U.S. patent application number 09/812799 was filed with the patent office on 2001-10-18 for biopolymer-resistant coatings, methods and articles related thereto.
Invention is credited to Laibinis, Paul E., Lee, Seok-Won.
Application Number | 20010031309 09/812799 |
Document ID | / |
Family ID | 22163822 |
Filed Date | 2001-10-18 |
United States Patent
Application |
20010031309 |
Kind Code |
A1 |
Lee, Seok-Won ; et
al. |
October 18, 2001 |
Biopolymer-resistant coatings, methods and articles related
thereto
Abstract
The present invention relates to new biopolymer resistant
coatings for materials that come in contact with such molecules in
solution. Additionally, the present invention discloses a process
for the fabrication of these coatings, under mild and scaleable
reaction conditions, from simple, low molecular weight molecular
components. Furthermore, the present invention teaches a general
conceptual strategy for the design of additional protein resistant
coatings.
Inventors: |
Lee, Seok-Won; (Cambridge,
MA) ; Laibinis, Paul E.; (Medford, MA) |
Correspondence
Address: |
FOLEY, HOAG & ELIOT, LLP
PATENT GROUP
ONE POST OFFICE SQUARE
BOSTON
MA
02109
US
|
Family ID: |
22163822 |
Appl. No.: |
09/812799 |
Filed: |
March 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09812799 |
Mar 20, 2001 |
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09289288 |
Apr 9, 1999 |
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6235340 |
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60081387 |
Apr 10, 1998 |
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Current U.S.
Class: |
427/2.1 |
Current CPC
Class: |
B82Y 30/00 20130101;
A61L 33/0082 20130101; A61L 27/34 20130101; B82Y 15/00 20130101;
A61L 27/28 20130101; B82Y 5/00 20130101; A61L 27/34 20130101; C08L
83/04 20130101 |
Class at
Publication: |
427/2.1 |
International
Class: |
A61L 002/00 |
Goverment Interests
[0002] Work described herein was supported in part with funding
from the Office of Naval Research. The United States Government has
certain rights in this invention.
Claims
We claim:
1. A method of rendering a surface of an object resistant to the
adhesion of a biopolymer, comprising the step of: treating a
surface of an object with a solution comprising a molecule that
adheres to said surface, thereby rendering said surface resistant
to the adhesion of a biopolymer.
2. The method of claim 1, wherein said surface is designed to
contact an aqueous solution comprising a biopolymer.
3. The method of claim 2, wherein said aqueous solution comprises a
protein.
4. The method of claim 2, wherein said aqueous solution is
blood.
5. The method of claim 1, wherein said surface is designed to
contact a biological fluid.
6. The method of claim 1, wherein said object is designed to be
implanted in the body of a mammal.
7. The method of claim 6, wherein said object is designed to be
implanted in the body of a primate.
8. The method of claim 7, wherein said object is designed to be
implanted in the body of a human.
9. The method of claim 1, 2, 3, 4, 5, 6, 7, or 8, wherein said
molecule comprises a Z moiety, wherein Z is selected from the group
consisting --CO.sub.2H, --PO.sub.3H.sub.2, --C(O)NHOH,
--Si(OR).sub.3, --SiCl.sub.3, --Sn(OR).sub.3, --SnCl.sub.3,
--Ge(OR).sub.3, and --GeCl.sub.3.
10. The method of claim 9, wherein said Z moiety adheres to said
surface.
11. The method of claim 9, wherein Z is --Si(halide).sub.3,
--Si(alkoxyl).sub.3 or --Si(acyloxy).sub.3.
12. The method of claim 9, wherein said molecule comprises a
biopolymer-resistant domain.
13. The method of claim 12, wherein said biopolymer-resistant
domain is an oligoether, oligoglycol, oligoalcohol, oligocarbonyl,
oligosulfide, oligosulfone or oligosaccharide domain.
14. The method of claim 9, wherein said molecule comprises a W
moiety, wherein W is H, a small alkyl group, an alkoxyl group, an
acyl group, an acyloxy group, a sulfone, a hydroxyl group, a
sulfhydryl group, or a thioalkyl group.
15. The method of claim 14, comprising the further step of:
treating said biopolymer-resistant surface of the object with a
solution comprising a second molecule, wherein said second molecule
adheres to said biopolymer-resistant surface of the object.
16. The method of claim 15, wherein said second molecule adheres to
said biopolymer-resistant surface of the object via a coulombic
interaction, a hydrophobic interaction, the formation of a covalent
bond, or a combination thereof.
17. The method of claim 14, wherein the presence of the second
molecule on said biopolymer-resistant surface of the object
modifies the biopolymer resistance of said surface.
18. The method of claim 17, wherein the presence of the second
molecule on said biopolymer-resistant surface of the object
enhances the biopolymer resistance of said surface.
19. The method of claim 1, wherein said molecule is represented by
general structure 1: 17wherein Z represents a domain, moiety, or
functional group which associates with the surface of the object;
tether represents a covalent attachment between Z and the
biopolymer-resistant domain; biopolymer-resistant domain represents
a molecular substructure to which biopolymers in solution do not
adhere well; and W represents H, a small alkyl group, or a small
hydrophilic group.
18. The method of claim 19, wherein: Z represents a functional
group which associates with the surface of the object selected from
the set comprising --CO.sub.2H, --PO.sub.3H.sub.2, --C(O)NHOH,
--Si(OR).sub.3, --SiCl.sub.3, --Sn(OR).sub.3, --SnCl.sub.3,
--Ge(OR).sub.3, and --GeCl.sub.3; tether represents a hydrophobic
covalent attachment, of approximately 5 to 20 bonds in length,
between Z and the biopolymer-resistant domain; biopolymer-resistant
domain represents a molecular substructure selected from the set
consisting of oligoethers, oligoglycols, oligoalcohols,
oligocarbonyls, oligosulfides, oligosulfones, and oligosaccharides;
and W represents H, a small alkyl group, an alkoxyl group, an acyl
group, an acyloxy group, a sulfone, a hydroxyl group, a sulfhydryl
group, or a thioalkyl group.
19. The method of claim 20, wherein: Z represents --Si(OR).sub.3,
--SiCl.sub.3, --Sn(OR).sub.3, --SnCl.sub.3, --Ge(OR).sub.3, or
--GeCl.sub.3.
20. The method of claim 21, wherein Z represents --Si(OR).sub.3 or
--SiCl.sub.3.
21. The method of claim 1, wherein said surface of said object
comprises SiO.sub.2.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of
U.S. Provisional Application No. 60/081,387, filed Apr. 10,
1998.
BACKGROUND OF THE INVENTION
[0003] The fouling of surfaces that come in contact with proteins
in solution, e.g., biological fluids and product streams from
biochemical reactors, is a pervasive problem. Transfer of solutions
of proteins via pipes is impeded by the non-specific binding of
proteinaceous material to the interior walls of the pipes; this
binding results in a continual decrease in the cross sectional area
of the pipe through which the solution may flow. Foreign objects
placed in mammals, e.g., during surgical procedures, accumulate
proteinaceous material rapidly. In certain cases, this non-specific
fouling can partially, or even completely, undermine the initial
positive outcome of a surgical procedure. For example, the gradual
accumulation of proteinaceous material in and around a artificial
heart valve can result in impaired function of the valve, or even
in its failure. Furthermore, the accumulation of proteinaceous
material around a stent in a blood vessel can result in partial or
complete blockage of that vessel. Contact lenses are rendered
opaque over time due to the non-specific adsorption of proteins to
their surfaces. The non-specific binding of proteins to surfaces
can attenuate the sensitivity of sensors exploited in the
qualitative and/or quantitative analysis of test samples. These
facts underscore the negative effects of non-specific protein
adsorption to various surfaces.
[0004] A number of strategies have been developed for suppressing
non-specific adhesion of proteins to surfaces. One of these
strategies involves the pretreatment of a given surface with a
specific protein, or proteins, whose effects on that surface are
either minimal, predictable, or both. A second strategy centers on
coatings which resist adsorption of proteins by presenting a
microscopic surface that lacks the structural characteristics
responsible for non-specific adhesion. A tremendous amount of
scientific resources has been applied to the design, development,
testing and the like of protein-resistant coatings, components
thereof, and methods for their application. Advances have been
achieved in these areas, but the need remains for further
improvements in the state-of-the-art in this field.
SUMMARY OF THE INVENTION
[0005] The present invention relates to new biopolymer resistant
coatings. Additionally, the present invention discloses a process
for the fabrication of these coatings, under mild and scaleable
reaction conditions, from simple low molecular weight molecular
components. Furthermore, the present invention teaches a general
conceptual strategy for the design of additional biopolymer
resistant coatings.
BRIEF DESCRIPTION OF THE FIGURES
[0006] FIG. 1 is a schematic illustration for formation of the
oligo(ethylene glycol)-terminated self-assembled monolayers
(SAMs).
[0007] FIG. 2 graphically depicts the ellipsometric thickness of
adsorbed films of insulin, lysozyme, albumin, hexokinase, and
fibrinogen on various SAM surfaces. EG represents an ethylene
glycol unit.
[0008] FIG. 3 depicts the synthesis of
.omega.-trichlorosilyl-oligo(ethyle- ne glycol) derivatives,
CH.sub.3COO(CH.sub.2CH.sub.2O).sub.n(CH.sub.2).sub-
.11SiCl.sub.3(n=3-4).
[0009] FIG. 4 depicts a schematic representation of on-surface
reactions.
[0010] FIG. 5 depicts XPS spectra of acetate- and
hydroxyl-terminated EG siloxane SAMs on a SiO.sub.2-covered silicon
substrate (top and bottom, respectively).
[0011] FIG. 6 depicts XPS survey spectra of a hydroxyl-terminated
EG SAM before (upper) and after (lower) attachment of biotin.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The immobilization of proteins on both organic and inorganic
surfaces is a well-established technique (see, e.g, Chapter 4,
Principles of Immobilization of Enzymes, Handbook of Enzyme
Biotechnology, Second Edition, Ellis Horwood Limited, 1985), and it
is possible to bond a large amount of protein to the surface while
retaining adequate biological activity.
[0013] However, it has been found that most solid surfaces are so
constituted that they adsorb proteins and other biopolymers
spontaneously. Such adsorption from aqueous solution is promoted
primarily by two types of physical forces, electrostatic
attraction, and hydrophobic interaction. Most surfaces, including
glass, at normal pH are negatively charged, but usually they also
contain hydrophobic domains. A protein usually has positive,
negative and hydrophobic seats, which means that a protein is
attracted to most surfaces, on the one hand by electrostatic
attraction between positive seats and negatively charged groups in
the surface, and, on the other hand, by hydrophobic interaction
between hydrophobic domains of the protein and the surface. This is
described in, for example, Surface and Interfacial Aspects of
Biomedical Polymers, Ed. J. D. Andrade, Plenum Press (1985), Vol.
2, p. 81.
[0014] Such nonspecific adsorption by electrostatic attraction and
hydrophobic interaction is an undesired phenomenon for many
applications involving, e.g., the contact of biological fluids or
preparation and storage of biopharmacutical products. In solid
phase diagnostics, for instance, it results in an impaired
sensitivity and a shorter life of the diagnostic kit. In both
extracorporeal therapy and in bio-organic synthesis, spontaneous
adsorption causes impaired activity and a shorter product life.
[0015] I. Overview
[0016] One aspect of the present invention relates to a method of
significantly reducing the adsorption proteins and other
biopolymers on solid oxide surfaces so as to provide the surfaces
with a layer of an uncharged hydrophilic polymer, preferbly a
thoroughly developed hydrophilic surface of low spontaneous
adsorption. By coating the surface with a layer of uncharged
hydrophilic polymer, such as polyethylene glycol side chains, both
electrostatic attraction and hydrophobic interaction can be
avoided. The hydrophilic surface does not attract the protein. On
the contrary, it acts as a repellent, because it is energetically
unfavorable for a protein in aqueous solution to approach such a
surface. Thus, the treated surfaces showing such advantageous
properties may be described as having improved biocompatability
compared to untreated surfaces.
[0017] In general, the subject coatings are fabricated by treating
an oxide surface, such as glass, with a mixture comprising an
excess of a molecule, or molecules, that adhere to the surface and
in so doing form the coating. The association between the
constituent molecules of the coating and the surface to be coated
may be based on hydrophobic interactions, electrostatic
interactions, covalent bonds, or a combination of any, or all, of
these classes of association.
[0018] In addition the present invention offers the further
advantages in many applications that the coated surfaces have
improved wettability and improved lubricity. This assists in, for
instance, avoiding the formation of gas bubbles in tubing and
facilitating insertion of catheters via surgical incisions.
[0019] The subject method can be applied in such fields as
ophthalmologic devices (activation of biochemical process, impaired
optical properties); blood bags and related devices for collection
and storage of blood and blood components; food processing and
storage, including dairy and meat industries; pharmaceutical
products (adsorption and denaturation of peptides or other active
agents); human hygiene products (such as diapers and sanitary
napkins); membranes (polarization and fouling); Sensors
(non-specific binding); separation processes, such as
chromatography, electrophoresis, and field flow fractionation.
[0020] It has also been found that surfaces coated by the process
of the present invention possess no net surface charge. Such
properties lead the process of the invention to have further
applications for instance in the electronics industry and in
electrochemical detection and analysis where electrostatic charge
or interfering background charge needs to be minimised.
[0021] The surface to be treated may be, merely to illustrate, a
blood-contacting surface, or it may be some other type of surface,
e.g. the surface of a biosensor, bioseparation chamber, or the
surface of an electronic device or component or of an
electrochemical detection or analysis device. It may be a surface
of a finished device such as a blood-contacting device or it may be
the surface of a material to be used in forming a finished device.
In the latter case subsequent forming steps are selected to avoid
disrupting the coating formed by the process of the invention in
portions of the device where the coating will protect the surface
in use and to avoid chemical damage, for instance due to high
temperatures, to the coating. The surface being treated may also be
refered to herein as the "substrate".
[0022] Such coated surfaces therefore have applications in blood
contacting devices and in devices where reduced non-specific
protein adsorption is desirable, for instance in diagnostic devices
which require a specific interaction of an analyte and detector
species, e.g. biosensors, bioseparation membranes and sight
correction devices.
[0023] In one embodiment, the subject method can be used for
improving medical or laboratory devices to increase
biocompatibility and resistance to protein binding.
[0024] II. Definitions
[0025] For convenience, certain terms employed in the
specification, examples, and appended claims are collected
here.
[0026] The term "heteroatom" as used herein means an atom of any
element other than carbon or hydrogen. Preferred heteroatoms are
boron, nitrogen, oxygen, phosphorus, sulfur and selenium.
[0027] The term "electron-withdrawing group" is recognized in the
art, and denotes the tendency of a substituent to attract valence
electrons from neighboring atoms, i.e., the substituent is
electronegative with respect to neighboring atoms. A quantification
of the level of electron-withdrawing capability is given by the
Hammett sigma (.sigma.) constant. This well known constant is
described in many references, for instance, J. March, Advanced
Organic Chemistry, McGraw Hill Book Company, New York, (1977
edition) pp. 251-259. The Hammett constant values are generally
negative for electron donating groups (.sigma.[P]=-0.66 for
NH.sub.2) and positive for electron withdrawing groups
(.sigma.[P]=0.78 for a nitro group), .sigma.[P] indicating para
substitution. Exemplary electron-withdrawing groups include nitro,
acyl, formyl, sulfonyl, trifluoromethyl, cyano, chloride, and the
like. Exemplary electron-donating groups include amino, methoxy,
and the like.
[0028] Herein, the term "aliphatic group" refers to a
straight-chain, branched-chain, or cyclic aliphatic hydrocarbon
group and includes saturated and unsaturated aliphatic groups, such
as an alkyl group, an alkenyl group, and an alkynyl group.
[0029] The term "alkyl" refers to the radical of saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. In preferred embodiments, a straight chain or branched
chain alkyl has 30 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.30 for straight chain, C.sub.3-C.sub.30 for branched
chain), and more preferably 20 or fewer. Likewise, preferred
cycloalkyls have from 3-10 carbon atoms in their ring structure,
and more preferably have 5, 6 or 7 carbons in the ring
structure.
[0030] Moreover, the term "alkyl" (or "lower alkyl") as used
throughout the specification, examples, and claims is intended to
include both "unsubstituted alkyls" and "substituted alkyls", the
latter of which refers to alkyl moieties having substituents
replacing a hydrogen on one or more carbons of the hydrocarbon
backbone. Such substituents can include, for example, a halogen, a
hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a
formyl, or an acyl), a thiocarbonyl (such as a thioester, a
thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a
phosphonate, a phosphinate, an amino, an amido, an amidine, an
imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a
sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a
heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety.
It will be understood by those skilled in the art that the moieties
substituted on the hydrocarbon chain can themselves be substituted,
if appropriate. For instance, the substituents of a substituted
alkyl may include substituted and unsubstituted forms of amino,
azido, imino, amido, phosphoryl (including phosphonate and
phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl
and sulfonate), and silyl groups, as well as ethers, alkylthios,
carbonyls (including ketones, aldehydes, carboxylates, and esters),
--CF.sub.3, --CN and the like. Exemplary substituted alkyls are
described below. Cycloalkyls can be further substituted with
alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls,
carbonyl-substituted alkyls, --CF.sub.3, --CN, and the like.
[0031] The term "aralkyl", as used herein, refers to an alkyl group
substituted with an aryl group (e.g., an aromatic or heteroaromatic
group).
[0032] The terms "alkenyl" and "alkynyl" refer to unsaturated
aliphatic groups analogous in length and possible substitution to
the alkyls described above, but that contain at least one double or
triple bond respectively.
[0033] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, more preferably from one to six
carbon atoms in its backbone structure. Likewise, "lower alkenyl"
and "lower alkynyl" have similar chain lengths. Throughout the
application, preferred alkyl groups are lower alkyls. In preferred
embodiments, a substituent designated herein as alkyl is a lower
alkyl.
[0034] The term "aryl" as used herein includes 5-, 6- and
7-membered single-ring aromatic groups that may include from zero
to four heteroatoms, for example, benzene, pyrrole, furan,
thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,
pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those
aryl groups having heteroatoms in the ring structure may also be
referred to as "aryl heterocycles" or "heteroaromatics." The
aromatic ring can be substituted at one or more ring positions with
such substituents as described above, for example, halogen, azide,
alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,
amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,
carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,
ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic
moieties, --CF.sub.3, --CN, or the like. The term "aryl" also
includes polycyclic ring systems having two or more cyclic rings in
which two or more carbons are common to two adjoining rings (the
rings are "fused rings") wherein at least one of the rings is
aromatic, e.g., the other cyclic rings can be cycloalkyls,
cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
[0035] The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent
methyl, ethyl, phenyl, trifluoromethanesulfonyl,
nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl,
respectively. A more comprehensive list of the abbreviations
utilized by organic chemists of ordinary skill in the art appears
in the first issue of each volume of the Journal of Organic
Chemistry; this list is typically presented in a table entitled
Standard List of Abbreviations. The abbreviations contained in said
list, and all abbreviations utilized by organic chemists of
ordinary skill in the art are hereby incorporated by reference.
[0036] The terms ortho, meta and para apply to 1,2-, 1,3- and
1,4-disubstituted benzenes, respectively. For example, the names
1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.
[0037] The terms "heterocyclyl" or "heterocyclic group" refer to 3-
to 10-membered ring structures, more preferably 3- to 7-membered
rings, whose ring structures include one to four heteroatoms.
Heterocycles can also be polycycles. Heterocyclyl groups include,
for example, thiophene, thianthrene, furan, pyran, isobenzofuran,
chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole,
isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine,
indolizine, isoindole, indole, indazole, purine, quinolizine,
isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline,
quinazoline, cinnoline, pteridine, carbazole, carboline,
phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,
phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine,
oxolane, thiolane, oxazole, piperidine, piperazine, morpholine,
lactones, lactams such as azetidinones and pyrrolidinones, sultams,
sultones, and the like. The heterocyclic ring can be substituted at
one or more positions with such substituents as described above, as
for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,
ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, --CF.sub.3, --CN, or the like.
[0038] The terms "polycyclyl" or "polycyclic group" refer to two or
more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls
and/or heterocyclyls) in which two or more carbons are common to
two adjoining rings, e.g., the rings are "fused rings". Rings that
are joined through non-adjacent atoms are termed "bridged" rings.
Each of the rings of the polycycle can be substituted with such
substituents as described above, as for example, halogen, alkyl,
aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,
sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,
carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde,
ester, a heterocyclyl, an aromatic or heteroaromatic moiety,
--CF.sub.3, --CN, or the like.
[0039] The term "carbocycle", as used herein, refers to an aromatic
or non-aromatic ring in which each atom of the ring is carbon.
[0040] As used herein, the term "nitro" means --NO.sub.2; the term
"halogen" designates --F, --Cl, --Br or --I; the term "sulfhydryl"
means --SH; the term "hydroxyl" means --OH; and the term "sulfonyl"
means --SO.sub.2.
[0041] The terms "amine" and "amino" are art-recognized and refer
to both unsubstituted and substituted amines, e.g., a moiety that
can be represented by the general formula: 1
[0042] wherein R.sub.9, R.sub.10 and R'.sub.10 each independently
represent a hydrogen, an alkyl, an alkenyl,
--(CH.sub.2).sub.m--R.sub.80, or R.sub.9 and R.sub.10 taken
together with the N atom to which they are attached complete a
heterocycle having from 4 to 8 atoms in the ring structure;
R.sub.80 represents an aryl, a cycloalkyl, a cycloalkenyl, a
heterocycle or a polycycle; and m is zero or an integer in the
range of 1 to 8. In preferred embodiments, only one of R.sub.9 or
R.sub.10 can be a carbonyl, e.g., R.sub.9, R.sub.10 and the
nitrogen together do not form an imide. In even more preferred
embodiments, R.sub.9 and R.sub.10 (and optionally R'.sub.10) each
independently represent a hydrogen, an alkyl, an alkenyl, or
--(CH.sub.2).sub.m--R.sub.80. Thus, the term "alkylamine" as used
herein means an amine group, as defined above, having a substituted
or unsubstituted alkyl attached thereto, i.e., at least one of
R.sub.9 and R.sub.10 is an alkyl group.
[0043] The term "acylamino" is art-recognized and refers to a
moiety that can be represented by the general formula: 2
[0044] wherein R.sub.9 is as defined above, and R'.sub.11
represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.2).sub.m--R.sub.80, where m and R.sub.80 are as defined
above.
[0045] The term "amido" is art recognized as an amino-substituted
carbonyl and includes a moiety that can be represented by the
general formula: 3
[0046] wherein R.sub.9, R.sub.10 are as defined above. Preferred
embodiments of the amide will not include imides which may be
unstable.
[0047] The term "alkylthio" refers to an alkyl group, as defined
above, having a sulfur radical attached thereto. In preferred
embodiments, the "alkylthio" moiety is represented by one of
--S-alkyl, --S-alkenyl, --S-alkynyl, and
--S--(CH.sub.2).sub.m--R.sub.80, wherein m and R.sub.80 are defined
above. Representative alkylthio groups include methylthio, ethyl
thio, and the like.
[0048] The term "carbonyl" is art recognized and includes such
moieties as can be represented by the general formula: 4
[0049] wherein X is a bond or represents an oxygen or a sulfur, and
R.sub.11 represents a hydrogen, an alkyl, an alkenyl,
--(CH.sub.2).sub.m--R.sub.80 or a pharmaceutically acceptable salt,
R'.sub.11 represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.2).sub.m--R.sub.80, where m and R.sub.80 are as defined
above. Where X is an oxygen and R.sub.11 or R'.sub.11 is not
hydrogen, the formula represents an "ester". Where X is an oxygen,
and R.sub.11 is as defined above, the moiety is referred to herein
as a carboxyl group, and particularly when R.sub.11 is a hydrogen,
the formula represents a "carboxylic acid". Where X is an oxygen,
and R'.sub.11 is hydrogen, the formula represents a "formate". In
general, where the oxygen atom of the above formula is replaced by
sulfur, the formula represents a "thiolcarbonyl" group. Where X is
a sulfur and R.sub.11 or R'.sub.11 is not hydrogen, the formula
represents a "thiolester." Where X is a sulfur and R.sub.11 is
hydrogen, the formula represents a "thiolcarboxylic acid." Where X
is a sulfur and R.sub.11' is hydrogen, the formula represents a
"thioformate." On the other hand, where X is a bond, and R.sub.11
is not hydrogen, the above formula represents a "ketone" group.
Where X is a bond, and R.sub.11 is hydrogen, the above formula
represents an "aldehyde" group.
[0050] The terms "alkoxyl" or "alkoxy" as used herein refers to an
alkyl group, as defined above, having an oxygen radical attached
thereto. Representative alkoxyl groups include methoxy, ethoxy,
propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons
covalently linked by an oxygen. Accordingly, the substituent of an
alkyl that renders that alkyl an ether is or resembles an alkoxyl,
such as can be represented by one of --O-alkyl, --O-alkenyl,
--O-alkynyl, --O--(CH.sub.2).sub.m--R.sub.80, where m and R.sub.80
are described above.
[0051] The term "sulfonate" is art recognized and includes a moiety
that can be represented by the general formula: 5
[0052] in which R.sub.41 is an electron pair, hydrogen, alkyl,
cycloalkyl, or aryl.
[0053] The terms triflyl, tosyl, mesyl, and nonaflyl are
art-recognized and refer to trifluoromethanesulfonyl,
p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl
groups, respectively. The terms triflate, tosylate, mesylate, and
nonaflate are art-recognized and refer to trifluoromethanesulfonate
ester, p-toluenesulfonate ester, methanesulfonate ester, and
nonafluorobutanesulfonate ester functional groups and molecules
that contain said groups, respectively.
[0054] The term "sulfate" is art recognized and includes a moiety
that can be represented by the general formula: 6
[0055] in which R.sub.41 is as defined above.
[0056] The term "sulfonamido" is art recognized and includes a
moiety that can be represented by the general formula: 7
[0057] in which R.sub.9 and R'.sub.11 are as defined above.
[0058] The term "sulfamoyl" is art-recognized and includes a moiety
that can be represented by the general formula: 8
[0059] in which R.sub.9 and R.sub.10 are as defined above.
[0060] The terms "sulfoxido" or "sulfinyl", as used herein, refers
to a moiety that can be represented by the general formula: 9
[0061] in which R.sub.44 is selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,
aralkyl, or aryl.
[0062] A "phosphoryl" can in general be represented by the formula:
10
[0063] wherein Q.sub.1 represented S or O, and R.sub.46 represents
hydrogen, a lower alkyl or an aryl. When used to substitute, e.g.,
an alkyl, the phosphoryl group of the phosphorylalkyl can be
represented by the general formula: 11
[0064] wherein Q.sub.1 represented S or O, and each R.sub.46
independently represents hydrogen, a lower alkyl or an aryl,
Q.sub.2 represents O, S or N. When Q.sub.1 is an S, the phosphoryl
moiety is a "phosphorothioate".
[0065] A "phosphoramidite" can be represented in the general
formula: 12
[0066] wherein R.sub.9 and R.sub.10 are as defined above, and
Q.sub.2 represents O, S or N.
[0067] A "phosphonamidite" can be represented in the general
formula: 13
[0068] wherein R.sub.9 and R.sub.10 are as defined above, Q.sub.2
represents O, S or N, and R.sub.48 represents a lower alkyl or an
aryl, Q.sub.2 represents O, S or N.
[0069] A "selenoalkyl" refers to an alkyl group having a
substituted seleno group attached thereto. Exemplary "selenoethers"
which may be substituted on the alkyl are selected from one of
--Se-alkyl, --Se-alkenyl, --Se-alkynyl, and
--Se--(CH.sub.2).sub.m--R.sub.80, m and R.sub.80 being defined
above.
[0070] Analogous substitutions can be made to alkenyl and alkynyl
groups to produce, for example, aminoalkenyls, aminoalkynyls,
amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls,
thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or
alkynyls.
[0071] As used herein, the definition of each expression, e.g.
alkyl, m, n, etc., when it occurs more than once in any structure,
is intended to be independent of its definition elsewhere in the
same structure.
[0072] Certain compounds of the present invention may exist in
particular geometric or stereoisomeric forms. The present invention
contemplates all such compounds, including cis- and trans-isomers,
R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the
racemic mixtures thereof, and other mixtures thereof, as falling
within the scope of the invention. Additional asymmetric carbon
atoms may be present in a substituent such as an alkyl group. All
such isomers, as well as mixtures thereof, are intended to be
included in this invention.
[0073] If, for instance, a particular enantiomer of a compound of
the present invention is desired, it may be prepared by asymmetric
synthesis, or by resolution, i.e., derivation with a chiral
auxiliary followed by separation of the resulting diastereomeric
mixture and cleavage of the auxiliary group to provide the pure
enantiomers. Alternatively, where the molecule contains a basic
functional group, such as amino, or an acidic functional group,
such as carboxyl, diastereomeric salts are formed with an
appropriate optically-active acid or base, followed by resolution
of the diastereomers thus formed by fractional crystallization or
chromatographic means well known in the art, and subsequent
recovery of the pure enantiomers.
[0074] Contemplated equivalents of the compounds described above
include compounds which otherwise correspond thereto, and which
have the same general properties thereof, wherein one or more
simple variations of substituents are made which do not adversely
affect the properties of the compound. In general, the compounds of
the present invention may be prepared by the methods illustrated in
the general reaction schemes as, for example, described below, or
by modifications thereof, using readily available starting
materials, reagents and conventional synthesis procedures. In these
reactions, it is also possible to make use of variants which are in
themselves known, but are not mentioned here.
[0075] It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, etc.
[0076] As used herein, the term "substituted" is contemplated to
include all permissible substituents of organic compounds. In a
broad aspect, the permissible substituents include acyclic and
cyclic, branched and unbranched, carbocyclic and heterocyclic,
aromatic and non-aromatic substituents of organic compounds.
Illustrative substituents include, for example, those described
herein above. The permissible substituents can be one or more and
the same or different for appropriate organic compounds. For
purposes of this invention, the heteroatoms such as nitrogen may
have hydrogen substituents and/or any permissible substituents of
organic compounds described herein which satisfy the valences of
the heteroatoms. This invention is not intended to be limited in
any manner by the permissible substituents of organic
compounds.
[0077] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87,
inside cover. Also for purposes of this invention, the term
"hydrocarbon" is contemplated to include all permissible compounds
having at least one hydrogen and one carbon atom. In a broad
aspect, the permissible hydrocarbons include acyclic and cyclic,
branched and unbranched, carbocyclic and heterocyclic, aromatic and
non-aromatic organic compounds which can be substituted or
unsubstituted.
[0078] The phrase "protecting group" as used herein means temporary
substituents which protect a potentially reactive functional group
from undesired chemical transformations. Examples of such
protecting groups include esters of carboxylic acids, silyl ethers
of alcohols, and acetals and ketals of aldehydes and ketones,
respectively. The field of protecting group chemistry has been
reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in
Organic Synthesis, 2.sup.nd ed.; Wiley: New York, 1991).
[0079] The term "protein-resistant domain" refers generally to
hydrophilic, uncharged moieties that render a coated surface
resistant to spontaneous association with a biopolymer, e.g.,
proteins, nucleic acid and/or carbohydrates.
[0080] In the context of the present invention, the term biopolymer
refers to any polypeptide (such as proteins, enzymes, antibodies,
etc.), polysaccharide, or polynucleic acid or conjugate of any of
these polymers.
[0081] By the term "resistance to biopolymer adsorption" it is
meant that the surface has a reduction in the amount of a
biopolymer adsorbed on the surface, when contacted with a medium
containing biopolymers available for adsorption, as compared to the
amount adsorbed on the same surface before treatment by the subject
method.
[0082] The term "biocompatibility", as used herein to describe the
coated surfaces of this invention, refers to the resistance to
adsorption of protein and to the lack of interactiveness with
physiological surfaces, e.g., as discussed herein.
[0083] III. Components and Fabrication of the Coatings
[0084] The subject coatings are fabricated by treating a surface
with a mixture comprising an excess of a molecule, or molecules,
that adhere to the surface and in so doing form the coating. The
association between the constituent molecules of the coating and
the surface to be coated may be based on hydrophobic interactions,
electrostatic interactions, covalent bonds, or a combination of
any, or all, of these classes of association.
[0085] Protein resistant coatings of the present invention may be
applied to myriad surfaces including, but not limited to, surfaces
comprising main group elements, alkali and alkaline earth metals,
transition metals, and heteroatoms. In certain embodiments, the
surface to be coated comprises main group and/or transition metal
oxides, main group and/or transition metal sulfides, or main group
and/or transition metal nitrides. In preferred embodiments, the
surface to be coated comprises --OH, --N(H)--, and/or --SH
moieties.
[0086] The adhesion of the constituent molecules of the coating to
the surface to be coated may be based on hydrophobic interactions,
electrostatic interactions, covalent bonds, or a combination of
any, or all, of these types of association. For example, in
instances of the present invention wherein electrostatic
interactions play a role in the adhesion of the coating to the
surface, when the surface to be coated includes cationic or anionic
moieties, the constituent molecules of the coating comprise anionic
or cationic moieties, respectively. In embodiments wherein
hydrophobic interactions play a role in the adhesion of the coating
to the surface, the surface and the constituent molecules of the
coating comprise complementary hydrophobic domains. In embodiments
wherein the adhesion of the coating to the surface comprises
covalent bonds, the surface comprises moieties, e.g. --OH,
--N(H)--, --SH, and the like, which react, to form a covalent bond,
with a functional group, e.g. C-halide, Si-halide, transition
metal-halide, and the like, of the constituent molecules of the
coating. In certain embodiments, the adhesion of the constituent
molecules of the coating to the coated surface will comprise more
than one point of contact, e.g. hydrophobic interaction,
electrostatic interaction, or covalent bond, between an individual
component molecule and the surface.
[0087] The constituent molecules of the coating comprise a
protein-resistant domain, e.g. a hydrophilic substructure or
moiety, situated such that said protein-resistant domain is located
on, or near, the surface of the coating that will be exposed to the
protein-containing sample or solution. The protein resistant domain
is selected from the set comprising hydrophilic groups and charged
groups that repel the surface charge of a protein. In preferred
embodiments, the protein-resistant domain is selected from the set
of charge-neutral hydrophilic moieties comprising alcohols, ethers,
carbonyls, esters, amides, hydroxamic acids, sulfones, and
sulfides.
[0088] While not limiting the effectiveness of this invention to
any specific theory, the qualities of these coatings are believed
to be superior when generated using predominantly or exclusively
ethylene oxide-based diols or polyols in the formulation of the
prepolymers and hydrated polymers. Representative examples of
polyols for forming these precursors include: ethylene glycol,
1,3-propylene glycol, 1,2-propylene glycol, 1,4-butylene glycol,
butene-1,4 diol, 1,5-pentane diol, 1,4-pentane diol, 1,6-hexane
diol, diethylene glycol, glycerine, trimethylol propane,
1,3,6-hexanetriol, trimethanolamine, pentaerythritol, sorbitol.
[0089] The diols and polyols used in the subject method may be made
up of ethylene oxide monomer units. Preferably, at least 75% of the
units should be ethylene oxide, more preferably at least 90%, and
even more preferably at least 95%. Most preferably, substantially
all or all of the units should be ethylene oxide.
[0090] The biopolymer-resistant coating is formed by treating the
surface to be coated with individual molecules represented by
general structure 1: 14
[0091] wherein
[0092] Z represents a domain, moiety, or functional group which
associates with the surface to be coated;
[0093] tether represents a covalent attachment between Z and the
biopolymer-resistant domain;
[0094] biopolymer-resistant domain represents a molecular
substructure to which biopolymers in solution do not adhere well;
and
[0095] W represents H, a small alkyl group, or a small hydrophilic
group.
[0096] In preferred embodiments, the biopolymer-resistant coating
is formed by treating the surface to be coated with individual
molecules represented by general structure 1, wherein:
[0097] Z represents a functional group which associates with the
surface to be coated selected from the set comprising --CO.sub.2H,
--PO.sub.3H.sub.2, --C(O)NHOH, --Si(OR).sub.3, --SiCl.sub.3,
--Sn(OR).sub.3, --SnCl.sub.3, --Ge(OR).sub.3, and --GeCl.sub.3;
[0098] tether represents a hydrophobic covalent attachment,
typically of approximately 5 to 20 bonds in length, between Z and
the protein-resistant domain;
[0099] biopolymer-resistant domain represents a molecular
substructure, to which biopolymers in solution do not adhere well,
selected from the set comprising oligoethers, oligoglycols,
oligoalcohols, oligocarbonyls, oligosulfides, oligosulfones, and
oligosaccharides; and
[0100] W represents H, a small alkyl group, an alkoxyl group, an
acyl group, an acyloxy group, a sulfone, a hydroxyl group, a
sulfhydryl group, or a thioalkyl group.
[0101] In highly preferred embodiments, the biopolymer-resistant
coating is formed by treating the surface to be coated with
individual molecules represented by general structure 1,
wherein:
[0102] Z represents a functional group which associates with the
surface to be coated selected from the set comprising
--Si(OR).sub.3, --SiCl.sub.3, --Sn(OR).sub.3, --SnCl.sub.3,
--Ge(OR).sub.3, and --GeCl.sub.3;
[0103] tether represents a hydrophobic covalent attachment,
typically of approximately 5 to 20 carbons in length, between Z and
the protein-resistant domain;
[0104] biopolymer-resistant domain represents a molecular
substructure, to which biopolymers in solution do not adhere well,
selected from the set comprising oligoethers, oligoglycols,
oligoalcohols, oligocarbonyls, oligosulfides, oligosulfones, and
oligosaccharides; and
[0105] W represents H, a small alkyl group, an alkoxyl group, an
acyl group, an acyloxy group, a sulfone, a hydroxyl group, a
sulfhydryl group, or a thioalkyl group.
[0106] A surface bearing a coating of the present invention on a
surface may be represented by 2. 15
[0107] In preferred embodiments, the thickness of the coating will
be selected according to the intended use of the device. Typical
thicknesses envisaged for coatings according to the invention are
in the order of 0.1 to 1000 nanometers, preferably 1 to 100
nanometers and most preferably about 1-10 nanometers.
[0108] The coating may may be applied to the substrate by any
conventional coating technique such as immersion in a coating bath,
spraying, painting or, for flat substrates, spin-coating, the
coating conditions being varied according to the desired thickness
of coating and the other characteristics of the substrate which
will be appreciated by the skilled artisan. Preferably coating is
achieved by immersion of the substrate in a bath of the coating at
an appropriate concentration and temperature for sufficient time to
cover the surfaces to be coated. The solvent may be removed by
conventional techniques, preferably by evaporation under reduced or
ambient pressure, in a gas stream and/or at elevated temperature.
By careful selection of the solvent, concentration of the solution
and coating and solvent removal techniques, the thickness of the
coating may be controlled within a desired range. Particularly
preferred solvents, concentrations and coating and solvent removal
techniques are described in detail and illustrated by the
Examples.
[0109] The coatings of the invention may be applied to a wide
variety of laboratory and medical care instruments and devices. The
coatings themselves may be, in certain instances, transparent and
do not interfere visually with any purpose of the coated substrate.
If desired, colorants or other compounds may be added. Moreover,
certain of the transparent coatings of this invention may remain
transparent and unclouded even after steam sterilization or
prolonged exposure to a protein-containing environment. Medical
devices may be coated, as may various types of labware which is
used in conjunction with tissue or cell cultures,
protein-containing fluids such as blood or serum, or the like. This
would include as appropriate, but not be limited to, assay plates,
supports or membranes, glassware, cell culture or bioreactor
devices or assemblies, tubing for blood transfer, blood cell
storage bags, filters, pharmaceutical manufacturing and packaging,
protein isolation, preparation and purification devices or systems,
etc. Any device or apparatus made of glass.
[0110] IV. Exemplary Uses of Coatings
[0111] In one aspect, it is the object of the present invention to
provide a method for modifying the surface of an apparatus or
device to create a surface that exhibits resistance to "biomolecule
adsorption." In the context of the present invention, the term
biomolecule refers to any polypeptide (such as proteins, enzymes,
antibodies, etc.), polysaccharide, or polynucleic acid. By the term
"resistance to biomolecule adsorption" it is meant that the surface
which exhibits a reduction in the amount of a biomolecule adsorbed
on the surface, when contacted with a medium containing
biomolecules available for adsorption, as compared to the amount of
biomolecules adsorbed on the same surface before being coated with
the coating composition of this invention.
[0112] Broadly, the coating composition of this invention is
desireable for the production of all surfaces which will be exposed
to environments containing biomolecules and which are desired to
exhibit a resistance to deposition by biomolecule adsorption. It is
known in the art that all devices which are used in contact with
protein-containing fluids or biological fluids must be selected on
the basis of acceptable physical and mechanical properties and
compatibility with the protein-containing or biological fluids.
Examples of these devices or apparatii include glassware and
experimental hardware used for conducting experiments. Examples of
such glassware include glassware used in protein purification
separation, blood bags, pipets, syringes, etc. Also included are
containers used for the storage of biologicals and transport of
biological material.
[0113] Medical devices which come in contact with biological fluids
and hence may be coated with the compositions disclosed herein
include catheters that are used surgically for insertion through
blood vessels, the urethrea, or body conduits, and guidewires used
with catheters for biopsy, balloon angioplasty and other medical
procedures. As used herein, the term "medical apparatus" means
apparatus suited for use in medical applications, particularly in
vivo applications. Such apparatus specifically includes, but are
not limited to, catheters, balloon catheters, guide wires,
endotracheal tubes, implants and other biomedical devices such as,
for example, the outer surface of an endoscope. Of particular note
for use with the invention are catheters having inflatable balloons
such as those developed for use in angioplasty and valvuloplasty,
guide catheters and guidewires. Contact lenses are another area
requiring biomolecule resistant coatings for preventing deposition
of undesirable materials.
[0114] For any given application of these materials it is usually
difficult to optimise all of these considerations simultaneously
and a compromise must be reached often resulting in less than
optimal performance. For example, major biological problems are
often encountered with materials which have otherwise optimal
mechanical and physical properties. These problems often manifest
themselves as undesirable deposition of biological components, in
particular, proteinaceous material. For instance, protein
adsorption results in blood clot formation in blood-contacting
materials, the adsorption of tear components onto contact lenses
resulting in deposit formation, formation of deposits on
intraocular lenses and in separation media resulting in blockage
and failure of separation devices. Such effects lead to significant
loss in operational performance and often complete rejection and
failure of devices.
[0115] In the case of medical devices, for example prostheses and
components of blood dialysis equipment, it is common practice to
employ biocompatible polymers to form at least the surface of the
devices to discourage protein adsorption. However, these materials
are not perfect and reaction with the living tissues still remains
a problem; for example surface-induced thrombosis is still a major
difficulty, particularly where large quantities of blood are
contacted with a foreign surface such as in artificial lungs and
kidneys. Formation of a clot in an artificial organ has a number of
adverse or even catastrophic effects including occlusion of the
blood pathway in the extracorporeal system, or embolism if the clot
breaks off the artificial surface and lodges in a host blood
vessel. Dialysis membranes, heart valves, circulatory-assist
devices, blood substitutes and artificial lungs all share this
problem.
[0116] It is known that materials for use as biocompatible coatings
should ideally:
[0117] (a) be capable of reproducible manufacture as pure
materials;
[0118] (b) be capable of being coated onto surfaces without being
degraded or adversely changed;
[0119] (c) have the requisite mechanical and permeability
properties required for the specific function of the device for
which they are intended;
[0120] (d) be sterilisable without adverse changes in, for example,
permeability and mechanical or surface properties;
[0121] (e) not be damaged or degraded by the biological
environment;
[0122] (f) not be carcinogenic.
[0123] In applications involving direct contact with blood further
restrictions exist. Materials should not:
[0124] (g) induce significant platelet adhesion;
[0125] (h) interfere with the normal clotting mechanism; or
[0126] (i) cause any significant damage to the cellular elements or
soluble components of the blood.
[0127] There have been many attempts to prepare biocompatible, and
specifically blood compatible (i.e, haemocompatible), surfaces,
which do not activate the blood coagulation process and do not
promote thrombus formation. Examples of such attempts include the
preparation of negatively charged surfaces, such as by use of
anionic polymers or suitably oriented polymers, preparation of
surfaces coated with the natural anticoagulant heparin or synthetic
heparin analogues, preparation of surfaces with inherently low
surface free energy such as by use of silicone rubber, preparation
of albumin-coated surfaces, and preparation of surfaces coated with
compounds such as some polymethanes which are thought to adsorb
albumin preferentially from blood. All of these however have had
limitations.
[0128] We have now devised new film-forming coating which can be
used to coat surfaces. It has been found that this coating may be
applied onto a wide variety of surfaces including, polyethylene,
PVC, steel, and poly(imide).
[0129] This invention also provides a coating composition which
when used to coat surfaces, do not swell, to any significant
extent, in aqueous environments; in some situations swelling in
aqueous environments can reduce the stability of the coatings. The
present invention seeks to provide a biocompatible coating which
reduces the deposition of proteins and cells at the substrate
surface when the coated substrate comes into contact with a
protein-containing solution or biological fluid. Additionally, such
coatings bind to surfaces with good adhesion and are not removable
in the environment in which the coated surfaces are used, e.g. in
use as a coating on a blood-contacting surface.
[0130] The extent to which a coating composition renders a surface
biocompatible may be assessed as a combination of factors such as
reduction in the extent to which the surface causes blood platelet
activation, protein adsorption, for instance, as judged by
absorption of fibrinogen from human plasma.
[0131] In other aspects, the invention relates to methods to
construct contact lenses using the compositions of the invention,
and to methods to prevent protein absorption by providing contact
lenses of the claimed compositions.
[0132] Additionally, it is apparent that protein resistance is a
valuable property in any apparatus or device which comes in contact
with the metabolism of the human or animal body. Accordingly, the
compositions of the invention are useful in the preparation of
materials for catheters or for medical tubing of any kind used to
carry body fluids or other fluids into and out of the body,
sutures, cannulas, surgical prostheses, vascular grafting materials
and materials used for reconstruction such as heart valves. These
compositions are also useful for apparatus used to store or
administer body fluids such as blood bags.
[0133] Accordingly, in another aspect, the invention relates to
medical devices constructed of the compositions of the invention.
In still another aspect, the invention relates to methods to coat
lenses and other medical devices so that they become more protein
resistant. Devices and lenses constructed of any polymeric material
can be used as substrates for such coating.
[0134] Thus, one embodiment of the present invention provides a
biocompatible, protein non-adsorptive medical or laboratory device
having a polymer coating on at least one surface thereof in which
the polymer of said polymer coating is a hydrophilic, biocompatible
hydrated polyurea-urethane polymer gel derived from prepolymer
units at least 75% of which are oxyethylene-based diols or polyols
having molecular weights of about 7000 to about 30,000, said diols
or polyols having essentially all of the hydroxyl groups capped
with polyisocyanate, said hydrated polymer gel characterized by
transparency and by a surface having improved resistance to
nonspecific protein adsorption, an formed by reacting said
prepolymer units with water.
[0135] To further illustrate one embodiment, the present invention
relates to the treatment of the surfaces of materials to prevent or
inhibit adsorption of protein or reduce thrombogenicity.
[0136] Many modern surgical and other medical procedures involve
the use of blood-contacting devices, such as surgical implants,
prostheses, catheters, drains and extra-corporeal circuitry. Such
devices are used and then discarded for hygiene reasons: such
devices must therefore be constructed from the most economical
materials available, usually polymeric plastics or glass. However
as already mentioned glass and most synthetic and natural polymers
tend to induce platelet adhesion and activation. Initiation of the
clotting cascade follows, leading to blockage of tubing and
clogging of other apparatus such as filtration and dialysis
membranes and interference with test procedures which, in certain
cases, may have disastrous consequences for patients. Moreover in
cases where a device is intended to be implanted into a patient and
to remain for a prolonged period, such platelet aggregation and
clotting must be avoided over a prolonged period as such a reaction
can lead to platelet adhesion and clotting, which can have severe,
sometimes disastrous, consequences. It is desirable therefore to
develop a treatment for such surfaces which reduces and preferably
avoids such a reaction.
[0137] The present application describes a simple process for
reducing the thrombogenicity of blood-contacting surfaces or
inhibiting or preventing the non-specific adsorption of protein
surfaces which may be used successfully with surfaces. Briefly, the
surface is coated with a stable coating, as described herein, so
that thrombogenicity or protein adsorption will be avoided over a
prolonged period. It is believed that the coatings used in the
present invention may be regarded as non-thrombogenic and involving
no interference with blood biochemistry rather than as
antithrombogenic.
[0138] Typical blood contacting devices whose blood contacting
surfaces may be coated using the process of the present invention
include glass tubing such as found in portions of catheters, for
instance central venous catheters, thoracic drain catheters, and
angioplasty balloon catheters, glass tubing used in extra-corporeal
circuitry such as in heart and/or lung bypasses and entire
extra-corporeal circuits such as whole blood oxygenators, cannulae,
vascular grafts, sutures, membranes such as those used in blood
separation, apheresis and donorpheresis units, gas exchange
membranes such as used in whole blood oxygenators, polycarbonate
membranes and haemodialysis membranes and membranes used in
diagnostic and biosensor devices, biosensors and other devices used
in diagnosis such as cuvettes used in blood clotting time
determinations, prostheses, artificial hearts and surgical
implants.
[0139] Problems can also arise from non-specific protein adsorption
at the surface of devices used in many applications, such as
medical devices. For instance, in many modern diagnostic devices,
such as many biosensors, a specific interaction between an analyte
and a detector species is relied upon. In such situations
non-specific protein adsorption can cause a dramatic loss in
sensitivity or even render the device inoperable. As above, the use
of the subject coatings can overcome, or at least alleviate, such
problems. Thus, other devices which may be treated to reduce
non-specific adsorption of proteins including diagnostic devices
such as biosensors, bioseparation membranes, sight correction
devices such as contact lenses.
[0140] Protein adsorption is also recognised as a problem in sight
correction devices such as contact lenses. Protein build up on such
devices leads to a loss in comfort to the wearer and a
deterioration in vision. It is contemplated that the subject method
can be used to render the surface of appropriate contact lenses
resistant to proteins, and reduce the rate of cloading of the
lenses.
[0141] In an exemplary embodiment, the subject method is used to
coat glass pharmaceutical vials. Briefly, glass vials can be
silylated, prior to treatment with the coating solution, to render
the surface very hydrophobic, resulting in a layer of silane on
which a very stable coating is formed. Silylation can be carried
out, e.g., by the addition of a solution (0.1% w/v in chloroform)
of monochlorodimethyloctadecylsila- ne to the vial.
[0142] V. Exemplification
[0143] The invention now being generally described, it will be more
readily understood by reference to the following examples which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention.
EXAMPLE 1
[0144] This example describes the preparation of oligo(ethylene
glycol)-terminated alkyltrichlorosilanes,
Cl.sub.3Si(CH.sub.2).sub.11(OCH- .sub.2CH.sub.2).sub.nOCH.sub.3
(n=2, 3), and their use in the formation of self-assembled
monolayers on an oxide surface. The adsorption of the
trichlorosilanes from solution produces densely packed, oriented
monolayer films that are 2-3 nm in thickness. The trichlorosilyl
group anchors the molecules to the surface, and the resulting film
exposes the ethylene glycol units at its surface, as noted by its
moderate hydrophilicity
(.theta..sub.a(H.sub.2O).apprxeq.68.degree.). The films are robust
with stabilities similar to those of other alkylsiloxane coatings.
These oligo(ethylene glycol)-terminated silane reagents produce
films that notably exhibit resistances against the non-specific
adsorption of proteins from solution that are better than for films
prepared from octadecyltrichlorosilane. With insulin, lysozyme,
albumin, and hexokinase, no adsorption was observed with the
oligo(ethylene glycol)-siloxane coatings whereas protein films of
approximately a monolayer formed on surfaces treated with
octadecyltrichlorosilane. With fibrinogen, complete resistance was
not possible with either coating; however, the oligo(ethylene
glycol)-siloxane coatings exhibited greater resistance against
non-specific adsorption. The oligo(ethylene glycol)-siloxane
coatings offer performance advantages over available systems and
could easily provide a direct and superior replacement in protocols
that presently use silane reagents to generate hydrophobic, "inert"
surfaces.
[0145] The non-specific adsorption of proteins and other
biomolecules onto surfaces is a problem common to biomedical
devices, biochemical processing, and biodiagnostics..sup.1, 2 This
problem is particularly acute for objects made of metal or glass as
proteins and other species will often adsorb in multilayer
quantities onto their corresponding metal oxide surfaces by
electrostatic attraction..sup.3 Common methods to retard the
adsorption include the use of alkyltrichlorosilanes (typically
n-C.sub.18H.sub.37SiCl.sub.3) to passivate the glass or metal oxide
surface with a covalent hydrocarbon film.sup.4 or the attachment or
grafting of poly(ethylene glycol) to the surface..sup.5-7 In the
former approach, molecular films from the silane are prepared on
the high energy oxide surface to produce a low energy, hydrophobic
surface..sup.8-10 The attached hydrocarbon chains reduce the
non-specific adsorption of proteins by screening the electrostatic
attraction between the underlying material and charged biomolecules
such as proteins; however, the hydrophobic surface-by nature of
having a relatively high interfacial free energy
(.gamma..sub.SL.apprxeq.50 mN/m) when contacted with water-will
routinely adsorb roughly a monolayer of protein. 1. Norde, W.,
Adsorption of Proteins From Solution at the Solid-Liquid Interface,
Adv. Colloid Interface Sci. 1986, 25, 267-340. 2. Andrade, J. D.
and Hlady, V., Protein Adsorption and Materials Biocompatibility,
Adv. Polym. Sci. 1986, 79, 1-63. 3. Vroman, L., Blood, Natural
History Press, 1966. 5. Nashabeh, W. and Rassi, Z. E., Capillary
Zone Electrophoresis of Proteins with Hydrophilic Fused-silica
Capillaries, J. of Chrom. 1991, 559, 367-383. 6. Herren, B. J.,
Shafer, S. G., Van Alstine, J., Harris, J. M. and Snyder, R. S.,
Control of Electroosmosis in Coated Quartz Capillaries, J. Colloid
Interface Sci. 1987, 115, 46-55. 7. Yang, Z. and Yu, H., Preserving
a Globular Protein Shape on Glass Slides: A Self-Assembled
Monolayer Approach, Adv. Mater. 1997, 9, 426-429. 8. Maoz, R. and
Sagiv, J., On the Formation and Structure of Self-Assembling
Monolayers I. A Comparative ATR-Wettability Study of
Langmuir-Blodgett and Adsorbed Films on Flat Substrates and Glass
Microbeads, J. Colloid Interface Sci. 1984, 100, 465-496. 9. Cohen,
S. R., Naaman, R. and Sagiv, J., Thermally Induced Disorder in
Organized Organic Monolayers on Solid Substrates, J. Phys. Chem.
1986, 90, 3054-3056. 10. Wasserman, S. R., Tao, Y. -T. and
Whitesides, G. M., Structure and Reactivity of Alkylsiloxane
Monolayers Formed by Reaction of Alkyltrichlorosilanes on Silicon
Substrates, Langmuir 1989, 5, 1074-1087.
[0146] Surface-bound poly(ethylene glycol) (PEG) is a common
strategy for retarding the non-specific adsorption of proteins and
other biological species..sup.11 Methods for covalently attaching
PEG to surfaces include the incorporation of PEG monomers into
polymer networks by graft polymerization.sup.12-18 and the direct
attachment of PEG chains to surfaces by various coupling
reactions..sup.11 In graft polymerization, the PEG chains are
incorporated as segments of a polymer backbone, and the
incorporated PEGs can have limited effect on non-specific
adsorption depending on the surface density of the PEG
chains..sup.19, 20 The direct attachment of PEG chains to the
surface provides a superior method for manipulating surface
properties; however, multiple processing steps are often required
for coupling the PEG molecules to the substrate..sup.21, 22 For
inorganic substrates, silane reagents are often used to present
reactive organic moieties (amines, epoxides, isocyanates, etc.)
that provide sites for the covalent attachment of PEG chains. In
these procedures, the molecules used for attach PEG chains to these
sites frequently include a variety of specialty PEG
derivatives.sup.23--PEG-mon- oacrylates, PEG--NH.sub.2, PEG--CHO,
CH.sub.3O--PEG, PEG epoxides, star-PEGs, etc.--whose availability
and cost can limit the utility of this approach. For these
procedures, the effectiveness of the resulting coated surface is
related to the surface density of PEG chains as uncoated regions
that expose the underlying material often provide sites that
undergo non-specific protein adsorption..sup.24 Objects with
complex morphologies offer particular challenges for this method of
surface modification due to difficulties in producing uniform,
defect-free coatings of PEG. Molecular precursors, such as analogs
of CH.sub.3(CH.sub.2).sub.17SiCl.sub.3 that produce densely packed
films spontaneously onto surfaces from solution with high
uniformity of coverage,.sup.10 could offer distinct advantages over
present methods if they exposed a PEG-type surface that retarded
the non-specific adsorption of proteins. 10. Wasserman, S. R., Tao,
Y. -T. and Whitesides, G. M., Structure and Reactivity of
Alkylsiloxane Monolayers Formed by Reaction of
Alkyltrichlorosilanes on Silicon Substrates, Langmuir 1989, 5,
1074-1087. 11. Harris, J. M., Poly(Ethylene Glycol) Chemistry,
Plenum Press, New York, 1992. 12. Mori, Y., Nagaoka, S., Takuichi,
T., et al., A New Antithrombogenic Material with Long Polyethylene
Oxide Chains, Trans. Am. Soc. Artif. Intern. Org. 1982, 28,
459-463. 13. Merrill, E. W. and Salzman, E. W., Polyethylene Oxide
as a Biomaterial, Am. Soc. Artif. Intern. Org. J. 1983, 6, 60-64.
14. Sun, Y. H., Gomboltz, W. R. and Hoffman, A. S., Synthesis and
Characterization of Non-fouling Polymer Surfaces: I. Radiation
Grafting of Hydroxyethyl Methacrylate and Polyethylene Glycol onto
Silastic Film, Compat. Polym. 1986, 1, 316-334. 15. Grasel, T. G.
and Cooper, S. L., Surface Properties and Blood Compatibility of
Polyurethaneureas, Biomaterials 1986, 7, 315-328. 16. Su, Y. H.,
S., H. A. and Gomboltz, W. R., Non-fouling Biomaterial Surfaces:
II. Protein Adsorption on Radiation Grafted Polyethylene Glycol
Methacrylate Copolymers, Polym. Prep. 1987, 28, 292-294. 17.
Grainger, D. W., Nojiri, C., Okano, T. and Kim, S. W., In vitro and
ex vitro Platelet Interactions with Hydrophilic-hydrophobic
Poly(ethylene oxide)-heparin Block Copolymers. I. Synthesis and
characterization, J. Biomed. Mater. Res. 1988, 22, 231-249. 18.
Grainger, D. W., Knutsen, K., Okano, T. and Feijin, J.,
Poly(dimethyl siloxane)-Poly(ethylene oxide)-heparin Block
Copolyers. II. Surface characterization and in vitro assessments,
J. Biomed. Mater. Res. 1990, 24, 403-431. 19. Jeon, S. I. and
Andrade, J. D., Protein-Surface Interactions in the Presence of
Polyethylene Oxide II. Effect of Protein Size, J. Colloid Interface
Sci. 1991, 142, 159-166. 20. Jeon, S. I., Lee, J. H., Andrade, J.
D. and De Gennes, P. G., Protein-Surface Interactions in the
Presence of Polyethylene Oxide I. Simplified Theory, J. Colloid
Interface Sci. 1991, 142, 149-158. 21. Lassen, B., Golander, C.
-G., Johansson, A. and Elwing, H., Some Model Surfaces Made by RF
Plasma Aimed for the Study of Biocompatibility, Clin. Mater. 1992,
11, 99-103. 22. Kiss, E. and Golander, C. -G., Chemical
Derivatization of Muscovite Mica Surfaces, Colloids and Surfaces
1990, 49, 335-342. 23. Harris, J. M., Laboratory Synthesis of
Polyethylene Glycol Derivatives, Rev. Macromol. Chem. Phys. 1985,
C25, 325-373. 24. Andrade, J. D., Hlady, V. and Jeon, S. -I.,
Poly(ethylene oxide) and Protein Resistance, in Hydrophilic
Polymers: Performance with Environmental Acceptance, (Ed. J. E.
Glass), American Chemical Society, 1996, 51-59.
[0147] To address this problem of surface modification, we have
developed two new reagents that combine the protocol of use of the
trichlorosilane-based adsorbates with the generation of
oligo(ethylene glycol)-based surfaces to generate robust coatings
for glass and metal oxide substrates that are resistant against the
non-specific adsorption of various proteins. These reagents are
based on the results of Prime and Whitesides who demonstrated the
effectiveness of films formed by the adsorption of
HS(CH.sub.2).sub.11(OCH.sub.2CH.sub.2).sub.nOR (R.dbd.H and n=0, 1,
2, 4, and 6; R.dbd.CH.sub.3 and n=6) onto gold to retard the
non-specific adsorption of proteins..sup.25 Alkanethiols,
HS(CH.sub.2).sub.mX, spontaneously assemble onto gold surfaces via
sulfur-gold interactions and form oriented, densely packed
molecular coatings ("self-assembled monolayers"=SAMs), where the
surface properties of the resulting films are controlled by
selection of tail group (X)..sup.26 Their observation that only a
few ethylene glycol units were required in these oriented
assemblies to retard protein adsorption and that methyl-terminated
ethylene glycol units were also effective provided the basis for
our exploration of thiol compounds that combine these two factors
and our development of the oligo(ethylene glycol)-terminated silane
reagents. The methyl cap is needed on the ethylene glycol group for
generation of a silane-based reagent that could be used on glass
and metal oxide substrates as the hydroxyl group of an ethylene
glycol cannot be accommodated within a molecule bearing a
trichlorosilyl group due to their cross reactivity. In general,
trichlorosilane reagents are useful for functionalizing a broader
class of substrates (metal oxides).sup.26 than the thiols (coinage
metals such as gold, silver, and copper),.sup.27 and they are
widely used in practical applications as they exhibit dramatically
superior levels of stability..sup.4, 26 4. Plueddemann, E. P.,
Silane Coupling Agents, Plenum Press, New York, 1982. 26. Ulman,
A., An Introduction to Ultrathin Organic Films: From
Langmuir-Blodgett to Self-Assembly, Academic Press, Boston, 1991.
27. Laibinis, P. E. and Whitesides, G. M., .omega.-Terminated
Alkanethiolate Monolayers on Surfaces of Copper, Silver and Gold
Have Similar Wettabilities, J. Am. Chem. Soc. 1992, 114,
1990-1995.
[0148] In this paper, we demonstrate the effectiveness of
methyl-capped di- and triethylene glycol-terminated silane
reagents,
CH.sub.3O(CH.sub.2CH.sub.2O).sub.2,3(CH.sub.2).sub.11SiCl.sub.3,
for producing robust molecular films that inhibit the non-specific
adsorption of proteins. In particular, we examined proteins with
molecular weights from 10,000 to 400,000 Da (insulin, lysozyme,
albumin, hexokinase, and fibrinogen). The reagents contained a
methyl cap and either two or three ethylene glycol units as their
tail group, where these functionalities become localized after
assembly of the coating at the outer surface. Our investigation
with these two compounds allowed examination of the effects of
oligo-ethylene glycol length and their thickness on the properties
of the coating; in this study, the thickness of the ethylene glycol
portion of the coating was .about.10 to 15 .ANG.. The ethylene
glycol-terminated silane reagents adsorb onto the surface of an
oxide spontaneously from solution and form a coating by methods
(FIG. 1) that are directly analogous to those commonly used to
hydrophobize glass with alkyltrichlorosilanes
(CH.sub.3(CH.sub.2).sub.mSiCl.sub.3). We compared the adsorptive
properties of the hydrocarbon and ethylene glycol-terminated
silane-based coatings with various proteins and examined the
abilities of films prepared from the ethylene glycol-terminated
silane reagents to maintain their non-adsorptive properties after
exposure to conditions of elevated temperatures and humidity.
Materials
[0149] Reagents were obtained from Aldrich and used as received
unless specified otherwise. Octadecyltrichlorosilane was distilled
under reduced pressure before use. 10-Undecylenic-1-bromide was
obtained from Pfaltz and Bauer (Waterbury, Conn.). Lysozyme
(chicken egg white, grade III), albumin (human, fraction V),
fibrinogen (bovine, type I-S), hexokinase (bakers yeast) and
insulin (bovine pancreas, type III) were obtained from Sigma (St.
Louis, Mo.) and used as received. Silicon wafers were test grade
and obtained from Silicon Sense (Nashua, N.H.). Gold shot (99.99%)
and chromium-coated tungsten filaments were obtained from Americana
Precious Metals (East Rutherford, N.J.) and R. D. Mathis Co. (Long
Beach, Calif.), respectively. Oligo(ethylene glycol)-undecenes and
undecanethiols were synthesized by reported procedures;.sup.25, 28
methyl-capped derivatives were synthesized by direct modifications
to these procedures. .sup.1H NMR spectra were obtained on a Bruker
250 MHz spectrometer in CDCl.sub.3 and referenced to residual
CHCl.sub.3 at 7.24 ppm. 25. Prime, K. L. and Whitesides, G. M.,
Adsorption of Proteins onto Surfaces Containing End-Attached
Oligo(ethylene oxide): A Model System Using Self-Assembled
Monolayers, J Am. Chem. Soc. 1993, 115, 10714-10721. 28.
Pale-Grosdemange, C., Simon, E. S., Prime, K. L. and Whitesides, G.
M., Formation of Self-Assembled Monolayers by Chemisorption of
Derivatives of Oligo(ethylene glycol) of Structure
HS(CH.sub.2).sub.11(OCH.sub.2CH.sub.2).sub.mOH on Gold, J. Am.
Chem. Soc. 1991, 113, 12-20.
Syntheses of Methyl [(1-trichlorosilyl)undec-11-yl] oligo(ethylene
glycol)s
[0150] Methyl (undec-10-en-1-yl) oligo(ethylene glycol)
[CH.sub.2=CH(CH.sub.2).sub.9(OCH.sub.2CH.sub.2).sub.nOCH.sub.3; n=2
and 3].sup.25 (9.5 mmol), HSiCl.sub.3 (28.5 mmol), and t-butyl
peroxide (0.14 mmol) were combined under a dry N.sub.2 atmosphere
in a glove box. The reaction mixture was stirred for 7 h under UV
irradiation by a medium pressure Hg lamp.sup.29 and concentrated
under reduced pressure to remove excess HSiCl.sub.3. The NMR
spectrum of the reaction mixture showed quantitative conversion of
the olefin to the trichlorosilane. Further purification was
performed by vacuum distillation. Methyl
[(1-trichlorosilyl)undec-11-yl] di(ethylene glycol): .sup.1H NMR
(250 MHz, CDCl.sub.3) .delta. 1.2-1.5 (m, 16 H), 1.56 (m, 4 H),
3.38 (s, 3 H), 3.45 (t, 2 H), 3.5-3.75 (m, 8 H). Methyl
[(1-trichlorosilyl)undec-11-yl] tri(ethylene glycol) was prepared
by a similar procedure. .sup.1H NMR (250 MHz, CDCl.sub.3) .delta.
1.2-1.5 (m, 16 H), 1.56 (m, 4 H), 3.38 (s, 3 H), 3.45 (t, 2 H),
3.5-3.75 (m, 12 H). 25. Prime, K. L. and Whitesides, G. M.,
Adsorption of Proteins onto Surfaces Containing End-Attached
Oligo(ethylene oxide): A Model System Using Self-Assembled
Monolayers, J. Am. Chem. Soc. 1993, 115, 10714-10721. 29. Eaborn,
C., Harrison, M. R. and Walton, M. R, Catalysis of Hydrosilylation
of Olefins by tert-Butyl Peroxide under UV Irradiation, J.
Organomet. Chem. 1971, 31, 43-46.
Preparation of Silicon Substrates
[0151] Si(100) test wafers were cut into strips of .about.1.times.3
cm.sup.2 that were subsequently cleaned by immersion in freshly
prepared "piranha" solution of 70% conc. H.sub.2SO.sub.4(aq)/30%
H.sub.2O.sub.2(aq) (v/v) for 0.5 to 1 h at 70.degree. C. (CAUTION:
"piranha" solution reacts violently with many organic materials and
should be handled with care). The substrates were immediately
rinsed with distilled water, dried in a stream of N.sub.2, and used
within 1 h of cleaning. This process produces a highly wettable,
hydrated oxide surface on silicon with similar properties to that
for glass.
Formation of Assemblies on SiO.sub.2 and Au
[0152] The piranha-cleaned silicon substrates were functionalized
by immersion in a 2 mM solution of the trichlorosilane in anhydrous
toluene. The solutions were prepared and kept in a dry nitrogen
atmosphere (glove box). After 6 to 24 h, the substrates were
removed from solution and rinsed in 20 mL of CH.sub.2Cl.sub.2. The
substrates were removed from the glove box, rinsed sequentially
with CHCl.sub.3 and ethanol to remove any residual organic
contaminants, and dried in a stream of N.sub.2. Optical constants
were measured on the bare substrates by ellipsometry for use in
determining thicknesses subsequently of the adsorbed silane films
and protein layers. The piranha-cleaned substrates were typically
exposed to the air for no more than 15 min before immersion in the
silane solution.
[0153] Gold substrates were prepared by the sequential evaporation
of Cr (100 .ANG.) and Au (1000 .ANG.) onto Si(100) wafers at
pressures of .about.10.sup.-6 torr. The wafers were cut into
.about.1.times.3 cm.sup.2 strips and immersed into .about.2 mM
solutions of the thiols in absolute ethanol for 24 h at room
temperature. These samples were rinsed with ethanol and blown dry
with N.sub.2 before use.
Protein Adsorption Experiments
[0154] The proteins were dissolved at a concentration of 0.25 mg/mL
in phosphate buffer saline (PBS) solution (10 mM phosphate buffer,
2.7 mM KCl, and 137 mM NaCl) that was adjusted to pH 7.4 and
contained sodium azide (0.2 mg/mL) as a bacteriostat. The coated
substrates were immersed in the PBS solutions for 24 h at
20-25.degree. C., rinsed with deionized water (Milli-Q), and dried
in a stream of N.sub.2. The amount of protein that remained on each
substrate after this procedure was determined by ellipsometry.
Experiments were also conducted using adsorption times of 3 to 6 h
and yielded similar thicknesses as those performed using adsorption
times of 24 h. Adsorption times were standardized to 24 h for
consistency.
Contact Angle Measurements
[0155] Contact angles were measured on a Ram-Hart goniometer
(Ram-Hart Inc., Mountain Lakes, N.J.) equipped with a video-imaging
system. Drops were placed on at least three locations on the
surface in the ambient environment and measured on both sides of
the drops. Contacting liquid drops were advanced and retreated with
an Electrapipette (Matrix Technologies, Lowell, Mass.) at
approximately 1 .mu.L/s. Angles were measured to
.about..+-.1.degree. and were reproducible from sample to sample
within .+-.2.
Ellipsometric Film-Thickness Measurements
[0156] The thicknesses of the films were determined with a Gaertner
L116A ellipsometer (Gaertner Scientific Corporation, Chicago,
Ill.). For each substrate, measurements were made before and after
derivatization with the trichlorosilanes, and after protein
adsorption. The thicknesses of the films were determined using a
three-phase model and a refractive index of 1.45.sup.30 and have an
error of .+-.2 .ANG.. The use of this value allows direct
comparison with data obtained by other groups and provides an
accurate relative measure of the amounts of materials adsorbed on
the various coatings. 30. Allara, D. L. and Nuzzo, R. G.,
Spontaneously Organized Molecular Assemblies. 2. Quantitative
Infrared Spectroscopic Determination of Equilibrium Structures of
Solution-Adsorbed n-Alkanoic Acids on an Oxidized Aluminum Surface,
Langmuir 1985, 1, 52-66.
Results and Discussions
Synthesis of Silane Reagents
[0157] We prepared the oligo(ethylene glycol)-terminated
alkyltrichlorosilanes via a two-step synthesis from commercially
available compounds (Scheme I). The monomethyl ether of an
oligo(ethylene glycol) was reacted under basic conditions with
11-bromo-undec-1-ene in dimethylformamide (DMF) to yield an
11-oligo(ethylene glycol)-undec-1-ene methyl ether in high yield.
Separation of the product from excess reagents was easily performed
by extraction. The transformation of the resulting olefin to a
trichlorosilane by photochemical addition of trichlorosilane
(HSiCl.sub.3) proceeded quantitatively. In this reaction, excess
HSiCl.sub.3 served as the solvent and was removed under reduced
pressure to yield the product silane in sufficient purity to
produce protein resistant coatings although distillation under
reduced pressure was used to produce the target silanes as purified
compounds. The process for synthesizing the silane reagents (Scheme
I) is amenable to scale-up as each reaction could be performed
quantitatively and excess reagents were easily separated from the
products by extraction and distillation procedures.
Scheme 1. Synthesis of .omega.-trichlorosilyl-oligo(ethyleneglycol)
derivatives (n=2-3)
[0158] 16
Preparation of Films
[0159] Siloxane films were prepared by a straightforward
solution-phase adsorption process onto silicon wafers that exposed
a hydrated oxide surface (FIG. 1)..sup.26 Silicon was used as a
substrate in these studies as its oxide surface is similar to glass
in reactivity and its reflective properties allowed measurement of
adsorbed protein films by ellipsometry. The semiconducting
properties of silicon additionally allowed direct analysis of the
surface by x-ray photoelectron spectroscopy to verify formation of
the coating and examine the levels of protein adsorption. 26.
Ulman, A., An Introduction to Ultrathin Organic Films: From
Langmuir-Blodgett to Self-Assembly, Academic Press, Boston,
1991.
[0160] To prepare the siloxane coatings, the silicon substrates
were immersed into unstirred solutions of the silanes in anhydrous
toluene for 6-24 h at room temperature. The silane solutions were
handled and stored under a dry N.sub.2 atmosphere and yielded
reproducible formation of films over several weeks of use. Similar
results may be obtained with trichlorosilane-based reagents when
the adsorbate solutions are used under ambient laboratory
conditions when the relative humidity is less than 40%;.sup.10
however, the compounds exhibit a cumulative sensitivity toward
moisture to produce insoluble polymerized aggregates that degrade
the properties of the coatings. This problem is common to all
trichlorosilane-based reagents (including alkytrichlorosilanes,
CH.sub.3(CH.sub.2).sub.mSiCl.sub.3, used for hydrophobizing glass)
due to the hydrolytic instability of the SiCl.sub.3 group that is
required for film formation. To avoid the possibility of
solution-phase hydrolysis and aggregate formation from the silane
reagents, we formed the siloxane coatings under a dry atmosphere of
nitrogen. 10. Wasserman, S. R., Tao, Y. -T. and Whitesides, G. M.,
Structure and Reactivity of Alkylsiloxane Monolayers Formed by
Reaction of Alkyltrichlorosilanes on Silicon Substrates, Langmuir
1989, 5, 1074-1087.
[0161] As a comparison to the trichlorosilane-based films,
oligo(ethylene glycol)-terminated monolayers on gold were prepared
by contacting gold-coated silicon wafers with 2 mM solutions of
HS(CH.sub.2).sub.11(OCH- .sub.2CH.sub.2).sub.nOCH.sub.3 (n=0, 2-4)
in ethanol for 6-24 h at room temperature. As the assembly of
thiols onto gold is not sensitive to humidity, we performed the
assembly of these films in the laboratory ambient.
Characterization of the Monolayer Films
[0162] Table I displays the wetting properties for films formed
upon adsorption of various n-alkanethiols and
n-alkyltrichlorosilanes onto Au and Si/SiO.sub.2 surfaces,
respectively. The wetting properties are compatible with the
formation of oriented monolayer films that expose the tail group at
the surface, with the thiol and silane-derived films exhibiting
similar wetting properties for a common tail group. The formation
of monolayer films was confirmed using ellipsometry where
thicknesses for the various ethylene glycol-terminated thiols
(n=2-4) ranged from 18 to 22 .ANG. and those derived from the
methyl-capped di- and triethylene glycol-terminated silanes were 18
and 20 .ANG., respectively. On gold, we observed that the
difference in wettability between the hydroxyl- and methyl-capped
ethylene glycol surfaces was .about.30.degree. and was much less
than the difference for similar substitutions on a purely
hydrocarbon chain (.about.100.degree.). This difference in behavior
probably reflects interaction by water with the ethylene glycol
framework that moderates that effect of the terminal group. For
both systems, water wets the methyl-capped ethylene
glycol-terminated films (.theta..sub.a(H.sub.2O)=62-72.degree.)
better than for the methyl-capped alkyl films
(.theta..sub.a(H.sub.2O)=110-115.d- egree.). This greater
wettability by the former surfaces implies a lower interfacial
energy (.gamma..sub.SL) between the film and water.
1TABLE 1 Contact Angles Measured on Films on Gold and Silicon
Obtained from HS(CH.sub.2).sub.11R and
Cl.sub.3Si(CH.sub.2).sub.11R.sup.a. Substrate R.sup.b
.theta..sub.a(H.sub.2O) .theta..sub.r(H.sub.2O) Gold CH.sub.3 110
99 (CH.sub.2).sub.6CH.sub.3 115 99 OH 10 <10 (EG).sub.3OH.sup.c
34 23 (EG).sub.4OH.sup.c 38 24 OCH.sub.3 81 68 (EG).sub.2OCH.sub.3
68 59 (EG).sub.3OCH.sub.3 62 52 (EG).sub.4OCH.sub.3 62 52 Silicon
(CH.sub.2).sub.6CH.sub.3 115 98 (EG).sub.2OCH.sub.3 72 55
(EG).sub.3OCH.sub.3 67 49 .sup.aAdvancing (.theta..sub.a) and
receding (.theta..sub.r) static contact angles of water. .sup.bEG =
--OCH.sub.2CH.sub.2-- .sup.cReference 28.
Protein Repellency of Films
[0163] We examined the adsorption properties of the methyl-capped
oligo(ethylene glycol)-terminated films on Au and Si/SiO.sub.2 by
immersing them into various protein-containing solutions at a
concentration of 0.25 mg/mL for 24 h at room temperature. We
performed concurrent experiments in these solutions using surfaces
coated with octadecyl chains to allow direct comparisons of the
performance of these films with available systems. The amount of
adsorbed protein was determined optically ex situ using
ellipsometry. We also used techniques such as x-ray photoelectron
spectroscopy (for siloxane and thiolate SAMs) and polarized
infrared external reflectance spectroscopy (for thiolate SAMs) to
determine the amount of adsorbed proteins. These techniques are
superior to ellipsometry because on their detection of specific
chemical signals-nitrogen composition or amide content--resulting
from the protein; however, they required much longer times for
characterizing each sample. In general, we found that the thickness
data from ellipsometry agreed with results from these other
methods, and we used it as our primary characterization method.
[0164] FIG. 2 summarizes the protein adsorption results for both
substrates. On gold, the five proteins adsorbed onto the
hydrophobic surfaces prepared from octadecanethiol, with the higher
molecular weight proteins forming thicker adsorbed films. These
thicknesses correspond to roughly a monolayer of adsorbed protein
suggesting that the proteins adsorb to lower the interfacial energy
between the hydrocarbon coating and water and the resulting protein
surface does not promote further adsorption. A surface expressing a
methoxy group (--OCH.sub.3) exhibits a lower surface energy than
the purely alkyl surface (as evidenced by its lower contact angle
by water, Table I); however, this difference did not have a large
effect on protein adsorption. This observation suggests that the
interfacial free energy for this surface when contacted with water
is sufficient to drive adsorption of a layer of protein. Again,
roughly a monolayer of protein appears to adsorb suggesting that
the protein surface does not promote further protein
adsorption.
[0165] The incorporation of two and four oligo(ethylene glycol)
units as linkers between the methoxy terminus and the alkyl chain
resulted in a reduction in the amount of protein adsorption onto
the gold surface (FIG. 2). For insulin, lysozyme, albumin, and
hexokinase, the SAMs resisted protein adsorption within the
experimental errors of ellipsometry. Complete resistance against
the adsorption of fibrinogen was not possible with the
EG.sub.2,4--CH.sub.3 surface; however, the SAM reduced the adsorbed
amount to roughly 10% of a monolayer. The difference in the
adsorption characteristics of the purely alkyl CH.sub.3--O-capped
monolayer and the EG-containing CH.sub.3-capped film can be
explained partially by the lower interfacial energy of the latter
system with water. Entropic effects may also be operative for the
oligo(ethylene glycol) system..sup.1, 24 1. Norde, W., Adsorption
of Proteins From Solution at the Solid-Liquid Interface, Adv.
Colloid Interface Sci. 1986, 25, 267-340. 24. Andrade, J. D.,
Hlady, V. and Jeon, S. -I., Poly(ethylene oxide) and Protein
Resistance, in Hydrophilic Polymers: Performance with Environmental
Acceptance, (Ed. J. E. Glass), American Chemical Society, 1996,
51-59.
[0166] With the silane reagents, the results mirrored those on the
gold substrates where the CH.sub.3-capped oligo(ethylene glycol)
monolayers adsorbed less protein than did the purely alkyl film.
Films derived from octadecyltrichlorosilane adsorbed roughly a
monolayer of protein. The CH.sub.3-capped di- and tri-(ethylene
glycol) films exhibited a resistance against adsorption by insulin,
lysozyme, albumin, and hexokinase, with the tri-(ethylene glycol)
films offering superior properties and being resistant against
adsorption for these proteins within the error of ellipsometry. As
for gold, the CH.sub.3-capped oligo-(ethylene glycol) films
adsorbed fibrinogen, with the adsorbed amounts being greater for
the silane-based films than for the thiols on gold. The superior
properties on gold may reflect the greater ease for forming
oriented, well-defined, self-assembled, thiol-based monolayer films
as silane reagents can form polymeric aggregates that can diminish
the surface properties of the film..sup.26 The presence of such
aggregates could provide local hydrophobic sites for the adsorption
of proteins. As we assembled the silane films under an inert
atmosphere and used physical methods to displace any physisorbed
materials from the surface, the amount of physisorbed material on
our surfaces should be low. Structural differences in the molecular
conformation of the CH.sub.3-capped tri(ethylene glycol)
layer--crystalline vs. amorphous--have been reported to affect the
protein resistance of such surfaces toward fibrinogen and such
differences may be operative here..sup.31 26. Ulman, A., An
Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to
Self-Assembly, Academic Press, Boston, 1991. 31. Harder, P.,
Grunze; M., Dahint, R., Whitesides, G. M. and Laibinis, P. E.,
Molecular Conformation in Oligo(ethylene glycol)-Terminated
Self-Assembled Monolayers on Gold and Silver Surfaces Determines
Their Ability to Resist Protein Adsorption, J. Phys. Chem. B, in
press.
[0167] In addition to the ellipsometric results, the wetting
properties of the surfaces provided a macroscopic (albeit
qualitative) indicator of protein adsorption. The initially
prepared surfaces were hydrophobic and emerged dry when rinsed with
water. After exposure to the protein solutions, the purely alkyl
systems became less hydrophobic, while the oligo(ethylene glycol)
surfaces maintained their hydrophobicity. In particular, the
receding contact angle of water on surfaces with an adsorbed layer
of protein was .about.25.degree. and was sufficiently low to
provide a visual indication of protein adsorption.
Stability of Films
[0168] The practical utility of a coating is based on both its
performance and its ability to maintain its useful properties. We
examined the stability of the siloxane films by exposing them to
various conditions including boiling water, hot hydrocarbon
solutions, oven drying, and autoclaving. The films retained their
protein resistant properties after immersion in boiling water at
100.degree. C. or decahydronaphthalene (DHN) at 90.degree. C. for 1
h and drying in an oven at 120.degree. C. for 1 h; however, they
exhibited significant deterioration after drying in an oven at
200.degree. C. for 1 h. These observations are compatible with the
literature regarding the thermal stabilities of siloxane monolayer
films as such films are reported to exhibit no detectable changes
in structure and wetting properties when heated to
.about.140.degree. C. and subsequently characterized at room
temperature..sup.9, 32 For our reagents and coatings, the presence
of the CH.sub.3-capped oligo(ethylene glycol) tail does not appear
to negatively impact the thermal stability of an alkylsiloxane
monolayer. For use in applications that require sterilized
glassware, we note the silane-based coatings maintained their
integrity and properties after an extended sterilization cycle (1
h) in an autoclave at .about.120.degree. C. (pressure=20 psi). This
ability may make these films suitable for numerous applications
where sterile conditions are required and there is a need to limit
the non-specific adsorption of proteins. 9. Cohen, S. R., Naaman,
R. and Sagiv, J., Thermally Induced Disorder in Organized Organic
Monolayers on Solid Substrates, J. Phys. Chem. 1986, 90, 3054-3056.
32. Calistri-Yeh, M., Kramer, E. J., Sharma, R., et al., Thermal
Stability of Self-Assembled Monolayers from Alkylchlorosilanes,
Langmuir 1996, 12, 2747-2755.
[0169] The thermal stabilities of the siloxane monolayers contrasts
with the rapid desorption under these conditions for their thiol
counterparts on gold. For comparison, .about.30% of a thiol-based
oligo(ethylene glycol)-terminated monolayer desorbed within 5 min
in boiling water and within 1 min in DHN at 90.degree. C. and lost
their abilities to resist the non-specific adsorption of proteins,
while the siloxane films were stable for at least an hour under
these conditions. The robust behavior of the siloxane monolayers
offers the needed stabilities required for practical application,
with the developed CH.sub.3-capped oligo-(ethylene
glycol)-terminated silane reagents providing easy access to robust,
protein resistant molecular coatings for glass and metal oxide
surfaces.
Conclusions
[0170] Molecular coatings that exhibit a resistance against the
non-specific adsorption of proteins such as insulin, lysozyme,
albumin, and hexokinase can be readily prepared on metal oxide
surfaces using a CH.sub.3 -capped oligo-(ethylene
glycol)-terminated silane reagent,
CH.sub.3[OCH.sub.2CH.sub.2].sub.2,3O(CH.sub.2).sub.11SiCl.sub.3.
These compounds are synthesized by a straightforward two-step
reaction sequence using commercially available precursors.
Solution-phase contact between a metal oxide surface and the silane
reagent results in the spontaneous formation of a densely packed,
oriented siloxane coating that expresses the oligo(ethylene glycol)
groups at its surface. These moderately hydrophilic surfaces
exhibit superior protein resistant properties than the more
hydrophobic surfaces prepared from the adsorption of
octadecyltrichlorosilane onto glass. The oligo-(ethylene
glycol)-terminated films maintain their integrity and protein
resistant properties after exposure to temperatures of
.about.100.degree. C. (including sterilization procedures in an
autoclave), suggesting that the parent reagents could be suitable
for producing coatings on various glassware and another implements
that contact protein-containing media and may be exposed to the
conditions used in sterilization procedures.
EXAMPLE 2
Preparation of Biotin-bearing SAMs and Measurements Thereon
Materials
[0171] Reagents were obtained from Aldrich and used as received
unless specified otherwise. Octadecyltrichlorosilane was distilled
under reduced pressure before use. 10-Undecylenic-1-bromide and
5-(biotinamido)pentylam- ine was obtained from Pfaltz and Bauer
(Waterbury, Conn.) and PIERCE (Rockford, Ill.), respectively.
Silicon wafers were test grade and obtained from Silicon Sense
(Nashua, N.H.). The methyl-capped tri(ethylene glycol)-terminated
undecyltrichlorosilane
(CH.sub.3O(CH.sub.2CH.sub.2O).sub.3(CH.sub.2).sub.11SiCl.sub.3) was
available from the previous study. The acetate-capped tri(ethylene
glycol)-terminated undecyltrichlorosilane was synthesized by the
procedure outlined in Scheme I. We protected the hydroxyl group as
acetate to prevent reaction of free hydroxyl groups with
chlorosilane groups. .sup.1H NMR spectra were obtained on a Bruker
250 MHz spectrometer in CDCl.sub.3 and referenced to residual
CHCl.sub.3 at 7.24 ppm.
Synthesis of Acetyl [(1-trichlorosilyl)undec-11-yl] tri(ethylene
glycol)
[0172] The acetyl (undec-10-1-yl) tri(ethylene glycol) precursor
[1,
CH.sub.2.dbd.CH(CH.sub.2).sub.9(OCH.sub.2CH.sub.2).sub.3OCOCH.sub.3]
was prepared by reported procedures.sup.1,2 and hydrosilated to add
a trichlorosilyl group..sup.3 The NMR spectrum of the reaction
mixture showed the quantitative conversion of the olefin to the
trichlorosilane by UV. The excess HSiCl.sub.3 was removed by vacuum
distillation. Acetyl [(1-trichlorosilyl)undec-11-yl] tri(ethylene
glycol): .sup.1H NMR (250 MHz, CDCl.sub.3, .delta.): 1.2-1.5 (m, 16
H), 1.55 (m, 4 H), 2.06 (s, 3 H), 3.43 (t, 2 H), 3.5-3.75 (m, 10
H), 4.20 (t, 2H). (1) Pale-Grosdemange, C.; Simon, E. S.; Prime, K.
L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12-20. (2)
Wenzler, L. A.; Moyes, G. L.; Raikar, G. N.; Hansen, R. L.; Harris,
J. M.; Beebe Jr., T. P. Langmuir 1997, 13, 3761-3768. (3)
Balachander, N.; Sukenik, C. Langmuir 1990, 6, 1621-1627.
Preparation of SiO.sub.2 Substrates
[0173] Si(100) test wafers and glass slides were cut into strips of
.about.1.times.3 cm.sup.2 that were subsequently cleaned by
immersion in freshly prepared "piranha" solution of 70% conc.
H.sub.2SO.sub.4(aq)/30% H.sub.2O.sub.2(aq) (v/v) for 0.5 to 1 h at
70.degree. C. (CAUTION: "piranha" solution reacts violently with
many organic materials and should be handled with care). The
substrates were immediately rinsed with distilled water, dried in a
stream of N.sub.2, and used within 1 h of cleaning. This process
produces a highly wettable, hydrated oxide surface on silicon with
similar properties to that for glass..sup.4 Optical constants were
measured on the bare substrates by ellipsometry for use in
determining thicknesses subsequently of the adsorbed silane films
and protein layers. The piranha-cleaned substrates were typically
exposed to the air for no more than 15 min before immersion in the
silane solution. (4) Pintchovski, F.; Price, J. B.; Tobin, P. J.;
Peavey, J.; Kobold, K. J. Electrochem. Soc. 1979, 126,
1428-1430.
Formation of Siloxane Films on SiO.sub.2
[0174] The piranha-cleaned silicon substrates were functionalized
by immersion in a .about.2 mM solution of the trichlorosilane in
anhydrous toluene. The solutions were prepared and kept in a dry
nitrogen atmosphere (glove box). After 6 to 24 h, the substrates
were removed from solution and rinsed in 20 mL of CH.sub.2Cl.sub.2.
The substrates were removed from the glove box, rinsed sequentially
with CHCl.sub.3 and ethanol to remove any residual organic
contaminants, and dried in a stream of N.sub.2.
Covalent Attachment of Biotin onto SAMs
[0175] The silanated substrates were sonicated in 0.1 mM
LiAlH.sub.4 in anhydrous diethyl ether for 10 mins to convert
terminal acetate groups to hydroxyl groups. The substrates were
then washed sequentially in .about.4% HCl, chloroform, acetone, and
deionized water and dried under a stream of N.sub.2. The substrates
were then tresylated by immersion to a 1.25 mg/mL tresyl chloride
solution in CH.sub.2Cl.sub.2 for 1 hr at room temperature, rinsed
with anhydrous methanol, and dried under a stream of N.sub.2..sup.5
The tresylated samples were then immediately transferred to a 1
mg/mL 5-(biotinamido)pentylamine solution in phosphate buffer
saline (PBS) solution (10 mM phosphate buffer, 2.7 mM KCl, and 137
mM NaCl) that was adjusted to pH 8.0..sup.5 (5) Liu, S. Q.; Liu, L.
S.; Ohno, T. Cytotechnology 1997, 26, 13-21.
Contact Angle Measurements
[0176] Contact angles were measured on a Ram-Hart goniometer
(Ram-Hart Inc., Mountain Lakes, N.J.) equipped with a video-imaging
system. Drops were placed on at least three locations on the
surface in the ambient environment and measured on both sides of
the drops. Contacting liquid drops were advanced and retreated with
an Electrapipette (Matrix Technologies, Lowell, Mass.) at
approximately 1 .mu.L/s. Angles were measured to
.about..+-.1.degree. and were reproducible from sample to sample
within .+-.2.degree..
Ellipsometric Film-Thickness Measurements
[0177] The thicknesses of the films were determined with a Gaertner
L116A ellipsometer (Gaertner Scientific Corporation, Chicago,
Ill.). For each substrate, measurements were made before and after
derivatization with the trichlorosilanes, and after protein
adsorption. The thicknesses of the films were determined using a
three-phase model and a refractive index of 1.45 and have an error
of .+-.2 .ANG.. The use of this value allows direct comparison with
data obtained by other groups and provides an accurate relative
measure of the amounts of materials adsorbed on the various
coatings.
X-ray Photoelectron Spectroscopy (XPS)
[0178] The XPS spectra were obtained with a surface Science
Instrument Model X-100 spectrometer using a monochromatized Al
K.alpha. x-ray source (elliptical spot of 1.0 mm.times.1.7 mm) and
a concentric hemispherical analyzer. The detector angle with
respect to the surface parallel was 35.degree.. The step width and
pass energy were set at 0.1 and 23 eV, respectively, giving an
experimental resolution of .about.1 eV. Peak positions were
referenced to C(1 s)=284.6 eV, and peaks were fit with 80%
Gaussian/20% Lorentzian profiles and a Shirley background.
[0179] All of the patents and publications cited herein are hereby
incorporated by reference.
Equivalents
[0180] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *