U.S. patent application number 15/067291 was filed with the patent office on 2018-05-31 for simple coacervates and methods of use thereof.
This patent application is currently assigned to UNIVERSITY OF UTAH RESEARCH FOUNDATION. The applicant listed for this patent is UNIVERSITY OF UTAH RESEARCH FOUNDATION. Invention is credited to Russell J. STEWART.
Application Number | 20180147316 15/067291 |
Document ID | / |
Family ID | 45094250 |
Filed Date | 2018-05-31 |
United States Patent
Application |
20180147316 |
Kind Code |
A1 |
STEWART; Russell J. |
May 31, 2018 |
SIMPLE COACERVATES AND METHODS OF USE THEREOF
Abstract
Described herein is the synthesis of adhesive from simple
adhesive coacervates and their uses thereof. The adhesives are
composed of (a) a polyanion in the absence of a polycation, wherein
the polyanion comprises at least one crosslinking group capable of
covalently crosslinking with itself, and (b) a sufficient amount of
a complimentary multivalent cation or multivalent anion to produce
the simple coacervate. The adhesives have numerous medical and
non-medical applications.
Inventors: |
STEWART; Russell J.; (Salt
Lake City, UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF UTAH RESEARCH FOUNDATION |
Salt Lake City |
UT |
US |
|
|
Assignee: |
UNIVERSITY OF UTAH RESEARCH
FOUNDATION
Salt Lake City
UT
|
Family ID: |
45094250 |
Appl. No.: |
15/067291 |
Filed: |
March 11, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14874854 |
Oct 5, 2015 |
9421300 |
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15067291 |
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13295061 |
Nov 12, 2011 |
9173971 |
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14874854 |
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61412834 |
Nov 12, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 6/30 20200101; A61L
31/16 20130101; Y10T 428/2852 20150115; A61L 31/10 20130101; A61L
24/0015 20130101; A61L 24/06 20130101; A61L 24/001 20130101; A61L
2400/18 20130101; C09J 143/02 20130101; A61L 2300/418 20130101 |
International
Class: |
A61L 24/06 20060101
A61L024/06; A61L 31/16 20060101 A61L031/16; A61L 24/00 20060101
A61L024/00; C09J 143/02 20060101 C09J143/02; A61L 31/10 20060101
A61L031/10; A61K 6/00 20060101 A61K006/00 |
Goverment Interests
ACKNOWLEDGEMENTS
[0002] This invention was made with government support under
N00014-10-1-0108 awarded by the Office of Naval Research. The
government has certain rights in the invention.
Claims
1. An adhesive comprising a simple coacervate, wherein the simple
coacervate comprises (a) a polyanion in the absence of a
polycation, wherein the polyanion comprises at least one
crosslinking group capable of covalently crosslinking with itself,
and (b) a sufficient amount of a complimentary multivalent cation
or multivalent anion to produce the simple coacervate.
2. The adhesive of claim 1, wherein the polyanion comprises a
synthetic or naturally-occurring polymer comprising one or more
sulfate, sulfonate, carboxylate, borate, boronate, phosphonate,
phosphate groups, or any combination thereof.
3. The adhesive of claim 1, wherein the polyanion comprises a
polysaccharide, a protein, or a synthetic polypeptide.
4. The adhesive of claim 1, wherein the polyanion comprises an
inorganic polyphosphate compound.
5. The adhesive of claim 1, wherein the polyanion comprises a
polymer comprising at least one fragment comprising the formula X:
##STR00007## wherein R.sup.4 is hydrogen or group; n is from 1 to
10; Y is oxygen, sulfur, or NR.sup.30, wherein R.sup.30 is
hydrogen, an alkyl group, or an aryl group; Z is sulfate,
sulfonate, carboxylate, borate, boronate, a substituted or
unsubstituted phosphate, or a phosphonate, or the
pharmaceutically-acceptable salt thereof.
6. The adhesive of claim 1, wherein the polyanion comprises a
polymer comprising at least one fragment comprising the formula II
##STR00008## wherein R.sup.4 is hydrogen or an alkyl group, n is
from 1 to 10, R.sup.40 is hydrogen, an alkyl group, or an aryl
group, or ##STR00009## wherein R.sup.41 is, independently,
hydrogen, an alkyl group, an aryl group, a phosphate group, or the
pharmaceutically-acceptable salt thereof.
7. The adhesive of claim 6, wherein R.sup.4 is methyl, R.sup.40 is
hydrogen, and n is 2.
8. The adhesive of claim 2, wherein the complimentary multivalent
cation is Mg.sup.+2.
9. The adhesive of claim 1, wherein the crosslinking group
comprises an actinically crosslinkable group.
10. The adhesive of claim 9, wherein the actinically crosslinkable
comprises an acryloyl group or a methacryloyl group.
11. The adhesive of claim 1, wherein the simple coacervate further
comprises one or more bioactive agents.
12. The adhesive of claim 1, wherein the simple coacervate further
comprises one or more contrast agents.
13. The adhesive of claim 1, wherein the simple coacervate further
comprises a polymerizable monomer, a water-insoluble filler, or a
combination thereof.
14. A method for producing an adhesive comprising covalently
crosslinking the polyanion in the simple coacervate of claim 2.
15. The method of claim 14, wherein the method comprises (a)
introducing into a subject the simple coacervate and (b) covalently
crosslinking the polyanion.
16. The method of claim 15 for closing or sealing a puncture in an
internal tissue or membrane of the subject comprising (a) applying
the simple coacervate to the puncture and (b) covalently
crosslinking the polyanion in the simple coacervate.
17. The method of claim 15 for closing or sealing a puncture in
internal tissue or membrane in the subject by adhering a scaffold
to or within the puncture with the simple coacervate and
subsequently covalently crosslinking the polyanion in the simple
coacervate.
18. The method of claim 15 wherein the simple coacervate is
introduced into a blood vessel and subsequently covalently
crosslinking the polyanion in the simple coacervate to partially or
completely block the vessel.
19. The method of claim 15 wherein the simple coacervate is applied
to a fractured bone and subsequently covalently crosslinking the
polyanion in the simple coacervate to repair the fractured
bone.
20. The method of claim 15 for adhering a bone-tissue scaffold to a
bone of the subject comprising (a) contacting the bone and/or
tissue with the simple coacervate, (b) applying the bone-tissue
scaffold to the bone and tissue, and (c) covalently crosslinking
the polyanion in the simple coacervate.
21. The method of claim 15 for method for securing a dental implant
in the subject, comprising (a) applying to an oral substrate and/or
dental implant the simple coacervate, (b) attaching the dental
implant to the substrate, and (c) covalently crosslinking the
polyanion in the simple coacervate.
22. The method of claim 15 for treating an ocular wound in a
subject comprising applying to the wound the simple coacervate and
subsequently covalently crosslinking the polyanion in the simple
coacervate.
23. A method for modifying one or more properties of a substrate,
the method comprising (a) applying on the surface of the substrate
the simple coacervate of claim 1, and (b) covalently crosslinking
the polyanion in the simple coacervate on the surface of the
substrate to modify one or more surface properties of the
substrate.
24. The method of claim 23, wherein the surface property is
wettability, corrosion resistance, anti-fouling, or promotion of
specific interactions on the surface of the substrate.
25. The method of claim 23, wherein the substrate is a metal
substrate, a foil, a fiber, a tape, a cloth, coral, or a device
that can be implanted in a subject.
26. A method for adhering two substrates together, the method
comprising (a) applying on the surface of a first substrate the
simple coacervate of claim 1, and (b) applying a second substrate
to the first substrate, wherein the second substrate is in contact
with the simple coacervate; and (c) covalently crosslinking the
polyanion in the simple coacervate on the surface of the substrate
to adhere the first substrate to the second substrate.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 14/874,854 filed on Oct. 5, 2015, which claims
priority upon divisional application of U.S. application Ser. No.
13/295,061 filed on Nov. 12, 2011, which claims priority upon U.S.
provisional application Ser. No. 61/412,834, filed Nov. 12, 2010.
These applications are hereby incorporated by reference in their
entireties.
CROSS REFERENCE TO SEQUENCE LISTING
[0003] Proteins described herein are referred to by a sequence
identifier number (SEQ ID NO). The SEQ ID NO corresponds
numerically to the sequence identifiers <400>1, <400>2,
etc. The Sequence Listing, in written computer readable format
(CFR), is incorporated by reference in its entirety.
BACKGROUND
[0004] There has always been a need for the development of better
adhesives, particularly adhesives that are exposed to aqueous
environments. For example, adhesives that can be administered to a
subject have numerous medical applications, such as with, bone
fractures which are a serious health concern in society today. In
addition to the fracture itself, a number of additional health
risks are associated with the fracture. Intra-articular fractures
are bony injuries that extend into a joint surface and fragment the
cartilage surface. Fractures of the cartilage surface often lead to
debilitating posttraumatic arthritis. Currently, stainless steel
and titanium implants are the primary methods of fixation, but
their size and the drilling necessary to place them frequently
interfere with the exact manipulation and reduction of smaller
pieces of bone and cartilage.
[0005] In additional to medical applications, adhesives that can be
used or exposed to aqueous environments can have several beneficial
applications in non-medical applications as well. For example, an
adhesive can be used to help restore marine ecosystems by adhering
coral and other materials to existing reefs to enhance the growth
and development of the reef. Described herein are adhesives that
address these needs.
SUMMARY
[0006] Described herein is the synthesis of adhesive from simple
adhesive coacervates and their uses thereof. The adhesives are
produced by (a) preparing a solution comprising (1) a
polyelectrolyte, wherein the polyelectrolyte comprises a polyanion
or polycation but not a combination thereof, the polyeletrolyte
comprises at least one crosslinking group, and (2) a sufficient
amount of a complimentary counterion to produce a simple adhesive
coacervate; and (b) crosslinking the simple adhesive coacervate to
produce the adhesive. The adhesives have numerous medical and
non-medical applications. The advantages of the invention will be
set forth in part in the description which follows, and in part
will be obvious from the description, or may be learned by practice
of the aspects described below. The advantages described below will
be realized and attained by means of the elements and combinations
particularly pointed out in the appended claims. It is to be
understood that both the foregoing general description and the
following detailed description are exemplary and explanatory only
and are not restrictive.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several aspects
described below.
[0008] FIGS. 1-6 shows several protein sequences produced by P.
californica that can be used as polycations in the present
invention as well as synthetic polycations and polyanions useful in
the present invention.
[0009] FIG. 7 provides the amino acid mole % of Pc1-Pc8.
[0010] FIG. 8 shows a chart displaying the underwater bond
strengths of adhesives prepared with polyphosphodopa on aluminum
adherends in a standard lap shear configuration (450 kPa is roughly
60 psi).
[0011] FIG. 9 shows a chart displaying the underwater bond
strengths of adhesives prepared with polyphosphodopa on aluminum
adherends in a standard lap shear configuration (450 kPa is roughly
60 psi), where iron (III) oxide nanoparticles are used as the
oxidant and filler.
DETAILED DESCRIPTION
[0012] Before the present compounds, compositions, articles,
devices, and/or methods are disclosed and described, it is to be
understood that the aspects described below are not limited to
specific compounds, synthetic methods, or uses as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular aspects only and
is not intended to be limiting.
[0013] In this specification and in the claims that follow,
reference will be made to a number of terms that shall be defined
to have the following meanings:
[0014] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a pharmaceutical carrier" includes
mixtures of two or more such carriers, and the like.
[0015] "Optional" or "optionally" means that the subsequently
described event or circumstance can or cannot occur, and that the
description includes instances where the event or circumstance
occurs and instances where it does not. For example, the phrase
"optionally substituted lower alkyl" means that the lower alkyl
group can or can not be substituted and that the description
includes both unsubstituted lower alkyl and lower alkyl where there
is substitution.
[0016] Ranges may be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another aspect includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another aspect. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint.
[0017] References in the specification and concluding claims to
parts by weight, of a particular element or component in a
composition or article, denotes the weight relationship between the
element or component and any other elements or components in the
composition or article for which a part by weight is expressed.
Thus, in a compound containing 2 parts by weight of component X and
5 parts by weight component Y, X and Y are present at a weight
ratio of 2:5, and are present in such ratio regardless of whether
additional components are contained in the compound.
[0018] A weight percent of a component, unless specifically stated
to the contrary, is based on the total weight of the formulation or
composition in which the component is included.
[0019] Variables such as R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.13-R.sup.22, R.sup.30, R.sup.40, R.sup.41, A, X, Z,
d, m, n, o, s, t, u, v, w, and x used throughout the application
are the same variables as previously defined unless stated to the
contrary.
[0020] The term "alkyl group" as used herein is a branched or
unbranched saturated hydrocarbon group of 1 to 25 carbon atoms,
such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl,
hexadecyl, eicosyl, tetracosyl and the like. Examples of longer
chain alkyl groups include, but are not limited to, a palmitate
group. A "lower alkyl" group is an alkyl group containing from one
to six carbon atoms.
[0021] The term "aryl group" as used herein is any carbon-based
aromatic group including, but not limited to, benzene, naphthalene,
etc. The term "aromatic" also includes "heteroaryl group," which is
defined as an aromatic group that has at least one heteroatom
incorporated within the ring of the aromatic group. Examples of
heteroatoms include, but are not limited to, nitrogen, oxygen,
sulfur, and phosphorus. The aryl group can be substituted or
unsubstituted. The aryl group can be substituted with one or more
groups including, but not limited to, alkyl, alkynyl, alkenyl,
aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy,
carboxylic acid, or alkoxy.
[0022] Any of the compounds described herein can be the
pharmaceutically-acceptable salt. In one aspect,
pharmaceutically-acceptable salts are prepared by treating the free
acid with an appropriate amount of a pharmaceutically-acceptable
base. Representative pharmaceutically-acceptable bases are ammonium
hydroxide, sodium hydroxide, potassium hydroxide, lithium
hydroxide, calcium hydroxide, magnesium hydroxide, ferrous
hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide,
ferric hydroxide, isopropylamine, trimethylamine, diethylamine,
triethylamine, tripropylamine, ethanolamine,
2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine,
histidine, and the like. In one aspect, the reaction is conducted
in water, alone or in combination with an inert, water-miscible
organic solvent, at a temperature of from about 0.degree. C. to
about 100.degree. C. such as at room temperature. In certain
aspects where applicable, the molar ratio of the compounds
described herein to base used are chosen to provide the ratio
desired for any particular salts. For preparing, for example, the
ammonium salts of the free acid starting material, the starting
material can be treated with approximately one equivalent of
pharmaceutically-acceptable base to yield a neutral salt.
[0023] In another aspect, if the compound possesses a basic group,
it can be protonated with an acid such as, for example, HCl, HBr,
or H.sub.2SO.sub.4, to produce the cationic salt. In one aspect,
the reaction of the compound with the acid or base is conducted in
water, alone or in combination with an inert, water-miscible
organic solvent, at a temperature of from about 0.degree. C. to
about 100.degree. C. such as at room temperature. In certain
aspects where applicable, the molar ratio of the compounds
described herein to base used are chosen to provide the ratio
desired for any particular salts. For preparing, for example, the
ammonium salts of the free acid starting material, the starting
material can be treated with approximately one equivalent of
pharmaceutically-acceptable base to yield a neutral salt.
[0024] Described herein are adhesives produced from simple adhesive
coacervate and their applications thereof. In general, the simple
adhesive coacervates are a mixture of one or more polyelectrolytes
(a polycation or a polyanion) capable of crosslinking with itself
and one or more complimentary counterions in balanced proportions
to produce stable aqueous coacervates at a desired pH. The simple
adhesive coacervate is an associative liquid with a dynamic
structure in which the individual polymer components diffuse
throughout the entire phase. The adhesive complex coacervates
exhibit low interfacial tension in water when applied to substrates
either under water or that are wet. In other words, the simple
adhesive coacervate spreads evenly over the interface rather than
beading up. Upon crosslinking of the polyelectrolyte, the adhesive
is produced.
[0025] In one aspect, the adhesive is produced by the process
comprising:
(a) preparing a solution comprising (1) a polyelectrolyte, wherein
the polyelectrolyte comprises a polyanion or polycation but not a
combination thereof, the polyeletrolyte comprises at least one
crosslinking group, and (2) a sufficient amount of a complimentary
counterion to produce a simple adhesive coacervate; and (b)
crosslinking the simple adhesive coacervate to produce the
adhesive.
[0026] Each component of the coacervate and methods for making the
same are described below.
[0027] The polyelectrolyte is composed of a polyanion or polycation
but not a combination thereof. The term "polycation" is any polymer
that has a net positive charge at the pH the simple adhesive
coacervate is formed. Conversely, a "polyanion" is any polymer that
has a net negative charge at the pH the simple adhesive coacervate
is formed. Unlike a complex adhesive coacervate, which includes a
polycation and polyanion, the simple adhesive coacervate useful
herein includes only a polycation or a polyanion but not a
combination thereof.
[0028] The polycation and polyanion are generally composed of a
polymer backbone with a plurality of chargeable groups at a
particular pH. The groups can be pendant to the polymer backbone
and/or incorporated within the polymer backbone. In certain
aspects, (e.g., biomedical applications), the polycation is any
biocompatible polymer possessing cationic groups or groups that can
be readily converted to cationic groups by adjusting the pH.
[0029] In one aspect, the polycation is a polyamine compound. The
amino groups of the polyamine can be branched or part of the
polymer backbone. The amino group can be a primary, secondary, or
tertiary amino group that can be protonated to produce a cationic
ammonium group at a selected pH. In general, the polyamine is a
polymer with a large excess of positive charges relative to
negative charges at the relevant pH, as reflected in its
isoelectric point (pI), which is the pH at which the polymer has a
net neutral charge. The number of amino groups present on the
polycation ultimately determines the charge of the polycation at a
particular pH. For example, the polycation can have from 10 to 90
mole %, 10 to 80 mole %, 10 to 70 mole %, 10 to 60 mole %, 10 to 50
mole %, 10 to 40 mole %, 10 to 30 mole %, or 10 to 20 mole % amino
groups. As will be discussed below, additional amino groups can be
incorporated into the polymer in order to increase the pI value. In
general, the pI of the polycation is greater than the pH used to
make the simple adhesive coacervate. In one aspect, the polycation
has a pI value greater than 7.
[0030] In one aspect, the amino group can be derived from a residue
of lysine, histidine, arginine, or imidazole attached to the
polycation. Any anionic counterions can be used in association with
the cationic polymers. The counterions should be physically and
chemically compatible with the essential components of the
composition and do not otherwise unduly impair product performance,
stability or aesthetics. Non-limiting examples of such counterions
include halides (e.g., chloride, fluoride, bromide, iodide),
sulfate and methylsulfate.
[0031] In one aspect, the polycation is naturally-occurring
polymer. For example, proteins produced by P. californica can be
used as the polycation. FIGS. 1-5 show the protein sequences of
several cement proteins produced by P. californica (Zhao et al.
"Cement Proteins of the tube building polychaete Phragmatopoma
californica" J. Biol. Chem. (2005) 280: 42938-42944). FIG. 20
provides the amino acid mole % of each protein. Referring to FIGS.
1-4, Pc1, Pc2, Pc4-Pc18 (SEQ ID NOS 1, 2, 5-19, respectively) are
polycations, where the polymers are cationic at neutral pH. The
type and number of amino acids present in the protein can vary in
order to achieve the desired solution properties. For example,
referring to FIG. 7 Pc1 is enriched with lysine (13.5 mole %) while
Pc4 and Pc5 are enriched with histidine (12.6 and 11.3 mole %,
respectively).
[0032] In another aspect, the polycation is a recombinant protein
produced by artificial expression of a gene or a modified gene or a
composite gene containing parts from several genes in a
heterologous host such as, for example, bacteria, yeast, cows,
goats, tobacco, and the like. In another aspect, the polycation can
be a genetically modified protein.
[0033] In another aspect, the polycation can be a biodegradable
polyamine. The biodegradable polyamine can be a synthetic polymer
or naturally-occurring polymer. The mechanism by which the
polyamine can degrade will vary depending upon the polyamine that
is used. In the case of natural polymers, they are biodegradable
because there are enzymes that can hydrolyze the polymers and break
the polymer chain. For example, proteases can hydrolyze natural
proteins like gelatin. In the case of synthetic biodegradable
polyamines, they also possess chemically labile bonds. For example,
.beta.-aminoesters have hydrolyzable ester groups. In addition to
the nature of the polyamine, other considerations such as the
molecular weight of the polyamine and crosslink density of the
adhesive can be varied in order to modify the degree of
biodegradability.
[0034] In one aspect, the polycation includes a polysaccharide, a
protein, a synthetic polyamine, or a synthetic polypeptide.
Polysaccharides bearing one or more amino groups can be used
herein. In one aspect, the polysaccharide is a natural
polysaccharide such as chitosan. Similarly, the protein can be a
synthetic or naturally-occurring compound. In another aspect, the
biodegradable polyamine is a synthetic random copolypeptide,
synthetic polyamine such as poly(.beta.-aminoesters), polyester
amines, poly(disulfide amines), mixed poly(ester and amide amines),
and peptide crosslinked polyamines. It is desirable in certain
aspects that the polycation as well as the polyanion be non-gelling
and low-endotoxin.
[0035] In the case when the polycation is a synthetic polymer, a
variety of different polymers can be used; however, in certain
applications such as, for example, biomedical applications, it is
desirable that the polymer be biocompatible and non-toxic to cells
and tissue. In one aspect, the biodegradable polyamine can be an
amine-modified natural polymer. The term "amine modified natural
polymer" is defined as any natural polymer that has been
subsequently manipulated or processed to change the natural state
of the polymer. For example, the natural polymer can be chemically
modified using the techniques described herein. Alternatively, the
natural polymer can be denatured or digested by an enzyme. In one
aspect, the amine-modified natural polymer can be an amine-modified
protein such as, for example, gelatin or collagen modified with one
or more alkylamino groups, heteroaryl groups, or an aromatic group
substituted with one or more amino groups. Examples of alkylamino
groups are depicted in Formulae IV-VI
##STR00001##
wherein R.sup.13-R.sup.22 are, independently, hydrogen, an alkyl
group, or a nitrogen containing substituent; s, t, u, v, w, and x
are an integer from 1 to 10; and A is an integer from 1 to 50,
where the alkylamino group is covalently attached to the natural
polymer. In one aspect, if the natural polymer has a carboxyl group
(e.g., acid or ester), the carboxyl group can be reacted with a
polyamine compound to produce an amide bond and incorporate the
alkylamino group into the polymer. Thus, referring to formulae
IV-VI, the amino group NR.sup.13 is covalently attached to the
carbonyl group of the natural polymer.
[0036] As shown in formula IV-VI, the number of amino groups can
vary. In one aspect, the alkylamino group is --NHCH.sub.2NH.sub.2,
--NHCH.sub.2CH.sub.2NH.sub.2, --NHCH.sub.2CH.sub.2CH.sub.2NH.sub.2,
--NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2,
--NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2,
--NHCH.sub.2NHCH.sub.2CH.sub.2CH.sub.2NH.sub.2,
--NHCH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2NH.sub.2,
--NHCH.sub.2CH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2NHCH.sub.2C-
H.sub.2CH.sub.2NH.sub.2,
--NHCH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2,
--NHCH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2N-
H.sub.2, or
--NHCH.sub.2CH.sub.2NH(CH.sub.2CH.sub.2NH).sub.dCH.sub.2CH.sub.2NH.sub.2,
where d is from 0 to 50.
[0037] In one aspect, the amine-modified natural polymer can
include an aryl group having one or more amino groups directly or
indirectly attached to the aromatic group. Alternatively, the amino
group can be incorporated in the aromatic ring. For example, the
aromatic amino group is a pyrrole, an isopyrrole, a pyrazole,
imidazole, a triazole, or an indole. In another aspect, the
aromatic amino group includes the isoimidazole group present in
histidine. In another aspect, the biodegradable polyamine can be
gelatin modified with ethylenediamine.
[0038] In one aspect, the polycation includes a polyacrylate having
one or more pendant amino groups. For example, the backbone can be
a homopolymer or copolymer derived from the polymerization of
acrylate monomers including, but not limited to, acrylates,
methacrylates, acrylamides, and the like. In one aspect, the
backbone of the polycation is polyacrylamide. In other aspects, the
polycation is a block co-polymer, where segments or portions of the
co-polymer possess cationic groups depending upon the selection of
the monomers used to produce the co-polymer.
[0039] In one aspect, the polycation is a polyamino compound. In
another aspect, the polyamino compound has 10 to 90 mole % tertiary
amino groups. In a further aspect, the polycation polymer has at
least one fragment of the formula I
##STR00002##
wherein R.sup.1, R.sup.2, and R.sup.3 are, independently, hydrogen
or an alkyl group, X is oxygen or NR.sup.5, where R.sup.5 is
hydrogen or an alkyl group, and m is from 1 to 10, or the
pharmaceutically-acceptable salt thereof. In another aspect,
R.sup.1, R.sup.2, and R.sup.3 are methyl and m is 2. Referring to
formula I, the polymer backbone is composed of CH.sub.2--CR.sup.1
units with pendant --C(O)X(CH.sub.2).sub.mNR.sup.2R.sup.3 units. In
this aspect, the fragment having the formula I is a residue of an
acrylate, methacrylate, acrylamide, or methacrylamide. FIG. 2
(structures C and D) and FIG. 5 (4 and 7) show examples of
polycations having the fragment of formula I, where the polymer
backbone is derived from acrylamide and methacrylate residues as
discussed above. In one aspect, the polycation is the free radical
polymerization product of a cationic tertiary amine monomer
(2-dimethylamino-ethyl methacrylate) and acrylamide, where the
molecular weight is from 10 to 20 kd and possesses tertiary monomer
concentrations from 15 to 30 mol %. FIG. 3 (structures E and F) and
FIG. 5 (5) provide examples of polycations useful herein, where
imidazole groups are directly attached to the polymer backbone
(structure F) or indirectly attached to the polymer backbone via a
linker (structure E via a methylene linker).
[0040] Similar to the polycation, the polyanion can be a synthetic
polymer or naturally-occurring. When the polyanion is a synthetic
polymer, it is generally any polymer possessing anionic groups or
groups that can be readily converted to anionic groups by adjusting
the pH. Examples of groups that can be converted to anionic groups
include, but are not limited to, carboxylate, sulfonate,
phosphonate, boronate, sulfate, borate, or a substituted or
unsubstituted phosphate. The term "substituted phosphate" is
defined herein as a phosphate group where one of the OH protons is
substituted with an organic group such as, for example, an alkyl or
aryl group. Any cationic counterions can be used in association
with the anionic polymers if the considerations discussed above are
met. Depending upon the selection of the anionic group, the group
can be pendant to the polymer backbone and/or incorporated in the
polymer backbone.
[0041] In one aspect, the polyanion is a polysaccharide. Examples
of polysaccharides useful herein include, but are not limited to, a
hyaluronate, arabic gum, an alginate, chondroitin sulfate,
dermatan, dermatan sulfate, heparan sulfate, or any combination
thereof. In another aspect, the polyanion comprises a
polysaccharide, a protein, or a synthetic polypeptide.
[0042] In one aspect, the polyanion is a polyphosphate. In another
aspect, the polyanion is a polyphosphate compound having from 10 to
90 mole % phosphate groups. For example, the polyphosphate can be a
naturally-occurring compound such as, for example, highly
phosphorylated proteins like phosvitin (an egg protein), dentin (a
natural tooth phosphoprotein), casein (a phosphorylated milk
protein), bone proteins (e.g. osteopontin), or DNA. In another
aspect, the polyphosphate is an inorganic polyphosphate such as,
for example, sodium polymetaphosphate (Graham's salt).
[0043] In other aspects, phosphorous containing polymers can be
converted to polyanions. For example, a phospholipid or
phosphosugar is not a polyanion but it can be converted into a
polyanion by creating a liposome or a micelle with it. Thus, in
this aspect, the complex coacervate is a charged colloid.
Alternatively, the colloid can be produced by any of the polyanions
or polycations described herein.
[0044] In another aspect, the polyphosphate can be a synthetic
compound. For example, the polyphosphate can be a polymer with
pendant phosphate groups attached to the polymer backbone and/or
present in the polymer backbone. (e.g., a phosphodiester backbone).
In one aspect, the polyphosphate can be produced by chemically or
enzymatically phosphorylating a natural compound. In another
aspect, a natural serine-rich protein can be phosphorylated to
incorporate phosphate groups into the protein. In a further aspect,
hydroxyl groups present on a polysaccharide can be phosphorylated
to produce a polyanion useful herein.
[0045] In one aspect, the polyanion includes a polyacrylate having
one or more pendant phosphate groups. For example, the backbone can
be a homopolymer or copolymer derived from the polymerization of
acrylate monomers including, but not limited to, acrylates,
methacrylates, acrylamides, and the like. In one aspect, the
backbone of the polyanion is derived from the polymerization of
polyacrylamide. In other aspects, the polyanion is a block
co-polymer, where segments or portions of the co-polymer possess
anionic groups depending upon the selection of the monomers used to
produce the co-polymer. In a further aspect, the polyanion can be
heparin sulfate, hyaluronic acid, chitosan, and other biocompatible
and biodegradable polymers typically used in the art.
[0046] In another aspect, the polyanion is a polymer having at
least one fragment having the formula X
##STR00003##
wherein R.sup.4 is hydrogen or an alkyl group; n is from 1 to 10; Y
is oxygen, sulfur, or NR.sup.30, wherein R.sup.30 is hydrogen, an
alkyl group, or an aryl group; Z is an anionic group or a group
that can be converted to an anionic group, or the
pharmaceutically-acceptable salt thereof.
[0047] In one aspect, Z is sulfate, sulfonate, carboxylate, borate,
boronate, a substituted or unsubstituted phosphate, or a
phosphonate.
[0048] In one aspect, the polyanion is a polyphosphate. In another
aspect, the polyanion is a polymer having at least one fragment
having the formula II
##STR00004##
wherein R.sup.4 is hydrogen or an alkyl group, n is from 1 to 10,
R.sup.40 is hydrogen, an alkyl group, or an aryl group, or
##STR00005##
wherein R.sup.41 is, independently, hydrogen, an alkyl group, an
aryl group, a phosphate group, or the pharmaceutically-acceptable
salt thereof.
[0049] In another aspect, wherein R.sup.4 is methyl, R.sup.40 is
hydrogen, and n is 2. Similar to formula I, the polymer backbone of
formula II is composed of a residue of an acrylate or methacrylate.
The remaining portion of formula II is the pendant phosphate group.
FIG. 6 (structure B), shows an example of a polyanion useful herein
that has the fragment of formula II, where the polymer backbone is
derived from acrylamide and methacrylate residues. In one aspect,
the polyanion is the copolymerization product of ethylene glycol
methacrylate phosphate and acrylamide, where the molecular weight
is from 10,000 to 50,000, preferably 30,000, and has phosphate
groups in the amount of 45 to 90 mol %.
[0050] In another aspect, the polycation or polyanion are
electrostatically associated block copolymers. The
electrostatically associated block copolymers are water-soluble
polymers composed of a polymer backbone with alternating
polycationic blocks (i.e., blocks having a net positive charge) and
polyanionic blocks (i.e., blocks having a net negative charge).
Individual positive or negative charged groups are present in each
block. The groups can be pendant to the polymer backbone and/or
incorporated within the polymer backbone. In certain aspects,
(e.g., biomedical applications), the polycationic blocks are
composed of a series of cationic groups or groups that can be
readily converted to cationic groups by adjusting the pH. In one
aspect, the polycationic block is a polyamine compound. The amino
groups of the polyamine can be branched or part of the polymer
backbone. The amino group can be a primary, secondary, tertiary, or
a guanidinium group that can be protonated to produce a cationic
ammonium group at a selected pH.
[0051] In one aspect, the polycationic block of the copolymer can
be derived from residues of lysine, histidine, arginine, and/or
imidazole. Any anionic counterions can be used in association with
the polycationic block. The counterions should be physically and
chemically compatible with the essential components of the
composition and do not otherwise unduly impair product performance,
stability or aesthetics. Non-limiting examples of such counterions
include halides (e.g., chloride, fluoride, bromide, iodide),
sulfate and methylsulfate.
[0052] In another aspect, the polycationic block can be a
biodegradable polyamine. The biodegradable polyamine can be any of
the synthetic polymers or naturally-occurring polymers described
above. In one aspect, when the polycationic block is an
amine-modified natural polymer, the amine-modified natural polymer
can include an aryl group having one or more amino groups directly
or indirectly attached to the aromatic group. Alternatively, the
amino group can be incorporated in the aromatic ring. For example,
the aromatic amino group is a pyrrole, an isopyrrole, a pyrazole,
imidazole, a triazole, or an indole. In another aspect, the
aromatic amino group includes the isoimidazole group present in
histidine. In another aspect, the biodegradable polyamine can be
gelatin modified with ethylenediamine.
[0053] In one aspect, the polycationic block includes a
polyacrylate having one or more pendant amino groups. For example,
the backbone of the polycationic block can be a homopolymer or
copolymer derived from the polymerization of acrylate or
methacrylate monomers.
[0054] In other aspects, the polycationic block can in itself be a
co-polymer (i.e., random or block), where segments or portions of
the co-polymer possess cationic groups depending upon the selection
of the monomers used to produce the co-polymer. In this aspect, the
number of positively charged groups present in the polycationic
block can vary from a few percent up to 100 percent (e.g., between
10 and 50%). In this aspect, the polycationic block can be the
polymerization product between a neutral monomer (i.e., no charged
groups) and a monomer possessing a positively charged group, where
the amount of each monomer will determine the overall positive
charge of the polycationic block. Thus, it is possible to produce
different polycationic blocks within the electrostatically
associated block copolymer.
[0055] Equations 1-3 below depict different embodiments regarding
the polyactionic block. In equation 1, the same polycationic block
(A) is incorporated into the block copolymer. In equation 2, two
different polycationic blocks (A and B) are present in each
polycationic block. In the case of the polycationic block AB in
equation 2, monomers possessing different cationic groups can be
used to produce the polycationic block AB. Thus, the polycationic
block can in itself be a block copolymer. This is depicted in
equation 2, where A depicts the first block in the polyactionic
block and B depicts the second block. In equation 3, there are two
different polycationic blocks, where each block (A and B) is the
polymerization product of the same monomer.
##STR00006##
[0056] In one aspect, the polycationic block has at least one
fragment of the formula I described above.
[0057] Similar to the polycationic block, the polyanionic block in
the copolymers described herein can be any synthetic polymer
described above. The polyanionic block can in itself be a
co-polymer (i.e., random or block), where segments or portions of
the co-polymer possess cationic groups depending upon the selection
of the monomers used to produce the co-polymer. In this aspect, the
number of negatively charged groups present in the polyanionic
block can vary from a few percent up to 100 percent (e.g., between
10 and 50%). In this aspect, the polyanionic block can be the
polymerization product between a neutral monomer (i.e., no charged
groups) and a monomer possessing a negatively charged group, where
the amount of each monomer will determine the overall negative
charge of the polyanionic block. Thus, it is possible to produce
different polyanionic blocks within the electrostatically
associated block copolymer.
[0058] In one aspect, the polyanionic block is a polyphosphate. In
another aspect, the polyanion is a polyphosphate compound having
from 10 to 90 mole % phosphate groups (i.e., a random co-polymer).
For example, the polyphosphate can be a polymer with pendant
phosphate groups attached to the polymer backbone of the
polyanionic block and/or present in the polymer backbone of the
polyanionic block (e.g., a phosphodiester backbone). In one aspect,
the polyphosphate can be produced by chemically or enzymatically
phosphorylating a protein (e.g., natural serine-rich proteins).
[0059] In one aspect, the polyanionic block includes a polyacrylate
having one or more pendant phosphate groups. For example, the
backbone of the polyanionic block can be a homopolymer or copolymer
derived from the polymerization of acrylate monomers including, but
not limited to, acrylates and methacrylates, Similar to above for
the polycationic blocks as shown in equations 1-3, the polycationic
blocks can be composed of the same or different blocks (A and
B).
[0060] In one aspect, the polyanionic block is a polyphosphate. In
another aspect, the polyanionic block is a polymer having at least
one fragment having the formula X or II described above.
[0061] The polycations and polyanions useful herein have at least
one crosslinking group. The mode of crosslinking between the
polyelectrolyte can vary depending upon the nature of the
crosslinking group. In one aspect, the crosslinking group can
crosslink with itself without the need for additional reagents or
chemistry. For example, a polycation that contains free thiol
groups can crosslink with itself to produce disulfide bonds.
Alternatively, a first polycation can have a nucleophilic group as
a crosslinking group and a second polyelectrolyte can have an
electrophilic group capable of reacting with the nucleophilic
group. Examples of nucleophilic groups that are useful include, but
are not limited to, hydroxyl, thiol, and nitrogen containing groups
such as substituted or unsubstituted amino groups and imidazole
groups. For example, residues of lysine, histidine, and/or cysteine
or chemical analogs can be incorporated into the polycationic block
and introduce nucleophilic groups. Examples of electrophilic groups
include, but are not limited to, anhydride groups, esters, ketones,
lactams (e.g., maleimides and succinimides), lactones, epoxide
groups, isocyanate groups, and aldehydes. For example, a thiol
group (nucleophile) on a first polyeletrolyte can react with an
olefinic group (electrophile) on a second polyeletrolyte via a
Michael addition to crosslink the two polyelectrolytes.
[0062] In other aspects, the crosslinking group present on the
polyeletrolyte can undergo crosslinking via catalytic reactions.
For example, the polyelectrolytes can possess alkynes and azides
capable of undergoing cyclization via a Click reaction.
Alternatively, crosslinking between the polyelectrolytes can be
performed enzymatically (e.g. transglutaminase).
[0063] In one aspect, the crosslinking group is an actinically
crosslinkable group. As used herein, "actinically crosslinkable
group" in reference to curing or polymerizing means that the
crosslinking of the polyelectrolyte is performed by actinic
irradiation, such as, for example, UV irradiation, visible light
irradiation, ionized radiation (e.g. gamma ray or X-ray
irradiation), microwave irradiation, and the like. Actinic curing
methods are well-known to a person skilled in the art. The
actinically crosslinkable group can be an unsaturated organic group
such as, for example, an olefinic group. Examples of olefinic
groups useful herein include, but are not limited to, an acrylate
group, a methacrylate group, an acrylamide group, a methacrylamide
group, an allyl group, a vinyl group, a vinylester group, or a
styrenyl group. In certain aspects, when the crosslinking group is
an actinically crosslinkable group, the polyelectrolyte is capable
of crosslinking with it self in the presence of an initiator.
Alternatively, the actinically crosslinkable groups can polymerize
with any of the polymerizable monomers described herein.
[0064] In another aspect, the crosslinking group includes a
dihydroxyl-substituted aromatic group capable of undergoing
oxidation in the presence of an oxidant. In one aspect, the
dihydroxyl-substituted aromatic group is a dihydroxyphenol or
halogenated dihydroxyphenol group such as, for example, DOPA and
catechol (3,4 dihydroxyphenol). For example, in the case of DOPA,
it can be oxidized to dopaquinone. Dopaquinone is an electrophilic
group that is capable of either reacting with a neighboring DOPA
group or another nucleophilic group. In the presence of an oxidant
such as oxygen or other additives including, but not limited to,
peroxides, periodates (e.g., NaIO.sub.4), persulfates,
permanganates, dichromates, transition metal oxidants (e.g., a
Fe.sup.+3 compound, osmium tetroxide), or enzymes (e.g., catechol
oxidase), the dihydroxyl-substituted aromatic group can be
oxidized. In another aspect, crosslinking can occur between the
polycation and polyanion via light activated crosslinking through
azido groups. Once again, new covalent bonds are formed during this
type of crosslinking.
[0065] In certain aspects, the oxidant can be stabilized. For
example, a compound that forms a coordination complex with
periodate that is not redox active can result in a stabilized
oxidant. In other words, the periodate is stabilized in a
non-oxidative form and cannot oxidize the dihydroxyl-substituted
aromatic group while in the complex. The coordination complex is
reversible and even if it has a very high stability constant there
is a small amount of uncomplexed periodate present. The
dihydroxyl-substituted aromatic group competes with the compound
for the small amount of free periodate. As the free periodate is
oxidized more is released from the reversible complex. In one
aspect, sugars possessing a cis,cis-1,2,3-triol grouping on a
six-membered ring can form competitive periodate complexes. An
example of a specific compound that forms stable periodate complex
is 1,2-O-isopropylidene-alpha-D-glucofuranose. The stabilized
oxidant can control the rate of crosslinking. Not wishing to be
bound by theory, the stabilized oxidant slows down the rate of
oxidation so that there is time to add the oxidant and position the
substrate before the fiber (i.e., adhesive) hardens
irreversibly.
[0066] The stability of the oxidized crosslinker can vary. For
example, polyanions described herein can contain oxidizable
crosslinkers that are stable in solution and do not crosslink with
each other. This permits nucleophilic groups present on another
polyanion to react with the oxidized crosslinker. This is a
desirable feature, which permits the formation of intermolecular
bonds and, ultimately, the formation of a strong adhesive.
[0067] Not wishing to be bound by theory, the polyelectrolyte with
the dihydroxyl aromatic group(s) are stable in that they react
slowly with itself in solution. Thus, the polyeletrolyte reacts
with itself primarily via intermolecular cross-linking (e.g.,
polycation has a nucleophilic group or a dihydroxyl aromatic group)
to produce a simple adhesive coacervate. This provides numerous
advantages with respect to the use and administration of the
adhesive. For example, the polyelectrolyte can be premixed and
administered to a subject instead of the sequential administration
of the polymers. This greatly simplifies administration of the
coacervate and ultimately the adhesive that is not an option with
currently available bioadhesives.
[0068] In other aspects, the crosslinking group present on the
polyeletrolyte can form coordination complexes with transition
metal ions. For example, a transition metal ion can be added to a
mixture of polyeletrolyte, where the polyeletrolyte contains
crosslinking groups capable of coordinating with the transition
metal ion. The rate of coordination and dissociation can be
controlled by the selection of the crosslinking group, the
transition metal ion, and the pH. Thus, in addition to covalent
crosslinking as described above, crosslinking can occur through
electrostatic, ionic, or other non-covalent bonding. Transition
metal ions such as, for example, iron, copper, vanadium, zinc, and
nickel can be used herein.
[0069] In order to produce the simple adhesive coacervate, a
sufficient amount a complimentary counterion is used. The nature
and amount of complimentary counterion that is used will vary
depending upon, among other things, the polyeletrolyte that is
selected, the pH used to make the coacervate, and the dielectric
constant of the solution used to prepare the coacervate. Methods
for producing the simple adhesive coacervate are described in
detail below.
[0070] In certain aspects, when the polyeletrolyte is a polyanion,
the complimentary counterion is a multivalent cation (i.e., cations
having a charge of +2 or greater). In one aspect, the multivalent
cation can be a divalent cation composed of one or more alkaline
earth metals. For example, the divalent cation can be Ca.sup.+2
and/or Mg.sup.+2. In other aspects, transition metal ions with a
charge of +2 or greater can be used as the multivalent cation. In
other aspects, when the polyeletrolyte is a polycation, the
complimentary counterion can be a sulfate, a sulfonate, a
carboxylate, a borate, a boronate, a substituted or unsubstituted
phosphate, or a phosphonate.
[0071] In certain aspects, prior to crosslinking the simple
adhesive coacervate, the coacervate can include one or more
polymerizable monomers capable of undergoing polymerization in
order to produce an internal network within the coacervate. The
selection of the polymerizable monomer can vary depending upon the
application. Factors such as molecular weight can be altered in
order to modify the solubility properties of the polymerizable
monomer in water.
[0072] The selection of the functional group on the polymerizable
monomer determines the mode of polymerization. For example, the
polymerizable monomer can be a polymerizable olefinic monomer that
can undergo polymerization through mechanisms such as, for example,
free radical polymerization and Michael addition reactions. In one
aspect, the polymerizable monomer has two or more olefinic groups.
In one aspect, the monomer comprises one or two actinically
crosslinkable groups as defined herein in the presence of a
photoinitiator. Alternatively, polymerization can be performed in
the presence of thermal or chemical initiators, which are also
discussed in detail below.
[0073] Examples of hydrophilic polymerizable monomers include, but
not limited to, hydroxyalkyl methacrylate (HEMA), hydroxyalkyl
acrylate, N-vinyl pyrrolidone, N-methyl-3-methylidene-pyrrolidone,
allyl alcohol, N-vinyl alkylamide, N-vinyl-N-alkylamide,
acrylamides, methacrylamide, (lower alkyl)acrylamides and
methacrylamides, and hydroxyl-substituted (lower alkyl)acrylamides
and -methacrylamides. In one aspect, the polymerizable monomer is a
diacrylate compound or dimethacrylate compound. In another aspect,
the polymerizable monomer is a polyalkylene oxide glycol diacrylate
or dimethacrylate. For example, the polyalkylene can be a polymer
of ethylene glycol, propylene glycol, or block co-polymers thereof.
In one aspect, the polymerizable monomer is polyethylene glycol
diacrylate or polyethylene glycol dimethacrylate. In one aspect,
the polyethylene glycol diacrylate or polyethylene glycol
dimethacrylate has a M.sub.n of 200 to 2,000, 400 to 1,500, 500 to
1,000, 500 to 750, or 500 to 600.
[0074] In other aspects, the simple adhesive coacervate can include
a water-insoluble filler. Not wishing to be bound by theory, stress
transfer from the polymeric matrix to the rigid filler can be
reasonably expected to provide greater strength and a higher
elastic modulus to the coacervate. The filler can have a variety of
different sizes and shapes, ranging from particles to fibrous
materials. In one aspect, the filler is a nano-sized particle.
Compared to micron-sized silica fillers, nanoscale fillers have
several desirable properties. First, with regard to toughening
properties, nano-particles have been shown to be more effective in
some cases than micro-particles. The higher specific surface area
of nano- vs. microparticles increases the stress transfer from the
polymer matrix to the rigid filler. Second, smaller volumes of
nanofiller are required than of the larger micron-sized particles
for a greater increase in toughness.
[0075] Additionally, an important consequence of the smaller
diameters and lower fill volumes of nanoparticles is reduced
viscosity of the uncured adhesive, which has direct benefits for
processability. This is advantageous, as the coacervate can retain
its injectable character while potentially increasing bond
strengths dramatically. Third, maximum toughening requires uniform
dispersion of the filler particles within the adhesive. Nanoscale
colloidal particles, again because of the small diameter, lend
themselves more readily to stable dispersions within the
coacervate. In the case of state-of-the-art filled epoxy adhesives,
gel-sol techniques create nearly perfect dispersions of
nanosilica.
[0076] In one aspect, the filler comprises a metal oxide, a ceramic
particle, or a water insoluble inorganic salt. Examples of the
nanoparticles or nanopowders useful herein include those
manufactured by SkySpring Nanomaterials, Inc., which are listed
below.
Metals and Non-Metal Elements
Ag, 99.95%, 100 nm
Ag, 99.95%, 20-30 nm
[0077] Ag, 99.95%, 20-30 nm, PVP coated
Ag, 99.9%, 50-60 nm
[0078] Ag, 99.99%, 30-50 nm, oleic acid coated Ag, 99.99%, 15 nm,
10 wt %, self-dispersible Ag, 99.99%, 15 nm, 25 wt %,
self-dispersible
Al, 99.9%, 18 nm
Al, 99.9%, 40-60 nm
Al, 99.9%, 60-80 nm
[0079] Al, 99.9%, 40-60 nm, low oxygen
Au, 99.9%, 100 nm
[0080] Au, 99.99%, 15 nm, 10 wt %, self-dispersible
B, 99.9999%
B, 99.999%
B, 99.99%
B, 99.9%
B, 99.9%, 80 nm
Diamond, 95%, 3-4 nm
Diamond, 93%, 3-4 nm
Diamond, 55-75%, 4-15 nm
Graphite, 93%, 3-4 nm
Super Activated Carbon, 100 nm
Co, 99.8%, 25-30 nm
Cr, 99.9%, 60-80 nm
Cu, 99.5%, 300 nm
Cu, 99.5%, 500 nm
Cu, 99.9%, 25 nm
Cu, 99.9%, 40-60 nm
Cu, 99.9%, 60-80 nm
[0081] Cu, 5-7 nm, dispersion, oil soluble
Fe, 99.9%, 20 nm
Fe, 99.9%, 40-60 nm
Fe, 99.9%, 60-80 nm
[0082] Carbonyl-Fe, micro-sized
Mo, 99.9%, 60-80 nm
Mo, 99.9%, 0.5-0.8 .mu.m
[0083] Ni, 99.9%, 500 nm (adjustable)
Ni, 99.9%, 20 nm
[0084] Ni coated with carbon, 99.9%, 20 nm
Ni, 99.9%, 40-60 nm
Ni, 99.9%, 60-80 nm
Carbonyl-Ni, 2-3 .mu.m
Carbonyl-Ni, 4-7 .mu.m
Carbonyl-Ni--Al (Ni Shell, Al Core)
Carbonyl-Ni--Fe Alloy
[0085] Pt, 99.95%, 5 nm, 10 wt %, self-dispersible
Si, Cubic, 99%, 50 nm
[0086] Si, Polycrystalline, 99.99995%, lumps
Sn, 99.9%, <100 nm
Ta, 99.9%, 60-80 nm
Ti, 99.9%, 40-60 nm
Ti, 99.9%, 60-80 nm
W, 99.9%, 40-60 nm
W, 99.9%, 80-100 nm
Zn, 99.9%, 40-60 nm
Zn, 99.9%, 80-100 nm
Metal Oxides
AlOOH, 10-20 nm, 99.99%
[0087] Al.sub.2O.sub.3 alpha, 98+%, 40 nm Al.sub.2O.sub.3 alpha,
99.999%, 0.5-10 .mu.m Al.sub.2O.sub.3 alpha, 99.99%, 50 nm
Al.sub.2O.sub.3 alpha, 99.99%, 0.3-0.8 .mu.m Al.sub.2O.sub.3 alpha,
99.99%, 0.8-1.5 .mu.m Al.sub.2O.sub.3 alpha, 99.99%, 1.5-3.5 .mu.m
Al.sub.2O.sub.3 alpha, 99.99%, 3.5-15 .mu.m Al.sub.2O.sub.3 gamma,
99.9%, 5 nm Al.sub.2O.sub.3 gamma, 99.99%, 20 nm Al.sub.2O.sub.3
gamma, 99.99%, 0.4-1.5 .mu.m Al.sub.2O.sub.3 gamma, 99.99%, 3-10
.mu.m Al.sub.2O.sub.3 gamma, Extrudate Al.sub.2O.sub.3 gamma,
Extrudate
Al(OH).sub.3, 99.99%, 30-100 nm
Al(OH).sub.3, 99.99%, 2-10 .mu.m
[0088] Aluminium Iso-Propoxide (AIP), C.sub.9H.sub.21O.sub.3Al,
99.9%
AlN, 99%, 40 nm
BaTiO3, 99.9%, 100 nm
BBr.sub.3, 99.9%
[0089] B.sub.2O.sub.3, 99.5%, 80 nm
BN, 99.99%, 3-4 .mu.m
BN, 99.9%, 3-4 .mu.m
B.sub.4C, 99%, 50 nm
[0090] Bi.sub.2O.sub.3, 99.9%, <200 nm
CaCO.sub.3, 97.5%, 15-40 nm
CaCO.sub.3, 15-40 nm
[0091] Ca.sub.3(PO.sub.4).sub.2, 20-40 nm
Ca.sub.10(PO.sub.4).sub.6(OH).sub.2, 98.5%, 40 nm
CeO.sub.2, 99.9%, 10-30 nm
CoO, <100 nm
[0092] Co.sub.2O.sub.3, <100 nm Co.sub.3O.sub.4, 50 nm
CuO, 99+%, 40 nm
[0093] Er.sub.2O.sub.3, 99.9%, 40-50 nm Fe.sub.2O.sub.3 alpha, 99%,
20-40 nm Fe.sub.2O.sub.3 gamma, 99%, 20-40 nm Fe.sub.3O.sub.4,
98+%, 20-30 nm Fe.sub.3O.sub.4, 98+%, 10-20 nm Gd.sub.2O.sub.3,
99.9%<100 nm
HfO.sub.2, 99.9%, 100 nm
[0094] In.sub.2O.sub.3:SnO.sub.2=90:10, 20-70 nm In.sub.2O.sub.3,
99.99%, 20-70 nm
In(OH).sub.3, 99.99%, 20-70 nm
LaB.sub.6, 99.0%, 50-80 nm
[0095] La.sub.2O.sub.3, 99.99%, 100 nm
LiFePO.sub.4, 40 nm
MgO, 99.9%, 10-30 nm
MgO, 99%, 20 nm
MgO, 99.9%, 10-30 nm
Mg(OH).sub.2, 99.8%, 50 nm
[0096] Mn.sub.2O.sub.3, 98+%, 40-60 nm
MoCl.sub.5, 99.0%
[0097] Nd.sub.2O.sub.3, 99.9%, <100 nm
NiO, <100 nm
[0098] Ni.sub.2O.sub.3, <100 nm Sb.sub.2O.sub.3, 99.9%, 150
nm
SiO.sub.2, 99.9%, 20-60 nm
[0099] SiO.sub.2, 99%, 10-30 nm, treated with Silane Coupling
Agents SiO.sub.2, 99%, 10-30 nm, treated with Hexamethyldisilazane
SiO.sub.2, 99%, 10-30 nm, treated with Titanium Ester SiO.sub.2,
99%, 10-30 nm, treated with Silanes SiO.sub.2, 10-20 nm, modified
with amino group, dispersible SiO.sub.2, 10-20 nm, modified with
epoxy group, dispersible SiO.sub.2, 10-20 nm, modified with double
bond, dispersible SiO.sub.2, 10-20 nm, surface modified with double
layer, dispersible SiO.sub.2, 10-20 nm, surface modified,
super-hydrophobic & oleophilic, dispersible SiO.sub.2, 99.8%,
5-15 nm, surface modified, hydrophobic & oleophilic,
dispersible SiO.sub.2, 99.8%, 10-25 nm, surface modified,
super-hydrophobic, dispersible SiC, beta, 99%, 40 nm SiC, beta,
whisker, 99.9% Si.sub.3N.sub.4, amorphous, 99%, 20 nm
Si.sub.3N.sub.4 alpha, 97.5-99%, fiber, 100 nm.times.800 nm
SnO.sub.2, 99.9%, 50-70 nm
[0100] ATO, SnO.sub.2:Sb.sub.2O.sub.3=90:10, 40 nm TiO.sub.2
anatase, 99.5%, 5-10 nm
TiO.sub.2 Rutile, 99.5%, 10-30 nm
[0101] TiO.sub.2 Rutile, 99%, 20-40 nm, coated with SiO.sub.2,
highly hydrophobic TiO.sub.2 Rutile, 99%, 20-40 nm, coated with
SiO.sub.2/Al.sub.2O.sub.3 TiO.sub.2 Rutile, 99%, 20-40 nm, coated
with Al.sub.2O.sub.3, hydrophilic TiO.sub.2 Rutile, 99%, 20-40 nm,
coated with SiO.sub.2/Al.sub.2O.sub.3/Stearic Acid TiO.sub.2
Rutile, 99%, 20-40 nm, coated with Silicone Oil, hydrophobic
TiC, 99%, 40 nm
TiN, 97+%, 20 nm
WO.sub.3, 99.5%, <100 nm
WS.sub.2, 99.9%, 0.8 .mu.m
WCl.sub.6, 99.0%
[0102] Y.sub.2O.sub.3, 99.995%, 30-50 nm
ZnO, 99.8%, 10-30 nm
[0103] ZnO, 99%, 10-30 nm, treated with silane coupling agents ZnO,
99%, 10-30 nm, treated with stearic acid ZnO, 99%, 10-30 nm,
treated with silicone oil
ZnO, 99.8%, 200 nm
ZrO.sub.2, 99.9%, 100 nm
ZrO.sub.2, 99.9%, 20-30 nm
ZrO.sub.2-3Y, 99.9%, 0.3-0.5 um
ZrO.sub.2-3Y, 25 nm
ZrO.sub.2-5Y, 20-30 nm
ZrO.sub.2-8Y, 99.9%, 0.3-0.5 .mu.m
ZrO.sub.2-8Y, 20 nm
ZrC, 97+%, 60 nm
[0104] In one aspect, the filler is nanosilica. Nanosilica is
commercially available from multiple sources in a broad size range.
For example, aqueous Nexsil colloidal silica is available in
diameters from 6-85 nm from Nyacol Nanotechnologies, Inc.
Amino-modified nanosilica is also commercially available, from
Sigma Aldrich for example, but in a narrower range of diameters
than unmodified silica. Nanosilica does not contribute to the
opacity of the adhesive, which is an important attribute of the
coacervates and glues produced therefrom.
[0105] In another aspect, the filler can be composed of calcium
phosphate. In one aspect, the filler can be hydroxyapatite, which
has the formula Ca.sub.5(PO.sub.4).sub.3OH. In another aspect, the
filler can be a substituted hydroxyapatite. A substituted
hydroxyapatite is hydroxyapatite with one or more atoms substituted
with another atom. The substituted hydroxyapatite is depicted by
the formula M.sub.5X.sub.3Y, where M is Ca, Mg, Na; X is PO.sub.4
or CO.sub.3; and Y is OH, F, Cl, or CO.sub.3. Minor impurities in
the hydroxyapatite structure may also be present from the following
ions: Zn, Sr, Al, Pb, Ba. In another aspect, the calcium phosphate
comprises a calcium orthophosphate. Examples of calcium
orthophosphates include, but are not limited to, monocalcium
phosphate anhydrate, monocalcium phosphate monohydrate, dicalcium
phosphate dihydrate, dicalcium phosphate anhydrous, octacalcium
phosphate, beta tricalcium phosphate, alpha tricalcium phosphate,
super alpha tricalcium phosphate, tetracalcium phosphate, amorphous
tricalcium phosphate, or any combination thereof. In certain
aspects, the calcium phosphate crystals include crystals possessing
carbonate groups (CO.sub.3), which can facilitate the adhesion of
the coacervate to certain types of cells such as, for example, bone
cells. In other aspects, the calcium phosphate can also include
calcium-deficient hydroxyapatite, which can preferentially adsorb
bone matrix proteins.
[0106] In certain aspects, the coacervate also includes one or more
initiators. For example, a photoinitiator can be entrapped in the
coacervate. Thus, when the photoinitiator is activated (e.g.,
exposed to light), polymerization of the polymerizable monomer also
entrapped in the coacervate occurs to produce the internal network.
Examples of photoinitiators include, but are not limited to a
phosphine oxide, a peroxide group, an azide group, an
.alpha.-hydroxyketone, or an .alpha.-aminoketone. In one aspect,
the photoinitiator includes, but is not limited to, camphorquinone,
benzoin methyl ether, 1-hydroxycyclohexylphenyl ketone, or
Darocure.RTM. or Irgacure.RTM. types, for example Darocure.RTM.
1173 or Irgacure.RTM. 2959. The photoinitiators disclosed in
European Patent No. 0632329, which are incorporated by reference,
can be used herein. In other aspects, the photoinitiator is a
water-soluble photoinitiator including, but not limited to,
riboflavin, eosin, eosin y, and rose Bengal.
[0107] In certain aspects, multiple initiators can be used to
broaden the absorption profile of the initiator system in order to
increase the initiation rate. For example, two different
photoinitiators can be employed that are activated by different
wavelengths of light. In another aspect, a chemical initiator can
be used in combination with a photoinitiator. In another aspect, a
co-initiator can be used in combination with any of the
polymerization initiators described herein. In one aspect, the
co-initiator is 2-(diethylamino)ethyl acrylate,
2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)ethyl benzoate,
2-(dimethylamino)ethyl methacrylate, 2-ethylhexyl
4-(dimethylamino)benzoate, 3-(dimethylamino)propyl acrylate,
4,4'-bis(diethylamino)benzophenone, or
4-(diethylamino)benzophenone.
[0108] In certain aspects, the photoinitiator and/or co-initiator
are covalently attached to the polyelectrolyte. For example, the
photoinitiator and/or co-initiator can be copolymerized with
monomers used to make the polyelectrolyte. In one aspect, the
photoinitiators and co-initiators can possess polymerizable
olefinic groups such as acrylate and methacrylate groups (e.g., see
examples of co-initiators above) that can be copolymerized with
monomers described used to make the polycation and polyanion. In
another aspect, the initiators can be chemically grafted onto the
backbone of the polyelectrolyte. Thus, in these aspects, the
photoinitiator and/or co-initiator are covalently attached to the
polymer and pendant to the polymer backbone. This approach will
simplify formulation and possibly enhance storage and
stability.
[0109] The simple adhesive coacervate can be synthesized a number
of different ways. In one aspect, coacervate is produced by
preparing a solution comprising (1) a polyelectrolyte, wherein the
polyelectrolyte comprises a polyanion or polycation but not a
combination thereof, the polyeletrolyte comprises at least one
crosslinking group, and (2) a sufficient amount of a complimentary
counterion to produce a simple adhesive coacervate. As discussed
above, the nature and amount of complimentary counterion used to
coacervate can vary depending upon. In general, the amount of
complimentary counterion is sufficient to produce a solution having
a net overall charge approaching zero. It is when the net charge is
neutral the simple adhesive coacervate is formed. The pH of the
solution can change the charge density on the polyeletrolyte, which
in turn changes the amount of complimentary counterion needed to
make the coacervate. Additionally, the dielectric constant of the
solution can also be modified to produce the coacervate. For
example, organic solvents such as alcohols, aldehydes, esters, and
carboxylic acids can be used herein. In one aspect, ethanol can be
used to make the coacervates described herein. In the case when the
adhesives are to be used in biomedical applications, it is
desirable that the organic solvent be biocompatible. Exemplary
methods for producing the simple adhesive coacervates described
herein are provided in the Examples.
[0110] After the simple adhesive coacervate, the coacervate is
crosslinked in order to produce an adhesive. The mode of
crosslinking will vary depending upon the nature of the
crosslinking groups present on the polyeletrolyte. Exemplary
methods for crosslinking simple adhesive coacervates described
herein to produce adhesives are provided in the Examples. In
certain aspects, the polyelectrolytes possess crosslinking groups
that are capable of crosslinking with each. These groups were
discussed in detail above and do not require the use of a
crosslinker. For example, if the crosslinking group is a
dihydroxyl-substituted aromatic group (e.g., DOPA) capable of
undergoing oxidation in the presence of an oxidant to produce
dopaquinone, the dopaquinone is an electrophilic group that is
capable of reacting with a neighboring DOPA group in the absence of
a crosslinker.
[0111] In other aspects, a crosslinker is used to crosslink the
polyelectrolytes. In one aspect, the crosslinker comprises at least
two nucleophilic groups. Examples of nucleophilic groups include,
but are not limited to, a hydroxyl group, a thiol group, an amino
group, or any combination thereof. This, the crosslinker can have
tow or more different nucleophilic groups present on the molecule.
In one aspect, the crosslinker includes an oligoamine, an
oligopeptide, or a polythiol. In the case of the oligoamine, this
is an amine compound possessing 2 to 10 substituted and/or
unsubstituted amino groups. Examples of suitable amino groups
include, but are not limited to, heterocyclic amines and aromatic
amines (e.g., imidazole). An oligopeptide is a peptide possessing
from 2 to 10 amino acid residues. In one aspect, the oligopeptide
can include enzyme cleavage sequences or cell adhesion sequences.
An oligothiol is a compound possessing from 2 to 10 thiol
groups.
[0112] In one aspect, the crosslinker comprises
H.sub.2NCH.sub.2NH.sub.2, H.sub.2NCH.sub.2CH.sub.2NH.sub.2,
H.sub.2NCH.sub.2CH.sub.2CH.sub.2NH.sub.2,
H.sub.2NCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2,
H.sub.2NCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2,
H.sub.2NCH.sub.2NHCH.sub.2CH.sub.2CH.sub.2NH.sub.2,
H.sub.2NCH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2NH.sub.2,
H.sub.2NCH.sub.2CH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2NHCH.su-
b.2CH.sub.2CH.sub.2NH.sub.2,
H.sub.2NCH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2,
or
H.sub.2NCH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.su-
b.2NH.sub.2.
[0113] In other aspects, the crosslinker comprises a
surface-modified nanoparticle. In certain aspects, any of the
fillers described above can be functionalized with one or more
functional groups that are capable of capable of reacting with a
crosslinkable group on the polyeletrolyte and, when applicable, the
polymerizable olefinic monomer. In this aspect, the filler is
covalently attaches to the polyelectrolyte in order to crosslink
the polyeletrolyte. For example, the filler particle can be
modified with surface amines or thiols (i.e., nucleophiles) that
can react with react with electrophilic groups present on the
polyelectrolyte as described above. In other aspects, the filler
can be modified to produce charged groups such that the filler can
form electrostatic bonds with the polyeletrolyte. For example,
aminated silica can be added to a solution and the pH adjusted so
that the amino groups are protonated and available for
electrostatic bonding.
[0114] In certain aspects, the polyelectrolyte can be crosslinked
using multiple reagents and steps. In one aspect, the crosslinking
step is performed in the presence of an oxidant and a crosslinker.
For example, if the crosslinking group is a dihydroxyl-substituted
aromatic group (e.g., DOPA) capable of undergoing oxidation in the
presence of an oxidant to produce dopaquinone, the dopaquinone is
an electrophilic group that is capable of reacting with a
crosslinker possessing two or more nucleophilic groups. In one
aspect, the crosslinker is an oligoamine, where an amino group
reacts with the dopaquinone to produce a new covalent bond.
Exemplary procedures for this aspect are provided in the
Examples.
[0115] In another aspect, the crosslinker comprises two or more
actinically crosslinkable groups. Any of the actinically
crosslinkable groups described herein can be used as the
crosslinker. Thus, when the polyelectrolyte has one or more
actinically crosslinkable groups, the polyelectrolytes can be
crosslinked with one with a crosslinker having two or more
actinically crosslinkable groups in the presence of an initiator.
Examples of such crosslinkers include, but are not limited to,
diacrylates, dimethacrylates, and the like. In one aspect, the
crosslinker can be a polyalkylene oxide glycol diacrylate or
dimethacrylate. For example, the polyalkylene can be a polymer of
ethylene glycol, propylene glycol, or block co-polymers thereof. In
one aspect, the polymerizable monomer is polyethylene glycol
diacrylate or polyethylene glycol dimethacrylate. In one aspect,
the polyethylene glycol diacrylate or polyethylene glycol
dimethacrylate has a M.sub.n of 200 to 2,000, 400 to 1,500, 500 to
1,000, 500 to 750, or 500 to 600.
[0116] Additional reaction conditions can be varied in order to
facilitate crosslinking and produce the adhesive. In one aspect,
the pH of the coacervate can be raised. For example, when the
coacervate is applied at a pH of 5 and subsequently exposed to
seawater at pH 8.2, the coacervate crosslinks spontaneously to
produce the adhesive. The polyelectrolytes described herein can be
stored as dry powders for extended periods of time. This feature is
very useful for preparing the coacervates and ultimately the
adhesives when desired. Thus, described herein are kits for making
the complex coacervates and adhesives described herein. In one
aspect, the kit comprises (1) a polyelectrolyte, wherein the
polyelectrolyte comprises a polyanion or polycation but not a
combination thereof, wherein the polyeletrolyte comprises at least
one crosslinking group; (2) a sufficient amount of a complimentary
counterion to produce a simple adhesive coacervate; and (3) a
crosslinker. In another aspect, the kit comprises (1) a simple
adhesive coacervate comprising (a) a polyelectrolyte, wherein the
polyelectrolyte comprises a polyanion or polycation but not a
combination thereof, the polyeletrolyte comprises at least one
crosslinking group, and (b) a sufficient amount of a complimentary
counterion to produce a simple adhesive coacervate, and (2) a
crosslinker. In this aspect, the simple adhesive coacervate is
pre-made.
[0117] When stored as dried powders, water can be added to the
polyelectrolyte and complimentary counterion to produce the
coacervate. In one aspect, prior to lyophilizing the
polyelectrolyte in order to produce a dry powder, the pH of the
polyelectrolyte can be adjusted such that when they are admixed in
water the desired pH is produced with the addition of acid or base.
For example, excess base can be present in the polyelectrolyte
powder which upon addition of water adjusts the pH accordingly. The
kits can include additional components as needed such as, for
example, an oxidant, a polymerizable monomer and/or water-insoluble
filler, and a polymerization initiator and optionally a
co-initiator.
[0118] The adhesives described herein have numerous applications,
particularly where the adhesive is to be used in aqueous
environments. For example, the simple adhesive coacervates have low
initial viscosity, specific gravity greater than one, and being
mostly water by weight, low interfacial tension in an aqueous
environment, all of which contribute to their ability to adhere to
a wet surface. The simple adhesive coacervates prior to
crosslinking can be applied to variety of substrates. The
substrates can be wet or dry. Additionally, in certain aspects, the
surface of the substrate can be primed prior to application of the
coacervate. For example, the substrate surface can be primed with a
separate solution before adding the coacervate to increase
interfacial adhesion. For example, the surface can be cleaned or
etched. Alternatively, the surface of the substrate can be modified
with groups that can crosslink with the polyelectrolyte. For
example, nucleophilic groups can be introduced to the surface of
the substrate that crosslink with the polyelectrolyte. Examples of
substrates that the coacervates can be applied to include, but are
not limited to, metal substrates, foils, fibers, a tapes, or cloth.
In certain aquatic applications (fresh or salt water), the
substrate can include coral, a marker, a beacon, an ordinance, or a
material for producing an artificial reef. The adhesives described
herein have particular relevance in restoring aquatic ecosystems
such reefs. Here, natural materials (e.g., coral) and other
synthetic materials (e.g., calcium carbonate rocks, dead coral,
reef plugs, coral plugs, coral mounting pieces, etc.) can be
adhered to existing reefs in order to promote the growth of the
reef.
[0119] Depending upon the application to be used, the adhesive can
be prepared a number of different ways on the substrate. For
example, when a crosslinker is used to produce the adhesive, the
coacervate can be applied to the substrate first followed by the
addition of the crosslinker to the coacervate. Alternatively, the
crosslinker can be applied to the substrate first followed by the
application of the coacervate. In another embodiment, the
coacervate and crosslinker can be applied to the substrate
simultaneously through a dual syringe. Here, the crosslinker and
coacervate react with one another to produce the adhesive when it
is applied to the substrate.
[0120] The adhesives produced herein have numerous biological
applications as well. In one aspect, the adhesives are useful in
adhering implantable devices in a subject. For example, stents,
pins, and screws can be adhered in a subject using the adhesives
described herein.
[0121] In other aspects, the substrate can be bone. For example,
the adhesives can be used to repair a number of different bone
fractures and breaks. Examples of such breaks include a complete
fracture, an incomplete fracture, a linear fracture, a transverse
fracture, an oblique fracture, a compression fracture, a spiral
fracture, a comminuted fracture, a compacted fracture, or an open
fracture. In one aspect, the fracture is an intra-articular
fracture or a craniofacial bone fracture. Fractures such as
intra-articular fractures are bony injuries that extend into and
fragment the cartilage surface. The adhesives may aid in the
maintenance of the reduction of such fractures, allow less invasive
surgery, reduce operating room time, reduce costs, and provide a
better outcome by reducing the risk of post-traumatic
arthritis.
[0122] In other aspects, the adhesives can be used to join small
fragments of highly comminuted fractures. In this aspect, small
pieces of fractured bone can be adhered to an existing bone. For
example, the coacervate can be applied to the fractured bone and/or
the existing bone. It is especially challenging to maintain
reduction of the small fragments by drilling them with mechanical
fixators. The smaller and greater number of fragments the greater
the problem. In one aspect, the adhesive may be injected in small
volumes to create spot welds in order to fix the fracture rather
than filling the entire crack. The small biocompatible spot welds
would minimize interference with healing of the surrounding tissue
and would not necessarily have to be biodegradable. In this respect
it would be similar to permanently implanted hardware.
[0123] In other aspects, the adhesives can be used to secure
scaffolds to bone and other tissues such as, for example,
cartilage, ligaments, tendons, soft tissues, organs, membranous
tissues (e.g., vaginal, nasal, amniotic membrane) and synthetic
derivatives of these materials. Using the adhesives and spot
welding techniques, the adhesive complex coacervates and adhesives
produced therefrom can be used to position biological scaffolds in
a subject. The adhesive can be applied to the biological scaffold
and/or the bone or tissue prior to securing the scaffold. Small
adhesive tacks would not interfere with migration of cells or
transport of small molecules into or out of the scaffold. In
certain aspects, the scaffold can contain one or more drugs that
facilitate growth or repair of the bone and tissue. In other
aspects, the scaffold can include drugs that prevent infection such
as, for example, antibiotics. For example, the scaffold can be
coated with the drug or, in the alternative, the drug can be
incorporated within the scaffold so that the drug elutes from the
scaffold over time.
[0124] The adhesives have numerous dental applications. Using the
spot weld techniques, the adhesive can be applied to specific
points in the mouth (e.g., jaw, sections of a tooth). For example,
the adhesives can be used in the treatment of recession defects,
increasing gingival tissue height and width, increase the amount of
attached gingival tissue at the gingival margin, and increase the
zone of attached gingival tissue. In oral surgery they could be
used to improve soft tissue outcomes and grow new bone in guided
bone regeneration procedures. Additionally, the adhesives can
facilitate wound healing of gums after a periodontal procedure and
help prevent or reduce bleeding. As will be discussed below, the
adhesives can be used to deliver bioactive agents. Thus, the
adhesives can be used to deliver bioactive agents to the gums and
roots of teeth. In other aspects, the adhesives can be used to
secure dental implants to teeth (e.g., crowns, dentures).
Alternatively, the adhesives can be used as a primer to prepare the
dentin or enamel surface of a tooth to bond dental cements.
[0125] In other aspects, the adhesives can adhere a substrate to
bone. Examples of substrates include metal substrates (e.g.,
plates, medical implants, etc.), fibers, foils, pieces of cloth, or
any other materials that can be implanted within a subject. The
coacervate can be applied to the substrate and/or bone prior to
use. For example, implants made from titanium oxide, stainless
steel, or other metals are commonly used to repair fractured bones.
The adhesives can be applied to the metal substrate, the bone, or
both prior to adhering the substrate to the bone. In certain
aspects, the crosslinking group present on the polyelectrolyte can
form a strong bond with titanium oxide. For example, it has been
shown that DOPA can strongly bind to wet titanium oxide surfaces
(Lee et al., PNAS 103:12999 (2006)). Thus, in addition to bonding
bone fragments, the adhesives described herein can facilitate the
bonding of metal substrates to bone, which can facilitate bone
repair and recovery.
[0126] It is also contemplated that the adhesives can include one
or more bioactive agents. The bioactive agents can be any drug that
will facilitate bone growth and repair when the complex is applied
to the bone. The rate of release can be controlled by the selection
of the materials used to prepare the complex as well as the charge
of the bioactive agent if the agent is a salt. In certain aspects,
when the adhesive is converted to an insoluble solid by a change in
temperature and/or pH, the adhesive can be administered to a
subject and produce an insoluble solid in situ. Thus, in this
aspect, the insoluble solid can perform as a localized controlled
drug release depot. It may be possible to simultaneously fix tissue
and bones as well as deliver bioactive agents to provide greater
patient comfort, accelerate bone healing, and/or prevent
infections. In other aspects, the adhesives can include contrast
agents typically used in imaging procedures such as MRI. In this
aspect, the position and amount of the adhesive in the subject can
be detected and monitored over time. Contrast agents typically used
in the art can be used herein.
[0127] The adhesives can be used in a variety of other surgical
procedures. For example, the adhesives can be used to treat ocular
wounds caused by trauma or by the surgical procedure itself. In one
aspect, the adhesives can be used to repair a corneal or schleral
laceration in a subject. In other aspects, the adhesives can be
used to facilitate healing of ocular tissue damaged from a surgical
procedure (e.g., glaucoma surgery or a corneal transplant). The
methods disclosed in U.S. Published Application No. 2007/0196454,
which are incorporated by reference, can be used to apply the
coacervates described herein to different regions of the eye.
[0128] In other aspects, the adhesives can be used to inhibit blood
flow in a blood vessel of a subject. In general, the adhesive is
injected into the vessel in order to partially or completely block
the vessel. This method has numerous applications including
hemostasis or the creation of an artificial embolism to inhibit
blood flow to a tumor or aneurysm.
[0129] The adhesives described herein can seal the junction between
skin and an inserted medical device such as catheters, electrode
leads, needles, cannulas, osseo-integrated prosthetics, and the
like. In this aspect, the adhesives prevent infection at the entry
site when the device is inserted in the subject. In other aspects,
the adhesives can be applied to the entry site of the skin after
the device has been removed in order to expedite wound healing and
prevent further infection.
[0130] In another aspect, the adhesives described herein can be
used to close or seal a puncture in an internal tissue or membrane.
In certain medical applications, internal tissues or membranes are
punctured, which subsequently have to be sealed in order to avoid
additional complications. Alternatively, the adhesives described
herein can be used to adhere a scaffold or patch to the tissue or
membrane in order to prevent further damage and facilitate wound
healing.
[0131] In one aspect, the coacervates described herein can modify
one or more the properties of a substrate. For example, the
coacervates prior to crosslinking can modify the wettability,
charge, or anti-fouling properties corrosion resistance,
anti-fouling, of the surface as well as promote specific
interactions on the surface (e.g. biomolecule attachment, cell
attachment, metal ion coordination, etc.).
EXAMPLES
[0132] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, and methods
described and claimed herein are made and evaluated, and are
intended to be purely exemplary and are not intended to limit the
scope of what the inventors regard as their invention. Efforts have
been made to ensure accuracy with respect to numbers (e.g.,
amounts, temperature, etc.) but some errors and deviations should
be accounted for. Unless indicated otherwise, parts are parts by
weight, temperature is in .degree. C. or is at ambient temperature,
and pressure is at or near atmospheric. There are numerous
variations and combinations of reaction conditions, e.g., component
concentrations, desired solvents, solvent mixtures, temperatures,
pressures and other reaction ranges and conditions that can be used
to optimize the product purity and yield obtained from the
described process. Only reasonable and routine experimentation will
be required to optimize such process conditions.
Preparation of Simple Adhesive Coacervate
[0133] The simple adhesive coacervates were prepared as follows.
Using multiple independent variables, coacervation of the MOEP-DOPA
copolymer was determined using a Design of Experiment (DOE) matrix
with JMP 8 software. Each independent variable (pH of the solution,
Mg:PO.sub.4 ratio, ratio of ethanol to deionized water) was chosen
and limits set to create a window where coacervation may occur.
Variables outside of the upper and lower limits would cause
MOEP-DOPA copolymer to either create a gel or remain in solution. A
design of experiment (DOE) was generated with 16 runs, varying pH,
Mg:PO.sub.4 ratio, and EtOH:H.sub.2O ratio. The results where rated
based on appearance using a scale of 1-3 (1--In solution,
2--appearance of coacervate, 3--Gel/Solid). The resulting data was
input into the DOE matrix and modeled using the software. The
resulting parameters, based on the highest desirability for each
parameter, resulted in a pH of 6.8, Mg: PO.sub.4 ratio of 0.45, and
EtOH:H.sub.2O ratio of 0.20.
[0134] The runs for the DOE where conducted at 5% wt MOEP-DOPA
copolymer in solution. Each run was done in a 1.7 mL Ependorf tube,
using a total 500 .mu.L of solution, at room temperature. MOEP-DOPA
copolymer (5% wt) was weighed into an empty tube. Deionized water
with and without 450 mM of NaCl (simulated ocean water) was added
and the tube vortexed on high until MOEP-DOPA copolymer went into
solution. The pH of the solution was adjusted using a 6M solution
of NaOH. The volume of NaOH used factored into total volume. The
appropriate volume of MgCl.sub.2 in deionized H.sub.2O with and
without 450 mM of NaCl was slowly added to the solution while
stirring. To this, ethanol was slowly added while stirring. The
final solution was vortexed, left on ice for 1 hr, and then left at
room temperature. The coacervate appeared within 2 hrs. Each run
was rated based on appearance.
Preparation of Adhesive
[0135] Testing was done to maximize the bond strength based on the
proportions of NaIO.sub.4 used compared to DOPA present and to
determine the affect of a diamine on the strength. Bond strengths
were tested with Al strips. The coacervate was applied to a wet
strip, the appropriate cross-linking solution (ethylenediamine
dihydrochloride) added and stirred on the strip, and then covered
with another wet strip and clamped together. Samples where
incubated for 24 hrs in deionized H.sub.2O with and without 450 mM
of NaCl at 37.degree. C. After 24 hrs, each Al strip was mounted on
the Instron in deionized H.sub.2O with and without 450 mM of NaCl
at 37.degree. C. and bond strengths were determined.
[0136] The crosslinking solution was prepared using sodium
m-periodate and 1,2-O-isopropylidene-D-glucofuranose vortexed in
deionized H.sub.2O. The solution was prepared based on the
NaIO.sub.4:DOPA ratio. It was then applied to the coacervate on the
strips. For samples requiring diamine, the NaIO.sub.4/sugar
solution was then added to ethylenediamine dihydrochloride and
vortexed. The solution was then applied to the coacervate on the
metal strips. FIG. 8 displays the underwater bond strengths of the
adhesives on the aluminum adherends in a standard lap shear
configuration (450 kPa is roughly 60 psi). Table 1 provides
additional parameters for preparing numerous adhesives: (1) EtOH
was 20% in experiments 1-13 and 30% in experiments 14-21; (2)
Mg/PO4 was 0.45 in experiments 1-13 and 0.5 in experiments 14-21;
(3) the amine was ethylenediamine; and (4) deionized water was used
in experiments 1-14 and 16, and deionized water with 450 mM NaCl
was used in experiments 16 and 18-21. Amine modified filler was
added in experiments 12 and 13. In experiments 18 and 19, 5 mg of
crushed coral was added.
[0137] In Experiments 22-25 use Fe(III) oxide nanoparticles were
used as the oxidant and filler, where wt % of Fe.sub.2O.sub.3:DOPA
is in the coacervate. The samples were incubated and tested in
instant ocean sea water (pH 8.4). The results are shown in Table 1
and FIG. 9.
[0138] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the compounds,
compositions and methods described herein.
[0139] Various modifications and variations can be made to the
compounds, compositions and methods described herein. Other aspects
of the compounds, compositions and methods described herein will be
apparent from consideration of the specification and practice of
the compounds, compositions and methods disclosed herein. It is
intended that the specification and examples be considered as
exemplary.
TABLE-US-00001 TABLE 1 Ex. Total Coacervate % DOPA in Strength
(kPa) PhosphoDOPA Amine No. DOPA DOPA Coacervate 1 2 3 4 5 (mg)
(mg) DOPA:NaIO4 pH 1 3.92E-05 2.98E-05 76.02 41 56 307 47.39 2.61
1:1 6.55 2 4.14E-05 2.98E-05 71.98 220 50 1:1 6.48 3 3.92E-05
2.55E-05 65.05 563 488 330 343 47.39 2.61 1:1 6.77 4 3.92E-05
2.55E-05 65.05 369 112 202 102 47.39 2.61 2:1 6.77 5 3.92E-05
1.32E-05 33.67 256 300 328 326 47.39 2.61 4:1 6.8 6 3.92E-05
1.32E-05 33.67 317 432 420 281 47.39 2.61 10:1 6.8 7 4.14E-05
1.20E-05 28.99 410 450 224 249 50 1:1 6.68 8 4.14E-05 1.20E-05
28.99 226 249 50 2:1 6.68 9 4.14E-05 1.33E-05 32.13 273 215 304 50
4:1 6.73 10 4.14E-05 1.33E-05 32.13 274 193 196 180 50 10:1 6.73 11
4.14E-05 1.89E-05 45.65 18 68 50 1:1 6.48 12 3.34E-05 1.88E-05
56.29 20 78 143 38.19 11.81 1:1 6.81 13 3.34E-05 1.60E-05 47.90 55
105 38.19 11.81 1:1 7.3 14 4.38E-05 1.51E-05 34.47 4 114 4 84 50
5.11 15 4.38E-05 3.34E-05 76.26 15 169 257 333 50 5.4 16 4.38E-05
1.51E-05 34.47 36 0.6 1.8 52 50 1:1 5.11 17 4.38E-05 3.34E-05 76.26
355 185 233 50 1:1 5.4 18 4.38E-05 3.36E-05 76.71 93 27 98 50 5.47
19 4.38E-05 3.36E-05 76.71 137 270 120 50 1:1 5.47 20 4.14E-05
3.25E-05 78.50 118 10 127 7.6 47.25 2.75 1:1 5.73 21 4.14E-05
3.27E-05 78.99 9.5 229 91 142 135 47.25 2.75 5.27 22 3.95E-05
3.30E-05 83.54 202.7 344 224 47.38 2.62 1% wt Fe.sub.2O.sub.3 6.16
23 4.16E-05 2.67E-05 64.18 4.23 0.75 219 126 50 1% wt
Fe.sub.2O.sub.3 5.63 24 3.95E-05 3.30E-05 83.54 158 12.7 161 56.9
47.38 2.62 10% wt Fe.sub.2O.sub.3 6.16 25 4.16E-05 2.67E-05 64.18
162 280 248 50 10% wt Fe.sub.2O.sub.3 5.63
Sequence CWU 1
1
191191PRTPhragmatopoma californica 1Met Lys Val Phe Ile Val Leu Ala
Leu Val Ser Ala Ala Tyr Gly Cys 1 5 10 15 Gly Val Gly Ile Gly Cys
Ala Gly Gly Arg Cys Gly Gly Ala Cys Gly 20 25 30 Gly Lys Gly Tyr
Gly Tyr Gly Gly Lys Leu Gly Tyr Gly Ala Tyr Gly 35 40 45 Lys Gly
Gly Ile Gly Gly Tyr Gly Tyr Gly Lys Gly Cys Val Gly Gly 50 55 60
Tyr Gly Tyr Gly Gly Leu Gly Ala Gly Lys Leu Gly Gly Tyr Gly Tyr 65
70 75 80 Gly Gly Ser Lys Cys Gly Gly Tyr Gly Tyr Gly Gly Gln Lys
Leu Gly 85 90 95 Gly Tyr Gly Tyr Gly Gly Lys Lys Leu Gly Gly Tyr
Gly Tyr Ala Ala 100 105 110 Lys Lys Val Gly Gly Tyr Gly Tyr Gly Ala
Lys Lys Val Gly Gly Tyr 115 120 125 Gly Tyr Gly Ala Lys Lys Val Gly
Gly Tyr Gly Tyr Gly Ala Lys Lys 130 135 140 Val Gly Gly Tyr Gly Tyr
Gly Ala Lys Lys Val Gly Gly Tyr Gly Tyr 145 150 155 160 Gly Ala Lys
Lys Val Gly Gly Tyr Gly Tyr Gly Ala Lys Lys Val Gly 165 170 175 Gly
Tyr Gly Tyr Gly Val Lys Lys Val Gly Gly Tyr Gly Tyr Gly 180 185 190
2210PRTPhragmatopoma californica 2Met Lys Val Leu Ile Phe Leu Ala
Thr Val Ala Ala Val Tyr Gly Cys 1 5 10 15 Gly Gly Ala Gly Gly Trp
Arg Ser Gly Ser Cys Gly Gly Arg Trp Gly 20 25 30 His Pro Ala Val
His Lys Ala Leu Gly Gly Tyr Gly Gly Tyr Gly Ala 35 40 45 His Pro
Ala Val His Ala Ala Val His Lys Ala Leu Gly Gly Tyr Gly 50 55 60
Ala Gly Ala Tyr Gly Ala Gly Ala Trp Gly His Pro Ala Val His Lys 65
70 75 80 Ala Leu Gly Gly Tyr Gly Ala Gly Ala Trp Gly His Pro Ala
Val His 85 90 95 Lys Ala Leu Gly Gly Tyr Gly Gly Tyr Gly Ala His
Pro Ala Val His 100 105 110 Val Ala Val His Lys Ala Leu Gly Gly Tyr
Gly Ala Gly Ala Cys Gly 115 120 125 His Lys Thr Gly Gly Tyr Gly Gly
Tyr Gly Ala His Pro Val Ala Val 130 135 140 Lys Ala Ala Tyr Asn His
Gly Phe Asn Tyr Gly Ala Asn Asn Ala Ile 145 150 155 160 Lys Ser Thr
Lys Arg Phe Gly Gly Tyr Gly Ala His Pro Val Val Lys 165 170 175 Lys
Ala Phe Ser Arg Gly Leu Ser His Gly Ala Tyr Ala Gly Ser Lys 180 185
190 Ala Ala Thr Gly Tyr Gly Tyr Gly Ser Gly Lys Ala Ala Gly Gly Tyr
195 200 205 Gly Tyr 210 3151PRTPhragmatopoma californica 3Met Lys
Leu Leu Ser Val Phe Ala Ile Val Val Leu Ala Val Tyr Ile 1 5 10 15
Thr His Val Glu Ala Asp Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser 20
25 30 Ser Tyr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser
Tyr 35 40 45 Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser Ser
Tyr Ser Ser 50 55 60 Ser Ser Ser Tyr Ser Ser Ser Ser Ser Tyr Ser
Ser Ser Ser Tyr Ser 65 70 75 80 Ser Ser Ser Tyr Ser Ser Ser Ser Tyr
Ser Ser Ser Ser Ile Leu Thr 85 90 95 Ser Thr Ser Ser Ser Asp Trp
Lys Arg Lys Val Pro Ala Arg Arg Val 100 105 110 Leu Arg Thr Arg Arg
Phe Leu Lys Cys Val Thr Arg Cys Thr Leu Arg 115 120 125 Cys Ile Leu
Phe Arg Ser Ala Lys Thr Cys Ala Arg Lys Cys Ser Arg 130 135 140 Arg
Cys Leu Lys Arg Val Phe 145 150 4342PRTPhragmatopoma californica
4Met Lys Ser Phe Thr Ile Phe Ala Ala Ile Leu Val Ala Leu Cys Tyr 1
5 10 15 Ile Gln Ile Ser Glu Ala Gly Cys Cys Lys Arg Tyr Ser Ser Ser
Ser 20 25 30 Tyr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Tyr Ser
Ser Ser Ser 35 40 45 Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser
Ser Ser Ser Tyr Ser 50 55 60 Ser Ser Ser Ser Ser Ser Ser Ser Ser
Tyr Ser Ser Ser Ser Ser Ser 65 70 75 80 Tyr Ser Ser Ser Ser Ser Ser
Ser Tyr Ser Ser Ser Ser Ser Ser Ser 85 90 95 Ser Ser Ser Tyr Ser
Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser 100 105 110 Ser Ser Ser
Ser Tyr Ser Ser Ser Ser Ser Ser Ser Ser Ser Tyr Ser 115 120 125 Ser
Ser Ser Ser Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser 130 135
140 Tyr Ser Ser Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser Ser
145 150 155 160 Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser Tyr Ser Ser
Ser Ser Ser 165 170 175 Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser
Ser Ser Ser Tyr Ser 180 185 190 Ser Ser Ser Ser Ser Ser Tyr Ser Ser
Ser Ser Ser Ser Tyr Ser Ser 195 200 205 Ser Ser Ser Ser Ser Ser Ser
Ser Ser Tyr Ser Ser Ser Ser Ser Ser 210 215 220 Ser Ser Ser Ser Tyr
Ser Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser 225 230 235 240 Ser Ser
Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser Ser Ser Ser Tyr 245 250 255
Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser 260
265 270 Ser Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser Tyr Ser
Ser 275 280 285 Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser Ser
Ser Ser Ser 290 295 300 Tyr Ser Ser Ser Ser Ser Ser Ser Tyr Ser Ser
Ser Ser Ser Ser Ser 305 310 315 320 Ser Ser Ser Ser Tyr Ser Ser Ser
Ser Ser Ser Ser Ser Ser Ser Ser 325 330 335 Ser Ser Tyr Ser Ser Ser
340 5272PRTPhragmatopoma californica 5Met Pro Thr Leu Tyr Lys Lys
Val Gly Lys Leu Val Ile Leu Ala Ile 1 5 10 15 Ile Val Thr Val Ala
Ser Val Ala Ser Ala Gly Tyr Pro Thr Tyr Ser 20 25 30 Pro Ser Gly
Gly Thr His Ser Gly Tyr Asn Gly Pro His Gly Asn Val 35 40 45 Val
Lys Lys Thr Tyr Arg Gly Pro Tyr Gly Ala Gly Ala Ala Lys Ala 50 55
60 Trp Asn Gly Tyr His Gly Ala Gly Tyr Thr Ser Val His His Gly Pro
65 70 75 80 Ala Ser Thr Ser Trp His Thr Ser Trp Ser Asn Lys Lys Gly
Gly Tyr 85 90 95 Gly Tyr Gly Leu Lys Asn Lys Gly Tyr Gly Tyr Gly
Leu Lys Lys Val 100 105 110 Gly Tyr Gly Val Gly Leu His Ala Ala Gly
Trp His Gly Val Gly Pro 115 120 125 Tyr Gly Ala Gly Tyr His Gly Ala
Gly Trp Asn Gly Leu Gly Tyr His 130 135 140 Gly Ala Gly Tyr Gly Val
His Gly Val Gly Leu His Gly Ala Gly Tyr 145 150 155 160 Gly Leu His
Gly Val Gly Leu His Gly Val Gly Tyr Gly Leu His Gly 165 170 175 Val
Gly Leu His Gly Ala Gly Tyr Gly Leu His Gly Val Gly Leu His 180 185
190 Gly Ala Gly Tyr Gly Ile His Gly Val Gly Leu His Gly Ala Gly Tyr
195 200 205 Gly Ile His Gly Val Gly Leu His Gly Val Gly Tyr Gly Leu
His Gly 210 215 220 Val Gly Leu His Gly Ala Gly Tyr Gly Leu His Gly
Val Gly Leu His 225 230 235 240 Gly Ala Gly Tyr Gly Ile His Gly Val
Gly Leu His Gly Ala Gly Cys 245 250 255 Gly Ile His Lys Thr Ala Cys
Tyr Gly Val Gly Leu His Gly His Tyr 260 265 270
6144PRTPhragmatopoma californica 6Met Lys Phe Leu Val Leu Leu Ala
Leu Val Ala Ser Ala Ser Ala Tyr 1 5 10 15 Tyr Pro Leu Met Gly Gly
Phe His Gly Gly Trp His Ala Pro Met Val 20 25 30 His Gly Gly Leu
Tyr His Gly Gly Trp His Ala Pro Met Val His Gly 35 40 45 Gly Leu
Tyr His Gly Gly Trp His Ala Pro Ile Val His Gly Gly Trp 50 55 60
His Ala Pro Val Phe His Ala Pro Ala Pro Ile His Thr Val Ser His 65
70 75 80 Ser Val Val Asn His Val Pro Met Met Pro Met Trp His His
Pro Ala 85 90 95 Pro Ala Pro Ala Pro Ala Pro Arg Pro Gly Arg Thr
Ile Ile Leu Gly 100 105 110 Gly Gly Lys Tyr Gly Pro Phe Gly Lys Tyr
Gly Gly Gly Ala Gly Leu 115 120 125 Leu Ala Leu Gly Ala Leu Gly Gly
Asn Gly Gly Phe Trp Lys Arg Arg 130 135 140 7328PRTPhragmatopoma
californica 7Met Leu Phe Tyr Asn Ala Asn Phe Val Gln Lys Ser Trp
Val Leu Ile 1 5 10 15 Leu Leu Gly Leu Ala Ala Val Val Ala Cys Ser
Glu Tyr Asp Lys Gly 20 25 30 Leu Gly Gly Tyr Gly Arg Pro Ser Tyr
Gly Gly Arg Arg Gly Tyr Gly 35 40 45 Gly Arg Arg Gly Leu Gln Tyr
His Gly Lys Tyr Gln Gly Arg Cys Glu 50 55 60 Tyr Asp Gly Leu Tyr
Phe Arg Asp Glu Lys Ser Phe Val Tyr Cys Ser 65 70 75 80 Asn Arg Asn
Ser Tyr Ile Gln Pro Cys Ala Pro Gly Thr Arg Asn Ser 85 90 95 Pro
Tyr Thr Lys Tyr Asn Arg Gly Ser Lys Tyr Asn Tyr Arg Asp Phe 100 105
110 Cys Glu Val Asn Leu Val Asp Ser Gly Tyr Val Pro Lys Pro Gly Tyr
115 120 125 Leu Pro Ala Pro Lys Lys Ala Tyr Pro Thr Lys Val Tyr Asp
Leu Lys 130 135 140 Val Asp Tyr Ala Pro Lys Val Asp Tyr Ala Pro Lys
Val Asp Tyr Ala 145 150 155 160 Pro Lys Val Asp Tyr Ala Pro Lys Val
Asp Tyr Val Ala Pro Lys Ala 165 170 175 Ser Tyr Val Pro Pro Lys Ala
Ser Tyr Val Asp Pro Thr Pro Thr Tyr 180 185 190 Gly Tyr Glu Ala Pro
Phe Lys Gly Gly Tyr Asp Lys Pro Ser Tyr Gly 195 200 205 Lys Asp Val
Asp Thr Ser Tyr Glu Ser Lys Thr Thr Tyr Thr Val Glu 210 215 220 Lys
Thr Ala Asp Lys Gly Tyr Gly Lys Gly Tyr Gly Asp Lys Glu Ile 225 230
235 240 Ser Ala Lys Lys Ser Tyr Thr Leu Thr Glu Lys Arg Asp Tyr Asp
Thr 245 250 255 Gly Tyr Asp Asn Ser Arg Ser Asp Glu Asp Ser Lys Glu
Tyr Gly Tyr 260 265 270 Asp Asn Asp Arg Ser Glu Ser Tyr Glu Arg Thr
Glu Ser Tyr Thr Asp 275 280 285 Glu Arg Thr Asp Gly Tyr Gly Thr Gln
Lys Val Glu Tyr Thr Gln Gln 290 295 300 Ser Glu Tyr Asp Arg Val Thr
Arg Arg Gly Ile Trp Leu His Lys Gly 305 310 315 320 Thr Glu Val Glu
His Val Leu Tyr 325 8136PRTPhragmatopoma
californicamisc_feature(49)..(49)Xaa can be any naturally occurring
amino acid 8Met Asn Thr Phe Val Val Leu Ala Ala Ile Val Ala Val Ala
Ala Cys 1 5 10 15 Ser Gly Gly Tyr Asp Gly Arg Gln Tyr Thr Tyr Arg
Gly Arg Tyr Asn 20 25 30 Asn Lys Cys Gly Asn Asp Gly Leu Tyr Phe
Lys Asp Asp Lys Asn Phe 35 40 45 Xaa Phe Cys Ser Asn Gly Asn Ser
Tyr Val Gln Pro Cys Ala Pro Gly 50 55 60 Thr Arg Asn Ser Gly Tyr
Asn Asn Tyr Lys Gln Gly Ser Ile Tyr Asn 65 70 75 80 Tyr Arg Asp Phe
Cys Asp Val Asn Leu Val Asp Glu Gly Tyr Gly Val 85 90 95 Gly Ala
Lys Pro Gly Tyr Asn Lys Gly Tyr Asn Pro Gly Tyr Asn Pro 100 105 110
Gly Tyr Gly Gly Tyr Asn Pro Gly Tyr Ser Thr Gly Tyr Gly Gly Tyr 115
120 125 Lys Ala Gly Pro Gly Pro Tyr Trp 130 135
9158PRTPhragmatopoma californica 9Met Lys Leu Ala Leu Leu Leu Leu
Val Ala Val Cys Ala Ala Val Ala 1 5 10 15 Val Asn Ala Cys Gly Pro
Leu Gly Cys Ser Gly Gly Tyr Gly Gly Val 20 25 30 Leu Lys Cys Gly
Val Gly Gly Cys Ala Leu Gly Gly Tyr Gly Gly Gly 35 40 45 Tyr Ser
Ala Gly Ile Gly Gly Tyr Gly Ile Lys Arg Leu Gly Cys Arg 50 55 60
Gly Gly Arg Cys Gly Leu Arg Arg Arg Val Gly Cys Arg Gly Gly Arg 65
70 75 80 Cys Gly Leu Arg Gly Arg Leu Gly Cys Arg Gly Gly Arg Cys
Gly Leu 85 90 95 Arg Lys Leu Gly Cys Arg Gly Gly Arg Cys Gly Leu
Arg Gly Arg Leu 100 105 110 Gly Cys Arg Gly Gly Arg Cys Gly Leu Arg
Lys Arg Leu Gly Cys Arg 115 120 125 Gly Gly Arg Cys Gly Arg Gly Gly
Tyr Gly Gly Gly Tyr Gly Gly Val 130 135 140 Cys Ser Lys Gly Val Cys
Gly Gly Tyr Pro Ala Tyr Gly Lys 145 150 155 10236PRTPhragmatopoma
californica 10Met Lys Val Ser Ile Ala Val Leu Ile Met Cys Cys Ile
Ala Ala Val 1 5 10 15 Leu Ala Asp Gly Tyr Lys Ser Lys Asn Gly Gly
Gln Ala Gly Gly Tyr 20 25 30 Gly Gly Tyr Gly Ser Gly Tyr Gly Gly
Gly Tyr Gly Gly Gly Tyr Asp 35 40 45 Gly Gly Tyr Gly Gly Glu Lys
Gly Lys Ser Gly Lys Gly Tyr Gly Asp 50 55 60 Arg Lys Gly Lys Ser
Glu Lys Gly Tyr Gly Asn Gly Lys Gly Lys Gly 65 70 75 80 Gly Ser Gly
Tyr Gly Gly Gly Tyr Asp Gly Gly Tyr Gly Gly Gly Lys 85 90 95 Gly
Lys Ser Gly Ser Gly Tyr Gly Gly Gly Tyr Asp Gly Gly Tyr Gly 100 105
110 Gly Gly Lys Gly Lys Ser Gly Ser Gly Tyr Gly Gly Gly Tyr Asp Gly
115 120 125 Gly Tyr Asp Gly Gly Tyr Gly Gly Gly Lys Gly Lys Ser Gly
Ser Gly 130 135 140 Phe Gly Gly Gly Tyr Asp Gly Gly Tyr Asp Gly Gly
Tyr Gly Gly Gly 145 150 155 160 Lys Gly Lys Ser Gly Ser Gly Tyr Gly
Gly Gly Tyr Asp Gly Gly Tyr 165 170 175 Asp Gly Gly Tyr Gly Gly Gly
Lys Gly Lys Ser Gly Ser Gly Tyr Gly 180 185 190 Gly Gly Tyr Asp Gly
Gly Tyr Asp Gly Gly Tyr Gly Gly Gly Lys Gly 195 200 205 Lys Ser Gly
Ser Gly Tyr Gly Gly Gly Tyr Asp Gly Gly Tyr Asp Gly 210 215 220 Arg
Tyr Gly Gly Gly Lys Gly Lys Ser Gly Ser Gly 225 230 235
11191PRTPhragmatopoma californica 11Met Lys Leu Ile Cys Leu Val Leu
Leu Ala Val Cys Ile Val Ala Val 1 5 10 15 Ser Ala Ser Ser Ser Ser
Gly Gly Arg Arg Arg Arg Val Ile Val Ile 20 25 30 Gly Asn Arg Gly
Arg Ala Pro Ala Arg Pro Arg Ser Asn Ile His Tyr 35 40 45 His Met
His Ala Pro Gln Pro Arg Met Met Met Ala Pro Arg Met Met 50 55 60
Met Ala Pro Met Met Met Ala Pro
Met Ala Met Pro Ala Thr Ser His 65 70 75 80 Val Tyr Gln Ser Val Ser
Tyr Pro Gly Ala Met Tyr Arg Tyr Gly Leu 85 90 95 Gly Ser Leu Gly
Gly Gly Phe Ile Ser Gly Gly Leu Gly Gly Ile Val 100 105 110 Gly Gly
Gly Leu His Gly Gly Val Val Thr Ser Gly Leu His Gly Gly 115 120 125
Val Val Thr Ser Gly Leu His Gly Gly Val Val Thr Ser Gly Leu His 130
135 140 Gly Gly Leu Val Ser Gly Gly Trp His Ser Gly Val Val Ser His
Gly 145 150 155 160 Gly Leu Ile Gly Gly Gly Ile His Thr Thr Tyr Gly
Gly Phe His Lys 165 170 175 Gly Val Val His Gly Gly Tyr Thr Gly His
Tyr Gly Lys Arg Arg 180 185 190 12102PRTPhragmatopoma californica
12Met Lys Leu Ala Val Phe Ala Leu Leu Val Ala Phe Ala Ile Val Tyr 1
5 10 15 Thr Ala Glu Gly Leu Val Tyr Gly Gly Gln Lys Gly Tyr Gly Tyr
Gly 20 25 30 Gly Lys Gly Tyr Gly Tyr Gly Cys Thr Gly Gly Tyr Gly
Leu Tyr Gly 35 40 45 Gly Lys Gly Tyr Gly Tyr Gly Lys Gly Tyr Gly
Tyr Gly Cys Arg Gly 50 55 60 Gly Tyr Gly Tyr Gly Lys Gly Tyr Gly
Tyr Gly Gly Lys Tyr Arg Gly 65 70 75 80 Tyr Gly Tyr Gly Asn Lys Val
Gly Tyr Gly Tyr Gly Gln Gln Leu Gly 85 90 95 Tyr Lys Asn Gly Arg
Lys 100 1391PRTPhragmatopoma californica 13Leu Asp Gly Gly Cys Lys
Pro Thr Gly Gly Phe Ile Lys Gly Ser Val 1 5 10 15 Gly Pro Cys Gly
Gly Tyr Asn His Gln His Val Val Gly Pro Asn Gly 20 25 30 Ala His
Gly Arg Arg Val Gly Tyr Gly Pro Asn Gly Lys Tyr Ser Gln 35 40 45
Ile Tyr Gly Asn Gly Pro Gly Gly Arg Tyr Ser His Thr Val Val Tyr 50
55 60 Pro Arg Val Arg Pro Tyr Gly Gly Tyr Gly Phe Lys Gly Gly Tyr
Gly 65 70 75 80 Gly Tyr His Gly Val Gly Tyr Lys Gly Gly Tyr 85 90
14145PRTPhragmatopoma californica 14Met Lys Val Phe Val Ala Ala Leu
Leu Leu Cys Cys Ile Ala Ala Ala 1 5 10 15 Ala Ala Glu Asp Gly Tyr
Gly Phe Gly Tyr Asp Gly Tyr Gly Ser Gly 20 25 30 Tyr Gly Tyr Asp
Gly Tyr Ser Tyr Gly Gly Asp Lys Gly Tyr Gly Tyr 35 40 45 Gly Lys
Gly Lys Gly Tyr Gly Tyr Glu Gly Gly Lys Gly Tyr Gly Tyr 50 55 60
Glu Gly Gly Lys Gly Tyr Gly His Glu Glu Gly Lys Gly Tyr Gly His 65
70 75 80 Glu Gly Gly Lys Gly Tyr Gly Tyr Glu Gly Gly Lys Gly Tyr
Gly Tyr 85 90 95 Gly Gly Gly Lys Gly Tyr Gly His Asp Gly Gly Lys
Gly Tyr Gly His 100 105 110 Asp Gly Gly Lys Gly Tyr Gly Tyr Gly Gly
Gly Lys Gly Tyr Gly His 115 120 125 Glu Gly Gly Lys Gly Tyr Gly Tyr
Glu Gly Gly Lys Gly Tyr Gly Lys 130 135 140 Tyr 145
15134PRTPhragmatopoma californica 15Met Arg Ile Val Ile Cys Leu Leu
Val Leu Val Ala Gly Ala Tyr Gly 1 5 10 15 Ile Gly Cys Gly Tyr Gly
Gly Tyr Gly Gly Tyr Gly Gly Gly Phe His 20 25 30 Gly Gly Tyr Ile
Gly Tyr His Gly Gly Tyr Pro Gly Tyr Ser Gly Gly 35 40 45 Phe Arg
Gly Tyr Gly Tyr Pro Gly Arg Val His Thr Asn Val Val His 50 55 60
His Asn Ile Pro Val Phe Met Pro Pro Pro Met Pro Arg Arg Ala Pro 65
70 75 80 Ala Pro Ala Pro Arg Gly Arg Thr Ile Ile Leu Gly Gly Gly
Lys Tyr 85 90 95 Gly Leu Phe Gly Lys Lys Ser Lys Asn Lys Gly Phe
Gly Gly Leu Gly 100 105 110 Val Leu Ser Leu Leu Gly Gly Leu Gly Gly
Lys Gly Gly Gly Gly Ile 115 120 125 Arg Phe Leu Gly Arg Lys 130
16226PRTPhragmatopoma californica 16Met Lys Val Ile Ile Leu Leu Ala
Thr Val Ala Ala Val Tyr Gly Cys 1 5 10 15 Gly Gly Trp Asn Gly Gly
Phe Gly Gly Gly Lys Ala Cys Gly Gly Gly 20 25 30 Trp Gly Ala Lys
Ala Leu Gly Gly Tyr Gly Ser Tyr Asn Gly Asn Gly 35 40 45 Tyr Gly
Ala His Pro Val Ala Val Lys Ser Ala Phe Asn Lys Gly Val 50 55 60
Ser Tyr Gly Ala Arg Ser Ala Val Lys Ala Thr Arg Gly Phe Ala Tyr 65
70 75 80 Gly Lys Gly Ser Ser Tyr Gly Tyr Gly Ala His Pro Ala Val
Lys Ser 85 90 95 Ala Phe Gly Asn Gly Phe Lys Thr Gly Ala His Ala
Ala Val Asn Gly 100 105 110 Tyr Gly Tyr Gly Ala Val Lys Ser Ala Leu
Ser Gly Gly Tyr Gly Tyr 115 120 125 Gly Ser Tyr Gly Ala His Pro Ala
Val Lys Ser Ala Tyr Arg Lys Gly 130 135 140 Leu Ser Tyr Gly Ala Lys
Ser Ala Val Lys Ala Thr Arg Gly Phe Ala 145 150 155 160 Tyr Gly Arg
Ser Gly Tyr Gly Ala His Pro Val Val Lys Ser Ala Phe 165 170 175 Ser
Asn Gly Phe Lys Tyr Gly Ala His Ala Ala Val Lys Ala Thr Asn 180 185
190 Gly Tyr Gly Tyr Gly Ala Val His Pro Ala Val Lys Ala Ala Val Lys
195 200 205 Gly Gly Tyr Gly Tyr Gly Asn Lys Gly Gly Tyr Gly Ala Gly
Tyr Ala 210 215 220 Ala Tyr 225 1789PRTPhragmatopoma californica
17Met Lys Val Phe Val Ala Thr Leu Leu Leu Cys Cys Ile Ala Ala Ala 1
5 10 15 Ala Ala Ala Gly Tyr Gly Asn Gly Tyr Ala Gly Tyr Gly Ser Gly
Tyr 20 25 30 Ala Gly Tyr Gly Thr Gly Tyr Ala Gly Tyr Gly Ser Gly
Tyr Gly Tyr 35 40 45 Asp Gly Tyr Gly Tyr Gly Gly Gly Lys Gly Tyr
Gly Tyr Gly Gly Asp 50 55 60 Lys Gly Tyr Gly Tyr Gly Gly Lys Gly
Tyr Gly Tyr Gly Gly Gln Lys 65 70 75 80 Gly Tyr Gly Tyr Gly Tyr Gly
Lys Tyr 85 18105PRTPhragmatopoma californica 18Met Lys Leu Leu Leu
Leu Phe Ala Leu Ala Ala Val Ala Val Ala Leu 1 5 10 15 Pro Tyr Gly
Tyr Ser Gly Lys Pro Gly Tyr Gly Tyr Asp Ala Val Asp 20 25 30 Ala
Val Tyr Asn Arg Leu Ala Asp Lys Gln Gln Ala Val Asn Arg Lys 35 40
45 Ala Glu Tyr Val Gly Ala Gly Thr Gly Thr Ala Lys Tyr Asn Gly Val
50 55 60 Pro Gly Ala Asn Tyr Gly Tyr Glu Asn Asp Arg Lys Tyr Gly
Tyr Asp 65 70 75 80 Asn Lys Gly Tyr Gly Gly Tyr Gly Asp Lys Gly Tyr
Gly Gly Tyr Gly 85 90 95 Asp Lys Gly Leu Tyr Asp Gly Tyr Tyr 100
105 19275PRTPhragmatopoma californica 19Lys Tyr Tyr Asp Asp Glu Lys
Arg Asp Ala Asp Lys Tyr Arg Lys Pro 1 5 10 15 Ser Tyr Asn Pro Tyr
Asn Thr Tyr Lys Asp Tyr Pro Pro Lys Lys Ile 20 25 30 Tyr Asn Asp
Asp Glu Lys Arg Asp Ala Asp Gln Tyr Arg Ile Ser Tyr 35 40 45 Asn
Pro Tyr Asn Thr Tyr Lys Asp Tyr Pro Pro Lys Lys Lys Tyr Tyr 50 55
60 Asp Asp Glu Lys Arg Asp Ala Tyr Lys Tyr Arg Asn Pro Ser Tyr Asn
65 70 75 80 Pro Tyr Asn Thr Tyr Lys Asp Tyr Pro Pro Lys Lys Ile Tyr
Tyr Asp 85 90 95 Asp Glu Lys Arg Asp Ala Asp Gln Tyr Arg Asn Pro
Ser Tyr Asn Pro 100 105 110 Tyr Asn Thr Tyr Lys Asp Tyr Pro Pro Lys
Lys Lys Tyr Tyr Asp Asp 115 120 125 Glu Lys Arg Asp Ala Asp Gln Tyr
Arg Asn Pro Ser Tyr Asn Pro Tyr 130 135 140 Asn Thr Tyr Lys Asp Tyr
Leu Pro Lys Lys Lys Tyr Tyr Asp Asp Glu 145 150 155 160 Lys Arg Asp
Ala Asp Gln Tyr Arg Lys Pro Ser Tyr Asn Pro Tyr Asn 165 170 175 Ser
Tyr Lys Asp Tyr Pro Pro Lys Lys Lys Tyr Tyr Asp Asp Glu Lys 180 185
190 Arg Asp Ala Asp Gln Tyr Arg Asn Pro Ser Tyr Asn Pro Tyr Asn Thr
195 200 205 Tyr Lys Asp Tyr Leu Pro Lys Lys Lys Tyr Tyr Asp Asp Glu
Lys Arg 210 215 220 Asp Ala Asp Gln Tyr Arg Asn Pro Ser Tyr Asn Pro
Tyr Asn Thr Tyr 225 230 235 240 Lys Asp Tyr Pro Pro Lys Lys Lys Tyr
Tyr Asp Asp Glu Lys Arg Asp 245 250 255 Ala Asp Gln Tyr Arg Asn Pro
Ser Tyr Asn Pro Tyr Asn Thr Tyr Lys 260 265 270 Asp Tyr Pro 275
* * * * *