U.S. patent application number 10/972781 was filed with the patent office on 2005-06-02 for functionalized porous materials and applications in medical devices.
This patent application is currently assigned to Porex Technologies Corp.. Invention is credited to Greene, George Warren IV, Li, Xingguo, Mao, Guoqiang, Yao, Li.
Application Number | 20050118407 10/972781 |
Document ID | / |
Family ID | 25348541 |
Filed Date | 2005-06-02 |
United States Patent
Application |
20050118407 |
Kind Code |
A1 |
Yao, Li ; et al. |
June 2, 2005 |
Functionalized porous materials and applications in medical
devices
Abstract
The invention relates to porous polymeric materials, methods of
making them, and applications in medical devices. A specific
embodiment of the invention encompasses a material comprising a
porous polyolefin substrate containing inclusions of a material to
which chemical or biological moieties are attached directly or via
a spacer.
Inventors: |
Yao, Li; (Peachtree City,
GA) ; Greene, George Warren IV; (Peachtree City,
GA) ; Mao, Guoqiang; (Smyrna, GA) ; Li,
Xingguo; (Peachtree City, GA) |
Correspondence
Address: |
JONES DAY
51 Louisiana Avenue, N.W.
Washington
DC
20001
US
|
Assignee: |
Porex Technologies Corp.
|
Family ID: |
25348541 |
Appl. No.: |
10/972781 |
Filed: |
October 26, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10972781 |
Oct 26, 2004 |
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09866842 |
May 30, 2001 |
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6808908 |
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Current U.S.
Class: |
428/304.4 ;
428/317.9 |
Current CPC
Class: |
Y10T 428/249953
20150401; Y10T 428/31855 20150401; A61L 27/56 20130101; Y10T
428/249986 20150401; Y10S 530/811 20130101; A61L 27/446 20130101;
A61L 31/128 20130101; Y10S 530/816 20130101; A61L 31/146 20130101;
Y10S 530/815 20130101 |
Class at
Publication: |
428/304.4 ;
428/317.9 |
International
Class: |
B32B 003/26; B32B
005/22 |
Claims
What is claimed is:
1. A material comprising: a porous substrate comprised of a polymer
and a functional additive and having a surface, wherein the surface
comprises a region defined by at least some of the functional
additive; and a biological or chemical moiety covalently or
non-covalently bound to the region.
2. The material of claim 1, wherein the surface comprises a
plurality of regions defined by at least some of the functional
additive, each of which is covalently or non-covalently bound to a
chemical or biological moiety.
3. A material comprising: a porous substrate comprised of a polymer
and a functional additive and having a surface, wherein the surface
comprises a region defined by at least some of the functional
additive; a spacer covalently bound to the region; and a biological
or chemical moiety covalently or non-covalently bound to the
spacer.
4. The material of claim 3, wherein the surface comprises a
plurality of regions defined by at least some of the functional
additive, each of which is covalently bound to a spacer, which in
turn is covalently or non-covalently bound to a biological
moiety.
5. The material of claim 1 or 3, wherein the polymer is a
polyolefin, polyether, nylon, polycarbonate, poly(ether sulfone),
or a mixture thereof.
6. The material of claim 5, wherein the polyolefin is ethylene
vinyl acetate; ethylene methyl acrylate; polyethylenes;
polypropylenes; ethylene-propylene rubbers;
ethylene-propylene-diene rubbers; poly(1-butene); polystyrene;
poly(2-butene); poly(1-pentene); poly(2-pentene);
poly(3-methyl-1-pentene); poly(4-methyl-1-pentene);
1,2-poly-1,3-butadiene; 1,4-poly-1,3-butadiene; polyisoprene;
polychloroprene; poly(vinyl acetate); poly(vinylidene chloride);
poly(tetrafluoroethylene) (PTFE); poly(vinylidene fluoride) (PVDF);
acrylonitrile-butadiene-styrene (ABS); or a mixture thereof.
7. The material of claim 5, wherein the polyolefin is polyethylene
or polypropylene.
8. The material of claim 5, wherein the polyether is polyether
ether ketone (PEEK,
poly(oxy-1,4-phenylene-oxy-1,4-phenylene-carbonyl-1,4-pheny-
lene)), polyether sulfone (PES), or a mixture thereof.
9. The material of claim 1 or 3, wherein the functional additive
comprises a hydroxyl, carboxylic acid, anhydride, acyl halide,
alkyl halide, aldehyde, alkene, amide, amine, guanidine, malimide,
thiol, sulfonate, sulfonic acid, sulfonyl ester, carbodiimide,
ester, cyano, epoxide, proline, disulfide, imidazole, imide, imine,
isocyanate, isothiocyanate, nitro, or azide functional group.
10. The material of claim 9, wherein the functional additive
comprises a hydroxyl, amine, aldehyde, or carboxylic acid
functional group.
11. The material of claim 10, wherein the functional additive
comprises a hydroxyl functional group.
12. The material of claim 1 or 3, wherein the functional additive
is silica powder, silica gel, chopped glass fiber, controlled
porous glass (CPG), glass beads, ground glass fiber, glass bubbles,
kaolin, alumina oxide, or a mixture thereof.
13. The material of claim 3, wherein the spacer is a silane,
functionalized silane, diamine, alcohol, ester, glycol, anhydride,
dialdehyde, terminal difunctionalized polyurethane, dione,
macromer, or a multifunctional polymer.
14. The material of claim 1, wherein the spacer to which the porous
substrate and biological or chemical moiety is attached is of
Formula I: 10wherein the substrate is bound to the oxygen atom and
the chemical or biological moiety is bound to R.sup.3; R and R'
each independently is hydrogen, substituted or unsubstituted alkyl,
aryl, or aralkyl; R.sup.3 is a substituted or unsubstituted
aliphatic chain or a bond; and n is an integer from about 1 to
about 18.
15. The material of claim 1 or 3, wherein the chemical or
biological moiety is a drug, hydrophilic moiety, catalyst,
antibiotic, antibody, antimycotic, carbohydrate, cytokine, enzyme,
glycoprotein, lipid, nucleic acid, nucleotide, oligonucleotide,
peptide, protein, ligand, cell, ribozyme, or a combination
thereof.
16. A material comprising a porous polyethylene substrate having a
surface in which a functional additive has been embedded, and a
spacer precursor of Formula II covalently attached to at least a
portion of said functional additive: 11wherein R.sup.1, R.sup.2 and
R.sup.4 each independently is hydrogen, substituted or
unsubstituted alkyl, aryl, or aralkyl; R.sup.3 is a substituted or
unsubstituted aliphatic chain or a bond; n is an integer from about
1 to about 18; and X is OH, NH.sub.2, CHO, CO.sub.2H, NCO or
epoxy.
17. A material comprising a porous polyethylene substrate having a
surface in which a functional additive has been embedded, and a
spacer of Formula I: 12wherein R and R' each independently is
hydrogen, substituted or unsubstituted alkyl, aryl, or aralkyl;
R.sup.3 is a substituted or unsubstituted aliphatic chain or a
bond; and n is an integer from about 1 to about 18, covalently
attached to at least a portion of said functional additive and to a
chemical or biological moiety.
18. The material of claim 17, wherein the chemical or biological
moiety is a nucleotide, oligonucleotide, polynucleotide, peptide,
cell, ligand, or protein.
19. A method of providing a material which comprises: forming a
porous substrate comprised of a polymer and a functional additive
and having a surface, wherein the surface comprises a region
defined by at least some of the functional additive, wherein the
region contains at least one functional group; contacting the
functional group with a spacer under reaction conditions suitable
for the formation of a covalent bond between an atom of the
functional group and an atom of the spacer; and contacting the
spacer with a chemical or biological moiety under reaction
conditions suitable for the formation of a covalent or non-covalent
bond between an atom of the spacer and an atom of the chemical or
biological moiety.
20. The method of claim 19, wherein the functional group is
hydroxyl, carboxylic acid, anhydride, acyl halide, alkyl halide,
aldehyde, alkene, amide, amine, guanidine, malimide, thiol,
sulfonate, sulfonic acid, sulfonyl ester, carbodiimide, ester,
cyano, epoxide, proline, disulfide, imidazole, imide, imine,
isocyanate, isothiocyanate, nitro, or azide.
21. The method of claim 19, wherein the porous substrate is formed
by sintering beads and then attaching the spacer, or attaching the
spacer to beads prior to sintering the beads.
22. A method of providing a material which comprises: forming a
porous substrate comprised of a polymer and a functional additive
and having a surface, wherein the surface comprises a region
defined by at least some of the functional additive, wherein the
region contains at least one hydroxyl group; and contacting the
hydroxyl group with a compound of Formula II: 13wherein each of
R.sup.1, R.sup.2, and R.sup.4 each independently is hydrogen,
substituted or unsubstituted alkyl, aryl, or aralkyl; R.sup.3 is a
substituted or unsubstituted aliphatic chain or a bond; n is an
integer from about 1 to about 18; and X is OH, NH.sub.2, CHO,
CO.sub.2H, NCO, or epoxy under conditions suitable for the
formation of a material of Formula m: 14wherein Surface is the
surface of the porous substrate.
23. The method of claim 22 wherein the material of Formula III is
contacted with a chemical or biological moiety having an amine
group if X is an aldehyde or carboxylic acid, or a chemical or
biological moiety having an aldehyde of carboxylic acid group if X
is an amine, under reaction conditions suitable for the formation
of a material of Formula V: 15wherein Moiety is the chemical or
biological moiety.
24. The method of claim 22 wherein the material of Formula III, X
is NH.sub.2 and is contacted with a compound of Formula V:
Z-Spacer-Z Formula V wherein Spacer is a hydrophilic segment and Z
is a terminal group capable of covalently or non-covalently bonding
to proteins, amino acids, oligonucleotides, under reaction
conditions suitable for the formation of a material of Formula VI:
16wherein R is the surface of the porous substrate.
25. The method of claim 24, wherein Spacer is a hydrophilic
polyurethane, polyethylene glycol, or polyelectrolyte and wherein Z
is isocyanurate, aldehyde, amino, carboxylic acid, or
N-hydroxysuccimide.
26. A method of controlling the functionalization of a sintered
polyolefin substrate which comprises: forming a mixture of
polyolefin particles and particles of a functional additive; and
sintering the mixture; wherein the functional additive comprises
functional groups and the concentration of functional additive in
the mixture is approximately proportional to the density of
functional groups on a surface of the sintered polyolefin
substrate.
27. The method of claim 26, wherein the functional additive is
silica powder, silica gel, chopped glass fiber, controlled porous
glass (CPG), glass beads, ground glass fiber, glass bubbles,
kaolin, alumina oxide, or a mixture thereof.
28. The material of claim 13 wherein the spacer to which the porous
substrate and biological or chemical moiety is attached is of
Formula VIII: 17wherein R is the surface of the porous substrate;
R.sup.1 and R.sup.2 each independently is hydrogen, substituted or
unsubstituted alkyl, aryl, or aralkyl; X is OH, NH.sub.2, CHO,
CO.sub.2H. NCO, or epoxy; n is an integer from about 1 to about 5;
m is an integer from about 2 to about 5; and o is an integer from 0
to about 3.
29. The material of claim 25 wherein X is CHO or NH.sub.2.
30. The material of claim 13 wherein the spacer to which the porous
substrate and biological or chemical moiety is attached is of
Formula IX: 18wherein R is the surface of the porous substrate;
R.sup.1 and R.sup.2 each independently is hydrogen, substituted or
unsubstituted alkyl, aryl, or aralkyl; and n is from about 1 to
about 18.
Description
1. FIELD OF THE INVENTION
[0001] The invention relates to porous polymeric materials to which
chemical or biological moieties have been attached, and methods for
making the same.
2. BACKGROUND OF THE INVENTION
[0002] Porous polymeric materials can be used in a variety of
applications. Their uses include medical devices that serve as
substitute blood vessels, synthetic and intraocular lenses,
electrodes, catheters, and extracorporeal devices such as those
that are connected to the body to assist in surgery or dialysis.
Porous polymeric materials can also be used as filters for the
separation of blood into component blood cells and plasma,
microfilters for removal of microorganisms from blood, and coatings
for opthalmic lenses to prevent endothelial damage upon
implantation.
[0003] Bonding materials other than polymers to porous polymeric
materials can alter the properties of porous polymers. A
combination of properties may provide porous polymers suitable for
the above mentioned purposes. This combination, however, has been
difficult to achieve because of the natural properties of
polymers.
[0004] The hydrophobic nature of typical polymers, however, has
limited the usefulness of porous materials made from them. For
example, proteins will often denature when placed in contact with
such materials. But contact lenses, implants, and related devices
that are in intimate contact with the body must have hydrophilic
surfaces that are biologically compatible.
[0005] The physical and/or chemical properties of a plastic surface
can be changed by adhering or bonding a different material to it.
Examples of this technique are disclosed by U.S. Pat. No.
4,619,897, which is directed to a porous resin membrane, and by
U.S. Pat. No. 4,973,493, which is directed to a device with a
biocompatible surface. Other examples of surface modification are
provided by U.S. Pat. Nos. 5,077,215, 5,183,545, and 5,203,997,
which disclose the adsorption of anionic and nonionic fluorocarbon
surfactants onto the surface of fluorocarbon support members.
[0006] Further examples of surface modification can be found in
U.S. Pat. No. 5,263,992, which discloses the adsorption of
polymeric chains onto a support member, and in U.S. Pat. No.
5,308,641, which discloses the covalent attachment of a
polyalkylimine to an aminated substrate.
[0007] Despite the different techniques available for modifying the
surface of polymeric materials, most are limited in their ability
to control the degree to which a surface is modified, and many are
expensive, inefficient, or cannot be use to modify porous surfaces
without coating or clogging their pores. A need exists for
polymeric materials that can alter their functionality depending
upon incorporation of additives and/or post treatment of these
additives. The present invention provides new porous polymeric
materials and methods of their manufacture that address this
need.
3. SUMMARY OF THE INVENTION
[0008] This invention encompasses novel porous materials and
methods of their manufacture. Materials of the invention comprise a
porous substrate to which chemical or biological moieties are bound
directly or by a spacer.
[0009] A first embodiment of the invention encompasses a material
comprising: a porous substrate comprised of a polymer and a
functional additive and having a surface wherein the surface
comprises a region defined by at least some of the functional
additive; and a biological or chemical moiety covalently or
non-covalently bound to the region. In a preferred material
encompassed by this embodiment, the surface comprises a plurality
of regions defined by at least some of the functional additive,
each of which is covalently bound to a chemical or biological
moiety.
[0010] A second embodiment of the invention encompasses a material
comprising: a porous substrate comprised of a polymer and a
functional additive and having a surface, wherein the surface
comprises a region defined by at least some of the functional
additive; a spacer covalently or non-covalently bound to the
region; and a biological or chemical moiety covalently or
non-covalently bound to the spacer. In a preferred material
encompassed by this embodiment, the surface comprises a plurality
of regions defined by at least some of the functional additive,
each of which is covalently bound a spacer, which in turn is bound
to a biological moiety.
[0011] Examples of polymers from which materials of the invention
can be made include, but are not limited to, polyolefins,
polyethers, nylons, polycarbonates, poly(ether sulfones), or
mixtures thereof. Polyethers include, but are not limited to,
polyether ether ketone (PEEK,
poly(oxy-1,4-phenylene-oxy-1,4-phenylene-carbonyl-1,4-phenylene)),
polyether sulfone (PES), or mixtures thereof. Polyolefins include,
but are not limited to, ethylene vinyl acetate; ethylene methyl
acrylate; polyethylenes; polypropylenes; ethylene-propylene
rubbers; ethylene-propylene-diene rubbers; poly(1-butene);
polystyrene; poly(2-butene); poly(1-pentene); poly(2-pentene);
poly(3-methyl-1-pentene- ); poly(4-methyl-1-pentene);
1,2-poly-1,3-butadiene; 1,4-poly-1,3-butadiene; polyisoprene;
polychloroprene; poly(vinyl acetate); poly(vinylidene chloride);
poly(tetrafluoroethylene) (PTFE); poly(vinylidene fluoride) (PVDF);
acrylonitrile-butadiene-styrene (ABS); or mixtures thereof.
Preferred polyolefins are polyethylene or polypropylene.
[0012] Functional additives are materials that contain functional
groups such as, but not limited to, hydroxyl, carboxylic acid,
anhydride, acyl halide, alkyl halide, aldehyde, alkene, amide,
amine, guanidine, malimide, thiol, sulfonate, sulfonic acid,
sulfonyl ester, carbodiimide, ester, cyano, epoxide, proline,
disulfide, imidazole, imide, imine, isocyanate, isothiocyanate,
nitro, or azide. Preferred functional groups are hydroxyl, amine,
aldehyde, or carboxylic acid. A particularly preferred functional
group is hydroxyl. Examples of functional additives include, but
are not limited to, silica powder, silica gel, chopped glass fiber,
controlled porous glass (CPG), glass beads, ground glass fiber,
glass bubbles, kaolin, alumina oxide, or other inorganic
oxides.
[0013] Examples of spacers useful in the second embodiment of the
invention include, but are not limited to, silanes, functionalized
silanes (functional groups such as aldehyde, amino, epoxy, halides,
etc.), diamines, alcohols, esters, glycols (such as polyethylene
glycol), anhydrides, dialdehydes, terminal difunctionalized
polyurethanes, diones, macromer, difunctional and multifunctional
polymers with end groups, including, but not limited to, amino,
carboxylic acid, ester, aldehyde, or mixtures thereof. In a
preferred material, the spacer to which the porous substrate and
biological or chemical moiety is attached is of Formula I: 1
[0014] wherein the bond broken by a wavy line are those between the
spacer and the substrate or other moieties; R.sup.1 and R.sup.2
each independently is hydrogen, substituted or unsubstituted alkyl,
aryl, or aralkyl; R.sup.3 is a substituted or unsubstituted
aliphatic chain or a bond; and n is an integer from about 1 to
about 18, preferably, n is an integer from about 1 to about 10, and
more preferably from about 2 to about 5.
[0015] A variety of chemical and biological moieties can be
attached to the porous substrate or spacer of materials of the
invention. Examples include, but are not limited to, drugs (e.g.,
pharmaceuticals), hydrophilic moieties, catalysts, antibiotics,
antibodies, antimycotics, carbohydrates, cytokines, enzymes,
glycoproteins, lipids, nucleic acids, nucleotides,
oligonucleotides, polynucleotides, proteins, peptides, ligand,
cells, ribozymes, or combinations thereof.
[0016] A specific material of the invention comprises a porous
polyethylene substrate having a surface in which a functional
additive has been embedded, and a spacer precursor of Formula II
covalently attached to at least a portion of said functional
additive: 2
[0017] wherein R.sup.1, R.sup.2, and R.sup.4 each independently is
hydrogen, substituted or unsubstituted alkyl, aryl, or aralkyl;
R.sup.3 is a substituted or unsubstituted aliphatic chain or a
bond; X is a group capable of bonding to a biological or chemical
moiety, such as OH, NH.sub.2, CHO, CO.sub.2H, NCO, epoxy, and the
like, preferably, X is NH.sub.2 or CHO; and n is an integer from
about 1 to about 18, preferably, n is an integer from about 1 to
about 10, and more preferably from about 2 to about 5.
[0018] Still another specific material of the invention comprises a
porous polyethylene substrate having a surface in which a
functional additive has been embedded, and a spacer of Formula I
covalently attached to at least a portion of said functional
additive and to a chemical or biological moiety. Preferably, the
chemical or biological moiety is a nucleotide, oligonucleotide,
polynucleotide, peptide, cell, ligand, or protein.
[0019] A third embodiment of the invention encompasses a method of
providing a material which comprises: forming a porous substrate
comprised of a polymer and a functional additive and having a
surface, wherein the surface comprises a region defined by at least
some of the functional additive, wherein the region contains at
least one functional group; contacting the functional group with a
spacer under reaction conditions suitable for the formation of a
covalent bond between an atom of the functional group arid an atom
of the spacer; and contacting the spacer with a chemical or
biological moiety under reaction conditions suitable for the
formation of a covalent bond or non-covalent bond between an atom
of the spacer and an atom of the chemical or biological moiety.
[0020] In a preferred method, the functional group is hydroxyl,
carboxylic acid, anhydride, acyl halide, alkyl halide, aldehyde,
alkene, amide, amine, guanidine, malimide, thiol, sulfonate,
sulfonyl halide, sulfonyl ester, carbodiimide, ester, cyano,
epoxide, proline, disulfide, imidazole, imide, imine, isocyanate,
isothiocyanate, nitro, or azide. Preferred functional groups are
hydroxyl, amine, aldehyde, and carboxylic acid.
[0021] A fourth embodiment of the invention encompasses a method of
providing a material which comprises: forming a porous substrate
comprised of a polymer and a functional additive and having a
surface, wherein the surface comprises a region defined by at least
some of the functional additive, wherein the region contains at
least one hydroxyl group; and contacting the hydroxyl group with a
compound of Formula II: 3
[0022] wherein R.sup.1, R.sup.2, R.sup.4, X, and n are as defined
above, under conditions suitable for the formation of a material of
Formula III: 4
[0023] wherein R.sup.1, R.sup.2, X, and n are as defined above for
Formula II; and Surface is the surface of the substrate.
[0024] The porous substrate may be formed by at least two methods.
In one method, beads are sintered together with other polymer beads
prior to attaching compounds of Formula II or IV. In another
method, compounds of Formula II or IV are attached to the surface
of beads prior to sintering the beads to form the porous
substrate.
[0025] In a specific method of this embodiment, the material of
Formula III is contacted with a chemical or biological moiety
having an amine group if X is an aldehyde or carboxylic acid, or a
chemical or biological moiety having an aldehyde or carboxylic acid
group if X is an amine, under reaction conditions suitable for the
formation of a material of Formula IV: 5
[0026] wherein R.sup.1, R.sup.2, R.sup.3, and n are defined as
above for Formula II; R is the porous substrate surface and Moiety
is a chemical or biological moiety.
[0027] In another specific method of this embodiment, the material
of Formula IV wherein X is NH.sub.2 is contacted with a compound of
Formula V:
Z-Spacer-Z Formula V
[0028] wherein Spacer is a hydrophilic segment and Z is a terminal
group capable of covalently or non-covalently bonding to proteins,
amino acids, oligonucleotides, and the like, under reaction
conditions suitable for the formation of a material of Formula VI:
6
[0029] wherein R is the surface of the porous substrate; and
R.sup.1, R.sup.2, and n are defined as above for Formula II.
[0030] Preferably, the Spacer is a hydrophilic polyurethane,
polyethylene glycol, or polyelectrolytes with terminal groups (Z)
capable of bonding to proteins, amino acids, oligonucleotides
wherein Z includes, but is not limited to, isocyanurate, aldehydes,
amines, carboxylic acids, N-hydroxysuccimide, and the like.
[0031] A sixth embodiment of the invention encompasses a method of
controlling the functionalization of a sintered polyolefin
substrate which comprises: forming a mixture of polyolefin
particles and particles of a functional additive; and sintering the
mixture; wherein the functional additive comprises functional
groups and the concentration of functional additive in the mixture
is approximately proportional to the density of functional groups
on a surface of the sintered polyolefin substrate. In a preferred
embodiment, the functional additive is silica powder, silica gel,
chopped glass fiber, controlled porous glass (CPG), glass beads,
ground glass fiber, glass bubbles, kaolin, alumina oxide, or other
inorganic oxides.
3.1. Definitions
[0032] As used herein and unless otherwise indicated, the term
"alkyl" includes saturated mono- or di-valent hydrocarbon radicals
having straight, cyclic or branched moieties, or a combination of
the foregoing moieties. An alkyl group can include one or two
double or triple bonds. It is understood that cyclic alkyl groups
comprise at least three carbon atoms.
[0033] As used herein and unless otherwise indicated, the term
"aralkyl" includes an aryl substituted with an alkyl group or an
alkyl substituted with an aryl group. An example of aralkyl is the
moiety --(CH.sub.2).sub.pAr, wherein p is an integer of from 1 to
about 4, 8, or 10.
[0034] As used herein and unless otherwise indicated, the term
"aryl" includes an organic radical derived from an aromatic
hydrocarbon by removal of one hydrogen, such as phenyl or
naphthyl.
[0035] As used herein and unless otherwise indicated, the term
"halo" means fluoro, chloro, bromo, or iodo. Preferred halo groups
are fluoro, chloro, or bromo.
[0036] As used herein and unless otherwise indicated, the term
"non-covalent" when used to describe a bond, means a bond formed by
ionic interactions, Van der Waals interactions, hydrogen bonding
interactions, steric interactions, hydrophilic interactions, or
hydrophobic interactions between two atoms or molecules.
[0037] As used herein and unless otherwise indicated, the term
"substituted," when used to describe a chemical moiety, means that
one or more hydrogen atoms of that moiety are replaced with a
substituent. Examples of substituents include, but are not limited
to, alkyl and halo.
[0038] As used herein and unless otherwise indicated, the term
"heteroaryl" means an aryl group wherein at least one carbon atom
has been replaced with an O, S, P, Si, or N atom.
[0039] As used herein and unless otherwise indicated, the terms
"heterocyclic group" and "heterocycle" include aromatic and
non-aromatic heterocyclic groups containing one or more heteroatoms
each selected from O, S, P, Si, or N. Non-aromatic heterocyclic
groups must have at least 3 atoms in their ring system, but
aromatic heterocyclic groups (i.e., heteroaryl groups) must have at
least 5 atoms in their ring system. Heterocyclic groups include
benzo-fused ring systems and ring systems substituted with one or
more oxo moieties. An example of a 3 membered heterocyclic group is
epoxide, and an example of a 4 membered heterocyclic group is
azetidinyl (derived from azetidine). An example of a 5 membered
heterocyclic group is thiazolyl, and an example of a 10 membered
heterocyclic group is quinolinyl. Examples of non-aromatic
heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl,
tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl,
piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl,
azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl,
thiepanyl, oxazepinyl, diazepinyl, thiazepinyl,
1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl,
2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl,
dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,
dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,
3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl,
3H-indolyl, and quinolizinyl. Examples of aromatic heterocyclic
groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, tiazolyl,
pyrazinyl, tetrazolyl, furyl, thienyl; isoxazolyl, thiazolyl,
oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl,
indolyl, benzimidazolyl, benzofuranyl, cinolinyl, indazolyl,
indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl,
pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl,
benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl,
quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The
foregoing groups, as derived from the compounds listed above, may
be C-attached or N-attached where such attachment is possible. For
instance, a group derived from pyrrole can be pyrrol-1-yl
(N-attached) or pyrrol-3-yl (C-attached).
[0040] As used herein, unless otherwise indicated, the term
"polyelectrolyte" means a polymer having electronic charges. The
polyelectrolyte may exist in a complex form, which is also called
symplexes. Polyelectrolytes are divided into polyacids, polybases,
and polyampholytes. Depending on the charge density in the chain,
polyelectrolytes are divided into weak and strong. The charge of
weak polyelectrolytes is determined by dissociation constants of
ionic groups and pH of the solution. Strong polyelectrolytes in
water solutions are mostly ionized independent of the solution's
pH. Typical weak polyacid polyelectrolytes include, but are not
limited to, poly(acrylic acid) and poly(methacrylic acid). Strong
polyacid polyelectrolytes include, but are not limited to,
poly(ethylenesulfonic acid), poly(styrenesulfoinic acid), and
poly(phosphoric acid). Weak polybase polyelectrolytes include, but
are not limited to, poly(4-vinylpyridine), polyethyleneimine (PEI),
and polyvinylamine. Strong polybase polyelectrolytes can be
obtained by alkylation of nitrogen, sulfur, or phosphorus atoms of
weak polybase polyelectrolytes. See "Concise Polymeric Materials
Encyclopedia" (Joseph C. Salamone, 1999 by CRC Press LLC, ISBN
0-84932-226-X, pages 1140-1141).
3.2. FIGURES
[0041] Various aspects of the invention are understood with
reference to the following figures:
[0042] FIG. 1 represents a side view of a material of the
inventions wherein regions of a functional additive are embedded
within a porous plastic substrate;
[0043] FIG. 2 provides a representation of the attachment of
spacers of varying lengths to a porous substrate, followed by the
attachment of chemical or biological moieties to those spacers;
[0044] FIG. 3 represents a material of the invention which
comprises a spacer covalently bound to a porous substrate, but
bound to a chemical or biological moiety by a non-covalent
bond;
[0045] FIG. 4 provides a synthetic scheme whereby a chemical or
biological moiety can be covalently attached to a porous substrate
of the invention via an amine-terminated silane spacer;
[0046] FIG. 5 provides a synthetic scheme whereby a chemical or
biological moiety can be covalently attached to a porous substrate
of the invention via an aldehyde-terminated silane spacer; and
[0047] FIG. 6 provides a synthetic scheme which allows the
lengthening of a spacer connecting a porous substrate and a
chemical or biological moiety.
4. DETAILED DESCRIPTION OF THE INVENTION
[0048] Many plastics do not contain reactive functional groups
(e.g., hydroxyl or amine groups) that can be used to attach
chemical or biological moieties to surfaces of materials made from
them. The surfaces of such materials can therefore be difficult to
modify. This invention is based on a discovery that the surface
properties of porous plastic materials can be altered in a
controllable fashion by incorporating varying amounts of functional
additives into those materials. Functional additives are materials
that contain functional groups to which biological and/or chemical
moieties can be covalently attached.
[0049] FIG. 1 depicts various aspects of materials of the
invention. In particular, it provides a representation of a porous
substrate made of a plastic and a functional additive. Portions of
the functional additive that are exposed on the surface of the
substrate provide functional groups (represented in FIG. 1 as "Fn")
to which chemical or biological moieties (represented by "CBM") can
be covalently bound. Alternatively, a spacer can connect a chemical
or biological moiety to the surface. Preferably, the plastic that
surrounds the regions of functional additives contains few, if any,
functional groups. Alternatively, the plastic may contain
functional groups that are less reactive under certain reaction
conditions than the functional groups of the functional additive,
such that chemical or biological moieties can be bound primarily to
exclusively to regions of functional additive.
[0050] As discussed elsewhere herein, the polymer (e.g., plastic)
from which the porous substrate is made is selected to provide a
substrate of desired strength, flexibility, porosity, and
resistance to degradation. Other factors, such as cost and
biocompatibility may also play a role in its selection. The
chemical or biological moiety(ies) bound to the substrate are
selected to provide a final material with desired chemical and/or
physical properties, while the type and amount of functional
additive(s) are selected to affect the number and/or density of
chemical or biological moieties bound to the substrate. For
example, the chemical reactivity of functional groups provided by
the functional additive can affect the number of moieties attached
to the substrate surface.
[0051] Examples of chemical and/or physical properties conveyed by
the covalent bonding of chemical or biological moieties to the
surface of a porous substrate include, but are not limited to,
increased hydrophilicity and biocompatibility. Indeed, the surfaces
of preferred materials of the invention are hydrophilic, and can
readily accept high surface tension fluids. Examples of high
surface tension fluids include, but are not limited to, water, and
aqueous salt and protein solutions. Hydrophilic substrates
typically have a surface energy of greater than about 40
dyn/cm.sup.2, more typically greater than about 60
dyn/cm.sup.2.
[0052] Materials of the invention can be used in a variety of
applications. For example, biocompatible materials can be used to
provide temporary or permanent implants such as, but not limited
to, soft or hard tissue prosthesis, artificial organs or organ
components, or lenses for the eye such as contact or intraocular
lenses. Biocompatible materials of the invention reduce or avoid
undesirable reactions such as, but not limited to, blood clotting,
tissue death, tumor formation, allergic reaction, foreign body
rejection, and inflammation reaction. Preferred implants of the
invention can be easily fabricated and sterilized, and will
substantially maintain their physical properties and function
during the time they remain implanted in, or in contact with,
tissues and biological fluids.
[0053] Materials of the invention can also be used as, and within,
medical devices. Examples of medical devices include, but are not
limited to, dialysis tubing and membranes, blood oxygenator tubing
and membranes, ultrafiltration membranes, diagnostic sensors (e.g.,
ELISA and sandwich assays), and drug delivery devices.
4.1. Porous Substrates
[0054] As shown in FIG. 1, typical materials of the invention
comprise a porous substrate made of at least one plastic and at
least one functional additive. The relative amounts and types of
plastic(s) and functional additive(s) used to provide a substrate
depend on the desired properties (e.g., strength, flexibility, and
utility) of that material. For example, the strength and
flexibility of a substrate will typically increase as the amount of
functional additive it contains decreases. But the number of
functional groups on the surface of a substrate will typically
increase as the amount of functional additive it contains
increases, although it is possible to concentration functional
additive at a particular surface of a substrate.
[0055] Polymers (e.g., plastics) used to provide typical substrates
have few--if any--functional groups to which chemical, biological,
and other moieties (e.g., spacers) can be attached. Typical
plastics are hydrophobic, and have a surface energy of less than
about 40 dyn/cm.sup.2, and more typically less than about 30
dyn/cm.sup.2. Preferred plastics can be easily sintered or
otherwise shaped to provide strong, durable, and/or flexible porous
materials.
[0056] Examples of plastics that can be used to provide suitable
substrates include, but are not limited to, polyolefins,
polyethers, nylons, polycarbonates, poly(ether sulfones)., or
mixtures thereof. Preferred plastics are polyolefins. Examples of
polyethers include, but are not limited to, polyether ether ketone
(PEEK, poly(oxy-1,4-phenylene--
oxy-1,4-phenylene-carbonyl-1,4-phenylene)), polyether sulfone
(PES), or mixtures thereof.
[0057] Examples of polyolefins include, but are not limited to,
ethylene vinyl acetate; ethylene methyl acrylate; polyethylenes;
polypropylenes; ethylene-propylene rubbers;
ethylene-propylene-diene rubbers; poly(1-butene); polystyrene;
poly(2-butene); poly(1-pentene); poly(2-pentene);
poly(3-methyl-1-pentene); poly(4-methyl-1-pentene);
1,2-poly-1,3-butadiene; 1,4-poly-1,3-butadiene; polyisoprene;
polychloroprene; poly(vinyl acetate); poly(vinylidene chloride);
poly(tetrafluorcethylene) (PTFE); poly(vinylidene fluoride) (PVDF);
acrylonitrile-butadiene-styrene (ABS); or mixtures thereof.
Preferred polyolefins are polyethylene or polypropylene. Examples
of suitable polyethylenes include, but are not limited to, low
density polyethylene, linear low density polyethylene, high density
polyethylene, ultra-high molecular weight polyethylene, or
derivatives thereof.
[0058] Because typical plastics possess no functionality and are
hydrophobic, porous substrates made from them according to this
invention comprise at least one type of functional additive.
Functional additives are materials that contain functional groups
to which biological and/or chemical moieties can be covalently
attached. Examples of functional groups include, but are not
limited to, hydroxyl, carboxylic acid, anhydride, acyl halide,
alkyl halide, aldehyde, alkene, amide, amine, guanidine, malimide,
thiol, sulfonate, sulfonyl halide, sulfonyl ester, carbodiimide,
ester, cyano, epoxide, proline, disulfide, imidazole, imide, imine,
isocyanate, isothiocyanate, nitro, or azide. Preferred functional
groups are hydroxyl, amine, aldehyde, or carboxylic acid. A
particularly preferred functional group is hydroxyl.
[0059] Mixtures of functional groups can be provided in controlled
ratios by including two or more additives within a substrate, or by
using an additive that contains more than one type of functional
group. The presence, types, and densities of functional groups on
the surface of a substrate can be readily determined by methods
such astitration, fourier transform infrared spectroscopy (FTIR),
attenuated total reflectance (ATR), and X-ray photoelectron
spectroscopy (XPS), or using molecular probes such as, D-1557
sulfonyl chloride, fluorescein isothiocyanate, fluorescein
dichlorotriazine, and the like.
[0060] Preferred functional additives are inexpensive, and can be
readily incolporated into porous substrates without degrading
(e.g., losing their functionality) during the thermal process.
Examples of functional additives include, but are not limited to,
silica powder, silica gel, chopped glass fiber, glass beads, ground
glass fiber, or glass bubbles. Preferred functional additives are
silica powder or glass fiber, which provide hydroxyl functional
groups.
[0061] Substrates used to provide materials of the invention are
porous, and consequently contain one or more channels through which
gas or liquid molecules can pass. Preferred substrates have an
average pore size of from about 10 .mu.m to about 200 .mu.m, more
preferably from about 15 .mu.m to about 50 .mu.m, and most
preferably from about 20 .mu.m to about 30 .mu.m. Mean pore size
and pore density can be determined using, for example, a mercury
porosimeter, or scanning electron microscopy.
[0062] Because porous substrates of the invention are made of a
porous polymeric matrix into which inclusions of functional
additive are trapped, a surface of a typical substrate will contain
regions of functional additive surrounded by regions of the
polymeric matrix. The surface density and size of the regions of
functional additive will depend on a variety of factors, including
the desired density of chemical or biological moieties to be
attached to it. In a typical substrate of the invention, functional
additive covers about 5% to about 60%, and more typically of about
10% to about 50% percent of the surface area of the substrate.
4.1.1. Preparation of Porous Substrates
[0063] A variety of methods known to those skilled in the art can
be used to make porous substrates. Some examples include sintering;
the use of blowing agents and/or leaching agents; microcell
formation methods such as those disclosed by U.S. Pat. Nos.
4,473,665 and 5,160,674, both of which are incorporated herein by
reference; drilling, including laser drilling; and reverse phase
precipitation. Depending on how it is made, a porous substrate can
thus contain regular arrangements of channels of random or
well-defined diameters and/or randomly situated pores of varying
shapes and sizes. Pore sizes are typically referred to in terms of
their average diameters, even though the pores themselves are not
necessarily spherical.
[0064] The particular method used to form the pores or channels of
a porous substrate and the resulting porosity (i.e., average pore
size and pore density) of the porous substrate can vary according
to the desired application to which the final porous material will
be put. The desired porosity of the substrate can also be affected
by the substrate material itself, as porosity can affect in
different ways the physical properties (e.g., tensile strength and
durability) of different materials.
[0065] Preferred substrates of the invention are made by sintering
a mixture comprising particles of at least one polymer (e.g.,
plastic) and particles of a functional additive (e.g., silica
powder or chopped glass fiber). Optional additional materials such
as, but not limited to, lubricants, colorants, and fillers can also
be added to the mixture. The relative amounts of polymer and
functional additive depend on the desired number and density of
functional groups on the substrate surface and on the desired
strength of the final material.
[0066] As discussed elsewhere herein, the strength of a substrate
may decrease as its functional additive content increases, although
if the functional additive can adhere to the plastic surrounding
it, this may not be the case.
[0067] The relative amounts of plastic and functional additive used
to provide a porous substrate will vary with the specific materials
used, the desired functionality of the substrate surface, and the
strength and flexibility of the substrate itself. Typically,
however, the mixture from which the porous substrate is made
comprises from about 5% to about 60%, more preferably from about
10% to about 40%, and most preferably from about 20% to about 30%
weight percent functional additive.
[0068] The polymer, functional additive, and optional additional
materials are blended to provide a uniform mixture, which is then
sintered. Depending on the desired size and shape of the final
product (e.g., a block, tube, cone, cylinder, sheet, or membrane),
this can be accomplished using a mold, a belt line such as that
disclosed by U.S. Pat. No. 3,405,206, which is incorporated herein
by reference, or other techniques known to those skilled in the
art. In a preferred embodiment, the mixture is sintered in a mold.
Suitable molds are commercially available and are well known to
those skilled in the art. Specific examples of molds include, but
are not limited to, flat sheets with thickness ranging from about
1/8 inch to about 0.5 inch and round cylinders of varying heights
and diameters. Suitable mold materials include, but are not limited
to, metals and alloys such as aluminum and stainless steel, high
temperature thermoplastics, and other materials both known in the
art and disclosed herein.
[0069] In a preferred embodiment, a compression mold is used to
provide the sintered material. In this embodiment, the mold is
heated to the sintering temperature of the plastic, allowed to
equilibrate, and then subjected to pressure. This pressure
typically ranges from about 1 psi to about 10 psi, depending on the
composition of the mixture being sintered and the desired porosity
of the final product. In general, the greater the pressure applied
to the mold, the smaller the average pore size and the greater the
mechanical strength of the final product. The duration of time
during which the pressure is applied also varies depending on the
desired porosity of the final product, and is typically from about
2 to about 10 minutes, more typically from about 4 to about 6
minutes. In another embodiment of the invention, the mixture is
sintered in a mold without the application of pressure.
[0070] Once the porous substrate has been formed, the mold is
allowed to cool. If pressure has been applied to the mold, the
cooling can occur while it is still being applied or after it has
been removed. The substrate is then removed from the mold and
optionally processed. Examples of optional processing include, but
are not limited to, sterilizing, cutting, milling, polishing,
encapsulating, and coating.
[0071] Using methods such as that described above, a variety of
materials of varying sizes and shapes can be used to provide a
suitable porous substrate. In one embodiment, similarly-sized
particles of plastic and/or functional additive are sintered. In
this embodiment, the particles' size distribution is preferably
narrow (e.g., as determined using commercially available screens).
This is because it has been found that particles of about the same
size can be consistently packed into molds, and because a narrow
particle size distribution allows the production of a substrate
with uniform porosity (i.e., a substrate comprising pores that are
evenly distributed throughout it and/or are of about the same
size). This is advantageous because solutions and gases tend to
flow more evenly through uniformly porous materials than those
which contain regions of high and low permeability. Uniformly
porous substrates are also less likely to have structural weak
spots than substrates which comprise unevenly distributed pores of
substantially different sizes. In view of these benefits, if a
polymer is commercially available in powder (i.e., particulate)
form, it is preferably screened prior to use to ensure a desired
average size and size distribution. However, many polymers are not
commercially available in powder form. Consequently, methods such
as cryogenic grinding and underwater pelletizing can be used to
prepare powders of them.
[0072] Cryogenic grinding is a well-known method, which can be used
to prepare particles of plastic and functional additive of varying
sizes. But because cryogenic grinding provides little control over
the sizes of the particles it produces, powders made by it may have
to be screened to ensure that the particles to be sintered are of a
desired average size and size distribution.
[0073] Plastic particles can also be made by underwater
pelletizing. Underwater pelletizing is described, for example, in
U.S. patent application Ser. No. 09/064,786, filed Apr. 23, 1998,
and U.S. Provisional Patent Application No. 60/044,238, filed Apr.
24, 1999, both of which are incorporated herein by reference.
Although this method is typically limited to the production of
particles with diameters of at least about 36 .mu.M, it offers
several advantages. First, underwater pelletizing provides accurate
control over the average size of the particles produced, in many
cases thereby eliminating the need for an additional screening step
and reducing the amount of wasted material. A second advantage of
underwater pelletizing is that it allows significant control over
the particles' shape.
[0074] Thermoplastic particle formation using underwater
pelletizing typically requires an extruder or melt pump, an
underwater pelletizer, and a drier. The thermoplastic resin is fed
into an extruder or a melt pump and heated until semi-molten. The
semi-molten material is then forced through a die. As the material
emerges from the die, at least one rotating blade cuts it into
pieces herein referred to as "pre-particles." The rate of extrusion
and the speed of the rotating blade(s) determine the shape of the
particles formed from the pre-particles, while the diameter of the
die holes determine their average size. Water, or some other liquid
or gas capable of increasing the rate at which the pre-particles
cool, flows over the cutting blade(s) and through the cutting
chamber. This coagulates the cut material (i.e., the pre-particles)
into particles, which are then separated from the coolant (e.g.,
water), dried, and expelled into a holding container.
[0075] The average size of particles produced by underwater
pelletizing can be accurately controlled and can range from about
0.014" (35.6 .mu.M) to about 0.125" (318 .mu.M) in diameter,
depending upon the porous substrate. Average particle size can be
adjusted simply by changing dies, with larger pore dies yielding
proportionally larger particles. The average shape of the particles
can be optimized by manipulating the extrusion rate and the
temperature of the water used in the process.
[0076] While the characteristics of a porous material can depend on
the average size and size distribution of the particles used to
make it, they can also be affected by the particles' average shape.
Consequently, in another embodiment of the invention, the particles
of plastic and functional additive particles are substantially
spherical. This shape facilitates the efficient packing of the
particles within a mold. Substantially spherical particles, and in
particular those with smooth edges, also tend to sinter evenly over
a well defined temperature range to provide a final product with
desirable mechanical properties and porosity.
[0077] In a specific embodiment of the invention, the particles of
plastic and/or functional additive are substantially spherical and
free of rough edges. Consequently, if the particles used in this
preferred method are commercially available or made by cryogenic
grinding, they are thermal fined to ensure smooth edges, and are
screened to ensure a proper average size and size distribution.
Thermal fining is a well-known process wherein particles are
rapidly mixed and optionally heated such that their rough edges
become smooth. Mixers suitable for thermal fining include the W
series high-intensity mixers available from Littleford Day, Inc.,
Florence, Ky.
[0078] Particles made by underwater pelletizing, which allows
precise control over particle size and can yield smooth,
substantially spherical particles, typically do not need to be
thermal fined or screened.
4.2. Substrate Surface Modification
[0079] Once the porous substrate has been formed, chemical and/or
biological moieties are bound directly or indirectly to its
surface. FIG. 2 provides a representation of this process wherein a
spacer molecule (e.g., an alkane substituted with at least one
functional group) is attached to the surface of a substrate to
provide an intermediate material. Chemical or biological moieties
such as proteins are then attached to the spacer. Also as shown in
FIG. 2, the length of the spacer can be optionally increased by
reacting the intermediate material with additional chemical
moieties to provide a second intermediate material, to which
chemical or biological moieties can be attached.
[0080] As used herein, spacers are differentiated from chemical or
biological moieties only by their use in a particular instance. To
be specific, a chemical or biological moiety is something that
provides, at least to a substantial degree, the useful physical or
chemical properties of a particular material. For example, the
chemical or biological moiety in a material to be used as a
biosensor will be a moiety that can recognize, bond, or associate
with the molecule(s) to be detected. By contrast, a spacer is a
chemical moiety that provides some distance between the surface of
the porous substrate and the chemical or biological moiety. Still,
a spacer can augment or facilitate the activity or utility of a
chemical or biological moiety by removing it from the surface of
the substrate. It will be readily apparent to those of skill in the
art, however, that a moiety used as a spacer in one instance can be
the chemical or biological moiety in another.
[0081] Spacers and biological or chemical moieties that are
directly bound to the substrate surface are bound covalently or
though strong multi-point non-covalent interactions. However, bonds
between moieties bound to the surface and other moieties not bound
to the surface need not be covalent. For example, FIG. 3 represents
a material comprised of a spacer covalently attached to a porous
substrate, wherein the spacer can form ligand-receptor or
hybridization-type bonding with a pharmacologically active chemical
or biological moiety. In a specific example of a material
encompassed by the representation of FIG. 3, the spacer is an
oligonucleotide, and the chemical or biological moiety comprises an
oligonucleotide or polynucleotide complementary to the spacer.
[0082] The invention further encompasses spacers that are capable
of selectively releasing an immobilized chemical or biological
moiety. For example, a drug may be attached to a spacer by a bond
that readily hydrolyzes under physiological conditions, or which
breaks when exposed to radiation of a particular energy. Such
materials can be useful for the controlled release of drugs.
[0083] In order to form a covalent bond between the substrate
surface and a moiety, a functional group on the surface must be
complementary to a functional group on the chemical precursor of
the moiety. In other words, functional groups on the surface and on
the precursor to whatever will be attached to it must be capable of
forming a covalent bond under suitable reaction conditions. An
example of complementary groups is amine and aldehyde, which can
react to form a bond under suitable conditions. Other complementary
pairs of functional groups will be readily apparent to those
skilled in the art.
[0084] Examples of spacers include, but are not limited to,
silanes, silane aldehydes, diamines, alcohols, esters, glycols
(such as polyethylene glycol), anhydrides, dialdehydes, terminal
difunctionalized polyurethanes, succinic acid, diaminohexanes,
glyoxylic acids, glycines, dentrimers, multifunctional polymers,
and diones. Preferred spacers are those of Formula I: 7
[0085] wherein the bond broken by a wavy line are those between the
spacer and the substrate or other moieties; R.sup.1 and R.sup.2
each independently is hydrogen, substituted or unsubstituted alkyl,
aryl, or aralkyl; R.sup.3 is a substituted or unsubstituted
aliphatic chain or a bond; and n is an integer from about 1 to
about 18, preferably n is an integer from about 1 to about 10, and
more preferably n is about 2 to about 5. More preferred spacers are
those of Formula VIII: 8
[0086] wherein R.sup.1, R.sup.2, and n are as defined above; R is
the surface of the porous substrate; X is a group capable of
bonding to a biological or chemical moiety, such as OH, NH.sub.2,
CHO, CO.sub.2H, NCO, epoxy, and the like; m is an integer from
about 1 to about 5; o is an integer from 0 to about 3; most
preferably, X is NH.sub.2 or CHO.
[0087] Other more preferred spacers are those of Formula IX: 9
[0088] wherein R, R.sup.1, R.sup.2, and n are as defined above.
[0089] A wide array of chemical and biological moieties can be
attached directly or via a spacer to the surface of a porous
substrate. Examples of such moieties include, but are not limited
to, drugs (e.g., pharmaceuticals), hydrophilic moieties, catalysts,
antibiotics, antibodies, antimycotics, carbohydrates, cytokines,
enzymes, glycoproteins, lipids, nucleic acids, nucleotides,
oligonucleotides, peptides, polynucleotides, proteins, cells,
ligands, ribozymes, or combinations thereof.
4.2.1. Functionalization of Substrates
[0090] A porous substrate is functionalized according to the
invention by the covalent bonding of a molecule (e.g., a spacer or
chemical or biological moiety) to its surface. The molecule can
often be directly attached to the surface of the substrate.
However, it is sometimes desirable to attach a chemical or
biological moiety to a substrate indirectly by means of a
spacer.
[0091] For example, if a biological moiety is to interact with
molecules in solution, it may interact with such molecules more
readily if positioned at some distance from the surface to which it
is attached. Alternatively, the shape and size of some biological
moieties can prevent the formation of covalent bonds between them
and functional groups on a substrate surface. In such cases, a
spacer is used to link the moiety to the surface.
[0092] FIG. 4 illustrates a method by which amine-functionalized
chemical or biological moieties can be attached to the surface of a
substrate of the invention. In this method, a silane with a
terminal amine reacts with a hydroxyl group provided by a
functional additive such as silica powder embedded in the surface.
The terminal amine of the resulting material can then be converted
to an aldehyde using, for example, an excess of glutaraldehyde. The
resulting product is then contacted with an amine-functionalized
chemical or biological moiety (e.g., a protein) the under suitable
conditions to form a material of the invention. FIG. 5 shows a
related method, wherein the silane initially reacted with the
substrate surface already has a terminal aldehyde, which can react
with amine-functionalized chemical or biological moieties.
[0093] In some cases, a spacer such as that shown in FIG. 4 and
FIG. 5 is of insufficient length to provide a useful material,
i.e., a desirable environment for the bioactive substance. In such
cases, the length of the spacer can be increased using methods
readily apparent to those skilled in the art. One example is shown
in FIG. 6. According to this method, a silane with a terminal amine
reacts with a hydroxyl group provided by a functional additive such
as silica powder embedded in the surface. The resulting material is
then contacted under suitable conditions with a compound of Formula
VI:
Z-Spacer-Z Formula VI
[0094] wherein Spacer is a hydrophilic segment and Z is a terminal
group capable of covalently or non-covalently bonding to proteins,
amino acids, oligonucleotides, and the like. Preferably, Spacer is
hydrophilic polyurethane, polyethylene glycol, or polyelectrolytes
with terminal groups (Z) capable of bonding to proteins, amino
acids, oligonucleotides wherein Z includes, but is not limited to,
isocyanurate, aldehydes, amino, carboxylic acids,
N-hydroxysuccimide, and the like. In a more preferred compound of
Formula VI, Spacer is hydrophilic polyurethane (NCO-hydrophilic
polyurethane-NCO) or N-hydroxysuccimide-PEG-N-hydroxysuc- cimide
(PEG-NHS). The resulting complex is then contacted with an
amine-functionalized chemical or biological moiety under reaction
conditions suitable for the formation of a covalent bond.
5. EXAMPLES
[0095] Certain embodiments of the invention, as well as certain
novel and unexpected advantages of the invention, are illustrated
by the following non-limiting examples. Materials were purchased
from Sigma-Aldrich Co. (Milwaukee, Wis.).
5.1 Example 1
Preparation of a Hydrophilic Polyurethane Spacer
[0096] A reaction flask was charged under nitrogen with
4,4'-methylenebis(cyclohexyl isocyanate) (5.8 g), dibutyltin
bis(ethyl hexanoate) (30 mg), and THF (10 g). Using a heating
mantle, the reaction flask was gently warmed to 65.degree. C.
Subsequently, a solution of polyethylene glycol (PEG 1000)
(MW=1000, 11.2 g) dissolved in 25 g of THF was added dropwise to
the reaction flask. After the addition was complete, the reaction
was allowed to proceed for 12 hours at 65.degree. C. under
nitrogen. The resulting solution, a light-yellow and transparent
gel, was poured out of the reaction flask and stored in a sealed
glass bottle filled with dry nitrogen.
5.2 Example 2
Preparation of a Porous Plastic with Glass Powder as Dopant
[0097] Glass bubble (amorphous silica, CAS No. 7631-86-9) supplied
by 3M (St. Paul, Minn.) was blended with an ultra high molecular
weight polyethylene powder GUR 2122 by TICONA (Summit, N.J.) at 20%
by weight. After tne mixture was well blended, it was placed into a
0.25 inch flat mold. Using an electrically heated plate, the mold
was heated to and was kept at 140.degree. C. for 4 minutes. A
skilled artisan can readily determine the amount of time to heat
the mold, depending upon the thickness of the final product. After
heating, the mold was cooled and the porous plastic with
immobilized glass bubbled was removed.
5.3 Example 3
Preparation of a Functionalized Porous Material (Method 1)
[0098] Binding buffer (10 mM phosphate; pH 7.5, 0.015 M NaCl).
0.192 g NaH.sub.2PO.sub.4, 2.252 g Na.sub.2HPO.sub.4.7H.sub.2O and
0.27 g of NaCl are dissolved in 800 ml deionized (DI) water. The pH
7.5 is adjusted with 1N HCl/1N NaOH, and the volume is brought to
1000 ml with DI water. The buffer is then purged with nitrogen
before coupling procedure.
[0099] Washing buffer: (10 mM phosphate, pH 7.5, 1.0 M NaCl). 5.844
g NaCl is dissolved in 100 ml of binding buffer.
[0100] Storage buffer: (10 mM phosphate, pH 7.5, 0.15M NaCl, 0.02%
NaN.sub.3).
[0101] Silica powder incorporated substrate, prepared in Example 2,
is washed with alcohol, filtered, and air dried. After the
substrate has been completely dried, they are immersed into a
silane solution (5% of 3-aminopropyltriethoxysilane/isopropanol
solution, CAS #: 919-30-2) until the substrate is completely wet.
The substrate is removed from the solution and air dried. After the
substrate is half-dried, it is placed into an oven at about 60 to
70.degree. C. for about 30 minutes. After the substrate is
completely dried it is submerged in a glutaraldehyde/alcohol
solution (20%) for about 20 minutes. The substrate is dried again
in the oven for 30 minutes. Finally, the reactive substrate is
dipped into protein/binding buffer solution (0.1 mg/ml IgG
solution) and mixed gently for 24 hours at 4.degree. C. The binding
buffer of unreacted protein solution is drained from the matrix.
The functionalized porous material is then immersed in a washing
buffer (pH 7.5), and sodium cyanoborohydride is added until the
final concentration is approximately 1.0 M. The functionalized
porous material is washed with washing buffer (pH 7.5) until all
excess sodium cyanoborohydride is removed and immersed in storage
buffer at 4.degree. C.
5.4 Example 4
Preparation of a Functionalized Porous Material (Method 2)
[0102] The silica powder incorporated substrate, prepared in
Example 2) is washed and dried as described above. The substrate is
immersed into a silane solution (5% of aldehyde
trimethoxysilane/isopropanol solution) until the substrate is
completely wet. Aldehyde methoxysilane was supplied by United
Chemical Corporation, Piru, Calif. The substrate is filtered out of
the solution and rinsed with anhydrous isopropanol. The substrate
is then air dried. After the substrate is half-dried, the substrate
is placed in an oven at about 60.degree. C. to 70.degree. C. for 30
minutes. The reactive substrate is dipped into protein/binding
buffer solution (0.1 mg/ml IgG solution) and mixed gently for 24
hours at 4.degree. C. The binding buffer of unreacted protein
solution is drained, and the substrate is immersed in washing
buffer (pH 7.5). Sodium cyanoborohydride is added until the final
concentration is approximately 1.0 M. The functionalized porous
material is then washed with washing buffer (pH 7.5) until all
excess sodium cyanoborohydride is removed and immersed in storage
buffer at 4.degree. C.
5.5 Example 5
Preparation of a Functionalized Porous Material (Method 3)
[0103] In a separate procedure, the silica powder incorporated
substrate is washed with alcohol, filtered, and air dried. After
the substrate has been completely dried, it is immersed into a
silane solution (5% of 3-aminopropyltriethoxysilane/isopropanol
solution, CAS #: 919-30-2) until completely wet. The substrate is
filtered out of the solution and air dried. The substrate is
half-dried and placed in an oven at about 60 to 70.degree. C. for
30 minutes. After the substrate is completely dried it is submerged
into hydrophilic polyurethane/tetra-hydrofuran (THF) solution for
about 6 hours. The preparation of the hydrophilic polyurethane was
described in Example 5.1. The synthesis of other suitable
hydrophilic polyurethanes was described in U.S. patent application
Ser. No. 09/375,383, which is incorporated herein by reference. The
substrate is then filtered out of the solution and rinsed with dry
THF. The washed substrate is then dried in a vacuum oven to drive
off the residual THF. Finally, the reactive substrate is dipped
into a protein/binding buffer solution (0.1 mg/ml IgG solution) and
mixed gently for 24 hours at 4.degree. C. The binding buffer of
unreacted protein solution is drained, and the substrate is
immersed in washing buffer (pH 7.5). Sodium cyanoborohydride is
added until the final concentration is approximately 1.0. The
functionalized porous material is washed with washing buffer (pH
7.5) until all excess sodium cyanoborohydride is removed and
immersed in storage buffer at 4.degree. C.
5.6. Example 6
Detection of Functional Groups
[0104] The hydroxyl functionality of the substrate can be
characterized in several ways.
[0105] For example, X-ray photoelectron spectroscopy (XPS) can
offer the elemental information about oxygen. Also, Fourier
Transform Infra-red Sprectroscopy (FTIR) can reveal certain
chemical structures, such as hydroxyl. Direct measurement of
surface R--OH concentrations can be carried out by using the
molecular probe D-1557 sulfonyl chloride from Molecular Probes
Inc., of Eugene, Oreg. after reaction of the base hydrolysis (which
releases the chromophore) of the sulfonic acid ester formed by
reaction of the D-1557 probe (1 mg/ml in cry acetone with 1.0%
pyridine) of the hydroxyl functionalized polymer surface.
[0106] Detection of amine groups can be carried out in several
ways. Amine selective molecular probes such as fluorescein
isothiocyanate (FITC) and fluorescein dichlorotriazine (DTAF) react
with all functionalized surfaces throughout the porous polyethylene
article, while being unreactive with the unmodified surfaces.
Cleavage of the isothiourea formed by reaction of FITC with alkyl
amine functions with aqueous 0.1 M NaOH releases fluorescein into
solution for direct spectrophotometric measurement and the number
of nicromoles of amine function per gram of porous solid can be
calculated. The amino groups on an aminated specimen can also be
determined chemically according to R. Allul (DNA Probes, H. G.
Keller et al. eds., Macmillan, N.Y. (1993)). Thus, the aminated
specimen is treated with
3-O-4-(nitrophenylsuccinylated)-5'-O-DMT-deoxyri- bonucleoside,
followed by blockage of unreached amines with pyridine/acetic
anhydride/N-methyl imidazole (8:1:1, v:v:v). The amount of bound
deoxyribonucleoside is determined by absorbance at 498 nm after
treatment with 70% aqueous perchloric acid, toluenesulfonic acid in
acetonitrile, commercial deblock preparations, or the like, to
release the DMT group from the support.
[0107] Another method for the detection of amino functional groups
is carried out as follows. Amino functional porous plastics (1 g)
cut into 1 mm particles are mixed with a 0.2 mM Sulfo-SDTB
solution, made by dissolving 3 mg of Sulfo-SDTB into 25 ml 0.025 M
NaHCO.sub.3 buffer solution (pH 8.5) at room temperature for one
hour. Remove the porous plastics and rinse three times with 15 ml
of deionized water for 10 min. Add the rinsed porous plastics to 10
ml of 50% perchloric acid water solution and shake at room
temperature for 15 min. Remove a 3 ml sample and measure the
absorbency at a 498 nm wavelength with a 1 cm path length UV
cuvette. The amino functional group density can be calculated by
C(M amino group/gram porous plastic)=3.3(As-Af)/70000.
[0108] It should be understood that variations and modifications
within the spirit and scope of the invention may occur to those
skilled in the art to which the invention pertains. Accordingly,
all expedient modifications readily attainable by one versed in the
art from the disclosure set forth herein that are within the scope
and spirit of the invention are to be included as further
embodiments of the invention. The scope of the invention
accordingly is to be defined as set forth in the appended
claims.
* * * * *