Sulfonated multiblock copolymer and uses therefor

Sulfonated multiblock copolymers, and uses thereof, are disclosed. The sulfonated copolymers are useful for providing non-thrombogenic coatings, e.g., for medical devices, and for promoting cell growth, differentiation, or production of normal cell products. The sulfonated copolymers are also useful for administration of therapeutic agents.

RELATED APPLICATIONS 
This application claims benefit of priority under 35 U.S.C. 119(e) to U.S. 
Provisional application Ser. No. 60/001,973, filed Jul. 28, 1995, the 
contents of which is hereby incorporated by reference. 
BACKGROUND OF THE INVENTION 
The need for biocompatible materials for use in medical applications is 
acute. Many materials used for medical devices are selected for mechanical 
strength or stability in the body, but are capable of causing 
thrombogenesis and other undesirable side effects when in contact with 
blood or blood products. Prior art approaches to preventing thrombogenesis 
include the covalent or non-covalent attachment of non-thrombogenic 
molecules to the surface of an implantable device. For example, heparin 
has been attached to the surface of implantable materials in an effort to 
reduce thrombogenicity of the material (see, e.g., U.S. Pat. No. 
3,826,678; 4,526,714; 4,613,517; 5,061,750). 
However, this approach has the disadvantage of providing a coating on the 
surface of the material only; thus, if the surface coating is abraded or 
washed off, the thrombogenic material will be exposed, possibly resulting 
in clot formation. Also, recent studies have concluded that heparinized 
surfaces are only modestly effective at preventing adverse outcomes in 
patients. 
In addition to the need for biocompatibility of materials, it is now 
generally recognized that there is a further need for bioactive materials, 
that is, materials that can stimulate or promote normal tissue functions 
such as conduction, growth and differentiation of cells, and the 
production of materials characteristic of normal cellular activity. For 
example, endothelial cells should be attracted (i.e., conducted) to the 
surface of an implanted material. Cells attracted to the material surface 
should also produce the products typically expressed by normal cells; for 
example, endothelial cells should produce natural clot inhibitors. 
SUMMARY OF THE INVENTION 
This invention pertains to methods of using an anionic multiblock copolymer 
which is biocompatible and which has desirable properties such as 
nonthrombogenicity. The anionic multiblock copolymer can also be used to 
deliver drugs, or to promote endothelialization or epithelialization, and 
other forms of conduction, growth, or differentiation of cells and 
tissues. In one aspect, the invention features a nonthrombogenic article 
for use in contact with blood or blood products, the article having at 
least one surface, the surface comprising an anionic multiblock copolymer. 
In a preferred embodiment, the multiblock copolymer is a sulfonated 
styrene-ethylene/butylene-styrene triblock copolymer. In preferred 
embodiments, the multiblock copolymer is at least 20%, 30%, 50%, 70% or 
90% sulfonated. In preferred embodiments, the nonthrombogenic article is a 
medical device. In preferred embodiments, the article comprises a hybrid 
material comprising a triblock copolymer and a material selected from the 
group consisting of Teflon.RTM., Dacron.RTM., titanium oxide, magnetic 
particles, and calcium phosphate. In preferred embodiments, the article is 
selected from the group consisting of stents, catheters, cannulae, tubing, 
vascular grafts, artificial hearts, heart valves, pacemakers, implants, 
artificial joints, and prostheses. In a preferred embodiments, the article 
is an electrical lead, e.g., for an implanted medical device. 
In another aspect, the invention features a method of manufacturing a 
thromboresistant article, the method comprising coating at least one 
surface of an article with an anionic multiblock copolymer. 
In still another aspect, the invention features a method of promoting cell 
growth, or adhesion, comprising contacting cells with an anionic 
multiblock copolymer, under conditions such that cell growth, 
differentiation, or production of normal cell products is promoted. In 
preferred embodiments, the cell are endothelial cells, epithelial cells, 
osteoblasts, or islet cells. 
In yet another aspect, the invention provides a method of administering a 
therapeutic agent to a subject, the method comprising contacting the 
subject with an anionic multiblock copolymer, wherein the copolymer 
entraps the therapeutic agent, such that the therapeutic agent is 
delivered to said subject. 
In another aspect, the invention provides a medical implant which can be 
modified in situ by application of an electric field. In this aspect, the 
implant comprises an anionic multiblock copolymer, preferably a sulfonated 
styrene-ethylene/butylene-styrene triblock copolymer.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention features methods of using an anionic copolymer. In 
one aspect, the invention provides a method of manufacturing a 
thromboresistant article. The method comprises coating at least one 
surface of an article with an anionic multiblock copolymer. A 
thromboresistant article can also be fabricated entirely from an anionic 
multiblock copolymer. The term "block copolymer" is known in the art, and 
refers to a copolymer of two or more monomers in which the polymeric 
chains contain long stretches (e.g., at least about 10 monomer units on 
average) of one kind of repeating unit linked covalently to one or more 
long stretches of repeating units of one or more different polymers. For 
example, a block copolymer of components A and B could have a partial 
structure (A).sub.n (B).sub.m (A).sub.p, where n, m, and p are, 
independently, integers which are generally greater than 10 on average, 
and are each preferably, on average, in the range between 5 and 1000, more 
preferably between 10 and 100. Preferred copolymers include multiblock 
(i.e., diblock, triblock, and the like) copolymers composed of hydrocarbon 
subunits (prior to sulfonation). For example, preferred blocks include 
styrene, ethylene/butylene, isoprene, butadiene, propylene, and the like. 
Preferred sulfonated copolymers are not sulfonated in all blocks (e.g., 
only styrene is sulfonated). A particularly preferred copolymer is 
styrene-ethylene/butylene-styrene, which is available from Shell Chemical. 
This copolymer can be sulfonated by methods known in the art. A suitable 
sulfonation method is described in U.S. Pat. No. 5,468,574 to Ehrenberg et 
al. This patent teaches, inter alia, the use of sulfur trioxide and 
triethyl phosphate in dichloroethane/cyclohexane solution for the 
sulfonation of styrene-ethylenelbutylene-styrene. Sulfonation according to 
this method sulfonates principally the styrene blocks. The copolymer can 
be sulfonated to a desired extent by controlling the sulfonation 
conditions; alternatively, the monomer units (e.g., the styrene monomer) 
can be separately sulfonated and then combined with the remaining monomer 
units and copolymerized. The skilled artisan will appreciate that the 
block lengths and other characteristics of the copolymer can be varied by 
changing the polymerization conditions; thus, it is possible to alter the 
copolymer morphology (e.g., microphase separation) and the bulk physical 
properties of the copolymer. Other copolymers can also be used in the 
methods of the invention, as taught in the above-referenced U.S. Pat. No. 
5,468,574. 
Although reference is made herein to sulfonated copolymers, other anionic 
copolymers may be used in the methods of the present invention. Thus, 
while copolymers with sulfonate functional groups are particularly 
preferred, copolymers comprising other anionic moieties such as sulfates, 
phosphates, phosphonates, carboxylates, phenolates and the like, or 
mixtures thereof, may be useful according to the present invention. More 
than one type of anionic moiety group may be employed in a particular 
copolymer. Such copolymers are known in the art and/or can be made 
according to known techniques. 
The sulfonated copolymers of the invention can be molded, cast, laminated, 
extruded, worked or shaped to provide a variety of useful forms, according 
to standard techniques for forming polymers. For example, the subject 
copolymers can be cast by dissolving the copolymer in a suitable solvent 
(for example, n-propanol and dichloroethane), casting the mixture into a 
form, and removing the solvent to yield the cast product, according to 
standard techniques. In a preferred embodiment, after casting, an IR lamp 
is used to dry and cure the copolymer. 
The copolymers of the invention can also be used as a coating to cover a 
substrate. Exemplary substrates include metals, ceramics, and polymers 
(natural or synthetic). In addition, the sulfonated 
styrene-ethylene/butylene-styrene copolymer can be effectively grafted to 
a variety of ceramic and polymer substrates, including polyvinyl polymers 
(such as polyvinylchloride and polyethylene), mylar and the like. 
Non-polymeric substrates can be employed by appropriate surface 
modification to facilitate grafting. Grafting can be by a variety of 
well-known techniques, including the use of corona discharge, UV 
irradiation, ionizing radiation, plasmas, and the like. The copolymers of 
the invention may be used to form the surface of a wide variety of medical 
devices, as described below. 
The sulfonated copolymer of the invention can also form hybrid materials 
with polymers or ceramic materials, thus combining the physical or 
chemical properties of those materials with the biological, chemical, and 
controlled-release characteristics of the copolymer, to create novel 
hybrids. Where the anionic copolymer is substantially soluble in organic 
solutions, porous structures are readily infiltrated. Exemplary materials 
which can be infiltrated in this manner are: glass fiber mat; porous 
Teflon.RTM. (tetrafluoroethylene) and Dacron.RTM. (polyethylene 
terephthalate), e.g., as currently used in vascular grafts; porous or 
pitted native titanium oxide and sintered and non-sintered calcium 
phosphate, e.g., as used in coatings of dental and orthopedic implant 
devices. By controlling the amount of polymer applied to a surface it is 
possible to only partially cover pore surfaces, thus producing a desired 
net porosity. 
Another method of hybrid formation consists in particles of another 
material being suspended in the dissolved or suspended sulfonated 
copolymer of the invention, the particles remaining suspended in the 
sulfonated copolymer solution or suspension. Alternatively, the inorganic 
material can be synthesized in situ, using, e.g., a sol-gel approach, to 
create a fine dispersion of the particles. As an extension of this method, 
it is possible to prepare the particulate material in the sulfonated 
domains of the sulfonated copolymer of this invention after casting the 
sulfonated copolymer into a film, for example, by using the sol-gel 
approach described above. The particulate material can be e.g., a polymer, 
ceramic, or metallic powder. The particle size can range, e.g., from about 
100 nanometers to about 1000 microns in diameter, more preferably from 
about 10 microns to about 100 microns in diameter. One illustrative hybrid 
formed in this manner is a hybrid of the sulfonated copolymer and 
suspended magnetic particles, metals, or metal oxides, with or without 
organic derivatization. Such a hybrid can be used, e.g., to coat 
orthopedic or dental implants. A magnetic field induced in this hybrid can 
improve bone healing in ways well-known in the art. 
In another aspect, the invention features a nonthrombogenic article for use 
in contact with blood or blood products, the article comprising at least 
one surface, said surface comprising a sulfonated multiblock copolymer. 
Articles, e.g., medical devices, for which the copolymer can form a 
surface include stents, catheters, cannulae, tubing (e.g., for use in 
kidney dialysis and heart-lung machines), vascular grafts, artificial 
hearts, heart valves, venous valves, pacemakers (including leads for 
pacemakers), implantable defibrillators, implants (for example, implants 
to be placed in bone), artificial joints, prostheses, and the like. Such 
medical devices have significant advantages over current devices, e.g., 
cardiovascular devices which are thrombogenic and non-endothelializing, or 
implants to be placed in bone which induce encapsulation with fibrous 
connective tissue, rather than with bone. For example, stents are often 
placed after balloon angioplasty to prevent restenosis of the blood 
vessel. The placement of the stents requires the use of clotting 
inhibitors for several weeks before and after angioplasty, resulting in 
long hospital stays, considerable expense and risk to the patient. 
Placement of a nonthrombogenic stent prepared according to the method of 
the present invention would reduce or eliminate the need for additional 
antithrombogenic measures. 
The subject sulfonated copolymers have several properties which make them 
valuable for use in medical devices. As described in Example 1, infra, the 
sulfonated copolymers possess antithrombogenic properties. The ability to 
prevent thrombogenesis makes the subject sulfonated copolymers useful in 
applications which require contact with blood or blood products. The 
copolymers have several advantages over coatings known in the art. For 
instance, a device composed of one of the subject copolymers is inherently 
antithrombogenic, that is, there is no antithrombogenic surface coating 
which can wash off or be abraded or degraded. Thus, the antithrombogenic 
properties of such a device are substantially permanent rather than 
temporary. Such properties is desirable in a permanently implanted device. 
Furthermore, the magnitude of the antithrombogenic quality of the subject 
copolymers can be controlled by controlling the degree of sulfonation of 
the copolymer. As described in Example 1, increasing the degree of 
sulfonation from 30% to 74% more than doubles the time required for blood 
to clot. Thus, a copolymer having a predetermined degree of sulfonation 
can be used to ensure a desired antithrombogenic effect of the copolymer. 
In preferred embodiments, the sulfonated triblock copolymer is at least 
20%, 30%, 50%, 70% or 90% sulfonated. Sulfonation is expressed as a 
percentage of available styrene units which are sulfonated. 
In another aspect of the invention, sulfonated multiblock copolymers can be 
used as promoters of endothelialization or epithelialization. It is well 
known that anionic glycosaminoglycans (GAGs) are associated with cell 
membrane or extracellular matrix (ECM), and that the growth (and, under 
certain conditions, differentiation and cell-type-specific functions) of 
cells is promoted by GAGs. The anionic nature of the subject sulfonated 
copolymers is analogous to GAGs; thus, articles which have a surface 
comprising the subject copolymers can promote cell growth, binding of the 
cells to the material, cell differentiation, and production of normal 
cell- or tissue-specific products. Accordingly, in one aspect, the 
invention features a method of promoting cell growth or adhesion. The 
method includes the step of contacting cells with a sulfonated multiblock 
copolymer, under conditions such that cell growth or adhesion is promoted. 
Endothelialization is the growth of endothelial cells on a surface. The 
endothelium, or lining of the blood vessels and the heart, has a vital 
role in resisting clotting and generally maintaining the integrity of the 
cardiovascular organs. One of the functions of endothelial cells is to 
produce anti-clotting factors. Implantation of a device into the blood 
vessels or the heart can cause foreign body responses, which can result in 
eventual organ failure, as well as clots produced by the disruption of 
normal circulation dynamics by the device. If a layer of endothelium could 
be induced to grow over the device, the implant would effectively cease to 
present a foreign surface to the bloodstream, and these problems would be 
reduced or eliminated. We have found that aortic endothelial cells are 
conducted or attracted to a sulfonated copolymer surface and grow in an in 
vitro tissue culture system (data not shown). Furthermore, the growing 
cells were assayed by an enzyme immunoassay (EIA) and were found to 
produce prostacyclin, a product of normal endothelium. Thus, in a 
preferred embodiment, the invention provides a method of promoting 
endothelialization or epithelialization. Such a method is valuable both 
for promoting the conduction and growth of epithelial or endothelial cells 
in vivo (for example, on an implant or other medical device), and in vitro 
(for example, to grow cells for use in grafting, e.g., skin grafts). 
Growth and conduction of other types of healthy cells, including 
osteoblasts, odontoblasts, chondrocytes, and other connective tissue 
cells, as well as their induction or differentiation from precursor cells, 
can also be promoted in a similar fashion. For example, the integration of 
a bone implant into a bone can be improved by use of a sulfonated 
copolymer surface on the implant, such that osteoblast conduction, 
differentiation, and growth on and around the implant is promoted. In 
periodontal disease, the ability of the gingival epithelium to grow on the 
surface of the material makes it useful in promoting bone and ligament 
healing in periodontal disease. 
In still another aspect, the invention provides methods of administering a 
therapeutic agent to a subject, i.e., controlled release. Polymers are 
useful as drug carriers for controlled release. Up to 5 years of 
relatively steady release has been achieved (the Norplant birth control 
implant system is an example). A first type of drug-release system is said 
to be `diffusional` since simple diffusion of the drug through the polymer 
is the release mechanism. In such a system, the polymer generally does not 
significantly dissolve or degrade and, if implanted, must be surgically 
removed after the drug has been delivered. Such controlled release 
materials are particularly suitable as permanent coatings on non-resorbed, 
implanted devices, such as tooth implants and cardiovascular stents. 
Another type of polymeric drug release devices involves bioerodible 
polymers, where the polymer matrix erodes with time (`erosional`) to 
non-toxic products which can be metabolized or eliminated, circumventing 
the need to surgically remove the polymer after release is complete. 
More recently, attention has focused on a third generation type of 
drug-release system. Development of these systems is driven by the 
recognition that in certain biomedical applications, continuous, low-level 
release may not be desirable. For example, for delivery of hormones such 
as insulin, pulsed release at specific times is desirable. 
In a preferred embodiment, the method comprises contacting the subject with 
a sulfonated multiblock copolymer, wherein the copolymer entraps a 
therapeutic agent, such that the therapeutic agent is delivered to the 
subject. It is believed that at least some of the subject sulfonated 
multiblock copolymers are hydrogels; thus, the copolymers can be made to 
entrap (or immobilize), and subsequently release, a variety of therapeutic 
agents. Data from microscopy and x-ray scattering experiments show that a 
film formed from a sulfonated styrene-ethylene-butylene-styrene block 
copolymer has a lamellar morphology where the sulfonated PS (polystyrene) 
and EB (ethylene/butylene) phases form alternating plates or layers with 
thicknesses of about 200-300 .ANG.. The high ionic conductivities observed 
in polymer films of sulfonated styrene-ethylene-butylene-styrene block 
copolymer suggest that sulfonated PS `sheets` extend through the thickness 
of the membrane. As with certain known block copolymers, there are 
distinct phases due to poor molecular compatibility (e.g., 
hydrophobicity/hydrophilicity) of the components, along with a thin 
interphase region at or near the block junctions where there is at least 
some small degree of mixing. As a result, there are least three locations 
for added molecules to reside in the film: largely in the EB phase if they 
are very non-polar or hydrophobic; largely in the S-PS (sulfonated 
polystyrene) phase if they are very hydrophilic; and at the interphase if 
the added molecule is, e.g., cationic with at least some hydrophobic 
character (e.g., a cationic surfactant). Another possible location for a 
guest molecule occurs within the S-PS domain near a block region having a 
sulfonated styrene flanked by unsulfonated styrenes, the latter being 
hydrophobic. For these reasons many types of molecular species can be 
trapped and immobilized in, or released from, the copolymeric matrix. 
In a preferred embodiment, the therapeutic agent is entrapped by including 
the guest, e.g., therapeutic, agent in the casting solvent when the 
copolymer is cast, or coated onto a substrate. In this way, the 
therapeutic agent is distributed throughout, and is integral with, the 
copolymer structure. Examples of therapeutic agents include enzymes (for 
example, glucose oxidase or lumbrikinase, a fibrinolytic enzyme), 
antiinflammatories, analgesics, growth factors, antibiotics, steroids, 
hormones, antiviral agents, neurotransmifters or neuroregulators, 
antibodies, antiplatelet agents (such as carbamoylpiperidines) and the 
like. In another embodiment, the therapeutic agent is immobilized or 
associated with the sulfonated copolymer by adsorption or through ionic 
(e.g., electrostatic) interactions. For example, neuroregulators such as 
dopamine are cationic and bind well to the subject copolymer. Since 
dopamine deficiency is linked to Parkinson's disease, an article 
comprising a sulfonated copolymer film that includes dopamine can be 
implanted in the brain to offer sustained doses of the neuroregulator. 
In certain embodiments, the therapeutic agent is primarily immobilized on 
the surface of the copolymer, while in other embodiments, the therapeutic 
agent is distributed throughout the copolymer structure. For example, the 
anionic nature of the sulfonated copolymers permits the immobilization of 
cationic species through ionic interactions. Thus, cationic species such 
as amines, amino acids, basic proteins, and the like can be immobilized on 
the copolymer surface or in the copolymer structure. It is believed that 
polycations are generally immobilized on the surface, whereas smaller 
cations are often found throughout the copolymer. For example, compound I 
(FIG. IA) is bound to about 80% of the sulfonate groups in a sulfonated 
copolymer film. Moreover, non-cationic species can be derivatized to yield 
a cationic derivative. For example, a non-cationic protein can be made 
cationic by conversion of carboxylates (Glu and Asp) to amines with 
ethylenediamine, or by addition of a polylysine "tail" to the protein. 
Other compounds can similarly be modified to provide cationic derivatives 
by methods known in the art. 
For diffusional release, the rate of release is determined, at least in 
part, by how strongly the drug is bound to polymer. There are several ways 
to control the rate of release. In a preferred embodiment, the sulfonation 
level of the subject copolymer is modified. Lower sulfonation increases 
the hydrophobicity of the sulfonated polystyrene (S-PS) phase and can lead 
to stronger binding of a hydrophobic, positively-charged drug. Also, the 
degree of swelling of the polymer is generally lower at lower sulfonation, 
making drug diffusion slower. It will be apparent to the skilled artisan 
that drugs can be selected or modified to obtain a desired (e.g., 
preselected) rate of release from a selected copolymer. 
Other methods of controlling the rate of diffusional release can also be 
employed. For example, in the method described above, S-PS lamellae can 
function, at least in part, as cation-exchange regions, where as a 
cationic drug is released, cations such as Na.sup.+ or K.sup.+ (e.g., 
from the serum) diffuse into the polymer to replace the released drug. In 
an alternative embodiment, the counter-ion for the S-PS phase can be a 
"modifier cation," such as a quaternary ammonium surfactant, which is 
tightly bound and not released from the polymer. The function of the 
"modifier cation" is to "tune" or adjust the hydrophilic/hydrophobic 
balance in the copolymeric lamellae for binding of various drugs which are 
not readily released from the native copolymer, in which a typical 
counter-ion is Na.sup.+. A partial exchange (e.g., 10-20%) of native 
Na.sup.+ with a modifier cation, followed by exchange of the remaining 
Na.sup.+ with a cationic drug, can produce a release rate considerably 
modified from the release rate of the native copolymer, permitting a 
fine-tuned rate of release. 
While a therapeutic agent can be released by diffusion, solubilization, and 
the like, it is also possible to cause release of an agent by applying an 
electric field to the copolymer entrapping the drug. The electric field 
can be weak, preferably in the range of 5-20 volts/cm. Modulation can be 
controlled by the hydrophobicity of molecular species lining the S-PS 
lamellae (as described above) and by controlling redox reactions that 
fine-tune the ionic conductivity (see, e.g., Example Three below). Thus 
the sulfonated copolymer can be used as a material for pulsatile release, 
in which periodic releases of a therapeutic agent can be controlled by 
application of pulses of electric current to the copolymer. In this way 
the sulfonated copolymer can be used as a field-stimulated gate, releasing 
chemicals on demand. 
Yet another method for controlling release derives from the electrostatic 
qualities of the inventive membrane. Ferrofluids (available from, e.g., 
Ferrofluidics Inc.) are single-domain magnetic particles (ca. 100 .ANG. in 
diameter) that have organic surface functionality. Immersing the polymer 
film in a solution of a cationic ferrofluid leads to electrostatic 
attachment of the magnetic particles to surface sulfonates, rendering the 
membrane deformable in a magnetic field. By oscillating a magnet field 
near the film, a rapid sinusoidal deflection of the polymer can produce 
pulsatile release. 
Furthermore, it is possible to immobilize compounds by "layering" materials 
on the sulfonated copolymer surface. For example, a polycation, such as 
polylysine, can be immobilized on the anionic copolymer surface, as 
described above. A polyanionic material, such as chondroitan sulfate, can 
then be immobilized to the polycation layer. In this fashion, a 
hydrophobic charged environment can be created which is similar to ECM. 
Such an environment could be used to promote the growth of islets cells, 
osteoblasts, and the like. Artificial organs, such as an artificial 
pancreas, can therefore be made according to the present invention. The 
sulfonated multiblock copolymer also functions as a size-exclusion 
membrane, and therefore could allow oxygen and nutrients to reach the 
cells, and insulin to pass out of the artificial organ, when implanted in 
vivo. In addition, the surface ionic charge density can be increased by 
"layering", e.g., by first binding a polycation, such as poly(lysine), to 
a lightly sulfonated surface, and then binding a more heavily sulfonated 
polymer, e.g., sulfonated polystyrene, to the polycation layer. 
In another preferred embodiment, the polyanion is a nucleic acid. In this 
embodiment, the invention provides a method of administering a nucleic 
acid construct to a cell, by contacting a cell with a nucleic acid 
construct immobilized on a polycationic surface which is, immobilized on a 
sulfonated copolymer. Gene therapy by delivery of nucleic acid/polycation 
complexes to cells is well known (see, e.g., U.S. Pat. No. 5,166,320 to 
Wu). 
It will be appreciated that the mode of immobilization of the therapeutic 
agent will be selected to ensure desirable characteristics of the 
resulting copolymer. For example, the skilled artisan will be able to 
incorporate the therapeutic agent in an amount or concentration sufficient 
to ensure that a therapeutically effective amount of the agent will be 
released to the subject when the copolymer is implanted into (or otherwise 
contacted with) the subject. Also, copolymer, therapeutic agent, and mode 
of immobilization or incorporation will be selected to ensure that the 
release of the agent occurs in a controlled fashion. 
In still other embodiments, the invention provides copolymers which are 
useful as biosensors. In Example 2, the immobilization of redox-active 
molecules in a sulfonated multiblock copolymer is demonstrated. A 
copolymer incorporating a redox-active molecule is useful as a biosensor 
where electrical communication between an electrode and a redox-active 
enzyme cofactor is important. 
It will also be appreciated that the above aspects of the invention can be 
combined. For example, a cardiovascular implant can be coated with a 
sulfonated copolymer which incorporates a therapeutic drug (for example, 
an antiinflammatory drug). Such an implant would combine 
antithrombogenicity with the ability to improve endothelial cell growth on 
the implant surface, while releasing an antiinflammatory drug to speed the 
healing process. Another example of a combination is an implant which 
features an immobilized TGF-.beta. superfamily factor (e.g., bone 
morphogenic protein (BMP)) in the sulfonated copolymer. Such an implant 
would act to stimulate the growth and morphogenesis of bone on the implant 
surface through the additive effect of the natural growth factor 
derivative of demineralized bone and the bone-matrix stimulating effect of 
the sulfonated copolymer. 
The mechanical strength or other qualities of the sulfonated multiblock 
copolymers of the invention is at least partly dependent on the degree of 
sulfonation of the copolymer, and can be adjusted by the inclusion of 
additives. For example, the addition of polyterpenes increases the 
stiffness or rigidity of the copolymer. Addition of multivalent cations, 
such as multivalent metal ions (e.g., Al.sup.3+, Zn.sup.2+, and the like) 
or organic cations or polycations, such as polylysine, also increases the 
stiffness of the copolymer. Other methods of stiffening the subject 
sulfonated copolymers include radiation cross-linking, for example with 
gamma rays or high-energy electrons. A stiffer copolymer can be desirable 
for those applications which require such stiffness for the production of 
the desired article, that is, where working of the article is more readily 
accomplished on a rigid workpiece. Furthermore, addition of appropriate 
additives can increase the mechanical strength of the copolymer 
sufficiently to permit the use of the copolymer to form a finished article 
without an underlying substrate. For example, a heart valve must be rigid 
and strong to function efficiently and to prevent failure when implanted; 
thus, a strong, rigid copolymer is necessary, and may be obtained by 
appropriate choice of additives. The skilled artisan will be able to 
determine additives appropriate for imparting desired qualities to the 
copolymers of the invention. 
The subject copolymers also exhibit electromechanical properties; that is, 
application of a weak electric field causes the copolymer to move, bend, 
or deform in response to the field. This property is useful for providing 
implantable devices which can be modified in situ by application of an 
electric field, thus providing an implant which can be customized, 
adapted, or modified after implantation without removing the implant and 
without need for surgical intervention. 
Other properties of the subject sulfonated copolymers useful in the present 
invention can also be optimized for particular applications. For example, 
the degree of sulfonation of the copolymer determines, at least in part, 
the relative hydrophobicity of the copolymer, and the charge density on 
the copolymer surface. That is, the greater the extent of sulfonation of 
the copolymer, the more hydrophilic the copolymer will be, and the more 
anionic groups will be present on the copolymer surface. The extent of 
sulfonation can thus determine properties such as the ability to interact 
with (e.g., retain on the surface or within the copolymer matrix) various 
molecules such as drugs, as well as determining cell affinity for the 
copolymer surface. In preferred embodiments, the copolymer is at least 
20%, 30%, 50%, 70%, or 90% sulfonated. 
EXEMPLIFICATION 
Example 1 
The antithrombogenic properties of the sulfonated multiblock copolymers of 
the invention were demonstrated in an in vitro system. Wells of a standard 
six-well tissue culture dish were coated with sulfonated 
styrene-ethylene/butylene-styrene triblock copolymers of the invention 
according to standard techniques. The copolymers differed in the extent of 
sulfonation; both 30% and 74% sulfonated copolymers were tested. Control 
wells were not coated with a copolymer; however, the control wells had an 
anionic (carboxylated) surface as a result of the standard manufacturing 
process. A glass tube was used as an uncharged control surface. 
To each well (and the glass tube) was added freshly drawn human blood. The 
time required for the blood in each well (or tube) to clot was measured; 
the wells were stirred periodically to determine fibrin and clot 
formation. The results are shown in the Table: 
TABLE 
______________________________________ 
Treat- 
Time (in munutes) 
ment 0 8 10 15 20 25 30 35 40 45 55 
65 75 
______________________________________ 
anionic 
NC NC NC F C C C C C C C 
C C 
(uncoat- 
ed well) 
glass 
NC C C C C C C C C C C C C 
tube 
30% NC NC NC NC NC F small C C C C C C 
sulfo- clot 
nation 
74% NC NC NC NC NC NC NC NC NC NC NC NC C 
sulfo- 
nation 
______________________________________ 
NC = No clot formed 
F = Fibrin appears 
C = Clot formed 
It can be seen from the Table that sulfonated 
styrene-ethylene-butylene-styrene block copolymer significantly retards 
fibrin formation and clotting. The more highly sulfonated copolymer shows 
greater antithrombogenic activity than the less sulfonated copolymer; both 
copolymers retard clotting more than the anionic control well or the 
untreated control surface. 
Example 2 
The ability of the sulfonated styrene-ethylene-butylene-styrene block 
copolymers of the invention to immobilize molecules of interest was 
demonstrated in the following in this experiment. 
The redox-active molecules shown in FIG. 1A were immobilized in the 
sulfonated triblock copolymer of styrene-ethylene/butylene-styrene. The 
copolymers thus formed were examined by cyclic voltammetry to determine 
whether the added molecules were incorporated and remained redox-active. 
In each case, the molecules were shown to be active by cyclic voltammetry. 
The compounds shown in FIG. 1B were also shown to be incorporated by the 
sulfonated triblock copolymer of styrene-ethylene/butylene-styrene. 
Example 3 
FIG. 2 depicts a cell which allows for the measurement of ionic 
conductivity through a membrane including various molecules. The cell 1 
includes cathode side 3 and anode side 5, each side having an electrode 
(10, 10'), e.g., a platinum electrode, and the two sides 3, 5 being 
separated from each other by a membrane (20) of a material to be tested. 
The electrodes 10, 10' are connected to a source of electric current (not 
shown) through leads 14, 14'. Each half 3, 5 of the cell 1 is filled with 
an electrolyte solution (12), and the cell is supported by a clamp (30). 
The following observations were made with this cell: a) The compound 
trimethylaminomethylferrocene, which was ion-exchanged into block 
copolymer film (styrene-ethylene/butylene-styrene, about 50% sulfonated), 
was released from the polymer when a field of about 10 V/cm was applied. 
It was released into the cathode (-) side of the cell, as evidenced by the 
appearance of a yellow color on the cathode side. This result may reflect 
electrophoretic movement of the cationic ferrocene compound toward the 
cathode, or it might be due to a change in the S-PS phase of the polymer, 
which may expand due to the applied field, facilitating release. b) When 
the membrane 20 described above contains trimethylaminomethyl-ferrocene, 
the membrane still allows ions to pass; however, a membrane containing the 
more hydrophobic dimethylheptylaminomethylferrocene appears to have lower 
ionic conductivity. This observation suggests that the S-PS lamellae have 
been lined or modified with hydrophobic molecules, leading to a 
hydrophobic environment that slows or prevents movement of aqueous salt 
ions through the membrane, and consequent low conductivity. This suggests 
that a "modifier cation," as described above, could be useful in 
controlling the permeability of the membrane and hence the rate at which 
drugs are released through the membrane. Moreover, it suggests that ionic 
conductivity can be increased by oxidizing ferrocene to the ferrocenium 
cation, which would increase the hydrophilicity and increase the ionic 
conductivity. Since this redox process is reversible, membrane 
permeability to aqueous solutions can be controlled simply by application 
of appropriate electric potential to the membrane, creating an intelligent 
membrane which acts as a gate for controlled drug delivery. 
Example 4 
The ability of endothelial cells to attach, grow, and express a 
differentiated phenotype was demonstrated in an in vitro system. In this 
experiment, standard 24-well tissue culture plates were utilized. Enough 
wells in each plate to provide six replicates of each condition were 
coated with 150 microliters of solutions of the sulfonated copolymer of 
the invention at each of three different sulfonation levels. After polymer 
treatment, the wells were rinsed with sterile distilled water for thirty 
minutes, followed by three rinses with Hank's Balance Salt Solution. 
Controls consisted of wells of normal culture ware (that is, culture ware 
that is treated to allow cell adhesion and cell growth, by carboxylation 
of the surface, thereby rendering the surface anionic) that were coated 
with the subject sulfonated styrene-ethylene/butylene-styrene copolymer, 
and, as a negative control, culture dishes obtained from the manufacturer 
that had not been treated to promote adhesion and growth of cells 
(untreated controls). Into each of these wells were introduced 100,000 
porcine aortic endothelial cells in 1 ml of M199 medium (Sigma Chemical 
Co., St. Louis, Mo.) with 10% fetal bovine serum (FBS), leaving at least 
two wells per condition inoculated just with medium to serve as a blank. 
Plates were then placed in an incubator at 37.degree. C., with an 
atmosphere containing 5% CO.sub.2, for 48 hours. After the incubation 
period the medium was removed from each well, spun down in a centrifuge, 
and frozen separately to use in the phenotype expression assays. 500 ml of 
medium were added back to each well to allow a cell proliferation assay 
(CellTiter AQueous, Promega, Inc., Madison, Wiss.) to be performed. 
Microscopic examination demonstrated that aortic endothelial cells become 
attached to surfaces, such as microtiter wells, treated with the subject 
copolymer, and have the appearance of normal cells. In contrast, untreated 
control wells do not promote endothelial cell attachment. FIGS. 3 and 4 
are graphs showing the results of the cell proliferation assay. As 
indicated in FIG. 3, the proliferation of cells on the sulfonated polymer 
is related to the degree of sulfonation in the polymer (21%, 57% or 72%; 
greater sulfonation of the polymer increases cell proliferation), and FIG. 
4 indicates that the proliferation of aortic endothelial cells at the 
highest percentage sulfonation utilized compares favorably to the 
commercially-available anionically charged polystyrene culture dish, which 
is designed to promote cell adhesion and growth (first column: uncharged 
polystyrene culture dish; second column: commercially-available anionic 
polystyrene culture dish; third column: 72% sulfonated 
styrene-ethylene/butylene-styrene block copolymer-treated culture dish). 
FIG. 5 shows the results of a phenotypic expression assay on the incubation 
media of the experimental and control conditions described above. The 
assay utilized was the Biotrak 6-keto-prostaglandin F 1a EIA. The compound 
6-keto-prostaglandin is normally produced by endothelial cells, but is not 
produced if the endothelial cells dedifferentiate. As can be seen from 
FIG. 5, the cells seeded on the sulfonated copolymer all continued to 
express the endothelial phenotype, and did not appear to dedifferentiate 
(first column: uncharged polystyrene culture dish; second column: 
commercially-available anionic polystyrene culture dish; third column: 72% 
sulfonated styrene-ethylene/butylene-styrene block copolymer-treated 
culture dish). 
These experiments illustrate the efficacy of the sulfonated copolymers of 
the invention in normal cell growth and adhesion where endothelialization 
is a desirable feature of the material. 
Example 5 
A piece of Teflon sheet (Xytex, Norton Co.) with 10 micron pore structure 
was dipped in an approximately 3% (w/w) solution of the sulfonated 
styrene-ethylene-butylene-styrene block copolymer (about 50% sulfonated), 
shaken to remove liquid droplets, and dried for several hours. The treated 
material was noticeably different to the touch compared to the untreated 
Teflon sheet. The wettability of the treated sheet was also altered 
compared to an untreated control: water beaded on the untreated porous 
Teflon, but wetted (and was absorbed into) the Teflon-sulfonated copolymer 
hybrid. The hybrid material retains the strength and resilience of porous 
Teflon, but acquires characteristics of the sulfonated polymer (e.g., a 
non-thrombogenic surface, ability to grow and develop viable and healthy 
tissues on the surface, and ability to immobilize or deliver biologically 
important substances). Where infiltration of the sulfonated copolymer 
phase into a second material is slow, as where the second material has 
small pores, the copolymer can be driven into the porous structure 
electrophoretically. 
The contents of all references and patents cited herein are hereby 
incorporated by reference. 
Those skilled in the art will recognize, or be able to ascertain using no 
more than routine experimentation, numerous equivalents to the specific 
procedures described herein. Such equivalents are considered to be within 
the scope of this invention and are covered by the following claims.