarily by promoting the activity of antithrombin III
(ATIII) to block the coagulation enzymes thrombin, factor Xa and,
to some extent, factors IXa, XIa and XIIa. Surfaces bearing bound
heparin have been shown to have anticoagulant activity, therefore,
heparinization tends to be a popular technique for improving the
thromboresistance of biomaterials. In fact, surface heparinization
through an ionic bond is one of the methods used to improve the
blood compatibility of a variety of biomaterial surfaces.
[0013] The original method of heparinization of surfaces was
described by Gott et al., "Heparin Binding On Colloidal Graphite
Surfaces", Science, 142, 1297-1298 (1963). They prepared a
graphite-benzalkonium-heparin surface and observed good
non-thrombogenic characteristics. Others followed, treating
materials with quaternary ammonium salts to ionically bind heparin.
Improving on Gott's technique, Grode et al., "Nonthrombogenic
Materials via a Simple Coating Process"; Trans. Amer. Soc. Artif.
Intern. Organs, 15, 1-6 (1969), eliminated the need for a graphite
coating by using tridodecyl methylammonium chloride (TDMAC).
Various other quaternary ammonium salts have also been used such as
benzalkonium chloride, cetylpyrrdinium chloride,
benzyldimethylstearyammonium chloride, benzylcetyldimethylammonium
chloride as set forth in U.S. Pat. No. 5,069,899 to Whitbourne and
Mangan.
[0014] Glutaraldehyde was even used by some investigators to
increase the stability of heparin bound ionically through various
ammonium groups. Rather than using a low molecular weight
quaternary salt or quaternary amine, many investigators
incorporated the quaternizable amine directly onto substrates by
copolymerization techniques. In another approach, Barbucci et al.,
"Surface-Grafted Heparinizable Materials", Polymer. 26, 1349-1352
(1985), grafted tertiary amino polymers of poly(amido-amine)
structure onto substrates for ionically coupling heparin. The
cationic amino groups are capable of interacting electrostatically
with the negatively charged groups present in the heparin molecule.
They found that the surface's capacity to retain heparin was
directly related to the basicity of the grafted cationic amino
groups. The greater the basicity of the surface amino groups on the
surface, the greater the capacity of the surface has to retain
heparin due to a greater percentage of the surface amino groups
being protonated at physiological pH.
[0015] Current techniques for immobilization of heparin or other
charged biomolecules by an ionic bond have been achieved by
introducing opposite charges on a biomaterial surface. The main
limit to utilization of ionically bonding methods is the creation
of opposing charges on either a biomolecule or a biomaterial
surface or both. Thus, what is needed are methods for creating
charges on a biomolecule or a biomaterial surface or both. These
newly formed charges can then be used to attach a biomolecule to a
medical device substrate surface.
SUMMARY OF THE INVENTION
[0016] The present invention provides methods for attaching a
biomolecule to a substrate surface and corresponding medical
devices. The present invention provides methods for making a
medical device having at least one biomolecule immobilized on a
biomaterial surface. One method of the present invention includes
converting a biomolecule comprising an unsubstituted amide moiety
(RCONH.sub.2) into an amine-functional material (RNH.sub.2);
combining the amine-functional material with a medical device
biomaterial surface comprising a chemical moiety (such as, for
example, an aldehyde moiety, an epoxide moiety, an isocyanate
moiety, a phosphate moiety, a sulphate moiety or a carboxylate
moiety) which is capable of forming a chemical bond with the
amine-functional material, to bond the two materials together to
form an immobilized biomolecule on a medical device biomaterial
surface.
[0017] The present invention provides another method for making a
medical device having at least one biomolecule immobilized on a
biomaterial surface. The method includes converting a medical
device biomaterial surface comprising an unsubstituted amide moiety
(RCONH.sub.2) into an amine-functional material (RNH.sub.2);
combining the amine-functional material with a biomolecule
comprising a chemical moiety (such as, for example, an aldehyde
moiety, an epoxide moiety, an isocyanate moiety, a phosphate
moiety, a sulphate moiety or a carboxylate moiety) which is capable
of forming a chemical bond with the amine-functional material, to
bond the two materials together to form an immobilized biomolecule
on a medical device biomaterial surface.
[0018] The present invention also provides a method for making a
medical device having at least one biomolecule immobilized on a
biomaterial surface. The method includes converting a biomolecule
comprising an unsubstituted amide moiety (RCONH.sub.2) into an
amine-functional material (RNH.sub.2); converting the
amine-functional material into a guanidino-functional material
(RNHC(NH)NH.sub.2); combining the guanidino-functional material
with a medical device biomaterial surface comprising a chemical
moiety (such as, for example, a 1,2-dicarbonyl moiety, a phosphate
moiety, a sulphate moiety or a carboxylate moiety) which is capable
of forming a chemical bond with the guanidino-functional material,
to bond the two materials together to form an immobilized
biomolecule on a medical device biomaterial surface.
[0019] Additionally, the present invention provides a method for
making a medical device having at least one biomolecule immobilized
on a biomaterial surface. The method includes converting a medical
device biomaterial surface comprising an unsubstituted amide moiety
(RCONH.sub.2) into an amine-functional material (RNH.sub.2);
converting the amine-functional material into a
guanidino-functional material (RNHC(NH)NH.sub.2); combining the
guanidino-functional material with a biomolecule comprising a
chemical moiety (such as, for example, a 1,2-dicarbonyl moiety, a
phosphate moiety, a sulphate moiety or a carboxylate moiety) which
is capable of forming a chemical bond with the guanidino-functional
material, to bond the two materials together to form an immobilized
biomolecule on a medical device biomaterial surface.
[0020] Another method of the present invention may be employed to
crosslink biomolecules, located in solution or on biomaterial
surfaces, comprising both an unsubstituted amide moiety and a
chemical moiety capable of forming a chemical bond with an amine
moiety. Such a method comprises converting a biomolecule comprising
an unsubstituted amide moiety into an amine-functional material;
allowing the amine-functional material to combine with the chemical
moiety capable of forming a chemical bond with an amine moiety to
form a chemical linkage and a crosslinked material. This
crosslinked material may be employed as a biomaterial or as a
biomaterial coating. In addition, such crosslinked materials may be
further modified to contain additional biomolecules. For example,
biomolecules comprising a chemical moiety capable of forming a
chemical bond with an amine moiety may be attached to residual
amine moieties present in or on the surface of the crosslinked
material. Alternatively, biomolecules comprising an amine moiety
may be attached to residual chemical moieties capable of forming
chemical bonds with amine moieties present in or on the surface of
the crosslinked material. Additionally, biomolecules coated onto a
biomaterial surface may be crosslinked according to still another
method of the present invention.
[0021] Another method of the present invention may be employed to
crosslink biomolecules, located in solution or on biomaterial
surfaces, comprising both an unsubstituted amide moiety and a
chemical moiety capable of forming a chemical bond with a guanidino
moiety. Such a method comprises converting a biomolecule comprising
an unsubstituted amide moiety into an amine-functional material;
converting the amine-functional material into a
guanidino-functional material (RNHC(NH)NH.sub.2); allowing the
guanidino-functional material to combine with the chemical moiety
capable of forming a chemical bond with an guanidino moiety to form
a chemical linkage and a crosslinked material. This crosslinked
material may be employed as a biomaterial or as a biomaterial
coating. In addition, such crosslinked materials may be further
modified to contain additional biomolecules. For example,
biomolecules comprising a chemical moiety capable of forming a
chemical bond with an guanidino moiety may be attached to residual
guanidino moieties present in or on the surface of the crosslinked
material. Alternatively, biomolecules comprising a guanidino moiety
may be attached to residual chemical moieties capable of forming
chemical bonds with guanidino moieties present in or on the surface
of the crosslinked material. Additionally, biomolecules coated onto
a biomaterial surface may be crosslinked according to still another
method of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0022] As used in the specification and claims hereof, the
following terms have the particular meanings and definitions set
forth below.
[0023] We define the term "chemical bond" appearing herein to be
interpreted broadly to encompass not only covalent bonding and
ionic bonding but also interactions, such as, for example, van der
Waals forces and hydrogen bonding.
[0024] We define the term "biomolecule" appearing herein as a
material that engages in a biological activity or which is
effective in modulating a biological activity such as eliminating,
reducing or enhancing various biological reactions that typically
accompany the exposure of human or animal bodily tissues or fluids
to a biomaterial. Biomaterial-associated reactions include
thrombosis, tissue death, tumor formation, allergic reaction,
foreign-body reaction (rejection), inflammatory reaction, infection
and cellular attachment and growth. Biomolecules suitable for use
in the present invention comprise either an unsubstituted amide
moiety, a 1,2-dihydroxy moiety, a 2-aminoalcohol moiety, a
1,2-dicarbonyl moiety, a guanidino moiety, a chemical moiety
capable of forming either a covalent bond with an amine moiety
(such as, for example, an aldehyde moiety, an epoxide moiety or an
isocyanate moiety) or a chemical moiety capable of forming an ionic
bond with an amine moiety (such as, for example, a phosphate
moiety, a sulphate moiety or a carboxylate moiety), or any possible
combination of any one or more of these moieties alone or in
combination. In addition, the term "biomolecule" appearing herein
may mean any one or more of a biomolecule alone or a combination of
different biomolecules.
[0025] Generally, biomolecules used according to this invention may
be, for example an anticoagulant agent such as heparin and heparan
sulfate, an antithrombotic agent, a clotting agent, a platelet
agent, an anti-inflammatory agent, an antibody, an antigen, an
immunoglobulin, a defense agent, an enzyme, a hormone, a growth
factor, a neurotransmitter, a cytokine, a blood agent, a regulatory
agent, a transport agent, a fibrous agent, a protein such as
avidin, a glycoprotein, a globular protein, a structural protein, a
membrane protein and a cell attachment protein, a peptide such as a
glycopeptide, a structural peptide, a membrane peptide and a cell
attachment peptide, a proteoglycan, a toxin, an antibiotic agent,
an antibacterial agent, an antimicrobial agent such as penicillin,
ticarcillin, carbenicillin, ampicillin, oxacillian, cefazolin,
bacitracin, cephalosporin, cephalothin, cefuroxime, cefoxitin,
norfloxacin, perfloxacin and sulfadiazine, hyaluronic acid, a
polysaccharide, a carbohydrate, a fatty acid, a catalyst, a drug,
biotin, a vitamin, a DNA segment, a RNA segment, a nucleic acid, a
lectin, a ligand and a dye (which acts as a biological ligand). The
biomolecules may be found in nature (naturally occurring) or may be
chemically synthesized.
[0026] Biomolecules may be chemically synthesized by a number of
methods well known to those skilled in the art. For example, a
number of methods are know for synthesizing proteins or peptides
from amino acids including solution (classical) synthesis methods
and solid phase (e.g., SPPS) synthesis methods. Peptides of varying
length may also be formed by the partial hydrolysis of very long
polypeptide chains of proteins. In addition, proteolytic enzymes
such as trypsin, chymotrypsin, and pepsin may be used to cleave
specific peptide bonds in proteins and peptides. Furthermore,
site-specific oxidative cleavage of peptide bonds using either
cupric ions or ferric ions may be used to create peptide and/or
polypeptide chains comprising unsubstituted amide moieties.
Peptides are short chains constructed of two or more amino acids
covalently joined through substituted amide linkages, termed
peptide bonds. Two amino acids joined by a peptide bond forms a
dipeptide. Three amino acids joined by two peptide bonds forms a
tripeptide; similarly, there are tripeptides and pentapeptides.
When there are many amino acids joined together, the structure is
termed a polypeptide. In general, polypeptides contain less than
100 amino acid residues and proteins contain 100 or more amino acid
residues.
[0027] Some biomolecules are susceptible to conformational changes
when brought into contact with a hydrophobic substrate surface.
These conformational changes can lead to the exposure of
internalized nonpolar groups which may lead to hydrophobic
interactions between the biomolecule and the surface. These
hydrophobic interactions may cause the exclusion of water molecules
that normally surround the biomolecule in solution. This exclusion
of water molecules between the biomolecule and the surface
strengthens the hydrophobic interaction and may cause further
conformational change of the biomolecule. The degree of
conformational change a biomolecule experiences may or may not
destroy its biological properties. Therefore, one must take into
account the hydrophobic nature of the substrate surface when
attaching biomolecules which are prone to hydrophobic interactions.
In such cases, it is preferred to create a hydrophilic environment
on the biomaterial surface, thereby preventing any unwanted
hydrophobic interactions between the biomolecule and the surface
which may destroy the biological properties of the biomolecule.
[0028] There are a number of surface-derivatization techniques
(e.g., grafting techniques) well known to those skilled in the art
for creating hydrophilic substrate surfaces. For example,
techniques based on ceric ion initiation, ozone exposure, corona
discharge, UV irradiation and ionizing radiation (.sup.60Co,
X-rays, high energy electrons, plasma gas discharge) are known.
[0029] We define the term "glycoprotein" appearing herein as a
conjugated protein which contains at least one carbohydrate group
which may comprise a 1,2-dihydroxy moiety. A typical glycoprotein
contains one or more oligosaccharide units linked to either
asparagine amino acid residues by N-glycosidic bonds or serine or
threonine amino acid residues by O-glycosidic bonds. The saccharide
unit directly bonded to asparagine is typically
N-acetylglucosamine, whereas N-acetylgalactosamine tends to be the
saccharide unit bonded to serine or threonine residues.
Oligosaccharides bound to glycoproteins may contain a variety of
carbohydrate units. They tend to be located at sites away from the
biologically active site of the protein. Thus, oligosaccharide
moieties of glycoproteins can typically be modified with little or
no effect on the biological properties of the protein
[0030] We define the term "glycopeptide" appearing herein as a
conjugated peptide which contains at least one carbohydrate group
which may comprise a 1,2-dihydroxy moiety. As mentioned earlier,
peptides are short chains constructed of two or more amino acids
covalently joined through substituted amide linkages, termed
peptide bonds. Two amino acids joined by a peptide bond forms a
dipeptide. Three amino acids joined by two peptide bonds forms a
tripeptide; similarly, there are tetrapeptides and pentapeptides.
When there are many amino acids joined together, the structure is
termed a polypeptide. In general, polypeptides contain less than
100 amino acid residues and proteins contain 100 or more amino acid
residues.
[0031] Glycoproteins and glycopeptides can be chemically
synthesized by a number of methods well known to those skilled in
the art. For example, glycoproteins and/or glycopeptides can be
formed from natural or chemically synthesized proteins and/or
peptides by glycosylation, which is the addition of carbohydrate
side chains. There are a number of methods well known to those
skilled in the art for glycosylating proteins or peptides. For
example, side-chain glycosylation can be performed chemically with
glycosylbromides for serine (Ser, S) and threonine (Thr, T) amino
acid residues and glycosylamines for aspartic acid (Asp, D) amino
acid residues, thereby producing glycosylated asparagine (Asn, N)
amino acid residues. In addition, glycosylating enzymes can be used
to attach carbohydrate side chains to proteins or peptides.
[0032] Proteins or peptides, chemically synthesized or naturally
occurring, also suitable for use in the present invention comprise
an asparagine (Asn, N) amino acid residue or a glutamine (Gln, Q)
amino acid residue, both of which comprise an unsubstituted amide
moiety. In addition, proteins or peptides, again chemically
synthesized or naturally occurring, which are also suitable for use
in the present invention comprise a N-terminal serine (Ser, S)
amino acid residue, a N-terminal threonine (Thr, T) amino acid
residue, or a 5-hydroxylysine (5-hydroxylysine is only known to
occur naturally in collagen, but in principal may be placed
anywhere in a synthetic peptide or protein) amino acid residue, all
of which comprise a 2-aminoalcohol moiety.
[0033] Biomolecules or biomaterials of the present invention
comprising an unsubstituted amide moiety may be converted into an
amine-functional material via a Hofmann rearrangement reaction,
also known as a Hofmann degradation of amides reaction. For
example, Wirsen et al., "Bioactive heparin surfaces from
derivatization of polyacrylamide-grafted LLDPE", Biomaterials, 17,
1881-1889 (1996), demonstrated the conversion of unsubstituted
amide moieties of a polyacrylamide-low density polyethylene film
into primary amine moieties using a Hofmann rearrangement reaction.
In another example, Sano et al., "Introduction of functional groups
onto the surface of polyethylene for protein immobilization",
Biomaterials, 14, 817-822 (1993), demonstrated the conversion of
unsubstituted amide moieties of a polyacrylamide-high density
polyethylene film into primary amine moieties using a Hofmann
rearrangement reaction. In another example, Fuller et al., "A new
class of amino acid based sweeteners", J. Am. Chem. Soc., 107,
5821-5822 (1985), demonstrated the conversion of an unsubstituted
amide moiety of an amino acid into a primary amine moiety using a
Hofmann rearrangement reaction. In another example, Loudon et al.,
"Conversion of aliphatic amides into amines with
[I,I-bis(trifluoroacetoxy)iodo]benzene. 1. Scope of the reaction",
J. Org. Chem., 49, 4272-4276 (1984), demonstrated the conversion of
various unsubstituted amide moieties, including the amide side
chain in a glutamine amino acid residue, into primary amine
moieties using Hofmann rearrangement reactions.
[0034] The Hofmann rearrangement reaction which converts an
unsubstituted amide moiety into a primary amine moiety may be
carried out with chemical reactants such as, for example, bromine,
bromide, bromite, hypobromite, chlorine, chloride, chlorite,
hypochlorite, lead tetraacetate, benzyltrimethylammonium tribromide
and hypervalent organoiodine compounds such as, for example,
[bis(trifluoroacetoxy)iodo]benzene, hydroxy(tosyloxy)iodobenzene
and iodosylbenzene. A general discussion of Hofmann rearrangement
reactions is contained in Comprehensive Organic Synthesis, Volume
6, 800-806, Pergamon Press. For example, Kajigaeshi et al., "An
efficient method for the Hofmann degradation of amides by use of
benzyltrimethylammonium tribromide", Chemistry Letters, 463-464
(1989), described various methods for obtaining amines from amides
using the Hofmann rearrangement reaction. These methods include,
for example, the use of bromine or chlorine in an alkaline
solution, the use of lead tetraacetate in an alcohol solution, the
use of [bis(trifluoroacetoxy)iodo]benzene in an aqueous
acetonitrile solution, the use of sodium bromite in an alkaline
solution, and the use of benzyltrimethylammonium tribromide in an
alkaline solution. Catalysts such as, for example, triethylamine,
tin(IV) chloride, dibutylstannyl dilaurate or pyridine are
sometimes used in a Hofmann rearrangement reaction. Typical
solvents include, for example, water, hydroxides, methoxides,
alcohols, dimethylformamide, acetonitrile, benzene, carboxylic
acids or combinations thereof.
[0035] Depending on the chemical reactants, the Hofmann
rearrangement reaction may be carried out under acidic, neutral or
basic conditions. Although applicants do not wish to be bound by
any single theory, it is generally believed that when the reaction
is carried out with particular amine forming agents in an aqueous
base a N-halo amide intermediate is formed. An isocyanate is then
formed from the N-halo amide intermediate. The formed isocyanate
then readily hydrolyzes into a primary amine. In contrast, when the
reaction is carried out in an alcohol a carbamate is generally
formed. The carbamate may then be hydrolyzed into a primary amine.
For example, when the reaction of an amide with bromine is carried
out in methanol containing sodium methoxide instead of in aqueous
base, the product is a carbamate which is easily converted to an
amine via hydrolysis. Depending on the reaction conditions,
side-reactions such as chain scission, hydrolysis and/or urea
formation may occur. However, side-reactions may be minimized by
changes in the reaction conditions. For example, changes in the
reaction conditions such as pH, time, temperature and/or the amount
of amine forming agent may minimize various side-reactions.
[0036] In general, the Hofmann rearrangement reaction is carried
out with an amine forming agent in amounts ranging from about 0.5
eq. to about 2 eq. based on the amide content of the biomolecule or
biomaterial. In addition, the reaction is generally carried out at
a temperature between about -10 and about 100 degrees Celsius,
preferably from about 0 and about 50 degrees Celsius. Depending on
the material, a Hofmann rearrangement reaction may be carried out
for as short as a few minutes to as long as many hours. Time,
temperature and pH limitations of the present invention are
generally governed by the stability of the materials imparted by
the Hofmann rearrangement process. Wide latitude may be employed in
determining the optimum conditions for a particular system. Such
conditions may be determined readily by one skilled in the art by
routine experimentation upon examination of the information
presented herein.
[0037] We define the term "amine forming agent" appearing herein to
include any chemical agent or combination of chemical agents
capable of forming an amine moiety upon its or their reaction with
an unsubstituted amide moiety. Examples of amine forming agents
include, for example, bromine, bromide, bromite, hypobromite,
chlorine, chloride, chlorite, hypochlorite, lead tetraacetate,
benzyltrimethylammonium tribromide and hypervalent organoiodine
compounds such as, for example, [bis(trifluoroacetoxy)iodo]benzene,
hydroxy(tosyloxy)iodobenzene and iodosylbenzene. Amine forming
agents include any of the many possible Hofmann rearrangement
reactants. As mentioned above, the term "amine forming agent"
appearing herein may mean any one or more of an amine forming agent
or a combination of different amine forming agents.
[0038] Biomolecules or biomaterials of the present invention
comprising, in addition to an unsubstituted amide moiety, a primary
amine moiety of which is desired to be left intact and unreacted
following coupling may be protected or blocked prior to the Hofmann
rearrangement reaction. For example, a protein may comprise both a
lysine amino acid residue and, for example, an asparagine amino
acid residue. As mentioned earlier, there are a number of
established coupling procedures which may couple a protein
comprising a lysine amino acid residue to a substrate surface
through the protein's lysine amino acid residue. However, a
protein's lysine amino acid residues are typically associated with
the protein's biologically active site. Therefore, coupling a
protein to a substrate surface via a protein's primary amine moiety
in the side chain of its lysine amino acid residue may destroy the
biological properties of the attached protein. However, a protein's
lysine amino acid residue may be protected or blocked by a number
of methods well known to those skilled in the art. For example, the
amine moiety may be protected using, for example, a
tert.butyloxycarbonyl (Boc) group which is typically cleaved with
acid, a benzyloxycarbonyl (Z) group which is typically cleaved by
hydrogenolysis, a biphenylisopropyloxycarbonyl (Bpoc) group which
is typically cleaved with acid, a triphenylmethyl (trityl) group
which is typically cleaved with acid, a
9-fluoroenylmethyloxycarbonyl (Fmoc) group which is typically
cleaved with base or a blocking group which is pH stable but is
cleaved by enzyme-catalyzed hydrolysis. The appropriate
amine-blocking group to use to protect the amine moiety will depend
highly on the entire sequence of reaction conditions chosen for
biomolecule attachment or crosslinking. Following blocking of the
amine moiety of the lysine residue, the amide moiety of the
asparagine residue may then be converted into an amine moiety via a
Hofmann rearrangement reaction. The protein may then be coupled to
the substrate via one method of the present invention through the
newly formed amine moiety. Following coupling, the amine-blocking
group may then be removed, thereby preserving the protein's
biological activity. This type of blocking scheme may be employed
on biomolecules and/or biomaterials of the present invention which
contain amine moieties which are desired to be left intact and
unreacted.
[0039] Biomaterials of the present invention not comprising
unsubstituted amides on their surface may be amidated readily
through a number of methods well known in the art. For example,
unsubstituted amides may be provided by ceric ion grafting
acrylamide to a biomaterial surface as set forth in U.S. Pat. No.
5,344,455 to Keogh et al. Alternatively, for example, a grafted
acrylamide-containing polymer may be attached by radiation grafting
as set forth in U.S. Pat. No. 3,826,678 to Hoffman et al. There are
a number of surface-derivatization techniques (e.g., grafting
techniques) well known in the art for creating substrate surfaces
comprising unsubstituted amide moieties. For example, techniques
based on ceric ion initiation, ozone exposure, corona discharge. UV
irradiation and ionizing radiation (.sup.60Co, X-rays, high energy
electrons, plasma gas discharge) are known. In addition, amides can
generally be prepared by reaction of ammonia with acid chlorides.
This reaction is commonly known as ammonolysis. Acid chlorides are
prepared by substitution of --Cl for the --OH group of a carboxylic
acid. Reagents commonly used to form acid chlorides from carboxylic
acids include thionyl chloride (SOCl.sub.2), phosphorus trichloride
(PCl.sub.3) and phosphorus pentachloride (PCl.sub.5). Two amino
acids comprising carboxylic acid moieties which may be converted
into acid chlorides are aspartic acid (Asp, D) amino acid and
glutamic acid (Glu, E) amino acid. The acid chloride moieties may
then be converted into amide moieties followed by conversion into
amine moieties. In addition, treatment of an ester moiety with
ammonia, generally in ethyl alcohol solution, will yield an amide
moiety.
[0040] Biomolecules or biomaterials suitable for use according to
one method of the present invention may comprise at least one
negatively charged moiety (also known as an anionic moiety) at
physiological pH, such as a phosphate moiety, a sulphate moiety or
a carboxylate moiety. A negatively charged moiety is capable of
interacting electrostatically with a positively charged moiety
(also known as a cationic moiety) thereby forming an ionic chemical
bond or linkage.
[0041] Biomaterials that do not contain a negative charge on their
surfaces may be furnished with a net negative and may be modified
readily through a number of methods well known in the art. For
example, polyethylene may be exposed to sulfuric acid comprising
potassium permanganate thereby creating a negative charge. Other
examples of furnishing biomaterials with negatively charged
surfaces are taught in U.S. Pat. No. 5,344,455 to Keogh et al. and
U.S. Pat. No. 5,429,618 to Keogh.
[0042] Biomolecules or biomaterials suitable for use according to
one method of the present invention may comprise at least one
positively charged moiety at physiological pH, such as an amine
moiety or a guanidino moiety. Biomolecules or biomaterials that do
not comprise a positive charge may be furnished with a net positive
charge by one method of the present invention. As mentioned
earlier, a positively charged moiety is capable of interacting
electrostatically with a negatively charged moiety thereby forming
an ionic chemical bond or linkage.
[0043] Biomolecules or biomaterials suitable for use according to
one method of the present invention may comprise at least one
epoxide moiety. Epoxide moieties are three-membered rings
comprising two carbon atoms and an oxygen atom. An epoxide ring is
also known as an oxirane ring. There are a number of techniques
well known in the art to produce epoxides. Epoxide moieties are
highly reactive due to the ease of opening of the highly strained
three-membered ring. An epoxide moiety will react readily with an
amine moiety, thereby forming a covalent bond or linkage. The
reaction of an epoxide moiety with an amine moiety is well known in
the art.
[0044] Biomolecules or biomaterials suitable for use according to
one method of the present invention may comprise at least one
isocyanate moiety. An isocyanate moiety (RNCO) will react readily
with an amine moiety, thereby forming a covalent bond or linkage
called a urea. There are a number of techniques well known in the
art to produce isocyanates. In addition, the reaction of an
isocyanate moiety with an amine moiety is well known in the
art.
[0045] We define the term "1,2-dihydroxy moiety" appearing herein
as a carbon-carbon bond bearing two adjacent hydroxyl moieties.
[0046] We define the term "2-aminoalcohol moiety" appearing herein
as a carbon-carbon bond bearing an amine moiety adjacent to a
hydroxyl moiety.
[0047] The 1,2-dihydroxy moiety and the 2-aminoalcohol moiety are
both oxidizable with periodate, which may be provided as periodic
acid or salts thereof, such as sodium periodate, potassium
periodate, or other alkali metal periodates. Typically, a
stoichiometric amount of periodate is used to oxidize the desired
number of 1,2-dihydroxy moieties or 2-aminoalcohol moieties to form
aldehyde moieties, however less than a stoichiometric amount or
more than a stoichiometric amount may be used.
[0048] Periodate oxidation of a 1,2-dihydroxy moiety or a
2-aminoalcohol moiety is generally carried out in an aqueous
solution, preferably an aqueous buffered solution, at a temperature
that does not destroy the desired properties of the material.
Generally, buffers having a pH in a range between about 4 and about
9 can be used, with a pH between about 6 and about 8 desired for
certain pH sensitive materials. Generally, the oxidation is carried
out at a temperature between about 0 and about 50 degrees Celsius,
and preferably at a temperature between about 4 and about 37
degrees Celsius. Depending on the material, oxidation reactions can
be carried out for as short as a few minutes to as long as many
days. Commonly, oxidation is complete within 24 hours. Long-term
oxidation reactions are preferably performed in the dark to prevent
"overoxidation."
[0049] Treatment times and temperatures for the oxidation process
tend to be inversely related. That is, higher treatment
temperatures require relatively shorter treatment times. Time and
temperature limitations of the present invention are generally
governed by the stability of the materials imparted by the
oxidation process. Wide latitude may be employed in determining the
optimum conditions for a particular system. Such conditions may be
determined readily by one skilled in the art by routine
experimentation upon examination of the information presented
herein.
[0050] Subsequent to oxidation, the reaction solution may be stored
prior to use at about 4 degrees Celsius. Typically, the storage
stability of the reaction solution at a neutral pH or slightly
acidic pH may extend between about one and about fourteen days and
sometimes even months when stored in the dark.
[0051] In general, an aldehyde moiety (RCHO) will react chemically
with a primary amine moiety (R'NH.sub.2) to form a relatively
unstable imine moiety (R'N.dbd.CHR). The reaction of an aldehyde
moiety with a primary amine moiety which is commonly referred to as
a Schiff base reaction may be carried out under the same conditions
as described above for the periodate oxidation reaction, which is
generally designed to protect a biomolecule from damage. To
stabilize the relatively unstable imine linkage, subsequent
reductive alkylation of the imine moiety is carried out using
reducing agents (i.e., stabilizing agents) such as, for example,
sodium borohydride, sodium is cyanoborohydride, and amine boranes,
to form a secondary amine (R'NH--CH.sub.2R). This reaction can also
be carried out under the same conditions as described above for the
periodate oxidation reaction. Typically, however, the coupling and
stabilizing reactions are carried out in a neutral or slightly
basic solution and at a temperature between about 0 and about 50
degrees Celsius. Preferably, the pH is between about 6 and about
10, and the temperature is between about 4 and about 37 degrees
Celsius, for the coupling and stabilizing reactions. These
reactions (coupling and stabilizing) may be allowed to proceed for
just a few minutes or for many hours. Commonly the reactions are
complete (i.e., coupled and stabilized) within 24 hours.
[0052] We define the term "guanidino moiety" appearing herein to
include guanidine, guanidinium, guanidine derivatives such as
(RNHC(NH)NHR'), monosubstituted guanidines, monoguanides,
biguanides, biguanide derivatives such as (RNHC(NH)NHC(NH)NHR''),
and the like. In addition, the term "guanidino moiety" appearing
herein may mean any one or more of a guanide alone or a combination
of different guanides.
[0053] Guanidine is the imide of urea, or the amidine of carbamic
acid. It is a very strong base with a pK.sub.a of 13.5 in water.
The great basicity of guanidine is a result of the stability of the
conjugated acid (guanidinium) in water. The positive charge on the
guanidinium ion can be spread equally among the three nitrogens by
resonance. The guanidinium ion is also quite hydrophilic and is
well solvated in aqueous media due to the extensive hydrogen
bonding of six potential hydrogen bond donors to the solvent. The
partial positive charge of the hydrogen bond donors increases their
strength for donation to the negative dipole of water. Crystal
structures of simple guanidinium derivatives have revealed several
common features. First, the C--N single bond length in an alkyl
guanidine is typically shorter than the usual C--N single bond
length. Usually, the three C--N bonds in the guanidinium group
itself are nearly equal in length with an average of 1.33 A. The
three N--C--N bond angles are almost always near 120.degree..
[0054] The guanidinium group's features make it a very attractive
moiety. For example, its high basicity (a pK.sub.a of 13.5 for
guanidinium itself) allows it to remain protonated over a much
wider range of pH than does the ammonium group. In fact, at
physiological pH, all but a small fraction of the guanidine
molecules will exist as positively charged species. The guanidinium
group's enhanced hydrogen bonding capabilities, typically two
linear hydrogen bonds, allow it to form tighter complexes with
anions that are capable of hydrogen bonding. In fact, the
guanidinium group may form characteristic pairs of zwitterionic
hydrogen bonds which provide binding strength by their charge and
structural organization by their arrangement. Another feature of
guanidines are their ability to react with 1,2-dicarbonyl moieties
under mild alkaline conditions to form covalent bonds. The reaction
of a guanidino moiety and a 1,2-dicarbonyl moiety is similar to a
Schiff base reaction (the reaction between an amine moiety and an
aldehyde moiety). In some cases, it may be desirable to use a
stabilizing agent such as borate ion (BO.sub.3.sup.-) to stabilize
the resultant compound.
[0055] We define the term "1,2-dicarbonyl moiety" appearing herein
as two carbonyl (C.dbd.O) groups located on adjacent carbon atoms.
A carbonyl group contains a carbon-oxygen double bond.
[0056] Biomolecules or biomaterials of the present invention
comprising an unsubstituted amide moiety may be modified to
comprise guanidino moieties.
[0057] The method of the present invention includes converting an
unsubstituted amide moiety (RCONH.sub.2) into an amine-functional
material (RNH.sub.2). The amine-functional material is then
modified to comprise guanidino moieties by reaction with compounds
such as S-ethylthiouronium bromide, S-ethylthiouronium chloride,
O-methylisourea, O-methylisouronium sulfate, O-methylisourea
hydrogen sulfate, S-methylisothiourea, 2-methyl-1-nitroisourea,
aminoiminomethanesulfonic acid, cyanamide, cyanoguanide,
dicyandiamide, 3,5-dimethyl-1-guanylpyrazole nitrate and
3,5-dimethyl pyrazole. For example, reaction of amines with
O-methylisourea, S-methylisourea, S-ethylthiouronium bromide or
S-ethylthiouronium chloride, thereby yielding guanidino moieties,
are generally completed after 8 hours at 70 degrees Celsius in a
solution of sodium hydroxide (NaOH) at pH 10. Reactions of amines
with aminoiminomethanesulfonic acid or cyanamide are generally
performed at room temperature. Another example is the reaction of
an amine with 2-methyl-1-nitroisourea in water to form a
nitroguanidine. The nitro group is then easily removed to form a
guanidino moiety by hydrogenolysis.
[0058] We define the term "guanidino forming agent" appearing
herein to include any chemical agent capable of forming a guanidino
moiety upon its reaction with a non-guanidino moiety. Examples of
guanidino forming agents include S-ethylthiouronium bromide,
S-ethylthiouronium chloride, O-methylisourea, O-methylisouronium
sulfate, O-methylisourea hydrogen sulfate, S-methylisothiourea,
2-methyl-1-nitroisourea, aminoiminomethanesulfonic acid, cyanamide,
cyanoguanide, dicyandiamide, 3,5-dimethyl-1-guanylpyrazole nitrate
and 3,5-dimethylpyrazole. In addition, the term "guanidino forming
agent" appearing herein may mean any one or more of a guanidino
forming agent or a combination of different guanidino forming
agents.
[0059] We define the term "biomaterial" appearing herein as a
material that is substantially insoluble in human or animal bodily
fluids and that is designed and constructed to be placed in or onto
the body or to contact fluid of the body. Ideally, a biomaterial
will not induce undesirable reactions in the body such as blood
clotting, tissue death, tumor formation, allergic reaction, foreign
body reaction rejection) or inflammatory reaction; will have the
physical properties such as strength, elasticity, permeability and
flexibility required to function for the intended purpose; may be
purified, fabricated and sterilized easily; will substantially
maintain its physical properties and function during the time that
it remains implanted in or in contact with the body. Biomaterials
suitable for use in the present invention comprise either an
unsubstituted amide moiety, a 1,2-dihydroxy moiety, a
2-aminoalcohol moiety, a 1,2-dicarbonyl moiety, a guanidino moiety,
a chemical moiety capable of forming either a covalent bond with an
amine moiety (such as an aldehyde moiety, an epoxide moiety or an
isocyanate moiety) or a chemical moiety capable of forming an ionic
bond with an amine moiety (such as a phosphate moiety, a sulphate
moiety or a carboxylate moiety), or any possible combination of any
one or more of these moieties alone or in combination.
Additionally, biomaterials comprising both an unsubstituted amide
moiety and a 1,2-dihydroxy moiety, a 2-aminoalcohol moiety, or a
chemical moiety capable of forming a chemical bond with an amine
moiety may by crosslinked, according to one method of the present
invention. Also, biomaterials may be fabricated by crosslinking
biomolecules, comprising both an unsubstituted amide moiety and a
1,2-dihydroxy moiety, a 2-aminoalcohol moiety, or a chemical moiety
capable of forming a chemical bond with an amine moiety, according
to another method of the present invention.
[0060] Biomaterials or substrates that may be modified according to
one method of the present invention include metals such as
titanium, titanium alloys, TiNi alloys, shape memory alloys, super
elastic alloys, aluminum oxide, platinum, platinum alloys,
stainless steels, stainless steel alloys, MP35N, elgiloy, haynes
25, stellite, pyrolytic carbon, silver carbon, glassy carbon,
polymers such as polyamides, polycarbonates, polyethers,
polyesters, polyolefins including polyethylenes or polypropylenes,
polystyrenes, polyurethanes, polyvinylchlorides,
polyvinylpyrrolidones, silicone elastomers, fluoropolymers,
polyacrylates, polyisoprenes, polytetrafluoroethylenes, rubber,
minerals or ceramics such as hydroxapatite, human or animal protein
or tissue such as bone, skin, teeth, collagen, laminin, elastin or
fibrin, organic materials such as wood, cellulose, or compressed
carbon, a string, a suture, a fiber, a mesh and other materials
such as glass and the like. Biomaterials of the present invention
made using these materials may be coated or uncoated, porous or
nonporous, permeable and nonpermeable, derivatized or
underivatized. We define the term "medical device" appearing herein
as a device having surfaces that contact human or animal bodily
tissue and/or fluids in the course of their operation. This
definition includes within its scope, for example, extracorporeal
devices for use in surgery such as blood oxygenators, blood pumps,
blood sensors, tubing used to carry blood and the like which
contact blood which is then returned to the patient. The definition
includes within its scope endoprostheses implanted in blood contact
in a human or animal body such as vascular grafts, stents,
pacemaker leads, heart valves, and the like that are implanted in
blood vessels or in the heart. The definition also includes within
its scope devices for temporary intravascular use such as
catheters, guide wires, and the like which are placed into the
blood vessels or the heart for purposes of monitoring or
repair.
[0061] One method of the invention may be used to modify substrates
of any shape or form including tubular, sheet, rod and articles of
proper shape for use in a number of medical devices such as
vascular grafts, aortic grafts, arterial, venous, or vascular
tubing, vascular stents, dialysis membranes, tubing or connectors,
blood oxygenator tubing or membranes, ultrafiltration membranes,
intra-aortic balloons, blood bags, catheters, sutures, soft or hard
tissue prostheses, synthetic prostheses, prosthetic heart valves,
tissue adhesives, cardiac pacemaker leads, artificial organs,
endotracheal tubes, lenses for the eye such as contact or
intraocular lenses, blood handling equipment, apheresis equipment,
diagnostic and monitoring catheters and sensors, biosensors, dental
devices, drug delivery systems, or bodily implants of any kind.
[0062] The present invention has an object of solving a number of
problems associated with the use of medical devices. The present
invention includes within its scope methods for attaching
biomolecules to biomaterial surfaces for use in medical devices.
The present invention further provides methods for fabricating
crosslinked biomaterials or crosslinked biomaterial coatings
comprising biomolecules.
[0063] One preferred method of the present invention may be
employed to immobilize a least one biomolecule comprising at least
one 2-aminoalcohol moiety on a biomaterial surface comprising at
least one unsubstituted amide moiety. The method comprises the
steps of: combining a periodate with a biomolecule comprising a
2-aminoalcohol moiety to form an aldehyde-functional material in an
aqueous solution having a pH between about 4 and about 9 and a
temperature between about 0 and about 50 degrees Celsius; providing
a biomaterial surface comprising an unsubstituted amide moiety,
converting the amide moiety into an amine moiety using an amine
forming agent to form an amine-functional material; combining the
aldehyde-functional material with the amine-functional material to
chemically bond the two materials together through an imine moiety;
and reacting the imine moiety with a reducing agent to form an
immobilized biomolecule on a medical device biomaterial surface
through a secondary amine linkage.
[0064] Another preferred method of the present invention may be
employed to immobilize a least one glycoprotein or glycopeptide
comprising at least one 1,2-dihydroxy moiety on a biomaterial
surface comprising at least one unsubstituted amide moiety. The
method includes the steps of: combining a periodate with a
glycoprotein or glycopeptide comprising a 1,2-dihydroxy moiety to
form an aldehyde-functional material in an aqueous solution having
a pH between about 4 and about 9 and a temperature between about 0
and about 50 degrees Celsius; providing a biomaterial surface
comprising an unsubstituted amide moiety, converting the amide
moiety into an amine moiety using an amine forming agent to form an
amine-functional material; combining the aldehyde-functional
material with the amine-functional material to chemically bond the
two materials together through an imine moiety; and reacting the
imine moiety with a reducing agent to form an immobilized
glycoprotein or glycopeptide on a medical device biomaterial
surface through a secondary amine linkage.
[0065] Still another preferred method of the present invention may
be employed to immobilize a least one biomolecule comprising at
least one epoxide moiety on a biomaterial surface comprising at
least one unsubstituted amide moiety. The method includes the steps
of: providing a biomaterial surface comprising an unsubstituted
amide moiety, converting the amide moiety into an amine moiety
using an amine forming agent to form an amine-functional material;
combining the epoxide-functional biomolecule with the
amine-functional material to chemically bond the two materials
together through a covalent linkage.
[0066] Yet another preferred method of the present invention may be
employed to immobilize a least one biomolecule comprising at least
one isocyanate moiety on a biomaterial surface comprising at least
one unsubstituted amide moiety. The method includes the steps of:
providing a biomaterial surface comprising an unsubstituted amide
moiety, converting the amide moiety into an amine moiety using an
amine forming agent to form an amine-functional material; combining
the isocyanate-functional biomolecule with the amine-functional
material to chemically bond the two materials together through a
covalent linkage.
[0067] Another preferred method of the present invention may be
employed to immobilize at least one biomolecule comprising at least
one negatively charged moiety, such as a phosphate moiety, a
sulphate moiety or a carboxylate moiety, on a biomaterial surface
comprising at least one unsubstituted amide moiety. The method
includes the steps of: providing a biomaterial surface comprising
an unsubstituted amide moiety, converting the amide moiety into an
amine moiety using an amine forming agent to form an
amine-functional material; combining the negatively charged
biomolecule with the amine-functional material to chemically bond
the two materials together through an ionic linkage.
[0068] One preferred method of the present invention may be
employed to immobilize at least one biomolecule comprising at least
one chemical moiety which is capable of forming a chemical bond
with a guanidino-functional material (such as, for example, a
1,2-dicarbonyl moiety, a phosphate moiety, a sulphate moiety or a
carboxylate moiety) on a biomaterial surface comprising at least
one unsubstituted amide moiety. The method includes the steps of:
providing a biomaterial surface comprising an unsubstituted amide
moiety, converting the amide moiety into an amine moiety using an
amine forming agent to form an amine-functional material;
converting the amine-functional material into a
guanidino-functional material using a guanidino forming agent;
combining the guanidino-functional material with a biomolecule
capable of forming a chemical bond with a guanidino-functional
material to chemically bond the two materials together through a
chemical linkage.
[0069] Still another preferred method of the present invention may
be employed to immobilize a least one biomolecule comprising a
least one unsubstituted amide moiety on a biomaterial surface
comprising a least one chemical moiety which is capable of forming
a chemical bond with an amine-functional material (such as, for
example, an aldehyde moiety, an epoxide moiety, an isocyanate
moiety, a phosphate moiety, a sulphate moiety or a carboxylate
moiety). The method includes the steps of: providing a biomolecule
comprising an unsubstituted amide moiety, converting the amide
moiety into an amine moiety using an amine forming agent to form an
amine-functional material; combining the amine-functional material
with a biomaterial surface capable of forming a chemical bond with
an amine-functional material to chemically bond the two materials
together through a chemical linkage.
[0070] Yet another preferred method of the present invention may be
employed to immobilize a least one biomolecule comprising a least
one unsubstituted amide moiety on a biomaterial surface comprising
a least one chemical moiety which is capable of forming a chemical
bond with a guanidino-functional material (such as, for example, a
1,2-dicarbonyl moiety, a phosphate moiety, a sulphate moiety or a
carboxylate moiety). The method includes the steps of: providing a
biomolecule comprising an unsubstituted amide moiety, converting
the amide moiety into an amine moiety using an amine forming agent
to form an amine-functional material; converting the
amine-functional material into a guanidino-functional material
using a guanidino forming agent; combining the guanidino-functional
material with a biomaterial capable of forming a chemical bond with
a guanidino-functional material to chemically bond the two
materials together through a chemical linkage.
[0071] One preferred method of the present invention may be
employed to crosslink biomolecules, located in solution or on
biomaterial surfaces, comprising at least one unsubstituted amide
moiety and at least one chemical moiety which is capable of forming
a chemical bond with an amine-functional material (such as, for
example, an aldehyde moiety, an epoxide moiety, an isocyanate
moiety, a phosphate moiety, a sulphate moiety or a carboxylate
moiety). This method comprises the steps of: providing a
biomolecule comprising an unsubstituted amide moiety, converting
the amide moiety into an amine moiety using an amine forming agent
to form an amine-functional biomolecule; allowing the
amine-functional biomolecule to combine with a biomolecule capable
of forming a chemical bond with an amine-functional biomolecule to
chemically bond the two biomolecules together through a chemical
linkage, thereby forming a crosslinked material. This crosslinked
material may be employed as a biomaterial or as a biomaterial
coating. In addition, such crosslinked material may be further
modified to contain additional biomolecules. For example,
biomolecules comprising aldehyde moieties may be attached to
residual amine moieties present in or on the surface of the
crosslinked material.
[0072] Another preferred method of the present invention may be
employed to crosslink biomolecules, located in solution or on
biomaterial surfaces, comprising at least one unsubstituted amide
moiety and at least one chemical moiety which is capable of forming
a chemical bond with a guanidino-functional material (such as, for
example, a 1,2-dicarbonyl moiety, a phosphate moiety, a sulphate
moiety or a carboxylate moiety). This method comprises the steps
of: providing a biomolecule comprising an unsubstituted amide
moiety, converting the amide moiety into an amine moiety using an
amine forming agent to form an amine-functional biomolecule;
converting the amine-functional biomolecule into a
guanidino-functional biomolecule using a guanidino forming agent;
allowing the guanidino-functional biomolecule to combine with a
biomolecule capable of forming a chemical bond with an
guanidino-functional biomolecule to chemically bond the two
biomolecules together through a chemical linkage, thereby forming a
crosslinked material. This crosslinked material may be employed as
a biomaterial or as a biomaterial coating. Additionally, such
crosslinked material may be further modified to contain additional
biomolecules. For example, biomolecules comprising 1,2-dicarbonyl
moieties may be attached to residual guanidino moieties present in
or on the surface of the crosslinked material.
[0073] One example of a biomolecule of the present invention is
collagen. Collagen, which is found in connective tissue, has
special amino acids, one of which is 5-hydroxylysine which may be
oxidized with a source of periodate, which may be provided as
periodic acid or salts thereof, such as sodium periodate, potassium
periodate, or other alkali metal periodates, to form a pendant
aldehyde moiety. The resultant aldehyde moieties may be used to
crosslink the collagen through bonds formed between the aldehydes
and amines, for example, lysine amino acid residues or modified
asparagine amino acid residues, contained on neighboring collagen
molecules. The resultant imine bonds may then be reduced using a
mild reducing agent like sodium borohydride, sodium
cyanoborohydride, or amine boranes. These crosslinks may endow the
collagen biomaterial or biomaterial coating with desirable
biological and/or physical properties such as mechanical strength,
anti-immunogenicity, biostability, among others, without the use of
a coupling agent. Thus, the method of the present invention
eliminates the need for using glutaraldehyde, a commonly used
cytotoxic coupling agent, to crosslink the collagen to control its
physical and biological properties.
[0074] The aldehyde moieties formed by oxidation of collagen may
also be used to couple a variety of amine-containing biomolecules
to the crosslinked collagen biomaterial or biomaterial coating.
Also, the ability to create aldehyde moieties along collagen
molecules enables them to be covalently attached to amine
containing biomaterial surfaces. Such collagen-coated biomaterial
surfaces may be used, for example, as cell seeding surfaces, cell
binding surfaces, cell separating surfaces, tissue fixation,
collagen-coated stents, collagen-coated vascular grafts or collagen
glues.
[0075] Other biomolecules, such as structural proteins, may be
crosslinked to form a material that may be used as a biomaterial or
a biomaterial coating. Also, additional biomolecules, as described
herein, may be attached to residual amine moieties contained in or
on a fabricated crosslinked biomaterial or biomaterial coating, as
described herein. Alternatively, amine containing biomolecules may
be attached to residual aldehyde moieties is contained in or on a
fabricated crosslinked biomolecule biomaterial or biomaterial
coating, as described herein.
[0076] An example of a glycoprotein that can be used in a number of
aspects of the present invention is fibrin(ogen). Fibrin(ogen),
which is a structural protein, has oligosaccharides which can be
oxidized with a source of periodate, which can be provided as
periodic acid or salts thereof, such as sodium periodate, potassium
periodate, or other alkali metal periodates, to form a pendant
aldehyde moiety. The resultant aldehyde moieties can be used to
crosslink the fibrin(ogen) through bonds formed between the
aldehydes and amines contained on neighboring fibrin(ogen)
molecules. The resultant imine bonds can then be reduced using a
mild reducing agent like sodium borohydride, sodium
cyanoborohydride, or amine boranes. These crosslinks can endow the
fibrinogen and/or fibrin (thrombin polymerized fibrinogen)
biomaterial or biomaterial coating with desirable biological and/or
physical properties such as mechanical strength,
anti-immunogenicity, biostability, among others, without the use of
a coupling agent. Thus, the method of the present invention
eliminates the need for using glutaraldehyde, a commonly used
cytotoxic coupling agent, to crosslink the fibrinogen and/or fibrin
to control its physical and biological properties.
[0077] The aldehyde moieties formed by oxidation of fibrin(ogen)
can also be used to couple a variety of amine-containing
biomolecules to the crosslinked fibrin(ogen) biomaterial or
biomaterial coating. Also, the ability to create aldehyde moieties
along fibrin(ogen) molecules enables them to be covalently attached
to amine containing biomaterial surfaces. Such fibrinogen/fibrin
coated biomaterial surfaces can be used, for example, as cell
seeding surfaces, cell binding surfaces, cell separating surfaces,
fibrinogen/fibrin-coated stents, fibrinogen/fibrin-coated vascular
grafts or fibrinogen/fibrin glues.
[0078] Although the examples described below relate generally to
treatment of polymeric films or tissue culture plates as substrate
surfaces, those examples are merely illustrative and are intended
to limit in no way the scope of the present invention.
EXAMPLE 1
Periodate Oxidation of a Peptide Containing an N-Terminal Serine
Amino Acid Residue
[0079] Two biomolecules, a tripeptide made of three serine amino
acid residues and a dipeptide made of two lysine amino acid
residues, both obtained from Sigma Chemical Co. (St. Louis, Mo.),
were incubated in sodium metaperiodate (NaIO.sub.4) also obtained
from Sigma Chemical Co. (St. Louis, Mo.). The tripeptide, 0.90
mmoles, was incubated in the dark while shaking at room temperature
for 3 hours in 10 ml deionized water containing 1.2 mmoles
NaIO.sub.4. The resultant solution, 2.5 ml, was added to 2 ml of a
solution containing 0.8 g NaOH, 0.2 g
4-amino-3-hydrazino-5-mercapto-1,2,4-triazole, which is available
under the trade designation PURPALD from Sigma Chemical Co. (St.
Louis, Mo.), in 20 ml deionized water, and shaken vigorously for 15
minutes at room temperature. The dipeptide, 0.72 mmoles, was
incubated in the dark while shaking at room temperature for 3 hours
in 10 ml deionized water containing 1.2 mmoles NaIO.sub.4. The
resultant solution, 10 ml (note that this amount is four times the
amount used for the tripeptide), was then added to 2 ml PURPALD
solution and shaken vigorously for 15 minutes at room temperature.
The resultant solutions were then analyzed spectrophotometrically
at 550 nm. Dickinson and Jacobsen, Chem. Commun., 1719 (1970),
described the specific and sensitive reaction of aldehydes with
PURPALD to yield purple-to-magenta-colored
6-mercapto-5-triazolo-(4,3-b)-s-tetrazines which can be measured
spectrophotometrically at 550 nm. Sample absorbances obtained at
550 nm were 0.04 for the dipeptide and 1.81 for the tripeptide,
which indicates that only the tripeptide which contained an
N-terminal serine was successfully oxidized using periodate. The
dipeptide of the two lysine amino acids lacked a 2-aminoalcohol
moiety, that is a carbon-carbon bond bearing an amine moiety
adjacent to a hydroxyl moiety.
EXAMPLE 2
Periodate Oxidation of a Peptide Containing an N-Terminal Threonine
Amino Acid Residue
[0080] A biomolecule, a dipeptide made of N-terminal threonine and
leucine amino acid residues obtained from Sigma Chemical Co. (St.
Louis, Mo.), was incubated in sodium metaperiodate (NaIO.sub.4)
also obtained from Sigma Chemical Co. (St. Louis, Mo.). The
dipeptide, 4.3 mmoles, was incubated in the dark while shaking at
room temperature for 3 hours in 10 ml deionized water containing
1.2 mmoles NaIO.sub.4. The resultant solution, 10 ml, was added to
2 ml of the PURPALD solution described in Example 1 and shaken
vigorously for 15 minutes at room temperature. After the 15 minutes
of shaking at room temperature, the resultant solution was analyzed
spectrophotometrically at 550 nm. Sample absorbance obtained at 550
nm was 0.62 indicating the periodate had successfully oxidized the
N-terminal threonine amino acid present in the dipeptide, thereby
forming an aldehyde moiety.
EXAMPLE 3
Periodate Oxidation of a Peptide Containing an N-Terminal Serine
Amino Acid Residue
[0081] A biomolecule, a pentapeptide made of N-terminal serine,
aspartic acid, glycine, arginine, and glycine amino acid residues
obtained from Sigma Chemical Co. (St. Louis, Mo.), was incubated in
sodium metaperiodate (NaIO.sub.4) also obtained from Sigma Chemical
Co. (St. Louis, Mo.). The pentapeptide, 0.01 mmoles, was incubated
in the dark while shaking at room temperature for 3 hours in 2 ml
deionized water containing 0.23 mmoles NaIO.sub.4. The resultant
solution, 10 ml, was added to 2 ml of the PURPALD solution
described in Example 1 and shaken vigorously for 15 minutes at room
temperature. After the 15 minutes of shaking at room temperature,
the resultant solution was analyzed spectrophotometrically at 550
nm. Sample absorbance obtained at 550 nm was 0.74, indicating the
periodate had successfully oxidized the N-terminal serine amino
acid residue present in the pentapeptide, thereby forming an
aldehyde moiety.
EXAMPLE 4
Oxidation of Collagen
[0082] The biomolecule, mouse collagen, type IV, obtained from
Sigma Chemical Co. (St. Louis, Mo.), was oxidized with sodium
metaperiodate (NaIO.sub.4). Collagen type IV is known to mediate
the attachment of epithelial, endothelial, myoblasts and nerve
cells in vivo and in vitro. Two collagen solutions were prepared by
i) mixing half a vial of collagen with 56 mg NaIO.sub.4 in 5 ml
deionized water and ii) mixing half a vial of collagen in 5 ml
deionized water. Both solutions were incubated in the dark for 2
hours while shaking at room temperature. The resultant solutions,
100 ml of each, were added to 2 ml the PURPALD solution described
in Example 1 and shaken vigorously for 30 minutes at room
temperature. After the 30 minutes of shaking at room temperature,
the resultant solutions were analyzed spectrophotometrically at 550
nm. The PURPALD solution was used as the blank. Sample absorbances
obtained at 550 nm were 0.03 for nonoxidized collagen and 0.25 for
oxidized collagen, indicating the periodate had successfully
oxidized the collagen, thereby forming aldehyde moieties.
EXAMPLE 5
Attachment of Periodate Oxidized Biomolecules to Aminated
Substrates
[0083] One method for creating amines on substrate surfaces entails
grafting substrate surfaces with acrylamide (AAm) and
N-(3-aminopropyl)methacrylamide (APMA) monomers using ceric
(Ce.sup.IV) ions. The Ce.sup.IV ions create free radicals on ozone
treated silicone and polystyrene surfaces and untreated
polyurethane surfaces which initiate the graft copolymerization of
the acrylamides. The amount of surface amination (the graft
copolymerization of APMA and AAm) that takes place on the substrate
surface may be measured via staining with ponceau S dye, a
negatively charged dye molecule. This dye ionically associates with
the primary amines on the aminated surface. Following grafting, a
periodate oxidized biomolecule may be coupled to the amine
containing derivatized substrate surface. A
2-aminoalcohol-containing biomolecule is first oxidized with sodium
metaperiodate (NaIO.sub.4) forming a reactive aldehyde moiety. The
aldehyde moiety is then used to covalently attached the biomolecule
to the primary amine moiety present on the substrate surface.
Sodium cyanoborohydride (NaCNBH.sub.3) is then used to stabilize
the imine linkages. Specific procedures required for each of these
steps are described below.
[0084] Polystyrene 24 well tissue culture plates were ozone treated
by placing the culture plates in an ozone reaction vessel for 30
minutes while oxygen, which contained ozone, was flowing at a rate
of 1.3 cm.sup.3/min. The oxygen containing ozone was created by
flowing the oxygen through a corona discharge apparatus, which
exposed the flowing oxygen to an 8000V electrical potential.
Following ozone treatment, the plates were soaked in nitrogen
purged deionized water for 30 minutes at room temperature.
Following the 30 minute soak in nitrogen purged deionized water,
the plates were grafted with acrylamide (AAm) and
N-(3-aminopropyl)methacrylamide (APMA) monomers using Ce.sup.IV
ion. The grafting solution consisted of 40 g AAm, 10 g APMA, 50 g
deionized water solution, and 20 g Ce.sup.IV ion solution. The
Ce.sup.IV ion solution consisted of 2.74 g ceric ammonium nitrate
and 3.15 g nitric acid in 50 ml deionized water. The plates were
allowed to graft for 3 hours in a 65 degrees Celsius nitrogen
purged oven. Following grafting, the plates are rinsed vigorously
with deionized water. The grafted plates were then tested with
ponceau S dye. Following staining, the ponceau S dye was released
from the surface using a 1% sodium dodecyl sulphate (SDS) solution
and quantified spectrophotometrically at 520 nm. Sample absorbances
obtained at 520 nm were 0.00 for nonderivatized plates and 1.44 for
surface-derivatized plates. As the results demonstrate, the
surface-derivatized plates contain primary amines on their
surfaces.
[0085] The 2-aminoalcohol moiety of a peptide may be oxidized using
the procedure of Example 1. Sodium cyanoborohydride (1 mg/ml) then
is added to the oxidized peptide solution. The resultant solution
then is immediately added to each of the amine containing
surface-derivatized tissue culture plate wells (approximately 1 ml
solution/well). The oxidized peptide is then incubated in the
derivatized tissue culture plate wells overnight at room
temperature. Following incubation, the wells are vigorously rinsed
with phosphate buffered saline (PBS) solution.
[0086] Polyurethane film samples were cut into 1.4 cm diameter
disks. Sample disks were grafted with AAm and APMA monomers using
Ce.sup.IV ion. The sample disks were allowed to graft 1 hour at
room temperature. Following grafting, the sample disks were rinsed
vigorously with deionized water. Again, the 2-aminoalcohol moiety
of a peptide can be oxidized as previously described. Sample disks
are then exposed to the oxidized peptide solution. Sodium
cyanoborohydride is then added (1 mg/ml) and the resultant solution
and sample disks are incubated overnight at room temperature.
Following incubation, the polyurethane sample disks are vigorously
rinsed with PBS.
EXAMPLE 6
Crosslinking of Collagen
[0087] A biomolecule such as collagen, type IV, may be oxidized
with sodium metaperiodate (NaIO.sub.4). A collagen solution may be
prepared by mixing half a vial of collagen with 56 mg NaIO.sub.4 in
5 ml deionized. The solution may be incubated in the dark for 2
hours while shaking at room temperature. The oxidized collagen
molecules are then allowed to form crosslinks, thereby bonding the
molecules together through imine moieties. An imine moiety is
formed from an aldehyde moiety of one collagen molecule reacting
with an amine moiety of a neighboring collagen molecule. The imine
linkages are then stabilized by reacting the imine moieties with
sodium cyanoborohydride (1 mg/ml) to form secondary amine linkages.
The resultant crosslinked material may be employed as a biomaterial
or as a biomaterial coating.
EXAMPLE 7
Periodate Oxidation of Bovine Fibrinogen
[0088] The glycoprotein bovine fibrinogen obtained from Sigma
Chemical Co. (St. Louis, Mo.) was incubated in sodium metaperiodate
(NaIO.sub.4) also obtained from Sigma Chemical Co. (St. Louis,
Mo.). The following four fibrinogen solutions were prepared to
investigate the oxidation of fibrinogen with varying amounts of
periodate: (1) 0.03 mM fibrinogen, 0.2 mM NaIO.sub.4, 0.008 M
Na.sub.2HPO.sub.4, 0.002 M KH.sub.2PO.sub.4, 0.14 M NaCl, pH 7.4;
(2) 0.03 mM fibrinogen, 0.1 mM NaIO.sub.4, 0.008 M
Na.sub.2HPO.sub.4, 0.002 M KH.sub.2PO.sub.4, 0.14 M NaCl, pH 7.4;
(3) 0.03 mM fibrinogen, 0.05 mM NaIO.sub.4, 0.008 M
Na.sub.2HPO.sub.4, 0.002 M KH.sub.2PO.sub.4, 0.14 M NaCl, pH 7.4;
and (4) 0.03 mM fibrinogen, 0.008 M Na.sub.2HPO.sub.4, 0.002 M
KH.sub.2PO.sub.4, 0.14 M NaCl, pH 7.4. The four fibrinogen
solutions were incubated in the dark for 2 hours while shaking at
room temperature. The resultant solutions, 500 .mu.l of each, were
added to 2 ml of a solution containing 0.8 g NaOH, 0.2 g
4-amino-3-hydrazino-5-mercato-1,2,4-triazole, which is available
under the trade designation PURPALD from Sigma Chemical Co. (St.
Louis, Mo.), in 20 ml deionized water, and shaken vigorously for 15
minutes at room temperature. Dickinson and Jacobsen, Cem. Commun.,
1719 (1970), described the specific and sensitive reaction of
aldehydes with PURPALD to yield purple-to-magenta-colored
6-mercapto-s-triazolo-(4,3-b)-s-tetrazines. After the 15 minutes of
shaking at room temperature, the resultant solutions were analyzed
spectrophotometrically at 550 nm. Sample 4 was used as the blank.
Sample absorbances obtained at 550 nm were 0.54 for sample 1, 0.53
for sample 2 and 0.51 for sample 3, which indicates that for all
samples the fibrinogen was successfully oxidized forming aldehyde
groups.
EXAMPLE 8
Periodate Oxidation of Bovine Vitronectin
[0089] The glycoprotein bovine vitronectin obtained from Sigma
Chemical Co. (St. Louis, Mo.) was incubated in sodium metaperiodate
(NaIO.sub.4) also obtained from Sigma Chemical Co. (St. Louis; MO).
The following two vitronectin solutions were prepared: (1) 0.001 mM
vitronectin, 0.05 M NaIO.sub.4 and (2) 0.001 mM vitronectin. Both
solutions were incubated in the dark for 2 hours while shaking at
room temperature. The resultant solutions, 100 .mu.l of each, were
added to 2 ml PURPALD solution described in example 1, and shaken
vigorously for 30 minutes at room temperature. After the 30 minutes
of shaking at room temperature, the resultant solutions were
analyzed spectrophotometrically at 550 nm. The PURPALD solution was
used as the blank. Sample absorbances obtained at 550 nm were 0.09
for sample 1 and 0.04 for sample 2, which indicated that
vitronectin was successfully oxidized with forming aldehyde
groups.
EXAMPLE 9
Periodate Oxidation of Bovine Fibronectin
[0090] The glycoprotein bovine fibronectin obtained from Sigma
Chemical Co. (St. Louis, Mo.) was incubated in sodium metaperiodate
(NaIO.sub.4) also obtained from Sigma Chemical Co. (St. Louis,
Mo.). The following two fibronectin solutions were prepared: (1)
0.002 mM fibronectin, 0.05 M NaIO.sub.4, 0.5 M NaCl, 0.05 M Tris,
pH 7.5; and (2) 0.002 mM fibronectin, 0.5 M NaCl, 0.05 M Tris, pH
7.5. Both solutions were incubated in the dark for 2 hours while
shaking at room temperature. The resultant solutions, 100 .mu.l of
each, were added to 2 ml PURPALD solution describe in Example 1 and
shaken vigorously for 30 minutes at room temperature. After 30
minutes of shaking at room temperature, the resultant solutions
were analyzed spectrophotometrically at 550 nm. Following an
initial analysis, sample 1 was observed to contain to many
aldehydes to measure. Therefore, sample 1 was diluted 1:50 in
deionized water to achieve a measurable amount of aldehydes
contained in the sample solution. The PURPALD solution was used as
the blank. Sample absorbances obtained at 550 nm were 0.81 for
sample 1 and 0.0 for sample 2, which indicate that the fibronectin
in sample 1 was successfully oxidized forming aldehyde groups.
Fibronectin in sample 2 was not oxidized due to the omission of
periodate.
EXAMPLE 10
Attachment of Fibronectin to Aminated Substrates
[0091] Fibronectin was covalently attached to a substrate surface.
The attachment technique began with the graft copolymerization of
acrylamide (AAm) and N-(3-aminopropyl)methacrylamide (APMA)
monomers onto an ozone treated polystyrene tissue culture plate
with ceric (Ce.sup.IV) ions. The Ce.sup.IV ions create free
radicals on the ozone treated surface which initiate the graft
copolymerization of the acrylamides. The amount of surface
amination (the graft copolymerization of APMA and AAm) that took
place on the substrate surface was measured via staining with
ponceau S dye, a negatively charged dye molecule. Following
grafting, fibronectin was coupled to the amine containing
derivatized substrate surface. Fibronectin was first oxidized with
sodium metaperiodate (NaIO.sub.4) forming reactive aldehyde groups.
These aldehyde groups were then used to covalently attached
fibronectin to the primary amino groups present on the substrate
surface. Sodium cyanoborohydride (NaCNBH.sub.3) was then used to
stabilize the imine linkages. The specific procedures for each of
these steps are described below.
[0092] Polystyrene 24 well tissue culture plates were ozone treated
by placing the culture plates in an ozone reaction vessel for 30
minutes while oxygen, which contained ozone, was flowing at a rate
of 1.3 cm.sup.3/min. The oxygen containing ozone was created by
flowing the oxygen through a corona discharge apparatus, which
exposes the flowing oxygen to an 8000V electrical potential.
Following ozone treatment, the plates were soaked in nitrogen
purged deionized water for 30 minutes at room temperature.
Following the 30 minute soak in nitrogen purged deionized water,
the plates were grafted with acrylamide (AAm) and
N-(3-aminopropyl)methacrylamide (APMA) monomers (Eastman Kodak Co.,
Rochester, N.Y.) using ammonium cerium (IV) nitrate (Aldrich
Chemical Co., Milwaukee, Wis.). The grafting solution consisted of
11.2 M AAm, 1.1 M APMA, 400 mM nitric acid and 40 mM ammonium
cerium (IV) nitrate in deionized water. The plates were allowed to
graft for 3 hours in a 65.degree. C. nitrogen purged oven.
Following grafting the plates are rinsed vigorously with deionized
water. The grafted plates were then tested with ponceau S dye.
Following staining, the ponceau S dye was released from the surface
using a 1% sodium dodecyl sulphate (SDS) solution and quantified
spectrophotometrically at 520 nm. Sample absorbances obtained at
520 nm were 0.00 for nonderivatized plates and 1.44 for
surface-derivatized plates. As the results demonstrate, the
surface-derivatized plates contain primary amines on their
surfaces.
[0093] Bovine fibronectin obtained from Sigma Chemical Co. (St.
Louis, Mo.) was then incubated in sodium metaperiodate (NaIO.sub.4)
also obtained from Sigma Chemical Co. (St. Louis, Mo.). The
following fibronectin solution was prepared: 0.002 mM fibronectin,
0.05 M NaIO.sub.4, 0.5 M NaCl, 0.05 M Tris, pH 7.5. The solution
was incubated in the dark for 2 hours while shaking at room
temperature. Sodium cyanoborohydride (1 mg/ml) was then added to
the fibronectin solution. The resultant solution was immediately
added to each of the amine containing surface-derivatized tissue
culture late wells (approximately 1 ml solution/well). The
fibronectin solution incubated in the derivatized tissue culture
plate wells overnight at room temperature. Following incubation,
the wells were then vigorously rinsed with phosphate buffered
saline (PBS) solution. The attachment of fibronectin to the amine
containing surface-derivatized tissue culture plate surfaces was
assessed using toluidine blue dye, a positively charged dye
molecule. This dye ionically associates with the negative charges
on a substrate surface. Therefore, the binding of toluidine blue
dye to the fibronectin-derivatized surface is due to fibronectin's
negative charges. The wells of each plate were filled with a 1%
toluidine blue dye in deionized water solution. After a 5 minute
incubation at room temperature, the dye solution was removed and
the wells were thoroughly rinsed with PBS. The surface associated
dye in each well was then eluted by mechanically shaking the plates
in a 1% SDS in deionized water solution overnight. The amount of
dye eluted from the wells was then determined
spectrophotometrically at 630 nm. Sample absorbances obtained at
630 nm were 0.05 for the nonderivatized sample plate, 0.54 for the
AAm/APMA-derivatized sample plate and 1.83 for the
fibronectin-derivatized sample plate, which indicate that the
fibronectin was successfully oxidized and then covalently attached
to the substrate surface.
EXAMPLE 11
ELISA and Cellular Adherence to Fibronectin Coupled Surfaces
[0094] Polyurethane in the form of Pellethane 2363-55D was obtained
from Dow Chemical Co. (Midland, Mich.) and extruded into film. The
film was then cut into 1 cm.sup.2 sample disks. Sample disks were
then cleansed with ethanol and surface grafted with AAm and APMA
monomers using Ce.sup.IV ion. The grafting solution consisted of
11.2 M AAm, 1.1 M APMA, 400 mM nitric acid and 40 mM ammonium
cerium (IV) nitrate in deionized water. The sample disks were
placed into the grafting solution and allowed to graft for 1 hour
at room temperature. Following grafting, the sample disks were
thoroughly washed with deionized water. Fibronectin was then
coupled to the resultant APMA/AAm surface-derivatized sample disks
via two methods.
[0095] The first method or peroxide method included the oxidation
of fibronectin by sodium metaperiodate. Fibronectin (0.1 mg/ml) was
exposed in the dark to a 1 .mu.g/ml sodium metaperiodate in
deionized water solution for 3 hours at room temperature. The
APMA/AAm-derivatized sample disks were then placed into the
oxidized fibronectin solution for 24 hours at room temperature.
Sample disks were then thoroughly rinsed with deionized water. The
samples were then incubated for 24 hours at room temperature in a 3
mg/ml sodium cyanoborohydride in deionized water solution. Sample
disks were then thoroughly rinsed with deionized water.
[0096] The second method used glutaraldehyde as a coupling agent.
The method included soaking the APMA/AAm-derivatized sample disks
in a 2% glutaraldehyde in deionized water solution for 2 hours at
room temperature. Sample disks were then thoroughly rinsed with
deionized water. Following rinsing, the sample disks were then
incubated in a 0.1 mg/ml fibronectin in deionized water solution
for 24 hours at room temperature. Sample disks were then thoroughly
rinsed with deionized water. The sample disks were then incubated
for 24 hours at room temperature in a 3 mg/ml sodium
cyanoborohydride in deionized water solution. Sample disks were
then thoroughly rinsed with deionized water.
[0097] An enzyme linked immunosorbent assay (ELISA) was then
performed to determine the ability of an antibody to recognize the
fibronectin which had been coupled to the sample surfaces. Sample
disks were washed for 20 minutes at room temperature with wash
buffer (pH 7.4) consisting of 10 mM Tris, 0.15 M NaCl and 0.05%
Tween. Sample disks were then incubated at 37.degree. C. for 30
minutes in blocking buffer (pH 7.4) consisting of 10 mM Tris, 0.15
M NaCl, 0.05% Tween and 0.05% gelatin followed by three 10 minute
washes with wash buffer. Next, sample disks were incubated at
37.degree. C. for 1 hour in a primary antibody solution (pH 7.4)
consisting of 10 mM Tris, 0.15 M NaCl and 2 .mu.g/ml mouse
monoclonal anti-fibronectin antibody (Sigma Chemical Co., St.
Louis, Mo.). Sample disks were then rinsed thrice (10 minutes per
wash) with wash buffer. Next, sample disks were incubated at
37.degree. C. for 1 hour in a peroxidase-labeled secondary antibody
solution (pH 7.4) consisting of 10 mM Tris, 0.15 M NaCl and 0.5
ng/ml anti-mouse IgG peroxidase antibody conjugate (Sigma Chemical
Co., St. Louis, Mo.). Sample disks were then rinsed thrice (10
minutes per wash) with wash buffer. Sample disks were then
incubated for 15 minutes at room temperature in a phosphate-citrate
buffer (pH 5.0) containing 0.4 mg/ml o-phenyldiamine
dihydrochloride and 0.2 .mu.l/ml 30% hydrogen peroxide. The
phosphate-citrate buffer consisted of 50 mM dibasic sodium
phosphate and 25 mM citric acid in deionized water. Following the
15 minute incubation, the peroxide reaction was stopped with 3 M
HCl and the absorbance of the resultant solution was measured
spectrophotometrically at 492 nm. The APMA/AAm-derivatized sample
disks were used as controls for this experiment. Sample absorbances
obtained from the spectrophotometric analysis were 0.016.+-.0.038
for APMA/AAm-derivatized samples which contained glutaraldehyde
coupled fibronectin and 0.204.+-.0.068 for APMA/AAm-derivatized
samples which contained periodate oxidized fibronectin. The results
indicate that the periodate oxidation method was more successful at
attaching fibronectin to the sample surfaces.
[0098] A cellular adherence assay was also performed to determine
the ability of cells to adhere to fibronectin-derivatized sample
surfaces. Sample disks were incubated for 1 hour at 37.degree. C.
in a blocking buffer consisting of 2 mg/ml ovalbumin in phosphate
buffered saline (PBS), pH 7.4. Mouse fibroblasts (C3T3) obtained
from American Type Culture Collection (Rockville, Md.) and
maintained in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum were harvested using
trypsin:EDTA and resuspended in serum-free DMEM containing 2 mg/ml
ovalbumin. The cells were then washed twice, counted and
resuspended to a final density of 5.times.10.sup.4 cells/ml in
serum-free DMEM containing 2 mg/ml ovalbumin. Sample disks were
then incubated in the cell suspension for 1 hour at 37.degree. C.
Nonadherent cells were removed by a PBS wash. Sample disks were
then fixed in 3% paraformaldehyde solution for 30 minutes. Adherent
cells were then stained with a staining solution consisting of 1%
toluidine blue dye and 3% paraformaldehyde in PBS. Following
staining, sample surfaces were then examined for cellular adherence
using a light microscope. Upon examination, APMA/AAm-derivatized
samples and APMA/AAm-derivatized samples which contained
glutaraldehyde coupled fibronectin appeared to have no adherent
cells. In contrast, cells appeared adherent to APMA/AAm-derivatized
samples which contained periodate oxidized fibronectin.
EXAMPLE 12
Crosslinking of Fibrinogen
[0099] Porcine fibrinogen obtained from Sigma Chemical Co. (St.
Louis, Mo.) was incubated in sodium metaperiodate (NaIO.sub.4) also
obtained from Sigma Chemical Co. (St. Louis, Mo.) and sodium
cyanoborohydride (NaCNBH.sub.3) obtained from Aldrich Chemical Co.
(Milwaukee, Wis.). The following fibrinogen solution was prepared:
0.03 mM fibrinogen, 0.02 M NaIO.sub.4, 0.02 M NaCNBH.sub.3, 0.008 M
Na.sub.2HPO.sub.4, 0.002 M KH.sub.2PO.sub.4, 0.14 M NaCl, pH 7.4.
The solution was then shaken vigorously and placed into a 24 well
tissue culture plate (approximately 1 ml of fibrinogen
solution/well). The plate was then incubated in the dark for 2
hours while shaking at room temperature. After 2 hours, the
solution was observed to have become cloudy and very viscous
indicating the fibrinogen had crosslinked. The sample was then
shaken for an additional 22 hours in the dark. Following
incubation, the crosslinked fibrinogen was tested for residual
aldehydes using the PURPALD solution describe in Example 1. The
results of the PURPALD assay demonstrated few residual aldehydes
were present which indicated the formation of covalent crosslinks
between the aldehydes and the amines present along the fibrinogen
molecules.
[0100] The following bovine fibrinogen (Sigma Chemical Co., St.
Louis, Mo.) solution was prepared: 0.02 mM fibrinogen, 0.008 M
Na.sub.2HPO.sub.4, 0.002 M KH.sub.2PO.sub.4, 0.14 M NaCl, pH 7.4.
Following preparation, the solution was divided into four equal
portions. Sodium metaperiodate (0.05 mM) was then added to samples
3 and 4. All four fibrinogen solutions were then incubated in the
dark for 2 hours while shaking at room temperature. Next, 0.02 mM
NaCNBH.sub.3 was added to samples 2 and 4. Again, all four
fibrinogen solutions were allowed to react for 2 hours while
shaking at room temperature. The samples, 50 .mu.l of each, were
then placed into 450 .mu.l of SDS-PAGE buffer solution consisting
of 62.5 mM Tris-HCL, 5% b-mercaptoethanol, 10% glycerol and 2.3%
SDS. Samples were then boiled for 3 minutes. The samples, 10 .mu.l
of each, were then loaded onto a 4-15% gradient gel and SDS-PAGE
was performed according to the procedures described in O'Farrell,
"High Resolution Two-dimensional Electrophoresis of Proteins", J.
Biol. Chem. 250, 4007-4021 (1974). Following electrophoresis, the
gel was stained with Coomassie Brilliant Blue, and the identity of
the eluted proteins was determined by reference to molecular weight
standards included on the gel. The results from SDS-PAGE indicated
that the fibrinogen molecules in sample 4 had formed stable
covalent crosslinks. In contrast, the results demonstrated that the
fibrinogen molecules in the samples which contained no NaIO.sub.4
had formed no crosslinks.
[0101] It will be appreciated by those skilled in the art that
while the invention has been described above in connection with
particular embodiments and examples, the invention is not
necessarily so limited, and that numerous other embodiments,
examples, uses, modifications and departures from the embodiments,
examples and uses are intended to be encompassed by the claims
attached hereto. The entire disclosure of each patent and
publication cited herein is incorporated by reference, as if each
such patent or publication were individually incorporated by
reference herein.
* * * * *