Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR SEALING TISSUE LEAKS
BACKGROUND OF THE INVENTION
Fluid and/or gaseous leaks can result from surgeries involving vascular,
pulmonary, thoracic, spinal, meningeal, neural, hepatic, lymphatic, digestive,
oncological, gynecological and renal tissues. The current standard of care
involves the
use of hemostats such as thrombin, gelatin and fibrin glue for diffuse
bleeding, or the
s placement of drains until wound resolution for thoracic surgery or lymph
node
dissections.
A number of surgical sealant compositions also exist but suffer from one or
more
disadvantages such as handling, biocompatibility, or toxicity. Currently
available
polymer-based bioadhesives and surgical sealant compositions may also cause
unwanted side effects at the tissue sites to which they are applied. Typical
side effects
include local inflammation, and encapsulation of the material, which results
in the
formation of fibrous or scar tissue. These side effects can be very
detrimental to the
health of the patient. For example, neural tissues in both the central and
peripheral
nervous systems are particularly sensitive to local inflammation, which can
result in
~5 permanent damage. There is therefore a need for tissue sealing methods and
compositions that are easy to handle and that do not elicit severe adverse
host
reactions.
SUMMARY OF THE INVENTION
The invention provides compositions and methods useful for bonding or sealing
2o tissue, including sealant and adhesive compositions, methods for
sealing/adhering fluid
and gas leaks, and methods for priming tissues to increase adhesion. The
invention
also provides methods for controlling the degradation of a sealant or
adhesive.
Accordingly, the invention provides methods and compositions for reducing the
severity
of an adverse host reaction to a sealant, which correlates not only with the
degree of
25 immunogenicity of the polymeric sealant material but also with the amount
of time the
material persists at a tissue locus. Useful kits for producing sealants and
adhesives are
also described.
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The present invention depends, in part, on the discovery that the crosslinking
of
a protein preparation can bond or seal a damaged tissue, and that the bonding
of the
tissue can be modified by an appropriate selection of proteins and
crosslinking agents,
by the presence of additives such as surfactants or lipids, and by modifying
the pH of
the tissue, as described herein. Thus, a leak in a tissue can be repaired
using the
methods and compositions of the invention, providing a rapid and efficient
means to
treat a serious or life-threatening condition such as a gas or fluid leak in a
tissue.
Useful surgical sealants meet certain performance characteristics. A sealant
preferably does not run off of the tissue surface to which it is applied, and
it should
adhere well to the tissue substrate, b~e cohesively strong, be compliant, and
degrade as
the wound heals.
The present invention discloses sealant and adhesive compositions, methods for
sealing/adhering fluid and gas leaks, methods for priming tissue to increase
adhesion of
a sealant or adhesive, and methods for controlling the degradation of the
sealant or
adhesive. Useful kits for producing sealants and adhesives are also described.
In one embodiment, a composition of the invention for use as a tissue sealant
or
adhesive comprises a solution of protein and a surfactant preparation, a lipid
preparation or a carbohydrate preparation. In preferred embodiments the
surfactant,
lipid, or carbohydrate are provided at between about 0.1 % (w/v), and 10%
(w/v) and
2o more preferably between about 0.1 % (w/w) and 10% (w/w). In another
embodiment,
the sealant comprises a protein, surfactant and lipid.
In one embodiment, a method of the invention includes the steps of: (1)
providing a lipid, surfactant, and protein in a liquid carrier; (2) providing
a crosslinker
capable of crosslinking the protein; (3) preparing a sealant by mixing the
protein with
2s the crosslinker; and (4) applying the sealant to a tissue, thereby to bond
the tissue or
seal a fluid or gas leak in the tissue.
In another embodiment, a method for bonding or sealing fluid or gas leaks in
tissue includes the steps of: (1 ) applying to the tissue: (a) a protein
preparation, (b) at
least one preparation selected from a surfactant preparation and a lipid
preparation, and
30 (c) a crosslinker preparation; and (2) permitting the preparation of (1) to
form crosslinks,
thereby to bond said tissue or seal a fluid or gas leak in said tissue.
In the present invention the protein is preferably albumin, collagen, or
globulin
and is preferably in solution at a concentration of 3-55% (w/w) of the
uncrosslinked
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solution. The most preferred protein is albumin at a concentration of 25-50%
(w/w) of
the uncrosslinked solution. The surfactants of the present invention can be
either ionic
or non-ionic. Preferred surfactants are the alkyl aryl polyetheralcohols,
alkanoic acids,
perfluoroalkanoic acids, and alkylsulfonic acids. (e.g. tyloxapol, octanoic
acid,
perfluorooctanoic acid, or sodium lauryl sulfate). The lipids of the present
invention may
include any natural or synthetic lipid. Preferred lipids are hydrophobically-
modified
glycerophosphocholines (e.g. dipalmitoylphoshpatidylcholine). The surfactants
and
lipids are used to modify such properties as adhesion, and physical and
chemical
characteristics such as, elongation/tensile moduli, viscosity (rheometry),
contact angle
and cure time.
A preferred crosslinker is a crosslinker capable of crosslinking a protein.
Preferred crosslinkers of the invention are zero-length crosslinkers, in
particular
carbodiimides. A most preferred carbodiimide is 1-ethyl-3-(3-
dimethylaminopropyl)
carbodiimide hydrochloride (EDC).
15 The present invention also describes methods for preparing a tissue to
react with
a protein-based tissue sealant or adhesive. One embodiment comprises the step
of:
applying a primer solution at a pH of between about 3.0 to 9.0 to a tissue
locus, thereby
to prepare said tissue locus for reaction with a protein-based tissue sealant
or adhesive.
The solution is preferably a buffer with a buffering capacity near the
reactive pH of the
2o sealant crosslinker.
In another embodiment, a tissue is prepared to react with a protein-based
tissue
sealant or adhesive by applying a primer solution containing crosslinker to a
tissue
locus, thereby to prepare said tissue locus for reaction with a protein-based
tissue
sealant or adhesive.
25 In another method, a tissue is prepared to react with a protein-based
tissue
sealant or adhesive comprising the step of: applying a primer solution
containing
molecules that promote increased interaction between the sealant and tissue
locus,
thereby increasing surface area for reaction with a protein-based tissue
sealant or
adhesive.
so The present invention also provides methods for increasing the degradation
rate,
or reducing the persistence of a polymer-based tissue sealant or adhesive.
One embodiment comprises the step of mixing a polymer degrading agent with a
polymer-based tissue sealant or adhesive before applying said polymer-based
tissue
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sealant or adhesive to a tissue locus, thereby increasing the degradation rate
of said
polymer-based tissue sealant or adhesive at said tissue locus.
In another embodiment for increasing the degradation rate, or reducing the
persistence of a polymer-based tissue sealant or adhesive, a polymer degrading
agent
is applied to a polymer-based tissue sealant or adhesive at a tissue locus,
thereby
increasing the degradation rate of said polymer-based tissue sealant or
adhesive at
said tissue locus.
In particular, the invention is useful to regulate the degradation rate of
protein or
carbohydrate based bioadhesives or sealants. In a preferred embodiment of the
invention, the degradation rate of a polymeric gel is increased in order to
increase its
degradation in vivo, thereby reducing unwanted side effects associated with
prolonged
persistence of the gel at a tissue site in a patient.
The current invention also provides a number of useful kits based on preferred
compositions and methods:
In one embodiment, a kit for producing a protein-based tissue adhesive or
sealant comprises: (1 ) a tissue primer, (2) a protein preparation, (3) at
least one
preparation selected from a surfactant preparation and a lipid preparation (4)
a cross-
linker preparation, and (5) a preparation of protein degrading agent.
In an alternative embodiment, a kit for producing a protein-based tissue
adhesive
or sealant comprises: (1) a protein preparation, (2) at least one preparation
selected
from a surfactant preparation or a lipid preparation, and (3) a cross-linker
preparation,
and that may further comprise at least one preparation selected from: (a) a
tissue
primer, and (b) a preparation of protein degrading agent.
In another embodiment, a kit for producing a protein-based tissue adhesive or
sealant comprising: (1) a protein preparation, (2) a cross-linker preparation.
In a further embodiment, a kit for producing a protein-based tissue adhesive
or
sealant comprising: (1 ) a protein preparation, (2) a preparation of protein
degrading
activity, and (3) a cross-linker preparation.
DETAILED DESCRIPTION OF THE INVENTION
3o The present invention provides sealant and adhesive compositions, methods
for
sealing or adhering fluid and gas leaks, methods for priming tissue to
increase adhesion
of a sealant or adhesive, and methods for controlling the degradation of the
sealant or
adhesive. Useful kits for producing sealants and adhesives are also described.
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The compositions, methods, and kits, are useful sealants and adhesives for
adhering and/or sealing fluid and/or gaseous leaks in biological and/or
synthetic tissues.
Such methods are particularly useful for surgical procedures such as vascular,
cardiovascular, pulmonary, renal, hepatic, general, digestive, neural, and
spinal
, procedures.
As used herein, "tissue" means a biological tissue or a synthetic tissue. A
biological tissue includes connective tissues, endothelial tissues, nervous
tissues,
muscle tissue and organs. Preferred biological tissues are selected from the
group
consisting of bone, skin, cartilage, spleen, muscle, lymphatic, renal,
hepatic, blood
vessels, lung, dura, bowel and digestive tissue. A synthetic tissue includes
synthetic
tissue made from biological material or synthetic tissue made from synthetic
material,
and may further include biological materials such as cells, or bioactive
molecules.
Examples of synthetic tissue include expanded poly(tetraflouroethylene)
(PTFE),
polyester, or other synthetic materials used to manufacture an implant such as
a
prosthesis.
COMPOSITION OF THE SEALANT
According to preferred embodiments of the invention, a sealant or implant is
based on a protein component. The protein component may comprise any natural
protein', peptide or polypeptide such as collagen, albumin, globulin, fibrin,
elastin,
2o histone, laminin, protamine, or serum fraction protein, or any combination
thereof. The
protein component may also comprise synthetic proteins, peptides, or
polypeptides or
any combination thereof. In this invention, a synthetic protein, peptide or
polypeptide is
defined as any protein, peptide or polypeptide that has been chemically or
recombinantly modified or produced.
In alternative embodiments of the invention, a sealant or implant is based on
a
carbohydrate component. Preferred carbohydrates include alginates and pectins.
In a
further embodiment a sealant or implant is based on a mixture of protein and
carbohydrate. Carbohydrates are useful to modify the viscosity and/or
elasticity of a
protein-based sealant. For example, the addition of pectin or alginate to an
albumin-
so based sealant, prior to cross-linking, increases the viscosity of the
reaction mixture and
results in increased elasticity of the final gel.
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Proteins
In accordance with the present invention, a choice of proteins) to use and the
concentration needed depend on well-defined factors described herein. Factors
include how the final product will be used, target tissue, desired degradation
rate, and
s physical/chemical properties. For example, a sealant must meet certain
specifications
determined by the specific needs of the substrate. The sealant will have to
withstand
the normal pressures the tissue is under (e.g. 120 mm Hg for blood vessels) as
well as
conform to the natural transitions at the tissue site (e.g. have a high
elastic modulus in
the case of lung tissue applications). Assays useful for optimizing the
compositions and
methods of the invention are disclosed herein, and exemplary protein-based
compositions and tissue specific applications are provided in the Examples.
The concentration of protein in the crosslinked sealant or adhesive ranges
from
about 3 to 55% (w/w). The actual concentration of protein used is dependent on
variables such as protein solubility, desired physical properties following
crosslinking
15 (e.g. tensile strength, elasticity, hardness), as described herein. The
concentration will
also have an affect on how long a sealant or adhesive persists in vivo.
Preferred proteins of this invention include albumin, collagen, gelatin,
protamine,
histones and globulins. The concentration of albumin is preferably between 10
and
55% w/w, and more preferably between 25 and 45% and most preferably between 30
2o and 40% w/w. The concentration of collagen is preferably between 3 and 12%
w/w,
and more preferably between 5 and 10%. The concentration of globulin is
preferably
between10 and 35% w/w, and more preferably between 15 and 30%, and most
preferably between 20 and 25% w/w.
In any one of the above embodiments, the sealant monomer may be in solution
25 with or covalently bound to a molecule selected from the group consisting
of
nucleotides, peptides, synthetic polymers, carbohydrates (e.g., alginates),
polysaccharides (e.g., glycosaminoglycans, dextrans, hyaluronic acid,
chondroitin
sulfate, heparan sulfates), polyethers (e.g., polyethylene glycol,
polypropylene glycol,
polybutylene glycol), polyesters (e.g., polylactic acid, polyglycolic acid,
polysalicylic
3o acid), aliphatic, alicyclic, aromatic, perfluorinated or non-
perfluorinated, and other
derivatizing agents. In addition, the monomer preparation may contain a
chlorinated,
fluorinated, brominated or iodinated derivative.
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Albumin
In some embodiments, albumin-based sealants are preferred. Preferably, the
albumin is of mammalian origin, but other sources of albumin also may be
employed. It
is believed that most albumins are readily cross-linked according to the
methods of the
s invention. However, an albumin with low immunogenicity is preferred for in
vivo
applications. Accordingly, for uses in humans, it is preferred that the
albumin is human
albumin. Bovine serum albumin (BSA) may also be used in humans, and is more
readily available. Alternatively, the albumin may be recombinant albumin,
isolated from
cells expressing a recombinant albumin gene, using methods known in the art.
When
1o produced recombinantly for use in humans, the albumin gene is preferably a
human or
bovine gene. However, other species or biosynthetic variants may be used.
Major
fragments of albumin, comprising at least 100 residues of an albumin sequence,
whether produced by partial proteolysis or by recombinant means, may also be
used
instead of intact albumin. Alternatively, useful fragments may contain at
least 50
~5 residues, and more preferably at least 75 residues of an albumin sequence.
Finally,
mixtures of different forms of albumin (e.g., human, bovine, recombinant,
fragmented),
and plasma fractions rich in albumin may also be employed.
Albumin may be purified directly from tissues or cells, using methods well
known
in the art (see, e.g., Cohn et al. (1946) J. Amer. Chem. Soc. 68:459; Cohn et
al. (1947)
2o J. Amer. Chem. Soc. 69:1753; Chen (1967) J. Biol. Chem. 242:173).
Alternatively,
albumin may be purchased from a commercial supplier. For example, albumin
preparations from various mammalian and avian species may be purchased from
Sigma Chemical Company (St. Louis, MO) in the form of solutions or lyophilized
powders. A preferred commercial supplier of albumin is Intergen (Purchase, New
2s York).
In preferred embodiments, albumin is provided as an aqueous solution of 10-
55%, preferably 25-45%, and most preferably about 30%-40% albumin by weight.
As
explained more fully below, lower concentrations of albumin may be employed
when
viscosity-enhancing agents are added. In some embodiments, the solution is
preferably
3o substantially purified to remove contaminants such as immunogens that would
disrupt
or interfere with the bioadhesive or sealant properties of the cross-linked
albumin. On
the other hand, the presence of many other proteins, such as collagen,
elastin, laminin,
fibrin, and thrombin, can be tolerated.
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Alternatively, albumin may be provided as a dry powder. In such embodiments,
the dry albumin is solubilized at the site of administration. Thus, body
fluids (such as
blood) present at the site of administration may be sufficient to solubilize
the protein.
Alternatively, additional fluids may be provided along with the dry albumin.
The cross-
linker may also be provided as a dry powder that is solubilized at the site of
administration. In a preferred embodiment, the dry protein and cross-linker
are mixed
prior to administration. In a most preferred embodiment, a wetting reagent is
added to
the protein and cross-linker mixture in order to increase fluid absorbance.
Preferably,
the wetting reagent absorbs water from the available body fluids and speeds up
1o solubilization of the protein and cross-linker.
Albumin may be modified or derivatized to increase viscosity. For example
albumin viscosity may be increased by adding in solution or covalently
attaching
relatively large (10-100 kD), substantially linear molecules such as
polysaccharides
(e.g., glycosaminoglycans, dextrans, hyaluronic acid, chondroitin sulfate,
heparan
~5 sulfates), polyethers (e.g., polyethylene glycol, polypropylene glycol,
polybutylene
glycol), polyesters (e.g., polylactic acid, polyglycolic acid, polysalicylic
acid), and
aliphatic, alicyclic or aromatic acylating or sulfonating agents. Preferred
acylating
agents including aliphatic, alicyclic and aromatic anhydrides or acid halides,
particularly
acid anhydrides of dicarboxylic acids. Non-limiting examples of these include
glutaric
2o anhydride, succinic anhydride, lauric.anhydride, diglycolic anhydride,
methacrylic
anhydride, phthalic anhydride, succinyl chloride, glutaryl chloride, and
lauroyl chloride.
The acylating agents may also include various substituents and secondary
functionalities such as aliphatic, alicyclic, aromatic and halogen
substituents, as well as
amino, carboxy, keto, ester, epoxy, and cyano functionalities, and
combinations thereof.
25 Similarly, preferred sulfonating agents useful in the invention include
aliphatic, alicyclic
and aromatic sulfonic acids and sulfonyl halides, which may also include
various
substituents and secondary functionalities as described above.
Albumin also may be modified or derivatized to increase its hydrophobicity in
order to promote interactions with hydrophobic tissues or prosthetic
materials.
so Specifically, albumin may be derivatized with branches or straight chain
alkyl, alkenyl,
or aromatic reagents, including long chain alkyl or alkenyl and alkyl
aldehydes or
carboxylic acids such as octyl or dodecyl aldehyde or carboxylic acid.
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Finally, in order to increase its hydrophobicity and its ability to interact
with
fluorine-containing prosthetic materials (e.g., PTFE-containing materials),
albumin or
modified albumin may be halogenated, preferably fluorinated, by standard
methods well
known in the art. For example, albumin may be derivatized with polyfluoro
dicarboxylic
acid anhydrides (e.g., hexafluoro glutaric anhydride), polyfluoro aklyl ethers
(e.g.,
perfluoroalkyl glycidyl ethers), or other halogen containing reagents.
Alternatively, a recombinant albumin may be produced by standard techniques of
site-directed mutagenesis in which one or more amino acid residues are
inserted,
deleted or substituted to increase the viscosity of the albumin, to alter the
hydrophobicity of the protein, to provide more side chains for derivatization,
or to
provide more free carboxyl or amine groups for the cross-linking reaction. As
a general
matter, under the conditions employed, albumin contains an adequate (and
roughly
equal) number of free carboxyl and amine groups for cross-linking. Therefore,
it is
anticipated that modifications of the albumin sequence will be most useful for
increasing
~5 the viscosity of the protein by replacing small or hydrophilic residues
(e.g., glycine,
alanine) with larger and/or more hydrophobic and/or charged residues which can
participate in non-covalent intermolecular bonds through charge-charge or
hydrophobic
interactions. Alternatively, however, one may produce two forms of modified
albumin
differing substantially in their free carboxyl and amine contents.
2o Carbohydrates
Preferred carbohydrates are natural or synthetic poly- and oligo-saccharides.
Preferred carbohydrates include amylose, amylopectin, alginate, agarose,
cellulose,
carboxymethyl cellulose, carboxymefhylamylose, chitin, chitosan, pectin, and
dextran.
Crosslinkers
25 According to the invention a polymer is crosslinked to form a sealant or
adhesive.
The crosslinking agent can be any crosslinker capable of crosslinking protein,
including
crosslinkers known in the art as well as any new crosslinkers discovered in
the future.
Crosslinkers useful in the invention can be divided into two general classes.
The first
class includes crosslinkers that activate functional groups on proteins to
react with each
3o other (e.g. carbodiimides, oxidants, deprotectants). The second class
includes
crosslinkers containing functional groups that react with other functional
groups on the
proteins to form molecular bonds (e.g. multielectrophilic PEG, multialdehyde).
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Crosslinkers include zero-length crosslinkers, homobifunctional crosslinkers,
heterobifunctional crosslinkers, and multifunctional crosslinkers, or any
other crosslinker
that produce any combination of ionic, covalent, intermolecular, or
intramolecular
bonds. Non-limiting examples of crosslinkers include carbodiimides,
isoxazolium salts,
s carbonyldiimidazole, electrophilic crosslinkers such as di- and multi-
aldehydes, di- and
multi succinimidyl esters, sulfhydryl oxidation. In addition, the crosslinker
may be
covalently bound to the protein or free as a secondary molecule.
In some embodiments, a crosslinker that forms reversible crosslinks may be
used to promote degradation of the sealant in the body. Thus, a crosslinker
may
1o contain or form an unstable bond including, for example, a disulfide,
lactone, lactam,
ester, thioester, acetal, ketal, thioacetal, thioketal, or imidoamide.
The preferred crosslinker of this invention is a carbodiimide, in particular
the
water soluble 1-ethyl-3-(3-dimethylaminopropyl carbodiimide) hydrochloride
(EDC-HCI).
Carbodiimides
15 Carbodiimides are cross-linking reagents having the general formula:
R~-N=C=N-R2.
In general, carbodiimides react with carboxyl groups to form a reactive
intermediate. This reactive intermediate subsequently reacts with a
nucleophile (e.g.
amines, hydroxyl, sulfhydryl, etc.) to form a bond (e.g, amide, ester,
thioester, etc.) and
2o a urea based byproduct. The chemistry is outlined in the following general
reaction:
R3-COOH + R~-N=C=N-R2 ~ R3-COOC-N- R~(=N-R2)
R3-COOC-N- R~(=N-R2) + R3-NH2 ~ R3-CONH- R4 + R~-NH-CO-NH-R2
Carbodiimides are reactive over a wide pH range (1-9.5). At alkaline pH (>8)
the
reaction is slow, but as the pH decreases the reaction rate increases.
However, at low
2s pH hydrolysis of the carbodiimide competes with the formation of
crosslinks. Thus, the
reaction is most efficient in a pH range of 5-7. The pH-sensitivity of the
reaction permits
control over the speed of the reaction and the density of crosslinking. The
reaction
speed can be measured using a cure-time assay, and crosslink density can be
measured indirectly using tensile strength as described in the analytical
methods
30 section.
According to the invention R1 and R2 may be the same or different and may be
any chemical group that does not react with the carbodiimide. R~ and R2 are
typically
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selected from the group consisting of any straight or branched chain,
saturated or
unsaturated, alkyl, alkenyl, aryl, aralkyl, or aralkenyl group, or variants
thereof with
halogen, tertiary amino, quaternary amino, ester, keto, polyalkylene oxide or
other
substituents. In addition, one or both of R~ and R2 may include an additional
carbodiimide group, such that the cross-linker is a polycarbodiimide.
Preferably, the carbodiimides employed are water-soluble. However, a
suspension of water insoluble carbodiimide may also be useful for cross-
linking if
sufficiently dispersed in the cross-linking reaction. By appropriate choice of
R groups,
the solubility and reactivity of the carbodiimide may be varied. In addition,
the choice of
R groups will affect the immunogenicity and toxicity of the cross-linker, as
well as its
ability to interact with the biopolymer molecules.
The carbodiimide may be provided as a solution or suspension. However, since
carbodiimides are subject to hydrolysis they are usually provided in dry form,
such as a
powder. The dry carbodiimide is solubilized or suspended before it is mixed
with the
device or administered to the tissue. It may also be solubilized by body
fluids present at
the site of administration, as may be the case in a sponge-based sealant, or
if being
used as a primer for tissue crosslinking activation.
In another embodiment, the carbodiimide may be provided in a solution of an
inert material. Examples of inert materials include tetrahydrofuran, glycerol,
2o triglycerides, polyvinyl alcohol), polyalkylene oxides (polyethylene
glycol, polypropylene
glycol), non-ionic surfactants, including PEG-based surfactants such as
pluronic
polymers and other inert polymers and soybean oil or tyloxapol.
Examples of useful carbodiimides include 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide; 1,3-di-p-tolylcarbodiimide; 1,3-diisopropylcarbodiimide;
1,3-
dicyclohexylcarbodiimide; 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide
metho-p-
toluenesulfonate; polycarbodiimide; 1-tert-butyl-3-ethylcarbodiimide; 1,3-
dicyclohexylcarbodiimide; 1,3-bis(trimethylsilyl)carbodiimide; 1,3-di-tert-
butylcarbodiimide; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide;
and 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide, all available from the Aldrich
Chemical
3o Company, Milwaukee, WI.
The preferred carbodiimide of~this invention is the water soluble 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCI).
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A typical sealant or adhesive is prepared by mixing a protein component with a
crosslinking component. The reaction conditions for crosslinking a protein
with a
carbodiimide depend on the desired properties of the crosslinking reaction and
the final
gel. Herein the term "gel" refers to the crosslinked sealant or adhesive.
Desired
s properties include time to gellation, time to complete cure and crosslink
density.
Reaction conditions include concentration of carbodiimide and protein, pH of
the mixed
protein/crosslinker solutions, and the ratio of carbodiimide solution volume
to protein
solution volume.
Reaction conditions using carbodiimides
1o The amount of carbodiimide needed to crosslink a protein to form a sealant
or
adhesive depends on well defined variables and may be optimized as described
herein.
Optimization may be based on the measurement of cure time, and the strength of
a gel
over time. In practice, estimating the amount of carbodiimide to be employed
in a
biopolymer system can be done by two methods. Following this the concentration
and
~5 pH of the crosslinker and sealant solution can be adjusted to obtain the
required gel
time and gel strength.
In the first method one equivalent of carbodiimide is used per equivalent of
limiting functional group (e.g. carboxylic acid or nucleophilic groups (amine,
sulfhydryl)
of the protein at a particular pH (usually 5-7). Depending on the gellation
time from this
2o experiment, the amount of carbodiimide may be adjusted and optimized for a
particular
application.
Following is an example demonstrating this method. The example is for
crosslinking 1 g of a bovine serum albumin (BSA) solution at a concentration
of 40%
w/w (represents 0.4 g BSA), where the crosslinker is EDC-HCI. BSA has a
molecular
25 weight of approximately 67,000 and contains 101 moles carboxylic acid/mole
BSA
(carboxylic acids being part of aspartic and glutamic acid residues and at the
C-
terminus) and 60 moles amine/mole BSA (amines being part of lysines and at the
N-
terminus), therefore the amine is the limiting functional group. The following
calculation
determines the amount of EDC-HCI to try first:
0.4 a x 1 mole x 60 mole x 191.7 g EDC- - 0.069
BSA BSA amine HCI g
67000 g mole BSA Mole EDC-HCI
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BSA
In the second method a weight ratio (carbodiimide:protein) of 1:20 is used as
a
starting point. This method would be useful if the exact chemistry of the
starting protein
is unknown and limiting functional groups cannot readily be determined.
It should be recognized that these two methods are only a means of arriving at
a
starting point for determining the final amount of carbodiimide needed. Other
variables
also effecting the amount of carbodiimide needed include the pH of the protein
solution,
effect of additives, ratio of crosslinker solution to protein solution and
viscosity/effectiveness of mixing. Accordingly, it may be necessary to adjust
the
amount of carbodiimide.
1o The above examples represent two methods for determining the ratio of
carbodiimide to protein, however it is recognized that other methods for
determining the
ratio exist and are known to those skilled in the art.
In the present invention useful ranges of weight carbodiimide:weight protein
include 1:80 to 1:1, with a more preferred range of 1:40 to 1:5. However, each
~5 individual polymer system may be different and optimal ratios may be
determined as
described herein. For example, a preferred range for albumin-based sealants is
1:10 to
1:20, and most preferably about 1:14 to 1:16.
The ratio of crosslinker solution volume (CSV) to protein solution volume
(PSV)
will affect the overall strength of the gel. For example, compare mixing a one
to one
2o ratio of 40% albumin to 2% EDC-HCI with a five to one ratio of the same
solutions. The
1:1 solution will result in a weaker, more easily deformed gel with only 20%
w/w
crosslinked albumin while the 5:1 will result in a stronger, more robust gel
with 32% w/w
crosslinked albumin. This dependency on the ratio of PSV:CSV on the final
properties
of the gel allow for some control over the final properties of the gel.
25 The concentration of the carbodiimide solution depends on the mixing and
delivery system and the effect it has on the final device's properties.
Crosslinking proteins using carbodiimide mediated reactive esters
In another embodiment a secondary crosslinking molecule may be added with
the carbodiimide to affect the rate, extent, and operational range of a
crosslinking
3o reaction. For example, in the case of carbodiimide crosslinking the
reactive
intermediate is short lived and works best in the pH range of 5-7. In some
circumstances it may be more advantageous to crosslink at a higher pH or to
have a
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reactive intermediate with a longer half life, especially if the concentration
of reactive
nucleophile (amine, sulfhydryl, hydroxyl) is limited. In one embodiment of the
invention
the additive is a molecule that will react in conjunction with a carbodiimide
to form
another reactive group. In a preferred embodiment the additive forms a
succinimidyl,
s nitrophenol, or maleimide reactive ester. The most preferred additives of
the present
invention are N-hydroxysuccinimide (NHS) and N-hydroxysulfosuccinimide
(suIfoNHS).
The concentration of NHS and suIfoNHS are preferably between 0.1 and 50%
(w/w),
more preferably between 1 and 25% (w/w) and most preferably between 5 and 15%
(w/w).
Lipids
According to the invention a lipid may be provided with the sealant or
adhesive.
Natural lipids are water-insoluble, oily or greasy organic substances that are
extractable
from cells and tissues by nonpolar solvents such as chloroform or ether.
Lipids also
include synthetic lipids and synthetic variants of natural lipids. In one
embodiment, a
~5 lipid may be added to a sealant or adhesive to increase wetting into a
hydrophobic
surface; in another embodiment the lipid may be added to increase the
elasticity of the
sealant or adhesive. For example, DPPC increases the elasticity of an albumin
gel
cross-linked with carbodiimide. Since lipids are insoluble in aqueous
solutions, it may
need to be used in conjunction with a surfactant. Exemplary lipids include
2o phospholipids such as phosphoglycerides or sphingomyelin, glycolipids, and
sterols.
Preferred lipids include phosphoglycerides such as phosphatidyl cholines,
phosphatidyl
serines, phosphatidyl ethanolamines, phosphatidyl inositols, and
diphosphatidyl
glycerol. For applications involving lung tissue, preferred lipids, if
present, include
phosphatidyl cholines. One particularly useful set of lipids is the set of
hydrophobically
2s substituted glycero-phosphocholines with a structure of R~-C(O)-O-CH2-(R2-
C(O)-
O)CH2-CH2-OP02(CH2)2-N(CH3)3, where R~ and R2 are typically saturated and/or
unsaturated alkyl groups ranging in size from C4 to C22 and may either be the
same or
different. For example, a useful device that interacts well with lung tissue
is composed
of dipalmitoylphosphotidylcholine (DPPC, R~ and R2 are C16) dispersed into an
albumin
3o solution containing the non-ionic surfactant tyloxapol.
Other insoluble modifiers that may also be used include but are not limited to
hydrophobically modified phosphatidic acid (e.g. dipalmitoylphospatidic acid,
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dilaurylphosphatidic acid), phosphotidylethanolamine, phosphotidylinositol,
alkyl-
glucopyranosides, long chain fatty alcohols, and bile acids.
The type and concentration of lipid is dependent on the application. The
preferred concentration of lipid is 0.1 to 10%, more preferably 2-8%, and most
preferably 3-7%.
S a rfa cta nts
According to the invention a surfactant may be provided with the sealant or
adhesive in order to effect some physical or chemical property of the sealant
or
bioadhesive or some other component. Surfactants of this invention are
compounds
that lower the surface tension of water. Surfactant molecules preferably
contain a
hydrophobic end of one or more hydrocarbon chains and a hydrophilic end.
In one embodiment of the invention, the surfactant is ionic. Ionic surfactants
are
charged. Ionic surfactants of this invention include fatty acids (linear and
branched
alkanoic acids), linear and branched alkylbenzenesulfonates, linear and
branched
alkanesulfonates, alkylamines, quaternary aminoalkanes, perfluoroalkanoic
acids,
perfluoroalkanesulfonates. Non-limiting examples of these include octanoic
acid,
dodecanoic acid, palmitic acid, sodium lauryl sulfate, perfluorooctanoic acid,
and
perfluorosuberic acid, all available from Sigma-Aldrich.
The preferred ionic surfactants are octanoic acid, palmitic acid,
perfluorooctanoic
2o acid, and sodium lauryl sulfate.
In another embodiment of the invention the surfactant is non-ionic. Non-ionic
surfactants contain a hydrophobic region with an uncharged hydrophilic region
to impart
aqueous solubility. Non-ionic surfactants of this invention include alkyl aryl
polyether
alcohols, and alkyl- or perfluoroalkyl- polyoxyethylene ethers,
polyoxyethylene esters,
polyoxyethylene sorbitan. Non-limiting examples of these include tyloxapol,
Brij 58,
Zonyl 100, all available from Sigma-Aldrich.
According to the invention a surfactant should be between 0.05 and 10%.
However, the choice of surfactant and the concentration needed depend on the
intended use. A surfactant may be chosen as a function of the specific sealant
3o composition or tissue application as described herein.
In one aspect of the invention the surfactant is added to promote wetting of
the
sealant or adhesive into the tissue. Wetting is defined as the ability of a
liquid to
interact with a solid surface, and is analogous to solvents where like
dissolves like. The
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affinity or interaction of a liquid for a solid can be measured indirectly
using contact
angle. In general .a lower contact angle indicates a higher degree of
interaction. In the
case of sealants and adhesives of this invention, it is advantageous to have
high
interaction between the target tissue and the sealant/adhesive, since good
wetting and
spreading increases the contact area providing more opportunity for bonding.
According to the invention a surfactant may be included with a sealant
composition to
increase the sealant's ability to interact with a target tissue by matching
the chemical
characteristics (e.g. hydrophobicity) of the tissue to promote wetting of the
sealant or
adhesive into the tissue substrate. For example, perfluorooctanoic acid can be
added
to a sealant solution to allow for better wetting into expanded PTFE. . In
another
example octanoic acid or sodium lauiyl sulfate is added to a sealant to allow
better
wetting into the hydrophobic lung surface.
In another aspect of the invention the surfactant is added to disperse an
insoluble lipid component. For example tyloxapol may be included in a sealant
to
~5 disperse an insoluble lipid.
In yet another aspect the surfactant is added to increase viscosity. For
example,
if the tyloxapol concentration of an albumin solution is increased the
viscosity increases.
This viscosity increase is due to a denaturation of the protein. It should be
recognized
that other means of denaturation (e.g. sodium lauryl sulfate, urea, heat,
etc.) would also
20 increase viscosity.
Degrading agents
Methods of controlling the degradation of polymer based tissue sealants or
adhesive
The current invention also provides methods related to regulating the rate of
degradation of polymeric gels used in medical applications. The invention is
particularly
25 useful to increase the rate of degradation of polymer-based bioadhesives or
sealants,
thereby increasing their rate of degradation in vivo. Faster degradation of
these
sealants or bioadhesives prevents or reduces unwanted adverse reactions
associated
with their presence (in the form of a bioadhesive or sealant) at a tissue site
in the body
of a patient.
3o According to the invention, one method for increasing the degradation rate,
or
reducing the persistence of a polymer-based tissue sealant or adhesive
comprising the
step of mixing a polymer degrading agent with a polymer-based tissue sealant
or
adhesive before applying said polymer-based tissue sealant or adhesive to a
tissue
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locus, thereby increasing the degradation rate of said polymer-based tissue
sealant or
adhesive at said tissue locus.
A method for increasing the degradation rate, or reducing the persistence of a
polymer-based tissue sealant or adhesive comprising the step of applying a
polymer
degrading agent to a polymer-based tissue sealant or adhesive at a tissue
locus,
thereby increasing the degradation rate of said polymer-based tissue sealant
or
adhesive at said tissue locus.
Polymeric gels used for medical applications are typically biocompatible.
However, most biocompatible polymeric gels are nonetheless antigenic (even if
only
1o minimally so) and do elicit a host immune response, especially if they are
present in
large amounts and have a prolonged persistence in the patient. A polymer may
also be
biocompatible, but the host may not have the physiology to break down the
polymer.
For example, plant derived polymers (e.g. polysaccharides) may not be readily
broken
down by metabolic mechanisms present in humans.
Therefore, the prolonged presence of useful polymer-based bioadhesives,
sealants, and implants may cause unwanted side effects at the tissue sites
they are
applied to. Typical side effects include local inflammation and encapsulation
of the
polymeric gel that results in the formation of scar tissue. These side effects
can be very
detrimental to the health of the patient. For example, neural tissue (nerves
and central
2o nervous system) is particularly sensitive to local inflammation. It will be
apparent to one
of ordinary skill in the art that the severity of an adverse host reaction at
a tissue locus
correlates not only with the level of bio-incompatibility of the polymeric gel
but also with
the amount of time the gel persists at the tissue locus.
The adverse host reaction to the presence of a polymeric gel is prevented or
reduced by increasing the degradation rate of the gel. In general, faster
degradation of
a bioadhesive, sealant, or implant results in less host reaction. In a
preferred
embodiment, the degradation rate of the gel is optimized to permit the gel to
persist for
a time sufficient to perform its function (e.g. binding, sealing), but no
longer than
necessary. For example, a sealant should degrade at the rate of healing. The
optimal
3o rate of gel degradation is a function of the wound healing rate. Methods
for measuring
and optimizing the rate of gel degradation are described herein, and
exemplified in
Example 2.
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Methods of the invention are particularly useful to increase the degradation
rates
of polymeric gels that degrade slowly in vivo. For example, the invention is
useful to
enhance the degradation of albumin-based gels, which are typically degraded
very
slowly in vivo.
According to the invention, methods for regulating the degradation of a
polymeric
gel comprise providing an additive that alters the gel's degradation rate. In
preferred
embodiments, the invention comprises 1) providing a degradation factor (for
example a
degradation enzyme), 2) providing a stimulatory factor that stimulates or
enhances a
natural tissue-associated gel degradation activity or 3) providing any
combination of
1o additives 1 and 2. In another preferred embodiment the invention comprises
providing
an inhibitory factor that inhibits or reduces a natural tissue associated gel
degradation
activity.
The type of additive that is provided is a function of the type of polymer
being
degraded. Polymers contemplated by the invention comprise those useful as
sealants
~5 or adhesives. Typical sealants are formed by mixing a structural (polymer)
component
with a cross-linking or polymerizing agent under conditions to promote cross-
linking or
polymerization of the structural component to generate a gel. Polymers used as
sealants or adhesives are generally cross-linked or polymerized in situ at the
site of a
wound or other tissue injury being treated. In preferred embodiments of the
invention,
2o the sealant or adhesive is protein-based. However, the invention also
contemplates
regulating the degradation of gels based on carbohydrates, nucleic acids,
synthetic
polymers, and any combination of above mentioned polymers.
In a preferred embodiment, a degradation factor or a stimulatory factor is
added
to either the structural component of the sealant or bioadhesive, the cross-
linking or
25 polymerizing agent, or both, before the sealant or bioadhesive is formed
via cross-
linking or polymerization. The degradation or stimulatory factor is thereby
incorporated
throughout the sealant or bioadhesive. The degradation or stimulatory factor
may be
cross-linked to the structural components of the sealant or adhesive, or may
remain in
solution. According to the invention, the additive is preferably provided in
solution, but
30 optionally in suspension or in dry powder form. In an alternative
embodiment, a
degradation or stimulatory factor is added to the formed gel after cross-
linking or
polymerization. The degradation or stimulatory factor is applied to the
surface of the
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gel. According to the invention, the additive is preferably applied as a
solution, but
optionally as a suspension or a dry powder.
One of ordinary skill in the art will appreciate that the amount of
degradation
agent or stimulatory factor provided to a sealant or bioadhesive is a function
of several
well-defined factors described herein, including the desired degradation rate,
the type
of polymer used, the activity of the added material, the crosslinking density,
and
antigenicity of a sealant, adhesive, or implant.
In preferred embodiments of the invention, the additive is provided in an
amount
sufficient to result in degradation of the sealant or bioadhesive when it is
no longer
needed. An adhesive or a sealant is typically provided to temporarily bond two
tissues
together, or seal a fluid or gas leak in a tissue. The adhesive or sealant
properties
preferably persist while the wound heals. According to the invention, once the
wound
has healed sufficiently to be structurally stable in the absence of the
adhesive or
sealant, then the sealant or bioadhesive is degraded. In most embodiments of
the
invention, the gel is degraded in less than about 100 days. In preferred
embodiments,
the gel is degraded in less than about 50 days, and most preferably in less
than about
30 days.
Accordingly, in one embodiment of the invention, an additive is provided in an
amount sufficient to increase the sealant or bioadhesive degradation. As used
herein
2o the term "degradation" means the breaking of molecular bonds (covalent,
ionic,
hydrogen) within the biopolymer or those formed by crosslinking or a
combination of
both, resulting in the breakdown of ttie structural integrity of the
crosslinked sealant or
bioadhesive. Preferably, degradation results in disappearance of the sealant,
adhesive,
or implant.
In the context of sealant. or tissue degradation at a tissue site in a
patient, the
sealant or bioadhesive degradation products may be soluble and removed from
the
tissue site as it is degraded. Alternatively, the degradation products may be
insoluble
sealant or bioadhesive fragments, or may form a precipitate at the tissue site
as the
sealant or bioadhesive is degraded. In preferred embodiments, these fragments
or
3o precipitated degradation products are removed by natural processes such as
phagocytosis.
In a preferred embodiment, degradation results from the cleavage of covalent
bonds in the sealant or bioadhesive structure. In a sealant or bioadhesive
that is
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formed by chemically cross-linking multiple gel subunits (e.g. proteins or
carbohydrates)
to form a cross-linked product, degradation can result from cleavage of
covalent bonds
within the subunits, or from cleavage of the bonds formed by the chemical
cross-linkers,
or from a combination of both.
Typically, there are many possible cleavage sites within a sealant or
bioadhesive
contemplated by the invention. For example, a protein-based sealant can
theoretically
be degraded by cleavage at any one or more peptide bonds within the protein
amino
acid sequence. However, in practice, only a subset of these sites are cleaved
as the
sealant is degraded. The cleavage sites are determined by the structure of the
sealant
and sealant subunits, and also by the substrate specificity of the cleavage
agent or
factor (for example, most proteases have preferred cleavage sites).
At a gross structural level, sealant degradation can be observed after
cleavage of
only a subset of the bonds holding the sealant together. From the point of
view of the
gross structure of the sealant, degradation can occur in different ways. The
outer layer
~5 or surface of the sealant may be degraded first thereby exposing inner
layers or
surfaces of the sealant to further degradation. Accordingly, the sealant
progressively
shrinks in size over time. Alternatively, bonds are cleaved throughout the
sealant
thereby progressively destabilizing the entire structure. Accordingly, the
sealant initially
weakens and eventually breaks up into small fragments. These fragments may
then be
2o further degraded. The type of degradation depends on a number of factors,
including
whether the degradation agent or factor is applied to the surface of the
sealant or is
present throughout the sealant; whether the agent is only active on the
surface, or is
active throughout the sealant; and the structure of the sealant.
In preferred embodiments of the invention the host response is reduced by
25 reducing the presence of the sealant; resulting in less inflammation,
hemorrhaging,
encapsulation and formation of scar tissue.
The amount of additive required is a function of the activity of the additive,
the
structure of the sealant, and the tissue application. Typical molar ratios of
the structural
components to the additive of the sealant are preferably from 1:10 and
20,000:1, more
3o preferably from 100:1 to 5000:1, and most preferably from 1000:1 and
2500:1.
In the case of protein based sealants the amount of protease to add will be a
function of the concentration of the protein in the sealant, the specific
activity of the
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protease sample, the amount of available cleavage sites on the protein, the
specific
turnover of the enzyme, and crosslink density of the final gel.
The specific activity of the protease is defined as the units per milligram of
solid
protein. The unit definition will vary depending on the enzyme-substrate
combination
used; the preferred definition is the one used in the US Pharmacopoeia
guidelines.
Once the specific activity has been determined, it will be necessary to
determine the
potential cleavage sites available on the protein. Proteases typically cleave
around one
or more specific amino acid residues and the potential sites will approximate
the
amount of those residues present in the sequence of the protein.
To calculate the amount of enzyme to add, the active sites will be calculated
per
ml of formulation used. It is not necessary to cleave all of the potential
sites, and the
amount of enzyme should cleave from 1 to 50% of the potential sites, depending
on the
desired time of degradation. The following formula can be used to determine
the
amount of enzyme: Moles of cleavage sites (MCS): (Moles of protein/ml) x
(Moles of
cleavage sites/Mol of protein). Moles used in crosslinking (MUC): grams of
crosslinker/molecular weight of crosslinker (assume 100% efficacy). Units of
protease:
(MCS-MUC)X% of potential sites (Where X=1-50).
The above calculation is affected by the following. Catalytic constants are
calculated based on small substrate analogs, so steric hindrance will not
affect its
2o activity. When the substrate is many sites on a protein some of them will
not be
available, resulting in a decrease in the substrate concentration.
The constants are also calculated in solution. In the case of sealants the
enzyme is immobilized, which will greatly reduce its turnover number since it
can only
catalyze the cleavage of neighboring peptide bonds, resulting in a loss of
catalytic
efficacy.
Its not necessary to cleave all the theoretical sites to get enough
degradation
since the in vivo processes will also be contributing to the degradation of
the gel.
If the crosslink density of the protein is increased, then there should be a
proportional increase in the amount of enzyme added to counter the increased
3o possibility of losing proteolytic activity due to inhibited enzyme.
It is contemplated, however, that the optimal amounts of additive provided,
and
the optimal modes of administering the additive may be determined by routine
experimentation well within the level of skill in the art. Example 2 describes
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experiments useful to optimize the amount of additive that is provided to a
sealant or
adhesive of the invention.
In embodiments where the sealant is particularly antigenic, or if the tissue
site of
application is particularly sensitive, the degradation of the gel is optimized
to be rapidly
degraded when it is no longer needed. In other embodiments, where the gel is
very
biocompatible, or where the tissue is.tolerant of a host immune response, the
degradation rate of the gel does not need to be as carefully optimized.
However, an
additive is preferably provided to ensure that the gel is eventually degraded.
In most preferred embodiments of the invention, the degradation rate of the
gel is
non-linear. Preferably, little or no degradation occurs when the gel is first
applied to the
tissue, and the degradation rate progressively increases over time. An
additive may be
provided in an inactive form, and subsequently activated to degrade the gel.
For
example, the additive may be an inactive form of a protease that is processed
(either
self-processed, or by a separate processing activity provided along with the
protease) to
15 produce an active protease. The amount of time before gel degradation
occurs is
controlled by controlling the processing rate of the protease.
Methods of the invention are preferably used to regulate the degradation of
sealants or bioadhesives based on the following structural elements: proteins,
carbohydrates, and nucleotides including naturally occurring and synthetic
variants, and
20 other synthetic polymers, or any combination thereof.
Proteases
According to one embodiment of the invention, including a protease in the
polymer reaction mixture regulates the degradation of protein-based polymers.
Naturally occurring proteases are preferred degrading agents for protein-based
25 polymers. However, modified proteases are also contemplated by the
invention.
Modified proteases include chemically derivatized proteases and recombinantly
modified proteases. In one embodiment, a modified protease with altered
proteolytic
activity [e.g. increased substrate specificity and catalytic activity is used
to obtain
optimal specificity and degradation rate.
3o Naturally occurring proteases contemplated by the invention include the
following
types of proteases:
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serine proteases, including but not limited to chymotrypsin, trypsin,
elastase,
pancreatic kallikrein, and subtilisin
cysteine proteases, including papain, actinidin, rat liver cathepsins B and H.
aspartic proteases, including penicillopepsin, Rhizopus chineses, Endothia
acid
proteases, and renin;
metalloproteases, including carboxypeptidase and Thermolysin;
unclassified proteases including proteases of unidentified mechanism such as
collagenases, aminopeptidases, signal peptidases; and,
exopeptidases and endopeptidases, including aminopeptidases (alpha-
aminoacyl peptide hydrolases), dipeptidylpeptidases (dipeptidyl peptide
hydrolases), tripeptidylpeptidases (tripeptidyl peptide hydrolases),
carboxypeptidases (peptidylamino acid hydrolase), peptidyldipeptidases
(peptidyl dipeptide hydrolases such as the angiotensin converting enzyme
(ACE) and cathepsin B), dipeptidases, tripeptidases (tripeptide
aminopeptidases), and omega peptidases.
Preferred proteases are active under physiological conditions and their
activity is
not significantly modified by the composition of the polymeric material. In
preferred
embodiments, the protease added to the polymer specifically degrades the major
protein-component of the polymer. The following examples indicate preferred
2o proteases and provide information about their substrate specificity.
Bromelain is a plant
derived cysteine protease. Chymotrypsin preferentially catalyzes the
hydrolysis of
peptide bonds involving tyrosine, phenylalanine, and tryptophan. Clostripain
(Endoproteinase-Arg-C) is a highly specific sulfhydryl proteinase that
hydrolyzes the
carboxyl peptide bond of arginine. This protease is particularly useful to
regulate the
degradation rate of arginine-rich proteins. Collagenase is particularly useful
in
formulations where collagen is the main component. Elastase is particularly
useful in
formulations where elastin is the main component. Papain is a sulfhydryl
protease of
broad specificity. Protease, S. aureus, V8 (Endoproteinase-Glu-C) specifically
cleaves
peptide bonds on the carboxy-terminal side of either aspartic or glutamic
acids.
3o Proteinase K is an endoproteinase with a broad spectrum of action.
Subtilisin is a mix
of proteases with a wide specificity. Trypsin preferentially catalyzes the
hydrolysis of
peptide bonds involving lysine or arginine. In one embodiment involving the
degradation of an albumin-EDC based sealant, trypsin at 1.2 mg/mL is
preferred.
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Glucanases
It is also contemplated by the invention that the degradation rate of
carbohydrate
based sealants or bioadhesives can be regulated. According to the invention
including
a glucanase in the polymer reaction mixture regulates the degradation of
carbohydrate-
s based polymers. Naturally occurring glucanases are preferred degrading
agents for
carbohydrate-based polymers. However, modified glucanases are also
contemplated
by the invention. Modified glucanases include chemically derivatized
glucanases and
recombinantly modified glucanases. In one embodiment, a modified glucanases
with
altered proteolytic activity [e.g. increased substrate specificity and
catalytic activity] is
used to obtain optimal specificity and degradation rate.
Naturally occurring glucanases contemplated by the invention include the
following types: agarases, amylases, cellulases, chitinases, dextranases,
hyaluranidases, lysozymes, pectinases, alginases (and other preferred
enzymes).
Stabilizing Agents
~5 In another embodiment of the invention additives would be used to decrease
the
rate of degradation. Stabilizing agents contemplated by the invention include
the
following: enzymatic inhibitors, chelators, allosteric modifiers, substrate-
based inhibitors
and others known in the art.
Degradation: Application-Specific Considerations
2o Different rates of degradation are appropriate for different tissue
applications, as
described herein. Appropriate amounts of enzyme or other degradation agents
will be
determined by in vivo implantation of the crosslinked gel with different molar
ratios of
the enzyme or degradation agents. In one embodiment, the sufficient amount of
enzyme or other agent is the amount that degrades the sealant, adhesive, or
implant at
25 a rate coinciding with the rate of healing.
Methods of the invention are useful to regulate the degradation rate of
polymeric
compositions at any tissue site in a patient's body. However, in preferred
embodiments,
the rate of degradation of a polymeric composition is adapted for use at a
specific tissue
locus. According to the invention, non-limiting examples of a tissue locus are
selected
3o from a group comprising connective tissues, endothelial tissues, nervous
tissues, and
organs. Preferred tissues are selected from the group consisting of bone,
skin,
cartilage, spleen, renal tissue, hepatic tissues, blood vessels, lung, dural,
menengeal,
bowel and digestive tissue.
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According to the invention, the rate of degradation is optimized to match both
the
requirements of the use (e.g. bonding tissues or sealing a hole in a tissue)
and of the
tissue (e.g. CNS, muscle, or liver). For example sealants contemplated by the
invention are used to seal fluid leaks in tissues or to bond a first tissue to
a second
s tissue. According to methods of the invention, a fluid or gaseous leak can
be sealed by
cross-linking the tissues surrounding the leak. Alternatively, a cross-linked
gel of the
invention can seal a leak by strongly adhering to the surrounding tissue and
physically
occluding the leak. Preferred methods of the invention are useful for sealing
incisions,
perforations, and/or fluid or gaseous leaks in biological tissues during a
surgical
procedure, and comprise contacting the tissue with an effective amount of a
sealant
preparation along with an appropriate amount of degradation agent under
conditions
that promote cross-linking of the sealant preparation to the tissue thereby
sealing the
incision, perforation, or fluid or gaseous leak. 'Subsequently, the cross-
linked polymer is
rapidly.degraded due to the presence of the degradation agent. In preferred
15 embodiments, the polymer degradation process lasts for a sufficient amount
of time to
allow the incision or other wound to heal.
Measuring Degradation in vitro and in vivo
Gel degradation can be measured in several ways. In general, the rate of gel
degradation is measured by monitoring either the disappearance of the starting
product,
2o the appearance of degradation product(s), including intermediate products
of
degradation, or the degradation reaction itself.
Degradation can be measured in vitro by preparing crosslinked gels containing
the degradation agent in molds of known volume. The gels are then dialyzed in
a low
molecular weight cut off dialysis bag against saline and any cofactors
necessary for
25 degradation agent activity. It is preferable that conditions be
physiologically relevant
with regard to pH, temperature and salts. The gel is monitored for a period of
several
days, with the end point being disappearance of the gel. Compared to control
gels with
no degradation agents the amount of time for disappearance will be reduced and
will
vary with the concentration of the degradation agent. In preferred
embodiments, the
3o size of the gel is monitored during the degradation assay. Alternatively or
additionally,
the structural integrity of the gel is monitored during the assay by testing
physical
properties of the gel at given time intervals.
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In a preferred embodiment, gel degradation is monitored
spectrophotometrically,
For example, for a protein based gel, fluid from the dialysis bag is analyzed
by taking
spectrophotometric measurements at 214 nm. As degradation proceeds and
additional
peptides are released, the absorbance at 214 nm will increase.
Degradation can be measured in vivo by preparing crosslinked gels containing
the degradation agent in molds of known volume. Pieces of known weight of
cured gel
can then be implanted into a host animal. Implantation may be intramuscular,
but it is
preferable to implant the gel at the specific tissue site of interest. Each
animal tested is
held set numbers of days (usually 15 and 30 days). At the end of the study all
1o implantation, sites are observed macroscopically for the presence of the
gel and then
explanted and examined microscopically after histological processing (fixed in
10%
neutral buffered formalin).
Buffers
In one embodiment of the invention a high buffer concentration in the sealant
is
~5 used to increase the reactivity of functional groups on the tissue surface
that can
participate in the bioadhesive/sealant crosslinking reaction. In the case of
carbodiimide
crosslinkers, the additive optimizes the pH of the tissue surface for reaction
with the
carbodiimide crosslinker. For example, a protein-based, carbodiimide
crosslinked
sealant's adhesion to biological tissues (e.g. dura, lung, vascular) can be
dramatically
2o improved by adding an acidic buffer to the protein solution. The additive
is preferably a
buffer with a buffering capacity in the pH range of 4-6.5, in the
concentration range of
0.1 to 1 Molar, more preferably pH 5-6 in the concentration range of 0.3 to
0.7M. The
most preferred buffer is one that will not interfere in the crosslinking, for
example buffers
that contain carboxyl groups (e.g. acetate, citrate) or amine groups (Tris-
HCI) which will
25 react with carbodiimides or intermediates of the reaction. An example of a
non-
interfering buffer is morpholino ethanesulfonic acid (MES) and N, N-bis[2-
Hydroxyethyl]-
2-aminoethane sulfonic acid (BES). The concentration of buffer should be
determined
experimentally, by analysis of tissue surface pH before and after application,
and by
examination of adhesion.
3o In preferred embodiments, the sealant preparation includes a buffer. The pH
of
the preparation should be compatible with biological tissues, the sealant
monomers and
the crosslinker. If the crosslinking reaction is pH-dependent, the pH of the
preparation
should be selected appropriately.
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In the embodiments where a buffer is present, the buffer should be effective
at
the desired pH of the preparation. For example, where EDC is used as the
crosslinker,
although the pH of the preparation may be between three and ten, the pH is
more
preferably between about five and about seven, and is most preferably about
6.4. In
this highly preferred embodiment, a buffer may be present and capable of
maintaining
the pH of the solution at or near 6.4. In this particular embodiment, a
preferred buffer
has a pKa within two pH units of the desired pH (i.e. between 4.4 and 8.4),
more
preferably within one pH unit of the desired pH, and even more preferably
within 0.5 pH
units of the desired pH.
If a buffer is present, a preferred buffer will not interfere in the
crosslinking
reaction. Buffers that contain carboxyl groups (e.g. acetate, citrate) or
amine groups
(Tris-HCI), for example, may react with carbodiimide crosslinkers, and are
less
desirable than non-competing buffers that do not contain carboxyl or amine
groups. An
example of a non-competing buffer is morpholino ethanesulfonic acid (MES).
Another
15 preferred buffer is phosphate buffer (e.g. between 10 mM and 250 mM
phosphate).
Adhesion modifiers
A preferred method of the invention comprises providing an additive to modify
the adhesiveness of a bioadhesive or sealant composition, a primer solution,
or an
implant. In one embodiment, the additive is provided to the target surface to
increase
2o its chemical affinity for the sealant, primer, or implant. In another
embodiment, the
additive chemically modifies the molecules of the sealant, the primer
solution, or the
implant to increase their chemical affinity for the target tissue. This can be
accomplished, for example, by modifying the hydrophobicity of the molecules,
or their
electrochemical properties, or any other physico-chemical property that
promotes
2s interaction between the molecules.
Adhesiveness of a surgical sealant, for example, can be assayed by using the
sealant to seal an end-to-side anastomosis on blood vessels. By applying
pressure to
the suture line, the pressure at which a detectable leak occurs can be
detected and
recorded. This pressure correlates with the adhesiveness of the sealant.
3o Adhesion of sealants or other compositions to specific surfaces can also be
carried out by other methods well known to those skilled in the art. Preferred
assays
include ASTM tests D903, D1062, D1781, D1876, D3167, D3433, D3762, D3807, and
D5041 of the Annual Book of ASTM Standards, published by the American Public
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Health Association, Inc. of l/Vashington, DC, the disclosure of which is
hereby
incorporated by reference.
Accessory Molecules
According to one embodiment of the invention, an accessory molecule or
additive may be used with a sealant or adhesive to modify its physical and/or
chemical
properties. The type of additive used is determined by the property
modification that is
most appropriate for a given tissue application. According to the invention,
an additive
is preferably mixed with a sealant prior to cross-linking or polymerization.
As a typical
device is prepared by mixing a protein preparation with a cross-linking
preparation, then
accordingly, the additive may be added to any one of the individual components
of the
sealant, or to the mixed components immediately prior to polymerization or
cross-
linking.. In another embodiment, the additive is covalently coupled to the
protein.
According to the invention, the sealant or adhesive comprises an additives) to
modify its adhesive, physical and chemical properties. This includes additives
that
~5 effect: chemical adhesion, viscosity, tensile, elongation, stability to
sterilization methods
(e.g. gamma irradiation sterilization and electron-beam), solubility,
crosslinking,
degradation, and wetting. It should be noted that a single additive may effect
more than
one property, and that multiple additives may have effects on one another. The
additive
may comprise a molecule selected from the group consisting of viscosity-
enhancing
2o agents, cross-linkers, buffers, hydrophilic agents, hydrophobic agents,
cationic and
anionic agents, hormones, growth factors, anesthetics, antibiotics,
surfactants, lipids,
fatty acids, anti-inflammatory agents, denaturants, degradation agents,
stabilizing
agents,
In some preferred embodiments, viscosity-enhancing agents are added to the
25 mixture and, therefore, the concentration of albumin that is employed may
be
decreased. However, the concentration of albumin is preferably at least 10%,
and more
preferably at least 20%. In preferred embodiments the viscosity-enhancing
agent is
itself cross-linked in the reaction. Viscosity-enhancing agents may include
substituted
or unsubstituted polysaccharides (e.g., glycosaminoglycans or heparin
sulfates), fibrous
3o proteins (e.g., collagen, elastin, fibrin, fibrinogen, thrombin, laminin),
or other
compounds which polymerize under physiological conditions or in the presence
of the
carbodiimides of the invention (e.g., polyacids and polyamines). Preferred
viscosity-
enhancing agents include glycosaminoglycans, dextran, hyaluronic acid,
collagen,
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chondroitin sulfate, pectin, carboxymethyl cellulose, alginic acid, elastin,
poly(ethyleneglycols) and poly(propyleneglycols).
The viscosity of a protein based surgical sealant or adhesive can be modified
by
adding a partially cross-linked protein to the formulation. In one embodiment,
a protein
is polymerized prior to formulation of.the sealant or adhesive to increase its
molecular
weight and thus increase its viscosity.
In another embodiment, the protein is partially cross-linked after formulation
of
the sealant or adhesive.
Partial cross-linking to increase weight can be accomplished using any
reactant
1o capable of forming a bond between protein molecules, and may include zero-
length
cross-linkers, bi-functional cross-linkers, and multi-functional cross-
linkers. By varying
the condition of the partial cross-linking (such as concentration of protein,
concentration
of cross-linker, reaction rate and time of reaction) the protein solution
viscosity can be
adjusted for any particular application.
In some preferred embodiments, accessory molecules are added in order to alter
the rate and/or degree of cross-linking. In general, a carboxylic acid may
reduce the
rate or degree of cross-linking by competing with a protein carboxylic group
in the first
step of the carbodiimide cross-linking reaction. Similarly, an amine may
reduce the rate
or degree of cross-linking by competing with a protein amine group in the
second step
of the carbodiimide cross-linking reaction. However, polycarboxylic acids,
polyamines,
poly(carboxy/amino) compounds (i.e., compounds having a multiplicity of
carboxyl and
amino groups), and mixtures thereof, may increase the rate of gel formation by
reacting
with carbodiimides to form cross-links with two or more protein molecules,
thereby
participating in the gel formation. Such polycarboxylic acids, polyamines,
and/or
poly(carboxyl/amino)compounds should have a relatively high density of carboxy
and/or
amino groups.
In further embodiments, the hydrophobicity of the albumin solution is
increased
by solubilizing the albumin in a solution that is more hydrophobic than water.
In a
preferred embodiment, the albumin is solubilized in a solution comprising a
secondary
or tertiary alcohol. Preferably, albumin is provided in a solution of
isopropyl alcohol
(IPA) or isobutyl alcohol (IBA). Most.preferably, a 30% solution of BSA is
prepared with
20% IPA or 8% IBA.
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In another aspect, the invention provides methods and compositions that bind
or
adhere to synthetic material such as artificial blood vessels (for example
PTFE material)
or biological implants (for example polyethylene material).
In another aspect, the invention provides a kit for producing a bioadhesive,
s surgical sealant or implantable device comprising, in separate containers,
an albumin
preparation, and a carbodiimide preparation. In a preferred embodiment, the
kit further
comprises an accessory molecule, preferably a molecule selected from the group
consisting of viscosity-enhancing agents, cross-linkers, buffers, hormones,
growth
factors, antibiotics, anesthetics, anti-inflammatory agents, hydrophobicity
increasing
1o agents, and surFactants.
In some embodiments, the albumin solution being cross-linked comprises
additional reagents to promote interaction with the tissues at the site of
application.
Mixing and Delive or r~holymer-crosslinker solutions
According to the invention the mixing of a protein component with the
crosslinker
15 component can occur just prior to the application by simple end to end
syringe mixing
through a connector. In another embodiment a binary delivery system, having
separate
compartments holding the protein component and the cross-linker component
prior to
dispensing and mixing, may be particularly useful. Thus, in one method, a
double-
barreled syringe that simultaneously dispenses and mixes the components is
used.
2o Such a double-barreled syringe may be quite convenient for in vivo
applications where
the bioadhesive or surgical sealant is applied to the site of tissue injury or
incision.
In another embodiment of the invention, a double barrel system comprises a
first
barrel containing a protein solution at a pH near or below where the cross-
linking
reaction may occur (e.g., pH 5.0-6.0 for carbodiimide crosslinking), and a
second barrel
25 containing the protein solution at alkaline pH (i.e. pH>_8) sufficient to
slow crosslinking to
an extended period of time. The crosslinker can then be mixed with this second
component and remain useable in a fluid state. Upon mixing the low and high pH
solutions crosslinking proceeds. In this embodiment, the pH and/or buffer
systems in
the two barrels must be selected such that, upon mixing, the pH of the
resultant solution
3o is optimized to permit the cross-linking reaction to proceed efficiently.
In an alternative embodiment, a single barreled syringe contains the protein
solution separated from a crosslinker by a breakable membrane. The
crosslinking
reaction is started by breaking the membrane, and the resulting mixture is
applied as
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described above. In another embodiment, the crosslinker is encapsulated within
a
microsphere which, when shear forces are applied, rupture allowing the
crosslinker to
mix with the sealant solution. Alternatively, the two components may be
applied as a
spray from a device with separate reservoirs for the two components. Finally,
although
s it is not preferred, the two components may be applied sequentially. This
method
suffers from the disadvantage that the components will not be as thoroughly
mixed, and
only a thin coat of cross-linked protein may form at their interface.
PRIMERS
Methods of priming tissues to promote adhesion of sealants and adhesives
1o The present invention also provides methods for preparing a tissue to react
with
a protein based tissue sealant or adhesive. According to the invention primers
are used
to promote adhesion between a tissue substrate and a sealant or adhesive. In
one
embodiment, the primer optimizes the tissue-sealant interface by matching one
or more
chemical and/or physical properties of the tissue surface to that of the
15 sealant/bioadhesive. In one embodiment a primer washes a tissue to remove
any weak
boundary layers at the surface. In another embodiment, the primer will contain
molecules that strongly bind to the tissue and will subsequently react and
bind with the
sealant. For example, a primer containing perfluorooctanoic acid (PFOA)
improves
adhesion of the sealant to expanded PTFE. The fluorinated tail binds strongly
to
2o expanded PTFE while the free carboxyl group can react with the sealant. In
another
embodiment the primer will optimize the tissue surface to participate in the
crosslinking
reaction of the bioadhesive or sealant. In another embodiment the primer may
do any
combination of things listed above. A primer is generally applied using a
brush,
sprayer, or by simple irrigation.
25 Primers are used to promote adhesion between a tissue substrate and a
device.
In one embodiment, the primer optimizes the tissue-sealant interface by
matching one
or more chemical and/or physical properties of the tissue surface to that of
the
sealant/bioadhesive. In one embodiment a primer washes a tissue to remove any
weak
boundary layers at the surface. In another embodiment, the primer will contain
so molecules that strongly bind to the tissue and will subsequently react and
bind with the
sealant. In another embodiment the primer will optimize the tissue surface to
participate in the crosslinking reaction of the bioadhesive or sealant. In
another
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embodiment the primer may do any combination of things listed above. A primer
is
generally applied using a brush, sprayer, or by simple irrigation.
Cleansing a tissue surface
Each tissue surface has differing properties (e.g. type of bodily fluids
present,
hydrophobicity, hydrophilicity, pH, and charge) thus, how a particular sealant
interacts
with the surface will differ among the various tissues to which they are
applied. These
fluids and/or chemical characteristics form a weak boundary layer that will
interfere with
the adhesion of the sealant to that surface and are preferably removed. A
primer may
be used to wash a tissue site prior to application of a surgical sealant to
remove body
fluids that could interfere with the sealant. A primer is generally a solution
that is
brushed, sprayed, or irrigated onto a tissue. An additive can be mixed with
the primer
solution to improve its compatibility with the tissue surface and ultimately
improve
washing efficiency. For example a lung sealant application may require a
hydrophobic
and/or .low pH primer to effectively remove the pleural fluid that bathes the
lung surface.
The primer could be an acidic solution of IPA (20%), or it may contain an
artificial lung
surfactant such as tyloxapol and dipalmitoylphosphatidylcholine.
In the case of synthetic tissue (e.g. expanded PTFE) the primer should lower
the
surface free energy of the substrate and subsequently the contact angle of the
sealant.
~ne example of this is priming the substrate with a solution of a
perfluorinated
2o compound such as perfluorooctanoate, or Zonyl FSN (Dupont). It will be
recognized by
any one of ordinary skill in the art that the choice of primer additive will
depend on the
composition of the particular tissue.
Primer interaction with tissue or sealant
According to the invention the primer may contain a molecule that will
strongly
bind to a tissue substrate. This strongly bound molecule should also have a
high affinity
for the sealant, or contain functional groups that can participate in the
sealant's
crossliriking reaction. For example, the primer could be perfluorooctanote for
a
carbodiimide crosslinked sealant on synthetic tissue (e.g. expanded PTFE). The
fluorinated tail strongly interacts to the expanded PTFE, while the carboxyl
group can
3o react with the sealant via carbodiimide crosslinking.
According to the invention the primer may contain a molecule that will
strongly
bind to a tissue substrate. This strongly bound molecule should also have a
high affinity
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for the sealant, or contain functional groups that can participate in the
sealant's
crosslinking reaction. For example, the primer could be perfluorooctanote for
a
carbodiimide crosslinked sealant on synthetic tissue (e.g. expanded PTFE). The
fluorinated tail strongly binds to the expanded PTFE, while the carboxyl group
can react
with the sealant via carbodiimide crosslinking.
' Modification of the tissue surface
According to the invention an additive is mixed with the primer solution to
modify
the tissue-sealant interface. In one embodiment the primer solution is a
dilute solution
of a crosslinker. For example, a protein-based sealant's adhesion can be
improved if
the tissue substrate is first primed either with a carbodiimide,
carbodiimide/Sulfo-N-
hydroxysuccinimide, glutaraldehyde or any combination thereof. One of ordinary
skill
would recognize that any crosslinking solution would produce similar results
providing
the tissue surface conditions were optimized for the reaction it catalyzes.
The
concentration of the crosslinker in the priming solution depends on the
reactivity and
lifetime of the crosslinker. For example carbodiimide intermediates are short
lived,
while reactive esters and glutaraldehyde are longer lived. The concentration
of
crosslinker may be determined by a series of priming experiments using either
in vitro
burst models or in vivo testing on the tissue. The preferred concentration of
the
carbodiimide solution is between 1 % and 50% w/w, more preferably between 10%
and
30% w/w, and most preferably between 15% and 25% w/w. The preferred
concentration of carbodiimide and suIfoNHS are between 1 % and 50% for each,
more
preferably 10 and 30%. For a glutaraldehyde primer the concentration can be
between
0.1 and 10 %, preferably between 0.5 and 5% and most preferably between 0.7
and
1.3%.
In another preferred embodiment, the primer solution modifies the tissue
surface
to participate in the crosslinking reaction of the sealant. The primer
optimizes the tissue
surface to the crosslinker being used (e.g. pH). For example, a carbodiimide
crosslinked device's adhesion can be dramatically improved by priming the
tissue with a
buffer solution to "activate" it. Activate is defined as optimizing the
surface chemistry of
3o the tissue so functional groups present at the surface can participate in
the crosslinking
reactioh. The primer can be as simple as a dilute HCI solution, but is
preferably a
biocompatible buffer with a pK in the range of 4-7. The most preferred buffer
is one that
will not interfere in the crosslinking reaction; such as morpholino
ethanesulfonic acid
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(MES). If it becomes necessary to use a buffer that contains groups that
interfere with
the crosslinking, for example buffers that contain carboxyl groups (e.g.
acetate, citrate)
or amine groups (Tris-HCI), which will react with carbodiimides or
intermediates of the
reaction, then priming may be done in two steps; the surface is primed with a
first buffer
at a high concentration, followed by water or the same buffer at a lower
concentration
dilute enough not to interfere with the crosslinking reaction. The
concentration of the
buffer is dependent on the volume of buffer to be used. For example, a dilute
solution
of buffer will require more volume to change the pH than a higher
concentration of the
same buffer. In one embodiment of the invention the concentration of the
buffer is
between 0.01 and 2 M. The preferred concentration is between 0.1 and 1 M, and
most
preferably between 0.3 and 0.7 M.
In the case of crosslinkers that function at a basic pH the buffers should
increase
the pH of the surface, while not interfering with the crosslinking reaction.
For example,
the adhesion of a polycarboxylated-based, reactive ester crosslinked sealant
can be
improved by priming the tissue with a buffer that has a basic pK (>7) such as
BES.
According to the invention, an additive is mixed with the primer solution to
modify
the tissue-sealant interface. In one embodiment the primer solution is a
dilute solution
of a crosslinker. For example, a protein-based sealant's adhesion can be
improved if
the tissue substrate is first primed either with a carbodiimide,
carbodiimide/Sulfo-N-
2o hydroxysuccinimide, glutaraldehyde or any combination thereof. Of course it
is
recognized in the art that any crosslinking solution would produce similar
results
providing the tissue surface conditions were optimized for the reaction it
catalyzes. The
concentration of the crosslinker in the priming solution depends on the
reactivity and
lifetime of the crosslinker. For example carbodiimide intermediates are short
lived,
while reactive esters and glutaraldehyde are longer lived. The concentration
of
crosslinker may be determined by a series of priming experiments using either
in vitro
burst models or in vivo testing at the tissue in question. The preferred
concentration of
the carbodiimide solution is between 1 % and 50% w/w, more preferably between
10%
and 30% w/w, and most preferably between 15% and 25% w/w. The preferred
3o concentration of carbodiimide and suIfoNHS are between 1 % and 50% for
each, more
preferably 10 and 30%. For a glutaraldehyde primer the concentration can be
between
0.1 and 10 %, preferably between 0.5 and 5% and most preferably between 0.7
and
1.3%.
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KITS
According to the invention a useful kit for producing a protein-based tissue
adhesive or sealant comprises: (1) a tissue primer (preferably a
morpholinoethanesulfonic acid buffer), (2) a protein preparation (preferably
albumin) (3)
at least one preparation selected from a surfactant preparation and a lipid
preparation
(preferably tyloxapol and dipalmitoylphosphatidylcholine) (4) a cross-linker
preparation
(preferably carbodiimide), and (5) a preparation of protein degrading agent
(preferably
trypsin).
In another embodiment a kit for producing a protein-based tissue adhesive or
sealant comprises: (1 ) a protein preparation, (2) at least one preparation
selected from
a surfactant preparation or a lipid preparation, and (3) a cross-linker
preparation, and
that may further comprise at least one preparation selected from: (a) a tissue
primer,
and (b) a preparation of protein degrading agent.
In another embodiment, a kit for producing a protein-based tissue adhesive or
sealant comprise: (1) a protein preparation, (2) a cross-linker preparation.
In a further embodiment, a kit for producing a protein-based tissue adhesive
or
sealant comprise: (1) a protein preparation, (2) a preparation of protein
degrading
activity, and (3) a cross-linker preparation.
According to a preferred embodiment of the invention, a useful kit comprises
at
least two of the following: a primer, a primer applicator (e.g. a brush), a
protein
preparation, a crosslinker preparation, a crosslinker diluent, a degrading
agent, a
degrading agent diluent, a syringe connector, a sealant applicator, or printed
instructions describing proper uses of the kit. Preferred kits comprise at
least four of the
above components. Highly preferred kits comprise at least six of the above
components. The most preferred kits comprise at least eight of the above
components.
A preferred kit provides a first protein preparation at an acidic pH,
preferably
between about 3.0 and 6.0, and a second protein preparation at a basic pH,
preferably
between about 6.5 and 10Ø In a preferred embodiment, an EDC crosslinker is
mixed
with the second protein preparation. When the first and second protein
preparations
3o are mixed, crosslinking is initiated, because the resulting pH is optimal
for crosslinker
activity.
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METHODS FOR PRIMING AND SEALING TISSUES
Using an additive for improved leak sealing in lung
A preferred method of the invention comprises providing an additive to a
sealant
for use in a lung. The additive modifies the surface tension and
hydrophobicity of the
sealant mixture to promote interaction between the sealant and the surface of
the lung
tissue. Preferably, in the presence of the additive, the sealant mixture
spreads evenly
over the surface of the lung tissue.
In a preferred embodiment, an albumin solution comprises a surfactant and/or a
lipid when it is used as a pulmonary sealant. Preferably, the surfactant and
lipid
1o component is similar to the natural surfactant and lipid composition of the
lung.
Alternatively, synthetic surfactants and lipids may be used.
Methods of bonding or sealing fluid or gas leaks in tissue
The invention also describes methods for using the preferred compositions of
this invention as surgical sealants and adhesives. According to the invention,
one
~5 method for bonding or sealing fluid or gas leaks in tissue comprise the
steps of mixing a
preferred composition (such as albumin, tyloxapol, and
dipalmitoylphospatidylcholine)
with a crosslinker capable of crosslinking the protein and then applying the
sealant to a
tissue wound, thereby to bond the tissue or seal a fluid or gas leak in the
tissue.
Another method for bonding or sealing fluid or gas leaks in tissue comprise
the
2o steps of applying to the tissue locus a preferred composition and a
crosslinker and
permitting the preparation to form crosslinks, thereby to bond said tissue or
seal a fluid
or gas leak in said tissue.
Using an additive for improved leak-sealing in synthetic tissue
A preferred method of the invention comprises providing an additive to a
sealant
25 or bioadhesive for use with synthetic material. In one embodiment, the
additive
modifies the surface tension of the sealant/bioadhesive to match that of the
synthetic
material, thereby promoting adhesion. In alternative embodiments, the additive
modifies the hydrophobicity or provides a moiety that specifically binds to
the synthetic
material. The additive is preferably covalently bound to the sealant monomers
prior to
30 or during the cross-linking reaction of the sealant or adhesive.
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e4nalytical methods
A number of analytical methods are employed in the present invention to
monitor
and optimize the effect of additives and measure the usefulness of the device
for
particular applications. The methods referred to in the invention are outlined
below.
In vitro assays
Adhesion
Adhesion of a surgical sealant or bioadhesive is defined as how well it bonds
to
a tissue substrate. One method of measuring a sealant's adhesion is to conduct
a burst
test. Two types of tests are outlined below.
Dural Burst Test
Defects including incisions, flaps, and expanded-polytetrafluoroethylene
(expanded PTFE) patches are made in porcine dura. The dura is then placed in a
apparatus, the sealant is applied, the chamber of the apparatus is filled with
saline and
pressurized. The pressure at which a detectable leak occurs (air bubbling in
saline) can
be measured and recorded. This pressure correlates with the adhesiveness and
effectiveness of the sealant or bioadhesive.
Vascular Burst Test
End-to-side anastomosis on blood vessels or expanded PTFE grafts are treated
with sealant and then pressurized using an aqueous dye solution. The pressure
at
2o which a detectable leak occurs (release of aqueous dye) can be measured and
recorded. This pressure correlates with the adhesiveness and effectiveness of
the
sealant or bioadhesive.
Skin adhesion test for seroma
In addition to the burst test, adhesion of sealants or other compositions to
specific surfaces can also be carried out by other methods well known to those
skilled in
the art. Preferred assays include ASTM tests D903, D1062, D1781, D1876, D3167,
D3433, D3762, D3807, and D5041 of the Annual Book of ASTM Standards, published
by the American Public Health Association, Inc. of Washington, DC, the
disclosure of
which is hereby incorporated by reference.
3o Rheometric properties
Viscosity and other rheometric properties (flow, effect of shear) were
measured
using a Brookfield rheometer.
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Tensile and Elon ap tion
Tensile and elongation were measured using a Chatillon force gauge.
Cure Time is a measurement of the time required for a sealant or bioadhesive
to
go from a liquid phase to a solid (or semisolid phase). In these experiments
it is defined
s as the point at which the force required to extrude the material through an
orifice rises
exponentially.
Wettin was measured using contact angle. Contact angle was determined
using a goniometer.
In vivo assays
1 o Lung sealant assays
Partial Lobectomy
A partial lobectomy model was created by removing an approximately 1-3"
section of lung tissue from an edge of a lobe. This created a 1-3" long by
~/4 - 1/2" wide exposed-parenchyma wound site. Wound site hemostasis
~s was achieved using electrocautery. Air leaks from the wound site were
verified before treatment by submerging the wound site in saline and
inflating the lung. Air leaks were verified by the presence of air bubbles in
the saline. The lung was then deflated for sealant application.
Planar Dissection
2o A planar dissection model was created by removing a section of pleural
tissue from the planar surface of the lung. The approximately'/2" diameter
by 1/8" deep exposed-parenchyma wound site was made using forceps
and a standard electrocautery instrument. Wound site hemostasis was
achieved using electrocautery. Air leaks from the wound site were verified
25 before treatment by submerging the wound site in saline and inflating the
lung. Air leaks were verified by the presence of air bubbles in the saline.
The lung was then deflated for sealant application.
Wedge Resection
A wedge resection model was created by removing a V-shaped section of
30 lung tissue from the edge of a lobe. An approximately 1/" incision was
made into the edge of a lobe at a 45 degree angle using surgical scissors.
A second'/Z", 45 degree angle incision was made, meeting the first to form
a "V", and the V-shaped section of tissue removed. This left an exposed-
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parenchyma wound site. Wound site hemostasis was achieved using
electrocautery. Air leaks from the wound site were verified before
treatment by submerging the wound site in saline and inflating the lung.
Air leaks were verified by the presence of air bubbles in the saline. The
lung was then deflated for sealant application.
Staple Line
A staple line model was created using a standard staple line instrument.
An approximately 3" incision into the side of a lobe. Every second staple
in the staple line was removed. Wound site hemostasis was achieved
using electrocautery. Air leaks from the wound site were verified before
treatment by submerging the wound site in saline and inflating the lung.
Air leaks were verified by the presence of air bubbles in the saline. The
lung was then deflated for sealant application.
Vascular sealant assays
Suture line
A suture line model was created by making an incision in a blood vessel
approximately 1 cm in length and suturing. The suture spacing could be
varied to increase the level of bleeding. Hemostasis was achieved using
clamps or loops. A sealant was considered effective if no visible bleeding
occurred after removal of the clamps.
Synthetic patch.
The synthetic patch model was created by sewing a 2X20 mm expanded
patch into the blood vessel. The sealant is used to cover the patch to
obtain a fluid tight seal.
End to side anastomoses
In order to create a vascular anastomosis that would leak consistently, a
hemorrhage model was developed by administering anticoagulants, employing
hemodilution and increasing the space between sutures from 1 mm (normally
used) to ~4 mm. Using this model, the following configurations of anastomoses
3o were created: 1) arterial suture line, 2) end-to-side with autogenous vein,
and 3)
end-to-side with an expanded PTFE graft. As expected, significant bleeding was
observed in 100% of the anastomoses after using sutures only (N=35). These
leaking anastomoses were assigned into either the treated {vascular sealant)
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group or the control (thrombin-soaked gelatin sponge) group. The number of
leak-free anastomoses and the time to hemostasis were recorded for both
groups.
The efficacy of the sealant was proven in terms of time to hemostasis and
the number of anastomoses sealed. The configuration of the anastomosis did
not affect efficacy.
Dural sealant assays
Incision
Following a craniotomy an incision was made in the dura of about 1 cm in
length and leakage of cerebrospinal fluid was detected. Fluid stasis was
achieved by tilting the animal and the sealant would be applied. A sealant
was considered effective if no visible leaking could be detected
Seroma prevention assays
Seroma prevention can be analyzed using rats that undergo a
~5 lymphadenectomy. The wound is dried with sterile gauze, treated with
sealant or bioadhesive, and closed using sutures. Animals are evaluated
7 days post operatively for serous drainage, adhesion formation and
histology.
EXAMPLES
2o The following non-limiting examples demonstrate the use of preferred
compositions and methods outlined above to form bioadhesives, sealants and
implants.
EXAMPLE 1. Examples of preferred compositions
According to the invention one useful sealant formulation consists of aqueous
bovine serum albumin (BSA), tyloxapol, and dipalmitoylphosphotidylcholine
(DPPC).
25 The albumin concentration can be between 15 and 55 wiw%, but is preferably
between
25 and 45 w/w% and most preferably between 30 and 40 wiw%. The tyloxapol is
added to increase viscosity, and disperse the insoluble DPPC. The
concentration of
tyloxapol can be between 0.05 and 15 w/w%, but is preferably between 3 and
10%.
The DPPC is added to increase hydrophobicity and interaction with hydrophobic
tissue,
3o as well as increase elongation properties of the final sealant. The
concentration of
DPPC can be between 0.5 and 10 wlw%, but is preferably between 3 and 8%. The
pH
of the final solution can be between 4.5-8.0, but is preferably between 5 and
7, and
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most preferably between 6 and 7. The overall strength of the lung sealant will
depend
on the final albumin concentration, crosslinking density, and effects of the
additives.
Another useful sealant formulation consists of aqueous bovine serum albumin
(BSA), and sodium dodecylsulfate (SDS). The albumin concentration is similar
to the
previously described formulation. The SDS is added to increase viscosity and
hydrophobicity. The concentration of SDS can be between 0.5 and 10 w/w%, but
is
preferably between 1 and 7%, and most preferably between 2 and 5%.
Another useful sealant formulation consists of aqueous bovine serum albumin
(BSA), and sodium octanoate. The albumin concentration is similar to the
previously
1o described formulation. The sodium octanoate is added to increase
hydrophobicity. The
concentration of sodium octanoate can be between 0.5 and 15 w/w%, but is
preferably
between 3 and 10%, and most preferably between 5 and 8%.
Another useful sealant formulation consists of collagen derivatized with
glutaric
anhydride and perfluorooctanoic acid (PFOA). The collagen has been derivatized
with
~5 glutaric anhydride. The derivatization is to increase the solubility of
collagen at
physiological pH and can be between 5 and 95% but is preferably between 10 and
60%
and most preferably between 25 and 45%. The derivatized collagen concentration
can
be between 2 and 15 w/w%, but is preferably between 5 and 10%. The PFOA is
added
to increase wetting and adhesion to expanded PTFE. The concentration is
between
20 0.05 and 5 w/w%, but is preferably between 0.2 and 2%, and most preferably
between
0.5 and 1 %.
Another useful sealant composition consists of aqueous bovine serum albumin
(BSA), polyethylene glycol 600) (PEG 600), perfluorooctanoic acid (PFOA). The
albumin concentration can be between 15 and 55 w/w%, but is preferably between
25
25 and 45 w/w% and most preferably between 30 and 40 w/w%. The PEG 600
concentration can be between 0.5 and 20 w/w%, but is preferably between 10 and
20
w/w%. The PFOA concentration can be between 0.5 and 10 w/w%, but is preferably
between 1 and 7 w/w% and most preferably between 2 and 4 w/w%. The pH of the
final solution can be about 4.5-8.0, but is preferably about 5-7 and most
preferably
3o about 6-7.
Another useful sealant composition consists of aqueous bovine serum albumin
(BSA), pectin, perfluorooctanoic acid (PFOA). The albumin concentration can be
between 15 and 55 w/w%, but is preferably between 25 and 45 w/w% and most
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preferably between 30 and 40 w/w%. The pectin concentration can be between 0.5
and
20 w/w%, but is preferably between 1 and 10 w/w% and most preferably between 2
and
4 w/w%. The PFOA concentration can be between 0.5 and 10 w/w%, but is
preferably
between 1 and 7 w/w% and most preferably between 2 and 4 w/w%. The pH of the
final solution can be about 4.5-3.0, but is preferably about 5-7 and most
preferably
about 6-7.
Another sealant formulation consists of perfluorooctanoic acid (PFOA)
derivatized albumin. The albumin is derivatized with PFOA, which will increase
viscosity and wetting of sealant into expanded PTFE. The substitution can be
between
0.5 and 95%,but is preferably between 5 and 50% and most preferably between 10
and
30%. The derivatized albumin concentration can be between 5 and 40 w/w%, but
is
preferably between 20 and 35%.
EXAMPLE 2: Demonstrating the regulation of degradation
I. Assayina carbodiimide cross-linked albumin degradation in vitro
~5 A protein solution (300 pl) was mixed with a solution of trypsin (20p1).
The
concentrations of the enzyme are in a certain relation to the concentration of
the
protein. The resulting solution is then mixed with 31 pl of a 250 mg/ml
solution of 1-
ethyl-3- (3-dimethylaminopropyl) carbodiimide -HCI (EDC). The protein-
crosslinker-
trypsin solution was poured into molds and allowed to set for 5 minutes. The
resulting
1 Ox3mm cylinders are removed from the molds and placed in a 12kDa molecular
weight
cut off dialysis bag. The bags are placed in 4L of 0.9% NaCI supplemented with
1 mM
CaCl2 with constant stirring and maintained at 37°C. The samples are
periodically
observed and degradation was equated to the disappearance of the cylinder.
Experiment 1:
SAMPLES: A 30% BSA solution pH 5.5 was mixed with a 225 mg/ml solution of
trypsin
(the enzyme was obtained from Sigma Chemical Corp. and had a S.A= 3100 USP
Units/mg of protein). The final mixture corresponds to a 20:1 (protein:enzyme)
weight
ratio. Control samples were made by replacing the trypsin solution with water.
RESULTS: The control degraded in 56 days, while the sample containing trypsin
3o disappeared in only two (2) days.
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Experiment 2:
SAMPLES: The same BSA and trypsin samples were used. The amount of trypsin
added was lowered resulting in weight ratios of 50:1, 100:1, 200:1, and 500:1
BSAarypsin (same enzyme source as in Experiment 1). Control contained water
instead
of enzyme.
RESULTS: The experimental results, summarized in the following table, show
that,
again the cured gel that contained the enzyme disappeared before the control.
The
results also show that by changing the protein:enzyme ratio we can regulate
the rate of
degradation within the gel.
BSA:Trypsin RatioDegradation Time
(Units) (Days)
50:1 (5580) 5
100:1 (2790) 7
200:1 (1395) 9
500:1 (558) 16
Control 41
(The values in parenthesis indicate the total amount of units of enzyme added
to the
sample. Units are standard USP units for trypsin)
Experiment 3:
SAMPLE: A solution of 35% BSA containing 5% Tyloxapol and 5% DPPC in 100mM
MES buffer at pH 6.3 was mixed with varying amounts of trypsin (obtained from
Worthington Biochemical Corp. S.A.= 3458 U/mg). The EDC solution was less
concentrated (200mg/ml) but was mixed in the same ratios. Controls contained
water
2o instead of enzyme solution.
RESULTS: The degradation times for these molds are shown in the following
table:
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Protein:Enzyme Degradation Time
Ratio (Days)
Units
12.5:1 (48412) 0.8
31:1 (19365) 2
63:1 (9682) 3.7
125:1 (4841 ) 6~
310:1 ( 1937) 8
Control 35
Again we observe a shorter half-life of the mold that contains more enzyme, as
compared to those with less enzyme, and controls without trypsin.
s Experiment 4:
SAMPLE: The BSA, tyloxapol and DPPC concentrations are the same as in the
previous experiment. The buffer utilized was also the same, except that it
also
contained 1mM CaCl2, which functions as a stabilizer of the trypsin molecule
and
facilitates the activation of any trypsinogen present in the enzyme solution.
Trypsin was
obtained from Intergen Corp., and the sample had a S.A.= 3194 USP U/mg.
RESULTS: The degradation of these samples is presented in the following table:
Protein:Enzyme Degradation Time
Ratio (Days)
Units
1966:1 (284) 2
3889:1 (144) 3
7609:1 (73) 5
15909:1 (35) 12
38889:1 (14) 17
Control 35
The differences observed among the samples is due mostly to the changing
conditions.
In experiments 1 and 2 the crosslinking density is higher resulting in a more
resistant
~ 5 control, and the need for more enzyme as compared to experiments 3 and 4.
The latter
difFer from each other in the amounts of enzyme included in the gel and the
presence of
calcium which stabilizes the enzymes and activates any proenzyme present.
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II. Assaying carbodiimide cross-linked albumin degradation in vivo
Cross-linked protein molds again were made containing different amounts of
trypsin. These were then implanted into rabbits and the tissue reaction was
observed
both macroscopically as well microscopically.
Preparation of the implant:
All .materials were prepared sterile. A 30% rabbit serum albumin (RSA)(Sigma
Chemical Corp., St. Louis, MO) solution was prepared at pH 5.7. A 0.5 ml
aliquot of this
solution was mixed with a 20 pL aliquot of a trypsin solution (1.5-150 mg/ml)
(Sigma
1o Chemical Corp., St. Louis, MO). This. solution was then mixed with a tenth
volume of the
crosslinking solution, EDC 250 mg/ml (Sigma Chemical Corp., St. Louis, MO).
The gel
was deposited in cubic molds (10x10x1 mm) and allowed to cure for 5 minutes.
The trypsin concentrations used and the protein:enzyme weight ratios obtained
were:
1s 1. 150 mg/ml for a 50:1 ratio
2. 15 mg/ml for a 500:1 ratio
3. 1.5 mg/ml for a 5000:1 ratio
Implantation method:
Pieces of the cured gel were implanted into the paravertebral muscle of New
2o Zealand white rabbits. From the cubes strips measuring 1 x1 x1 Omm were
aseptically
cut. Each animal received three intramuscular implants, corresponding to each
gel. For
each gel one animal was. held for 15 days and another for 30 days. At the end
of each
time period the implantation sites were observed macroscopically and scored
for
hemorrhaging, erythema, necrosis, purulence and encapsulation. The sites were
then
2s retrieved and examined microscopically after histological processing. The
table
summarizes the results.
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Albumin:Trypsin RatioDays ~ Macroscopic Observation
15 No material observed
50:1 at site
30 No material observed
at site
15 Trace material observed
500:1
30 No material observed
at site
15 Material observed at
5000:1 site
30 No material observed
at site
Histopathology of the implant sites showed that the microscopic observations
correlate with these results. The 50:1 dilution had a very limited
inflammatory response
at 15 and 30 days, while the 5000:1 implantation sites presented increased
inflammation and microscopic particles of the cured gel. The 500:1 'sites
showed an
intermediate response, with a decrease in inflammation and implant from 15 to
30 days.
III. Assa r~ ing carbodiimide cross-linked carbohydrate degradation in vitro
Plant derived carbohydrates are not very susceptible to degradation in the
1o human body. A classic example is cellulose, which cannot be degraded or
metabolized
in the body, and if implanted would have to be small or would have to be
degraded by
enzymes supplied with the implant. To utilize a derivatized form of cellulose
as an
implant, we incorporated cellulase, which are enzymes capable of degrading
cellulose,
into the formulation to affect the degradation rate if the sealant.
SAMPLE: A solution was made that contained 16.5% Carboxymethylcellulose (CMC)
and 2.5 mol% Polyoxyethylene bis(Amine) at a final pH of 6.3. To affect the
degradation of the crosslinked solution, it was mixed with varying amounts of
cellulase
(Worthington Biochemical Corp.) that had a S.A.= 62.9 Units/mg. To crosslink
the
solution a 400pL aliquot of this solution is mixed with 100pL of a solution
containing 44
2o mg of EDC and 4.4 mg of N-hydroxysulfosuccinimide (the crosslinker
solution). Both
were mixed and applied into the same cylindrical mold described previously and
allowed to cure for 3 minutes.
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The cylinders were put into dialysis tubing (12kDa MWCO) and left in a 0.9%
NaCI
solution bath at 37°C, with constant stirring. The cylinders were
observed on a daily
basis and degradation was equated to the disappearance of the crosslinked
cylinder.
RESULTS: The following table illustrates the degradation of the crosslinked
CMC gels
containing cellulase:
CMC:Cellulose Degradation Times
Ratio (Days)
(Units
50:1 (84) 0.05
100:1 (42) 0.05
200:1 (21 ) 0.75
500:1 (8.4) 1.00
1000:1 (4.2) 1.70
2000:1 (2.1 ) 5
5000:1 (0.84) 16
10000:1 (0.42) 40
. Control >60
The control has continued past 60 days, but what is significant is the
dramatic
effect that the inclusion of cellulase has on the degradation of the
crosslinked gel. Only
small amounts of enzyme need to be incorporated to rapidly degrade the gel,
which
may be due to the large amount of sites that are available for the enzyme(s).
EXAMPLE 3. Preparation of a protein-surfactant-lipid sealant composition
A sealant composition based on 35% albumin, 5% tyloxapol, and 5%
dipalmitoylphosphotidylcholine (DPPC) is prepared as follows: For a 100 g
scale, 5 g of
Tyloxapol (Sigma) is dissolved in 55 g water, followed by 5g of DPPC
(Genzyme). The
solution is stirred until the DPPC is well dispersed. To this solution 35 g of
bovine
serum albumin (Intergen) is slowly added and mixed until fully dissolved. The
pH of the
final solution is adjusted to 6.3-6.6 using 6N HCI.
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EXAMPLE 4: Preparation of a protein-surfactant sealant composition
A sealant composition based on 35% albumin, 5% tyloxapol, and 4.5%
perfluorooctanoic acid (PFOA), and 0.5 % morpholinoethanesulfonic acid (MES)
is
prepared as follows: For a 100 g scale, 5 g of Tyloxapol (Sigma) is dissolved
in 55 g
water, followed by 0.5 g of MES and 4.5g of PFOA (Aldrich). The solution is
stirred and
titrated with 6N HCI to a pH of 5.5-6Ø To this solution 35 g of bovine serum
albumin
(Intergen) is slowly added and mixed until fully dissolved. The pH of the
final solution is
adjusted to 6.3-6.6 using 6N HCI.
EXAMPLE 5: Preparation of a protein-surfactant sealant composition
A sealant composition based on 7% collagen and 0.5% perfluorooctanoic acid
(PFOA) is prepared as follows for a 100 g scale: 0.5 g of PFOA is suspended in
92.5g
of 25 mM phosphate buffer and pH is adjusted to 5-6 using 10 M NaOH. This is
followed by the addition of 7 g of 20-45% derivatized collagen (derivatized
with glutaric
using methods known in the art). The resulting solution is then titrated to a
pH of 6.5-
7.5 using 10M NaOH.
EXAMPLE 6: Preparation of a protein-surfactant-viscosity modifier composition
A sealant composition based on 36.6% albumin, 2.5% perfluorooctanoic acid
(PFOA), 15.2% poly(ehtyleneglycol (600)), and 0.9% MES is prepared as follows:
For a
100 gram scale, 2.5 g PFOA, 0.9 g MES and 15.2 g PEG 600 are dissolved in 44.8
g
water and titrated with 10 M NaOH to a pH of about 6.5. To this solution 36.6
g of
bovine serum albumin (Intergen) is slowly added and mixed until fully
dissolved.
A sealant composition based on 40% albumin, 2.8% perfluorooctanoic acid
(PFOA), 2.3% Pectin, and 0.9% MES is prepared as follows: For a 100 gram
scale, 2.8
g PFOA, 0.9 g MES and 2.3 g pectin are dissolved in 54 g water and titrated
with 10 M
NaOH to a pH of about 6.2. To this solution 40 g bovine serum albumin
(Intergen) is
slowly added and mixed until fully dissolved.
3o EXAMPLE 7: Use of a two component sealant system using carbodiimide as
crosslinker
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Two sealant compositions similar to example 1 were prepared, but the first
solution was made up in 50 mM phosphate and the pH was adjusted to pH 8.1
(part I).
The second solution was made up in 0.5M MES and the pH was adjusted to 5.4
(part
II). 1.8 mL of part I was mixed with 0.2 mL of 40% w/v EDC resulting in an
activated
solution of part I. This material will remain liquid for 15 minutes. The 2.0
mL of active
part I solution was then mixed equally with part II through a static mixer
nozzle. The
resulting mixture rapidly gelled (in about 18 seconds).
EXAMPLE 8: Effect of different primers on adhesion to lung tissue
A series of studies were conducted to determine an effective primer system for
lung tissue. Experiments were carried out on an anesthetized dog. The first
experiments were conducted on a model for thoracic surgical complicatioris on
the lung
including pulmonary air leaks.
Wound sites were made at various points on the lobe of the lung using
scissors.
The wound resulted in bleeding and loss of air. Hemostasis was achieved by
cauterization and an air leak was confirmed as evidenced by air bubbles from
the
submerged lung.
One single formulation was used for all experiments. The formula consisted of
BSA (35%), tyloxapol (5%), DPPC (5%), and EDC-HCI (2%).
2o Priming was done using a brush or by spraying (the method used will be
indicated). The following Table outlines the results.
PrimervType1 Application Results
Method
fVo primer Brush Poor adhesion to smooth
pluera.
Good adhesion to peranchaemal
tissue
Saline Brush Poor adhesion to smooth
pluera.
Good adhesion to peranchaemal
tissue
Dilute crosslinkerBrush Excellent adhesion to smooth
luera and eranchaemal tissue
Acetate buffer Brush Excellent adhesion to smooth
luera and eranchaemal tissue
MES buffer Brush Excellent adhesion to smooth
luera and eranchaemal tissue
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~lutaraldehyde Brush Excellent adhesion to smooth
pluera and peranchaemal tissue
1. Saline primer The saline primer was a 0.15M solution of NaCI.
Dilute crosslinker solution A solution of 10% EDC
Buffers: Acetate 0.5M acetate followed by 25 mM acetate both at pH 5.5.
Buffers: MES 0.5M solution of MES at pH 5
s Glutaraldehyde 0.5% w/v glutaraldehyde solution
EXAMPLE 9. Effect of primers on adhesion to expanded PTFE
In this experiment expanded PTFE was brush primed using either saline or a 1
w/w perfluorooctanoic acid (PFOA) solution at pH 6.3 and then dried with
gauze.
sealant consisting of glutaric anhydride derivatized collagen (5.6% w/w), and
EDC/Sulfo-NHS (3.8 and 1.5% w/w respectively) at pH 7 was applied. After
curing for a
total of 3 minutes the adhesion to expanded PTFE examined. The sealant applied
to
the saline treated expanded PTFE could be pulled off entirely with no cohesive
failure.
The sealant applied to the PFOA treated expanded PTFE could not be removed
without
15 cohesive failure.
EXAMPLE 10: lJse of sealant compositions
Use of compositions as a Lung Sealants.
An anesthetized dog was used as an experimental model for thoracic surgical
2o complications including pulmonary leaks.
Wound sites were made at various points on the lobe of the lung using
scissors.
The wound resulted in bleeding and loss of air. Bleeding was terminated by
cauterization and an air leak was confirmed as evidenced by air bubbles from
the
submerged lung.
25 In a first experiment, the wound site and surrounding tissue (parenchyma
and
pluera) were brush primed using 0.5M MES. A device consisting of BSA (35%
w/w),
tyloxapol (5% w/w), DPPC (5% wlw),. and EDC (2% w/w) at a pH of 6.3 was then
applied. The sealant viscosity was such that the material easily spread on the
lung
surface, but did not run off the wound site. After a total curing time of 3
minutes the
30 lung was inflated. No air leaks were observed.
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In a second experiment, the wound site and surrounding tissue (parenchyma and
pluera) were brush primed using 0.5M MES and the device was then applied. The
device consisted of BSA (40% w/w), sodium dodecylsulfate (SDS) and EDC (2%
w/w)
at a pH of 7. After curing for a total of 7 minutes the lung was inflated. No
air leaks
were observed.
In one experiment a 40% solution of BSA was prepared by using a 25/10 ratio of
BSA and glutaric anhydride derivatized BSA. This solution (pH 6) was used with
a
16.67% aqueous solution of EDC~HCI at a ratio of 8:1 (vol/vol) on a porcine
lung (ex
vivo) to seal a planar wedge resection. The solution on curing adhered to the
lung
tissue and withstood a static air pressure in excess of 60 mm of Hg (average
lung
pressure during surgery is in the range of 20-25 mm of Hg).
In another experiment, a 40% (pH 6) BSA solution was mixed with Tyloxapol and
dipalmitoyl phosphatidyl choline (DPPC) such that they were 1 mg/ml and 14
mg/ml,
respectively. The dispersion was mixed (10: 1 ) with an aqueous solution of
EDC HCI
(20%) and applied to a porcine lung in a planar wedge resection model. The
site was
previously primed with a 30% solution of BSA (pH 5.5). The material was
allowed to
attain maximum strength (about 4-5 minutes), and then tested. The material
withstood
a dynamic pressure of about 100mm of Hg before lung tissue rupture occurred.
In another experiment, Gelatin (300 Bloom) was mixed with DPPC and tyloxapol
2o in a similar ratio. The material was a gel at room temperature. The
material was
warmed to about 45°C and mixed appropriately with an aqueous EDC~HCI
solution. On
applying to the lung tissue, the material gelled rapidly and adhered
satisfactorily to the
wound site.
A preferred sealant composition for lung applications is prepared by mixing a
35% (w/w) of albumin with an EDC cross-linker as described herein.
Use of compositions as vascular sealants
An anesthetized, heparinized, hemodiluted dog was used as an experimental
model for vascular surgical complications including anastomoses and suture
hole leaks.
Natural-to-synthetic end-to-side anastomoses were created in the femoral and
so carotid arteries using 6 mm diameter expanded PTFE with suture spacing
between 2
and 2.5 mm. The artery was pressurized by removal of clamps and blood leakage
was
confirmed.
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In the first experiment the anastomosis was brush primed using a saline
solution
to remove excess blood and the cleaned anastomoses was dried with gauze. A
device
consisting of glutaric anhydride derivatized collagen (5.6% w/w),
perflurooctanoic acid
(0.5% w/w), and EDC/Sulfo-NHS (3.8 and 1.5% w/w respectively) at pH 7 was
applied
s using an applicator tip. After curing for a total of 3 minutes the
anastomosis was
pressurized and no blood leakage was observed.
In a second experiment the anastomosis was brush primed using a saline
solution to remove excess blood and the cleaned anastomoses was dried with
gauze.
A device consisting of BSA-perfluorooctanoamide (25% derivatization, 30% w/w),
and
EDC (2.5% w/w) at pH 6.5 was applied using a splayed applicator tip. After
curing for a
total of 3 minutes the anastomosis was pressurized and no blood leakage was
observed.
In another experiment the anastomosis was brush primed with 0.5 M MES (pH
5). A device consisting of aqueous BSA (36 w.w%), PEG 600 (15 w/w%), PFOA (2.5
15 w.w%), MES (0.9 w/w%), and EDC (2 w/w%) at pH 6.7 was then applied. After
curing
for a total of 3 minutes the anastomosis was pressurized and no blood leakage
was
observed.
In another experiment an anastomosis was brush primed with 0.5 M MES (pH 5).
A device consisting of aqueous BSA (40 w/w%), pectin (2.3 w/w%), PFOA (2.8
w/w%),
2o MES (0.9 w/w/%, and EDC (2 w/w%) at pH 6.7 was then applied. After curing
for a total
of 3 minutes the anastomosis was pressurized and no blood leakage was
observed.
A preferred sealant composition for vascular applications is prepared by
mixing a
35% (w/w) of albumin with an EDC cross-linker as described herein.
Use of compositions as dural sealants
25 An anesthetized dog was used as an experimental model for dural surgical
complications including cerebrospinal fluid leaks.
Following a craniotomy an incision was made in the dura of about 1 cm in
length
and leakage of cerebrospinal fluid was detected. Fluid stasis was achieved by
tilting the
animal and then sealant was applied. A sealant was considered effective if no
visible
30 leaking could be detected.
In a first experiment the incision was brush primed using a saline solution to
remove excess blood, and the cleaned dura was dried with gauze. A mixed
sealant
composition consisting of approximately 18% alpha-globulin (w/w) and 2% EDC
(w/w)
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at pH 5.7 was applied to the incision. After curing for a total of 3 minutes
the dog was
tilted head down and observations made for leaks. No leakage was observed. The
dura was further pressurized using saline through a catheter and no leakage
was
observed.
In a second experiment the incision was spray primed using a 0.25 M MES
solution at pH 5 to remove excess blood, and the cleaned dura was dried with
gauze. A
mixed sealant composition consisting of approximately 18% BSA (w/w), 1.8%
alginate
(w/w) and 2% EDC (w/w) at pH 5.7 was applied to the incision. After curing for
a total of
3 minutes the dog was tilted head down and observations made for leaks. No
leakage
was observed. The dura was further pressurized using saline through a catheter
and
no leakage was observed.
A preferred sealant composition for dural applications is prepared by mixing a
20% (vir/w) of albumin with an EDC cross-linker as described herein. '
Use of compositions in seroma prevention
A preferred sealant composition for seroma prevention is prepared by mixing a
40% (w/w) solution of albumin with an EDC cross-linker as described herein.
The
resulting sealant forms a strong thin gel that is useful to adhere tissue
planes together.
This composition is particularly useful to adhere tissue planes together after
a surgical
removal of tissue matter, for example after a mastectomy.
2o Use of compositions for bonding tissue
In a preferred embodiment, a composition of the invention is used to bond a
first
tissue to a second tissue. One or both tissues may be primed as described
herein. A
composition of the invention may be applied to either one or both of the
tissues to be
bonded. The first and second tissues are then held together for a sufficient
time to
allow the cross-linking reaction to create a stable bond or adhesion between
the first
and second tissues.
EXAMPLE 11. Adhering to End-to-Side Arterial Anastomosis of expanded PTFE
Graft
to Artery
3o In a first experiment, a sealant mixture of BSA and EDC~HCI was delivered
in
vitro onto an end-to-side anastomosis of an expanded PTFE (expanded
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polytetrafluoroethylene) graft onto a porcine aorta. The mixture was delivered
through a
static mixing nozzle, and contained a 9.3:1 ratio of 35% BSA (pH 5.55) : 40%
EDC~HCI.
The adhesive mixture was slowly extruded onto all sides of the anastomosis.
The
mixture cross-linked fairly rapidly. Indeed, the gel could be neatly cut with
scissors ~ 30
seconds after its application. The artery was pressurized by introducing
saline via a
large syringe, and the adhesive treated graft provided a good seal.
In a second experiment, the same composition was used on a 2 x 20 mm
expanded PTFE patch sewn into a porcine carotid artery in vivo. The albumin
sealant
adhered to the vessel and sealed the leaking suture line.
1 o Use of PTFE adhesion-enhancing additives v~rith surgical sealants
A PTFE adhesion-enhancing additive is provided to a sealant consisting of two
components A and B. Component A is a mixture of crosslinkable polymer (e.g.,
protein)
and a PTFE adhesion-enhancing additive (e.g., perfluorooctanoic acid).
Component B
is a solution of crosslinking agent(s). The concentration of component A
depends on
15 the protein, the desired handling characteristics, and desired final gel
properties. When
these two components are mixed the resulting solution is activated and starts
to cure.
The rate of cure is dependent on the precise composition of the two components
and
thus provides the ability to slow the cure sufficiently to allow for
application followed by
a short set time.
2o In one example, Component A consists of collagen or modified collagen plus
a
perfluorinated alkanoic acid. As used herein, "modified collagen" is collagen
with 10-50
mole% of its accessible primary amines modified with glutaric anhydride. The
collagen
or modified collagen is at a concentration of from 40 to 100 mg/mL and at a pH
of from
to 8.5. The perfluorinated compound is at a concentration of from I to 20
mg/ml.
25 Component B comprises from 1 to 100 mg of a carbodiimide and from 1 to 40
mg
of N-hydroxysuccinimide dissolved in water. The volume of water used to
dissolve the
crosslinker is between 1 and 1/10 the volume of component A.
Preferably, component A is a solution of modified collagen (25-40%) at 60-80
mg/mL.and with a pH between 6.8 and 7.2 containing 4-6 mg/mL perfluorooctanoic
3o acid. This is mixed with a solution of 25-45 mg of 1-ethyl-3-trimethyl
propyl
carbodiimide (EDC) and 10-20 mg of sulfo-N-hydroxysuccinimide (sulfo-NHS)
dissolved
in a volume of water from 1/2 to 2/3 of the volume of component A.
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In one experiment, Component A was prepared by dissolving 600 mg of freeze
dried, modified collagen (27% derivatized according to methods known in the
art) in 10
ml of an aqueous solution of 160 mM sodium hydroxide. This resulted in
slightly
viscous solution with a pH of 7.15. 50 mg of perfluorooctanoic acid and were
added
and mixed until dissolved. The final pH of the solution was 6.9. Addition of
the
perfluorooctanoic acid caused aeration of the solution. 0.6cc of aliquots of
component A
were transferred to 1 cc syringes. The samples were centrifuged to remove air.
In the same experiment, component B was prepared as follows. The
crosslinkers (EDC and sulfo-NHS) were stored dry, under nitrogen, in capped
vials and
reconstituted just prior to use. 37~5 mg EDC and 15+3 mg sulfo-NHS were added
to
each vial. Each vial was reconstituted with 0.15 cc ultrapure water just prior
to mixing
with component A.
After reconstitution, component B was drawn into a 1 cc syringe and the air
was
removed. The syringes containing components A and B were connected by a luer
lock
connector. The components were mixed for five to ten seconds. The sealant
cured to
an unworkable gel in 60~10 seconds and was fully cured in 180 seconds.
Evaluation of the effectiveness of additives
The effectiveness of an additive can be evaluated by qualitative assessment of
the adhesive properties of the resulting mixture. These properties are
generally
2o categorized as follows:
Poor No adhesion
Fair Resists pulling off, but adhesion fails
Good Cohesive failure before adhesive
For example, whereas a sealant without an additive such as perfluorooctanoic
25 acid has been found to have "Good" adhesive properties when assayed with
tissue but
"Poor" when assayed with expanded PTFE, a sealant with perfluorooctanoic acid
was
found to have "Good" adhesive properties in either assay.
A second testing method involves comparing the contact angle of the solution
without the additive to the contact angle observed in the presence of the
additive.
3o Generally, smaller contact angles are preferred when increased wetting and
adhesion
are desired. Greater contact angles are preferred when adhesion is not
desired. For
example, deionized water has a 119 degree contact angle with PTFE. An aqueous
solution of 5% octanoic acid, however, has a 25 degree contact angle. The
presence of
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octanoic acid can therefore improve "wetting" of an aqueous solution on PTFE.
Similarly, one mL of a 38% solution of bovine serum albumin in 5% octanoic
acid, when
crosslinked with 50 mg EDC produces a gel with "Good" adherence to PTFE.
A third testing method involves applying the sealant to end-to-side
anastomoses
(natural to expanded PTFE), filling the graft with liquid, and subjecting it
to pressure.
The pressure at which it starts to leak is recorded as its burst pressure. An
effective
additive wil( increase the observed burst pressure when used in conjunction
with an
appropriate sealant and graft material.
The final method of testing is in surgery using dogs. End-to-side anastomoses
1o and suture line models were tested.
In Vivo testina (results from suraervl:
Formulation Results
see text below .
All applications are on end-to-side
anastomoses-natural to expanded PTFE-
unless otherwise noted
162-117B Femoral artery-profuse bleeding before
a lication. No bleedin after a lication
162-111 B Carotid artery-suture weeping before
a lication. No bleedin after a lication.
162-143A Femoral artery-profuse bleeding before
a lication. Sto s 95% of bleedin
162-143A Femoral artery-profuse bleeding before
a lication. Sto s 95% of bleedin
.
162-143A Reapplication on top of previous.
100% of
bleedin sto ed.
162-143A Carotid artery- profuse bleeding
before
a lication. No bleedin after a lication
162-14.3A Carotid artery- profuse bleeding
before
a lication. No bleedin after a lication
162-143A Carotid artery-suture line (1 cm,
5 sutures)-
excessive bleeding. 100% of bleeding
sto ed.
162-117B: 0.8 mL of a solution of 80 mg/mL collagen (27% derivatized) and 0.5
mg/mL perfluorooctanoic acid, pH=6.88, were mixed with 0.4 mL of a solution of
125
mg/mL EDC and 37.5 mg/mL suIfoNHS. The vessel was rinsed with saline and dried
with gauze. The sealant was applied using a splayed applicator tip.
162-111 B: 0.8 mL of a solution of 80 mg/mL collagen (27% derivatized),
pH=6.9,
were mixed with 0.4 mL of a solution of 125 mg/mL EDC and 37.5 mg/mL suIfoNHS.
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The vessel was rinsed with a 1 % perfluorooctanic acid solution (pH=7) and
dried with
gauze. The sealant was applied using a splayed applicator tip.
162-143A: 0.6 mL of a solution of 80 mg/mL collagen (27% derivatized), pH=6.9,
were mixed with 0.4 mL of a solution of 125 mg/mL EDC, 37.5 mg/mL suIfoNHS,
and 10
mg/mL of perfluorooctanoic acid. The vessel was rinsed with saline and dried
with
gauze. The sealant was applied using a splayed applicator tip.
EXAMPLE 12. lJse of additives to promote the adhesion of BSA-based sealants to
PTFE
1o Cross-linking experiments with different additives were performed using 30,
33,
and 38% (weight/volume) solutions of BSA. Cross-linking was initiated by
mixing
approximately 10-20 mg of EDC per ml of BSA solution.
The sealant properties of the cross-linked reaction products were evaluated by
assessing the adhesion of the cross-linked product to PTFE and vascular
tissue.
15 The addition of octanoic acid (approximately 5% final concentration)
resulted in a
cross-linked product with good adhesion to PTFE whereas using octanoic acid
(approximately 1 % final concentration), isopropanol (approximately 20% final
concentration), isobutanol (approximately 8% final concentration) as an
additive
resulted in poor adhesion to PTFE.
2o These results correlate to average contact angles measured for these
additives
on PTFE:
SolventlSam le Avera a Contact An le de rees
DI H20 119
__
20% Isopropanol 95
8% Isobutanol 95
1 % Octanoic acid 88
5% Octanoic acid 25
1 % Perfluoro octanoic acid 75
1 % Perfluoro sebacic acid 90
33% BSA/5% Octanoic acid 24
A low contact angle on PTFE for the additive correlates with good adhesion to
PTFE.
25 EXAMPLE 13. Effect of Derivatization of BSA
BSA was derivatized to increase its hydrophobicity with reactive molecules
with
hydrophobic tails.
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In one experiment, 10 g of BSA was dissolved in 200 ml of 0.05N phosphate
buffer and pH adjusted to 8.5. 6.89 ml of hexanoic acid anhydride was added in
an
acetone solution (the acetone solution is saturated with hexanoic acid
anhydride) at
once. There was no obvious change in the pH of the solution. The reaction was
allowed to stir for 2 days at low temperature. The mixture was diafiltered, pH
adjusted to
6.0, and dried. Solutions of the derivatized BSA exhibited a more hydrophobic
nature
as evidenced from contact angle studies.
In one experiment, 10 g of BSA was dissolved in 90 ml of de-ionized water and
100 ml of 0.05N phosphate buffer and pH adjusted to 8.5. 13g of pyromellitic
1o dianhydride was added in an acetone solution dropwise. The pH was
maintained at 8-9
using dilute NaOH. The reaction was allowed to stir overnight at low
temperature
(approximately 4 °C). The mixture was diafiltered, pH adjusted to 6.0,
and dried.
Solutions of the derivatized BSA exhibited a more hydrophobic nature as
evidenced
from contact angle studies. The solution was also highly viscous as compared
to BSA
~5 solutions of similar concentrations.
In one experiment, 10 g of BSA was dissolved in 67 ml of 0.05N phosphate
buffer. . The pH of the resulting solution was adjusted to 8.5, and 6 g of
tetra fluoro
phthalic anhydride was added as a solution in acetone. The pH was maintained
at 8-9
using dilute NaOH. After several hours of stirring, the reaction mixture was
diafiltered,
2o pH adjusted to 6.0, and dried. Solutions of the derivatized BSA exhibited a
more
hydrophobic nature as evidenced from contact angle studies.
In one experiment, 20 g of BSA was dissolved in 500m1 of a 65/35 mixture of de-
ionized
water and methanol. The pH of the pale yellow solution was adjusted to 9.0,
and 8 ml
of (2, 2, 3, 3, 4, 4, 5, 5, 6, 6, 7, 7, 8, 8, 9, 9, 9-heptadecafluorononyl)-
oxirane was added
25 all at once in a 50% acetone solution. The reaction was allowed to stir for
2 days,
maintaining the pH at 9. The slightly turbid solution was centrifuged,
dialyzed, pH
adjusted to 6.0, and allowed to dry. Solutions of the modified BSA solution
exhibited
higher viscosity and an improved wettability towards expanded PTFE graft, and
on
cross-linking with the appropriate amount of EDC~HCI, adhered very well to the
graft
3o and natural tissue.
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EXAMPLE 14: Effect of partially cross-linked protein on sealant viscosity
The viscosity of a sealant comprising bovine serum albumin (BSA), tyloxapol,
and dipalmitoyl-phosphatidyl choline was modified by replacing bovine serum
albumin
with partially cross-linked bovine serum albumin. The partially cross-linked
albumin
samples were prepared as follows: A 40% w/w BSA solution (pH 6.85) and a 4%
w/w
EDC solution were mixed at room temperature at a ratio of 9 parts BSA solution
to 1
part EDC solution. The solutions were stirred for set periods to allow the
viscosity to
build and then the reaction was quenched by diluting by four volumes of water
and
adjusting the pH of the resulting solution to 10. The resulting solution was
purified by
exhaustive dialysis and lyophillized. The resulting products were formulated
at the
concentrations described above and viscosity was measured using a Brookfield
cone
and plate viscometer (spindle number S52, 25° C, 20PRM).
The following table outlines the results:
Sample Description Viscosity (cps) 1
Control ( normal albumin) 427
Reaction time: 20 minutes 970
Reaction Time: 24 minutes 1300
Reaction Time: 32 minutes 2900
INCORPORATION BY REFERENCE
The disclosures of the following patents and patent applications are
incorporated
herein by reference: US Patent Nos. 5,219,895, 5,354,336, and 5,874,537,
issued on
2o June 15, 1993, October 11, 1994, and February 23, 1999, respectively; USSN
09/180,687 based on PCT/US97/08124 filed on May 14, 1997; USSN 60/090,609
filed
on June 23, 1999; PCT/US99/14232 filed on June 23, 1999 designating the US;
and
60/171,859 filed on December 22, 1999.
