U.S. patent application number 12/911578 was filed with the patent office on 2011-02-17 for ligand-coupled initiator polymers and methods of use.
Invention is credited to Stephen J. Chudzik, Dale G. Swan.
Application Number | 20110040057 12/911578 |
Document ID | / |
Family ID | 33131143 |
Filed Date | 2011-02-17 |
United States Patent
Application |
20110040057 |
Kind Code |
A1 |
Chudzik; Stephen J. ; et
al. |
February 17, 2011 |
LIGAND-COUPLED INITIATOR POLYMERS AND METHODS OF USE
Abstract
Initiator polymers having an initiator group and a ligand group
are provided. The initiator polymers are capable of specifically
binding to a receptor on a surface. Using a macromer system, the
initiator polymers are useful for the formation of a polymeric
matrix on the surface of a material. In particular, initiator
polymers are provided that have specificity to pancreatic .beta.
cells and can be used to encapsulate cells for transplantation and
the treatment of diabetes.
Inventors: |
Chudzik; Stephen J.; (St.
Paul, MN) ; Swan; Dale G.; (St. Louis Park,
MN) |
Correspondence
Address: |
Kagan Binder, PLLC
221 Main Street North, Suite 200
Stillwater
MN
55082
US
|
Family ID: |
33131143 |
Appl. No.: |
12/911578 |
Filed: |
October 25, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10412063 |
Apr 10, 2003 |
7820158 |
|
|
12911578 |
|
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|
|
Current U.S.
Class: |
526/268 ;
526/288; 528/69 |
Current CPC
Class: |
Y10S 623/92 20130101;
A61K 47/32 20130101; A61K 9/1635 20130101; A61K 9/5026 20130101;
Y10S 522/904 20130101; Y10S 516/911 20130101 |
Class at
Publication: |
526/268 ;
526/288; 528/69 |
International
Class: |
C08F 124/00 20060101
C08F124/00; C08F 128/02 20060101 C08F128/02; C08G 18/71 20060101
C08G018/71 |
Claims
1. A ligand-coupled initiator polymer comprising: a) a hydrophilic
polymeric backbone; b) a photoinitiator group selected from the
group consisting of acridine orange, camphorquinone, ethyl eosin,
eosin Y, erythrosine, fluorescein, methylene green, methylene blue,
phloxime, riboflavin, rose bengal, thionine, and xanthine dyes; and
c) a ligand group that can specifically bind to a receptor.
2. A kit comprising: a) a ligand-coupled initiator polymer
comprising: i) an initiator group; and ii) a ligand group able to
specifically bind to a receptor on a surface; and b) polymerizable
material able to form a polymeric layer on the surface upon
activation of the initiator group.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present non-provisional patent application is a
divisional of U.S. patent application Ser. No. 10/412,063, filed on
Apr. 10, 2003, now U.S. Pat. No. 7,820,158, entitled LIGAND-COUPLED
INITIATOR POLYMERS AND METHODS OF USE, which is fully incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The current invention relates to compounds useful for
forming a polymeric matrix on the surface of a substrate. More
specifically, the invention relates to initiator polymers that can
specifically bind to a target surface and promote formation of a
polymeric matrix on the surface.
BACKGROUND
[0003] The use of polymeric material for the encapsulation of cells
and tissue offers great potential for the treatment of diseases and
other medical indications. Particularly useful applications involve
utilizing polymeric material for encapsulating tissues or cells for
transplantation into a patient in order to provide therapy.
Although various techniques for encapsulating mammalian cells have
been known for a number of decades and have been used in research
settings, only more recently cell encapsulation technologies have
been applied for the potential treatment of diseases.
[0004] Cell encapsulation methods are generally aimed at
surrounding a cell or group of cells with a material barrier in
order to protect the transplanted encapsulated cells from host
immune rejection. The material barrier around the cells ideally
allows the cells to remain viable and to function properly in order
to provide therapeutic value to the host. In order to perform this
function, the material that is used to encapsulate the cells, which
typically includes a polymeric compound, should be resistant to
biodegradation and should be sufficiently permeable to allow for
diffusion of cellular waste products, nutrients, and molecules
involved in cellular responses. Preferably, the material barrier is
not permeable to certain host molecules, such as immunoglobulins
and complement factors that could contribute to the destruction of
the foreign cells.
[0005] Advances in cell encapsulation technologies have been
focused on improving the permselectivity, mechanical properties,
immune protectivity, and biocompatibility of the material barrier
that is formed around the cells. Various micro- and
macroencapsulation techniques, including microencapsulation by
polyelectrolyte complexation, thermoreversible gelation,
interfacial precipitation, interfacial polymerization, and flat
sheet and hollow fiber-based macroencapsulation have been studied
and are reviewed by Uludag et al. Adv. Drug Deliv. Rev. 42:29-64
(2000).
[0006] One commonly used method for the encapsulation of cells is
the alginate crosslinking method, which utilizes polyanionic
alginate and polycationic polylysine polymers. Encapsulation by the
alginate method typically occurs by the crosslinking of alginate
via the Ca.sup.2+ ion and the interaction of polylysine with the
alginate molecules. Unfortunately, there are a number of problems
associated with this approach to cell encapsulation. Such problems
include the swelling of alginate microcapsules due to the presence
of Ca.sup.2+ in the inner alginate core, insufficient
biocompatibility due to guluronic acid content in
alginate/polylysine capsules, and insufficient mechanical strength
of the alginate coating. Moreover, the process of alginate
encapsulation is nonspecific and can result in the formation of
microcapsules that do not contain the cells or cell groups intended
to be encapsulated or that contain other non-target biological
materials. Due to these problems, alternative methods for cell
encapsulation have been investigated.
[0007] One promising alternative to alginate crosslinking is a
method termed interfacial polymerization. Interfacial
polymerization has the possibility of offering all of the
advantages of the alginate encapsulation method for cellular
encapsulation and its therapeutic applications, although there has
been little done to investigate its potential. Interfacial
polymerization generally involves the formation of a layer of
polymerized material, such as synthetic or natural polymerizable
polymers, on the surface of a biological substrate. The formation
of the layer of polymeric material is generally promoted by the
activation of a polymerization initiator, which is deposited on the
surface of the biological substrate, in the presence of the
polymerizable polymers.
[0008] Some polymerization initiators for use in interfacial
polymerization methods have been demonstrated in U.S. Pat. No.
5,410,016 and U.S. Pat. No. 5,529,914. These patents describe
depositing the polymerization initiator, eosin Y, on a cell
membrane and then activating the initiator to promote
polymerization of a macromer solution. However, the use of eosin Y,
which is a relatively nonpolar, low molecular weight
light-activated initiator dye, or compounds similar to eosin,
presents many disadvantages for interfacial polymerization methods
and also presents potential problems to subjects receiving
transplanted encapsulated cells. For example, these dyes and other
similar low molecular weight compounds present toxicity problems as
they can penetrate into a cell and interfere with normal
biochemical pathways. If penetrated into the cell, these dyes can
cause free radical damage when activated by external sources of
energy. Other drawbacks arise if the dye is able to diffuse out of
the formed polymeric layer, thereby producing potential toxicity to
a host organism. Dyes such as eosin also tend to aggregate in
aqueous solution, thereby reducing the efficiency of the
encapsulation process and introducing problems with
reproducibility. Finally, in view of the limited efficiency of
these dyes in initiating sufficient radical chain polymerization,
it is often necessary to add one or more monomeric polymerization
"accelerators" to the polymerization mixture. These accelerators
also tend to be small molecules which are capable of penetrating
the cellular membrane and have the potential to be cytotoxic or
carcinogenic. Therefore, it is also desirable to minimize the use
of these accelerators. In attempts to overcome the above problems,
applicants have previously introduced novel interfacial
polymerization reagents and techniques (see U.S. Pat. Nos.
6,007,833 and 6,410,044; herein incorporated by reference in their
entirety).
[0009] Despite these teachings, improved initiators for interfacial
polymerization methods are desired. The cell surface, to which the
initiator polymer is targeted, is very complex and presents a
challenge for the design of initiators that function in a desired
manner. For example, the cell surface contains numerous surface
proteins, some of which have carbohydrate groups containing charged
moieties, such as sulfated proteoglycans and glycosaminoglycans. It
is desirable to design initiators that localize to the biological
surface but do not affect the physiology of the cell in a negative
manner. For example, improved initiators should preferably promote
the formation of a polymeric layer on the cell surface in an
efficient manner without triggering any detrimental cellular
processes, such as signaling pathways that lead to cell death.
[0010] In another aspect, it may also be desirable to have the
interfacial polymerization reagents or polymeric layers formed by
the initiators impart a desired effect on the cell. For example,
having encapsulated pancreatic cells that produce insulin or having
encapsulated thyroid cells produce parathyroid hormone can be of
value to a patient in need of such a therapy. Such action may
reduce or eliminate the need for the patient to take drugs that
promote such an effect in vivo.
SUMMARY OF THE INVENTION
[0011] In one aspect, the invention provides a method for coating a
surface. The method includes contacting the surface with a
ligand-coupled initiator polymer. The initiator polymer includes a
polymerization initiator group and a ligand group, and the ligand
group can specifically bind to a receptor on the surface. The
method also includes contacting the surface with a polymerizable
material and then activating the initiator group of the
ligand-coupled initiator polymer to cause polymerization of the
polymerizable material on at least a portion of the biological
surface.
[0012] The method typically involves coating a biological surface,
such as the outer membrane of a cell. In one aspect, the invention
provides a method for encapsulating pancreatic islets with a
polymeric coating. According to this embodiment, the ligand group
used for this purpose can be a sulfonylurea derivative, which can
also be useful for stimulating the pancreatic .beta. cells to
secrete insulin.
[0013] In another aspect, the invention provides a ligand-coupled
initiator polymer that includes a photoinitiator group that is
selected from the group of light activated dyes, and a ligand
group. The light activated dyes can be selected from the group
consisting of acridine orange, camphorquinone, ethyl eosin, eosin
Y, erythrosine, fluorescein, methylene green, methylene blue,
phloxime, riboflavin, rose bengal, thionine, xanthine dyes, and the
like. In another aspect, the initiator polymer comprises a
hydrophilic backbone, such as a polyacrylamide backbone or a
backbone having similar hydrophilic properties.
[0014] The invention further provides a kit that includes an
initiator polymer and a polymerizable material such as a
macromer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 illustrates a synthetic scheme for the preparation of
a sulfonylurea monomer, SUM (Compound I).
[0016] FIG. 2 illustrates a synthetic scheme for the preparation of
an EITC Monomer, EITCM (Compound II).
[0017] FIG. 3 illustrates a synthetic scheme for the preparation of
a sulfonylurea derivative, SUNCS (Compound IV).
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention provides ligand-coupled initiator
polymers, herein referred to as "initiator polymers", compositions
and systems including the initiator polymers, and methods for
performing the interfacial polymerization of polymerizable material
on a surface using these initiator polymers. "Polymer" refers to a
compound having one or more different repeating monomeric units and
includes linear polymers and copolymers, branched polymers and
copolymers, such as highly branched dendrimer polymers and
copolymers, herein referred to as "dendrimers", graft polymers and
copolymers, and the like. The surface to which the initiator
polymer binds generally bears a receptor that is able to associate
with the ligand. In one embodiment of the invention the surface is
a biological surface. As used herein "biological surface" broadly
refers to the surface of any sort of biological material that has a
surface receptor, such as, for example, the surface of cells, the
surface of a group of cells, the surface of biological particles
such as viral particles, or the surface of tissue. The initiator
polymers of the invention are able to specifically interact with
the receptor on the surface of the biological material and promote
the polymerization of polymerizable material to form a polymeric
layer, also referred to herein as a "polymeric matrix", on or near
the biological surface.
[0019] The initiator polymers are particularly useful for cell
encapsulation methods, although they can also be used to form a
matrix of polymerized material on a biological surface having a
receptor in any sort of ex vivo or in vivo method. Cell
encapsulation involves the formation of a polymeric layer typically
over the entire surface of the cell or cells, and this polymeric
layer typically has certain physical and functional properties,
such as thickness, permselectivity, strength, and protectivity. In
other embodiments, the initiator polymers can be used to form a
polymeric matrix of polymerized material on the surface of any type
of natural or synthetic material that has specific receptors that
can interact with the ligand group of the initiator polymer.
[0020] According to the invention, initiator polymers useful for
providing a coating of a polymeric material on a surface include an
initiator group and a ligand group. The initiator group refers to a
portion of the initiator polymer that can specifically accept
energy and generate a free radical species, directly or indirectly,
and is sufficient to promote free radical polymerization of the
polymerizable material. The ligand group refers to a portion of the
initiator polymer that is able to specifically associate with a
distinct receptor (for example, a ligand-binding member) on the
surface of the material targeted for coating. The affinity between
the ligand group and the receptor is generally high, typically
having a dissociation constant (K.sub.d) in the range of 10.sup.-6
to 10.sup.-12 M.
[0021] In one embodiment, the ligand group of the initiator polymer
is a molecule that can specifically associate with a
therapeutically relevant receptor on the surface of a cell.
Typically, a therapeutically relevant receptor is a receptor that
can affect, either directly or indirectly, the function of a cell
wherein the function is associated with changing a physical
condition in a subject having the cell. For example, the binding of
the ligand group to the receptor can trigger the production of a
useful compound or can block the release of an undesirable
compound. "Production" is used in its broadest sense and includes
any cellular function that causes or increases the release of the
therapeutic compound from the cell. In this particular embodiment,
the ligand group of the initiator polymer serves a dual function.
First, the ligand group specifically binds the initiator polymer to
a specific receptor on the cellular surface, and second, the ligand
group provokes a cellular response from the cellular material it is
encapsulating. The cellular response can be initiated by the
binding of the ligand group to its receptor, wherein the receptor
moiety triggers the cell to produce a desired compound or compounds
(or to elicit a desired cellular response). The invention also
provides a novel way of initiating and maintaining a cellular
response since the ligand group of the initiator polymer, which
becomes incorporated into the polymer matrix formed via interfacial
polymerization, remains in contact with the receptor on the cell
surface following encapsulation. Therefore, the encapsulated cell
can be continuously stimulated to produce the therapeutically
useful compound. In one aspect, the ligand group is a molecule
which can bind to the surface receptor on an endocrine cell and the
binding causes the release of a compound that has an endocrine
function in the body.
[0022] In a specific embodiment, the ligand group is a molecule
that can bind a surface receptor on a pancreatic .beta. cell. In
some aspects, the binding of the molecule on the surface of the
.beta. cell can elicit a cellular response from the .beta. cell,
such as the production of insulin. In a preferred embodiment the
ligand group is a sulfonylurea derivative, such as glyburide.
"Sulfonyl derivatives", as used herein, refers to compounds having
a sulfonylurea portion and that are able to produce an
insulinotropic effect. Sulfonylureas such as glyburide are ligands
which can bind to potassium (ATP) channel proteins on the surface
of pancreatic .beta. cells.
[0023] The ligand-coupled initiator polymer of the invention is
arranged to be soluble in an aqueous solution and able to associate
with the receptor based on high affinity interactions between the
ligand group and the receptor. In some embodiments the initiator
group is non-polar and in some embodiments the ligand group is also
non-polar. Therefore, typically, the initiator group and the ligand
group will confer hydrophobic properties to the initiator polymer.
In a preferred embodiment the initiator polymer can include a
polymer backbone that is highly hydrophilic. A highly hydrophilic
backbone can allow the initiator polymer to maintain its solubility
and its receptor-binding properties in an aqueous environment.
[0024] In one embodiment, the initiator polymer can be used in a
method for coating a biological surface, such as a cell
encapsulation method. In these types of methods, the initiator
polymer is used with a polymerizable material, such as macromers,
that can form a matrix on the surface. In some embodiments the
initiator polymer is placed in contact with the surface separately
from the macromer component; in other embodiments the initiator
polymer and the macromer component are placed in contact with the
surface together as a polymerizable composition to the cells.
Therefore, the invention also provides compositions that include a
ligand-coupled initiator polymer and a polymerizable component.
Other compounds useful for cell encapsulation, such as
reductants/acceptors and viscosity enhancing agents can be
introduced into the polymerization method in existing steps or in
additional steps. Such reagents are described in detail below.
Therefore, the invention also provides polymerizable compositions
and kits for forming a polymer coating on a surface that can
include a ligand-coupled initiator polymer, a polymerizable
component, and other components that can enhance or that are useful
for coating a surface, particularly for cell encapsulation.
[0025] In a more specific embodiment the invention provides for the
ligand-coupled initiator polymer as a component in a group of
compounds used for interfacial polymerization methods, and
applicable for the treatment of particular diseases. These
compounds and methods can be implemented for the encapsulation of
cells or tissue, wherein the encapsulated cells or tissue are
therapeutically useful. Cells or tissue of a particular type can be
encapsulated and introduced into a subject in need of a certain
type of cell or tissue. Endocrine cells, for example, are one class
of cells that can be encapsulated using the initiator polymer of
the invention and that can be therapeutically useful following
administration to a patient having an endocrine-related disorder.
Specific types of endocrine cells such as pancreatic islets can be
encapsulated using the initiator polymer of the invention and
transplanted to a diabetic patient in need of functional pancreatic
tissue.
[0026] The ligand-coupled initiator polymer of the invention
includes one or more ligand groups. As used herein, "ligand group"
refers to any sort of chemical moiety that displays a specific
binding interaction with a receptor on a surface. The receptor can
be a molecule on a biological surface (e.g., a cell surface), for
example, a protein or a carbohydrate. Ligand:receptor interactions
exhibit binding specificity and typically exhibit effector
specificity. Specific binding interactions of a ligand to a
receptor are generally characterized as saturable. According to the
invention, ligand:receptor dissociation constants (K.sub.d) on the
order of 10.sup.-6 to 10.sup.-12 M are typical of most specific
binding interactions between the ligand and receptors as described
herein.
[0027] The ligand group of the initiator polymer can allow for the
specific localization and binding of the initiator polymer to the
surface of a biological substrate such as a cell, group of cells,
or tissue. Use of ligand groups allows for cell- or tissue-specific
surface localization of the initiator polymer and the formation of
a polymeric matrix on the surface of these specific target cells or
tissues. In another aspect the ligand group can serve to promote a
biological response as a consequence of the ligand:receptor
interaction.
[0028] Examples of specific ligand:receptor interactions include
small molecule:cell-surface receptor interactions such as
sulfonylurea:sulfonylurea receptor and
amiloride:amiloride-sensitive sodium channel protein (ENaC)
interactions; and protein or peptide:cell-surface receptor
interactions such as thyroid-stimulating hormone (TSH):thyroid
plasma membrane receptor, vasopressin:vasopressin receptor, and
antibody or antibody fragment:cell-surface antigen interactions. A
receptor molecule can be any sort of surface determinant on a
biological material, such as a portion of a membrane protein or a
portion of a carbohydrate moiety attached to membrane proteins. The
ligand can be chosen to bind various classes of cell surface
receptors. Such classes include, for example, G-coupled receptors,
ion-channel receptors, tyrosine kinase-linked receptors, and
receptors with intrinsic enzymatic activity having one or multiple
transmembrane domains.
[0029] The ligand group of the initiator polymer can be derived
from any low molecular weight hydrophilic or lipophilic molecules;
small charged molecules; water soluble peptides (peptide hormones);
lipophilic hormones including erconsanoid hormones; antibodies or
antibody fragments; proteins; and derivatives of any of the
above.
[0030] The ligand group can have either an agonistic or
antagonistic effect on the biological substrate. In one embodiment,
the ligand group of the initiator polymer can bind to the receptor
and elicit one or more biological responses, such as intercellular
signal transduction and gene expression. Intercellular signal
transduction can lead to, for example, changes in gene or protein
expression, or changes in the modification or secretion of a
particular compound, such as a protein, from the cell. In a
preferred embodiment, the ligand is chosen to promote a
biologically useful response from the biological material that it
is in contact with. For example, the ligand group of the initiator
polymer can bind a cell surface receptor and elicit production of a
compound that is physiologically useful, or that is therapeutic for
a particular physical condition. The ligand group pendent from the
initiator polymer can exert its biological effect alone and/or when
incorporated into the polymerized matrix that is formed after the
initiator polymer is activated.
[0031] In one particular embodiment of the invention, the ligand
group of the initiator polymer is a molecule capable of binding to
a receptor on the surface of a pancreatic .beta. cell. In some
preferred embodiments, the ligand group is able to both bind the
pancreatic .beta. cell cell-surface receptor and stimulate an
insulinotropic cellular response from the cells (for example, the
production of insulin). The ligand group can be an insulinotropic
agent able to cause the production and or release of insulin from
the .beta. cell. Therefore, according to the invention, an
initiator polymer having a pancreatic cell-binding ligand can be
placed in contact with and associated with a preparation of
pancreatic islets and used to promote the formation of a matrix
around the islets. Incorporated in the formed matrix is the
initiator polymer containing the ligand group. The islets
encapsulated within the matrix can be transplanted to a subject,
and, because of the matrix, are immunoprotected and able to produce
therapeutically useful compounds, such as insulin, that are able to
produce an effect in the subject.
[0032] In one embodiment, the ligand group can associate with a
portion of an ATP-sensitive potassium (K.sup.+-ATP) channel, and
can be, therefore, a K.sup.+-ATP channel-binding ligand. Portions
of the K.sup.+-ATP channel can include, for example, K.sup.+-ATP
channel proteins such as sulfonylurea receptor proteins SUR1, SUR2,
and pore-forming subunits such as KIR6.1 and KIR6.2. Particularly
relevant portions of these proteins are those that can bind ligands
which function to close the K.sup.+-ATP channel. In pancreatic
.beta. cells K.sup.+-ATP channel-closing ligands, a subgroup of
K.sup.+-ATP channel-binding ligands, can function to trigger
insulin secretion from the cells. Typically this insulin secretion
is caused by the K.sup.+-ATP channel-closing ligands binding and
preventing potassium efflux resulting in membrane depolarization
and calcium influx causing release of the insulin from the
cells.
[0033] K.sup.+-ATP channel-closing ligands include first generation
sulfonylureas such as tolbutamide, tolazamide, chlorpropamide, and
acetohexamide; second generation sulfonylureas such as glimepiride,
glipizide, and glyburide; insulin secretagogues such as
meglitinide, repaglinide, nateglinide, prandin, and starlix;
imidazoline-derived drugs such as midaglizole, LY397364, and
LY389382; mitiglinide and analogues such as
5-chloro-N-(2-(4-hydroxyphenyl)ethyl)-2-methoxybenzamide and
4-(2-(5-chloro-2-methoxybenzamido)ethyl)phenyl phosphate (Hastedt
and Panten, Biochem. Pharmacol. 65:599 (2003));
9-(3,4-dichlorophenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydro-1,8(2H,-
5H)-acridinedione (Gopalakrishnan, M. et al. Br. J. Pharmacol.
138:393 (2003)); and functional derivatives thereof.
[0034] In another embodiment, the invention provides polymerizable
monomers having a ligand group that can associate with a portion of
a K.sup.+-ATP channel. In particular, polymerizable monomers having
an ethylenically unsaturated group and a ligand group having a
sulfonylurea portion are provided by the invention.
[0035] The ligand groups can be coupled to the backbone of the
initiator polymer in any suitable manner. For example, the ligand
groups can be coupled to the backbone by preparing ligand-monomers
and polymerizing the ligand-monomers with initiator-monomers.
Synthesis of ligand-monomers can be readily accomplished using
standard chemical reactions. Another method for preparing the
initiator polymer involves preparing a reactive ligand moiety and
reacting the ligand moiety with a reactive group on a preformed
polymer. For example, an isocyanate or isothiocyanate derivative of
a ligand group can be reacted with a polymer containing pendent
amine groups thereby forming an initiator polymer bearing pendent
ligand groups. The ligand groups can be coupled to and spaced in
any suitable manner along the length of the polymer backbone, for
example the ligand groups can be spaced in a random or ordered
pattern along the length of the polymer backbone chosen or can be
present primarily on one end of the polymer backbone.
[0036] The number of ligand groups coupled to the polymer backbone
can be arranged to provide an initiator polymer that associates
with the cell surface in a manner suitable to allow the formation
of a polymeric material on the surface when the initiator polymer
is activated. In one embodiment, the initiator polymer includes at
least one ligand group. In another embodiment of the invention, the
initiator polymer has up to about 5% of the monomeric units of the
polymer coupled to ligand groups. In yet another embodiment the
initiator polymer has up to about 10% of the monomeric units of the
polymer coupled to ligand groups.
[0037] According to the invention, the ligand-coupled initiator
polymer includes one or more initiator groups, which are coupled to
the backbone of the initiator polymer. The initiator groups are
able to promote free radical polymerization of polymerizable
material, such as macromers, when energy capable of activating the
initiator group is applied to the initiator polymer. Activated
initiator groups can cause free radical polymerization of the
polymerizable material either directly or indirectly. Indirect
methods typically include the transfer of energy from the activated
initiator to an acceptor or reductant, a chemical species that can
form a free radical and can act to cause polymerization of the
polymerizable material. In direct methods the initiator group
provides the free radical itself.
[0038] According to the invention, the initiator polymer can be
localized to a surface, such as the surface of a cell, via
interaction of the ligand groups with the receptor on the surface.
Upon activation of the initiator groups, polymerizable material
that is in proximity to the initiator polymer polymerizes, leading
to the formation of a layer of polymeric material, or a matrix, on
the surface. This type of polymerization is typically referred to
as interfacial polymerization.
[0039] The initiator polymer can include light-activated
photoinitiator groups, thermally activated initiator groups,
chemically activated initiator groups, or combinations thereof.
Suitable thermally activated initiator groups include
4,4'azobis(4-cyanopentanoic) acid and
2,2-azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride or other
thermally activated initiators provided these initiators can be
incorporated into an initiator polymer. Chemically activated
initiation is often referred to as redox initiation, redox
catalysis, or redox activation. In general, combinations of organic
and inorganic oxidizers, and organic and inorganic reducing agents
are used to generate radicals for polymerization. A description of
redox initiation can be found in Principles of Polymermization,
2.sup.nd Edition, Odian G., John Wiley and Sons, pgs 201-204
(1981). Redox initiators that are not damaging to biological
systems are preferably used. Photoinitiator groups and thermally
activated initiator groups that utilize energy that is not damaging
to biological systems are preferably used. In one embodiment,
photoinitiator groups having long wavelength UV and visible
light-activated frequencies are coupled to the backbone of the
initiator polymer. In a preferred embodiment, visible
light-activated photoinitiators are coupled to the polymer
backbone.
[0040] Photoinitiation can occur by various mechanisms, including
Norrish type I reactions, intra- or intermolecular hydrogen
abstraction reactions, and photosensitization reactions utilizing
photoreducible or photo-oxidizable dyes. The latter two types of
reactions are commonly used with an energy transfer acceptor or a
reductant, which can be, for example, a tertiary amine. Such
tertiary amines can be incorporated into the polymeric backbone of
the macromer. In a preferred embodiment, the initiator polymer
includes one or more initiator groups that allow for intra- or
intermolecular hydrogen abstraction reactions or photosensitization
reactions utilizing photoreducible or photo-oxidizable dyes when
activated. Useful energy transfer acceptors or reductants for use
with these types of initiators include, but are not limited to,
tertiary amines such as triethanolamine, triethylamine, N-methyl
diethanolamine, N,N-dimethyl benzylamine, tetramethyl
ethylenediamine; secondary amines such as dibenzyl amine, N-benzyl
ethanolamine, N-isopropyl benzylamine; and primary amines such as
ethanolamine, lysine, and ornithine.
[0041] In one embodiment, photoinitiator groups having an
absorbance of 350 nm and greater are used. More preferably,
photoinitiator groups having an absorbance of 500 nm and greater
are used. Suitable photoinitiator groups include light-activated
initiator groups, such as long-wave ultra violet (LWUV)
light-activatable molecules and visible light activatable
molecules. Suitable long-wave ultra violet (LWUV) light-activatable
molecules include, but are not limited to,
[(9-oxo-2-thioxanthanyl)-oxy]acetic acid, 2-hydroxythioxanthone,
and vinyloxymethylbenzoin methyl ether. Suitable visible light
activatable molecules include, but are not limited to acridine
orange, camphorquinone, ethyl eosin, eosin Y, erythrosine,
fluorescein, methylene green, methylene blue, phloxime, riboflavin,
rose bengal, thionine, xanthine dyes, and the like.
[0042] One common feature of these visible light activatable
photoinitiator groups, and photoinitiator groups in general, is
that of having a nonpolar portion. Due to the presence of this
nonpolar portion, these photoinitiator groups generally have a low
solubility in aqueous solutions. When these photoinitiator groups
are coupled to another molecule, such as a polymer, the
photoinitiator groups can confer nonpolar characteristics to the
polymer conjugate and can generally reduce the solubility of the
polymer conjugate in an aqueous solution.
[0043] The initiator polymer is coupled to a number of initiator
groups in an amount sufficient to promote free radical
polymerization of polymerizable material on a surface, such as the
surface of a cell. The initiator polymer contains at least one and
more typically a plurality of initiator groups. In some cases, the
initiator polymer is highly loaded with initiator groups and can
provide a high level of polymerization initiator activity. This may
be desirable in cases wherein the number of receptor molecules on
the surface of a cell is low and the highest polymerization
potential per initiator polymer is desired. In another aspect, an
initiator polymer highly loaded with initiator groups can be
prepared and used in methods or compositions that include macromers
that do not readily polymerize to form a polymeric layer.
Accordingly, the invention provides ligand-coupled initiator
polymers that are highly loaded with initiator groups.
[0044] According to the invention, the initiator polymer includes
at least one initiator group. In another embodiment of the
invention, the initiator polymer has up to about 5% of the
monomeric units of the initiator polymer coupled to initiator
groups. In yet another embodiment, about 10% of the monomeric units
of the polymer are coupled to initiator groups. The initiator
groups can be coupled to and pendent along the polymer backbone at
any position and can be spaced in a random or ordered manner. The
initiator groups preferably do not interfere with the ability of
the initiator polymer to specifically associate with its receptor
on a surface, such as a cell surface.
[0045] The initiator group can be coupled to the initiator polymer
using any suitable method. In one method, for example,
polymerizable monomers having initiator groups can be synthesized
and subsequently used in a polymerization reaction to create an
initiator polymer with pendent initiator groups. Synthesis of
initiator-derivatized monomers can be readily accomplished using
standard chemical reactions. For example, an isothiocyanate or an
acid chloride analog of a photoinitiator group, such as a
light-activated dye, can be reacted with an ethylenically
unsaturated amine-containing monomer to form an
initiator-derivatized monomer. In another method of preparing the
initiator polymer, preformed polymers having reactive groups are
reacted with initiator groups to attach the initiator groups to the
preformed polymer. For example, an isothiocyanate analog of a
photoinitiator can be reacted with a polymer having pendent amine
groups thereby forming an initiator polymer having pendent
initiator groups. Other synthetic schemes known to those skilled in
the art can be employed to prepare the initiator polymer. These
schemes are contemplated but will not be discussed in further
detail.
[0046] In preferred embodiments the initiator polymer includes a
plurality of initiator groups that are typically nonpolar. The
presence of a plurality of initiator groups can confer substantial
hydrophobic properties to the initiator polymer. Accordingly, this
substantial hydrophobic property can be counter balanced by
providing the initiator polymer with a hydrophilic backbone, which
is discussed in detail below.
[0047] In a preferred embodiment of the invention, the
ligand-coupled initiator polymer includes a ligand group, an
initiator group, and is soluble in an aqueous solution. Generally,
the initiator polymer includes a hydrophilic polymer backbone. The
polymer backbone, which generally refers to the polymer chain
without addition of any initiator group or ligand group, typically
includes carbon and preferably one or more atoms selected from
nitrogen, oxygen, and sulfur. The backbone can include
carbon-carbon linkages and, in some preferred embodiments, can also
include one or more of amide, amine, ester, ether, ketone, peptide,
or sulfide linkages, or combinations thereof.
[0048] The polymeric backbone of the initiator polymer can include
chemical groups useful for coupling the ligand group and the
initiator group to the backbone to form the initiator polymer.
Suitable chemical groups include acid (or acyl) halide groups,
alcohol groups, aldehyde groups, alkyl and aryl halide groups,
amine groups, amide groups, carboxyl groups, and the like. These
chemical groups can be present either on a preformed polymer or on
monomers used to create the ligand-coupled initiator polymer.
Examples of polymers having suitable reactive or charged side group
include polymers having reactive amine groups such as polylysine,
polyornithine, polyethylenimine, and polyamidoamine dendrimers.
[0049] In one embodiment of the invention, the backbone of the
initiator polymer provides the initiator polymer with hydrophilic
properties. Preferred hydrophilic backbones include highly
water-soluble polymers such as polyacrylamide. Examples of suitable
polymer backbones include polyesters, polycarbonates, polyamides,
polyethers (such as polyoxyethylene), polysulfones, polyurethanes,
and copolymers containing representative monomer groups. Other
suitable polymers include polyamines such as polyethylenimine,
polypropylenimine, and the like, and polyamine polymers or
copolymers formed from monomers such as 2-aminoethylacrylate,
N-(3-aminopropyl)methacrylamide, and diallyl amine. In one
preferred embodiment the backbone of the initiator polymer contains
relatively few or no aromatic groups. Therefore, in one preferred
embodiment of the invention, the initiator polymer includes (i) a
polymerization initiator group, (ii) a ligand group, and (iii) a
hydrophilic backbone.
[0050] In another aspect, the hydrophilic character of the
initiator polymer can be improved by coupling charged groups to the
polymer backbone. In these embodiments it is preferable that the
initiator polymer is configured so that the presence of the charged
groups does not interfere with the ability of the initiator polymer
to associate with the target receptor on the surface of the
substrate to be coated. Suitable charged groups include cationic
groups such as quaternary ammonium, quaternary phosphonium, and
ternary sulfonium groups. Suitable anionic groups that can be
coupled to the initiator polymer include, but are not limited to,
sulfonate, phosphonate, and carboxylate groups.
[0051] An initiator polymer having at least one initiator group and
at least one ligand group can be prepared a variety of ways. For
example, the initiator group and the ligand group can be attached
to a "preformed" polymer or a copolymer that is reactive with the
initiator and ligand groups. The preformed polymer or copolymer can
be obtained from a commercial source or be synthesized from the
polymerization of a desired monomer or combination of different
monomers. In one example of preparing the initiator polymer, the
initiator groups and the ligand groups are reacted with and
attached to, for example, by covalent bonding, chemical groups
pendent from the backbone of the polymer or copolymer. Such
attachments of the initiator groups and the ligand groups can be
achieved by, for example, substitution or addition reactions.
[0052] In another method of preparing the initiator polymer,
monomers having initiator and monomers having ligand groups are
first prepared. These initiator and ligand group-containing
monomers are then co-polymerized to create an initiator polymer
having both initiator and ligand groups. In some embodiments an
individual monomer having both an initiator group and a ligand
group can be used to prepare the initiator polymer. Optionally,
other monomers that are not coupled to either an initiator or
cationic groups can be polymerized with the ligand and
initiator-coupled monomers to create the initiator polymer. A
useful mixture of monomers for preparation of the initiator polymer
includes up to about 10 wt % of a ligand-monomer, up to about 90 wt
% of a hydrophilic monomer, and up to about 20 wt % of a monomer
having a charged group. Methods of preparing the initiator polymer
are exemplified below. Other standard methods known to those of
skill in the art to prepare the initiator polymer are contemplated
and will not be discussed further.
[0053] In one embodiment, the initiator polymer has (i) an amount
of ligand groups that allow the initiator polymer to specifically
associate with a receptor on a surface, (ii) an amount of initiator
groups that can promote polymerization of a macromer on a surface,
and (iii) a hydrophilic backbone of a size sufficient to solubilize
the initiator polymer in an aqueous solution. In various
embodiments, the initiator polymer has a weight average molecular
weight (M.sub.w) of greater than about 50K Da, 100K Da, 250K Da,
500K Da, 750K Da, and 10,000 K Da. In some embodiments it is
preferable that the initiator polymer has a M.sub.w, in the higher
ranges of these molecular weights recited.
[0054] As used herein "weight average molecular weight" or M.sub.w,
is an absolute method of measuring molecular weight and is
particularly useful for measuring the molecular weight of a polymer
(preparation), such as preparations of initiator polymers and
macromers. Polymer preparations typically include polymers that
individually have minor variations in molecular weight. Polymers
are molecules that have a relatively high molecular weight and such
minor variations within the polymer preparation do not affect the
overall properties of the polymer preparation (for example, the
characteristics of an initiator polymer preparation). The weight
average molecular weight (M.sub.w) can be defined by the following
formula:
M w = i N i M i 2 i N i M i ##EQU00001##
[0055] wherein N represents the number of moles of a polymer in the
sample with a mass of M, and .SIGMA..sub.i is the sum of all
N.sub.iM.sub.i (species) in a preparation. The M.sub.w can be
measured using common techniques, such as light scattering or
ultracentrifugation. Discussion of M.sub.w and other terms used to
define the molecular weight of polymer preparations can be found
in, for example, Allcock, H. R. and Lampe, F. W., Contemporary
Polymer Chemistry; pg 271 (1990).
[0056] Therefore, in one specific embodiment of the invention, the
initiator polymer includes (i) a plurality of polymerization
initiator groups, (ii) a ligand group, and (iii) a hydrophilic
backbone, wherein the M.sub.w of the initiator polymer is greater
than about 50K Da, more preferably greater than about 100K Da, and
most preferably greater than about 250K Da.
[0057] In another specific embodiment of the invention, the
initiator polymer includes (i) a plurality of photoinitiator groups
selected from the group of visible light-activated dyes, (ii) a
ligand group, and (iii) a hydrophilic backbone, wherein the M.sub.w
of the initiator polymer is greater than about 50K Da, more
preferably greater than about 100K Da, and most preferably greater
than about 250K Da.
[0058] In yet another specific embodiment of the invention, the
initiator polymer includes (i) a plurality of photoinitiator groups
selected from the group of visible light-activated dyes, (ii) a
K.sup.+-ATP channel-binding ligand group, and (iii) a hydrophilic
backbone, wherein the M.sub.w of the initiator polymer is greater
than about 50K Da, more preferably greater than about 100K Da, and
most preferably greater than about 250K Da.
[0059] The ligand-coupled initiator polymer can promote the
polymerization of polymerizable material, such as macromers, on a
surface having a ligand-binding receptor. A matrix of polymeric
material is formed on the surface after the initiator polymer is
activated. The polymerizable material can be any sort of compound,
including monomers and polymers having one or more polymerizable
groups. Polymerizable groups are portions of the polymerizable
compounds that are able to propagate free radical polymerization,
such as carbon-carbon double bonds. Preferred polymerizable groups
are found in polymerizable compounds having vinyl or acrylate
groups. More specific polymerizable portions include acrylate
groups, methacrylate groups, ethacrylate groups, 2-phenyl acrylate
groups, acrylamide groups, methacrylamide groups, itaconate groups,
and styrene groups. Preferred materials for the encapsulation of
cellular material are biocompatible polymerizable polymers (also
referred to as macromers). Such macromers can be straight chain or
branched polymers or copolymers, or graft copolymers. Synthetic
polymeric macromers, polysaccharide macromers, and protein
macromers suitable for use with the initiator polymer of the
current invention are described in U.S. Pat. No. 5,573,934 (Hubbell
et al.), the teaching of which is incorporated in its entirety by
reference.
[0060] Preferred macromers include, but are not limited to,
polymerizable poly(vinylpyrrolidone) (PVP), poly(ethylene glycol)
(PEG), poly(ethylene oxide) poly(ethyloxazoline), poly(propylene
oxide), polyacrylamide (PAA), poly(vinyl alcohol) (PVA), copolymers
thereof, and the like. In particular, PEG and PAA are more
preferred macromers. These types of macromers are typically soluble
in water and are more stable in vivo as compared to biodegradable
polymers.
[0061] In some cases it may be desirable to use naturally occurring
or synthetic macromers as the polymerizable material. Suitable
macromers include naturally occurring polymers such as
polysaccharides, examples of which include, but are not limited to,
hyaluronic acid (HA), starch, dextran, heparin, and chitosan; and
proteins (and other polyamino acids), examples of which include,
but are not limited to, gelatin, collagen, fibronectin, laminin,
albumin, and active peptides thereof. In order to make these
naturally occurring or synthetic macromers polymerizable,
polymerizable groups can be incorporated into a polymer using
standard thermochemical reactions. For example, polymerizable
groups can be added to collagen via reaction of amine containing
lysine residues with acryloyl chloride. These reactions result in
collagen containing polymerizable moieties. Similarly, when
synthesizing a macromer, monomers containing reactive groups can be
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. Suitable synthetic polymers include
hydrophilic monomers containing degradable segments as described in
U.S. Pat. No. 5,410,016 supra, the teaching of which is
incorporated in its entirety by reference.
[0062] In another aspect, the invention provides a polymerizable
composition that includes a ligand-coupled initiator polymer and a
macromer. The polymerizable composition can also include other
compounds useful cell encapsulation methods such as
reductant/acceptors and viscosity-enhancing agents, for example,
polyethylene glycols, and glycerol. Therefore, in one embodiment,
the invention provides a polymerizable composition that includes:
(i) an initiator polymer having at least one polymerization
initiator group and a ligand group that is capable of interacting
with a receptor on a surface, and (ii) a macromer. In a more
specific embodiment the invention provides a polymerizable
composition that includes: (i) an initiator polymer having
photoinitiator group selected from the group of visible
light-activated dyes, and a ligand group able to interact with a
receptor on a surface, and (ii) a macromer.
Cell Encapsulation Methods
[0063] As previously indicated, the initiator polymer of the
invention is typically used with macromers and, in some cases, a
reductant/acceptor in a method to provide a coating to a biological
surface. The reagents are particularly suitable for cell
encapsulation processes.
[0064] Cells or tissue to be encapsulated can be obtained from an
organism, for example, a human donor, or obtained from a cell
culture, which can be transformed or otherwise modified. Specific
types of cells and tissue that can be encapsulated and used for the
treatment of diseases are discussed below. "Cells" refers to
individual membrane-bound biological units that can be present as
part of a tissue or organ, or can function independently as
micro-organisms. "Tissue" refers to a biological mass that includes
groups of similar cells, and also typically includes extracellular
material that is associated with the cells. Cells, or tissue in
particular, can be subject to treatment prior to the encapsulation
process. For example, tissue can be treated with enzymatic or other
suitable reagents, such as trypsin, hyaluronidase, or collagenase,
to obtain individual cells or cell groups of a suitable size for
the encapsulation process. Alternatively, tissue can be subject to
mechanical processes in order to prepare suitable cellular starting
material. Prior to encapsulation cells can also be treated with
drugs, prodrugs, hormones, or the like, or can be cultured to
provide cells that display a desired expression pattern or have a
certain morphological features. Technical references that provide
detailed instructions for the preparation of cells or tissue and
the treatment of prepared cells or tissue are available and can be
found in, for example, in Basic Cell Culture Protocols, Pollard, J.
W. and Walker, J. M., Ed. (1997).
[0065] Alternatively, cells or tissue suitable for encapsulation
and intended for use with the ligand-coupled initiator of the
invention can be commercially obtained. For example, viable human
liver preparations such as microsomes and hepatocytes, and viable
human pancreatic preparations such as pancreatic islets, can be
obtained from commercial sources such as CellzDirect, Inc. (Tucson,
Ariz.).
[0066] With information available in technical literature, one can
utilize the ligand-coupled initiator polymer in methods for coating
a surface, and in particular, in the novel and inventive methods as
described herein for encapsulating cells and tissue. For example,
the teaching Cruise, et al., Cell Transplantation 8:293 (1999), can
provide a basis for the cell encapsulation methods using the
ligand-coupled initiator polymer of the invention. Cells or tissue
suitable for the encapsulation process, prepared as indicated above
or obtained from a commercial source, can be suspended in a
suitable solution, such as a biocompatible buffered aqueous
solution, such as, for example Roswell Park Memorial Institute
(RPMI) media. Other reagents can be added to this solution, such as
animal serum; proteins such as albumin; oxidants; reductants;
vitamins; minerals; growth factors; or other components that can
have an impact on the viability and function of the cells or
tissues.
[0067] The ligand-coupled initiator polymer can be added to this
solution before or after contacting the cells or tissue with the
solution. The initiator polymer can be brought into contact with
the cells in an amount that is sufficient for formation of a matrix
around the cells or tissue. In one embodiment, the concentration of
the initiator polymer is from 0.001 to 0.5 wt %. In yet another
embodiment, the concentration of the initiator polymer is from 0.1
to 0.25 wt %. In one embodiment the initiator polymer is brought in
contact with the cells for a period of time that is sufficient for
the initiator polymer to associate with the surface of the cells.
Optionally, a washing step can be performed. This washing step can
be used, for example, to remove excess unbound initiator or other
material in contact with the cells. After the initiator polymer is
brought in contact with the cells or tissue, the polymerizable
material, such as macromers, can be brought in contact with the
cells. In another embodiment, the initiator polymer is brought into
contact with the cells or tissue together with the polymerizable
material. In yet another embodiment the polymerizable material is
brought into contact with the cells prior to bringing the initiator
polymer into contact with the cells.
[0068] The polymerizable material (e.g., macromers) can be brought
into contact with the cell or tissue in an amount that allows
formation of a matrix of a desired thickness. A concentration of
macromer in solution useful for cell encapsulation can be in the
range of 5-50 wt %, and more preferably in the range of 10-30 wt %.
In some embodiments, the polymerizable material can be placed in
contact with the cells for a period of time prior to activating the
ligand-coupled initiator polymer.
[0069] Other reagents can be brought in contact with the cells or
tissue during the encapsulation process. As previously mentioned,
such reagents include acceptors or reductants, such as tertiary
amines (e.g., triethanolamine) that can form a free radical and
cause free radical polymerization of the polymerizable material.
Suitable acceptors or reductants are known in the art and are
commercially available. These acceptors or reductants are typically
used in indirect polymerization methods wherein the initiator group
transfers energy to the acceptors or reductants to promote free
radical polymerization of the polymerizable material. Reagents such
as viscosity-enhancing reagents can also be used in the method of
the invention. Viscosity-enhancing reagents can improve the process
of polymerization. Suitable viscosity-enhancing reagents are known
in the art and are commercially available. One of skill in the art
can determine suitable amounts of any of these additional reagents
for performing the encapsulation process.
[0070] After the reagents necessary to promote formation of a
matrix are brought in contact with the surface to be coated, a
source of energy, such as a thermal or electromagnetic energy
sufficient to activate the initiator group, is applied to initiate
polymerization of the polymerizable material. Long-wave ultra
violet (LWUV) and visible wavelengths in range of 350 nm to 900 nm
are preferred and can be supplied by lamps and laser light sources.
Lamps or laser light sources that can provide these wavelengths of
light are commercially available and can be obtained from, for
example, EFOS Inc. (Mississauga, Ontario, Canada). A particularly
suitable wavelength for use with the preferred initiator polymers
of the invention is about 520 nm. The time and temperature of the
reaction are maintained to provide a desired coating. For example,
the cells or tissue in contact with the initiator polymer and
macromer can be treated with light for a period in the range of
seconds to minutes. The polymerization reaction can be terminated
by removing the light source. The encapsulated cells or tissue can
then be subject to further treatment if desired. For example, it
may be desirable to concentrate the encapsulated material, for
example, by centrifugation, prior to introducing the encapsulated
material into a subject.
[0071] As indicated, a number of technical references that provide
detailed procedures for encapsulating cells are available and can
provide a framework for which the ligand-coupled initiator polymer
can be used. Therefore using the available information, one can
perform surface coating of a material, more specifically, the
encapsulation of cellular material and tissue using the
ligand-coupled initiator polymer and reagents described herein or
in other references.
Treatment
[0072] According to the invention, the initiator polymer can be
used to promote the formation of a matrix of polymerized material
on a biological surface. Polymerization using the initiator polymer
can be performed in vivo by applying an initiator polymer and
polymerizable material, either together or separately, to a subject
in either an invasive or in a noninvasive procedure. Other
particularly useful applications involve the ex vivo encapsulation
of cells or tissue. In this application cells or tissue can be
obtained from a suitable source, encapsulated with a matrix of
polymeric material using a composition including the initiator
polymer described herein, and then introduced into a subject in
need of the encapsulated cells or tissue. In some cases, after
receiving the transplanted encapsulated cells, the subject can be
administered a pharmaceutical agent, such as a compound that is
different than the compound used as the ligand group of the
initiator polymer, that can penetrate the matrix that encapsulates
the cells and can provoke a cellular response which is of
therapeutic value to the subject. This type of ex vivo
encapsulation and transplantation procedure is advantageous as it
can provide a matrix coating affording the transplanted cells
protection from host immune rejection while allowing the
encapsulated cells to provide a therapeutic value to the host.
[0073] In one aspect of the invention, the initiator polymer is
used to encapsulate cells or tissue from glands and organs of the
endocrine system, which include cells from the pituitary gland;
cells from the adrenal gland; cells from the thyroid/parathyroid
glands; cells from the pancreatic islets, such as beta cells, alpha
cells, delta cells, and pancreatic polypeptide (PP) cells; cells
from the liver; and cells from reproductive glands such as the
testis and ovary. Endocrine cells can be removed from a donor
individual and encapsulated with polymeric material using the
initiator polymer as described herein.
[0074] Encapsulated endocrine cells can be transplanted to an
individual having any of the following conditions or needs: a
pituitary disorder and in need of growth hormone (GH),
adrenocorticotropic hormone (ACTH), follicle stimulating hormone
(FSH), leutinizing hormone (LH), thyroid stimulating hormone (TSH),
oxytocin, or antidiuretic hormone (ADH); an adrenal disorder and in
need of mineralcorticoids (for example, aldosterone)
glucocorticoids (for example, cortisol), androgenic steroids, or
catecholamines such as epinephrine or norepinephrine; a thyroid or
parathyroid disorder and in need of thyroxin, calcitonin, or
parathyroid hormone (PTH); a pancreatic disorder such as diabetes
and in need of insulin, glucagon, somatostatin, or pancreatic
polypeptide; a liver disorder and in need of bile or plasma
proteins, including clotting factors; a reproductive gland disorder
and in need of male hormones such as testosterone or female
hormones such as estrogen.
[0075] Other types of cells that can be encapsulated include
immature and mature cells from the cardiovascular, respiratory,
renal, nervous, muscular, and skeletal systems. In some aspects
cells that have been transformed or genetically modified can be
encapsulated and transplanted into a host. For example, cells that
have been transformed or modified to produce a therapeutically
useful compound, such as a peptide hormone or an enzyme can be
encapsulated and introduced into an individual.
[0076] The invention also specifically provides interfacial
polymerization compounds, compositions, and methods for the
treatment of diabetes. In particular, the invention provides for
initiator polymers useful for the binding to and promoting the
interfacial polymerization of a biocompatible polymeric layer
around pancreatic .beta. cells and islets. At the same time, the
use of the initiator polymer to provide the polymeric layer
stimulates a desirable cellular response by potentiating an
increased insulin production from the cells.
[0077] As stated above, in some instances, a pharmaceutical agent
can be administered to the subject after transplantation of the
encapsulated cells. The pharmaceutical agent can provoke a
therapeutically useful cellular response from the encapsulated
cells if needed. Other drugs that can stimulate insulin production
and that can be coadministered with the transplanted encapsulated
.beta. cells include metformin, acarbose, and troglitazone. Other
useful drugs include that can be administered to subjects having
encapsulated cells include antithrombogenic, anti-inflammatory,
antimicrobial, antiproliferative, and anticancer compounds, as well
as growth factors, morphogenic proteins, and the like.
[0078] In another aspect, the initiator polymer and polymerizable
material can also be used in in vivo applications to provide
artificial barriers, for example, barriers to prevent tissue
adhesion following surgery. For this application, the initiator
polymer along with polymerizable material is applied to the surface
of the tissue. The composition is then illuminated to initiate
polymerization and a bather matrix is formed. The polymeric matrix
prevents other tissue from adhering to the coated tissue. In some
procedures a polymeric matrix can be formed on the surface of a
blood vessel to prevent blood factors or cells, such as platelets,
from interacting with or adhering to the blood vessel wall. Both
degradable and non-degradable macromer systems can be used for this
purpose.
[0079] The initiator polymer of the invention can also be utilized
for other medically useful purposes. For example, the initiator
polymer can be a component used for forming adhesives for tissue
and other surfaces. In another example, the initiator polymer can
be applied to a surface bearing a receptor, to which adhesion is
desired. The surface can be washed to remove any unbound or excess
initiator polymer and a polymerizable material can be added.
Another surface can be contacted with the initiator polymer-coated
surface and then a source of energy can be applied to activate the
initiator polymer and to polymerize the polymerizable material,
thereby forming a surface-to-surface junction. If a temporary
adhesive is desired, the polymerizable material can include a
degradable material, for example, biodegradable macromers.
[0080] The initiator polymer can also be used for the formation of
barriers on surfaces bearing a receptor. An example of such an
application is a barrier for the prevention of tissue adhesion
following surgery. For this application, an initiator polymer can
be applied to the surface of damaged tissue. The surface can be
washed to remove unbound or excess initiator polymer, and a
polymerizable material can then be added. The initiator polymer can
then be activated on the surface to polymerize the polymerizable
material. The polymeric matrix formed by this polymerization can
prevent other tissue from adhering to the damaged tissue. Both
degradable and/or non-degradable macromers can be used in this
barrier formation method.
[0081] The invention will now be demonstrated referring to the
following non-limiting examples.
TABLE-US-00001 TABLE I ##STR00001## (Compound I: SUM) ##STR00002##
(Compound II: EITCM) ##STR00003## (Compound III: SEAA Initiator
Polymer) ##STR00004## (Compound IV: SUNCS) ##STR00005## (Compound
V: SEAP Initiator Polymer)
EXAMPLES
Example 1
Synthesis of a Sulfonylurea Monomer (SUM)
[0082] Preparation of a monomer having a sulfonylurea ligand
portion is achieved according to the synthetic scheme as
illustrated in FIG. 1. A solution of
4-(2-aminoethyl)benzenesulfonamide (AEBS) and triethylamine (TEA)
in chloroform (or acetonitrile) is cooled in an ice bath. As
illustrated in step 1 of FIG. 1, to the cooled stirred AEBS
solution is added a solution of methacryloyl chloride (MAC), in
chloroform (or acetonitrile). After the addition is completed, the
reaction is stirred at room temperature for 2 hours. The volatile
organic materials are removed under vacuum with an air bleed to
avoid polymerization. The residue (sulfamoyl monomer intermediate
(SMI): 2-methyl-N-(2-(4-sulfamoyl-phenyl)-ethyl)-acrylamide) is
dissolved in an aqueous sodium hydroxide solution. As illustrated
in step 2, to the aqueous solution is added a solution of
cyclohexyl isocyanate (CI) in acetone (or acetonitrile), and the
resultant reaction is stirred at room temperature for 16 hours.
Finally, the reaction is acidified with HCl and the precipitate is
isolated and dried to give the sulfonylurea monomer (Compound I:
SUM), which is also shown in Table I.
Example 2
Synthesis of an EITC Monomer (EITCM)
[0083] Preparation of a monomer having an EITC photoinitiator
portion is achieved according to the synthetic scheme as
illustrated in FIG. 2. To a solution of eosin isothiocyanate (EITC;
4-Isothiocyanato-2-(2,4,5,7-tetrabromo-6-hydroxy-3-oxo-3H-xanthen-9-yl)-b-
enzoic acid methyl ester) in dimethylsulfoxide (DMSO) is added a
solution of N-(3-aminopropyl)methacrylamide (APMA) in chloroform.
The solution is stirred at room temperature for 16 hours. The
chloroform is removed under vacuum with an air bleed. EITCM in a
DMSO solution is used in the preparation of an initiator polymer in
Example 3. EITCM (Compound II) is also shown in Table I.
Example 3
Preparation of a SUM-EITCM-AMPS-Acrylamide (SEAA) Initiator
Polymer
[0084] The initiator polymer, which can be represented by Compound
III as illustrated in Table I, is prepared by placing SUM, EITCM,
AMPS (sodium 2-acrylamido 2-methyl propane sulfonate),
mercaptoethanol, AIBN (2,2'-azobisisobutyronitrile) and DMSO
(dimethylsulfoxide) in a glass vessel and polymerizing the mixture.
The solution is degassed (deoxygenated), blanketed with argon and
heated at 55.degree. C. with stirring for 16 hours. The DMSO
solution containing the polymer product is placed in 12-14 kDa
molecular weight cut off (MWCO) dialysis tubing and dialyzed
against deionized water. The product is then isolated by
lyophilization.
Example 4
Synthesis of a Sulfonylurea-Isothiocyanate (SUNCS)
[0085] A sulfonylurea derivative (SUNCS) is synthesized in
preparation for making another sulfonylurea-containing initiator
polymer. The preparation of SUNCS is achieved according to the
synthetic scheme as illustrated in FIG. 3.
4-(2-Amino-ethyl)-benzenesulfonamide (AEBS) is first dissolved in
acetonitrile (or chloroform). In step 1 the AEBS solution is
reacted with carbon disulfide (CS.sub.2) and
dicyclohexylcarbodiimide (DCC). Dicyclohexylurea (DCU) product is
removed from sulfamoyl-isothiocyanate (S-NCS) by filtration. The
solvents are then removed to give the S-NCS intermediate. In step 2
the S-NCS, cyclohexyl isocyanate, and tetrabutylammonium bromide
(TBAB) are placed in a glass vessel with tetrahydrofuran (THF) and
the mixture is stirred under an inert dry atmosphere during the
slow addition of sodium hydride (NaH). The SUNCS product (Compound
IV, also shown in Table I) is then isolated and purified by flash
chromatography.
Example 5
Preparation of a SUNCS-EITC-APTAC-PEI (SEAP) Initiator Polymer
[0086] A SEAP initiator polymer is prepared according to the
following procedure. DMSO solutions containing eosin isothiocyanate
(EITCNCS; Sigma-Aldrich Corp., St. Louis, Mo.), acrylamido
propyltrimethyl ammonium chloride (APTAC; Sigma-Aldrich Corp., St.
Louis, Mo.) and SUNCS are first individually prepared. A solution
containing polyamine polyethylenimine (PEI) having a M.sub.w of
10,000 Da is prepared by dissolving the PEI in DMSO. To the PEI
solution is added the EITCNCS, APTAC, and SUNCS solutions. The
reaction is stirred for 16 hours at room temperature. The
polymerization product is purified using 5,000 MWCO dialysis tubing
and the product is isolated by lyophilization. The final product
can be represented by Compound V in Table I wherein EITC represents
eosin, APTAC represents acrylamido propyltrimethyl ammonium
chloride, and SU represents sulfonylurea.
* * * * *