Note: Descriptions are shown in the official language in which they were submitted.
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CROSSLINKABLE MACROMERS
Technical Field
The present invention relates to the preparation of matrices by the
polymerization of
macromers. In another aspect, the invention relates to the use of such
matrices for such
purposes as cell immobilization, tissue adherence, and controlled drug
delivery.
Background of the Invention
Matrices are polymeric networks characterized by insolubility in water. One
type of
polymeric matrix is a hydrogel, which can be defined as a water-containing
polymeric
network. The polymers used to prepare hydrogels can be based on a variety of
monomer
types, such as those based on methacrylic and acrylic ester monomers,
acrylamide
(methacrylamide) monomers, and N-vinyl-2-pyrrolidone. To form the gel, these
monomer
classes are typically crosslinlced with such crosslinking agents as ethylene
dimethacrylate,
N,N'-methylenebisacrylamide, methylenebis(4-phenyl isocyanate), ethylene
dimethacrylate,
divinylbenzene, and allyl methacrylate.
Another type of polymeric network can be formed from more hydrophobic monomers
and/or macromers. Matrices formed from these materials generally exclude
water. Polymers
used to prepare hydrophobic matrices can be based on a variety of monomer
types such as
alkyl acrylates and methacrylates, and polyester-forming monomers such as s-
caprolactone
and lactide. When formulated for use in an aqueous environment, these
materials do not need
to be crosslinked, but they can be crosslinlced with standard agents such as
divinyl benzene.
Hydrophobic matrices can also be formed from reactions of macromers bearing
the
appropriate reactive groups such as the reaction of diisocyanate macromers
with dihydroxy
macromers, and the reaction of diepoxy-containing macromers with dianhydride
or diamine-
containing macromers.
Although there exist a variety of methods for producing polymeric networks,
when
these networlcs are intended to be created in the presence of viable tissue,
and/or to contain a
bioactive compound, the number of acceptable methods of producing polymeric
networks is
extremely limited.
It is nevertheless desirable to form both hydrogel and non-hydrogel polymeric
matrices in the presence of viable tissue or bioactive agents for the purposes
of drug delivery,
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2
cellular immune isolation, prevention of post-surgical adhesions, tissue
repair, and the like.
These polymeric matrices can be divided into two categories: biodegradable or
bioresorbable
polymer networks and biostable polymer networks.
Biodegradable polymeric matrices have been previously suggested for a variety
of
purposes, including controlled release carriers, adhesives and sealers. When
used as
controlled release carriers, for instance, polymeric matrices can contain and
release drugs or
other therapeutic agents over time. Such matrices can be formed, for instance,
by a number
of different processes, including solvent casting hydrophobic polymers.
Solvent casting,
however, typically involves the use of orga~lic solvents and/or high
temperatures which can
be detrimental to the activity of biological materials and can complicate
production methods.
Solvent casting of polymers out of solution also results in the formation of
uncrosslinlced
matrices. Such matrices have less structure than crosslinked matrices and it
is more difficult
to control the release of bioactive agents from such matrices. Yet another
process, which
involves the polymerization of monomers in or around the desired materials,
suffers from
cytotoxicity of monomers, oxygen inhibition and heat of polymerization
complications.
Another process used in the past to prepare biodegradable and biostable
hydrogels
involves the polymerization of preformed macromers using low molecular weight
iutiators.
This process involves a number of drawbacks as well, however, including
toxicity, efficacy,
and solubility considerations. For instance, when using a macromer solution
containing a low
molecular weight soluble initiator to encapsulate viable cellular material,
the initiator can
penetrate the cellular membrane and diffuse into the cells. The presence of
the initiator may
involve some toxic consequence to the cells. When activated, however, these
initiators
produce free radicals having distinct cytotoxic potential. Other drawbacks
arise if the initiator
is able to diffuse out of the formed matrix, thereby producing toxicity and
other issues. Such
initiators also tend to aggregate in aqueous solution, causing efficiency and
reproducibility
problems. Finally, in view of the limited efficiency of many initiators for
initiating the
necessary radical chain polymerization, it is often necessary to add one or
more monomeric
polymerization "accelerators" to the polymerization mixture. Such accelerators
tend to be
small molecules capable of penetrating the cellular membrane, and often raise
cytotoxic or
carcinogenic concerns.
U.S. Patent Nos. 5,410,016 (Hubbell, et al.) and 5,529,914 (Hubbell, et al.)
for
instance, relate to hydrogels prepared from biodegradable and biostable
polymerizable
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3
macromers. The hydrogels are prepared from these polymerizable macromers by
the use of
soluble, low molecular weight initiators. Such initiators can be combined with
the
macromers, and irradiated in the presence of cells, in order to form a gel
that encapsulates the
cells. A considerable number of similar and related patents have arisen over
recent years.
See, for instance, US Patent Nos. 5,232,984; 5,380,536; 5,573,934; 5,612,050;
5,837,747;
5,846,530; and 5,858,746.
Hydrogels often suffer from similar or other drawbacks in use as biological
adhesives
or sealants, e.g., for use as tissue adhesives, endovascular paving,
prevention of post-surgical
adhesions, etc. In each of the applications, the hydrogel matrix must
generally "adhere" to
one or more tissue surfaces. Current methods rely upon physical "adhesion" or
the tendency
of hydrogels to "stick" to a surface. A superior adhesive would provide both
physical and
chemical adhesion to surfaces utilizing the same physical characteristics as
current hydrogel
adhesives, but also providing chemical, covalent coupling of the matrix
material to the tissue
surface. Covalent bonds are generally much stronger than physical adhesive
forces, such as
hydrogen bonding and van der Waals forces.
As described above, when various techniques are used to form polymeric
matrices via
photoinitiation of macromers, the photoinitiators utilized tend to be low
molecular weight.
Polymeric photoinitiators have been described as well, although for
applications and systems
quite distinct from those described above. See, for instance, "Radical
Polymerization", C.H.
Bamford, pp. 940-957 in Kroschwitz, ed., Concise Encyclopedia of Polymer
Science and
Engineering, 1990. In the subsection entitled "Photosensitized Initiation:
Polymeric
Photosensitizers and Photoinitiators", the author states that "[p]olymeric
photosensitizers and
photoinitiators have been described. Many of these are polymers based on
benzophenone,
e.g., poly(p-divinylbenzophenone) (DVBP). Such rigid polymers are reported to
be effective
sensitizers since hydrogen abstraction from the backbone by excited
benzophenone is less
likely."
U.S. Patent No. 4,315,998 (Neckers) describes polymer-bound photosensitizing
catalysts for use in the heterogeneous catalysis of photosensitized chemical
reactions such as
photo-oxidation, photodimerization, and photocyclo addition reactions. The
polymer-bound
photosensitizing catalysts are insoluble in water and common organic solvents,
and therefore
can be readily separated from the reaction medium and reaction products by
simple filtration.
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What is clearly needed are macromers and macromer systems that avoid the
problems
associated with conventional polymeric matrices, and in particular, those
drawbacl~s that arise
when polymeric matrices are formed in the presence of viable tissue or
bioactive agents.
Summary of the Invention
The present invention provides a method of improving the performance, within a
host
tissue site, of an implanted article (e.g., implanted tissue or implanted
prosthetic device
providing a porous surface), by promoting the growth of continuous tissue
between host
tissue and the implant. An implanted article of this invention can be
provided, for instance,
in the fore of cultured tissue and/or native tissue (e.g., transplanted
tissue), or can be
fabricated from polymeric and/or metallic materials. The method employs a
crosslinl~able
macromer system adapted to form an interface between the article and the host
tissue, in order
to facilitate (e.g., promote and/or permit) tissue integration by the body
between the implant
and the tissue site, e.g., into and tluough the crosslinl~ed macromer and into
the porosity of a
porous device surface.
Applicant's parent application Serial No. 09/121,248 (now US Patent No.
6,007,33)
provides examples of preferred macromer systems useful in the method of the
present
invention. The parent application teaches the use of such systems for various
applications,
including cellular encapsulation, adhesives and sealants, barriers, controlled
release carriers,
tissue replacement/scaffolding, wound dressings, and in situ device formation.
For use in
tissue replacement, for instance, the parent application teaches the placement
of a macromer
system in a mold or cavities in a device.
Polymeric matrices of this invention (as formed by crosslinlcing the macromer
system)
can be used, for instance, to improve the tissue response to implanted medical
devices.
Examples of improved tissue response include: 1) increased tissue ingrowth
(e.g., into the
pores of cementless hip implants), and, 2) decreased fibrosis (e.g., around
breast implants and
hernia repair meshes). In addition to tissue integration, the macromer system
can provide a
variety of advantages, including a reduction in both immediate and chronic
adverse reactions
to the device (e.g., thereby preventing the accumulation of fluid and/or
undesirable cells) by
virtue of the immediate and desirable space filling characteristics.
A macromer system of this invention can be provided between the implant and
tissue
site in any suitable manner, e.g., the system can be provided upon the surface
of the implant
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before, during and/or after placement of the implant in the tissue site.
Similarly, the
macromer systems) can be polymerized to form a matrix at any suitable point,
e.g., a system
can be coated on an implant, and/or delivered to the tissue site, and
crosslinked either before,
during and/ or after positioning the implant in or upon the tissue site. For
example, a system
5 can be provided and crosslinked on the surface of a prosthetic device, e.g.,
by either the
manufacturer or surgeon, and implanted into the tissue site. As another
example, a macromer
system can be applied to the tissue site itself, polymerized in situ (e.g., by
the application of
illumination either directly to the system or through translucent tissue), and
an implant article
(with or without a macromer system or matrix on its surface) positioned on or
in the site.
The resulting combination of implant and matrix within the tissue site,
permits the
formation of continuous tissue growth, over time, through the matrix and
between the implant
and tissue site. Such growth can be unidirectional (e.g., originating from the
tissue site
toward the implant) and/or bidirectional (e.g., originating from both the
tissue site and from
an implanted tissue).
A system, or resultant matrix, can be applied with or without additional
components,
such as growth factors, morphogenic factors, or DNA. Both degradable and non-
degradable
macromer systems are useful, but matrices adapted to degrade with desired
kinetics, are
preferred.
hl another aspect, the present invention provides a combination comprising an
implant
article, such as a tissue implant or prosthetic device having one or more
porous surfaces, and
a matrix formed of a crosslinked macromer system as described herein. In one
embodiment,
the article and uncrosslinked macromer system are initially combined (e.g.,
the system coated
on the article) outside the body, thereby permitting the system to be
crosslinlced prior to,
during and/or following placement of the implant within the body site. In an
alternative
embodiment, the combination is provided by positioning an article within
and/or in
apposition to the body site, and delivering the macromer system to the body
site (before,
during and/or after placement of the article itself), where it is crosslinlced
as a sufficiently
stable coating or other suitable interface in its position between the article
and the tissue site.
For instance, a macromer system in liquid (e.g., substantially flowable) form
can be
applied to a tissue defect, before, during or after which a tissue implant or
preformed device
is pressed into the defect. The system can be provided, for instance, to
occupy a space of
between about 0.1 mm to about 10 mm between the implant and the tissue site.
The system is
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illuminated in order to activate the initiator groups and thus polymerize the
macromer system
into a solid matrix. The resultant combination of tissue implant or preformed
device, together
with crosslinked macromer system, completely and conformally fills the defect
such that no
gaps remain for the accumulation of undesirable fluids or cells.
As described herein, the macromer system can thus be used in the manner of a
"grout", for instance, to fill the spaces between a tissue implant or
preformed device (itself
either tissue-based or non-tissue based) and adjacent tissue. Current tissue
implants include,
for instance, both those obtained as transplants (e.g., autografts, allografts
or xenografts) and
those provided by tissue engineering. Current tissue engineering products
often consist of
cultured tissues that are implanted into tissue defects. Such products do not
typically conform
well to adjacent native tissue, however, thus leaving spaces into which
undesirable fluids and
cells can accumulate and produce adverse tissue responses. For example, when
cultured
cartilage is implanted into cartilage defects, synovial fluid and macrophages
can enter the
unfilled space and lead to fibrous tissue formation, which prevents
integration of the
implanted cartilage with the native cartilage. Other cultured tissues that are
implanted into
tissue defects, and that would benefit from the present macromer system
applied as a grout
include, but are not limited to, skin, bone, ligaments, blood vessels, and
heart valves.
Implants, e.g., prosthetic devices, useful in a combination or method of this
invention
include those in which tissue integration is desired, and that themselves
provide (or can be
provided with) a sufficiently porous surface that permits or facilitates
tissue integration once
positioned in vivo. Examples of suitable porous prosthetic devices include,
but are not
limited to; joint implants (e.g., for hip or l~nee reconstruction), dental
implants, soft tissue
cosmetic prostheses (e.g., breast implants), wound dressings, vascular
prostheses (e.g.,
vascular grafts and stems), and ophthalmic prostheses (e.g., intracorneal
lenses). The
macromer system of this invention, in turn, can be used in any suitable
manner, e.g., to coat
and/or fill voids within or upon the surface of the prosthetic device.
Such devices preferably are themselves forned of or otherwise provide (or can
be
provided with) a surface having sufficient porosity to permit tissue ingrowth
ifa vivo. As used
herein, the word "porous", and inflections thereof, will refer to one or more
portions of the
device surface that are designed for direct or indirect contact with the
surrounding natural
tissue, sufficient to permit tissue integration into the porosity thereof.
Porous surfaces can be
provided in a variety of ways, e.g., as sintered particles on a surface, such
as titanium particles
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on the surface of cementless hip implants, as are available from a variety of
orthopedic
companies. Porous surfaces can also be provided in the form of cavities that
remain from
mixing salt crystals with silicone rubber oligomers, then solidifying
(vulcanizing) the silicone
rubber, and finally dissolving the salt crystals (as currently done for a
variety of breast
implants). Yet other porous surfaces can be fabricated from fibrous materials
or produced as
porous sponges via solvent casting and particulate leaching, phase separation,
or gas foaming
(see, e.g., B.S. Kim and D.J. Mooney, Development of Biocompatible Synthetic
Extracellular
Matricies for Tissue Engineering TIBTECH 16:224-230 (1990.
Such porous surfaces typically provide a three-dimensional structure of spaces
into
which tissue can grow and mature. Porous regions of an implantable device
preferably have a
high pore density, in that the pores themselves occupy a greater relative
volume than the
material forming and separating those pores. Desirably, the pores have
interconnected
passages that allow direct contact between the tissue growing in adjacent
pores. The
minimum average pore size is preferably sufficient to accommodate capillaries
(e.g., of about
5 micron diameter) and the maximum average pore size is about lmm. Preferable
pore sizes
will vary from tissue to tissue and typically range from about 20 microns to
about 600
microns, and more preferably from about 50 microns to about 400 microns.
A crossliucable macromer system useful in the present invention composes two
or
more polymer-pendent polymerizable groups and one or more initiators,
preferably in the
form of polymer-pendent initiator groups. In a preferred embodiment, the
polymerizable
groups and the initiator groups) are pendent on the same polymeric backbone.
In an
alternative preferred embodiment, the polymerizable groups and initiator
groups) are pendent
on different pol~nneric backbones.
In the first embodiment, the macromer system comprises a polymeric backbone to
which are covalently bonded both the polymerizable groups and initiator
group(s). Pendent
initiator groups can be provided by bonding the groups to the backbone at any
suitable time,
e.g., either prior to the formation of the macromer (for instance, to monomers
used to prepare
the macromer), or to the fully formed macromer itself. The macromer system
itself will
typically comprise but a small percentage of macromers bearing both initiator
groups and
polymerizable groups. The majority of macromers will provide only pendent
polymerizable
groups, since the initiator groups are typically sufficient if present at far
less than 1:1
stoichiometric ratio with macromer molecules.
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In an alternative prefeiTed embodiment, the macromer system comprises both
polymerizable macromers, generally without pendent initiator groups, in
combination with a
polymeric initiator. In either embodiment, the initiator will be referred to
herein as a
"polymeric initiator", by virtue of the attachment of such initiator groups to
a polymeric
backbone. Yet another embodiment of this invention includes the macromer
system having
free (non-polymer bound) initiator molecules.
Macromer systems of the present invention, employing polymeric initiators,
provide a
number of unexpected advantages over the use of polymerizable macromers and
separate, low
molecular weight initiators. Such systems, for instance, provide an optimal
combination of
such properties as nontoxicity, efficiency, and solubility. Solubility, for
instance, can be
improved by virtue of the ability to control the aqueous or organic solubility
of the
polylnerizable macromer by controlling the backbone. Toxicity can also be
improved, since
the polymeric initiators of this invention typically cannot diffuse into cells
in the course of
immobilization.
In a preferred embodiment, the pendent initiator groups are selected from the
group
consisting of long-wave ultra violet (LWUV) light-activatable molecules such
as; 4-
benzoylbenzoic acid, [(9-oxo-2-thioxanthanyl)-oxy]acetic acid, 2-hydroxy
thioxanthone, and
vinyloxymethylbenzoin methyl ether; visible light activatable molecules; eosin
Y, rose
Bengal, camphorquinone and erythrosin, and thermally activatable molecules;
4,4' azobis(4-
cyanopentanoic) acid and 2,2-azobis[2-(2-imidazolin-2-yl) propane]
dihydrochloride. An
important characteristic of the initiator group being the ability to be
coupled to a preformed
macromer containing polymerizable groups, or to be modified to form a monomer
which can
take part in the macromer synthesis, which is subsequently followed by the
addition of
polymerizable groups.
In such an embodiment, the pendent polymerizable groups are preferably
selected
from the group consisting of pendent vinyl groups, acrylate groups,
methacrylate groups,
ethacrylate groups, 2-phenyl acrylate groups, acrylamide groups,
methacrylamide groups,
itaconate groups, and styrene groups.
In a further preferred embodiment, the polymeric backbone is selected from the
group
consisting of synthetic macromers, such as polyvinylpyrrolidone (PVP),
polyethylene oxide
(PEO), and polyethylene glycol (PEG); derivatizable naturally occurring
polymers such as
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cellulose; polysaccharides, such as hyaluronic acid, dextran, and heparin; and
proteins, such
as collagen, gelatin, and albumin.
The macromers of the present invention can be used in a variety of
applications,
including controlled drug release, the preparation of tissue adhesives and
sealants, the
immobilization of cells, and the preparation of three-dimensional bodies for
implants. In one
aspect, for instance, the invention provides a method for immobilizing cells,
the method
comprising the steps of combining a polymeric initiator of the present
invention with one or
more polyrnerizable macromers and in the presence of cells, under conditions
suitable to
polymerize the macromer in a mamler that immobilizes the cells.
Detailed Description
As used herein the following words and terms shall have the meaning ascribed
below:
"macromer system" shall refer to a polymerizable polymer system comprising one
or
more polymers providing pendent polymerizable and initiator groups. Groups can
be present
either on the same or different polymeric backbones, e.g., on either a
polymerizable
macromer or a non-polymerizable polymeric backbone;
"polymerizable macromer" shall refer to a polymeric backbone bearing two or
more
polymerizable (e.g., vinyl) groups;
"initiator group" shall refer to a chemical group capable of initiating a free
radical
reaction, present as either a pendent group on a polymerizable macromer or
pendent on a
separate, non-polylnerizable polymer backbone; and
"polymeric initiator" shall refer to a polymeric backbone (polymerizable or
non-
polymerizable) comprising one or more initiator groups and optionally
containing one or
more other thermochemically reactive groups or affinity groups.
The polymeric backbone of this invention can be either synthetic or naturally-
occurring, and includes a number of macromers previously described as useful
for the
preparation of polymeric matrices. Generally, the backbone is one that is
soluble, or nearly
soluble, in aqueous solutions such as water, or water with added organic
solvent (e.g.,
dimethylsulfoxide) or can be rendered soluble using an appropriate solvent or
combination of
solvents. Alternatively, the polymeric backbone can be a material which is a
liquid under
ambient physiological conditions. Backbones for use in preparing biodegradable
gels are
preferably hydrolyzable under i~c vivo conditions.
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In general, the polymeric backbones of this invention can be divided into two
categories: biodegradable or bioresorbable, and biostable reagents. These can
be further
divided into reagents which form hydrophilic, hydrogel matricies and reagents
which form
non-hydrogel matricies.
Bioresorbable hydrogel-forming backbones are generally naturally occurring
polymers
such as polysaccharides, examples of which include, but are not limited to,
hyaluronic acid
(HA), starch, dextran, heparin, chondroitin sulfate, dermatan sulfate, heparan
sulfate, lceratan
sulfate, dextran sulfate, pentosan polysulfate, and chitosan; and proteins
(and other polyamino
acids), examples of which include but are not limited to gelatin, collagen,
fibronectin,
10 laminin, albumin, elastin, and active peptide domains thereof. Matrices
formed from these
materials degrade under physiological conditions, generally via enzyme-
mediated hydrolysis.
Hyaluronic acid, when derivatized with polymerizable groups in the manner
described
herein, provides a variety of advantages and benefits not heretofore known or
achievable.
Hyaluronic acid has conventionally been derivatized in aqueous conditions
(see, e.g., Soon-
Shiong (US Pat. No. 5,837,747), Salcurai (US Pat. No. 4,716,224), and Matsuda
(US Pat. No.
5,763,504). Occasional references have described the ability to derivatize
hyaluronic acid
under organic conditions (see, e.g., Della Valle, US Pat. No. 5,676,964). In
most, if not all,
such approaches, however, the reaction mixtures are either suspensions, as
opposed to true
solutions, or the hyaluronic acid is itself pre-reacted (typically in a
predominantly aqueous
mixture) to enhance its solubility in organic solvents.
Applicants have discovered the manner in which hyaluronic acid, as well as
other
polysaccharides and polyamino acids (such as collagen) can be effectively
derivatized in
organic, polar, anhydrous solvents and solvent combinations. A particularly
preferred solvent
is formarnide, and combinations of other solvents therewith. Functionally, the
solvent or
solvent system is one in which the polymer is sufficiently soluble and that
permits its
derivatization to the desired extent. The ability to derivatize such polymers
in the manner of
this invention provides a variety of unexpected benefits, and an optimal
combination of such
properties as preparation cost, controllability, and yield.
As exemplified below, for instance, hyaluronic acid is reacted in fonnamide
(and
TEA, for pH control) with a reactive moiety in the form of glycidyl acrylate
in order to
derivatize the hyaluronic acid molecules with acrylate groups. The number
and/or density of
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acrylate groups can be controlled using the present method, e.g., by
controlling the relative
concentration of reactive moiety to saccharide group content.
Bioresorbable matrix-forming backbones are generally synthetic polymers
prepared
via condensation polymerization of one or more monomers. Matrix-forming
polymers of this
type include polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL),
as well as
copolymers of these materials, polyanhydrides, and polyortho esters.
Biostable hydrogel matrix-forming backbones are generally synthetic or
naturally
occurring polymers which are soluble in water, matrices of which are hydrogels
or water-
containing gels. Examples of this type of baclcbone include
polyvinylpyrrolidone (PVP),
polyethylene glycol (PEG), polyacrylamide (PAA), polyvinyl alcohol (PVA), and
the lilce.
Biostable matrix-forming backbones are generally synthetic polymers formed
from
hydrophobic monomers such as methyl methacrylate, butyl methacrylate, dimethyl
siloxanes,
and the like. These backbone materials generally do not possess significant
water solubility
but can be formulated as neat liquids which form strong matrices upon
activation. It is also
possible to synthesize baclcbone polymers which contain both hydrophilic and
hydrophobic
monomers.
Polymeric backbones of polynerizable macromers can optionally provide a number
of
desirable functions or attributes, e.g., as described in the above-captioned
Hubbell patents, the
disclosures of which are incorporated herein by reference. Backbones can be
provided with
water soluble regions, biodegradable regions, hydrophobic regions, as well as
polymerizable
regions.
As used herein, the term "polymerizable group" will generally refer to a group
that is
polyrnerizable by initiation by free radical generation, most preferably by
photoinitiators
activated by visible or long wavelength ultraviolet radiation. Preferred
polymerizable groups
include acrylates, methacrylates, ethacrylates, itaconates, acrylamides,
methacrylamide, and
styrene.
Typically, polymerizable groups are incorporated into a macromer subsequent to
the
initial macromer formation using standard thennochemical reactions. Thus, for
example,
polymerizable groups can be added to collagen via reaction of amine containing
lysine
residues with acryloyl chloride or glycidyl acrylate. These reactions result
in collagen
containing pendent polymerizable moieties. Similarly, when synthesizing a
macromer for use
as described in the present invention, monomers containing reactive groups can
be
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incorporated into the synthetic scheme. For example, hydroxyethylmethacrylate
(HEMA) or
aminopropylmethacrylamide (APMA) can be copolymerized with N-vinylpyrrolidone
or
acrylamide yielding a water-soluble polymer with pendent hydroxyl or amine
groups. These
pendent groups can subsequently be reacted with acryloyl chloride or glycidyl
acrylate to
form water-soluble polymers with pendent polymerizable groups.
Initiator groups usefixl in the system of the present invention include those
that can be
used to initiate, by free radical generation, polymerization of the macromers
to a desired
extent and within a desired time frame. Crosslinlcing and polymerization are
generally
initiated among macromers by a light-activated free-radical polymerization
initiator.
Preferred initiators for long wave UV and visible light initiation include
ethyl eosin, 2,2-
dimethoxy-2-phenyl acetophenone, other acetophenone derivatives, thioxanthone,
benzophenone, and camphorquinone.
Preferred polymeric initiators are photosensitive molecules which capture
light energy
and initiate polymerization of the macromers. Other preferred polymeric
initiators are
thermosensitive molecules which capture thermal energy and initiate
polymerization of the
macromers.
Photoinitiation of the free radical polymerization of macromers of the present
invention will generally occur by one of three mechanisms. The first mechanism
involves a
homolytic alpha cleavage reaction between a carbonyl group and an adjacent
carbon atom.
This type of reaction is generally referred to as a Norrish type I reaction.
Examples of
molecules exhibiting Norrish type I reactivity and useful in a polymeric
initiating system
include derivatives of benzoin ether and acetophenone.
The second mechanism involves a hydrogen abstraction reaction, either infra-
or
intermolecular. This initiation system can be used without additional energy
transfer acceptor
molecules and utilizing nonspecific hydrogen abstraction, but is more commonly
used with an
energy transfer acceptor, typically a tertiary amine, which results in the
formation of both
aminoalkyl radicals and lcetyl radicals. Examples of molecules exhibiting
hydrogen
abstraction reactivity and useful in a polymeric initiating system, include
analogs of
benzophenone, thioxanthone, and camphorquinone.
When using a polymeric initiator of the hydrogen abstraction variety, pendent
tertiary
amine groups can be incorporated into the polymeric backbone of the macromer.
This will
insure that all free radicals formed are polymer-bound.
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13
The third mechanism involves photosensitization reactions utilizing
photoreducible or
photo-oxidizable dyes. In most instances, photoreducible dyes are used in
conjunction with a
reluctant, typically, a tertiary amine. The reluctant intercepts the induced
triplet producing
the radical anion of the dye and the radical canon of the reluctant. Examples
of molecules
exhibiting photosensitization reactivity and useful in a polymeric initiating
system include
eosin Y, rose bengal, and erythrosin. Reductants can be incorporated into the
polymer
backbone, thereby assuring that all free radicals will be polymer-bound.
Thermally reactive polyneric initiators are also useful for the polymerization
of
macromers. Examples of thermally reactive initiators usable in a polymeric
initiating system
include 4,4' azobis(4-cyanopentanoic acid) and analogs of benzoyl peroxide.
A surprisingly beneficial effect of the use of polymeric initiators to
polymerize
macromers is the increased efficiency of polymerization exhibited by these
polymeric
initiators as compared to their low molecular weight counterparts. This
increased efficiency
is seen in all three photoinitiation mechanisms useful for the polymerization
of macromers.
Polymeric initiation of monomer solutions has been investigated for its
application in
the field of UV-curable coatings for industrial uses, c.f. U.S. Patent No.
4,315,998 (Neckers)
and PCT Application, W ternational Publication No. WO 97/24376 (Kuester, et
aL) but there
have been no reports of the adaptation of the use of polymeric initiators for
the
polymerization of macromers in the presence of biologic material or for the
creation of drug-
releasing matrices.
High efficiency of initiation is particularly important in systems such as
these. It is
generally desirable, when forming polymeric matrices in the presence of
biologic or bioactive
materials, to minimize the exposure time of the material to the energy source
used to initiate
polymerization. It is therefore imperative that the initiation system utilized
possess optimum
initiation efficiency.
When matrix strength or durability are required for a particular application,
high
efficiency is again a necessary characteristic of an initiation system. When a
matrix-forming
system is initiated, the free radical polymerization of the system is
propagated until gelation
and vitrification of the polymerizing system render the diffusion of the
elements of the
matrix-forming system too difficult. Therefore, the higher the efficiency of
the initiation
system, the more complete the polymerization resulting in the formation of
stronger, more
durable matrices. The polymeric initiation systems described in this invention
provide a
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14
higher degree of efficiency, with or without the use of accelerants, than is
attainable using
nonpolymer-bound, low molecular weight initiators.
Another beneficial effect is realized when the initiating groups on the
polymeric
initiators consist of groups exhibiting hydrogen abstraction reactivity, i.e.,
the ability to
abstract hydrogens intermolecularly. The beneficial effect is important when
macromer
systems containing these initiators are used as tissue adhesives, endovascular
paving,
formation of barriers to prevent post-surgical adhesions, or any application
involving the
"adhesion" of the matrix to one or more surfaces. Since initiators exhibiting
this type of
reactivity can abstract hydrogens from adjacent molecules, when a macromer
system
containing polymeric initiators of this type is applied to a substrate,
photoactivation of the
system causes the abstraction of hydrogens from the substrate by the
initiators, thus forming a
free radical on the substrate and a free radical on the initiator. This
diradical can
subsequently collapse forming a covalent bond between the macromer system and
the
substrate.
Other initiator groups on the same macromer initiate free radical reactions
with other
macromers resulting in the formation of a crosslinked matrix covalently bound
to the surface.
Initiator groups exhibiting this type of reactivity include analogs of
benzophenone and
thioxanthone. As can be readily understood, only polymeric initiators are
capable of
accomplishing this adhesion of the matrix to a surface, low molecular weight
analogs of these
initiators camlot produce this phenomenon.
Optionally, the use of initiators for initiating the necessary radical chain
polymerization, can include the addition of one or more monomeric
polymerization
accelerants to the polymerization mixture, the accelerants serving to enhance
the efficiency of
polymerization. Polymerization accelerants useful in the present invention are
typically
monomers, which improve the reactivity of the macromer systems. Polymerization
accelerants that have found particular utility for this application are N-
vinyl compounds,
particularly N-vinyl pyrrolidone and N-vinyl caprolactam. Such accelerants can
be used, for
instance, at a concentration of between about 0.01 % and about 5%, and
preferably between
about 0.05% and about 0.5%, by weight, based on the volume of the macromer
system.
In another embodiment, the polymeric initiator comprises a polymeric backbone
with
pendent initiator groups and pendent reactive or affinity groups. These
reactive or affinity
groups enable the polymeric initiator to bind to target groups on surfaces of
interest. This
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allows the polymeric initiator to bind to the surface of interest. In this
manner, interfacial
polymerization of macromers can be accomplished. A solution of polymeric
initiator-
containing pendent reactive or affinity groups is applied to a surface with
target sites. The
reactive or affinity groups on the polymeric initiator react with the sites on
the surface causing
5 the polymeric initiator to bind to the surface. Excess polymeric initiator
can then be washed
away. A solution of a polymerizable macromer is then applied to the surface.
When light
energy in applied to the system, a free radical polymerization reaction is
iiutiated only at the
surface of interest. By varying the concentration of the polyrnerizable
macromer and the
illumination time, the thickness and crosslink density of the resulting matrix
on the surface
10 can be manipulated.
Generally, there are two methods by which an initiator group can be
incorporated into
a polymeric backbone. The first method involves the formation of a monomer
which includes
the initiator. This can be accomplished readily using standard chemical
reactions. For
example, the acid chloride analog of an initiator can be reacted with an amine-
containing
15 monomer, to form a monomer which contains the initiator.
The second method of incorporating initiator groups into a polymeric backbone
involves coupling a reactive analog of the initiator with a preformed polymer.
For example,
an acid chloride analog of an initiator can be reacted with a polyner
containing pendent
amine groups forming a polymer bearing pendent initiator groups.
Macromer systems can be applied to a tissue site and/or implant article in any
suitable
manner, including by spraying, dipping, inj ecting or brushing the macromer
system.
Polymeric matrices prepared from macromer systems can be used in a variety of
applications,
including:
Cellular Encapsulation. The use of hydrogels to form micro- or macrocapsules
contaiung cells and other tissue, is well documented in the literature.
Applications include
the treatment of diabetes, Parkinson's disease, Alzheimer's disease, ALS,
chronic pain, and
others. Descriptions of cellular encapsulation methods can be found throughout
the patent
and scientific literature. The use of the instant invention provides methods
of encapsulating
cells in two basic ways.
1) Bulk polymerization
In this embodiment, cellular material is mixed in a solution of the macromer
system
and energy subsequently added to activate initiation of free radical
polymerization. Prior to
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initiation, the solution containing the macromer system with suspended
cellular material, can
be placed in molds, shaped in particular geometric shapes, or placed inside a
preformed
membrane system, such as a hollow fiber. Upon illumination or other energy
addition, the
initiation of free radical polymerization causes the macromer system to gel,
forming a cell-
s containing matrix in the desired shape. When formed into free-standing
geometric shapes,
the formulation of the macromer system can be designed to provide the desired
degrees of
durability and permselectivity to the subsequently formed matrix. When formed
inside
membrane structures, such as hollow fibers designed to provide the desired
permselectivity,
the macromer system can be formulated to provide the desired characteristics
of the cell-
suspending matrix, such as biocompatibility, etc.
2) Interfacial polymerization
In this embodiment, a membrane is formed directly on the surface of the
cellular
material. A solution of polyrnerizable or non-polymerizable polymeric
initiator-containing
pendent affinity groups (e.g., positively charged groups) is mixed with the
cellular material.
The affinity groups bind to the sites on the surface of the cellular material.
The excess
polymeric initiator is subsequently washed away and the cellular material
suspended in a
solution of polymerizable macromer. Since initiator groups are present only at
the surface of
the cellular material, when light energy is applied, polymerization is
initiated only at the
surface:macromer interface. By manipulating the duration of illumination and
macromer
formulation, a polymeric matrix exhibiting the desired characteristics of
thickness, durability,
permselectivity, etc. is formed directly on the surface of the cellular
material.
Adhesives and sealants. Polymeric matrix systems have also found extensive use
as adhesives for tissue and other surfaces. For this application, a solution
of a macromer
system is applied to a surface to which adhesion is desired, another surface
is contacted with
this surface, and illumination is applied forming a surface-to-surface
junction. If a temporary
adhesive is desired, the macromer system can be composed of degradable
macromers.
Barners. Polymeric matrices can be used for the formation of barriers on
surfaces for various applications. One such application is a barrier for the
prevention of
tissue adhesions following surgery. For this application, a macromer system in
liquid form is
applied to the surface of damaged tissue. The liquid is illuminated to
polymerize the
macromers. The polymeric matrix prevents other tissue from adhering to the
damaged tissue.
Both degradable and non-degradable macromer systems can be used for this
purpose. As
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described above, both bulk polymerization and interfacial polymerization
methods can be
used to prepare surface coatings of this type.
Controlled Release Garners. Polymeric matrices fmd wide application as
controlled
release vehicles. For this application, a solution of a macromer system and
drug, protein, or
other active substance is applied to a surface. The solution is illuminated to
polymerize the
macromers. The polymeric matrix contains the drug, when exposed to a
physiological or
other liquid-containing environment, the drug is slowly released into the
environment. The
release profile of the entrained drug can be manipulated by varying the
formulation of the
macromer system. Both degradable and non-degradable macromer systems can be
utilized
for this purpose. Likewise, both bulk and interfacial polymerization
techniques can be used
to prepare controlled drug-releasing surfaces. In an alternative embodiment, a
drug or other
active substance can be imbibed by a preformed matrix on a surface. The
absorption and
release characteristics of the matrix can be manipulated by varying the
crosslink density, the
hydrophobicity of the matrix, and the solvent used for imbibition.
Alternatively, drug-containing polymeric microspheres can be prepared using
standard
techniques. A wide range of drugs and bioactive materials can be delivered
using the
invention which include but are not limited to, antithrombogenic, anti-
inflammatory,
antimicrobial, antiproliferative, and anticancer agents, as well as growth
factors, morphogenic
proteins, and the like.
Tissue Replacement/Scaffoldin~. Polymeric matrices have found utility as three-
dimensional scaffolding for hybrid tissues and organs. For this application, a
macromer
system in liquid form is applied to a tissue defect and subsequently
illuminated to polymerize
the macromers forming a matrix upon which ingrowing cells can migrate and
organize into a
functional tissue. In one embodiment, the macromer system additionally
includes a growth
factor which is slowly released and stimulates the ingrowth of desired cell
types. hl another
embodiment, the macromers include pendent extracellular matrix peptides which
can
stimulate the ingrowth of desired cell types. A third embodiment would include
both of the
above features. An alternative embodiment includes cells included in the
matrix with or
without additional growth factor. The scaffolding can be generated if2
vitr°o by placing the
liquid macromer system in a mold or cavities in a device, or can be generated
in vivo by
applying the liquid macromer system to a tissue defect. Both degradable and
non-degradable
macromer systems could be used for this application, but degradable matrices
are preferred.
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Wound Dressing. Polymeric matrices have been used extensively as superior
wound dressing preparations. Currently, hydrogel and hydrocolloid wound
dressing materials
are being increasingly used due to their superior wound healing properties.
For this
application, a macromer system in liquid form is applied to the wound site and
subsequently
formed into a flexible polymeric matrix upon exposure to light. When applied
as a liquid, the
macromer preparation conforms to the irregular surface of the wound. Upon
illumination, a
flexible matrix is formed which is completely confonnal to the surface of the
wound; no
fluid-filled pocpets which can act as sites of bacterial infiltration can
exist. In one
embodiment, the macromer system additionally includes one or more therapeutic
agents, such
as growth factors or antimicrobial agents which are slowly released into the
wound. Both
degradable and non-degradable macromer systems can be used for this
application.
In Situ Device Formation. Polymeric materials can be implanted into the body
to
replace or support the function of diseased or damaged tissues. One example of
this is the use
of hollow cylindrical polymeric devices to support the structure of a coronary
artery following
percutaneous transluminal coronary angioplasty (PTCA). Currently, pre-formed
cylindrical
devices are implanted via catheter insertion followed by balloon expansion to
secure the
device. The expanded device supports the structure of the artery and prevents
the reversion of
the artery to the closed position (restenosis).
For this application, a liquid macromer preparation could be applied to an
injured
artery via a multi-lumen catheter containing an illumination element. After
application of the
liquid macromer system to the injured tissue, a semi-rigid polymeric matrix
can be formed by
a brief illumination. Upon removal of the catheter, a hollow, cylindrical,
conformal
polymeric device remains to support the artery and prevent restenosis. In one
embodiment,
the macromer system additionally includes a releasable therapeutic agent or
agents, such as
antiproliferative and/or antithrombotic drugs. These agents are slowly
released from the
formed matrix, to provide additional therapeutic benefit to the injured
tissues. Both
degradable and non-degradable macromer systems can be used for this
application.
The invention will be further described with reference to the following non-
limiting
Examples. It will be apparent to those spilled in the art that many changes
can be made in the
embodiments described without departing from the scope of the present
invention. Thus the
scope of the present invention should not be limited to the embodiments
described in this
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19
application, but only by embodiments described by the language of the claims
and the
equivalents of those embodiments. Unless otherwise indicated, all percentages
are by weight
EXAMPLES
Example 1
Synthesis of 7-Methyl-9-oxothioxanthene-3-carboxylic Acid Chloride (MTA-Cl)
The 7-methyl-9-oxothioxanthene-3-carboxylic acid (MTA), 50.0 g (0.185 mol),
was
dissolved in 350 ml of toluene and 415 ml (5.69 mol) of thionyl chloride using
an overhead
stirrer in a 2 liter 3-neclc round bottom flash. N,N-Dimethylformamide (DMF),
2 ml, was
added and the reaction was brought to reflux for 2 hours. After this time, the
mixture was
stirred at room temperature for 16 hours. The solvent was removed under
vacutun and the
product was azeotroped with 3 x 350 ml of toluene to remove the excess thionyl
chloride.
The product was recrystallized from 800 ml of chloroform and the resulting
solid was placed
in a vacuum oven for 16 hours at 45°C to complete removal of solvent.
The isolated product
weighed 45.31 g (85% yield) and nuclear magnetic resonance spectroscopy (NMR)
confirmed
the desired structure. This product was used for the preparation of a
photoreactive monomer
as described in Example 2.
Example 2
Synthesis of N-[3-(7-Methyl-9-oxothioxanthene-3-
carboxamido)propyl]methacrylamide
~ (MTA-APMA)
The N-(3-aminopropyl)methacrylamide hydrochloride (APMA), 4.53 g (25.4 rmnol),
was suspended in 100 ml of anhydrous chloroform in a 250 ml round bottom
flaslc equipped
with a drying tube. After cooling the slurry in an ice bath, the MTA-Cl, 7.69
g (26.6 mmol),
was added as a solid with stirring. A solution of 7.42 ml (53.2 mmol) of
triethylamine (TEA)
in 20 ml of chloroform was then added over a 1.5 hour time period, followed by
a slow
warming to room temperature. The mixture was allowed to stir 16 hours at room
temperature
under a drying tube. After this time, the reaction was washed with 0.1 N HCl
and the solvent
was removed under vacuum after adding a small amount of phenothiazine as an
inhibitor.
The resulting product was recrystallized from tetrahydrofuran (THF)/toluene
(3/1) and gave
8.87 g (88.7 % yield) of product after air drying. The structure of the
compound was
confirmed by NMR analysis.
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Example 3
Preparation of N-Succinmidyl 6-Maleimidohexanoate (MAL-EAC-NOS)
6-Aminohexanoic acid, 100.0 g (0.762 moles), was dissolved in 300 ml of acetic
acid
in a three-neck, 3 liter flaslc equipped with an overhead stirrer and drying
tube. Malefic
5 aWydride, 78.5 g (0.801 moles), was dissolved in 200 ml of acetic acid and
added to the 6-
aminohexanoic acid solution. The mixture was stirred one hour while heating on
a boiling
water bath, resulting in the formation of a white solid. After cooling
overnight at room
temperature, the solid was collected by filtration and rinsed with 2 x 50 ml
of hexane. After
drying, the typical yield of the (Z)-4-oxo-5-aza-2-undecendioic acid was 158-
165 g (90-95%)
10 with a melting point of 160-165°C. Analysis on an NMR spectrometer
was consistent with
the desired product.
(Z)-4-Oxo-5-aza-2-undecendioic acid, 150.0 g (0.654 moles), acetic anhydride,
68 ml
(73.5 g, 0.721 moles), and phenothiazine, S00 mg, were added to a 2 liter
three-neck round
bottom flask equipped with an overhead stirrer. Triethylamine, 91 ml (0.653
moles), and 600
15 ml of THF were added and the mixture was heated to reflux while stirring.
After a total of 4
hours of reflux, the dark mixture was cooled to <60°C and poured into a
solution of 250 ml of
12 N HCl in 3 liters of water. The mixture was stirred 3 hours at room
temperature and then
was filtered through a Celite 545 pad to remove solids. The filtrate was
extracted with 4 x
500 ml of chloroform and the combined extracts were dried over sodium sulfate.
After
20 adding 15 mg of phenothiazine to prevent polymerization, the solvent was
removed under
reduced pressure. The 6-maleimidohexanoic acid was recrystallized from
hexane/chloroform
(2/1) to give typical yields of 76-83 g (55-60%) with a melting point of 81-
85°C. Analysis on
a NMR spectrometer was consistent with the desired product.
The 6-maleimidohexanoic acid, 20.0 g (94.7 mmol), was dissolved in 100 ml of
chloroform under an argon atmosphere, followed by the addition of 41 ml (0.47
mol) of
oxalyl chloride. After stirring for 2 hours at room temperature, the solvent
was removed
under reduced pressure with 4 x 25 ml of additional chloroform used to remove
the last of the
excess oxalyl chloride. The acid chloride was dissolved in 100 ml of
chloroform, followed by
the addition of 12.0 g (0.104 mol) of N-hydroxysuccinimide and 16.0 ml (0.114
mol) of
triethylamine. After stirring overnight at room temperature, the product was
washed with 4 x
100 ml of water and dried over sodium sulfate. Removal of solvent gave 24.0 g
(82%) of
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21
MAL-EAC-NOS which was used without further purification. Analysis on an NMR
spectrometer was consistent with the desired product.
Example 4
Preparation of a Copolymer of MTA-APMA, MAL-EAC-NOS, and N-Vinylpyrrolidone
A polymeric initiator is prepared by copolymerization of a monomer charge
consisting
of 5 mole % MTA-APMA, 10 mole % MAL-EAC-NOS, and 85 mole % N-vinylpyrrolidone
(VP). The polymerization is run in formamide or other suitable solvent using
2,2'-
azobisisobutyronitrile (AIBN) as an initiator and N,N,N',N'-
tetramethylethylenediamine
(TEMED) as an oxygen scavenger. Mercaptoethanol is added as a chain transfer
reagent at a
concentration designed to give a molecular weight between 2,000 and 20,000
daltons. Upon
completion of the polymerization, the copolymer is precipitated by addition of
ether or other
non-solvent for the polymer. After isolation by filtration, the product is
washed extensively
with the precipitating solvent to remove residual monomers and low molecular
weight
oligomers. The copolymer is dried under vacuum and is stored desiccated to
protect the
hydrolyzable N-oxysuccinimide (NOS) esters.
Example 5
Synthesis of a Photoreactive Macromer Derived from a Poly(caprolactone-co-
lactide)
Derivative of Pentaerythritol Ethoxylate
A 15 gram scale reaction was performed by charging a thicl~-walled tube with
8.147 g
(56.5 mmol) of 1-lactide (3,6-dimethyl-1,4-dioxane-2,5-dione) and 6.450 g
(56.5 mmol) of E-
caprolactone. To this mixture was added 0.402 g (1.49 mmol) of pentaerythritol
ethoxylate
(ave. MW appprox. 270) to provide polymerization sites and control molecular
weight. This
mixture was warmed gently until dissolution of all reagents was complete. The
catalyst,
stannous 2-ethylhexanoate (0.015 ml) was added and the reaction vessel sealed.
The reaction
mixture was warmed to 150°C and stirred for 20 hours. The resulting
polymer was dissolved
in chloroform and dialyzed against methanol using 1000 MWCO dialysis tubing.
After
dialysis, the solvent was removed in vacuo. The purified polymer was dissolved
in
chloroform and treated with 2.41 g (23.8 mmol) of TEA. To this reaction
mixture was added
292 mg (1.19 mmol) of 4-benzoylbenzoyl chloride (BBA-Cl) and the resulting
mixture was
stirred for 16 hours. To this reaction mixture was added 0.734 g (8.11 mmol)
of acryloyl
chloride and the reaction was stined an additional 8 hours. The modified
polymer was
purified by dialysis against methanol using 1000 MWCO dialysis tubing. After
dialysis, the
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solvent was removed in vacuo and the polymer (15.36 grams) stored desiccated
at room
temperature.
Example 6
Synthesis of Water Soluble Siloxane Macromer with Pendent Initiator Groups
Fifty grams of a water-soluble siloxane macromer with pendent initiator groups
were
synthesized by first dissolving 50 grams of commercially available
poly[dimethylsiloxane-co-
methyl (3-hydroxypropyl)siloxane]-graft-polyethylene glycol) 3-aminopropyl
ether (Aldrich
Chemical) in 50 ml of methylene chloride. To this solution was added 5.0 g (49
mmol) of
TEA. The reaction solution was cooled to -50°C, then transferred to a
stir plate at room
temperature. MTA-Cl, 1.0 g (3.5 mmol), prepared according to the general
method in
Example 1, and 5.0 g (55 mmol) of acryloyl chloride were added and the
solution was stirred
for 6 hours at room temperature. The solution was dialyzed against deionized
water using
3500 MWCO dialysis tubing and the water was subsequently removed in vacuo. The
product
(48.4 grams) was stored desiccated at room temperature.
Example 7
Synthesis of a Polymerizable Hyaluronic Acid
Two grams of hyaluronic acid (Lifecore Biomedical, Chaska, MIA were dissolved
in
100 ml of dry formamide. To this solution were added 1.0 g (9.9 mmol) of TEA
and 4.0 g
(31 rmnol) of glycidyl acrylate. The reaction mixture was stirred at
37°C for 72 hours. After
exhaustive dialysis against deionized water using 12-14k MWCO dialysis tubing,
the product
(2.89 grams) was isolated by lyophilization.
Example 8
Preparation of a Photoderivatized Polyacrylamide (Photo-PAA)
Acrylamide, 10.24 g (0.144 mol), was dissolved in 200 ml of deionized water.
To the
solution was added 0.279 g ( 1.56 mmol) of APMA, 0.33 g (1.45 mmol) of
ammonium
persulfate and 0.155 g (1.33 mmol) of TEMED. The solution was evacuated in a
filter flask
with a water aspirator for 10 minutes. The tubing was clamped and the solution
left under
vacuum for one hour. The resulting polymer solution was dialyzed against
deionized water
using 12-14k MWCO dialysis tubing. To 150 ml of polymer solution in a PTFE
bottle
containing 3.0 grams of polymer was added 0.504 ml (3.62 mmol) of TEA. To this
solution
was added 30 ml of 28.4 mg/ml (3.48 mmol) 4-benzoylbenzoyl chloride in CHCl3.
The bottle
was capped tightly and shaken for one hour. The bottle was then centrifuged
for 10 minutes
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23
to separate the phases after which the aqueous layer was removed, dialyzed
against deinoized
water using 12-14k MWCO dialysis tubing, and lyophilized. The product (3.21
grams) was
stored, dessicated at room temperature.
Example 9
Synthesis of the N-Hydroxysuccinimide Ester of Eosin Y
Eosin Y, 1.00 g (1.54 rmnol), was dissolved in 10 ml dry dioxane with stirnng,
gentle
warming and some sonication. After the solution was complete, the orange
solution was
cooled to room temperature under argon. N-Hydroxysuccinimide, 0.195 g (1.69
mmol), and
1,3-dicyclohexylcarbodiimide, 0.635 g (3.08 mmol), were added as solids. The
resulting red
mixture was stirred at room temperature for 48 hours under an inert
atmosphere. After this
time the solid was removed by filtration and washed with dioxane. The filtrate
was
concentrated in vacuo to give 1.08 g (94% yield) of a glassy red solid.
Example 10
Synthesis of a Copolymer of APMA, Methyl Methacrylate, and N-Vinylpyrrolidone
Followed
by Addition of Acryloyl Groups
The following ingredients for the copolymer were placed in a glass vessel and
dissolved in 20 ml DMSO: APMA (2.68 g, 15.0 mmol), VP (6.74 ml, 63.1 mmol),
methyl
methacrylate (mMA) (0.334 ml, 3.12 mmol), mercaptoethanol (0.053 ml, 0.76
mmol), AIBN
(0.041 g, 0.25 mmol), and TEMED (0.057 ml, 0.38 mmol). After solution was
complete, the
monomer solution was degassed, blanketed with argon and placed in an agitating
incubator at
55° C. The copolymer was dialyzed against deionized water in 6-8,000
MWCO dialysis
tubing. The dialyzed solution (~ 400 ml) was loaded with acrylate groups. TEA,
5.0 ml (35.9
mmol), was added with stirring. The solution was placed in a freezer for 5-10
minutes to
cool. After this time, 5.0 ml (61.5 mmol) of acryloyl chloride in 5 ml of
chloroform were
added with stirring. The reaction mixture was stirred at room temperature for
16 hrs. After
this time the acrylated polymer was dialyzed against deionized water using 6-
8,000 MWCO
tubing. The product was lyophilized and 7.10 g were obtained.
Example 11
Synthesis of a Copolymer of MTA-APMA, APMA, Methyl Methacrylate, and N-
Vinylpyrrolidone Followed by Addition of Acryloyl Groups
The following ingredients for the copolymer were placed in a glass vessel and
dissolved in 20 ml DMSO: MTA-APMA (0.613 g, 1.55 mmol), APMA (2.578 g, 14.4
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24
mmol), VP (6.27 ml, 58.7 mmol), mMA (0.319 ml, 2.98 mmol), mercaptoethanol
(0.054 ml,
0.77 mmol), AIBN (0.039 g, 0.24 mmol), and TEMED (0.053 ml, 0.35 mmol). After
solution
was complete, the monomer solution was degassed, blanketed with argon and
placed in an
agitating incubator at 55° C. The copolymer was dialyzed against
deionized water in 6-8,000
MWCO dialysis tubing. The dialyzed solution was protected from light and
loaded with
acrylate groups. TEA, 5.0 ml (35.9 mmol), was added with stirring. The
solution was placed
in a freezer for 5-10 minutes to cool. After tlus time, 5.0 ml (61.5 mmol) of
acryloyl chloride
in 5 ml of chloroform were added with stirring. The reaction mixture was
stirred at room
temperature for 9 hrs. After this time the acrylated polymer was dialyzed
against deionized
water using 6-8,000 MWCO tubing and protected from light. The product (8.88
grams) was
isolated by lyophilization.
Example 12
Evaluation of Matrix Formation
A 15 % solution of the co-polymer from Example 11 was prepared in 10% DMSO/
water. The MTA content of the solution was estimated by measuring the
absorbance of the
solution at 395nm(A@395nm=42.6).A 15 % solution of the co-polymer from
Examplel0(same co- polymer as that described in Example 11 but with no MTA-
APMA)
was prepared in 10 %DMSO/water. MTA was added to this solution until its
absorbance at
395nm matched that of the solution described above. The two solutions were
identical in
concentration of co-polymer and photoinitiator, the only difference between
them being that
in one solution the photoinitiator was present in polymeric form(POLY) and in
the other the
photoinitiator was present in non-polymeric fonn(NON).
In order to compare the matrix forming ability of the two solutions the
following
evaluation was undertaken: the indentations in the lid of a 96 well microtiter
plate were used
as miniature molds to evaluate the ability of the photoreactive polymer
solutions to form solid
hydrogel discs upon illumination. The indentations are eight millimeters in
diameter and
approximately 0. 6 millimeters deep. 30 microliters of polymer solution will
just fill the
indentatation. Thirty microliters of both the (POLY) and (NON) solutions were
added to
wells. After addition of the polymer solutions, the lids were illuminated
using an EFOS
Ultracure 100 SS illumination system equipped with a 400-SOOnm filter, for
varying lengths
of time. After illumination the lid was flooded with water and each polymer
formulation
rated for its ability to form solid discs using the following arbitrary scale:
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0 = liquid, no gelation
1 = soft gel, unable to remove from mold
2 = firm gel, removable from mold with slight difficulty
3 = very firm gel, easily removed from mold
5 4 = very firm gel, elastomeric properties evident
Results:
Polymer Time(sec)
2 5 10 30 60 120
(POLY) 1 2 3 4 4 4
10 (NON) 0 0 1 2 3 3
Matrix formation
The polymer solution containing the polymer-bound initiator(POLY) formed
matrices
more rapidly and more completely than the polymer solution containing non-
polymer-bound
initiator(NON) when exposed to light energy.
15 Example 13
Synthesis of an Eosin Substituted Polymer
N-Vinylpyrrolidone, 10.0 g (90.0 mmol), was dissolved in 50 ml DMSO. To the
solution was added 0.30 g (1.68 nnnol) of APMA, 0.15 g (0.91 mmol) of AIBN,
and 0.10 g
(0.86 mmol) of TEMED. The solution was sparged with nitrogen for 20 minutes
and ,
20 incubated at 55°C for 20 hours. The resulting polymer was pun~ified
by dialysis against water
and isolated by lyophiliaztion.
Three grams of the polymer were dissolved in 150 mls dny dioxane. To this
solution
was added 0.504 ml (3.62 mmoles) of TEA. Subsequently, 2.74 grams (3.5 mmoles)
of the
N-hydroxysuccinimide ester of Eosin Y was added and the reaction mixture
stirred for two
25 hours at room temperature. The solution was dialyzed against dH20 using 12-
141cda cut-off
dialysis tubing and lyophilized to isolate the product. The reaction yielded
3.96 grams of red
polymer.
Example 14
A Biodegradable Tissue Adhesive.
A solution was prepared consisting of 5% polyrnen-izable hyaluronic acid
(Example 7)
and 2% photodenivatized polyacrylamide (Example 8) in water. This reagent was
evaluated
for use as a tissue adhesive using cellulose dialysis tubing as a tissue
model.
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Shear strength testing was performed on dialysis tubing. The tubing was slit
and cut
into 2 cm x 4 cm pieces. The pieces were soaked in water briefly, removed, and
tested while
still damp. One piece was laid flat on a surface and 10 ~,l of adhesive
applied to one end of
the strip. Another piece was laid over this piece with a 1 cm overlap between
pieces. When
evaluating the photoactivatable adhesive (2/5 HA), the overlap area was
illuminated for 10
seconds. When evaluating a control adhesive, the adhesive was allowed to set
for five
minutes. The bonded samples were mounted in a tensiometer lengthwise by the
ends such
that the plane of the area of adhesive was parallel to the axis of the
tensiometer. The samples
were extended at the rate of 1 cm/minute until adhesive or substrate failure,
and the force at
failure recorded. Substrate-only, and, for photoactivatable adhesive, non-
illuminated
samples, were included as controls in the evaluations.
Adhesive Maximum Force Adhesive Failed Substrate Failed
Generated Kg Before SubstrateBefore Adhesive
2/5 HA 0.53 O/4 4/4
2/5 HA (no illumination)0.0~ 1 4/4 0/4
Fibrin glue 0.045 4/4 0/4
Cyanoacrylate 0.49 0/4 4/4
Example 15
Formation of an in situ hydrogel wound dressing.
Photopolyrnerizable, matrix-forming reagents were evaluated for efficacy as in
situ
wound dressings.
Preparation of reagents:
An experimental in situ forming wound dressing was prepared by:
1) Dissolving reactive macromer from Example 10 at 20% into a sterile 6%
glycerin
solution in water.
2) Preparing a sterile solution of polymeric eosin reagent from Example 12 at
4% in
water and a sterile solution of 2M triethanolamine (TEA) in water.
3) Transporting the three sterile solutions to a surgical suite for
application to wound
sites created on porcine skin.
Four young female China White swine weighing between 15-20 lcg were
anesthetized
and 12 wounds inflicted on one side of each pig. Wounds were 1" x 2" and
0.015" deep and
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were inflicted by a calibrated electrodermatome (Padgett). The wounds were
inflicted in two
rows of six on the thoracic and paravertebral area of each pig, leaving
approximately two
inches between adjacent wounds. The wounds were randomized and received one of
three
treatments:
1) No treatment (control)
2) Application of OpSite~, a semi-occlusive wound dressing from Smith and
Nephew, Inc.
3) Experimental photo-curable dressing
To apply the experimental dressing, 0.5 mls of the polymeric-eosin solution
and 0.5
mls of the TEA solution were added to the macromer/glycerin solution yielding
a photo-
wound dressing solution. The solution was transferred to 16 three ml sterile
syringes (2
ml/syringe) and one syringe was used to application to each wound site. The
solutions were
applied to each assigned wound site (approximately 1.5 mls solutions/site) and
allowed to
flow over the site. The solutions were fixed by illumination with a 150 W
incandescent light
bulb positioned four inches from the wound surface for 30 seconds. The
dressing solution
readily formed into a durable, rubbery hydrogel which adhered very well to the
wound sites.
Sterile 4 x 4 gauze pads were placed over the entire wounded area of each pig,
and the pigs
placed in sterile stocl~inettes. On selected days (3, 4, 5, and 7), one pig
was euthanized and
the effect of dressing on wound epithelialization and repair evaluated.
Evaluation of effect of dressing on wound epithelialization and repair:
Following euthanasia, slcin wounds were removed from the underlying deep
subcutaneous tissue and fixed in 10% neutral buffered formalin solution. After
fixation, five
biopsy sites from each wound were obtained with a 6 mm Keys shin biopsy punch.
Each
biopsy was pacl~aged, labeled and submitted for histological sectioning.
Histological sections
were sectioned at 4 microns and stained with hematoxylin and eosin.
Histological sections
were examined with the microscope without l~nowing the type of covering placed
over the
wound site. The following criteria were evaluated and scored in microscopic
examination:
Degree of epithelialization of the wound
Magnitude of the inflarninatory reaction
Degree of fibroplasia in the wound
Degree of damage to subcutaneous tissue:
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Morphometric analysis of cell types in the histological sections were used to
help
differentiate the degree of inflammatory reaction present. The number of
polymorphonuclear
cells, lymphocytic cells, and fibroblasts was evaluated. Each histological
biopsy was graded
on a scale of 1-5.
Degree of inflammatory reaction:
1. No or borderline cellular inflammatory reaction
2. Minimal inflammation
3. Moderate density of inflammatory cells with some exudate
4. Severe, high density of inflammatory cells in or on the wound tissue with
thicker layer of
exudate
5. Excessive inflammation, with signs of dense foci of inflammatory cells
infiltrating the
wound tissue or on the wound and forming a thick layer of inflammatory
exudate.
Degree of wound epithelialization:
1. Stratum corneum present at least 4 layers of cells and entire epidermal
surface is present.
2. Stratum corneum is present at least 1 layer of cells and entire epidermal
surface is present.
3. Stratum corneum is present at least 1 layer of cells and 1/2 of epidermal
surface is
covered.
4. No stratum corneum is present; minimal inflammation of the subepidennal
tissue.
5. No stratum corneum is present; moderate inflammation in subepidermal
tissue.
Degree of fibroplasia in the wound:
1. No fibroplasia in the wound
2. Mild fibroplasia in the wound involving 1/3 to 1/2 wound surface
3. Mild fibroplasia in the wound involving 2/3 or more of the wound
4. Moderate fibroplasia involving 1/3 to 1/2 of the wound
5. Severe fibroplasia involving 1/2 or more of the wound
Degree of damage to the subcutaneous tissue:
1. No damage to the subcutaneous tissue
2. Mild damage to the subcutaneous tissue with mild edema and few inflammatory
cells.
3. Moderate damage to the subcutaneous tissue with moderate edema and moderate
accumulation of inflammatory cells
4. Severe damage to the subcutaneous tissue with severe edema and large number
of
inflammatory cells
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5. Excessive damage to the subcutaneous tissue with dense foci of inflammatory
cells
Results:
Each biopsy was graded blindly using the criteria listed above. When the
histological
examination was completed, the graded biopsies were correlated with the wound
sites. A
single average score for each dressing was calculated by adding all the scores
for every site
for each dressing and dividing by the number or scores.
The total scores for each type of wound dressing on days 3, 4, 5, and 7 were
evaluated
with an ANOVA SAS program for data intervals to statistically evaluate if
there was any
difference between the three types of wound treatments administered. Only two
scores were
found to be statistically significant:
1. On day 4 following wound creation the mean for the OpSite~ dressing was 2.4
and
was found to be statistically significant when compared to the control and
experimental wound sites.
2. On day 7 following the creation of the wounds the mean for the experimental
dressing, 1.8 was found to be statistically significant when compared to the
control
and the OpSite~ wound dressings.
On day 7 post-wound creation, the wound sites treated with the experimental
photocurable dressing showed significantly superior healing to those that were
untreated or
treated with OpSite~ dressing, as judged by the criteria described.
Example 16
A Bioresorbable Drug Delivery Coating.
A solution of 33% of the macromer from Example 5 was prepared in ethanol. Ten
centimeter lengths of polyurethane rod (PU) were dipped into the macromer
solutions and
illuminated for six minutes to form a matrix. This procedure resulted in the
formation of a
very durable, tenacious, and flexible coating on the rod. One gram of
chlorhexidine diacetate
(an antimicrobial agent) was dissolved in 10 mls of the macromer solution and
the coating
process repeated on additional PU rods. This also resulted in a tenacious,
durable, and
flexible coating on the rods. The rods were cut into one centimeter pieces and
evaluated in a
zone of inhibition analysis.
Coated dye-containing pieces, coated no-drug controls, and uncoated pieces
were
placed in Mueller-Hinton agar plates which were swabbed with a 106 suspension
of
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Staphylococcus epidermidis (Christensen RP62A). These pieces functioned as
unwashed
controls and were transferred to freshly swabbed agar plates each day for 60
days.
Additional pieces, no-drug controls (both coated and uncoated) and drug-
incorporated
coated, were placed in snap-cap vials and washed with 50% Normal Calf Serum in
PBS. The
5 tubes were placed on an orbital shaker and incubated at 37°C and 200
rpm for 20 days. Each
day the wash solution was removed and replaced with fresh solution.
Periodically, pieces
were removed from the serum/PBS and placed in agar as described above. Zones
of
inhibition resulting from these pieces were recorded and compared to the zones
produced by
unwashed pieces.
10 The no-drug coated control pieces, both coated and mlcoated, produced no
zones. On
day 0, both washed and unwashed drug-incorporated pieces produced zone of 24.5
mm. On
day 20, when the final washed pieces were evaluated, the unwashed pieces were
producing
zones of 17.5 mm, and the washed pieces were producing zones of 9.5 mm. On day
60, when
the experiment was terminated, the unwashed pieces were still producing zones
of 17 mm.
15 This experiment demonstrates the utility of this matrix-forming polymer at
producing
drug delivery coatings which provide a long-term delivery of a bioactive
agent.
Example 17
A Biostable Drug Delivery Coating.
A solution of 25% of the macromer from Example 6 was prepared in 50% IPA/HZO.
20 Ten centimeter lengths of polyurethane rod were dipped into the macromer
solution and
illuminated for six minutes to form matrix. This procedure resulted in the
formation of a very
durable, tenacious, and flexible coating on the rod. Five hundred milligrams
of chlorhexidine
diacetate was dissolved in 10 mls ethanol. Half of the coated rods were soaped
in this
solution for 60 minutes at room temperature, and half of the rods were soaked
in neat ethanol
25 under the same conditions. After soaping, the rods were removed from the
ethanol and
allowed to dry for 20 hours at room temperature. The rods were cut into one
centimeter
pieces and evaluated in a zone of inhibition analysis.
Uncoated control, coated control, and coated drug-incorporated pieces were
placed in
Mueller-Hinton agar plates which were swabbed with a 10~ suspension of
Staphylococcus
30 epidermidis (Christensen RP62A). These plates were incubated for 20 hours
at 37°C. The
zone where no bacterial growth was evident around each piece was measured and
the piece
transferred to a freshly swabbed agar plate each day for 14 days.
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The uncoated control pieces and the coated control pieces produced no zones.
On day
0, the dnig-incorporated coated pieces produced average zones of 25 mm. These
pieces
continued to produce zones each day. On day 14, when the experiment was
terminated, the
pieces produced average zones of 6 mm.
Example 18
Formation of a Three-Dimensional Device.
One end of a 3 mm diameter teflon-coated rod was dipped to a level of 1.5 cm
in neat
BBA-acryloylpolytetra(caprolactone-c~-lactide)pentaerythritol ethoxylate (see
Example 5)
and irmnediately illuminated, with rotation, for 10 seconds suspended between
opposed
Dymax lamps. After illumination, a semi-rigid elastomeric coating had formed
on the rod.
The rod was cooled to facilitate removal of the polymeric coating. The closed
end of the
cylinder was removed with a razor blade, thus forming a hollow cylindrical
device of 1.25 cm
in length and 3.5 mm in diameter.
Example 19
Synthesis of a Polymerizable Collagen.
One gram of soluble collagen (Semed-S, Kensey-Nash Corp.) (a mixture of Types
I
and III) was dissolved in 50 mls of 0.01 N HCI. When dissolved, 1.25 gms
triethylamine
(12.4 mmoles) was added to the reaction mixture. One gram of acryloyl chloride
(11.0
mmoles) dissolved in one milliliter of methylene chloride was added to the
reaction vessel
and the mixture was stirred for 20 hours at room temperature.
The reaction mixture was dialyzed exhaustively against dH20, and the product
isolated by lyophilization. A yield of 1.17 grams of polymerizable collagen
was realized.
Example 20
A Collagen Scaffolding that Contains a Bone Morphogenic Protein.
A. Preparation of the solidified scaffolding.
A solution of liquid macromer is prepared which consists of 5% (w/v) of
polymerizable collagen (Example 19) plus 1 % (w/v) of photoderivatized
polyacrylamide
(prepared as described in Example 8) in phosphate buffered saline, pH 7.4. To
this is added
50 ~g/ml (0.005% w/v) of bone morphogenic protein (BMP-7 from a private
source).
Aliquots of the above solution (150 ~,1) are then placed in molds (8 mm
diameter and 3 mm
high) and are illuminated for 10 seconds with a Dymax lamp (as described in
Example 13) to
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solidify the collagen scaffolding. Control disks of solidified collagen
scaffolding are prepared
via the same protocol except that BMP-7 is not added.
B. Evaluation of the solidified scaffolding.
Disks of solidified collagen scaffolding with BMP-7 are evaluated for
stimulation of
bone growth in a rat cranial onlay implant model. In this model, the
periosteal membrane is
removed and the collagen disks are implanted on the cranium. After 30 days,
the implants
and adjacent cranial bone are removed, fixed in cold methanol, embedded in
PMMA,
sectioned, ground to 50-100 ~,m thickness, stained with Sandersons Rapid Bone
Stain, and
counterstained with Van Gieson's picro-fuchsin. This protocol evaluates
nondecalcified
bone, with mature bone staining red, immature bone staining pinlc, cartilage
staining blue-
gray~ and undegraded collagen appearing acellular and pale yellow.
One control consists of disks of solidified collagen scaffolding lacking BMP-
7. A
second control consists of 150 ~1 of nonilluminated liquid macromer solution
which contains
BMP-7 (the same solution composition that was placed in molds and illuminated
to produce
the solidified collagen scaffolding containing BMP-7).
When evaluated histologically at 30 days as described above, the experimental
disks
(solidified collagen scaffolding containing BMP-7) show extensive bone
formation in the
space originally occupied by the collagen disk. In contrast, both controls
(the solidified
collagen scaffolding lacking BMP-7 and the nonilluminated liquid control
solution containing
BMP-7) show little or no bone formation. The amount of bone that forms with
the controls is
less than 25% of that observed with the experimental disks, therefore
demonstrating that the
solidified collagen scaffolding greatly enhances BMP-stimulated bone
formation.
Example 21
Synthesis of a Polymerizable Collagen
Dissolved 0.5 gram collagen (insoluble bovine tendon collagen, Type I, ReGen
Corp.)
in 20 mls dry formamide by incubating for 20 hours on an orbital shaker at 37
degrees C.
With stirring, added 1.0 gram (9.8mmo1) TEA and equilibrated for 60 minutes in
ice water
bath. With stirring, added 1.0 gram (11 mmol) acryloyl chloride, in 0.25 gram
aliquots (1
aliquot/min). After the final addition, stirred in ice water bath for 2 hours.
Removed from ice
water bath and continued to stir at room temperature for 18 hours. The product
was purified
by dialysis against deionized water using 6-8K MWCO dialysis tubing, and
isolated by
lyophilization.
