U.S. patent application number 11/201606 was filed with the patent office on 2006-02-09 for methods for treating body tissue.
Invention is credited to Joseph Cappello, Erwin R. Stedronsky.
Application Number | 20060029638 11/201606 |
Document ID | / |
Family ID | 23729204 |
Filed Date | 2006-02-09 |
United States Patent
Application |
20060029638 |
Kind Code |
A1 |
Stedronsky; Erwin R. ; et
al. |
February 9, 2006 |
Methods for treating body tissue
Abstract
Methods of treating body tissue including repairing defects in
body tissue as well as augmenting body tissue. Body tissue defects
are repaired by injecting a polymeric adhesive composition through
an injector into the region of the defect and allowing the adhesive
composition to cure to repair the defect or to form an implant that
adheres to at least one surface tissue in the region of the defect.
Body tissue is augmented by filling a defect void with a polymeric
adhesive composition and allowing it to cure.
Inventors: |
Stedronsky; Erwin R.; (San
Clemente, CA) ; Cappello; Joseph; (San Diego,
CA) |
Correspondence
Address: |
DORSEY & WHITNEY LLP
555 CALIFORNIA STREET, SUITE 1000
SUITE 1000
SAN FRANCISCO
CA
94104
US
|
Family ID: |
23729204 |
Appl. No.: |
11/201606 |
Filed: |
August 10, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10117931 |
Apr 5, 2002 |
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11201606 |
Aug 10, 2005 |
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09451206 |
Nov 29, 1999 |
6423333 |
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10117931 |
Apr 5, 2002 |
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08642246 |
May 2, 1996 |
6033654 |
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09451206 |
Nov 29, 1999 |
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08435641 |
May 5, 1995 |
5817303 |
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08642246 |
May 2, 1996 |
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Current U.S.
Class: |
424/423 ;
604/500 |
Current CPC
Class: |
A61L 24/106 20130101;
A61P 17/02 20180101; A61K 38/00 20130101; C07K 14/001 20130101;
A61P 17/00 20180101; A61L 24/108 20130101; A61P 43/00 20180101;
A61L 27/227 20130101; Y10S 530/81 20130101; A61P 41/00
20180101 |
Class at
Publication: |
424/423 ;
604/500 |
International
Class: |
A61F 2/00 20060101
A61F002/00; A61M 31/00 20060101 A61M031/00 |
Claims
1: A method of repairing a defect in a body tissue, the method
comprising: positioning an injector proximate to a defect in a body
tissue; injecting a curable polymeric adhesive composition through
the injector into a region of the defect; and allowing the adhesive
composition to cure at the site of the defect, thereby repairing at
least a portion of the defect.
2: The method of claim 1, wherein the curable polymeric adhesive
composition repairs at least a portion of the defect by adhering to
tissue during curing.
3: The method of claim 1, wherein the curable polymeric adhesive
composition comprises a curable polymer and a chemical
crosslinker.
4: The method of claim 3, wherein the chemical crosslinker is
pre-mixed with the curable polymer before the injecting step.
5: The method of claim 1, further comprising injecting a chemical
crosslinker into a region of the defect.
6: The method of claim 3, wherein the chemical crosslinker is
selected from the group consisting of dialdehydes, diisocyanates,
acid anhydrides, diamines and combinations thereof.
7: The method of claim 3, wherein the chemical crosslinker is
hexamethylene diisocyanate (HMDI).
8: The method of claim 1, wherein the curable polymeric adhesive
composition comprises a curable protein copolymer solution.
9: The method of claim 1, wherein the curable polymeric adhesive
composition comprises repeating blocks of amino acid sequence.
10: The method of claim 1, wherein the curable polymeric adhesive
composition comprises a network of polymer chains including
repeating elastin-like and fibroin-like sequences.
11: The method of claim 10, wherein the repeating fibroin-like
sequences of the polymer chains of the curable polymeric adhesive
composition comprise the sequence: GAGAGS (SEQ ID NO:1).
12: The method of claim 10, wherein the repeating elastin-like
sequences of the polymer chains of the curable polymeric adhesive
composition comprise the sequence: GVGVP (SEQ ID NO:2).
13: The method of claim 10, wherein the repeating elastin-like
sequences of the polymer chains of the curable polymeric adhesive
composition comprise the sequence: GKGVP (SEQ ID NO:3), wherein the
K serves as a crosslinking site.
14: The method of claim 10, wherein the curable polymeric adhesive
composition is cured by chemically crosslinking the elastin-like
sequences.
15: The method of claim 1, wherein the polymeric adhesive
composition has a lap shear tensile strength of at least about 250
g/cm.sup.2 when cured.
16: The method of claim 15, wherein the polymeric adhesive
composition has said lap shear tensile strength of at least about
300 g/cm.sup.2 within a cure time of about 5 to about 30
minutes.
17: The method of claim 1, wherein the polymeric adhesive
composition has a lap shear tensile strength of about 100
g/cm.sup.2 to about 4000 g/cm.sup.2 when cured.
18: The method of claim 1, wherein the defect is a hole or void in
the body tissue.
19: The method of claim 1, wherein the curable polymeric adhesive
composition further comprises a drug.
20: The method of claim 1, wherein the adhesive composition is
bioresorbable.
21: The method of claim 1, wherein the injector is selected from
the group consisting of syringe, catheter and cannula.
22: The method of claim 1, wherein said injection is done to
augment tissue mass.
23: A method of augmenting body tissue, comprising: filling a
defect void in body tissue with a curable polymeric adhesive
composition; and allowing the adhesive composition to cure, thereby
augmenting the body tissue.
24: The method of claim 23, wherein the curable polymeric adhesive
composition comprises a curable polymer and a chemical
crosslinker.
25: The method of claim 24, wherein the chemical crosslinker is
pre-mixed with the curable polymer before the filling step.
26: The method of claim 23, further comprising applying a chemical
crosslinker to the defect void.
27: The method of claim 24, wherein the chemical crosslinker is a
diisocyanate.
28: The method of claim 23, wherein the curable polymeric adhesive
composition comprises a curable protein copolymer solution.
29: The method of claim 23, wherein the curable polymeric adhesive
composition comprises repeating blocks of amino acid sequence.
30: The method of claim 23, wherein the curable polymeric adhesive
composition comprises a network of repeating polymer chains
including elastin-like and fibroin-like sequences.
31: The method of claim 30, wherein the curable polymeric adhesive
composition is cured by chemically crosslinking the elastin-like
sequences.
32: The method of claim 23, wherein the polymeric adhesive
composition has a lap shear tensile strength of at least about 250
g/cm.sup.2 when cured.
33: The method of claim 32, wherein the polymeric adhesive
composition has said lap shear tensile strength of at least about
300 g/cm.sup.2 within a cure time of about 5 to about 30
minutes.
34: The method of claim 23, wherein the polymeric adhesive
composition has a lap shear tensile strength of about 100
g/cm.sup.2 to about 4000 g/cm.sup.2 when cured.
35: A method of repairing a defect in a body tissue, the method
comprising: positioning an injector proximate to a defect in a body
tissue; injecting a curable polymeric adhesive composition through
the injector into a region of the defect; and allowing the adhesive
composition to form an implant adherent to at least one tissue
surface in the region of the defect, thereby repairing at least a
portion of the defect.
36: The method of claim 35, wherein the implant is a hydrophilic
implant.
37: The method of claim 35, wherein the curable polymer polymeric
adhesive composition comprises a curable polymer and a chemical
crosslinker.
38: The method of claim 37, wherein the chemical crosslinker is
pre-mixed with the curable polymer before the injecting step.
39: The method of claim 35, further comprising injecting a chemical
crosslinker into a region of the defect.
40: The method of claim 35, wherein the curable polymeric adhesive
composition comprises a curable protein copolymer solution.
41: The method of claim 35, wherein the curable polymeric adhesive
composition comprises repeating blocks of amino acid sequence.
42: The method of claim 35, wherein the curable polymeric adhesive
composition comprises a network of polymer chains including
repeating elastin-like and fibroin-like sequences.
43: The method of claim 35, wherein the curable polymeric adhesive
composition comprises an additive.
44: The method of claim 43, wherein the additive includes an
extender selected from the group of polymers consisting of
synthetic polymers and naturally occurring polymers.
45: The method of claim 44, wherein the extender comprises a
naturally occurring polymer including hyaluronic acid.
46: The method of claim 44, wherein the extender comprises a
synthetic polymer including polyethylene glycol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
10/117,931 filed Apr. 5, 2002, which is a continuation of
application Ser. No. 09/451,206 filed Nov. 29, 1999, now U.S. Pat.
No. 6,423,333, which is a continuation of application Ser. No.
08/642,246 filed May 2, 1996, now U.S. Pat. No. 6,033,654, which is
a continuation-in-part of application Ser. No. 08/435,641 filed May
5, 1995, now U.S. Pat. No. 5,817,303, all of which are incorporated
herein by reference.
INTRODUCTION
TECHNICAL FIELD
[0002] The field of this invention is physiologically acceptable
compositions for use as tissue adhesives and sealants.
BACKGROUND
[0003] In many situations, there is a need to bond separated
tissues. Sutures and staples are effective and well established
wound closure devices. However, there are surgical procedures where
classical repair procedures are unsatisfactory, limited to highly
trained specialists (e.g. microsurgery), or not applicable due to
tissue or organ fragility, inaccessibility (e.g. endoscopy
procedures), or fluid loss, including capillary "weeping". Tissue
adhesives and sealants have been developed to meet these needs.
They may be used to seal or reinforce wounds that have been sutured
or stapled, as well as finding independent use. The leading
commercial products are fibrin glues and cyanoacrylates. However,
both products have significant limitations which have prevented
their widespread use.
[0004] Cyanoacrylates are mainly used for cutaneous wound closure
in facial and reconstructive surgery. The appeal of cyanoacrylates
is their speed of bonding, which is almost immediate, and its great
bond strength. However, its speed of bonding can be a disadvantage,
since glued tissue must be cut again in order to reshape it to the
desired conformation. Additionally, it can only be used on dry
substrates since its mode of action is through a mechanical
interlock, limiting its use as a sealant, and it is relatively
inflexible compared to surrounding tissue. Cyanoacrylates are also
known to be toxic to some tissues and although it is not considered
to be biodegradable, potential degradation products are suspected
to be carcinogenic.
[0005] Fibrin glues comprising blood-derived fibrinogen, factor
XIII and thrombin function primarily as a sealant and hemostat and
have been used in many different surgical procedures within the
body. They have been shown to be non-toxic, biocompatible and
biodegradable. They are able to control excessive bleeding and
decrease fibrosis. However, tissues bonded with fibrin cannot be
subjected to even moderate tensile stress without rupturing the
bond. It takes about three to ten minutes for an initial bond to
develop, but requires about 30 minutes to several hours for full
strength to develop. Depending upon the application, the product
may also resorb too quickly. Use of recombinantly produced
fibrinogen, factor XIII, thrombin and related components (e.g.
fibrin, activated factor XIII) has not been demonstrated to improve
the setting time or strength of fibrin glues. Fibrin glues derived
from heterologous, human and animal, serum may provoke undesirable
immune responses, and expose the patient to the potential risk of
viral infection. Autologous fibrin glues may be impractical to
obtain and use and may compromise patient safety.
[0006] There is, therefore, substantial interest in developing
products which have the biocompatibility of fibrin glues, but which
set more quickly and have enhanced strength. These products should
be readily available, desirably from other than natural sources, be
easily administered and capable of resorption over time.
Relevant Literature
[0007] Tissue adhesives are described in: Tissue Adhesives in
Surgery, Matsumoto, T., Medical Examination Publishing Co., Inc.
1972 and Sierra, D. H., J. Biomat. App. 7:309-352, 1993. Methods of
preparation of protein polymers having blocks of repetitive units
are described in U.S. Pat. No. 5,243,038 and EPA 89.913054.3.
SUMMARY OF THE INVENTION
[0008] Polymeric compositions and methods for their use are
provided, where the polymeric compositions are capable of in situ
chemical crosslinking to provide novel crosslinked polymeric
products, which have good mechanical and biological properties, as
exemplified by strong adherent bonds to tissue. The compositions
can be used in a variety of applications related to their physical,
chemical and biological properties, to bond to separated tissue to
provide at least one of the characterstics of a stable, flexible,
resorbable bond.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0009] The subject compositions comprise high molecular weight
recombinant polymers having one or a combination of repeating units
related to naturally occurring structural proteins. Of particular
interest are the repeating units of fibroin, elastin, collagen, and
keratin, particularly collagen and combinations of fibroin and
elastin. The polymers have functional groups which can be
chemically crosslinked under physiological conditions with
physiologically acceptable crosslinkers, so as to form a
composition which has strong adherent properties to a variety of
substrates, has strong mechanical properties in maintaining the
joint between the substrates, and can be formulated to have good
resorption properties.
[0010] Of particular interest, the subject compositions provide
strongly adherent bonds to tissue to maintain separated tissue in a
contiguous spatial relationship. The subject compositions may also
be employed as sealants, where the compositions may serve to fill a
defect void in tissue, to augment tissue mass or bond synthetic
materials to tissues. The subject compositions may also serve as
depots in vivo by being mixed with a drug composition, either when
used as an adhesive for bonding tissue together or for other
bonding or solely as a slow release source of the drug.
[0011] The functionalities for crosslinking may be all the same or
combinations of functionalities and may include the functionalities
of naturally occurring amino acids, such as amino, e.g. lysine,
carboxyl, e.g. aspartate and glutamate, guanidine, e.g. arginine,
hydroxyl, e.g. serine and threonine, and thiol, e.g. cysteine.
Preferably, the functionality is amino (including guanidine).
[0012] The polymers will have molecular weights of at least about
15 kD, generally at least about 30 kD, preferably at least about 50
kD and usually not more than 250 kD, more usually not more than
about 150 kD. The polymers will have at least two functionalities,
more usually at least about four functionalities, generally having
an equivalent weight per functionality in the range of about 1 kD
to 40 kD, more usually in the range of about 3 kD to 20 kD,
preferably in the range of about RD to 10 kD, there being at least
3, usually at least 6, functionalities available for crosslinking.
If desired, one may use mixtures of polymers, where the polymers
have combinations of functionalities or have different
functionalities present e.g. carboxyl and amino, thiol and
aldehyde, hydroxyl and amino, etc. Thus, depending upon the
functionalities and the crosslinking agent, one can form amides,
imines, ureas, esters, ethers, urethanes, thioethers, disulfides,
and the like.
[0013] The individual units in the polymer may be selected from
fibroin, GAGAGS (SEQ ID NO:01); elastin, GVGVP (SEQ ID NO:02);
collagen GXX, where the X's may be the same or different, and at
least 10 number % and not more than 60 number % of the X's are
proline, and keratin, AKLK/ELAE (SEQ ID NO:3). The desired
functionality may be substituted for one of the amino acids of an
individual unit or be present as an individual amino acid or part
of an intervening group of not more than about 30 amino acids,
usually not more than about 16 amino acids. In the former case,
within a block of repeats, one or more of the repeats is modified
to introduce a crosslinking functionality which would otherwise not
normally be present. Thus a valine may be replaced with a lysine, a
glycine with an arginine, an alanine with a serine, and the like.
In the latter case, there would be an intervening functionality
between a block of repeat units, where the number of intervening
functionalities would be based on the ranges indicated
previously.
[0014] Of particular interest are copolymers, either block or
random, preferably block, where in the case of elastin and fibroin,
the ratio of elastin units to fibroin units is in the range of
16-1:1, preferably 8-1:1, where blocks may have different ratios.
Normally, in block copolymers, each block will have at least two
units and not more than about 32 units, usually not more than about
24 units. By substituting an amino acid in the unit with an amino
acid having the appropriate functionality, one can provide for the
appropriate number of functionalities present in the polymer or
employ intervening groups between blocks.
[0015] The individual amino acid repeat units will have from about
3 to 30 amino acids, usually 3 to 25 amino acids, more usually 3 to
15 amino acids, particularly 3 to 12 amino acids, more particularly
about 3 to 9 amino acids. At least 40 weight %, usually at least 50
weight %, more usually at least 70 weight %, of the protein polymer
will be composed of segments of repetitive units containing at
least 2 identical contiguous repetitive units. Generally repeat
blocks will comprise at least 2, 4, 7 or 8 units, and combinations
thereof, where copolymers are employed, where the unit which is
modified with the crosslinking functionality is counted as a
unit.
[0016] While for the most part, the polymers of the subject
invention will have the active functionality of a naturally
occurring amino acid in the chain of the polymer, if desired,
pendent groups may be employed to provide the desired
functionalities. For example, carboxyl groups may be reacted with
polyamines so as to exchange a carboxyl functionality for a single
amino or plurality of amino groups. An amino group may be
substituted with a polycarboxylic acid, so that the amino group
will be replaced with a plurality of carboxylic groups. A thiol may
be replaced with an aldehyde, by reaction with an aldehydic olefin,
e.g. acrolein, so as to provide for an aldehyde functionality.
Other functionalities which may be introduced, if desired, include
phosphate esters, activated olefins, e.g. maleimido,
thioisocyanato, and the like. The functionalities may be greatly
varied from those which naturally occur to provide opportunities
for crosslinking. In some instances, this may be desirable to
increase the number of functionalities per unit molecular weight,
while not increasing the number of functionalities along the chain,
for replacing one functionality with another, e.g. thiol with
aldehyde, allowing for greater variation in the choice of
crosslinking agent.
[0017] The crosslinking agent will normally be difunctional, where
the functionalities may be the same or different, although higher
functionality may be present, usually not exceeding four
functionalities. Depending upon the particular functionalities
available on the polymers, various crosslinking agents may be
employed. The crosslinking agents will usually be at least about
three carbon atoms and not more than about 50 carbon atoms,
generally ranging from about 3 to 30 carbon atoms, more usually
from about 3 to 16 carbon atoms. The chain joining the two
functionalities will be at least one atom and not more than about
100 atoms, usually not more than about 60 atoms, preferably not
more than about 40 atoms, particularly not more than about 20
atoms, `where the atoms may be carbon, oxygen, nitrogen, sulfur,
phosphorous, or the like. The linking group may be aliphatically
saturated or unsaturated, preferably aliphatic, and may include
such functionalities as oxy, ester, amide, thioether, amino, and
phosphorous ester. The crosslinking group may be hydrophobic or
hydrophilic.
[0018] Various reactive functionalities may be employed, such as
aldehyde, isocyanate, mixed carboxylic acid anhydride, e.g.
ethoxycarbonyl anhydride, activated olefin, activated halo, amino,
and the like. By appropriate choice of the functionalities on the
protein polymer, and the crosslinking agent, rate of reaction and
degree of cross linking can be controlled.
[0019] Various crosslinking agents may be employed, particularly
those which have been used previously and have been found to be
physiologically acceptable. Crosslinking agents which may be used
include dialdehydes, such as glutaraldehyde, activated diolefins,
diisocyanates such as, tetramethylene diisocyanate, hexamethylene
diisocyanate, octamethylene diisocyanate, acid anhydrides, such as
succinic acid dianhydride, ethylene diamine tetraacetic acid
dianhydride, diamines, such as hexamethylene diamine,
cyclo(L-lysyl-L-lysine), etc. The crosslinking agent may also
contain unsymmetrical functionalities, for example, activated
olefin aldehydes, e.g. acrolein and quinoid aldehydes, activated
halocarboxylic acid anhydride, and the like. The crosslinking
agents will usually be commercially available or may be readily
synthesized in accordance with conventional ways, either prior to
application of the adhesive or by synthesis in situ.
[0020] In some instances it may be desirable to react a
physiologically acceptable second compound, which serves as a
modifying unit, with a polyfunctional, usually bifunctional,
compound to change the nature of the crosslinking. The addition of
the second compound may be to enhance the rate of crosslinking,
change the solubility properties of the crosslinker, enhance or
reduce the strength of the crosslinked polymer, enhance or reduce
the resorption rate, or provide for other physical, chemical or
biological properties of interest. The polyfunctional second
compound may be reacted with the crosslinking compound prior to
reaction with the protein or concurrently with the reaction with
the protein. Where the reaction is prior, the resulting
crosslinking product will be physiologically acceptable and when
concurrent, the polyfunctional second compound, the crosslinking
compound and the resulting crosslinking product will be
physiologically acceptable, when used in vivo. The ratio of the
polyfunctional second compound to the crosslinking compound will
generally be in the range of about 0.1-2:1, more usually in the
range of about 0.11:1, depending on the reactivity of the
polyfunctional second compound when the polyfunctional second
compound and crosslinking agent are brought together, the number of
crosslinks desired in the final protein composition, the size of
the bridge between protein molecules, and the like.
[0021] The nature of the polyfunctional second compound may vary
widely. The functional groups present may be the same or different
from the functional groups present on the polymer, but will be
reactive with the functionalities of the crosslinking compound. For
example, the polyfunctional second compound may have amino and/or
hydroxyl groups, where the protein has amino or hydroxyl
functionalities. By employing a diisocyanate with a diol,
diurethanes will be produced. Thus, the chain crosslinking the
proteins will comprise 2 or more urethanes.
[0022] In many instances, the polyfunctional second compound will
include an internal functionality that does not participate in the
reaction, but provides various other characteristics to the
crosslinking agent or the crosslinked protein product.
Characteristics of interest include hydrophilicity, hydrolytic
instability, sensitivity to enzymatic degradation,
biocompatibility, shear strength, and the like. For the most part
internal functionalities will comprise oxygen, sulfur and nitrogen
atoms, such as ethers, carboxylic acid esters, including urethanes,
amino groups, amides, ketones, dithiols and the like. To enhance
the rate of resorption, ester groups are of interest, while to
enhance hydrophilicity, the same groups maybe employed as well as
ethers, such as polyoxyalkylene groups.
[0023] The polyfunctional second compound will generally have at
least 2 carbon atoms and not more than 50 carbon atoms, usually not
more than about 30 carbon atoms, desirably having not more than
about 16 carbon atoms per heteroatom. Naturally occurring or
synthetic bifunctional compounds may be employed. Illustrative
compounds include lysine, arginine, di-(2'-aminoethyl) malonate,
citrate, lysyl lysine, 2'-aminoethyl glycinate, O,N-diglycinyl
ethanolamine, diethylene glycol diglycinate, cystine, and the like.
To provide terminal amino groups, various low molecular weight
amino acids may be used, particularly glycine and alanine bonded to
an intervening difunctional compound, such as ethylene glycol,
diethylene glycol, and tetraethylene glycol, propanediol,
1,4-butyn-2-diol, ascorbic acid, etc.
[0024] The subject compositions may be prepared prior to the use of
the adhesive by combining the protein polymer and the crosslinking
agent, where one or both may have extenders. The two compositions
may be readily mixed in accordance with conventional ways, for
example, using syringes which can inject the ingredients into a
central reactor and the mixture mixed by drawing the mixture back
into the syringes and moving the mixture back and forth.
Alternatively, the two compositions may be dispensed simultaneously
at the site of application. In some instances it may be desirable
to allow the crosslinking agent to partially react with the protein
prior to adding the polyfunctional second compound. Alternatively,
one may mix the polyfunctional second compound with the protein
prior to mixing with the crosslinking agent.
[0025] Usually, the polymer will be available as a dispersion or
solution, particularly aqueous, generally the concentration of the
protein polymer being in the range of about 50 mg to 1 g/ml, more
usually from about 100 to 800 mg/ml. The solution may be buffered
at a pH which enhances or retards the rate of crosslinking. Usually
the pH will be in the range of about 2 to 12, more usually 8 to 11.
Various buffers may be used, such as phosphate, borate, carbonate,
etc. The cation can have an effect on the nature of the product,
and to that extent, the alkali metals potassium and sodium, are
preferred. The protein composition will generally be about 5 to 40,
more usually from about 5 to 20, preferably from about 10 to 20
weight %, to provide for a composition which may be readily
handled, will set up within the desired time limit, and the like.
The buffer concentration will generally be in the range of about 50
to 500 mM. Other agents may be present in the protein solution,
such as stabilizers, surfactants, and the like. If the
polyfunctional second compound is present, its concentration will
be determined in accordance with its ratio to the crosslinking
agent and the polymer.
[0026] The ratio of crosslinking agent to polymer will vary widely,
depending upon the crosslinking agent, the number of
functionalities present on the polymer, the desired rate of curing,
and the like. Generally, the weight ratio of polymer to
crosslinking agent will be at least about 1:1 and not greater than
about 100:1, usually not greater than about 50:1, generally being
in the range of about 2 to 50:1, but in some instances may not be
more than 30:1. The equivalent ratio of protein to crosslinking
agent will generally be in the range of about 0.1-1:3, more usually
in the range of about 0.5-2:2. Considerations in selecting the
protein-crosslinking agent equivalent ratio will be the rate of
setup, reactivity of the crosslinking agent, relative solubility of
the crosslinking agent in the mixture, physiological properties of
the crosslinking agent, desired degree of stability of the
crosslinked product, and the like.
[0027] If desired, various extenders or extending agents may be
used, particularly naturally occurring proteins. Such extenders
will usually not exceed 50 weight percent of the composition,
generally not exceeding about 20 weight percent, more usually not
exceeding about 10 weight percent. Extenders which may be employed
include, but are not limited to: synthetic polymers, both addition
and condensation polymers, both protein and non-protein, such as
polylactides, polyglycolides, polyanhydrides, polyorthoesters,
polyvinyl compounds, polyolefins, polyacrylates, polyethylene
glycol, polyesters, polyvinyl alcohol, polyethers, copolymers and
derivatives thereof, and naturally occurring polymers, such as
proteins and nonproteins, including collagen, fibrinogen,
fibronectin, laminin, keratin, chitosan, heparin, dextran,
alginates, cellulose, glycosoaminoglycans, hyaluronic acid,
polysaccharides, derivatives thereof, and the like. The extenders
may modulate the setting time and provide for desirable physical or
physiological properties of the adhesive.
[0028] Based on the lap shear tensile strength test described in
the experimental section, within 30 minutes, usually within 15
minutes, more usually within 5 minutes, the lap shear tensile
strength will be at least 100, preferably at least about 250, more
preferably at least about 300, usually not exceeding about 4000,
more usually not exceeding about 3000 g/cm.sup.2.
[0029] The subject compositions may be applied to the tissue in any
convenient way, for example by using a syringe, catheter, cannula,
manually applying the composition, spraying or the like. The
subject compositions may be applied to the tissue prior to or
during the time the tissue segments are held in contiguous
relationship. The subject compositions will rapidly develop
substantial shear strength, so as to maintain the tissue in
proximity. In some situations there will be an interest in having
the composition decompose after some reasonable period of time,
usually at least one week and generally not more than about four
weeks.
[0030] Tissues of interest include vascular vessels such as an
artery, vein or capillary, muscel, nerve, organs, e.g. liver,
spleen, etc., lung, dura, colon, and the like.
[0031] In addition to their use as adhesives, the subject
compositions may be used to seal or fill defects, e.g. voids or
holes, in tissue, and therefore find use as sealants. Thus, the
compositions may serve to stop or staunch the flow of fluid, e.g.
blood, through ruptured vessels, e.g. arteries, veins, capillaries
and the like. In using the subject compositions as sealants, the
composition will be applied, as described above, at the site of the
defect, whereby it will set and seal the defect. The compositions
may be injected into normal or abnormal tissues to augment the
tissue mass, e.g. dermis.
[0032] The subject compositions may also find use in the formation
of articles of manufacture, by themselves or in combination with
other materials. In one application, articles may be produced for
use internally to a mammalian host, where there is an interest in
biocompatibility, reabsorption rate, ability to vascularize, tissue
adhesive and/or bonding capability, and the like. Various articles
can be prepared, such as gels, films, threads, coatings, formed
objects such as pins and screws, or injectable compositions which
are flowable, where the injectable composition may set up and bond
or seal tissues, form a depot for a drug, augment tissue or be a
filler, coating or the like. The formed objects may be prepared in
accordance with conventional ways, such as molding, extrusion,
precipitation from a solvent, solvent evaporation, and the like.
The flowable depot can be obtained by using a molecular dispersion,
fine particles in a medium saturated with a polymer, using a melt,
where the melting temperature may be achieved by adding
physiologically acceptable additives, and the like.
[0033] The articles may find use in a variety of situations
associated with the implantation of the article into a mammalian
host or the application of the article to the surface of a
mammalian host, e.g. wound healing, burn dressing, etc. Those
situations, where the performance of the articles may be retained
for a predetermined time and replaced by natural materials through
natural processes, desirably employ materials which will be
resorbed after having fulfilled their function in maintaining their
role until the natural process has reestablished a natural
structure. Thus, the compositions may find use in holding tissue
together, covering tissue, encapsulating cells for organs,
providing a coating that cells can invade and replace the
composition with natural composition, e.g., bone, soft tissues and
the like.
[0034] To enhance the rate of curing of the polymeric composition,
the composition may be partially prepolymerized. When
prepolymerized, the polymer will usually have at least about 3% of
the total number of crosslinks and not more than about 75% of the
total number of crosslinks, as compared to completion of the
crosslinking action. The number of crosslinks should allow the
resulting product to be workable and provide sufficient time prior
to set up for it to be manipulated and used. Alternatively, one may
react the functional groups with an excess of the crosslinking
reagent, so that the effect is to substitute the functionality of
the protein with the functionality of the crosslinking agent. The
protein with the substituted functionality may then be used to
crosslink protein with the original functionality or with a
polyfunctional second compound.
[0035] The subject compositions may also be used as depots to
provide for a relatively uniform release of a physiologically
active product, e.g., a drug. The drug may be mixed with a subject
composition at an appropriate concentration prior to crosslinking.
As the crosslinked polymer is degraded, the drug will be released
due to diffusion as well as erosion of the external surface of the
depot. By controlling the form or shape of the depot, the degree of
crosslinking, the concentration of the drug and the like, a
physiologically therapeutic level of the drug may be maintained
over extended periods of time. The period required for absorption
can be as short as 0.5 day and may exceed 4, 6 or 8 weeks or more,
depending upon the particular composition and the application.
[0036] The protein polymer compositions may be prepared in
accordance with conventional ways. See, for example, U.S. Pat. No.
5,243,038, which disclosure is incorporated herein by reference.
Briefly, sequences may be synthesized comprising a plurality of
repeating units, where complementary sequences result in dsDNA
having overhangs. A series of dsDNA molecules may be prepared and
stepwise introduced into a cloning vector as the gene for the
protein is constructed. A monomer can be obtained in this way,
which may be sequenced to ensure that there have been no changes in
the sequence, followed by multimerization of the monomer, cloning
and expression. For further details, see the above indicated
patent.
[0037] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
Example 1
Methods
[0038] The construction of synthetic DNA and its use in large
polypeptide synthesis is described in U.S. Pat. No. 5,243,038;
PCT/US89/05016 and PCT/US92/09485, the disclosures of which are
herein incorporated by reference. Modifications to these methods
and additional methods used are described below.
[0039] 1. Use of Filters and Columns for DNA Purification
[0040] A. Ultrafree.RTM.-Probind filter unit ("Probind",
Millipore): the DNA containing solution was applied to the filter
unit and spun at 12,000 RPM for 30 seconds in a Sorvall Microspin
24S.
[0041] B. Microcon-30 filter (Amicon): the DNA containing solution
was washed by applying to the filter and exchanging twice with H2O
by spinning at 12,000 RPM for 6 minutes in a microfuge.
[0042] C. Bio-Spin 6 column ("Bio-Spin", BioRad): Salts and
glycerol were removed from the DNA solution by applying to the
column, previously equilibrated in TEAB (triethyl ammonium
bicarbonate pH 7.0), and spinning in a Sorvall RC5B centrifuge
using an HB4 rotor at 2,500 RPM for 4 minutes.
[0043] 2. Phosphatase Treatment of DNA
[0044] Phosphatase treatment of DNA was also performed by
resuspending ethanol precipitated DNA from the restriction enzyme
digest in 20 mM Tris-HCl pH 8.0, 10 MM MgCl.sub.2 to a final DNA
concentration of 20 .mu.g/ml. Shrimp Alkaline Phosphatase (SAP) was
added at 2 U/.mu.g of DNA and the mixture was incubated at
37.degree. C. for one hour, heat inactivated for 20 minutes at
65.degree. C. and then passed through a Probind filter and
subsequently a Bio-Spin column.
[0045] 3. Preparative Agarose Gel Electrophoresis
[0046] For agarose ligation, the buffer used was 1.times.TAE (50 mM
Tris-acetate, pH 7.8).
[0047] 4. Agarose DNA Ligation
[0048] The agarose was melted at 65.degree. C., the temperature was
then lowered to 37.degree. C. and ligation buffer (5.times.=100 mM
Tris-HCl, pH 7.5, 50 MM MgCl.sub.2, 50 mM DTT, 1 mM ATP) was added;
the tube was then placed at room temperature and ligase was added
(1000 units T4 DNA ligase (NEB)). The reaction volume was usually
50 .mu.l. The reaction was incubated at 15.degree. C. for 16-18
hours.
[0049] 5. Agarose DNA Purification Using an Ultrafree.RTM.-MC
Filter Unit
[0050] This procedure can be used for agarose slices up to 400
.mu.l in size. After agarose gel electrophoresis, the DNA is
visualized by ethidium bromide staining and the agarose block
containing the DNA band of interest is excised. The agarose is then
frozen at -20.degree. C. for 1 hour, then quickly thawed at
37.degree. C. for 5 minutes. The agarose is then thoroughly
macerated. The pieces are then transferred into the sample cup of
the filter unit and spun at 5,000.times.g in a standard microfuge
for 20 minutes. The agarose is then resuspended in 200 .mu.l of
Tris-EDTA, or other buffer, and incubated at room temperature for
30 minutes to allow for elution of additional DNA from the gel. The
mixture is then centrifuged for an additional 20 minutes at 10,000
RPM. The DNA is, at this point, in the filtrate tube separated from
the agarose fragments and ready for subsequent DNA
manipulations.
[0051] 6. Preparation of Antibody to Artificially Synthesized
Peptides
[0052] The same procedures were used as described in U.S. Pat. No.
5,243,038, PCT/US89/05016 and PCT/US92/09485.
[0053] 7. Immunoblotting of Proteins in Gels
[0054] An alternative to the .sup.125I-Protein A detection method
was used. This method relied on a chemiluminescent signal activated
by horseradish peroxidase (HRP). The chemiluminescent reagents are
readily available from several suppliers such as Amersham and
DuPont NEN. The western blot was prepared and blocked with BLOTTO.
A number of methods were used to introduce the HRP reporter enzyme
including, for example, a hapten/anti-hapten-HRP, a biotinylated
antibody/streptavidin-HRP, a secondary reporter such as a goat or
mouse anti-rabbit IgG-biotinylated/streptavidin-HRP, or a goat or
mouse-anti rabbit IgG-HRP. These reagents were bought from
different sources such as BioRad or Amersham and occasionally
biotinylated antibodies were prepared in our laboratory using
Biotin NHS from Vector Laboratories, Burlingame, Calif. (Cat.
#SP-1200) following the procedure accompanying the product. The
following is an example of a procedure used to detect the
expression of protein polymers.
[0055] The blot was placed in 15 ml of BLOTTO solution containing
biotinylated goat anti-rabbit IgG (BioRad) diluted in BLOTTO
(1:7500) and gently agitated for 2 hours at room temperature. The
filter was then washed for 30 minutes with 3 changes of TSA (50 mM
Tris-HCl pH 7.4, 0.9% NaCl, 0.2% sodium azide) and then for 5
minutes each in TBS with 0.1% TWEEN.RTM.20. The blot was then
incubated for 20 minutes at room temperature with gentle rotation,
in 20 ml of TBS (100 mM Tris Base, 150 mM NaCl, pH 7.5)
HRP-Streptavidin (Amersham) diluted 1:1000 in TBS with 0.1% Tween
20. The blot was then washed three times for 5 minutes each in TBS
with 0.3% Tween 20 and then three times for 5 minutes each in TBS
with 0.1% Tween 20. The blot was then incubated for 1 minute with
gentle agitation in 12 ml of development solutions #1 an #2
(Amersham) equally mixed. The blot was removed from the development
solution and autoradiographed.
[0056] 8. Protein Expression Analysis
[0057] An overnight culture which had been grown at 30.degree. C.
was used to inoculate 50 ml of LB media contained in a 250 ml
flask. Kanamycin was added at a final concentration of 50 .mu.g per
ml and the culture was incubated with agitation (200 rpm) at
30.degree. C. When the culture reached an OD.sub.600, of 0.8, 40 ml
were transferred to a new flask prewarmed at 42.degree. C. and
incubated at the same temperature for approximately 2 hours. The
cultures (30.degree. and 42.degree.) were chilled on ice and
OD.sub.600. was taken. Cells were collected by centrifugation and
then divided in 1.0 OD.sub.600 aliquots and used to perform western
analysis using the appropriate antibodies.
[0058] 9. Amino Acid Analysis
[0059] Amino acid derivatives were analyzed by reverse phase HPLC
using a Waters 600E system.
[0060] 10. Peptide Synthesis
[0061] Synthetic peptides were also prepared on a Rainin/Protein
Technologies PS3 FMOC peptide synthesizer. Both the synthesis and
cleavage were accomplished using the methods supplied by the
manufacturer in the instrument manual.
[0062] 11. In Vitro DNA Synthesis
[0063] The .beta.-cyanoethyl phosphoramidites, controlled-pore
glass columns and all synthesis reagents were obtained from Applied
Biosystems, Foster City, Calif. Synthetic oligonucleotides were
prepared by the phosphite triester method with an Applied
Biosystems Model 381A DNA synthesizer using a 10-fold excess of
protected phosphoramidites and 0.2 mole of nucleotide bound to the
synthesis support column. The chemistries used for synthesis are
the standard protocols recommended for use with the synthesizer and
have been described (Matteucci et al., J. Amer. Chem. Soc.,
103:3185-3319 (1981)). Deprotection and cleavage of the oligomers
from the solid support were performed according to standard
procedures as provided by Applied Biosystems. The repetitive yield
of the synthesis as measured by the optical density of the removed
protecting group as recommended by Applied Biosystems was greater
than 97.5%.
[0064] The crude oligonucleotide mixture was purified by
preparative gel electrophoresis as described by the Applied
Biosystems protocols in Evaluating and Isolating Synthetic
Oligonucleotides, 1992 (Formerly: User Bulletin 13, 1987). The
acrylamide gel concentration varied from 10 to 20% depending upon
the length of the oligomer. If necessary, the purified oligomer was
identified by UV shadowing, excised from the gel and extracted by
the crush and soak procedure (Smith, Methods in Enzymology,
65:371-379 (1980)).
[0065] For DNA synthesis of oligonucleotides longer then 100 bases,
the synthesis cycle was changed from the protocol recommended by
Applied Biosystems for the 381A DNA synthesizer. All the reagents
used were fresh. All the reagents were supplied by Applied
Biosystems except for the acetonitrile (Burdick and Jackson Cat
#017-4 with water content less then 0.001%) and the 2000 A pore
size column (Glen Research). Due to the length of the oligo,
interrupt pauses had to be inserted during the synthesis to allow
changing the reagent bottles that emptied during synthesis. This
interrupt pause was done at the cycle entry step and the pause was
kept as short as possible. The washes after detritylation by TCA,
through the beginning of each synthesis cycle, were increased from
about 2.times. to 3.times. over the recommended time. The time
allocated for the capping was also increased to limit truncated
failure sequences. After the synthesis the deprotection was done at
55.degree. C. for 6 hours. After desalting the synthesized DNA was
amplified using PCR.
[0066] 12. Sequencing of DNA
[0067] Storage and analysis of data utilized software from DNA
Strider, DNA Inspection Ile or DNAid for Apple Macintosh personal
computer.
[0068] 13. Dideoxy DNA Sequencing of Double Stranded Plasmid
DNA
[0069] As described in U.S. Pat. No. 5,243,038, plasmid DNA was
prepared on a small scale. Primers were synthesized using a DNA
synthesizer and were annealed to the plasmid DNA following the
procedure described for M13 sequencing. The sequencing reactions
were done using Sequenase (United States Biochemicals) and the
conditions were as recommended by the supplier. All sequences were
run on polyacrylamide gels.
[0070] 14. PCR Amplification
[0071] The PCR reaction was performed in a 100 .mu.l volume in a
Perkin Elmer thinwalled Gene Amps" reaction tube. Approximately 1
.mu.M of each primer DNA was added to 1.times.PCR buffer (supplied
by Perkin Elmer as 10.times. solution), 200 .mu.M of each dNT, 5 U
AmpliTaq, and several concentrations of the target DNA.
Amplification was performed in a Perkin Elmer DNA Thermal cycler
model 480 for 30 cycles with the following step cycles of 12
minutes each: 95.degree. C., 62.degree. C., and 72.degree. C.
Aliquots from the different reactions were analyzed by agarose gel
electrophoresis using 1.5% low melting point agarose in
0.5.times.TA buffer. The reaction mixtures that gave the desired
band were pooled and spun through a Probind filter to remove the
AmpliTaq enzyme, then a Microcon-30 filter and a Bio-Spin column.
The DNA was then concentrated in vacuo.
[0072] 15. Diamine Synthesis
[0073] 2-Aminoethyl Glycinate:
[0074] Concentrated sulfuric acid (9.90 g, 0.101 mole) was diluted
into 10 ml, of water. Glycine (7.50 g, 0.100 mole), 2-aminoethanol
(6.10 g, 0.100 mole) and the diluted sulfuric acid were placed in a
250 mL, 3-neck, round bottom flask fitted with a stopper, a
mechanical stirrer, a heating mantle, and a Dean-Stark water trap.
The contents of the apparatus were protected from atmospheric
moisture with a nitrogen blanket. Toluene (100 mL) was added and
the contents of the apparatus refluxed until no further evolution
of water occurred. The apparatus was disassembled and the toluene
was decanted before the flask was connected to a vacuum line to
strip off toluene entrapped in the reaction mass. The product was
used without further purification. The FTIR spectrum of the
reaction product shows strong carbonyl adsorptions at 736 cm.sup.-1
and 1672 cm.sup.-1. The reaction product is estimated to be an
approximately 4:1 mixture of 2-aminoethyl glycinate and
N,O-diglycyl ethanolamine by comparison with the spectra of ethyl
glycinate hydrochloride and glycyl glycine hydrochloride.
[0075] Cholinyl Lysinate:
[0076] Concentrated sulfuric acid (11.40 g, 0.120 mole) was diluted
into water (10 mL). Lysine monohydrochloride (13.69 g, 0.075 mole),
choline chloride (10.47 g, 0.075 mole), and the diluted sulfuric
acid was placed into a 250 mL 1-neck round bottom flask fitted with
a magnetic stirring bar, heated in a thermostatted oil bath, and
connected to a vacuum line. Vacuum was gradually applied to the
flask and then heat gradually increased in order to remove
volatiles into a trap cooled in liquid nitrogen. The reaction was
terminated when the bath temperature reached 110.degree. C. and the
pressure decreased to 0.024 mm-Hg. The product is homogeneous by
thin layer chromatography (cellulose, acetic
acid/acetonitrile/water 5:65:30 v/v/v, developed with ninhydrin
spray, Rf=0.25). The product was used directly.
[0077] 1,3-Propanediyl Diglycinate:
[0078] Concentrated sulfuric acid (10.78 g, 0.110 mole) was diluted
into water (10 mL). 1,3-Propanediol (7.61 g, 0.100 mole), glycine
(15.0 g, 0.200 mole), and the diluted sulfuric acid were placed in
a 250 mL, 3-neck, round bottom flask fitted with a stopper, a
mechanical stirrer, a thermostatted oil bath, and a Dean-Stark
water trap. The contents of the apparatus were protected from
atmospheric moisture with a nitrogen blanket. Toluene (100 mL) was
added, the oil bath thermostatted at 130.degree. C., and the
contents of the apparatus refluxed until no further evolution of
water occurred (ca. 9 hours). The apparatus was disassembled and
the toluene decanted. The reaction mass was dissolved in water (29
mL) by stirring at room temperature. Upon cooling to -20.degree. C.
for 18 hours, the solution deposits fine white crystals which are
removed by filtration. The filtrate is poured into methanol (250
mL), precooled to 3.degree. C., to deposit a semi-solid paste. The
supernatant was decanted, and the paste triturated in several
portions in a mortar and pestle with methanol (50 mL) to yield a
granular solid (12.55 g). A sample of solid (9.19 g) was boiled
with methanol (18.4 mL) plus water (7.9 mL), filtered while hot,
and allowed to crystallize at 4.degree. C. for 18 hours. The
precipitate was filtered while cold, compacted on the funnel under
a dam, rinsed with methanol, acetone, and air dried, to yield a
white crystalline solid (6.89 g). A sample of this material was
titrated with aqueous KOH using a pH meter. The apparent equivalent
weight per amine is 201 g/mole; an acidic contaminant with an
apparent equivalent weight of 601 g/mole was also present. The FTIR
shows a single carbonyl absorption at 1744 cm.sup.-1
[0079] 16. Fermentation Conditions
[0080] The fermentors used for the expression of protein polymers
were usually a 15 L MBR, 10 L working volume, or a 13 L Braun
Biostat E, 8.5 L working volume. The choice of the fermentor and
its size is not critical. Any media used for the growth of E. coli
can be used. The nitrogen source ranged from NZAmine to inorganic
salts and the carbon source generally used was glycerol or glucose.
All fermentations were done with the appropriate selection
conditions imposed by the plasmid requirements (e.g. kanamycin,
ampicillin, etc.). The fermentation method used to express protein
polymers in E. coli was the fed-batch method. This is the preferred
method for the fermentation of recombinant organisms even if other
methods can be used.
[0081] The fed-batch method exploits the stage of cell growth where
the organisms make a transition from exponential to stationary
phase. This transition is often the result of either depletion of
an essential nutrient or accumulation of a metabolic byproduct.
When the transition is the result of nutrient depletion, the
addition of nutrients to the system causes cell division to
continue. One or more essential nutrients can incrementally be
added to the fermentation vessel during the run, with the net
volume increasing during the fermentation process. The result is a
controlled growth rate where biomass and expression levels can be
optimized. When the cell number in the culture has reached or is
approaching a maximum, protein polymer production is induced by
providing an appropriate physical or chemical signal, depending
upon the expression system used. Production will then continue
until the accumulated product reaches maximum levels (Fiestcliko,
J., and Ritch, T., Chem. Eng. Commun. 1986, 45:229-240; Seo, J. H.;
Bailey, J. E., Biotechnol. Bioeng. 1986, 28:1590-1594).
Example 2
[0082] Construction of SELP8K, SELP8E and CLP6
[0083] Polymers were prepared designated SELP8K and SELP8E, which
are characterized by having functional groups for cross-linking.
The construction of these polymers is described below starting from
the previous gene monomer, SELPO (see U.S. Pat. No. 5,243,038,
pSY1298).
[0084] SELP8K and SELP8E Amino Acid Monomer Sequence Design:
TABLE-US-00001 (SEQ ID NO:04) SELP8K MONOMER (GAGAGS).sub.4
(GVGVP).sub.4 GKGVP (GVGVP).sub.3 (SEQ ID NO:05) SELP8E MONOMER
(GAGAGS).sub.4 (GVGVP).sub.4 GEGVP (GVGVP).sub.3
SELP8 Construction
[0085] Plasmid pSY1378 (see U.S. Pat. No. 5,243,038) was digested
with Banl REN, purified using agarose gel electrophoresis followed
by NACS column, and the DNA was then ethanol precipitated in 2.5 M
ammonium acetate and ligated with pPT0134 (See PCT\US92\09485)
previously digested with Fold REN, phenol/chloroform extracted and
ethanol precipitated.
[0086] The products of the ligation mixture were transformed into
E. coli strain HB 101. Plasmid DNA from transformants was purified
and analyzed by digestion using Nrul and XmnI RENs. Plasmid pPT0255
containing the desired restriction pattern was obtained and was
used for subsequent constructions.
[0087] Plasmid DNA pPT0255 was treated with Cfr10I REN followed by
RNAse. The digestion fragments were separated by agarose gel
electrophoresis, the DNA was excised and self-ligated. The products
of the ligation mixture were transformed into E. coli strain HB
101. Plasmid DNA from transformants was purified and analyzed by
digestion using NaeI and Stal RENs. Plasmid pPT0267 containing the
desired deletion was used for subsequent constructions.
[0088] Two oligonucleotide strands as shown in Table 1 were
synthesized and purified as described in Example 1. TABLE-US-00002
TABLE 1 5'-CTGGAGCGGGTGCCI'GCATGTACATCCGA (SEQ ID NO:06) GT-3'
3'-CCGAGACCTCGCCCACGGACGTACATGTAG (SEQ ID NO:07) GCTCA-5'
[0089] The two oligonucleotide strands were annealed and ligated
with the DNA of plasmid pPT0267 which had been previously digested
with Banll and Scal RENs, and purified by agarose gel
electrophoresis followed by NACS column.
[0090] The products of this ligation reaction were transformed into
E. coli strain HB101. Plasmid DNA from transformants was purified
and digested with Dral. Plasmid DNA from two clones that gave the
correct digestion pattern was sequenced. One plasmid DNA,
designated pPT0287, was found to be correct and chosen for further
constructions.
[0091] Plasmid DNA pSY1298 (see U.S. Pat. No. 5,243,038) was
digested with BanlI REN, and the SELPO gene fragment was purified
by agarose gel electrophoresis followed by NACS and then ligated to
pPT0287 digested with Banll. The enzyme was then removed using
phenol/chloroform extraction and ethanol precipitation.
[0092] The products of the ligation mixture were transformed into
E. coli strain HB101. Plasmid DNA from transformants was purified
and analyzed by digestion using Dral REN. Plasmid DNA from the
clones showing the correct restriction pattern was further digested
with BanlI, Ahall and Stul RENs. Plasmid pPT0289 contained the
desired SELP8 monomer sequence (see Table 2). TABLE-US-00003 TABLE
2 SELP8 Gene Monomer Sequence (SEQ ID NOS: 08 & 09) BanI BanII
GGTGCCGGTTCTGGA GCTGGCGCGGGCTCTGGA GTAGGTGTGCCAGGT CCACGGCCAAGA
CCTCGACCGCGCCCCAGA CCTCATCCACACGGTCCA G A G S G A G A C S G V G V P
G GTAGGA GTTCCGGCTGTAGGCGTTCCGGGA GTTGGTGTACCTGGAGTG
CATCCTCAAGGCCCACATCCGCAAGGCCCTCAACCACATGGA CCTCAC V G V P G V G V P
G V G V P G V Smal GGTGTTCCAGGCGTAGGTGTGCCCGGG GTAGGA
GTACCACOGGTAGGC CCA CAACGI CCG CAT CCA CAC CCC CCC CAT CCI CAT GCT
CCC CAT CCG G V P G V G V P G V G V P G V G BanII GTCCCTGGA
GCCGGTGCTGGTAGCGGCGCAGGCGCGGGCTCTGGA GCG CAGGGA
CCTCGCCCACGACCATCGCCGCGTCCGCGCCCGAGA CCTCGC V P G A G A G S G A G A
G S G A
[0093] Construction of SELP8K and SELP8E Gene Monomers
[0094] One oligonucleotide strand coding for a portion of the SELP8
gene monomer was synthesized with a single base polymorphism at
position 90. The use of both adenine and guanidine at this position
produced oligonucleotides from a single synthesis that encoded the
amino acids lysine and glutamic acid (see Table 3). The synthesis
was conducted using an Applied Biosystems DNA synthesizer model
381A and a 2000A synthesis column supplied by Glen Research. During
the synthesis the required interrupt-pauses for bottle changes were
minimized. After the synthesis the 202 base DNA fragment was
deprotected and cleaved from the column support by treatment in 30%
ammonium hydroxide at 55.degree. C. for 6 hours. TABLE-US-00004
TABLE 3 (SEQ ID NO:10)
5'-ATGGCAGCGAAAGGGGACCGGGCTCTGGTGTTGGAGTGCCAGGTGTC
GGTGTTCCGGGTGTAGGCGTTGGTGTACCTGGA(A/G)AAGGTGTTCCGG
GGGTAGGTGTGCCGGGCGTTGGAGTACCAGGTGTAGGCGTCCCGGGAGCG
GGTGCTGGTAGCGGCGCAGGCGCGGGCTCTTTCCGCTAAAGTCCTGCCGT -3'
[0095] Two additional DNA strands were used as primers for PCR
amplification. The two strands were: TABLE-US-00005 (SEQ ID NO:11)
1. 5'-AAGAAGGAGATATCATATGGCAGCGAAAGGGGACC-3' (SEQ ID NO:12) 2.
5'-CGCAGATCTTTAAATTACGGCAGGACTTTAGCGGAAA-3'
[0096] The PCR reaction was carried out and the reaction product
was purified as described in Example 1.
[0097] The DNA was resuspended and digested with BanlI REN as
described in Example 1. The digested DNA was then separated by
low-melting agarose gel electrophoresis and ligated with pPT0289
previously digested with Banll RENs and purified by NACS column.
The products of the ligation reaction were transformed into E. coli
strain HB 101. Plasmid DNA from isolated transformants was purified
and analyzed by digestion using ApaLI and EcoNI RENs. Plasmid DNA
from the clones showing the correct restriction pattern were
further analyzed by digestion using Asp700 REN to distinguish
between clones encoding a lysine or glutamic acid at the
polymorphic position. Plasmid DNA from clones containing each of
the polymorphs was purified and analyzed by DNA sequencing. Plasmid
pPT0340 contained the desired SELP8K monomer sequence (see Table 4)
and pPT0350 contained the desired SELP8E monomer sequence.
TABLE-US-00006 TABLE 4 SELP8K Gene Monomer Sequence (SEQ ID NO: 13
&14) BanI BanII GGTGCCGGTTCTGGA GCTGGCGCGGGCTCTGGTGTTGGA
GTGCCAGGT CCACGGCCAAGA CCTCGACCGCGCCCGAGA CCACAACCTCACGGTCCA G A G
S G A G A G S G V G V P G EcoNI GTCGGTGTTCCGGGTGTAGGCGTTCCGGGA
GTTGGTGTACCTGGA AAA CAGCCACAAGGCCCACATCCGCAAGGCCCTCAACCACATGGA
CCTTTT V G V P G V G V P G V G V P G K GGTGTTCCGGGG
GTAGGTGTGCCGGGCGTTGGA GTACCAGGTGTAGGC
CCACAAGGCCCCCATCCACACGGCCCGCAACCTCATGGTCCACATCCG G V P G V G V P G
V G V P G V G. SmalBanII GTCCCGGGA
GCGGGTGCTGGTAGCGGCGCAGGCGCGGGCTCTGGA GCG
CAGGGCCCTCGCCCACGACCATCGCCGCGTCCGCGCCCGAGA CCTCGC V P G A G A G S G
A G A G S G A
[0098] SELP8K Polymer Construction
[0099] Plasmid DNA from pPT0340 was digested with Banl REN and the
digestion fragments were separated by agarose gel electrophoresis.
The SELP8K gene fragment, 192 bp, was excised and purified by NACS
column. The purified fragment was ligated with plasmid pPT0317
which had been digested with Banl REN, passed through a Millipore
Probind and a Bio-Spin 6 column. The DNA was then treated with
shrimp alkaline phosphatase (SAP) as described in Example 1.
[0100] The products of this ligation reaction were transformed into
E. coli strain HB101. Transformants were selected for resistance to
kanamycin. Plasmid DNA from individual transformants was purified
and analyzed for increase size due to SELP8K monomer multiple DNA
insertion. Several clones were obtained with insert sizes ranging
from 200 by to approximately 7 kb. Clones containing from 6 to 32
repeats, were used for expression of the SELP8K protein polymer
(pPT0341, pPT0343, pPT0344, pPT0345 and pPT0347).
[0101] SELP8K Expression Analysis
[0102] An overnight culture which had been grown at 30.degree. C.
was used to inoculate 50 ml of LB media contained in a 250 ml
flask. Kanamycin was added at a final concentration of 50 .mu.g per
ml and the culture was incubated with agitation (200 rpm) at
30.degree. C. When the culture reached an OD600 of 0.8, 40 ml were
transferred to a new flask prewarmed at 42.degree. C. and incubated
at the same temperature for approximately 2 hours. The cultures
(30.degree. and 42.degree.) were chilled on ice and OD600 was
taken. Cells were collected by centrifugation and divided in 1.0
OD600 aliquots and used to perform western analysis using anti-SLP
antibody.
[0103] E. coli strain HB101 containing plasmids pPT0341, pPT0343,
pPT0344, pPT0345 and pPT0347 were grown as described above. The
proteins produced by these cells were analyzed by Western blot for
detection of proteins reactive to SLP antibodies. Each clone
produced a strongly reactive band. The apparent molecular weights
of the products ranged from approximately 35 kD to greater than 250
kD. Strain pPT0345 produced an SLP antibody reactive band of
apparent molecular weight 80,000. The expected amino acid sequence
of the SELP8K polymer encoded by plasmid pPT0345 is shown below.
TABLE-US-00007 (SEQ ID NO:15) pPT0345 SELP8K 884 AA MW 69,772
MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPMGAGSGAGAGS ((GVGVP).sub.4 GKGVP
(GVGVP).sub.3 (GAGAGS).sub.4].sub.32 (GVGVP).sub.4 GKGVP
(GVGVP).sub.3 (GAGAGS).sub.2 GAGAMDPGRYQDLRSHHHHHH
[0104] SELP8K Purification
[0105] SELP8K was produced in E. coli strain pPT0345 by
fermentation. The product was purified from the cellular biomass by
means of cellular lysis, clearance of insoluble debris by
centrifugation, and affinity chromatography. The purified product
was analyzed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis, immunoreactivity with a polyclonal antisera which
reacts with silk-like peptide blocks (SLP antibody), and amino acid
analysis. A protein band of apparent molecular weight 80,000 was
observed by amido black staining of SDS-PAGE separated and
transferred samples and the same band reacted with the SLP antibody
on Western blots. As expected, amino acid analysis (shown in Table
5) indicated that the product was enriched for the amino acids
glycine (43.7%), alanine (12.3%), serine (5.3%), proline (11.7%),
and valine (21.2%). The product also contained 1.5% lysine. The
amino acid composition table below shows the correlation between
the composition of the purified product and the expected
theoretical compositions as deduced from the synthetic gene
sequence. TABLE-US-00008 TABLE 5 Amino Acid Analysis of Purified
SELP8K Actual % Theoretical % Amino Acid pmoles composition
composition Ala 1623.14 12.3 12.2 Asx 122.20 0.9 0.8 Glx nd nd 0.4
Phe 58.16 0.4 0.1 Gly 5759.31 43.7 41.5 His 46.75 0.4 0.8 Ile 43.87
0.3 0 Lys 198.21 1.5 1.5 Leu 39.54 0.3 0.5 Met 36.01 0.3 0.3 Pro
1534.21 11.7 12.4 Arg 70.84 0.5 0.6 Ser 703.83 5.3 6.1 Thr nd nd
0.1 Val 2797.47 21.2 22.4 nd = none detected
[0106] CLP6 Preparation
[0107] CLP6 was prepared as described in PCT/US92/09485 using
strain pPT0246 (CLP6 referred to as DCP6). The protein polymer was
purified in multigram quantities using standard protein
purification, extraction, and separation methods. The lyophilized
product was a white, spongy material, extremely soluble in water.
TABLE-US-00009 (SEQ ID NO:16) CLP6 pPT0246 1,065 AA MW 85,386
MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM
[(GAHGPAGPK).sub.2(GAQGPAGPG).sub.24(GAHGPAGPK).sub.2].sub.4
GAMDPGRYQLSAGRYHYQLVWCCK
Example 3
The Construction of SELPOK Polymers
[0108] Polymer Design Elements
[0109] The copolymer structure of SELP8K consists of silk-like
blocks (SLP block) and elastin-like blocks (ELP block) in the
following sequence: [(SLP block)4 (ELP block).sub.8]. Additional
polymers were designed to have different resorption and solution
properties by adjusting their silk-like to elastin-like block
lengths while maintaining their adhesive properties. SELPOK
contains half the length of crystallizable silk-like blocks than
SELP8K while maintaining the dispersion frequency with respect to
the elastin-like segments.
[0110] Polymers with intervening sequences to promote in vivo
resorption through proteolytic cleavage by collagenase (92 kd) and
cathepsins were also designed. SELPOK is used as the backbone for
these designs, but these sites can be used in many different
polymer backbone sequences. The insert location is chosen to permit
accessibility of the site to the catalytic groove of the protease.
Most proteases will bind up to 4 upstream amino acids from the
cleavage site. Therefore, the insert sequences should be free of
hydrogen bonding and crystallization that may be induced by, for
example, silk-like blocks.
[0111] The beta structure of the SELPOK will break after the
proline of the first elastin-like block. SELPOK-CS 1 contains two
adjacent cleavage sites for collagenase (PLGP) (SEQ ID NO: 17)
within a six amino acid insert. The insertion site was chosen to be
removed from the silk-like blocks by at least one proline amino
acid (GAGAGS GVGVP L G P L G P GVGVP) (SEQ ID NO:18). SELPOK-CS2
contains multiple cleavage sites for cathepsins B (ARR), L (FF), S
and H (FVR) and plasmin (R) within an eight amino acid insert. The
insertion site was chosen to be removed from the silk-like blocks
by at least one proline amino acid (GAGAGS GVGVP G F F V R A R R
GVGVP)(SEQ ID NO:19).
[0112] Construction of Plasmid pPT0317
[0113] Plasmid DNA pSY1262 (see U.S. Pat. No. 5,243,038) was
linearized with PvuII REN, then passed through a Probind filter and
a Bio-Spin 6 column. The DNA was then treated with Shrimp Alkaline
Phosphatase (SAP). The linearized pSY1262 DNA was then ligated with
a DNA fragment from pQE-17 (QIAGEN .Catalog #33173) prepared as
follows. Plasmid DNA pQE-17 was digested with BglII and HindIII
RENs and the 36 by fragment shown in Table 6 was purified using a
Probind filter and a Biospin column. The DNA was purified further
using a Microcon-30 filter and the filtrate, containing the 36 by
fragment, was kept. The DNA was then treated with DNA Polymerase I
and purified using a Probind filter and a Biospin column (see
Example 1). TABLE-US-00010 TABLE 6
5'-GATCTTCGATCTCATCACCATCACCATCACTA (SEQ ID NO:20)
3'-AAGCTAGAGTAGTGGTAGTGGTAGTGATTCG (SEQ ID NO:21)
[0114] The product of the ligation reaction was transformed into E.
coli strain HB 101. Plasmid DNA from transformants was purified and
analyzed by digestion using Bstl 1071 and EcoRV RENs. The clones
containing the desired DNA fragment were further digested with Bstl
1071 and BstYI RENs to determine the orientation of the insert.
Plasmid DNA from the clones showing the correct restriction pattern
was purified and analyzed by DNA sequencing. Plasmid pPT0317
contained the desired DNA insert and was used for further DNA
constructions.
[0115] SELPOK Polymer Construction
[0116] One oligonucleotide strand as shown in Table 7 was
synthesized using an Applied Biosystems DNA synthesizer model 381A
and a 2000A synthesis column supplied by Glen Research. After the
synthesis the 93 base DNA fragment was deprotected and cleaved from
the column support by treatment in ammonium hydroxide at 55.degree.
C. for 6 hours. TABLE-US-00011 TABLE 7 (SEQ ID NO:22)
5'-ATGGCAGCGAAAGGGGACCGGTGCCGGCGCAGGTAGCGGAGCCGGTG
CGGGCTCAAAAAGGGCTCTGGTGCCTTTCCGCTAAAGTCCTGCCGT -3'
[0117] The PCR reaction was performed using the same two DNA primer
strands as described for the construction of the SELP8K gene
monomer and the reaction product was purified. The DNA was
resuspended and digested with Banl REN. The digested DNA was then
separated by low-melting agarose gel and ligated with pPT0285 (see
PCT/US92/09485) previously digested with Banl REN and purified by
NACS column. The product of the ligation reaction was: transformed
into E. coli strain H13101. Plasmid DNA from transformants was
purified and analyzed by digestion using EcoRI and BaniI RENs.
Plasmid DNA from the clones showing the correct restriction pattern
was then purified and analyzed by DNA sequencing. Plasmid pPT0358
contained the desired sequence and was used for subsequent DNA
constructions.
[0118] Plasmid DNA from pPT0340 was digested with Banll REN and the
digestion fragments were separated by agarose gel electrophoresis.
The SELPOK gene fragment, 156 bp, (see Table 8), was excised and
purified using an Ultrafree-MC filter followed by Bio-Spin 6
column. TABLE-US-00012 TABLE 8 (SEQ ID NOS:23&24) BanII G GGC
TCT GGT GTT GGA GTG CCA GGT GTC GGT GTT CCG GGT GTA GGC GTT C CCG
AGA CCA CAA CCT CAC GGT CCA CAG CCA CAA GGC CCA CAT CCG CAA G S G V
G V P G V G V P G V G V CCG GGA GTT GGT GTA CCT GGA AAA GGT GTT CCG
GGG GTA GGT GTG CCG GGC CCT CAA CCA CAT GGA CCT TTT CCA CAA GGC CCC
CAT CCA CAC GGC P G V G V P G K G V P G V G V P GGC GTT GGA GTA CCA
GGT GTA GGC GTC CCG GGA GCG GGT GCT GGT AGC CCG CAA CCT CAT GGT CCA
CAT CCG CAG GGC CCT CGC CCA CGA CCA TCG G V G V P G V G V P G A G A
G S BanII GGC GCA GGC GCG GGC TC CCG CGT CCG CGC CCG AG G A G A G
S
[0119] The purified fragment was ligated with plasmid pPT0358 which
had been digested with Banll REN, then passed through a Probind
filter and a Microcon-30 filter. The digestion fragments were then
separated by agarose gel electrophoresis. The plasmid DNA was then
excised and purified using an Ultrafree-MC filter followed by
Bio-Spin 6 column (see Example 1).
[0120] The product of this ligation reaction was transformed into
E. coli strain HB 101. Transformants were selected for resistance
to chloramphenicol. Plasmid DNA from individual transformants was
purified and analyzed for increased size due to SELPOK multiple DNA
insertion. Several clones were obtained with inserts of different
sizes. Plasmid pPT0359, pPT0360 and pPT0374 containing respectively
18, 2 and 6 repeats of the SELPOK gene monomer were used for
subsequent instructions.
[0121] Plasmid DNA from pPT0359 and pPT0374 was digested with BanI
REN and the digestion fragments were separated by agarose gel
electrophoresis. The SELPOK gene fragments, approximately 2800 by
and 1000 bp, were excised and purified by NACS column. The purified
fragments were then ligated with plasmid pPT0317 which had been
digested with BanI REN, then passed through a Probind filter and
Bio-Spin 6 column. The DNA was then treated with Shrimp Alkaline
Phosphatase (SAP), passed through a Probind filter and then a
Bio-Spin 6 column (see Example 1).
[0122] The product of these ligation reactions was transformed into
E. coli strain 1113101. Transformants were selected for resistance
to kanamycin. Plasmid DNA from individual transformants was
purified and analyzed for increased size due to SELPOK multiple DNA
insertion. Several clones were obtained. Plasmid pPT0364 and
pPT0375 were chosen to be used for expression of SELPOK.
[0123] SELPOK Expression Analysis
[0124] E. coli strain HB 101 containing plasmid pPT0364 and pPT0375
were grown as described in Example 1. The proteins produced by
these cells were analysed by SDS-PAGE for detection of reactivity
to ELP antibodies. In every analysis a strong reactive band was
observed of an apparent molecular weight of approximately 95 kD and
35 kD respectively. TABLE-US-00013 (SEQ ID NO:25) PT0364 SELPOK
1000 AA MW 80,684 MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM [(GAGAGS).sub.2
(GVGVP).sub.4GKGVP (GVGVP).sub.3].sub.10 (GAGAGS).sub.2
GAGAMDPGRYQDLRSHHHHHH (SEQ ID NO:26) pPT0375 SELPOK 376 AA MW
31,445 MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM [(GAGAGS).sub.2
(GVGVP).sub.4GKGVP (GVGVP)31.sub.6 (GAGAGS).sub.2
GAGAMDPGRYQDLRSHHHHHH
[0125] SELPOK-CS 1 Polymer Construction
[0126] Plasmid pPT0360 was digested with Banl REN and the digestion
fragments were separated by agarose gel electrophoresis. The SELPOK
gene fragment, approximately 300 bp, was excised and purified using
an Ultrafree-MC filter followed by Bio-Spin 6 column. The purified
fragment was ligated with plasmid pPT0134 (see PCT/US92/09485)
which had been digested with Fold REN. The enzyme was heat
inactivated at 65.degree. C. for 20 minutes and the ligation
mixture was then passed through a Probind filter. The DNA was then
treated with Shrimp Alkaline Phosphatase (SAP), passed through a
Probind filter and then a Bio-Spin 6 column.
[0127] The product of this ligation reaction was transformed into
E. coli strain HB101. Transformants were selected for resistance to
chloramphenicol. Plasmid DNA from individual transformants was
purified and analyzed by digestion using Dral REN. One plasmid,
pPT0363, showed the correct restriction pattern and was used for
subsequent DNA constructions.
[0128] One oligonucleotide strand as shown in Table 9 was
synthesized using an Applied Biosystems DNA synthesizer model 381A
and a 2000A synthesis column supplied by Glen Research. After the
synthesis the 141 base DNA fragment was deprotected and cleaved
from the column support by treatment in ammonium hydroxide at
55.degree. C. for 6 hours. TABLE-US-00014 TABLE 9 (SEQ ID NO:27)
5'-ATGGCAGCGAAAGGGGACCGCCGGTGCGGGCTCTGGTGTTGGAGTGC
CGCTGGGTCCTCTTGGCCCAGGTGTCGGTGTTCCGGGTGTAGGCGTTCCG
GGAGTTGGTGTACCTGGAAAAGGTTTCCGCTAAAGTCCTGCCGT-3'
[0129] The PCR reaction was performed using the same two DNA primer
strands as described for the construction of the SELP8K gene
monomer and the reaction product was purified. The DNA was then
resuspended and digested with BsrFI and EcoNI RENs. The digested
DNA was treated with Probind and Microcon-30 filters, a Bio-Spin 6
column, and then ligated with pPT0363 previously digested with
BsrFI REN, treated with a ProBind filter and a Bio-Spin 6 column
and then further digested with EcoNI REN. The digestion fragments
were separated by agarose gel electrophoresis. The larger DNA band,
approximately 2000 bp, was excised and purified using an
Ultrafree-MC filter followed by Bio-Spin 6 column (see Example
1).
[0130] The product of the ligation reaction was transformed into E.
coli strain HB 101. Plasmid DNA from individual transformants was
purified and analyzed by digestion using Asp7001 and EcoO 1091
RENs. Plasmid DNA from the clones showing the correct restriction
pattern was then purified and analyzed by DNA sequencing. Plasmid
pPT0368 (see Table 10) contained the desired sequence and was used
for subsequent DNA constructions. TABLE-US-00015 TABLE 10 (SEQ ID
NOS:28 & 29) BanII G GGC TCT GGT GTT GGA GTG CCG CTG GGT CCT
CTT GGC CCA GGT GTC C CCG AGA CCA CAA CCT CAC GGC GAC CCA GGA GAA
CCG GGT CCA CAG G S G V G V P L G P L G P G V GGT GTT CCG GGT GTA
GGC GTT CCG GGA GTT GGT GTA CCT GGA AAA CCA CAA GGC CCA CAT CCG CAA
GGC CCT CAA CCA CAT GGA CCT TTT G V P G V G V P G V G V P G K GGT
GTT CCG GGG GTA GGT GTG CCG GGC GTT GGA GTA CCA GGT GTA CCA CAA GGC
CCC CAT CCA CAC GGC CCG CAA CCT CAT GGT CCA CAT G V P G V G V P G V
G V P G V BanII GGC GTC CCG GGA GCG GGT GCT GGT AGC GGC GCA GGC GCG
GGC TCT CCG CAG GGC CCT CGC CCA CGA CCA TCG CCG CCT CCG CGC CCG AGA
G V P G A G A G S G A G A G S
[0131] Plasmid DNA pPT0368 was digested with BanlI REN, and the
digestion fragments were separeted by agarose gel electrophoresis.
The SELPOK-CS1 gene fragment, 174 bp, was excised and purified
using an Ultrafree-MC filter followed by Bio-Spin 6 column. The
purified fragment was ligated with plasmid pPT0358 which had been
digested with BanlI REN, then passed through a Probind filter and a
Microcon-30 filter. Subsequently the digestion fragments were
separated by agarose gel electrophoresis. The plasmid DNA was then
excised and purified using an Ultrafree-MC filter followed by
Bio-Spin 6 column (see Example 1).
[0132] The product of this ligation reaction was transformed into
E. coli strain HB101. Transformants were selected for resistance to
chloramphenicol. Plasmid DNA from individual transformants was
purified and analyzed for increased size due to SELPOK-CS 1
multiple DNA insertion. Several clones were obtained with insert
sizes ranging from 1000 by to approximately 3000 bp. Plasmid
pPT0369 containing 16 repeats of the SELPOK-CS 1 gene monomer was
used for subsequent constructions.
[0133] Plasmid DNA from pPT0369 was digested with BanI REN,
followed by a Probind filter and then the digestion fragments were
separated by agarose gel electrophoresis. The SELPOK-CS1 gene
fragment, approximately 2800 bp, was excised and purified by an
Ultrafree-MC filter and desalted using a Bio-Spin 6 column. The
purified fragments were then ligated with plasmid pPT0317 which had
been digested with BanI REN and then passed through a Probind
filter and a BioSpin 6 column. The DNA was then treated with Shrimp
Alkaline Phosphatase (SAP), passed through a Probind filter and
then a Bio-Spin 6 column (see Example 1).
[0134] The product of these ligation reactions was transformed into
E. coli strain HB 101. Transformants were selected for resistance
to kanamycin. Plasmid DNA from individual transformants was
purified and analyzed for increased size due to SELPOK-CS 1
multiple DNA insertion. Several clones were obtained. Plasmid
pPT0370 was chosen to be used for expression of SELPOK-CS 1.
[0135] SELPOK-CS1 Expression Analysis
[0136] E. coli strain HB 101 containing plasmid pPT0370 was grown
as described in Example 1. The proteins produced by these cells
were analysed by SDS-PAGE for detection of reactivity to ELP
antibodies. In every analysis a strong reactive band was observed
with an apparent molecular weight of approximately 90 kD.
TABLE-US-00016 (SEQ ID NO:30) pPT0370 SELPOK-CS1 934 AA MW 76,389
MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM [(GAGAGS)Z (GVGVP).sub.1LGPLGP
(GVGVP).sub.3GKGVP(GVGVP).sub.3].sub.15 (GAGAGS).sub.2
GAGAMDPGRYQDLRSHHHHHH
[0137] SELPOK-CS2 Polymer Construction
[0138] One oligonucleotide strand as shown in Table 11 was
synthesized using an Applied Biosystems DNA synthesizer model 381A
and a 2000A synthesis column supplied by Glen Research. After the
synthesis the 147 base DNA fragment was deprotected and cleaved
from the column support by treatment in ammonium hydroxide at
55.degree. C. for 6 hours. TABLE-US-00017 TABLE 11 (SEQ ID NO:31)
5'-ATGGCAGCGAAAGGGGACCGCCGGTGCGGGCTCTGGTGTTGGAGTGC
CAGGCTTCTTTGTACGTGCACGCCGTGGTGTCGGTGTTCCGGGTGTAGGC
GTTCCGGGAGTTGGTGTACCTGGAAAAGGTTTCCGCTAAAGTCCTGCCGT -3'
[0139] The PCR reaction was performed using the same two DNA primer
strands as described for the construction of the SELP8K gene
monomer and the reaction product was purified. The DNA was then
resuspended and digested with BsrFI and EcoNI RENs. The digested
DNA was treated with ProBind and Microcon-30 filters, a Bio-Spin 6
column, and then ligated with pPT0363 previously digested with
BsrFI REN, treated with a ProBind filter and a Bio-Spin 6 column
and then further digested with EcoNI REN. The digestion fragments
were separated by agarose gel electrophoresis. The larger DNA band,
approximately 2000 bp, was excised and purified using an
Ultrafree-MC filter followed by Bio-Spin 6 column.
[0140] The product of the ligation reaction was transformed into E.
coli strain HB101. Plasmid DNA from individual transformants was
purified and analyzed by digestion using Asp7001 and DraIII RENs.
Plasmid DNA from the clones showing the correct restriction pattern
was then purified and analyzed by DNA sequencing. Plasmid pPT0367
(see Table 12) contained the desired sequence and was used for
subsequent DNA constructions. TABLE-US-00018 TABLE 12 (SEQ ID
NOS:32&33) BanII G GGC TCT GGT GTT GGA GTG CCA GGC TTC TTT GTA
CGT GCA CGC CGT C CCG AGA CCA CAA CCT GAG GGT CCG AAG AAA CAT GCA
GGT GCG GCA G S G V G V P G F F V R A R R GGT GTC GGT GTT CCG GGT
GTA GGC GTT CCG GGA GTT GGT GTA CCT GGA GCA GAG CCA CAA GGC CCA CAT
CGG CAA GGC CTT CAA CCA CAT GGA CCT G V G V P G V G V P G V G V P G
AAA GGT GTT CCG GGG GTA GGT GTG CCG GGC GTT GGA GTA CCA GGT GTA TTT
CCA CAA GGC CCC CAT CCA GAG GGC CCG CAA CCT CAT GGT CCA CAT K G V P
G V G V P G V G V P G V BanII GGC GTG CCG GGA GCG GGT GGT GGT AGC
GGC GCA GGC GGG GGG TC GCG GAG GGC CCT CGC CCA CGA GGA TCG GCG GGT
CCG CGC CCG AG G V P G A G A G S G A G A G S
[0141] Plasmid DNA pPT0367 was digested with Banll REN, treated
with a Probind filter and a Bio-Spin6 column and then the digestion
fragments were separated by agarose gel electrophoresis. The
SELPOK-CS2 gene fragment, 180 bp, was excised and purified using an
Ultrafree-MC filter followed by Bio-Spin 6 column. The purified
fragment was ligated with plasmid pPT0358 which had been digested
with Banll REN and then passed through a Probind filter and a
Microcon-30 filter. Subsequently the digestion fragments were
separated by agarose gel electrophoresis. The plasmid DNA was then
excised and purified using an Ultrafree-MC filter followed by
Bio-Spin 6 column (see Example 1).
[0142] The product of this ligation reaction was transformed into
E. coli strain HB 101. Transformants were selected for resistance
to chloramphenicol. Plasmid DNA from individual transformants was
purified and analyzed for increased size due to SELPOK-CS2 multiple
DNA insertion. Several clones were obtained with insert sizes
ranging from 200 by to approximately 3000 bp. Plasmid pPT0371 and
pPT0372, containing 18 and 15 repeats respectively of the
SELPOK-CS2 gene monomer were used for subsequent constructions.
[0143] Plasmid DNA from pPT0372 was digested with Banl REN,
followed by a Probind filter, and then the digestion fragments were
separated by agarose gel electrophoresis. The SELPOK-CS2 gene
fragment, approximately 2800 bp, was excised and purified by an
Ultrafree-MC filter and desalted using a Bio-Spin 6 column. The
purified fragments were then ligated with plasmid pPT0317 which had
been digested with Banl REN, passed through a Probind filter and
then a Bio-Spin 6 column. The DNA was treated with Shrimp Alkaline
Phosphatase (SAP), passed through a Probind filter and then a
Bio-Spin 6 column (see Example 1).
[0144] The product of these ligation reactions was transformed into
E. coli strain HD 101. Transformants were selected for resistance
to kanamycin. Plasmid DNA from individual transformants was
purified and analyzed for increased size due to SELPOK-CS2 multiple
DNA insertion. Several clones were obtained. Plasmid pPT0373 was
chosen to be used for the expression of SELPOK-CS2.
[0145] SELPOK-CS2 Expression Analysis
[0146] E. coli strain HB 101 containing plasmid pPT0373 was grown
as described in Example 1. The proteins produced by these cells
were analysed by SDS-PAGE for detection of reactivity to ELP
antibodies. In every analysis a strong reactive band was observed
of an apparent molecular weight of approximately 90 kD.
TABLE-US-00019 (SEQ ID NO:34) pPT0373 SELPOK-CS2 964 AA MW 83,218
MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM [(GAGAGS).sub.2 (GVGVP).sub.1
GFFVRARR(GVGVP).sub.3 GKGVP (GVGVP).sub.3].sub.15 (GAGAGS).sub.2
GAGAMDPGRYQDLRSHHHHHH
[0147] SELPOK and SELPOK-CS1 Purification
[0148] SELPOK and SELPOK-CS 1 were produced in E. coli strains
pPT0364 and pPT0370, respectively. The products were purified from
the cellular biomass by means of cellular lysis, clearance of
insoluble debris by polyethylene imine precipitation and
centrifugation, ammonium sulfate precipitation, and anion exchange
chromatography. The purified products were analyzed by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),
immunoreactivity with a polyclonal antisera which reacts with
elastin-like peptide blocks (ELP antibody), and amino acid
analysis.
[0149] For SELPOK, a protein band of apparent molecular weight
95,000 was observed by amido black staining of SDS-PAGE separated
and transferred samples and the same band reacted with the ELP
antibody on Western blots. As expected, amino acid analysis (shown
in Table 13) indicated that the product was enriched for the amino
acids glycine (41.0%), alanine (8.0%), serine (4.5%), proline
(14.1%), and valine (26.8%). The product also contained 1.9%
lysine. The amino acid composition table below shows the
correlation between the composition of the purified product and the
expected theoretical composition as deduced from the synthetic gene
sequence. TABLE-US-00020 TABLE 13 Amino Acid Analysis of Purified
SELPOK pMoles Mole % Theoretical Mole ASX 28.10 0.6 0.7 GLX 26.90
0.6 0.4 SER 199.84 4.5 4.0 GLY 1812.07 41.0 40.5 HIS 28.45 0.6 0.7
ARG 20.49 0.5 0.5 THR 0 0.0 0.1 ALA 355.29 8.0 8.0 PRO 623.22 14.1
15.0 TYR 8.47 0.2 0.1 VAL 1183.63 26.8 27.3 MET 17.21 0.4 0.3 ILE
4.83 0.1 0.0 LEU 20.66 0.5 0.4 PHE 7.57 0.2 0.1 LYS 84.02 1.9 1.8
Total 4420.75
[0150] For SELPOK-CS1, a protein band of apparent molecular weight
90,000 was observed by amido black staining of SDS-PAGE separated
and transferred samples and the same band reacted with the ELP
antibody on Western blots. As expected, amino acid analysis (shown
in Table 14) indicated that the product was enriched for the amino
acids glycine (40.0%), alanine (7.6%), serine (5.2%), proline
(16.3%), and valine (23.3%). The product also contained 1.5%
lysine. The amino acid composition table below shows the
correlation between the composition of the purified product and the
expected theoretical composition as deduced from the synthetic gene
sequence. TABLE-US-00021 TABLE 14 Amino Acid Analysis of Purified
SELPOK-CS1 pMoles Mole % Theoretical Mole ASX 16.43 0.7 0.7 GLX
10.59 0.5 0.4 SER 119.96 5.2 3.6 GLY 924.51 40 0 39.6 HIS 13.85 0.6
0.7 ARG 11.26 0.5 0.5 THR 0 0.0 0.1 ALA 175.07 7.6 7.3 PRO 376.40
16.3 16.7 TYR 2.49 0.1 0.1 VAL 537.96 23.3 24.5 MET 5.19 0.2 0.3
ILE 0 0.0 0.0 LEU 76.62 3.3 0.4 PHE 2.58 0.1 0.1 LYS 35.68 1.5 1.6
Total 2308.59
Example 4
Evaluation of CLP6 and SELP8K Properties
[0151] Test Procedures
[0152] Tiseel Adhesive Systems. Rat skins were washed with water,
blotted dry and cut into strips about 1 cm.times.4 cm. Adhesive
from Tiseel Kit VH (Osterreiches Institute Fur Haemoderivate, GmbH,
A-1220, Vienna, Austria) was applied according to the
manufacturer's specifications.
[0153] Rat Skin Lap Shear Tensile Strength Assay. Adhesive
formulations were tested for their ability to bond skin together
using an in vitro rat skin lap shear tensile strength assay.
Adhesives were applied to the subcutaneous side of a strip of
harvested rat skin. A second skin strip was overlapped in order to
produce an approximate bonding surface of 1 cm2. A 100 gram weight
was applied to the lap joint and the adhesive was allowed to cure,
usually at room temperature for a period of 2 hours and wrapped in
plastic to prevent desiccation. The lap joint was mounted on an
Instron Tensile Tester or similar apparatus and tensile force
applied. With the Instron, tensile force was typically applied at a
constant strain rate of 2 inches per minute. The load at failure
was recorded and normalized to the measured area of overlap.
[0154] Adhesive Systems with Glutaraldehyde. Rat skins were washed
with water, blotted dry, and cut into strips about 1 cm.times.4 cm.
Glutaraldehyde was distilled, stored frozen and thawed immediately
before use. Bovine serum albumin was dissolved according to
Goldman's specifications (Goldman, W094/01508). CLP6 was dissolved
at 600 mg/mL in 150 mM HEPES+30 mM NaCl and adjusted to pH 7.5.
SELP8K was dissolved at the concentrations indicated in Table 15 in
150 mM HEPES+45 mM NaCl and adjusted to pH 8. The indicated
aliquots of the solution of protein was spread over both skins
before the addition of the glutaraldehyde solution. The second skin
was overlaid, rubbed across the lower skin to distribute the
components, adjusted to an overlap area of ca. 1 cm2, covered with
plastic wrap to prevent drying, and cured for 2 hours at 25.degree.
C. under a compressive force of 100 g/cmZ.
[0155] Adhesive S stems with 1 6-(Diisocyanto)hexane. Rat skins
were washed with water, blotted dry, and cut into strips about 1
cm.times.4 cm. A solution of SELP8K was made up in the specified
buffer at a concentration of ca. 50% w/w. A 1:1 v/v mixture of
hexamethylene diisocyanate (HMDI) and Pluronic L-61 surfactant was
prepared. A 20 .mu.L aliquot of SELP8K solution was applied to one
skin followed by a 2 .mu.L aliquot of the diluted HMDI. The second
skin was overlaid, rubbed across the lower skin to mix the
components, adjusted to ca. 1 cm.sup.2 overlap, covered with
plastic wrap to prevent drying, and cured for 2 hours at 25.degree.
C. under a compressive force of 100 g/cm.sup.2.
[0156] Results
[0157] In order to provide a baseline for subsequent adhesive
experiments, ethyl cyanoacrylate and Tiseel fibrin glue were
evaluated. These results are reported in the following table.
TABLE-US-00022 TABLE 15 Base Case Lap Shear Tensile Strengths
Tensile Strength Reagent Dose g/cm.sup.2 Normal Saline not
applicable 13 .+-. 4 Tiseel Fibrin Glue .about.25 mg 261 .+-. 51
Ethyl cyanoacrylate 25 mg 385 .+-. 119
[0158] All data reported are based on at least three test
specimens. All test results are based on a two hour cure time.
[0159] The subject compositions were compared to the proteinaceous
adhesive system described by Goldman (W094/01508). Ten microliters
of glutaraldehyde solution of the indicated concentration was added
in all cases. The following table indicates the results.
TABLE-US-00023 TABLE 16 Lap Shear Tensile Strength of
Glutaraldehyde Cured Adhesive Systems Tensile Strength Reagent Dose
g/cm.sup.2 Ovalbumin + Glutaraldehyde 6 mg/2.5 mg 50 .+-. 10
(30.mu.) 200 mg/mL 10 .mu.L 2.5N Atelocollagen(denat) +
Glutaraldehyde 3 mg/2.5 mg 148 .+-. 47 (25 .mu.L) 125 mg/mL 10
.mu.L 2.5N CLP6 + Glutaraldehyde 24 mg/2.5 mg 306 .+-. 98 (40
.mu.L) 600 mg/mL 10 .mu.L 2.5N CLP6 + Glutaraldehyde 12 mg/2.5 mg
171 .+-. 42 (20 .mu.L) 600 mg/mL 10 .mu.L 2.5N SELP8K (30 .mu.L) +
Glutaraldehyde 600 mg/mL 1.0N 18 mg/1 mg 545 .+-. 153 300 mg/mL
1.0N 9 mg/1 mg 452 .+-. 54 300 mg/mL(impure) 1.0N 9 mg/1 mg 234
.+-. 51* 300 mg/mL(impure) 0.1N 9 mg/0.1 mg 210 .+-. 57* 287 mg/mL
2.5N 7 mg/2.5 mg 374 .+-. 90 100 mg/mL 1.0N 3 mg/1 mg 361 .+-. 47
100 mg/mL 2.5N 3 mg 12.5 mg 274 .+-. 17 *This preparation of SELP8K
was known to be impure and is estimated to yield adhesive strength
about one-half of that of the more completely purified
material.
[0160] This preparation of SELP8K was known to be impure and is
estimated to yield adhesive strength about one-half of that of the
more completely purified material.
[0161] The data in the above table demonstrate that the subject
polymers are able to provide superior adhesive capabilities when
used in the glutaraldehyde cured system under conditions comparable
to collagen and ovalbumin. Despite the lower number of amino groups
available for crosslinking, the SELP8K polymer provides the highest
tensile strengths in the rat skin lap shear results. The above
results demonstrate that significant adhesion can be obtained at
even low doses of glutaraldehyde down to 100 .mu.g/cm2. The quality
and purity of the glutaraldehyde is known to be critical to obtain
good crosslinking (Rujigrok, DeWijn, Boon, J. Matr. Sci. Matr. Med.
5:80-87 (1994); Whipple, Ruta, J. Org. Chem. 39:1666-1668 (1974).
The glutaraldehyde used in these experiments was distilled, diluted
to 2.5N and stored at -20.degree. C. until used.
[0162] In the next study, hexamethylene diisocyanate was employed.
It was found necessary to add an equal volume of diluent to obtain
good adhesion, since the curing was otherwise too fast. The
following table indicates the results, where n=12. TABLE-US-00024
TABLE 17 Lap Shear Tensile Strength of HMDI Derived Adhesive System
Tensile Reagent Dose Strength g/cm2 SELP8K 20 .mu.L .times. 50% w/w
10 mg 585 .+-. 203 HMDI/L-61 1:1 v/v 2 .mu.L .times. 50% v/v 1 mg
Buffer: (100 .mu.L water + 10 .mu.L 1M KHCO.sub.3) SELP8K 20 .mu.L
.times. 50% w/w 10 mg 503 .+-. 21 HMDI/L-61 1:1 v/v 2 .mu.L .times.
50% v/v 1 mg Buffer: (100 .mu.L 50 mM PO, (pH 6.8) + 5 .mu.L 1M
KHCO.sub.3) SELP8K 20 .mu.L .times. 50% w/w 10 mg 451 .+-. 67
HMDI/L-61 1:1 v/v 2 .mu.L .times. 50% v/v 1 mg Buffer: (100 .mu.L
50 mM PO.sub.4 (pH 6.8) + 10 .mu.L 1M KHCO.sub.3) SELP8K 20 .mu.L
.times. 50% w/w 10 mg 362 .+-. 71 HMDI/L-61 1:1 v/v 2 .mu.L .times.
50% v/v 1 mg Buffer: (100 .mu.L 50 mM P0.sub.4 (pH 6.8))
Example 5
Evaluation of SELPOK (SEOK) and SELPOK-CS 1 Properties
[0163] A number of formulations were prepared using different
components for the formulation and determining the lap shear
strength. In addition a variety of protocols were used to prepare
the protein dope to provide adhesion. These protocols are set forth
as follows: TABLE-US-00025 Protocol A. SEOK 17% w/w Lysine
hydrochloride 1:2 Potassium carbonate 1:1 Isocyanate:Amine
13.1:1
[0164] Preparation of Protein Dope
[0165] The designation 1:2 refers to the nominal ratio of amino
groups derived from lysine to amino groups derived from SEOK. The
designation 1:1 refers to the nominal ratio of carbonate ions per
amino group from SEOK plus lysine. The designation 13.1:1 refers to
the nominal ratio of isocyanate groups to amine groups from SEOK
plus lysine
[0166] A stock buffer solution was prepared by dissolving lysine
hydrochloride 0.0157 g, potassium carbonate 0.0710 g, and Evans
Blue dye 0.00371 g in 7.526 mL of deionized water. Stock buffer,
620.6 .mu.L, was added to SEOK, 127.1 mg in an Eppendorf tube. The
mixture was agitated on a vortex mixer until complete dissolution
occurred. The solution was centrifuged at about 5000 rpm for 30-60
seconds to separate air bubbles. The solution was then loaded into
a 1 mL syringe for dispensing onto the test skins. The optional
inclusion of dye in the protein dope serves to more readily
visualize the distribution of the dope on the test skins.
[0167] Preparation of HMDI Setting Agent
[0168] The HMDI setting agent was prepared by dissolving Sudan Red
dye, 1.75 mg, in neat 1,6-diisocyanatohexane, 1.00 g. The optional
inclusion of dye in the setting agent serves to more readily
visualize the distribution of the setting agent on the test
skins.
[0169] Preparation of Rat Skins
[0170] Freshly harvested rat hides were stored frozen at
-20.degree. C. Just before use the hides were thawed and cut into 1
cm.times.3 cm strips. All fascia was removed from the strips of
skin with a razor blade. Strips of skin were selected which were
uniform in width and thickness. Prepared rat skin samples were
temporarily stored at 37.degree. C. between gauze pads soaked with
PBS and contained in a plastic bag to prevent drying.
[0171] Application of Adhesive
[0172] In a 37.degree. C. warm room, protein dope, 15 .mu.L, was
applied to each of two strips of rat skin, 30 .mu.L total, and
aggressively worked into about a 1 cm.sup.2 area of each piece of
skin with a stainless steel spatula. A total of 1.8 .mu.L of HMDI
setting agent was applied to the skins, apportioned so that 3
parallel stripes of HMDI were applied to the first skin and two
stripes in an X-pattern were applied to the second skin. The skins
were immediately assembled to form the lap joint, covered with a
piece of plastic film to prevent drying, and compressed under a 100
g weight. The joint was allowed to cure for 15 minutes at
37.degree. C. The length and width of the lap joint was measured to
1 mm using a ruler immediately before tensile testing on an Instron
Model 55 test machine. The crosshead speed was set at 25 mm per
minute. Lap shear tensile strengths were reported in units of
g/cm.sup.2. Means and standard deviations were calculated for
measurements conducted at least in triplicate. TABLE-US-00026
Protocol B. SEOK 17% w/w Lysine hydrochloride 1:2 Potassium
carbonate 1:1 Isocyanate:Amine 14.5:1
[0173] The steps of Protocol A were followed, except that bubbles
were removed from the protein dope in a two stage process. After
centrifugation, the dope was exposed to reduced pressure, 26 in-Hg,
for 30 minutes. The volume of HMDI setting agent applied to the lap
joint was 2.0 .mu.L. TABLE-US-00027 Protocol C. SEOK 17% w/w Lysine
hydrochloride 1:2 Potassium carbonate 1:1 Isocyanate:Amine
7.3:1
[0174] The steps of Protocol B were followed, except that the
composition of the HMDI setting agent was altered. Sudan Red dye,
5.2 mg was dissolved in 10.735 g of neat Pluronic surfactant L-31
by beating to 100.degree. C. for 10 minutes. After cooling to room
temperature, an equal weight of 1,6-diisocyanatohexane was added to
this mixture. The mixture was prepared immediately before use.
TABLE-US-00028 Protocol D1 SEOK 17% w/w Lysine hydrochloride 1:2
Potassium carbonate 1:1 Isocyanate:Amine 14.5:1
[0175] The steps of Protocol B were followed, except that Pluronic
surfactant L-3 1, 4.57 mg, was added to SEOK, 74.6 mg. The ratio of
SEOK to lysine buffer solution remained as described in Protocol B.
TABLE-US-00029 Protocol D2 SEOK 17% w/w Lysine hydrochloride 1:2
Potassium carbonate 1:1 Isocyanate:Amine 14.5:1
[0176] The steps of Protocol B were followed, except that Pluronic
surfactant L-3 1, 1.07 mg, was added to SEOK, 74.7 mg. The ratio of
SEOK to lysine buffer solution remained as described in Protocol B.
TABLE-US-00030 Protocol E. SEOK 17% w/w Lysine hydrochloride 1:2
Potassium carbonate 1:1 Isocyanate:Amine 7.3:1
[0177] The steps of Protocol B were followed, except that in
Protocol E, 5.1 mg Pluronic L-31 was added to 85.0 mg SEOK. The
composition of the HMDI setting agent was also altered. Sudan Red
dye, 5.2 mg was dissolved in 10.735 g neat Pluronic surfactant L-31
by heating to 100.degree. C. for 10 minutes. After cooling to room
temperature, an equal weight of 1,6-diisocyanatohexane was added to
this mixture. The mixture was prepared immediately before use.
TABLE-US-00031 Protocol F. SEOK 17% w/w Lysine hydrochloride 1:2
Sodium Borate pH 9.5 Isocyanate:Amine 14.5:1
[0178] The steps of Protocol B were followed, except that a stock
buffer solution was prepared by dissolving lysine hydrochloride
0.46 g, boric acid 1.24 g in 92.2 mL of deionized water. The pH of
this solution was adjusted to pH 9.52 by the addition of 7.8 ml, of
2 N sodium hydroxide solution. Evans Blue dye, 0.50 mg/mL, was
dissolved in this buffer, and the solution filtered through a 0.45
micron syringe filter. Stock buffer, 333.5 .mu.L, was added to
SEOK, 68.3 mg in an Eppendorf tube. The mixture was agitated on a
vortex mixer until complete dissolution occurred. TABLE-US-00032
Protocol G. SEOK 17% w/w Lysine hydrochloride 1:2 Potassium Borate
pH 9.5 Isocyanate:Amine 14.5:1
[0179] The steps of Protocol B were followed, except that a stock
buffer solution was prepared by dissolving lysine hydrochloride,
0.46 g, and boric acid, 1.24 g in 99.1 mL of deionized water. The
pH of this solution was adjusted to pH 9.52 by the addition of 0.9
ml, of 10 N potassium hydroxide solution. Evans Blue dye, 0.50
mg/mL, was dissolved in this buffer, and the solution filtered
through a 0.45 micron syringe filter. Stock buffer, 367.2 .mu.L,
was added to SEOK, 75.2 mg in an Eppendorf tube. The mixture was
agitated on a vortex mixer until complete dissolution occurred.
TABLE-US-00033 Protocol H. SEOK 17% w/w Lysine hydrochloride 1:2
Lithium Carbonate pH 9.5 Isocyanate:Amine 14.5:1
[0180] The steps of Protocol B were followed, except that the stock
buffer solution was prepared by dissolving lysine hydrochloride,
42.7 mg, in deionized water, 10.0 mL, and adding lithium carbonate,
14.3 mg, to pH 9.55. Evans Blue dye, 0.50 mg/mL, was dissolved in
this buffer, and the solution filtered through a 0.45 micron
syringe filter TABLE-US-00034 Protocol I. SEOK 17% w/w Lysine
hydrochloride 1:2 Sodium Carbonate pH 9.5 Isocyanate:Amine
14.5:1
[0181] The steps of Protocol B were followed, except that the stock
buffer solution was prepared by dissolving sodium carbonate, 1.06
g, and lysine hydrochloride, 0.46 g, in 99.2 ml, of deionized
water. Using concentrated hydrochloric acid solution 0.8 mL, the
solution was adjusted to pH 9.54. Evans Blue dye, 0.50 mg/mL, was
dissolved in this buffer, and the solution filtered through a 0.45
micron syringe filter TABLE-US-00035 Protocol J. SEOK 17% w/w
Lysine hydrochloride 1:2 Potassium Carbonate pH 9.5
Isocyanate:Amine 14.5:1
[0182] The steps of Protocol B were followed, except that the stock
buffer solution was prepared by dissolving potassium carbonate,
1.38 g, and lysine hydrochloride, 0.46 g, in 99.1 mL of deionized
water. Using concentrated hydrochloric acid solution, 0.9 mL, the
solution was adjusted to pH 9.53. Evans Blue dye, 0.50 mg/mL, was
dissolved in this buffer, and the solution filtered through a 0.45
micron syringe filter. TABLE-US-00036 Protocol K. SEOK 17% w/w
Lysine hydrochloride 1:2 Cesium Carbonate pH 9.5 Isocyanate:Amine
14.5:1
[0183] The steps of Protocol B were followed, except that the stock
buffer solution was prepared by dissolving lysine hydrochloride,
42.7 mg, in deionized water, 10.0 mL, and adding cesium carbonate,
55.2 mg, to pH 9.52. Evans Blue dye, 0.50 mg/mL, was dissolved in
this buffer, and the solution filtered through a 0.45 micron
syringe filter TABLE-US-00037 Protocol L. SEOK 17% w/w Lysine
hydrochloride 1:2 Calcium Carbonate 1:1 Isocyanate:Amine 14.5:1
[0184] The steps of Protocol B were followed, except that the stock
buffer solution was prepared by dissolving lysine hydrochloride,
20.8 mg, in deionized water, 10.0 mL, and adding calcium carbonate,
68.6 mg. Evans Blue dye, 0.50 mg/mL, was dissolved in this buffer,
and the solution filtered through a 0.45 micron syringe filter.
TABLE-US-00038 Protocol M. SEOK 17% w/w Lysine hydrochloride 1:2
Potassium Carbonate pH 9.0 Isocyanate:Amine 14.5:1
[0185] The steps of Protocol B were followed, except that the stock
buffer solution was prepared by dissolving lysine hydrochloride,
103.9 mg, in deionized water, 50.0 mL, and adding potassium
carbonate, 473.3 mg. Using concentrated hydrochloric acid, this
buffer was adjusted to pH 9.00. TABLE-US-00039 Protocol N1. SEOK
17% w/w Lysine hydrochloride 1:2 Potassium Carbonate 1:1
Isocyanate:Amine 14.5:1 Iodide:Carbonate 1:2
[0186] The steps of Protocol B were followed, except that potassium
iodide, 5.70 mg/mL was added to the stock buffer solution. The
nominal pH of this dope was about pH 11. TABLE-US-00040 Protocol
N2. SEOK 17% w/w Lysine hydrochloride 1:2 Potassium Carbonate pH
9.0 Isocyanate:Amine 14.5:1 Iodide:Carbonate 1:2
[0187] The steps of Protocol B were followed, except that potassium
iodide, 5.70 mg/mL was added to the stock buffer solution. Using
concentrated hydrochloric acid, this buffer was adjusted to pH
9.00. TABLE-US-00041 Protocol 01. SEOK 17% w/w Lysine hydrochloride
1:2 Potassium Carbonate 1:1 Isocyanate:Amine 14.5:1 Glucose 1.09
M
[0188] The steps of Protocol B were followed, except that glucose,
1.2755 g, was added to the stock buffer solution, 6.495 mL. This
solution was pH 10.5. TABLE-US-00042 Protocol 02. SEOK 17% w/w
Lysine hydrochloride 1:2 Potassium Carbonate pH 9.0
Isocyanate:Amine 14.5:1 Glucose 1.09 M
[0189] The steps of Protocol B were followed, except that glucose,
1.2755 g, was added to the stock buffer solution, 6.495 mL. Using
concentrated hydrochloric acid, this buffer was adjusted to pH
9.00. TABLE-US-00043 Protocol P1. SEOK 17% w/w Lysine hydrochloride
1:2 Potassium Carbonate 1:1 Isocyanate:Amine 14.5:1 Urea 1.5 M
[0190] The steps of Protocol B were followed, except that urea,
0.5226 g, was added to the stock buffer solution, 5.807 mL,
nominally 1.5 M. This solution was pH 11. TABLE-US-00044 Protocol
P2. SEOK 17% w/w Lysine hydrochloride 1:2 Potassium Carbonate pH
9.00 Isocyanate:Amine 14.5:1 Urea 1.5 M
[0191] The steps of Protocol B were followed, except that urea,
0.5226 g, was added to the stock buffer solution, 5.807 mL. Using
concentrated hydrochloric acid, this buffer was adjusted to pH
9.00. TABLE-US-00045 Protocol Q. SEOK 17% w/w Lysine hydrochloride
1:2 Potassium carbonate 1:1 Isocyanate:Amine 7.3
[0192] The steps of Protocol B were followed, except that the
volume of the HMDI setting agent was reduced to 1.0 .mu.L.
TABLE-US-00046 Protocol R. SEOK 17% w/w Lysine hydrochloride 1:2
Potassium carbonate 1:1 Isocyanate:Amine 6.7; 6.5; 6.1:1
[0193] The steps of Protocol B were followed, except that the HMDI
setting agent was diluted 1:1 w/w, 1:3 w/w, or 1:5 w/w with
toluene. The volume of diluted HMDI setting agent applied to the
lap joint was 2 .mu.L, 4 .mu.L, or 6 .mu.L, respectively.
TABLE-US-00047 Protocol S. SEOK 17% w/w Lysine hydrochloride 1:2
Potassium carbonate 1:1 Isocyanate:Amine 6.1; 5.7; 5.6:1
[0194] The steps of Protocol B were followed, except that the HMDI
setting agent was diluted 1:1 w/w, 1:3 w/w, or 1:5 w/w with
methylcyclohexane. The volume of diluted HMDI setting agent applied
to the lap joint was 2 .mu.L, 4 .mu.L, or 6 .mu.L, respectively.
TABLE-US-00048 Protocol T. SEOK 17% w/w Lysine hydrochloride 1:2
Potassium carbonate 1:1 Isocyanate:Amine 8.5; 9.4; 9.7:1
[0195] The steps of Protocol B were followed, except that the HMDI
setting agent was diluted 1:1 w/w, 1:3 w/w, or 1:5 w/w with
chloroform. The volume of diluted HMDI setting agent applied to the
lap joint was 2 .mu.L, 4 .mu.L, or 6 .mu.L, respectively.
TABLE-US-00049 Protocol U. SEOK 17% w/w Lysine hydrochloride 1:2
Potassium carbonate 1:1 Isocyanate:Amine 8.0; 8.7; 7.8:1
[0196] The steps of Protocol B were followed, except that the HMDI
setting agent was diluted 1:1 w/w, 1:3 w/w, or 1:5 w/w with
methylene chloride. The volume of diluted HMDI setting agent
applied to the lap joint was 2 .mu.L, 4 .mu.L, or 6 .mu.L,
respectively. TABLE-US-00050 Protocol V1. SEEK 33% w/w Lysine
hydrochloride 1:1 Potassium carbonate 3:2 Isocyanate:Amine
5.0:1
[0197] Preparation of Protein Dope
[0198] A stock solution of buffer was prepared by dissolving lysine
hydrochloride, 9.14 mg/mL, potassium carbonate 41.6 mg/mL, and
Evans Blue dye, 0.50 mg/mL deionized water. The mixture was
filtered through a glass wool plug before use. SEEK, 113.9 mg, and
227.8 mg of stock buffer were placed in a Eppendorf vial and
agitated on a vortex mixer until dissolved. Bubbles were removed
from this solution by centrifugation at 5000 rpm for 30 seconds.
This protein dope solution was then loaded into a 1.00 mL syringe
and let stand for 20 minutes at room temperature before dispensing
to the lap joint test specimens.
[0199] Preparation of HMDI Setting Agent
[0200] The HMDI setting agent was prepared by dissolving Sudan Red
dye, 3.75 mg, in Pluronic surfactant L-61, and adding an equal
weight of 1,6-diisocyanatohexane.
[0201] Preparation of Rat Skins
[0202] Freshly harvested rat hides were stored frozen at
-20.degree. C. Just before use the hides were thawed and cut into 1
cm.times.3 cm strips. Strips were. selected which were uniform in
width and thickness and which were devoid of loose fascia and
muscle tissue. These rat skin samples were temporarily stored at
37.degree. C. between gauze pads soaked with PBS and contained in a
plastic bag to prevent drying prior to use.
[0203] Application of Adhesive
[0204] Protein dope, 35 .mu.L, was applied to one end of a rat skin
and worked into about a 1 cm2 area with 5-10 strokes of a stainless
steel spatula. The excess protein dope was transferred with the
stainless steel spatula to the second strip of rat skin and worked
in similarily. A total of 3.8 .mu.L of HMDI setting agent was
applied to the skins, apportioned so that 3 parallel stripes of
HMDI were applied to the first skin. The skins were immediately
assembled to form the lap joint, rubbed against each other to
distribute the HMDI setting agent, covered with a piece of plastic
film to prevent drying, and compressed under a 100 g weight. The
joint was allowed to cure for 15 minutes at 37.degree. C. The
length and width of the lap joint was measured to 1 mm using a
ruler immediately before tensile testing on an Instron Model 55
test machine. The crosshead speed was set at 25 mm per minute. Lap
shear tensile strengths were reported in units of g/cm2. Means and
standard deviations were calculated for measurements conducted at
least in triplicate. TABLE-US-00051 Protocol V2. SEW 33% w/w Lysine
hydrochloride 1:2 Potassium carbonate 3:2 Isocyanate:Amine
5.0:1
[0205] The method of Protocol V I was followed except that a stock
solution of buffer was prepared by dissolving lysine hydrochloride,
4.59 mg/mL, potassium carbonate 31.2 mg/mL, and Evans Blue dye,
0.50 mg/mL in deionized water. TABLE-US-00052 Protocol V3. SEW 33%
w/w Lysine hydrochloride 0:2 Potassium carbonate 3:2
Isocyanate:Amine 5.0:1
[0206] The method of Protocol V 1 was followed except that a stock
solution of buffer was prepared by dissolving potassium carbonate
13.83 mg/mL, and Evans Blue dye, 0.50 mg/mL in deionized water.
TABLE-US-00053 Protocol W1. SEOK 17% w/w Arginine 1:4 Potassium
carbonate 1.2:1 Isocyanate:Amine 13.1:1
[0207] The method of Protocol A was followed except that the buffer
was prepared using arginine, 2.4 mg/mL, potassium carbonate, 9.46
mg/mL, and Evans Blue dye, 0.50 mg/mL in deionized water. The
volume of HMDI setting agent applied to the lap joint was 2.0
.mu.L. Only the alpha amino group of the arginine is assume to
participate in the stoichoimetry of the setting reaction.
TABLE-US-00054 Protocol W2. SEOK 17% w/w Cysteine 1:2 Potassium
carbonate 1:1 Isocyanate:Amine 13.1:1
[0208] The method of Protocol A was followed except that the buffer
was prepared using cysteine, 1.38 mg/mL, and potassium carbonate,
9.46 mg/mL, and Evans Blue dye, 0.50 mg/mL in deionized water. The
volume of HMDI setting agent applied to the lap joint was 2.0
.mu.L. TABLE-US-00055 Protocol W3. SEOK 17% w/w Tyrosine 1:2
Potassium carbonate 1:1 Isocyanate:Amine 13.1:1
[0209] The method of Protocol A was followed except that the buffer
was prepared using tyrosine, 2.07 mg/mL, and potassium carbonate,
9.46 mg/mL, and Evans Blue dye, 0.50 mg/mL in deionized water. The
volume of HMDI setting agent applied to the lap joint was 2.0
.mu.L. TABLE-US-00056 Protocol W4. SEOK 17% w/w 1,3-BDSA 1:2
Potassium carbonate 1:1 Isocyanate:Amine 13.1:1
[0210] The method of Protocol A was followed except that the buffer
was prepared using 1,3-benzene disulfonic acid disodium salt
monohydrate (1,3-BDSA), 3.79 mg/mL, and potassium carbonate, 9.46
mg/mL, and Evans Blue dye, 0.50 mg/mL in deionized water. The
volume of HMDI setting agent applied to the lap joint was 2.0
.mu.L. TABLE-US-00057 Protocol X SEOK 17% w/w Peptide 1:2 (SEQ ID
NO: 35) RGRGRGKGKGK Potassium carbonate 1:1 Isocyanate:Amine
14.5:1
[0211] The method of Protocol A was followed except that the buffer
was prepared using synthetic peptide RGRGRGKGKGK (SEQ ID NO:35),
4.4 mg/mL, potassium carbonate, 9.46 mg/mL, and Evans Blue dye,
0.50 mg/mL in deionized water. The volume of HMDI setting agent
applied to the lap joint was 2.0 .mu.L. TABLE-US-00058 Protocol Y.
SEOK 17% w/w Cholinyl lysinate 1:4 Potassium carbonate 1.12:1
Isocyanate:Amine 14.5:1
[0212] The method of Protocol B was followed except that the buffer
was prepared by dissolving cholinyl lysinate, 29.2 mg, potassium
carbonate, 123.9 mg, and Evans Blue dye, 6.0 mg, in deionized
water, 13.09 mL. The buffer solution was filtered through a 0.45
micron syringe filter before use. The protein dope was prepared by
dissolving SEOK, 63.5 mg, in 310.0 .mu.L of buffer. TABLE-US-00059
Protocol Z. SEOK 17% w/w AEGIy 1:4 Potassium carbonate 1:1
Isocyanate:Amine 14.5:1
[0213] The method of Protocol B was followed except that the buffer
was prepared using 2-aminoethyl glycinate, AEGIy (see Example 1,
diamine synthesis). An aliquot, 28.9 mg, of 2-aminoethyl glycinate
in water, 236 mg/mL, and potassium carbonate, 48.6 mg, was
dissolved in water, 5.127 mL. Evans Blue dye, 2.20 mg was added,
and the solution filtered through a 0.45 micron syringe filter.
TABLE-US-00060 Protocol AA. SEEK 17% w/w Lysine hydrochloride 1:1
Potassium carbonate 1:1 Isocyanate:Amine 12.0:1
[0214] The method of Protocol V 1 was followed except that the
buffer was prepared using lysine hydrochloride, 3.73 mg/mL,
potassium carbonate, 11.33 mg/mL, and Evans Blue dye, 0.50 mg/mL in
deionized water. Pluronic surfactant L-61 was not added to the HMDI
setting agent. The protein polymer used was SE8K. The volume of
setting agent applied to the joint was 2.0 .mu.L. TABLE-US-00061
Protocol AB. SEOK-CS 1 17% w/w Lysine hydrochloride 0.94:2
Potassium carbonate 0.96:1 Isocyanate:Amine 16.4:1
[0215] The method of Protocol A was followed except that the buffer
was prepared using lysine hydrochloride, 1.84 mg/mL, potassium
carbonate, 8.37 mg/mL, and Evans Blue dye, 0.50 mg/mL in deionized
water. The protein polymer used was SEOK-CS1. The volume of setting
agent applied to the joint was 2.0 .mu.L. TABLE-US-00062 Protocol
AC. SEOK 17% w/w Lysine hydrochloride 1:2 Sodium Borate pH 9.5
Isocyanate:Amine 7.3:1
[0216] The steps of Protocol F were followed, except that the
volume of HMDI setting agent applied to the lap joint was 1.0
.mu.L. TABLE-US-00063 Protocol AD. SEOK 17% w/w Lysine
hydrochloride 1:2 Sodium Borate pH 9.5 Isocyanate:Amine 7.3:1
[0217] The steps of Protocol F were followed, except that the HMDI
setting agent was diluted 1:1 v/v, 1:3 v/v, or 1:5 v/v using
cyclohexane. The volume of diluted HMDI setting agent applied to
the lap joint was 2 .mu.L, 4 .mu.L, or 6 .mu.L, respectively.
TABLE-US-00064 Protocol AE. SEOK 17% w/w Lysine hydrochloride 1:2
Sodium Borate pH 9.5 Isocyanate:Amine 7.3:1
[0218] The steps of Protocol F were followed, except that the HMDI
setting agent was diluted *1:1 v/v, 1:3 v/v, or 1:5 v/v using
1,1,1-trichloroethane. The volume of diluted HMDI setting agent
applied to the lap joint was 2 gL, 4 gL, or 6 AL, respectively.
TABLE-US-00065 Protocol AF. SEOK 17% w/w Lysine hydrochloride 1:2
Sodium Borate pH 9.5 Isocyanate:Amine 7.3:1
[0219] The steps of Protocol F were followed, except that the HMDI
setting agent was diluted 1:1 v/v, 1:3 v/v, 1:5 v/v, or 1:9 v/v
using ethyl acetate. The volume of diluted HMDI setting agent
applied to the lap joint was 2 gL, 4 gL, 6 ItL, or 10/.LL,
respectively. TABLE-US-00066 Protocol AG. SEOK-CS1 17% w/w 1,3-PG
0.39:2 Potassium carbonate 0.496:1 Potassium bicarbonate 0.496:1
Isocyanate:Amine 2.5:1
[0220] The methods of Protocol B were followed with the following
modifications.
[0221] Preparation of Protein Dope
[0222] Protein polymer SEOK-CS 1, 81.6 mg, was added to 311 AL of
deionized water in an Eppendorf tube and agitated on a vortex
mixter until dissolved. To this solution was added 11.43 gL of a
solution of 1,3-propanediyldiglycinate (1,3-PG) in water, 10% w/w;
14.73 .mu.L of a solution of potassium carbonate in water, 10% w/w;
and 9.43 .mu.L of a solution of potassium bicarbonate in water, 10%
w/w. The contents were again agitated on the vortex mixer until
homogeneous. The solution was centrifuged at about 5000 rpm for
30-60 seconds to separate air bubbles, and i then the dope was
exposed to reduced pressure, 26 in-Hg, for 30 minutes.
[0223] Preparation of HMDI Setting Agent
[0224] Neat 1,6-diisocyanatohexane, 100 mg, and Sudan Red dye, 2.4
mg, were dissolved in 1-chloro-2,2,2-trifluoroethyl diflurormethyl
ether, 2.342 g. The volume i of diluted HMDI setting agent applied
to the lap joint was 4 .mu.L. TABLE-US-00067 Protocol AH. SEOK 17%
w/w Lysine 0:1 Potassium carbonate 1:1 Isocyanate:Amine -5.0:1
[0225] Preparation of Protein Dope
[0226] Protein dope was prepared by dissolving protein polymer SEOK
at 17% w/w in 10 mMolar aqueous lactic acid, 0.90 mg/mL. This
protein dope tested approximately pH 3.5 with wide range pH test
paper. A solution to initiate curing was prepared by dissolving
potassium carbonate, 1.66 g, in deionized water, 10 mL.
[0227] Preparation of HMDI Setting Agent
[0228] An HMDI setting agent was prepared by dissolving
1,6-diisocyanatohexane, 5.04 g, Pluronic surfactant F-127, 0.0033
g, and Sudan Red dye, 0.0020 g in chloroform, 2.24 g.
[0229] Preparation of Rat Skins
[0230] Freshly harvested rat hides were stored frozen at
-20.degree. C. Just before use the hides were thawed and cut into 1
cm.times.3 cm strips. All fascia was removed from the strips of
skin with a razor blade. Strips of skin were selected which were
uniform in width and thickness. Prepared rat skin samples were
temporarily stored at 37.degree. C. between gauze pads soaked with
PBS and contained in a plastic bag to prevent drying.
[0231] Application of Adhesive
[0232] The strips of skins were arranged on a glass plate in a
37.degree. C. warm room. Protein dope, 15 AL, was worked into an
approximately 1 cm2 area at the end of each of two rat skins using
a stainless steel spatula, 30 .mu.L total. The HMDI setting agent,
1.0 AL, was worked into an approximately 1 cm2 area at the end of
each of two rat skins using a stainless steel spatula, 2.0 .mu.L
total. The potassium carbonate curing solution, 2.0 .mu.L, was
added as 6 drops, 3 drops applied to each of the two rat skins and
the skins immediately assembled to form a lap joint. A 100 g weight
was applied to the joint and the adhesive allowed to cure for 15
minutes at 37.degree. C. The lap joint was tested to failure on an
Instron tensile testing machine as described herein.
[0233] The first study employed the surfactant Pluronic L-3 1.
TABLE-US-00068 TABLE 18 Role of Pluronic L-31 Surfactant. Lap L-
HMDI + Shear SEOK Dope K.sub.2C0.sub.3 Lys Pluronic g/cm.sub.2
Protocol 1 SEOK 17% 1:1 1:2 none 2143 .+-. 328 A w/w 2 SEOK 17% 1:1
1:2 none 2901 .+-. 685 B w/w (degassed) 3 SEOK 17% 1:1 1:2 HMDI/
1248 .+-. 370 C w/w (degassed) L-31 1:1 (3.3% w/w Wrt total dope) 4
SEOK 17% 1:1 1:2 none 745 .+-. 209 D1 w/w (degassed) 5 SEOK 17% 1:1
1:2 none 1499 .+-. 159 D2 w/w (degassed) + L-31 (0.24% w/w wrt
total dope) (1.4% w/w wrt SEOK) 6 SEOK 17% 1:1 1:2 HMDI/ 677 .+-.
284 E w/w L-31 (degassed) + 1:1 L-31 (0.17% (3.3% w/w wrt total w/w
dope) (1.0% wrt w/w wrt total SEOK) dope)
[0234] In the next sudy, various buffers were employed in
conjunction with lysine as the polyfunctional group. TABLE-US-00069
TABLE 19 Adhesive Performance Using 17% SELPOK (SEOK) in Various
Buffers at pH 9.5. Lap Shear Buffer g/cm.sup.2 CV % Protocol 1
Na3BO.sub.3/L-Lys (1:2)* 2360 .+-. 475 20% F 2 K3B0.sub.3/L-Lys
(1:2) 2037 .+-. 338 17% G 3 Li2CO.sub.3/L-Lys (1:2) 188 .+-. 38 20%
H 4 Na.sub.2CO.sub.3/L-Lys (1:2) 1168 .+-. 274 23% I 5
K2C0.sub.3/L-Lys (1:2) 2393 .+-. 631 26% J 6 Cs.sub.2CO.sub.3/L-Lys
(1:2) 233 .+-. 56 24% K 7 CaCO.sub.3/L-Lys (1:2) 88 .+-. 2 2% L
*The mole ratio of amino groups derived from lysine to amino groups
derived from SELPOK
[0235] In the next study various chemically unreactive and reactive
additives were added to the formulation to determine the effect of
the additives on shear strength. TABLE-US-00070 TABLE 20 Adhesive
Performance Using 17% SELPOK in Carbonate Buffers with Additives
Lap Shear Buffer pH g/cm.sup.2 CV % Protocol 1 K.sub.2C0.sub.3
(2:2)/L-Lys (1:2) 10.5 9.0 2901 .+-. 685 24% B M no additive 1617
.+-. 293 18% 2 K.sub.2C03 (2:2)/L-Lys (1:2) 11 9.0 2538 .+-. 441
17% N1 N2 plus KI (0.043 Mole/L) 826 .+-. 211 26% 3 K2C03
(2:2)/L-Lys 10.5 9.0 1896 .+-. 557 29% 01 02 (1:2) 1398 .+-. 358
26% plus Glucose (1.09 Mole/L) 4 K.sub.2C03 (2:2)/L-Lys (1:2) 11
9.0 2674 .+-. 846 32% PI P2 plus Urea (1.50 Mole/L) 239 .+-. 38
16%
[0236] In the next study various organic solvents were employed,
where the crosslinking agent was dissolved in the solvent prior to
mixing with the aqueous buffered protein solution. TABLE-US-00071
TABLE 21 Adhesive Performance Using HMDI Plus Volatile Diluents
with 17% SELPOK in Lysine-Borate Buffer pH 9.5. Dilution Setting
Ratio Agent Lap Shear CV Diluent bp [v/v] Volume g/cm.sup.2 %
Protocol 1 None n.a. 1:0 1 .mu.L 852 .+-. 173 20% AC 2 Cyclohexane
81.degree. 1:1 2 .mu.L 900 n.a. AD 1:3 4 .mu.L 953 1:5 6 .mu.L 1053
3 1,1,1- 75.degree. 1:1 2 .mu.L 781 .+-. 13 2% AE Trichloro 1:3 4
.mu.L 943 .+-. 119 13% ethane- 1:5 6 .mu.L 816 .+-. 51 6% 4 Ethyl
77.degree. 1:1 2 .mu.L 869 .+-. 41 40% AF Acetate 1:3 4 .mu.L 741
.+-. 296 21% 1:5 1:9 6 .mu.L 685 .+-. 147 20% 10 .mu.L 658 .+-.
131
[0237] TABLE-US-00072 TABLE 22 Adhesive Performance Using HMDI Plus
Volatile Diluents with 17% SELPOK in Lysine-Carbonate Buffer pH 10.
Setting Ratio agent Lap Shear CV Diluent by [w/w] Volume g/cm.sup.2
% Protocol 1 None n.a. 1:0 1 .mu.L 2713 .+-. 234 6% Q 2 Toluene
110.degree. 1:1 2 .mu.L 2303 .+-. 502 22% R 1:3 4 .mu.L 2295 .+-.
210 9% 1:5 6 .mu.L 1178 .+-. 282 24% 3 Methyl 101.degree. 1:1 2
.mu.L 2508 .+-. 234 9% S Cyclohexane 1:3 4 .mu.L 2160 .+-. 111 5%
1:5 6 .mu.L 1364 .+-. 395 29% 4 Chloroform 61.degree. 1:1 2 .mu.L
2075 .+-. 370 18% T 1:3 4 .mu.L 2836 .+-. 620 22% 1:5 6 .mu.L 1493
.+-. 223 15% 5 Methylene 400 1:1 2 .mu.L 2389 f 542 23% U Chloride
1:3 4 .mu.L 2636 .+-. 504 19% 1:5 6 .mu.L 2511 .+-. 493 20%
[0238] In the next study various polyfunctional agents were
employed using a variety of functionalities to crosslink the
polymer, where the functionalities were symmetrical or
unsymmetrical and the intervening chains were aliphatic or
aromatic, with different functional groups as side chains. In some
instances, the polyfunctional agents used for crosslinking were
hydrolytically unstable, having a hydrolytically susceptible bond
in the linking chain. TABLE-US-00073 TABLE 23 Adhesive Performance
Using SELP8K, SELPOK or SELPOK-CS1 With Different Polyfunctional
Agents Protein and Polyfunctional Lap Shear Buffer Ratio* Agent
Tensile g/cm.sup.2 Protocol SE8K 33% 2:2 Lysine 1328 + 203 V1 w/w
1:2 1212 .+-. 241 V2 K.sub.2C0.sub.3 3:2 0:2 1161 .+-. 383 V3 SEOK
17% 1:2 Lysine 2143 .+-. 328 W1 w/w K2C03 Arginine 1176 .+-. 748 W2
1:1 Cysteine 741 .+-. 66 W3 Tyrosine 622 .+-. 339 W4 3,5- 339
Disulfonatocatecho SEOK 17% 1:2 Peptide 850 .+-. 184 X w/w
K.sub.2C03 RGRGRGKGKGK 1:1 SEOK 17% 1:2 Lysine 2901 .+-. 685 B w/w
K.sub.2C0.sub.3 1:1 SEOK 17% 1:4 cholinyl lysinate 2385 + 502 Y w/w
K2C03 1.12:1 SEOK 17% 1:4 2-aminoethyl 3269 t 422 Z w/w K2C03
glycinate N,O- 1.12:1 Diglycyl ethanolamine 4:1 mixture as sulfate
sal SEOK-CS1 1:2 Lysine 2196 .+-. 275 AB 17% w/w K2C03 1:1 SEOK-CS1
0.77:2 1,3-Propanediyl 3028 .+-. 392 AG 17% w/w Diglycinate K2C03
1:1 *Ratio of added nucleophilic groups to amino groups available
on the protein backbone.
[0239] In Table 24, various formulations comprising either SELP8K,
SELPOK, SELPOK-CS 1 are compared. TABLE-US-00074 TABLE 24 Adhesive
Performance Using SELP8K, SELPOK, and SELPOK-CS1. K2C03 to Lap
Shear Amine Amine Tensile Polymer Ratio Ratio* g/cm.sup.2 % CV
Protocol SEEK 17% 1:1 2:2 2854 .+-. 1027 36% AA w/w SEOK 17% 1:1
1:2 2143 .+-. 328 15% A w/w SEOK 17% 1:1 0:2 532 .+-. 207 39% AH
w/w in 10 mM aq. Lactic Acid SEOK-CS1 1:1 1:2 2196 .+-. 275 13% AB
17% w/w *Ratio of amine groups derived from lysine to amine groups
derived from the protein.
[0240] It is evident from the above results, that the subject
invention provides for compositions which can set rapidly to
provide compositions having a broad range of properties. The
subject compositions can provide for strongly adhering compositions
with good shear strength, the shear strength being realized within
a short period of time. The subject invention also provides for
compositions that are capable of filling voids or holes in tissue
or otherwise augmenting the tissue. Thus, the subject proteinaceous
polymers may be employed as tissue adhesives, providing
physiologically compatible compositions which maintain their
strength for extended periods of time, while being capable of
resorption, as well as sealants, among other uses.
[0241] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0242] The invention now being fully described, it will be apparent
to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
Sequence CWU 1
1
* * * * *