U.S. patent application number 08/806029 was filed with the patent office on 2002-04-18 for synthetic proteins for in vivo drug delivery and tissue augmentation.
Invention is credited to CAPPELLO, JOSEPH, STEDRONSKY, ERWIN R..
Application Number | 20020045567 08/806029 |
Document ID | / |
Family ID | 22790163 |
Filed Date | 2002-04-18 |
United States Patent
Application |
20020045567 |
Kind Code |
A1 |
CAPPELLO, JOSEPH ; et
al. |
April 18, 2002 |
SYNTHETIC PROTEINS FOR IN VIVO DRUG DELIVERY AND TISSUE
AUGMENTATION
Abstract
Methods and compositions are provided which are useful for
delivering a biologically active substance to a localized site in
vivo and for altering the physical dimensions of a body tissue.
These methods and compositions employ protein polymers having
varying ratios of elastin-like, collagen-like, keratin-like
repeating units and repeating units which promote protein
crystallization such as silk-like repeating units. By varying the
length of segments of the repeating units and/or the concentration
of the protein polymers in the composition, the rate of delivery of
a biologically active substance to a localized site can be greatly
varied. Moreover, because the compositions are capable of acquiring
a non-liquid form under normal physiological conditions, they find
use as biocompatible tissue augmentation products.
Inventors: |
CAPPELLO, JOSEPH; (SAN
DIEGO, CA) ; STEDRONSKY, ERWIN R.; (SAN DIEGO,
CA) |
Correspondence
Address: |
FLEHR HOHBACH TEST ALBRITTON AND HERBERT
SUITE 3400
FOUR EMBARCADERO CENTER
SAN FRANCISCO
CA
94111
|
Family ID: |
22790163 |
Appl. No.: |
08/806029 |
Filed: |
February 24, 1997 |
Current U.S.
Class: |
514/17.2 ;
435/69.1; 530/329; 530/330; 530/331; 530/332 |
Current CPC
Class: |
C07K 14/00 20130101;
C07K 14/78 20130101; C07K 2319/02 20130101; A61L 31/047 20130101;
C07K 2319/61 20130101; A61K 38/00 20130101; A61L 27/227 20130101;
C07K 2319/00 20130101; D01F 4/00 20130101; A61L 17/10 20130101;
C08J 5/18 20130101; C08J 2389/00 20130101; C12N 15/11 20130101 |
Class at
Publication: |
514/2 ; 514/17;
435/69.1; 530/329; 530/330; 530/331; 530/332 |
International
Class: |
A61K 038/17; C07K
014/78; C12P 021/02 |
Claims
What is claimed is:
1. A method for delivering a biologically active substance to a
localized site in vivo, said method comprising: administering a
composition to said localized site, said composition comprising (i)
a protein polymer of at least 15 kD which comprises alternating
blocks of at least 2 units each of (a) an amino acid sequence of
from about 3 to 30 amino acids which promotes protein
crystallization and (b) an amino acid sequence element selected
from the group consisting of an elastin-like element, a
collagen-like element or a keratin-like element, and (ii) a
biologically active substance; wherein said composition acquires a
non-liquid form under physiological conditions and wherein said
biologically active substance is delivered from said non-liquid to
said localized site.
2. The method according to claim 1, wherein said amino acid
sequence which promotes protein crystallization is GAGAGS or
SGAGAG.
3. The method according to claim 1, wherein said amino acid
sequence element (b) is the amino acid sequence VPGG, APGVGV, GXGVP
or VPGXG, where X is valine, lysine, histidine, glutamic acid,
arginine, aspartic acid, serine, tryptophan, tyrosine,
phenylalanine, leucine, glutamine, asparagine, cysteine or
methionine.
4. The method according to claim 3, wherein amino acid X is valine
or lysine.
5. The method according to claim 1, wherein the delivery of said
biologically active substance occurs over an extended period of
time.
6. The method according to claim 1, wherein the step of
administering comprises injecting said composition in liquid form
which acquires a non-liquid form subsequent to injection.
7. The method according to claim 6, wherein the rate at which said
liquid form acquires a non-liquid form is decreased by the addition
to said liquid form of a compound which inhibits hydrogen
bonding.
8. The method according to claim 7, wherein said compound which
inhibits hydrogen bonding is selected from the group consisting of
urea, guanidine hydrochloride, dimethyl formamide, colloidal gold
sol, aqueous lithium bromide and formic acid.
9. The method according to claim 6, wherein the rate at which said
liquid form acquires a non-liquid form is increased by the addition
to said liquid form of a nucleating agent or accelerator.
10. The method according to claim 9, wherein said nucleating agent
is said protein polymer in precrystallized form.
11. The method according to claim 10, wherein said protein polymer
is SLP3 or SLP4.
12. The method according to claim 1, wherein said protein polymer
is about 10% (w/w) to about 50% (w/w) of said composition.
13. The method according to claim 1, wherein said biologically
active substance is selected from the group consisting of a protein
or a nucleic acid.
14. The method according to claim 13, wherein said protein has a
molecular weight of from about 350 daltons to about 500,000
daltons.
15. The method according to claim 13, wherein said nucleic acid is
from about 60 to about 22,000 bases in length.
16. The method according to claim 1, wherein said biologically
active substance is selected from the group consisting of an
anti-tumor agent, an analgesic, an antibiotic, an anti-inflammatory
compound, a hormone or a vaccine.
17. The method according to claim 1, wherein said biologically
active substance is labeled.
18. The method according to claim 2, wherein said amino acid
sequence GAGAGS or SGAGAG is repeated from 2 to 16 times per
alternating block.
19. The method according to claim 1, wherein said protein polymer
comprises an amino acid sequence selected from the group consisting
of: (a) [(VPGVG).sub.8(GAGAGS).sub.8].sub.12; (b)
[(VPGVG).sub.12(GAGAGS).sub- .8].sub.9; (c)
[(VPGVG).sub.16(GAGAGS).sub.8].sub.8; (d)
[(VPGVG).sub.32(GAGAGS).sub.8].sub.5; (e)
[(VPGVG).sub.8(GAGAGS).sub.6].s- ub.13; (f)
[(VPGVG).sub.8(GAGAGS).sub.4].sub.13; (g) [(GVGVP).sub.4GKGVP
(GVGVP).sub.3(GAGAGS).sub.4].sub.12; or (h) [GAGAGS
(GVGVP).sub.4GKGVP (GVGVP).sub.3(GAGAGS).sub.2].sub.12.
20. A composition comprising (i) a protein polymer of at least 15
kD which comprises alternating blocks of at least 2 units each of
(a) an amino acid sequence of from about 3 to 30 amino acids which
promotes protein crystallization and (b) an amino acid sequence
element selected from the group consisting of an elastin-like
element, a collagen-like element or a keratin-like element, and
(ii) a biologically active substance; wherein said composition
acquires a non-liquid form under physiological conditions.
21. The composition according to claim 20, wherein said amino acid
sequence which promotes protein crystallization is GAGAGS or
SGAGAG.
22. The composition according to claim 20, wherein said amino acid
sequence element (b) is the amino acid sequence VPGG, APGVGV, GXGVP
or VPGXG, where X is valine, lysine, histidine, glutamic acid,
arginine, aspartic acid, serine, tryptophan, tyrosine,
phenylalanine, leucine, glutamine, asparagine, cysteine or
methionine.
23. The composition according to claim 22, wherein amino acid X is
valine or lysine.
24. The composition according to claim 20, wherein said
biologically active substance is selected from the group consisting
of a protein or a nucleic acid.
25. The composition according to claim 24, wherein said protein has
a molecular weight of from about 350 daltons to about 500,000
daltons.
26. The composition according to claim 24, wherein said nucleic
acid is from about 60 to about 22,000 bases in length.
27. The composition according to claim 20, wherein said
biologically active substance is selected from the group consisting
of an anti-tumor agent, an analgesic, an antibiotic, an
anti-inflammatory compound, a hormone or a vaccine.
28. The composition according to claim 21, wherein the amino acid
sequence GAGAGS or SGAGAG is repeated from 2 to 16 times per
alternating block.
29. The composition according to claim 20, wherein said protein
polymer comprises an amino acid sequence selected from the group
consisting of: (a) [(VPGVG).sub.8(GAGAGS).sub.8].sub.12; (b)
[(VPGVG).sub.12(GAGAGS).sub- .8].sub.9; (c)
[(VPGVG).sub.16(GAGAGS).sub.8].sub.8; (d)
[(VPGVG).sub.32(GAGAGS).sub.8].sub.5; (e)
[(VPGVG).sub.8(GAGAGS).sub.6].s- ub.13; (f)
[(VPGVG).sub.8(GAGAGS).sub.4].sub.13; (g) [(GVGVP).sub.4GKGVP
(GVGVP).sub.3(GAGAGS).sub.4].sub.12; or (h) [GAGAGS
(GVGVP).sub.4GKGVP (GVGVP).sub.3(GAGAGS).sub.2].sub.12.
30. A method for altering the physical dimensions of a body tissue
in a mammal, said method comprising: introducing into or onto said
body tissue a composition comprising a protein polymer of at least
15 kD which comprises alternating blocks of at least 2 units each
of (a) an amino acid sequence of from about 3 to 30 amino acids
which promotes protein crystallization and (b) an amino acid
sequence element selected from the group consisting of an
elastin-like element, a collagen-like element or a keratin-like
element; wherein said composition acquires a non-liquid form under
physiological conditions.
31. The method according to claim 30, wherein said amino acid
sequence which promotes protein crystallization is GAGAGS or
SGAGAG.
32. The method according to claim 30, wherein said amino acid
sequence element (b) is the amino acid sequence VPGG, APGVGV, GXGVP
or VPGXG, where X is valine, lysine, histidine, glutamic acid,
arginine, aspartic acid, serine, tryptophan, tyrosine,
phenylalanine, leucine, glutamine, asparagine, cysteine or
methionine.
33. The method according to claim 32, wherein amino acid X is
valine or lysine.
34. The method according to claim 30, wherein the step of
introducing comprises injecting said composition in liquid form
which acquires a non-liquid form subsequent to injection.
35. The method according to claim 30, wherein said protein polymer
is about 10% (w/w) to about 50% (w/w) of said composition.
36. The method according to claim 31, wherein said amino acid
sequence GAGAGS or SGAGAG is repeated from 2 to 16 times per
alternating block.
37. The method according to claim 30, wherein said protein polymer
comprises an amino acid sequence selected from the group consisting
of: (a) [(VPGVG).sub.8(GAGAGS).sub.8].sub.12; (b)
[(VPGVG).sub.12(GAGAGS).sub- .8].sub.9; (c)
[(VPGVG).sub.16(GAGAGS).sub.8].sub.8; (d)
[(VPGVG).sub.32(GAGAGS).sub.8].sub.5; (e)
[(VPGVG).sub.8(GAGAGS).sub.6].s- ub.13; (f)
[(VPGVG).sub.8(GAGAGS).sub.4].sub.13; (g) [(GVGVP).sub.4GKGVP
(GVGVP).sub.3(GAGAGS)4].sub.12; or (h) [GAGAGS (GVGVP).sub.4GKGVP
(GVGVP).sub.3(GAGAGS).sub.2].sub.12.
38. The method according to claim 30, wherein said composition
further comprises a biologically active substance.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 08/212,237, filed Mar. 11, 1994, pending.
FIELD OF THE INVENTION
[0002] The field of this invention is the production and use of
bioresorbable polypeptide polymers. More specifically, the present
invention is directed to the use of bioresorbable polypeptide
polymers for the controlled release of biologically active
compounds in vivo and for altering the physical dimensions of a
body tissue.
BACKGROUND OF THE INVENTION
[0003] The rate at which an implanted material resorbs or
biodegrades within the body can be a major factor in determining
its utility as a biomaterial. So called inert materials, such as
metals, ceramics and plastics have been shown to be useful for
permanent implants. However, in applications in which a device
serves as an aid to healing or as a temporary aid in surgical
repair, a resorbable material has the advantage of not having to be
removed, once healing has occurred. Resorbable sutures and staples,
bone pins and screws, wound dressings, and injectable drug delivery
systems or depots are examples of such devices. However, there are
very few materials available today which have the physical,
chemical and biological properties necessary for the fabrication of
medical devices, which must degrade and resorb in the body without
detrimental consequences.
[0004] Various synthetic organic polymers have found use, such as
polylactides, polyglycolides, polyanhydrides and polyorthoesters,
which degrade in the body by hydrolysis. Collagen,
glycosaminoglycans and hyaluronic acid are examples of natural
implantable materials which resorb at least partially by enzymatic
degradation. However, the rates of resorption are limited to the
nature of the particular material and modifications can change the
rate of resorption, but at the same time may adversely affect the
desired properties of the product.
[0005] Illustrative of efforts to vary resorption characteristics
by compositional changes are synthetic resorbable sutures composed
of copolymers of lactide and glycolide. By varying the ratio of
lactic acid to glycolic acid, the rate of resorption may be varied.
Unfortunately, rapidly resorbing compositions tend to be soft and
weak. Slow resorbing compositions are stiff and string. However,
the hydrolytic resorption of these sutures produces acid buffered
by the tissue medium, where erosion occurs at the polymer surface.
In addition, hydrolysis may occur internally, where the resulting
acid catalyzes and accelerates the degradation of the polymer.
Thus, internal pockets of degradation can lead to rapid and
catastrophic failure of mechanical properties.
[0006] There is, therefore, a need for products which can be used
in the production of implantable devices. Such products should have
the desired mechanical properties of tensile strength, elasticity,
formability, and the like, provide for controlled resorption, and
be physiologically acceptable. Moreover, such products should allow
for ease of administration for a variety of in vivo indications
including drug delivery and tissue augmentation.
[0007] Relevant Literature
[0008] U.S. Pat. No. 5,243,038 describes the preparation of high
molecular weight, protein polymers and copolymers comprising long
segments of small repeating units. Bioactive Polymeric Systems,
Gebelein, C. G. and Carraher, C. E., eds., Plenum Press, New York,
1985; Contemporary Biomaterials, Boretos, John W. and Eden, Murray,
eds., Noyes Publications, New Jersey, 1984; and Concise Guide to
Biomedical Polymers: Their Design, Fabrication and Molding,
Boretos, John W., Thomas pub., Illinois, 1973, describe
compositions, characteristics, and applications of
biomaterials.
SUMMARY OF THE INVENTION
[0009] In one aspect, the present invention is directed to a method
for delivering a biologically active substance to a localized site
in vivo, wherein the method comprises:
[0010] administering a composition to the localized site, said
composition comprising (i) a protein polymer of at least 15 kD
which comprises alternating blocks of at least 2 units each of (a)
an amino acid sequence of from about 3 to 30 amino acids which
promotes protein crystallization and (b) an amino acid sequence
element selected from the group consisting of an elastin-like
element, a collagen-like element or a keratin-like element and (ii)
a biologically active substance; wherein said composition acquires
a non-liquid form under physiological conditions and wherein said
biologically active substance is delivered from said non-liquid
form to said localized site. In preferred embodiments of the
described method, the amino acid sequence which promotes protein
crystallization is GAGAGS or SGAGAG and/or the amino acid sequence
element (b) above is the amino acid sequence VPGG, APGVGV, GXGVP or
VPGXG where the amino acid X is valine, lysine, histidine, glutamic
acid, arginine, aspartic acid, serine, tryptophan, tyrosine,
phenylalanine, leucine, glutamine, asparagine, cysteine or
methionine, more preferably valine or lysine.
[0011] The method may provide for delivery of a biologically active
substance over an extended period of time. Biologically active
substances which find use herein are formulated into compositions
comprising the protein polymer of interest and include, for
example, proteins, nucleic acids, anti-tumor agents, analgesics,
antibiotics, anti-inflammatory compounds (both steroidal and
non-steroidal), hormones, vaccines, labeled substances, and the
like. The use of additional components in the compositions which,
for example, affect the rate at which the polymer composition
polymerizes into a non-liquid form is also provided.
[0012] In another aspect, the present invention provides
compositions which are useful in the above described method.
[0013] In yet another aspect, the present invention provides a
method for altering the physical dimensions of a body tissue in a
mammal, wherein the method comprises:
[0014] introducing into or onto said body tissue a composition
comprising a protein polymer of at least 15 kD which comprises
alternating blocks of at least 2 units each of (a) an amino acid
sequence of from about 3 to 30 amino acids which promotes protein
crystallization and (b) an amino acid sequence element selected
from the group consisting of an elastin-like element, a
collagen-like element or a keratin-like element;
[0015] wherein said composition acquires a non-liquid form under
physiological conditions.
[0016] In preferred embodiments of the described method, the amino
acid sequence which promotes protein crystallization is GAGAGS or
SGAGAG and/or the amino acid sequence element (b) above is the
amino acid sequence VPGG, APGVGV, GXGVP or VPGXG where the amino
acid X is valine, lysine, histidine, glutamic acid, arginine,
aspartic acid, serine, tryptophan, tyrosine, phenylalanine,
leucine, glutamine, asparagine, cysteine or methionine. more
preferably valine or lysine.
[0017] Other aspects will be readily apparent to the skilled
artisan upon a reading of the present specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1. Modulated Differential Scanning Calorimetry of 33%
(w/w) SELP8K Solution at 37.degree. C. The amplitude and period of
the sinusoidal heating function was 1.0.degree. C. and 60 seconds,
respectively. Upper trace corresponds to MDSC cell temperature
shown on the right-side ordinate axis. Lower curve corresponds to
the absolute magnitude of the non-reversing heat flow (left-side
ordinate axis) and shows integration of crystallization exotherm
using a heat capacity-corrected baseline. This integration includes
the mass of both the polymer and the 1.times.PBS, 20.4780 mg.
[0019] FIG. 2. Viscosity Versus Time of 20% (w/w) Solutions of
SELP5, SELP8K and SELP0K. Presented is viscosity versus time of 20%
(w/w) solutions of SELP5 (.quadrature.), SELP8K (.largecircle.),
and SELP0K (.tangle-solidup.) at 37.degree. C.
[0020] FIGS. 3A and 3B. Viscosity Versus Time of 20% (w/w)
Solutions of SELP8K and SELP5 at Various Temperatures. A. Presented
is viscosity versus time of 20% (w/w) solutions of SELP8K at
4.degree. C. (.tangle-solidup.), 23.degree. C. (.largecircle.) and
37.degree. C. (.quadrature.). B. Presented is viscosity versus time
of 20% (w/w) solutions of SELP5 at 4.degree. C. (.tangle-solidup.),
23.degree. C. (.largecircle.) and 37.degree. C. (.quadrature.).
[0021] FIG. 4. Viscosity Versus Time of 20% (w/w) Solutions of
SELP8K Containing Various Additives. Presented is viscosity versus
time of 20% (w/w) solutions of SELP8K in 1.times.PBS
(.largecircle.), 1.times.PBS containing 6M urea (.tangle-solidup.)
and 1.times.PBS containing precrystallized SELP8K (.quadrature.) at
37.degree. C.
[0022] FIG. 5. Release of Various Molecular Weight Compounds From
SELP8K Gels in 1.times.PBSA at 37.degree. C. Presented is the
release of various molecular weight compounds from SELP8K gels in
1.times.PBSA at 37.degree. C. wherein (.box-solid.) represents 20%
(w/w) SELP8K containing 0.25% dansyl-lysine (mw 379.5),
(.circle-solid.) represents 20% (w/w) SELP8K containing 0.004%
fluorescein-dextran (mw 10,000), (.tangle-solidup.) represents 20%
(w/w) SELP8K containing 1% dansyl-dextran (mw 40,000),
(.diamond-solid.) represents 20% (w/w) SELP8K containing 0.02%
fluorescein-dextran (mw 500,000) and (.quadrature.) represents 33%
(w/w) SELP8K (mw 69,814).
[0023] FIGS. 6A and 6B. Effect of SELP Composition on the Release
of 70 Kd Dansyl-Dextran. A. Presented is the rate of release of 70
Kd dansyl-dextran from 20% (w/w) SELP8 (.circle-solid.), SELP9K
(.box-solid.), SELP5 (.diamond-solid.) and SELP8K
(.tangle-solidup.) gels incubated in 1.times.PBSA at 37.degree. C.
Error bars represent one standard deviation (n=3). B. Presented is
the rate of release of 70 Kd dansyl-dextran from 40% (w/w) SELP8
(.circle-solid.), SELP9K (.box-solid.), SELP5 (.diamond-solid.) and
SELP8K (.tangle-solidup.) gels incubated in 1.times.PBSA at
37.degree. C. Error bars represent one standard deviation
(n=3).
[0024] FIG. 7. Release of Pantarin at 37.degree. C. from 33% (w/w)
SELP8K Gels. Presented is the rate of release of Pantarin at
37.degree. C. from 33% (w/w) SELP8K gels after a 4 hour
(.box-solid.) and a 24 hour (.circle-solid.) preincubation at
37.degree. C. Error bars represent one standard deviation
(n=3).
[0025] FIG. 8. DNA Release From SELP9K Gels. Presented is DNA
release from SELP9K gels. BanlI REN digested plasmid DNA containing
fragment sizes of 1374, 1097, 520, and 114 bp was codissolved with
SELP9K protein to yield a gel consisting of 20% (w/w) protein and
0.5% (w/w) DNA. Elutions were collected incrementally in TSAE
buffer at 37.degree. C. at specific time periods. Samples of the
elution medium were analyzed by PAGE on a 6% polyacrylamide gel and
stained with ethidium bromide. Lane 0, 0.5 ug of plasmid digest;
lanes 1-8, 25 ul of elution medium collected from 0-2 hours, 2-4
hours, 4-24 hours, 24-48 hours, 48-72 hours, 96-120 hours, 120-144
hours and 144-168 hours, respectively. The sizes of the bands are
given in bp.
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0026] Compositions and methods are provided for delivering a
biologically active substance of interest to a localized site in
vivo, wherein the methods employ compositions comprising
recombinant novel repetitive protein polymers which have
alternating blocks of amino acid sequences which (1) promote
protein crystallization or (2) are selected from either
elastin-like, collagen-like or keratin-like amino acid sequence
elements. Preferably, the alternating blocks are amino acid
seqeunce units which are identical or similar to those found in
natural silks and elastins; these latter polymers being referred to
herein as "SELPs" in that they are comprised of silk-like and
elastin-like amino acid sequence blocks. Also provided herein are
methods for altering the physical dimensions of a body tissue which
comprise employing the above described novel protein polymers. By
"protein polymer" is meant a polypeptide chain comprised of amino
acids which are arranged in a sequential manner within a block or
set of blocks that are repeated in tandem producing a high
molecular weight repetitive protein. Particularly, the units
employed in the preferred SELP protein polymers described herein
have the "silk-like" amino acid sequences GAGAGS or SGAGAG and the
"elastin-like" amino acid sequences VPGG, APGVGV, VPGVG or GVGVP,
although some variations are permitted as will be described below,
such as the particular order of the amino acids in the sequence and
conservative substitutions, such as, but not limited to, replacing
serine with threonine and glycine with alanine.
[0027] High molecular weight protein polymers are constructed from
"monomer" segments which appear in a repetitive fashion in the
polymer. Each monomer segment is comprised from alternating blocks
of the above described amino acid sequence units. As such, a
protein polymer as defined herein may generally be illustrated by
the following formulas:
[(C).sub.a(X).sub.b].sub.c or [(X).sub.b(C).sub.a].sub.c
[0028] where "X" represents an amino acid sequence element selected
from the group consisting of an elastin-like, collagen-like or
keratin-like unit and "b" represents the number of such units
present in the monomer segment, "C" represents an amino acid
sequence unit of from about 3 to 30 amino acid which promotes
protein crystallization and "a" represents the number of such units
present in the monomer segment and "c" represents the number of
monomer units which are repeated in the protein polymer.
[0029] The protein polymers employed in the present invention will
be at least about 15 kDa and generally not more than about 250 kDa,
usually not more than about 175 kDa, more usually not more than 125
kDa, preferably ranging from about 15 to 100 kDa and more
preferably from about 50 to 90 kDa in size. In order to achieve
repetitive protein polymers within these molecular weight ranges,
the number of repetitive monomer segments incorporated into the
polymer will provide for the desired molecular weight. In this
regard, the number of monomer segments in the polymer can vary
widely, depending upon the size of each individual monomer. Thus,
the number of monomers may vary generally from about 2 to 100,
usually from about 2 to 40, more usually ranging from about 6 to 20
and preferably from about 8 to 13.
[0030] Based upon the method of preparation, there may also be
non-repetitive amino acid units at the N- and C-termini of the
protein polymer. Usually, the terminal sequences will contribute
fewer than ten number percent of the total amino acids, more
usually fewer than five number percent of the total amino acids
present in the polymer. Generally, the terminal amino acid
sequences will range from about 0-125 amino acids, more usually
from about 0-60 amino acids, where the total number of amino acids
will generally not exceed about 100 amino acids, more usually not
exceed about 50 amino acids.
[0031] For special applications, the protein polymers may also be
modified by introducing intervening amino acid sequences between
one or more monomer segments or the alternating block units which
make up the monomer segment or by otherwise modifying one or more
amino acid residues present in the polymer. Intervening sequences
may include from about 1 to 60, usually about 3 to 40 amino acids,
and may provide for a wide variety of properties including
promotion of polymer chain interactions mediated by such things as
hydrogen bonding, salt bridges and/or hydrophobic interactions. For
example, by including amino acids which have chemically reactive
sidechains, one may provide sites for linking a variety of
chemically or physiologically active compounds, for cross-linking,
for covalently bonding compounds which may change the rate of
resorption, tensile properties, rate of release of an incorporated
biologically active compound, or the like. Thus, amino acids such
as cysteine, aspartic acid, glutamic acid, lysine and arginine may
be incorporated in these intervening sequences. Alternatively,
intervening sequences may provide for sequences that have
physiological activity, such as cell binding, specific protein
binding, enzyme substrates, specific receptor binding, and the
like. In addition, one or more amino acid residues in the polymer
may be modified, either chemically or otherwise, to provide for
novel characteristics, such as novel polymerization rates, novel
tensile strengths, novel rates of resorption in vivo, and the like.
For example, hydroxyalkylation at various amino acid sites in the
polymer may provide for such novel characteristics. In this manner,
the useful properties of the basic protein may be greatly varied in
accordance with the intended use, being tailored for specific
applications.
[0032] For the protein polymers employed herein, the ratio of the
average number of amino acid sequences elements selected from the
group consisting of elastin-like, collagen-like and silk-like units
to the average number of amino acid sequences which promote protein
crystallization per monomer segment will be in the range of about
0.5, usually about 1 to 5. For the most part, there will be at
least two protein crystallization units per monomer segment and not
more than about 16, usually not more than about 12, preferably
ranging from about 2 to 8 and more preferably from about 4 to 8.
For the elastin-like, collagen-like or keratin-like amino acid
sequence elements, there will usually be at least two per monomer
segment, more usually at least about four, generally ranging from
about 6 to 32, more usually from about 6 to 18 and preferably from
about 6 to 16. Monomer segments are generally composed of multiple
protein crystallization units followed by multiple elastin-like,
collagen-like or keratin-like units or vice versa (as shown in the
above formulas), however, insertion of one or more of one unit type
within a run of multiple units of the other unit type may also be
employed. The protein polymers which find use in the invention will
generally range from about 15-80% of amino acids provided by the
protein crystallization units.
[0033] For the most part, the "elastin-like" units employed herein
have the amino acid sequence VPGG, APGVGV, GXGVP or VPGXG, where X
is valine, lysine, histidine, glutamic acid, arginine, aspartic
acid, serine, tryptophan, tyrosine, phenylalanine, leucine,
glutamine, asparagine, cysteine or methionine, usually valine or
lysine, preferably valine.
[0034] "Collagen-like" units contain the tandemly repeated amino
acid triad GXO, where G is glycine and X and O are any amino acid
except that X and O are selected such that the proline content in
the triads of the polymer is less than about 45 number % (see U.S.
patent application Ser. No. 08/642,255, filed May 2, 1996). A
single "collagen-like unit" may comprise at least about 2 and not
more than about 100 tandemly repeated triads, more usually not more
than about 75, frequently not more than about 50, more frequently
not more than about 25. Preferably, amino acids X and O are
selected from the group consisting of alanine, isoleucine, valine,
leucine, serine, threonine, asparagine, glutamine, lysine,
arginine, aspartic acid, glutamic acid, histidine or proline.
[0035] "Keratin-like" units as defined herein have the so-called
"heptad" repeat unit consisting of a seven amino acid long stretch
with two positions separated by two amino acids, usually positions
three and six, occupied consistently with hydrophobic, aliphatic or
aromatic residues, e.g., AKLKLAE or AKLELAE (see U.S. Pat. No.
5,514,581, issued May 7, 1996).
[0036] Amino acid sequence units which promote protein
crystallization are sequences from about 3 to 30 amino acids in
length which, for the most part, possess relatively simple amino
acids with relatively low molecular weight side chains including,
for example, glycine, alanine, serine, threonine, cysteine and
valine. Because these "protein crystallization units" possess, for
the most part, relatively small molecular weight amino acids, they
are capable of forming extended chain conformations such as
.beta.-sheets or ,.beta.-strands that allow chains of the
polypeptide to come into close proximity where hydrogen bonding may
occur. These units allow the formation of ordered structures.
Different protein crystallization units as such are known in the
art and will find use in the present invention (e.g., Fossey et
al., Biopolymers 31(13):1529-1542 (1991)). Preferably, these
protein crystallization units are "silk-like" units which generally
possess the amino acid sequence GAGAGS or SGAGAG.
[0037] The amino acid sequence units and elements employed herein
can also be modified by conservative substitution of amino acids at
various positions in their sequences. For example, extensive
examples of modified elastin-like blocks have been reported
(Temperature of the inverse temperature transition for
poly[(VPGVG)n(VPGXG)m], Urry et al., Biopolymers 32:1243-1250
(1992)). Substitutions of amino acids can impart changes in the
chemical nature of the protein within which these blocks reside.
For example, the replacement of the first valine in GVGVP with a
more hydrophobic amino acid such as phenylalanine will decrease the
lower critical solution temperature at which the elastin-like
protein polymer is soluble. Replacing this valine with a more
hydrophilic amino acid such as lysine will increase the lower
critical solution temperature of the polymer solution. While these
modified elastin-like blocks may affect certain chemical or
physical properties, they can be readily chosen so as not to
destroy the ability of protein polymers containing crystallizable
silk-like blocks to acquire a non-liquid form, i.e., by gellation,
solidification, and the like.
[0038] In the protein polymers which find use herein, by varying
the ratio of the elastin-like, collagen-like or keratin-like
elements and protein crystallization units, the length of the
monomer segments comprising each of the units, the molecular
weight, any intervening sequences, modifications to the individual
repeating units, and the like, one can vary the tensile properties
of the product only moderately, such as elasticity, stiffness,
hardness, ease of processing, flexibility, the rate of resorption
after in vivo administration and the rate at which a liquid
composition of the SELP polymer acquires a non-liquid form. For
example, faster resorption can be achieved by reducing the number
of repeating silk-like units in the monomer segment below about 8
units or increasing the number of elastin-like units per monomer
segment to greater than 8, individually or in combination. Faster
gellation or crystallization of a liquid composition comprising the
protein polymer can be obtained by increasing the concentration of
the polymer in the liquid or increasing the relative number of
protein crystallization units in the protein.
[0039] The protein polymers described herein may be prepared in
accordance with the manner described in U.S. Pat. No. 5,243,038
and/or in PCT/US96/15306, both of which are expressly incorporated
herein by reference. For example, one procedure involves
synthesizing small segments of single stranded DNA of from about 15
to 150 nucleotides to provide a plurality of fragments which have
cohesive ends, which may be ligated together to form a segment or a
plurality of segments. The first dsDNA fragment is cloned to ensure
the appropriate sequence, followed by the addition of successive
fragments, which are in turn cloned and characterized, to ensure
that the integrity of the sequence is retained. The fragments are
joined together to form a monomer segment which, as described
above, then becomes the major repeating building block of the
polymer gene.
[0040] Alternatively, long single strands may be prepared, cloned
and characterized, generally being of at least 100 nucleotides and
up to about 300 nucleotides, where the two single strands are
hybridized, cloned and characterized and may then serve as the
monomer segment. The monomers may then be multimerized, having
complementary termini, particularly cohesive ends, so that the SELP
polymer will have two or more monomers present. The multimers may
then be cloned in an appropriate vector and characterized to
determine the number of monomers and the desired size polymer
selected. Expression can be achieved in an expression host using
transcriptional regulatory regions functional in the expression
host. The expression host can be prokaryotic or eukaryotic,
particularly bacterial, e.g. E. coli, B. subtilis, etc.; yeast,
e.g. Saccharomyces, Neurospora, etc.; insect cells, plant cells,
mammalian cells, and the like. If desired, a signal sequence may be
provided for secretion of the polymer. A wide variety of signal
sequences are known and have been used extensively for secreting
proteins which are not normally secreted by the expression
host.
[0041] After completion of expression, where the protein is
retained in the host, the cells are disrupted and the product
extracted from the lysate. Where the product is secreted, the
product may be isolated from the supernatant. In either case,
various techniques for purifying the products may be employed,
depending upon whether the products are soluble or insoluble in the
medium. Where insoluble, impurities may be extracted from the
polymer, leaving the polymer intact. Where soluble, the polymer may
be purified in accordance with conventional ways, such as
extraction, chromatography, or the like.
[0042] The protein polymers described herein are useful for a
variety of purposes. For example, one embodiment of the present
invention is directed to use of the herein described protein
polymers in methods for delivering biologically active substances
to localized sites in vivo. Such methods take advantage of the fact
that protein polymer compositions can by prepared in combination
with a biologically active substance of interest in a liquid
formulation which is capable of irreversibly acquiring a non-liquid
form under physiological conditions (e.g., at normal body
temperature after administration in vivo). Because the compositions
acquire a non-liquid form in vivo, they are useful for releasing a
biologically active substance incorporated therein to a localized
site. Release of the biologically active substance from the
non-liquid form appear to be a result of Fickian diffusion of the
substance from the non-liquid form. By "non-liquid form" is meant a
form which is recognized by those skilled in the art as a gel, a
solid or other form which substantially lacks the properties of
flow.
[0043] The transition from a liquid form to a non-liquid form
occurs without the need for chemical crosslinking via chemical
reaction or irradiation. In this way, no chemical changes to the
protein polymer composition or to any biologically active substance
contained in a composition thereof will occur. The rate of
gelation, solidification or crystallization may be influenced by
such things as the number of protein crystallization units in the
polymer (the greater the relative number of protein crystallization
units, the greater the rate of acquiring a non-liquid form in
vivo), the concentration of the polymer (the greater the
concentration of the protein polymer in the liquid composition, the
greater the rate of acquiring a non-liquid form in vivo),
temperature (the greater the temperature, the greater the rate of
acquiring a non-liquid form in vivo) and other solution conditions.
Generally, liquid protein polymer compositions will exhibit
sufficient working time as a liquid to allow them to be loaded into
a syringe and injected or otherwise introduced into the body. For
the most part, compositions which acquire a non-liquid form in from
about 30 seconds to about 500 minutes are preferred, more usually
from about 1 minute to about 250 minutes, more usually from about 5
minutes to about 125 minutes. The rate of release of biologically
active substances from the non-liquid form may depend on the
molecular weight of the substance, its solubility in the polymer
matrix, its charge, the composition of the polymer, including the
relative number of protein crystallization units present therein
and the conditions under which release takes place.
[0044] As such, using the present disclosure for guidance, polymer
compositions may be routinely selected to provide for varying rates
of release (i.e., quick release or sustained release over an
extended period of time) of virtually any biologically active
substance in vivo. Generally, polymer compositions which find use
in the present invention have from about 5% (w/w) to about 50%
(w/w) of the composition being protein polymer, usually from about
10% (w/w) to about 50% (w/w), more usually about 20% (w/w) to about
35% (w/w), preferably about 20% (w/w).
[0045] Because the transition from the liquid form to a non-liquid
form is believed to be mediated by hydrogen bonding occurring
between protein crystallization units present in the protein
polymers, compounds which inhibit hydrogen bonding may be employed
to decrease the rate at which the liquid form acquires a non-liquid
form. Such compounds include, for example, urea, guanidine
hydrochloride, dimethyl formamide, colloidal gold sol, aqueous
lithium bromide and formic acid. The concentrations of such
compounds which find use can be readily determined by the skilled
artisan.
[0046] Moreover, additives which increase the rate at which the
liquid composition acquires a non-liquid form may also find use in
the present invention. Such "nucleating agents" or "accelerators"
include, for example, pre-gelled protein polymers such as the SELP
or SLP protein polymers described herein, preferably SLP3 or SLP4,
aqueous solvents including, for example, ethanol, and the like. The
construction and expression of protein polymers SLP3 and SLP4 are
described in U.S. Pat. No. 5,243,038, issued Sep. 7, 1993 and have
the following amino acid sequences.
[0047] SLP 3
[0048] DPVVLQRRDWENPGVTQLNRLAAHPPFASDPMGAGS
[0049] [(GAGAGS).sub.9 GAAGY].sub.18
[0050] GAGAGSGAGAGSGAGAMDPGRYQLSAGRYHYQLVWCQK
[0051] SLP4
[0052] MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPMGAGS
[0053] [(GAGAGS).sub.6].sub.27 (GAGAGS).sub.4
[0054] GAGAMDPGRYQLSAGRYHYQLVWCQK
[0055] The concentrations of such compounds which find use can be
readily determined by the skilled artisan.
[0056] Protein polymer compositions which find use for delivering
biologically active substances to a localized site in vivo and for
altering the physical dimensions of a body tissue as described
below generally comprise alternating blocks of at least two
repeated units each of elastin-like, collagen-like or keratin-like
units and protein crystallization amino acid units per monomer.
[0057] By "biologically active substance" is meant any substance,
agent or chemical which is capable of being released from a
non-liquid form of a protein polymer-containing composition and
which performs some desired function in vivo. Biologically active
substances which find use in the present invention include, for
example, such things as oligopeptides, proteins, immunoglobulins,
growth factors, hormones, analgesics, antibiotics, vaccines,
anti-inflammatory compounds (both steroidal and non-steroidal),
nucleic acids including oligonucleotides, DNA, RNA, expression
vectors, and the like, labeled compounds, chemical compounds,
including anti-tumor and chemotherapeutic agents, liposomes, live
cells, cellular organelles and subfractions of cells, and the like.
For the most part, when proteins are employed as biologically
active substances, those proteins will generally range in size from
about 350 Da to about 500 kDa and will include oligopeptides from
about 350 Da to about 10,000 Da, usually from about 1kDa to 500 kDa
and more usually from about 10 kDa to about 400 kDa. When nucleic
acids are employed as biologically active substances, those nucleic
acids will generally be from about 10 to 22,000 bases in length,
usually from about 60 to 22,000 bases in length and preferably from
about 150 to 10,000 bases in length.
[0058] When administered in vivo, the biologically active substance
may exhibit a variety of therapeutic or diagnostic functions. For
example, the biologically active substance may be labeled with a
radioactive, fluorescein or other detectable marker and may be
employed for both diagnostic and therapeutic purposes. Labels which
find use in the present invention include, for example, contrast
agents, radioisotopes of such elements as iodine (l), including
.sup.123I, .sup.125I, .sup.131I, etc., barium (Ba), gadolinium
(Gd), technetium (Tc), including .sup.99Tc, phosphorus (P),
including 31P, iron (Fe), manganese (Mn), thallium (Tl), chromium
(Cr), including .sup.51Cr, carbon (C), including .sup.11C, or the
like, fluorescently labeled compounds, etc. Methodology for
attaching labels to different biologically active substances are
well known in the art.
[0059] The protein polymer-containing compositions may also be used
in combination with other materials, such as natural collagen,
fibrinogen, and other natural proteins, hyaluronic acid, dextran,
or other polysaccharides, or polyethylene oxide,
polyhydroxyalkanoates, or other polyesters, to produce blended
materials to provide a larger range of physical and biological
properties, for applications, such as wound dressings or membranes
for the prevention of surgical adhesions. For example, the protein
polymer SELP3 combined with sodium hyaluronate, in equal
proportions by weight, may be used to prepare a film, which
compared to pure hyaluronate gels, exhibits greater mechanical
toughness and a decreased resorption rate.
[0060] The present invention is also directed to the use of protein
polymer-containing compositions for altering the physical
dimensions of a body tissue in a mammal. For example, because of
the ability of the herein described protein polymers and
compositions thereof to dissolve in a biocompatible liquid which
may irreversibly acquire a non-liquid form under normal
physiological conditions, a liquid composition comprising one or
more different protein polymers may be administered to virtually
any site of interest in the body where it irreversibly acquires a
non-liquid form for altering the physical dimensions of a body
tissue including, for example, to fill a void, to augment an
external physical feature or to otherwise alter the size and/or
shape of a body tissue. Use of the above described protein
polymer-containing compositions for altering the physical
dimensions of a body tissue is advantageous in that the herein
described polymers are biocompatible, display little or no adverse
immunological reactivity, cause little or no undue tissue reaction
and ultimately resorb harmlessly into the body. Their degradation
products are for the most part simple amino acids which, as basic
nutrients, can be reutilized.
[0061] The presently described protein polymers have good
mechanical properties to form a wide variety of products. The
protein polymers may be drawn, molded, cast, spun, extruded, or the
like, in accordance with known ways for forming structures such as
films, formed objects, fibers, or unformed structures, such as
amorphous masses, and the like. Also, gels may be formed which may
be shaped in a variety of ways, depending upon the particular
application. The compositions can be sterilized by conventional
ways to provide sterile products.
[0062] The subject protein polymer-containing compositions can be
used to provide a wide variety of devices, such as membranes,
sutures, staples, bone pins, screws, wound dressings, and the like,
where the products may be formed prior to introduction into the
body or in situ. The compositions as formed are found to provide
the necessary mechanical properties for the particular
applications, the resorption times can be controlled so as to
ensure mechanical maintenance during the time required for
structure integrity, and at the same time ensuring that the device
or material need not be manually removed, since the material
undergoes resorption.
[0063] The SELP polymers described herein can be formulated into
liquid compositions by dissolving a polymer or a mixture thereof in
a liquid which is preferably biocompatible. As such, protein
polymer-containing solutions may be prepared in, for example,
water, saline, phosphate buffered saline or other isotonic aqueous
solution with or without other additives which may include, for
example, mannitol, glucose, alcohol, vegetable oil, and the
like.
[0064] The above described compositions may be administered or
introduced to virtually any in vivo site by a number of means.
Examples of administration techniques include, for example,
injection by syringe into a site of interest, use of trocar or
catheter, surgical implantation, placement into open wounds or
other cavities, and the like.
[0065] The following examples are offered by way of illustration
and not by limitation.
Experimental
A. Materials and Methods
[0066] E. coli strain EC3 harboring plasmids encoding each polymer
employed herein were prepared in accordance with the methods
described in U.S. Pat. No. 5,243,038 and PCT/US96/15306 (each of
which is expressly incorporated herein by reference) with the
following additions.
[0067] (1) Large Scale Plasmid Preparation
[0068] Large scale plasmid preparations from bacterial strains were
obtained from overnight cultures using Qiagen Plasmid Kits
(Qiagen-tips) and following the purification procedure recommended
by the supplier. Phosphatase treatment of DNA was performed by
resuspending ethanol precipitated DNA from a restriction enzyme
digest in 20 mM Tris-HCI 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 hr, heat inactivated for 20 min at 65.degree.
C. and then passed through a Probind filter (Millipore) and
subsequently a Bio-Spin column. The DNA was then ethanol
precipitated and resuspended in suitable buffer.
[0069] (2) Restriction Endonuclease Digestion
[0070] Restriction endonuclease (REN) digestion often employed the
restriction endonuclease buffer supplied by the enzyme
manufacturer. Whenever possible, the concentration of DNA was kept
below 1 .mu.g/25 .mu.l. Incubation was at 37.degree. C. for 1-4 hrs
for most restriction endonucleases except for BaII, Banl and Nael
digestions which were incubated overnight.
[0071] (3) Agarose DNA Ligation
[0072] Agarose DNA ligation was performed as follows. The agarose
sample was melted at 65.degree. C., the temperature was then
lowered to 37.degree. C. and ligation buffer (5.times.=100 mM
Tris-HCI, 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
hrs.
[0073] (4) DNA Purification with Filters and Columns
[0074] DNA purification employed various filters and columns. For
use of the 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. For use of the Microcon-30 filter (Amicon), the DNA containing
solution was washed by applying to the filter and exchanging twice
with H.sub.2O by spinning at 12,000 RPM for 6 min in a microfuge.
Finally, for use of the 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 min.
[0075] Purification of DNA using Ultrafree.RTM.-MC Filter Unit was
performed as follows. 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 hr; then quickly thawed at
37.degree. C. for 5 min. 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 min.
The agarose is then resuspended in 200 .mu.l of Tris-EDTA, or other
buffer, and incubated at room temperature for 30 min to allow for
elution of additional DNA from the gel. The mixture is then
centrifuged for an additional 20 min 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.
[0076] (5) Protein Expression Analysis
[0077] An overnight culture which had been grown at 30.degree. C.
was used to inoculate 50 ml of the LB media contained in a 250 ml
flask. Kanamycin was added at a final concentration of 50 .mu.g/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 hrs. The
cultures (30.degree. C. and 42.degree. C.) 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. All SELP protein polymer
molecular weights as specified herein were deduced from the gene
sequence using the computer program DNA Strider for Apple Macintosh
personal computer.
[0078] (6) Immunoblotting of Protein Gels
[0079] An alternative to the .sup.125I-Protein A detection method
described in U.S. Pat. No. 5,243,038 was also 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 lgG-biotinylated/streptavidin-HRP, or a goat or
mouse-anti rabbit lgG-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, CA. (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.
[0080] The blot was placed in 15 ml of BLOTTO solution containing
biotinylated goat anti-rabbit lgG (BioRad) diluted in BLOTTO
(1:7500) and gently agitated for 2 hrs at room temperature. The
filter was then washed for 30 min with 3 changes of TSA (50 mM
Tris-HCl pH 7.4, 0.9% NaCl, 0.2% sodium azide). The blot was then
incubated for 20 min 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 min each in TBS with
0.3% Tween 20 and then three times for 5 min each in TBS with 0.1 %
Tween 20. The blot was then incubated for 1 min 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.
[0081] (7) Amino Acid Analysis
[0082] As an alternative to the method described in U.S. Pat. No.
5,243,038, a modified Waters' Pico-Tag method was also used.
Protein samples were hydrolyzed with 6 N constant boiling HCl at
110.degree. C. for 24 hrs in vacuo. After reaction with PITC, amino
acid derivatives were detected at 254 nm by HPLC reverse phase
chromatography using ABI 140 B Dual Syringe Solvent Delivery System
with Supelco C18 column (15 um, 2.1 mm.times.25 cm) and analyzed by
Waters' Maxima 820 Data Acquisition System. The eluate A buffer is
0.14 M sodium acetate and eluate B buffer is 60% acetonitrile.
Reference to these procedures is found in User's Manuals of Waters'
Pico-Tag Method (1989 Millipore Corporation WM02, Rev.1) and ABI
140 B Dual Syringe Solvent Delivery System (1992 Applied
Biosystems, Inc. Part No. 1000-0567, Rev. C).
[0083] (8) In Vitro DNA Synthesis
[0084] In addition to the methods described in U.S. Pat. No.
5,243,038, 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 .ANG.
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 hrs. After desalting, the synthesized DNA
was amplified using PCR.
[0085] (9) Dideoxy DNA Sequencing of Double Stranded Plasmid
DNA
[0086] Plasmid DNA was prepared as described previously
(Preparation of plasmid DNA from E. coli, Small Scale, Maniatis et
al.) and sequenced by the primer extension method (Sanger et al.,
Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977) and Biggin et al.,
Proc. Natl. Acad. Sci. USA 80:3963-3965 (1983)) using
.sup.35S-deoxyadenosine 5'-(alpha-thio)-triphosphate (New England
Nuclear) as label. Primers were synthesized using a DNA synthesizer
as described previously. The sequencing reactions were done using
Sequenase (United States Biochemicals) and the conditions were as
recommended by the supplier. All sequences were run on 6 or 8%
polyacrylamide gels containing 8 M urea (Sanger et al., FEBS
Letters 87:107-110 (1978)). Storage and analysis of data utilized
software from DNA Inspection lie, DNAid, MacVector DNA Strider or
MacDNAsis for Apple Macintosh personal computer.
[0087] (10) PCR amplification:
[0088] The PCR reaction was performed in a 100 .mu.l volume in a
Perkin Elmer thin-walled Gene Amp.TM. reaction tube. Approximately
1 .mu.l of each primer DNA (corresponding to a 0.1 .mu.M final
concentration) was added to 1.times.PCR buffer (supplied by Perkin
Elmer as 10.times.solution), 200 .mu.M of each dNT, 5U 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 min 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 an
Ultrafree-Probind filter unit (Millipore) at 12,000 rpm for 30
seconds in a Sorvall Microspin 24S to remove the AmpliTaq enzyme.
The buffer was then exchanged with H.sub.2O two times, using a
Microcon-30 filter (Amicon) by spinning at 12,000 RPM for 6 min in
a microfuge. Salts and glycerol were removed from the amplified
dsDNA using a Bio-Spin 6 column (from BioRad) equilibrated in TEAB,
in a Sorvall RC5B centrifuge using an HB4 rotor at 2,500 RPM for 4
min. The DNA was then concentrated in vacuo.
[0089] (11) Fermentation Conditions
[0090] In addition to the method disclosed in U.S. Pat. No.
5,243,038, medium A as shown in Table 1 was also employed as the
fermentor medium. The starting volume in the case of 10 liter
fermentation, is no less than 3 L, and in the case of an 80 liter
fermentation, is no less than 30 liters.
[0091] If the fermentor starting volume is less than the final
volume desired, then when the carbon source concentration reaches
1%, a concentrated solution (5.times.) of medium A is added to the
fermentor in order to keep the carbon source concentration
approximately 1%.
[0092] Other fermentors used for the expression of protein polymers
were usually a 15 l MBR, 10 l working volume, or a 100 l New
Brunswick Scientific, NBS Fermacell, model F-130 fermentor, 80 l
working volume. The choice of the fermentor and its size is not
critical. Any media used for the growth of E. coil 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.).
[0093] The fermentation method used to express protein polymers in
E. coli was the fed-batch method. 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
(Fiestchko and Ritch, Chem. Eng. Commun. 45:229-240 (1986) and Seo
and Bailey, Biotechnol. Bioeng. 28:1590-1594 (1986).
1TABLE 1 Fermentation Media Composition Medium A Constituent g/l
(NH.sub.4)SO.sub.4 5.6 K.sub.2HPO.sub.4 6.7 MgSO.sub.4.7H.sub.2O
7.8 NaH.sub.2PO.sub.4.H.sub.2O 3.8 EDTA 0.98 Trace Elements 1 ml
Yeast Extract or NZ Amine 50 Glucose or glycerol 20 Kanamycin or
ampicillin 5 .times. 10.sup.-3
B. EXAMPLE 1
Specific SELP Gene Design, Construction and Expression
[0094] SELP polymers having at least one modified elastin-like unit
in the repeating monomer having an amino acid sequence GKGVP have
the designation "K" placed after the polymer number.
[0095] (1) SELP0K
[0096] The design, construction and expression of the SELP0K
polymer is described in WO 96/34618, which is expressly
incorporated herein by reference.
[0097] (2) SELP5
[0098] (a) Gene Construction
[0099] Plasmid pPSY1393 (U.S. Pat. No. 5,243,038) was digested with
Aval REN and the 60 bp fragment containing the ELP gene monomer was
purified using agarose gel electrophoresis followed by NACS column
purification and ligated in agarose with pSY1255 (U.S. Pat. No.
5,243,038) previously digested with Aval REN. The products of the
ligation mixture were transformed into E. col strain HB101. Plasmid
DNA from transformants was purified and analyzed by digestion using
EcoRV and Nrul RENs. Clones matching the correct restriction
pattern were further analyzed by treatment with BanlI with and
without Banl RENs. Plasmid pPT0257 containing 3 repeats of the ELP
gene fragment was used for subsequent constructions.
[0100] Plasmid DNA pSY1398 (U.S. Pat. No. 5,243,038) was treated
with Banl REN. The digestion fragments were purified using agarose
gel electrophoresis followed by NACS column purification. The DNA
fragment containing the SLP4 gene monomer was ligated, using
Hexamine Cobalt Chloride (HCC), with pPT0134 (U.S. Pat. No.
5,496,712) previously digested with Fokl REN, then treated with
phenol/chloroform followed by chloroform and then ethanol
precipitated. The products of the ligation mixture were transformed
into E. coli strain HB101. Plasmid DNA from transformants was
purified and analyzed by digestion using Nrul and Xmnl RENs.
Plasmid pPT0255 containing the SLP4 DNA gene fragment was used for
subsequent constructions.
[0101] Plasmid DNA pPT0257 was treated with BanlI REN. The
digestion fragments were purified using low melting point agarose
gel electrophoresis. The DNA fragment containing the SELP gene
monomer was ligated, using agarose ligation conditions, with
pPT0255 previously digested with BanlI REN. The products of the
ligation mixture were transformed into E. coli strain HB101.
Plasmid DNA from transformants was purified and analyzed by
digestion using Nrul and EcoRV RENs. Plasmid pPT0260 containing the
SELP5 DNA gene monomer (see Table 2) was used for subsequent
polymerization.
2TABLE 2 Nucleotide and Amino Acid Sequence of SELP5 Gene Monomer
GGT GCC GGC AGC GGT GCA GGA GCC GGT TCT GGA GCT GGC GCG GGC TCT G A
G S G A G A G S G A G A G S GGA GTA GGT GTG CCA GGT GTA GGA GTT CCG
GGT GTA GGC GTT CCG GGA G V G V P G V G V P G V G V P G GTT GGT GTA
CCT GGA GTG GGT GTT CCA GGC GTA GGT GTG CCC GGG GTA V G V P G V G V
P G V G V P G V GGC GTT CCG GGA GTA GGG GTG CCA GGT GTA GGA GTT CCG
GGT GTA GGC G V P G V G V P G V G V P G V G GTT CCC GGG GTA GGC GTT
CCG GGA GTA GGG GTG CCA GGT GTA GGA GTT V P G V G V P G V G V P G V
G V CCG GGT GTA GGC GTT CCC GGG GTA GGA GTA CCA GGG GTA GGC GTC CCT
P G V G V P G V G V P G V G V P GGA GCG GGT GCT GGT AGC GGC GCA GGC
GCG GGC TCT GGC GCG GGC GCA G A G A G S G A G A G S G A G A GGA TCC
GGC GCA GGC GCT GGC TCA GGT GCT GGA GCA GGA AGC GGA GCG G S G A G A
G S G A G A G S G A
[0102] (b) Polymer Gene Construction
[0103] Plasmid DNA from pPT0260 was digested with Fokl REN and the
digestion fragments were separated by agarose gel electrophoresis.
The SELP5 gene monomer, 384 bp, was excised and purified by NACS
column purification. The purified fragment was ligated, using HCC,
with plasmid pSY1262 which has been described previously (U.S. Pat.
No. 5,243,038). Plasmid DNA pSY1262 was digested with BanI REN,
then treated with calf intestinal phosphatase (CIP) followed by
phenol/chloroform and chloroform extractions and finally ethanol
precipitated as previously described.
[0104] 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 SELP5 multiple DNA
insertions. Several clones were obtained. Three clones were used
for expression analysis. pPT0263, pPT0264, and pPT0265 contained
polymer gene inserts of approximately 3.0, 2.6, and 2.2 kb,
respectively.
[0105] (c) Expression Analysis
[0106] E. Coli strain HB101 containing plasmids pPT0263, pPT0264,
and pPT0265 were grown as described. The proteins produced by these
cells were analyzed by SDS-PAGE for detection of bands
immunoreactive with anti-SLP or anti-ELP antibodies. Each clone
produced a strong reactive band with observed apparent molecular
weights of approximately 105, 90 and 70 Kd.
[0107] Amino Acid Sequence of SELP5 pPT0263
[0108] Amino Acids: 953 MW: 74,808 Dalton
[0109] MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM
[0110] GAGS (GAGAGS).sub.2
[0111] [(GVGVP).sub.16 (GAGAGS).sub.8].sub.6
[0112] (GVGVP).sub.16 (GAGAGS).sub.5 GAGA
[0113] MDPGRYQLSAGRYHYQLVWCQK
[0114] (3) SELP8
[0115] (a) Gene Construction
[0116] Plasmid pSY1378 (U.S. Pat. No. 5,243,038) was digested with
BanI REN and a fragment of the SLP4 gene was purified using agarose
gel electrophoresis followed by NACS column purification. The DNA
was ethanol precipitated in 2.5 M ammonium acetate and ligated with
pPT0134 previously digested with Fokl REN, extracted with
phenol/chloroform and ethanol precipitated. The products of the
ligation mixture were transformed into E. coli strain HB101.
Plasmid DNA from transformants was purified and analyzed by
digestion using Nrul and Xmnl RENs. Plasmid pPT0255 contained the
desired restriction pattern and was used for subsequent
constructions.
[0117] Plasmid DNA pPT0255 was treated with Cfr10l 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 HB101.
Plasmid DNA from transformants was purified and analyzed by
digestion using Nael and Stul RENs. Plasmid pPT0267 containing the
desired deletion was used for subsequent constructions.
[0118] The following two oligonucleotide strands were synthesized
and purified as described in Materials and Methods:
[0119] 5'-CTGGAGCGGGAGCCTGCATGTACATCCGAGT-3'
[0120] 3'-CGAGACCTCGCCCACGGACGTACATGTAGGCTCA-5'
[0121] The two oligonucleotide strands were annealed and ligated
with the DNA of plasmid pPT0267 which has been previously digested
with Banll and Scal RENs, and purified by agarose gel
electrophoresis followed by NACS column purification. The products
of this ligation reaction were transformed into E. coil 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.
[0122] Plasmid DNA pSY1298 (U.S. Pat. No. 5,243,038) was digested
with Banl REN, and the SELP0 gene fragment was purified by agarose
gel electrophoresis followed by NACS column purification and then
ligated to pPT0287 digested with Banl REN. The enzyme was removed
using phenol/chloroform extraction and the DNA was concentrated by
ethanol precipitation. 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 Banl, AhalI and Stul RENs.
Plasmid pPT0289, contained the desired SELP8 monomer sequence (see
Table 3).
3TABLE 3 Nucleotide and Amino Acid Sequence of SELP8 Gene Monomer
BanI BanII GGT GCC GGT TCT GGA GCT GGC GCG GGC TCT GGA GTA GGT GTG
CCA GGT G A G S G A G A G S G V G V P G GTA GGA GTT CCG GGT GTA GGC
GTT CCG GGA GTT GGT GTA CCT GGA GTG V G V P G V G V P G V G V P G V
SmalI GGT GTT CCA GGC GTA GGT GTG CCC GGG GTA GGA GTA CCA GGG GTA
GGC G V P G V G V P G V G V P G V G BanII GTC CCT GGA GCG GGT GCT
GGT AGC GGC GCA GGC GCG GGC TCT GGA GCG V P G A G A G S G A G A G S
G A
[0123] (b) Polymer Gene Construction
[0124] Plasmid DNA from pPT0289 was digested with BanI REN and the
digestion fragments were separated by agarose gel electrophoresis.
The SELP8 gene fragment, 192 bp, was excised and purified by NACS
column. The purified fragment was ligated with plasmid pSY1262 that
was digested with BanI REN. The DNA was then treated with SAP and
then passed through a Millipore Probind filter by microfuging for
30 min at 12,000 rpm and ethanol precipitated.
[0125] The product of this ligation reaction was transformed into
E. coli strain HB101. Transformants were selected for resistance to
kanamycin. Plasmid DNA from individual transformants was purified
and analyzed for increased size due to SELP8 multiple DNA
insertions. Several clones were obtained. Three clones were used
for expression analysis, pPT0301, pPT0302, and pPT0303 containing
4.4, 4.0 and 2.5 kb polymer gene inserts, respectively.
[0126] (c) Expression Analysis
[0127] E. coli strain HB101 containing plasmid pPT0303 was grown as
described above. The proteins produced by these cells were analyzed
by SDS-PAGE for detection of reactivity to SLP antibodies as
described in Materials and Methods. A strong reactive band was
observed with an apparent molecular weight of approximately 75 kD.
The expected amino acid sequence of the SELP8 polymer gene product
encoded by pPT0303 as deduced from the gene sequence is shown
below:
[0128] Amino Acid Sequence of SELP8 pPT0303
[0129] Amino Acids: 889 MW: 69,977 daltons
[0130] MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM
[0131] GAGSGAGAGS
[0132] [(GVGVP).sub.8 (GAGAGS).sub.4].sub.12
[0133] (GVGVP).sub.8 (GAGAGS).sub.2
[0134] GAGAMDPGRYQLSAGRYHYQLVWCQK
[0135] (4) SELP8K
[0136] The design, construction and expression of the SELP8K
polymer is described in WO 95/23611, which is expressly
incorporated herein by reference.
[0137] (5) SELP9K
[0138] (a) Gene Construction
[0139] A 93 base oligonucleotide strand coding for a portion of the
gene monomer (Table 4) was synthesized using an Applied Biosystems
DNA synthesizer model 381A and a 2000 .ANG. synthesis column
supplied by Glen Research. During the synthesis, the required
interrupt-pause steps, for reagent bottle changes, were minimized.
After the synthesis, the 93 base oligonucleotide strand was
deprotected and cleaved from the column support by treatment with
ammonium hydroxide at 55.degree. C. for 6 hrs.
[0140] Table 4: Synthetic DNA Used in Construction of SELP9K
Monomer
[0141] 5' ATGGCAGCGAAAGGGGACCGGTGCCGGCGC
AGGTAGCGGAGCCGGTGCGGGCTCAAAAAG GGCTCTGGTGCCTTTCCGCTAAAGTCCTGCCGT
3'
[0142] Two additional strands were synthesized and used as primers
for PCR amplification. The synthesis and purification of these DNA
primers was performed as described in Materials and Methods. The
sequences of the two primers are:
[0143] 5'-AAGAAGGAGATATCATATGGCAGCGAAAGGGGACC-3'
[0144] 5'-CGCAGATCTTTAAATTACGGCAGGACTTTAGCGGAAA-3'
[0145] PCR amplification and the reaction product purification were
conducted as described in Materials and Methods.
[0146] The DNA was resuspended and digested with BanI REN. The
digested DNA was separated by low-melting agarose gel and ligated
with pPT0285 (U.S. Pat. No. 5,496,712) which has been previously
digested with Banl REN and purified by agarose gel electrophoresis
followed by NACS column purification. The products of this ligation
reaction were transformed into E. coli strain HB101. Plasmid DNA
from transformants was purified and digested with BanlI REN.
Plasmid DNA from two clones that gave the correct digestion pattern
was sequenced. One plasmid DNA, designated pPT0358, was found to be
correct and chosen for further constructions.
[0147] Plasmid pPT0340 was digested with BanII REN, and the 156bp
fragment containing the SELP0K gene monomer was purified using
agarose gel electrophoresis followed by an Ultrafree MC spinfilter
and removal of salt on a Biospin column. The fragment was ligated
with pPT0358 previously digested with Banl REN and the larger
fragment was purified by agarose gel electrophoresis followed by
purification on an Ultrafree MC spinfilter and a Biospin column.
The products of the ligation mixture were transformed into E. coli
strain HB101. Plasmid DNA from transformants was purified and
analyzed by digestion using Banl REN. Plasmid pPT0360 containing a
dimer of the SELP0K gene fragment was used for subsequent
constructions.
[0148] Plasmid DNA pPT0360 was treated with BanI REN. The SELP0K
dimer gene fragment was purified using agarose gel electrophoresis
followed by an Ultrafree MC spinfilter and desalted on a Biospin
column. The fragment was ligated with pPT0134 previously digested
with Fokl REN, passed through a Millipore Probind filter by
microfuging for 3 min at 12,000 rpm, treated with SAP, passed
through another Millipore Probind filter and desalted on a Biospin
column. 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 pPT0363
containing the desired DNA fragment was used for subsequent
constructions.
[0149] Plasmid DNA pPT0363 was treated with EcoNI REN. The
digestion was passed through a Millipore Probind filter by
microfuging for 3 min at 12,000 rpm, desalted on a Biospin and
self-ligated. 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 pPT0365
containing the desired deletion was treated with BsrFl REN. The
digestion was passed through a Millipore Probind filter by
microfuging for 3 min at 12,000 rpm, desalted on a Biospin column
and self-ligated. The products of the ligation mixture were
transformed into E. coli strain HB101. Plasmid DNA from
transformants was purified and analyzed by digestion using
Asp700and NgoMI RENs. Plasmid pPT0366 containing the desired SELP9K
gene monomer (Table 5) was used for the polymer gene
construction.
4TABLE 5 Nucleotide and Amino Acid Sequence of SELP9K Gene Monomer
GGT GCC GGT GCG GGC TCT GGT GTT GGA GTG CCA GGT GTC GGT GTT CCG G A
G A G S G V G V P G V G V P GGT GTA GGC GTT CCG GGA GTT GGT GTA CCT
GGA AAA GGT GTT CGG GGG G V G V P G V G V P G K G V P G GTA GGT GTG
CCG GGC GTT GGA GTA CCA GGT GTA GGC GTC CCG GGA GCG V G V P G V G V
P G V G V P G A GGT GCT GGT AGC GGC GCA GGC GCG GGC TCT GGT GCA G A
G S G A G A G S G A
[0150] (b) Polymer Gene Construction
[0151] Plasmid DNA from pPT0366 was digested with Fokl REN and then
passed through a Millipore Probind filter by microfuging for 3 min
at 12,000 rpm. The digestion fragments were separated by agarose
gel electrophoresis. The SELP9K gene fragment, 174 bp, was excised
and purified by Ultrafree-MC 0.45 micron spin filter (Millipore)
and followed by Bio-Spin 6 column (BioRad) equilibrated with Dl
water, in a Sorvall RC5B centrifuge using an HB4 rotor at 2,500 RPM
for 4 min. The purified fragment was ligated with plasmid pPT0317
previously digested with BanI REN, then passed through a Millipore
Probind filter and followed by a Bio-Spin 6 column. The DNA was
then treated with SAP and passed through a Millipore Probind filter
and a Bio-Spin 6 column.
[0152] The product of this ligation reaction was transformed into
E. coli strain MM 294 (CGSC 6315) RecA. Transformants were selected
for resistance to kanamycin. Plasmid DNA from individual
transformants was purified and analyzed for increased size due to
SELP9K multiple DNA insertion. Several clones were obtained. Four
clones were used for expression analysis. pPT0377 was shown to
contain a 2.08 kb polymer gene insert.
[0153] (c) Expression Analysis
[0154] E. coli strain MM 294 containing plasmid pPT0377 was grown
as described in Materials and Methods. The proteins produced by
these cells were analyzed by SDS-PAGE for immunoreactivity with
anti-ELP antibodies. A strong reactive band was observed with an
apparent molecular weight of 62 Kd.
[0155] Amino Acid Sequence of SELP9K pPT0377
[0156] Amino Acids: 749 MW: 60,066 daltons
[0157] MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM
[0158] [GAGAGS(GVGVP).sub.4GKGVP (GVGVP).sub.3
(GAGAGS).sub.2].sub.12
[0159] GAGAMDPGRYQDLRSHHHHHH
C. EXAMPLE 2
Polymer Production
[0160] E. coli strain EC3 containing the respective plasmid
encoding each polymer shown in Table 6 below was prepared in
accordance with the methods described in U.S. Pat. No. 5,243,038.
Each strain was then fermented using a fed-batch method. These
polymer samples were used for the production of SELP polymer films,
sponges and fibrous meshes as described below (see Examples 3, 4
and 5).
[0161] Biomass for each polymer was harvested from the fermentation
broth by centrifugation in a Sorval RC3B using a H6000A rotor at
5,000 rpm for 30 min at 10.degree. C. to yield a packed cell paste.
500 grams of cell paste was resuspended in 2 liters of 50 mM Tris
buffer (pH=8.0). The cell slurry was homogenized using a Manton
Gaulin cell disrupter at 7,000-8,000 psi with three complete passes
of the liquid. The cell homogenate was passed through a chilled
heat exchanger to maintain the temperature at 15.degree. C. or
less. Pancreatic DNAse was added to the homogenate to a final
concentration of 1 .mu.g/ml and stirred at room temperature for 2
hrs. The homogenate was centrifuged in a Sorval RC3B centrifuge
using a H6000A rotor at 5,000 rpm for 1 hr at 10.degree. C.
[0162] For SELP0, 3, 7 and 8 preparations, the supernatant was
placed into 12-14,000 molecular weight cut-off dialysis bags and
dialyzed against 2 changes of 100.times.volume of 20 mM sodium
acetate buffer (pH=4.7) for 24 hrs. The contents of the bags were
transferred to centrifuge bottles and centrifuged in a Sorval RC3B
centrifuge using a H6000A rotor at 5,000 rpm for 1 hr at 10.degree.
C. The supernatant was removed to a large beaker and the pH
adjusted to 8.0 by addition of 30% ammonium hydroxide. Saturated
ammonium sulfate was then added to reach a final concentration of
20% for SELP0, 25% for SELP3 and 8 and 33% for SELP7. The solution
was stirred at room temperature for 1 hr. The solution was
centrifuged in a Sorval RC3B using a H6000A rotor at 5,000 rpm for
30 min at 10.degree. C. The pellet was resuspended in 2 liters of
deionized water, placed in dialysis bags and dialyzed against 3
changes of deionized water of 100.times.volume over 48 hrs. The
contents of the bags were shell frozen and lyophilized to
dryness.
[0163] For preparations of SELP4 and 5, the centrifuged homogenate
supernatant was directly precipitated with ammonium sulfate at a
concentration of 25%. The solution was then centrifuged in a Sorval
RC3B using a H6000A rotor at 5,000 rpm for 1 hr at 10.degree. C.
The pellet was resuspended in 5 liters of 4M LiBr and stirred at
4.degree. C. for 16 hrs. The solution was centrifuged in a Sorval
RC3B centrifuge using a H6000A rotor at 5,000 rpm at 10.degree. C.
for 1 hr. The pH of the supernatant was adjusted to pH 3.7 by slow
addition of 1 M acetic acid at 4.degree. C. The solution was
centrifuged in a Sorval RC3B using a H6000A rotor at 5,000 rpm at
10.degree. C. for 1 hr. The supernatant pH was adjusted to 8.0 by
addition of ammonium hydroxide and then dialyzed against 3 changes
of 100.times.volume deionized water over 48 hrs. The solution was
removed from dialysis and centrifuged in a Sorval RC3B using a
H6000A rotor at 5,000 rpm at 10.degree. C. for 1 hr. Saturated
ammonium sulfate was added to the supernatant to reach 25% of
saturation and stirred for 1 hr. The solution was centrifuged in a
Sorval RC3B using a H6000A rotor at 5,000 rpm at 10.degree. C. for
1 hr. The pellet was dissolved in 4.5M LiBr, placed in dialysis
bags and dialyzed against 3 changes of 100.times.volume of
deionized water. The contents of the bags were shell frozen and
lyophilized to dryness.
[0164] All reagent solutions used in the following procedures were
depyrogenated prior to use by filtration through a 10,000 nominal
molecular weight cut-off hollow fiber cartridge (AG Technologies).
All glassware and utensils used were sterilized and depyrogenated
by heating at 180.degree. C. for 4 hrs. 4-5 grams of all SELP dried
polymers were dissolved in 1.2 liters of 10M urea. 20 mis of 2M
Tris pH 8.0 and 780 mis of milli-Q water were added. The solution
was sonicated to promote full dissolution of the protein. 500 grams
of Whatman DE52 ion exchange resin was prepared by precycling
through acid and base treatment as recommended by manufacturer
prior to and in between each usage. The resin was finally
equilibrated with 6M urea, 20 mM Tris pH 8.0 in a beaker with
gentle stirring. The resin was filtered in a buchner funnel until
excessive liquid was removed. The cake of resin was placed in a
beaker and the protein solution was added. The slurry was stirred
gently for 1 hr. The slurry was filtered in a Buchner funnel and
the liquid was collected in a cleaned vacuum flask. 500 grams of
fresh precycled and equilibrated resin was added to a depyrogenated
vacuum beaker and the filtered solution was added. The slurry was
stirred gently for 1 hr and filtered again. The filtered solution
was once more combined with 500 grams of freshly precycled and
equilibrated resin, stirred for 1 hr, and filtered. The final
filtered solution was placed in 6,000 molecular weight cut-off
dialysis bags which had been soaked in 0.5N NaOH for at least 24
hrs. The solution was dialyzed against 3 changes of
100.times.volume of deionized water. The dialyzed solution was
removed from the bags, placed in depyrogenated lyophilization
flasks and lyophilized to dryness. Employing the above procedure,
the following polymers were prepared.
5TABLE 6 Protein Polymers Domain Polymer (MW) Polymer Block
Sequence.sup.1 Abbr..sup.2 E/S.sup.3 % S.sup.4 SELP0 (80,502)
[(VPGVG).sub.8(GAGAGS).sub.2].sub.18 E8S2 4.0 21.9 SELP8 (69,934)
[(VPGVG).sub.8(GAGAGS).sub.4].sub.13 E8S4 2.0 35.3 SELP7 (80,338)
[(VPGVG).sub.8(GAGAGS).sub.6].sub.13 E8S6 1.33 45.0 SELP3 (84,267)
[(VPGVG).sub.8(GAGAGS).sub.8].sub.12 E8S8 1.0 51.9 SELP4 (79,574)
[(VPGVG).sub.12(GAGAGS).sub.8].sub.9 E12S8 1.5 42.2 SELP5 (84,557)
[(VPGVG).sub.16(GAGAGS).sub.8].sub.8 E16S8 2.0 35.7 .sup.1The first
and last block domain of each polymer is split within the silk
blocks such that both parts sum to a whole domain. All polymers
also contain an additional head and tail sequence which constitutes
approximately 6% of the total amino acids. .sup.2Designates the
number of consecutive blocks per repeating domain (E = elastin-like
block, S = silk-like block) .sup.3Ratio of blocks per polymer.
.sup.4% of total amino acids in polymer contributed by silk-like
blocks.
[0165] Other polymers which were prepared include:
[0166] [(VPGVG).sub.32 (GAGAGS).sub.8].sub.5, referred to as SELP6;
and
[0167] {(GAGAGS).sub.12 GAAVTGRGDSPASAAGY
(GAGAGS).sub.5(GVGVGP).sub.8].su- b.7, referred to as SELPF.
[0168] Additional polymer samples including:
[0169] [(GAGAGS).sub.2 (GVGVP).sub.4 GKGVP (GVGVP).sub.3].sub.6,
referred to as SELP0K;
[0170] [(GVGVP).sub.4 GKGVP (GVGVP).sub.3 (GAGAGS).sub.4].sub.12,
referred to as SELP8K; and
[0171] [GAGAGS (GVGVP).sub.4 GKGVP (GVGVP).sub.3
(GAGAGS).sub.2].sub.12, referred to as SELP9K, were prepared
according to the following methods. These polymer samples were used
for the production of SELP gels as described below in Examples 6,
7, 8, 9 and 10. These protein polymer samples were produced from
fermented biomass conducted under a glucose fed-batch fermentation
process with minimal salts medium as described in Materials and
Methods. SELP8, SELP8K, SELP9K and SELP5 polymers were produced
using E. coli strains pPT0303, pPT0345, pPT0377 and pPT0263,
respectively. SELP8, SELP8K and SELP5 were fermented using an NBS
Fermacell, model F-130 fermentor (New Brunswick Scientific, Inc.)
and SELP9K was fermented using an MBR LAB 15L GLAS STAHL NR264
fermentor (MBR Bioreactor AG). The biomass obtained from each
fermentation run was separated from the medium by centrifugation,
labeled, and stored frozen at -20.degree. C. prior to
purification.
[0172] SELP8 batch 96083
[0173] The SELP8 batch 96083 was produced by combining two SELP8
preparations, 96071 and 96073, during the final steps of
purification. Each preparation started with 1500 grams of frozen
biomass that was resuspended in 10 liters of 20 mM Tris pH 8.0 with
10 mM EDTA. The suspension was mixed with a polytron mixer (Janke
& Kunkel, Kika-werk, Ultra Turrex model T45-S4) until
homogeneous. The suspension was passed three times through a Gaulin
Model 15 M homogenizer at 8000 psi and the temperature was
maintained below 15.degree. C. throughout the lysis by means of a
heat exchanger. After the first pass, 200 mis of 0.2 M
phenylmethylsulfonyl fluoride (PMSF) in isopropanol was added. The
lysate was centrifuged for 30 min at 8000 rpm at 4.degree. C. in a
Sorvall RC5B. The supernatant (8.3 liters) was decanted and then
placed in Spectrapore dialysis bags (MWCO=12-14 kdco) and dialyzed
for 3 days at 4.degree. C. against three changes of 100 liters of
20 mM sodium acetate, pH 4.7. The dialysate was centrifuged in a
DuPont/Sorvall RC5B centrifuge for 1 hr at 8000 rpm at 10.degree.
C. 1300 mls of saturated ammonium sulfate was added dropwise to the
supernant (5.2 liters) at room temperature while stirring. This
mixture was allowed to stir for 1 hr, then it was centrifuged for
30 min at 8000 rpm at 10.degree. C. in the RC5B. The pellet was
redissolved in 500 mls of milli-Q water and dialyzed in Spectrapore
dialysis bags (MWCO=12-14 kdco) against three changes of 100 liters
of Milli-Q water over three days.
[0174] This polymer solution was then applied to a Poros HQ50
(Perseptive) anion exchange column prepared as follows. Each step
using the column, including the application and elution of the
polymer solution, was done at 40 psi. An Amicon VA-180 column
containing 4.0 liters of Poros HQ50 resin was depyrogenated by
flushing 15 liters of 1.0 M NaOH/1.0 M NaCl through the column and
downstream apparatus. The column was left in contact with this
solution overnight. Tris buffer and Dl water, used to equilibrate
the column, were filtered through an AG/T hollow fiber
ultrafiltration module (10,000 MWCO) which had previously been
soaked in 0.5 N NaOH overnight and thoroughly flushed with Dl
water. Glass sample collection vessels were baked in a dry heat
oven at 180.degree. C. for a minimum of 4 hrs. After
depyrogenation, the column was then flushed with 15 liters of
ultrafiltered Dl water, followed by 15 liters of ultrafiltered 200
mM Tris-HCl, pH 8.0, followed by 10 liters of ultrafiltered 20 mM
Tris-HCl, pH 8.0.
[0175] The polymer solution was diluted to 4 liters and to a final
concentration of 20 mM Tris-HCL, pH 8.0, using ultrafiltered 200 mM
Tris-HCl, pH 8.0. The sample was then allowed to flow through the
column. The eluate was monitored for absorbance at 280 using an
Isco UA-5 absorbance detector with a 280-310 nm filter. After the
sample was applied, the column was flushed with ultrafiltered 20 mM
Tris-HCl, pH 8.0. The fractions containing protein were collected
and dialyzed in Spectrapore dialysis bags (6-8 kdco) which had
previously been soaked overnight in 0.5 N NaOH and then rinsed
thoroughly with ultrafiltered Dl water. The product was dialyzed
against three changes of 100 liters of Dl water. The final
dialysate obtained was lyophilized in thermally depyrogenated
lyophilization flasks.
[0176] The yields of both batches were combined and repassed over
the Poros HQ50 column as described above. The final dialyzed
product was lyophilized and stored in a thermally depyrogenated
container at -20.degree. C.
[0177] SELP9K batch 96084
[0178] 1572 grams of frozen biomass was resuspended in 6 liters of
Dl water. When completely thawed the suspension was mixed with a
polytron mixer (Janke & Kunkel, Kika-werk, Ultra Turrex model
T45-S4) and the cells were lysed using a Gaulin Model 15M
homogenizer (three passes at 8000 psig). The temperature of the
homogenate was maintained below 8.degree. C. by means of a heat
exchanger. The resultant cell lysate was mixed with an equal volume
of polyethyleneimine (PEI) solution (0.04% w/v, pH 8.0, Amresco
High Purity Grade, MW 50,000) and mixed for 15 min. The PEI mixture
was centrifuged in a DuPont/Sorvall RC3B centrifuge for 30 min at
5000 rpm, 8.degree. C. The supernatant was filtered through Whatman
#4 filter paper in a Buchner funnel and saturated ammonium sulfate
solution was slowly added to a final concentration of 25% of
saturation. The precipitated fraction was allowed to settle to the
bottom of the container while stored at 4.degree. C. overnight. The
supernatant was carefully decanted and the precipitate was
resolubilized in 2.0 liters of Dl water and dialyzed in Spectrapore
dialysis bags (MWCO=12-14 kdco) against two changes of 200 liters
of cold Dl water. The resultant dialysate was centrifuged in a
DuPont/Sorvall RC5B centrifuge, GS2 rotor, at 8000 rpm, 8.degree.
C. for 60 min. The recovered supernatant was filtered through a
Whatman #4 filter paper in a Buchner funnel. The filtrate was
diluted to a total protein concentration (as determined by Lowry
protein assay) of 1.0 mg/ml and saturated ammonium sulfate solution
was added to a final concentration of 20% of saturation. The
precipitate was collected after settling as described above and
resolubilized in 1.0 liter of cold Dl water. The polymer solution
was passed through a Gelman Spiralcap 0.2 .mu.m filter cartridge.
The solution was diafiltered against Milli-Q water using a
Millipore Pellicon filtration apparatus containing a 0.5 square
meter BIOMAX filter cassette (30K MWCO) until a conductivity of
<20 .mu.S/cm was obtained. The final volume was 2 liters.
[0179] The sample was brought up to 4 liters with 400 mis of 200 mM
Tris pH 8.0 and 1.6 liters of milli-Q water. This was passed
through a Poros HQ50 anion exchange column as described above. The
flow through and column wash fractions were dialyzed in Spectrapore
dialysis bags (6-8 kdco) which had previously been soaked overnight
in 0.5 N NaOH and then rinsed thoroughly with ultrafiltered Dl
water. The product was dialyzed against three changes of 180 liters
of Dl water. The final dialysate obtained was lyophilized in baked
lyophilization flasks and stored in thermally depyrogenated
containers at -20.degree. C.
[0180] SELP8K Batch 96072
[0181] Three kilograms of frozen biomass were resuspended to 12
liters with Dl water. When completely thawed and well mixed, cell
lysis was accomplished by passing it through a Gaulin Model 15M
homogenizer (three passes at 8000 psig). The solution temperature
was maintained below 8.degree. C. by means of a heat exchanger. The
resultant cell lysate was mixed with an equal volume of
polyethyleneimine (PEI) solution (0.04% w/v, pH 5.0)(Amresco High
Purity Grade, MW 50,000) and mixed for 15 min. The PEI mixture was
centrifuged in a RC3B centrifuge for 30 min at 5000 rpm, 8.degree.
C. The supernatant was collected and saturated ammonium sulfate
solution was slowly added to a final concentration of 20% of
saturation. The precipitated fraction was allowed to settle to the
bottom of the container while stored at 4.degree. C. overnight. The
supernatant was carefully drawn off and the ammonium sulfate
precipitate was resolubilized in 4.0 liters of Dl water. The
polymer solution was dialyzed in Spectrapor dialysis bags
(MWCO=12-14 kdco) against two changes of 200 liters of cold Dl
water. The resultant dialysate was centrifuged in a RC5B
centrifuge, GS2 rotor, at 8000 rpm, 8.degree. C. for 60 min. The
recovered supernatant was diluted to a total protein concentration
(as determined by Lowry protein assay) of 1.0 mg/ml and saturated
ammonium sulfate solution was again added to 20% of saturation. The
second ammonium sulfate precipitate was collected as described
above and resolubilized in 5.0 liters cold Dl water. The polymer
solution was passed through a Gelman Spiralcap 0.2 .mu.m filter
cartridge and then diafiltered against Milli-Q water using a
Millipore Pellicon cassette containing a 0.5 square meter BIOMAX
filter cassette (30K MWCO) until a conductivity of <20 .mu.S/cm
was obtained.
[0182] Six liters of sample at 5.7 mg/ml protein concentration was
passed through a Poros HQ50 anion exchange column as described
above. The collected product fractions were dialyzed against four
changes of 200 liters of Dl water. The final dialysate obtained was
lyophilized in baked lyophilization flasks, and stored in thermally
depyrogenated containers at -20.degree. C.
[0183] Using similar methods, the polymer SELP0K was prepared.
[0184] SELP5 5VA-1
[0185] Twelve kilograms of frozen biomass was thawed and
resuspended in 60 liters of 50 mM Tris-HCl, pH 8.0. The cell slurry
was lysed as described above with the additional step of adding
PMSF (as a 0.2 M solution in isopropanol) to a final concentration
of 2.5 mM after the first pass through the Gaulin homogenizer. The
cell lysate was centrifuged in a RC3B centrifuge for 60 min at 5000
rpm, 8.degree. C. Saturated ammonium sulfate solution was slowly
added to the resultant cell lysate supernatant to 25% of
saturation. This was mixed for 60 min and centrifuged in the RC3B
for 30 min at 5000 rpm, 8.degree. C.
[0186] The precipitate was resuspended in 40 liters of 4.0 M
lithium bromide and stirred overnight at 4.degree. C. The solution
was centrifuged in a Sharples AS14 centrifuge at a flowrate of 120
ml/min at approximately 14,000.times.g. The recovered supernatant
was adjusted to a pH of 3.7 by slow addition of concentrated
glacial acetic acid and stirred overnight at 4.degree. C. to
produce a precipitate. The precipitate was removed by centrifuging
in the Sharpies AS14 as described above. The supernatant (35
liters) was adjusted to pH 8.0 with concentrated ammonium hydroxide
and diafiltered using a Millipore Pellicon filtration apparatus
containing a 30K MWCO filter cassette (0.5 square meter) to a final
conductivity less than 100 .mu.S/cm. The retentate was centrifuged
in the Sharples AS14 centrifuge at 100 ml/min flow rate. Saturated
ammonium sulfate solution was added to the recovered supernatant to
25% of saturation and stirred for 60 min. The solution was
centrifuged in the RC3B centrifuge for 30 min at 5000 rpm,
8.degree. C. The precipitate was resolubilized in 4.0 liters of 4.5
M lithium bromide solution, diluted to 200 liters with Dl water,
and again diafiltered with the 30,000 MWCO ultrafilter as described
above. When a conductivity less than 350 .mu.S/cm was obtained, the
solution was concentrated with the ultrafilter to a volume of 10
liters and lyophilized.
[0187] The material was dissolved in 10 liters of 6.0 M urea/20 mM
Tris-HCl, pH 8.0 in preparation for application to the Poros HQ
anion exchange column as described above. The column and downstream
apparatus was depyrogenated and equilibrated as described above
except that the final equilibration of the column was performed
with 10 liters of ultrafiltered 6.0 M urea/20 mM Tris-HCl, pH 8.0.
The sample was applied to the column and collected in sterile
pyrogen-free plastic bottles (1000 ml). The column was flushed with
additional depyrogenated 6.0 M urea/20 mM Tris-HCl, pH 8.0.
Fractions containing protein were dialyzed in Spectrapor dialysis
bags (6-8 kdco) against four changes of 200 liters of Dl water. The
final dialysate was lyophilized in dry-heat depyrogenated
lyophilization flasks, collected in thermally depyrogenated
containers and stored at -20.degree. C.
[0188] Sample Characterization
[0189] The polymer samples prepared as described above were
characterized using standard analytical methods. The samples were
evaluated for moisture content, amino acid composition and
elemental composition (carbon, hydrogen, and nitrogen). Using the
theoretical values for amino acid and elemental composition as
expected from a 100% pure product, it is possible to estimate the
purity of the samples based on the actual composition of the
samples compared to theoretical.
[0190] Lyophilized SELP samples contain various amounts of water
depending on the lyophilization conditions and their subsequent
exposure history to ambient atmospheres. Therefore, it is important
to determine the moisture content of SELP samples both at the time
of production and again when critical experiments and measurements
are made. Moisture content is determined by measuring the weight
loss after drying to constant weight at 110.degree. C.
[0191] Elemental composition can be used to determine the weight
percent carbon and nitrogen contained in a sample. Because proteins
are more abundant in nitrogen than almost all other macromolecules
(C/N ratio of SELP8K is 3.25), and making the assumption that any
contaminants contained in the sample will contribute to the carbon
content to a greater degree than the nitrogen content, we may use
the excess carbon with respect to nitrogen in the sample as a
maximum estimate of chemical purity. It should be noted, however,
as is the case for the SELP5 sample below, C/N values less than
theoretical denote excess nitrogen in the sample which might be due
to residual processing chemicals such as urea, ammonium sulfate or
Tris buffer each of which have high nitrogen contents.
[0192] Amino acid composition analysis (AA Comp) determines the
amount of each amino acid contained in the sample. AA Comp does not
evaluate any other chemical species in the sample besides protein.
Because the SELP polymers consist of only a limited number of amino
acids (G, A, S, V, P, and K are the main ones) and completely lack
one amino acid (isoleucine, l), AA Comp data can be used to
estimate the protein purity of the sample (ie. the fraction of
protein in the sample which is SELP). E. Coli proteins that might
contaminate SELP samples were determined by amino acid composition
analysis to contain 4.8 weight % isoleucine. The amount of
isoleucine detected in SELP samples, as derived from E. Coli
proteins, divided by 0.048 represents the amount of protein in the
sample contributed by such contaminants. The detection limit of our
analysis for any single amino acid is 0.1%. Therefore, the limit of
contaminant protein detection based on isoleucine will be
0.001/0.048=0.021 or 2.1%.
[0193] Because SELPs consist almost exclusively of the six amino
acids listed above, the fraction of the total amino acids
constituted by these amino acids as compared to the theoretical
content of a 100% pure sample of the product is also an estimate of
purity. This estimate is only good for relatively pure samples
where contaminating proteins contribute only small amounts of the
amino acids used in the analysis. E. coli proteins contain 22.9% by
weight G+A+S+P+V and 31.1% by weight G+A+S+P+V+K. If a SELP sample
is 98% pure, for example, the E. coli protein contaminants would
contribute 0.02.times.0.311=0.006 or 0.6% of the total G+A+S+P+V+K
in the sample (+0.6% error). At 95% purity, the error of this
estimate may be +1.6%. For SELP8 and SELP5, which contain very
little lysine, K is not included in the analysis. Therefore, the
potential errors at 95% and 98% purity for these two polymers are
1.15% and 0.5%, respectively. Table 7 lists the results of these
analyses from several SELP sample preparations.
6TABLE 7 Characterization of SELP Samples Amino Acid Elemental
Analysis Composition Protein Moisture Purity Purity based on:
Analysis C/N ratio based on Isoleucine % G + A + Polymer Weight
(Theor. Excess C Content S + V + P Batch (%) C/N) (%) (%) (+K)* (%)
SELP8K 16.1 3.28 99.1 .+-. 0.1 None 100.8 96072 (3.25) Detected
>97.9 SELP8 12.1 3.46 95.7 .+-. 0.2 None 100.7 96083 (3.32)
Detected >97.9 SELP9K 18.0 3.36 98.8 .+-. 0.7 None 100.9 96084
(3.32) Detected >97.9 SELP5 5.6 3.25 NA** 95.8 98.3 5VA1 (3.32)
*lysine was not used in the calculation of protein purity of SELP8
**purity estimate was not possible because this sample contains
excess nitrogen (see discussion above)
D. EXAMPLE 3
SELP films
[0194] SELP films that were approximately 0.05 mm thickness were
produced by solvent evaporation.
[0195] Approximately 1.7 grams of each polymer, except for SELP7
where only 1.05 grams was used, were solubilized in 34 mls of 88%
formic acid. The solution was stirred for 7 hrs at room temperature
to insure complete solubilization. The solution was then poured
into a film casting apparatus consisting essentially of a
rectangular polyethylene trough with a removable polyethylene
bottom. The casting apparatus was placed in a vacuum oven attached
to a nitrogen gas source for sparging the atmosphere. The films
were dried in the sealed oven drawing a 10-15 micron vacuum with a
slow continual influx of nitrogen gas at 60-75.degree. C. After
15-18 hrs of drying, the apparatus was disassembled and the film
was peeled off the polyethylene bottom. The films were exposed for
5 min to a basic atmosphere (5% open solution of ammonium hydroxide
in a sealed desiccator) to neutralize any residual formic acid.
[0196] A polyethylene sheet of the same area dimensions as the
protein film was roughened by hand using fine grit sand paper and a
thin film of cyanoacrylate glue was spread over its surface. The
protein film was applied to the wet surface. A teflon sheet was
placed on top and bottom of the polyethylene and protein layers and
stainless steel plates were placed around those. The entire
assembly was pressed in a Carver laboratory press at a force of 0.8
metric tons for 18 hrs at room temperature. The
polyethylene/protein film laminated sheet was placed on a cutting
board and 1.3 cm diameter discs were punched out using a stainless
steel punch and rubber mallet. The discs were placed individually
in stoppered glass vials.
[0197] Specimens were produced from each of the polymers as well as
denatured collagen protein (DCP) produced identically as described
for the SELP films. Bovine collagen (fibrillar form, lot number
921101) was obtained from Colla-Tec, Inc. (Plainsboro, N.J.). It
was completely solubilized in 88% formic acid producing a clear but
viscous solution. All specimens were sterilized by electron beam
irradiation at 2.5+/-0.2 Mrads. Each disk was implanted
subcutaneously in the back of rats such that the protein film was
in direct contact with the muscle tissue. The specimens remained in
the animals for different periods of time: one, four and seven
weeks post implantation. At each time interval six specimens per
polymer group were retrieved for protein analysis. Additional
specimens from each group were evaluated for tissue reaction by
histology.
[0198] Non-implanted and retrieved specimens were analyzed to
determine the mass of SELP film contained per specimen. Amino acid
analysis was performed on each specimen by sealing them
individually in an hydrolysis vial with constant boiling
hydrochloric acid and heating for 24 hr at 100-110.degree. C. After
hydrolysis, the specimen was extracted and an aliquot of the
extract was derivatized with PTC. The derivatized amino acids were
separated by reverse phase HPLC and quantified by their absorbance
at 254 nm according to the methods of Henrickson and Meredith,
Anal. Biochem. 137:65-74 (1984).
[0199] The mass of the SELP film present on each specimen was
determined. The amino acid contribution of the SELP protein was
estimated based on the total content of the amino acids G, A, S, V
and P which for the pure polymers is >95%. Other amino acids
potentially contributed by extraneous protein deposited onto the
specimens during residence in the body were excluded from these
analyses. Average SELP film mass for non-implanted specimens was
determined from the same batch of specimens used for implantation.
Average SELP film mass for retrieved specimens was similarly
calculated except that replicates having values greater than two
standard deviations from the mean were discarded. Deviations in
many cases were due to partial retrieval of specimens that had
fragmented in the tissue after implantation and may not reflect
true resorption.
[0200] Resorption Analysis and Results
[0201] Resorption analysis was conducted statistically by analyzing
four specimen population treatment groups. These were: (1)
non-implanted; (2) one week post-implantation; (3) four weeks
post-implantation; and (4) seven weeks post-implantation.
7TABLE 8 Polymer Film Mass Remaining as Determined by AA
Composition Analysis (in mg) SELP0 SELP3 SELP4 SELP5 SELP7 SELP8
DCP Initial Film 12.21 +/-1.41 5.99 +/-0.46 8.19 +/-0.86 8.51
+/-1.04 3.27 +/-0.34 8.43 +/-0.59 6.6 +/-1.04 Mass 1 Week Film 0.53
+/-0.31 5.93 +/-0.73 7.89 +/-0.55 7.72 +/-1.57 4.67 +/-1.33 11.13
+/-1.40 0.15 +/-0.07 Mass 4 Week Film 0.27 +/-0.13 6.24 +/-0.61
9.20 +/-1.08 7.49 +/-0.75 0.19 +/-0.16 8.26 +/-1.21 0.09 +/-0.03
Mass 7 Week Film 0.10 +/-0.02 3.49 +/-1.60 8.56 +/-0.67 8.77
+/-0.97 0.08 +/-0.03 1.52 +/-1.40 0.07 +/-0.03 Mass
[0202]
8TABLE 9 Polymer Film Remaining as Percent of Non-implanted Mass
SELP0 SELP3 SELP4 SELP5 SELP7 SELP8 DCP Initial 100.0% 100.0%
100.0% 100.0% 100.0% 100.0% 100.0% Film Mass 1 4.3% 98.9% 96.3%
90.7% 142.8% 132.0% 2.3% Week Film Mass 4 2.2% 104.1% 112.4% 88.0%
5.8% 98.0% 1.3% Week Film Mass 7 0.8% 58.2% 104.5% 103.1% 2.6%
18.1% 1.1% Week Film Mass
[0203] The results from Table 8 are the values for the mass of
protein film contained on specimens after implantation. Each value
is the mean of at least five specimen masses as determined by amino
acid composition. Table 9 displays the same values as a percent of
the initial weight prior to implantation as determined by the mean
mass of six specimens of the non-implanted specimens. The results
indicate that upon implantation, SELP0 and DCP are substantially
resorbed by one week, falling below 5% of their non-implanted
masses. SELP7 is substantially resorbed by four weeks with only
5.8% remaining. SELP8 and SELP3 are resorbing by seven weeks with
mean values of 18.1 % and 58.2% remaining, respectively. SELP4 and
SELP5 films show no evidence of resorption at seven weeks.
[0204] From the above results one may conclude the following.
Faster resorption correlates with compositions containing domains
of silk-like blocks fewer than eight. The polymers containing eight
silk-like blocks have substantially reduced rates of resorption.
However, the total content of silk-like blocks in the copolymer
composition does not correlate with resorption rate. While very
similar compositionally, SELP7 and SELP8 resorbed quickly, while
SELP4 and SELP5 do not resorb in seven weeks. The lack of
resorption of SELP4 and SELP5 films at seven weeks
post-implantation corresponds with repeating domains containing
greater than eight elastin-like blocks. Although their silk-like
block lengths are identical at eight, SELP4 and 5 with elastin-like
block lengths of 12 and 16 resorb to a lesser degree than SELP3,
which has an elastin-like block length of 8.
[0205] The subject polymers, regardless of their composition, form
free-standing films with strength enough to allow easy handling.
SELP7 and SELP4 films have tensile strengths of 19+/-1 and 21+/-8
MPa, respectively. The compositional difference between them that
causes SELP7 to resorb in four weeks and SELP4 to remain intact
beyond seven weeks makes little apparent difference in their
tensile properties. These strengths are adequate for their use in
surgical and wound healing applications.
[0206] The observed resorption of these polymers occurs via surface
erosion. This is consistent with the mechanism of degradation of
SELP proteins within the body. At physiological conditions,
proteins will degrade only through the action of proteases. Because
endogenous proteases are high molecular weight compounds of
approximately 20 kDa or greater, their diffusion into the dense
SELP films will be limited. The degradation of SELP films is,
therefore, progressive from the external surfaces of the material.
The subject materials therefore should undergo a slow loss of
mechanical integrity while being reduced in mass.
E. EXAMPLE 4
SELP Porous Sponges
[0207] The function of an implanted material depends greatly on its
form, morphology and mechanical strength. SELP polymers have been
fashioned into a variety of forms; dense films, porous sponges, and
fibrillar mats. Dense films or sheets, as described above, are
semi-permeable barriers which may have utility in surgical repairs
by restricting fluid or gas flow, blocking cellular migration,
maintaining tissue separations, and confining and protecting
implanted organs or devices. Their properties will vary depending
on their permeability and their thickness which may range from 0.05
mm to greater than 1 mm. For example varying their thickness will
effect their mechanical strength, their resistance to abrasion, and
their ultimate resorption.
[0208] SELP polymers have been produced as follows as three
dimensional, porous sponges to serve as implantable materials that
will support cell and tissue ingrowth.
[0209] All glassware to come in contact with the protein polymer
was depyrogenated by heating to 180.degree. C. for 6 hrs. SELP5
(0.978 g) was stirred in LAL reagent grade water until dissolved to
yield a 1.0% w/v aqueous solution. This solution was aseptically
transferred to a 100 ml S.sub.T 24/40 pear shaped flask and tared.
This flask was fitted with a spray trap, attached to a rotary
evaporator, and 65.2 g of water was evaporated using a bath
temperature of 39.degree. C., a system pressure of 42 mbar, and a
rotation rate of 125 rpm, to yield a solution of 3.0% w/v
concentration. This solution was poured 6 mm deep into six standard
sterilized Petri dishes (mm diameter); covered with standard lids;
placed on a small plastic tray; and placed in a -8.degree. C.
freezer overnight. After freezing, the lids were removed from the
Petri dishes; the Petri dishes were placed into a 1200 ml wide
mouth lyophilization flask and lyophilized to dryness. After
completion of lyophilization, the sponges were removed from their
Petri dishes and placed, individually, into a 100 ml wide mouth
flask containing 75 ml of methanol at room temperature. The head
space was evacuated to less than the vapor pressure of the methanol
to induce eubulation and insure compete displacement of air
entrained within the sponge by the methanol. The sponge, wetted
with methanol was allowed to stand for 5 min at room temperature at
room temperature, methanol was removed from the sponge by washing 6
times with LAL reagent grade water (175 ml per wash) and allowing
each to stand for 5 min. The sponges, wetted by water, were
returned to 35 mm diameter Petri dishes, frozen at -8.degree. C.,
and again lyophilized. The lyophilized sponges were placed into new
35 mm diameter Petri dishes, lids applied and sealed with
parafilm.RTM., placed into a plastic instrument bag, heat sealed,
and sterilized using an electron beam irradiation at 2.8 Mrads.
[0210] The sponges were dimensionally stable when immersed in
saline or water. When engorged with saline, the sponge turned from
white to grey and was somewhat translucent. The engorged sponge
retained its original dimensions. Minimal swelling was observed.
The geometry and edges of the wet sponge remained unchanged. The
observed aqueous stability of the SELP5 sponges is different from
the properties of collagen hemostatic sponges (Helistat, Marion
Laboratories, Kansas City, Mo.) which almost immediately collapse
when exposed to liquid.
[0211] SELP5 sponges were cut into 2.times.2.times.0.4 cm specimens
and applied to 2.times.2 cm full thickness dermal wounds in pigs.
2.times.2.times.0.3 cm specimens of Helistat were similarly applied
to wounds. After bleeding was controlled and the wound flushed with
saline, the specimens were laid into the tissue void such that they
would firmly contact the wound bed. The Helistat specimens became
completely or partially engorged within a few seconds to several
min after application depending on the amount of the blood in the
wound. The engorged Helistat specimens collapsed and shrunk
resulting in nonuniform coverage of the wound, in some cases,
exposing part of the wound beds.
[0212] The SELP5 sponges remained substantially white during the 5
min observation period after application indicating that they did
not immediately absorb blood. One corner of one specimen turned red
within a minute after application. It remained physically
unchanged. The SELP5 sponges adhered well to the wound bed and
could not be lifted out of the wound with forceps using mild
tension. The SELP5 sponges did not shrink upon contact with the
bloody tissue and continued to completely cover the wound during
observation.
[0213] All wounds were covered with petrolatum gauze pads and
bandaged. After 7 days, the wounds were undressed and observed to
determine the extent of healing. Wounds containing SELP5 sponges
had progressed normally through the healing process as compared to
wounds to which no material was applied. The sponge material had
not been extruded from the wound as there was no evidence of
extraneous material on the gauze pads. No evidence of excessive
inflammation was observed. Epithelialization of the wound was in
progress.
F. EXAMPLE 5
SELP Fibrous Meshes
[0214] SELP polymers can be fabricated as non-woven fibrous meshes
to produce fibrillar mats which are flexible, have good
drapability, and are stable in wet environments. Fibrous meshes
with similar physical properties were produced from SELP5, SELP7
and SELPF using the following procedure. 1 gram of polymer was
dissolved in 88% formic acid with stirring at room temperature
until homogenous. For SELP5, 5 mls of formic acid were used to
dissolve the lyophilized polymer. For SELP7 and SELPF, 4 and 3 mis
of formic acid were used, respectively.
[0215] The polymer dope was drawn into a 1 cc polypropylene
syringe, affixed with a 75 mm.times.20 gauge stainless steel
hypodermic needle, and mounted on a Sage Instruments syringe pump
(model 341B). The pump was set to deliver approximately 0.05 to
0.07 cc/min. The tip of the needle was placed at 90.degree. C. to a
gas stream delivered from a stainless steel needle (25 mm.times.20
gauge). A more acute angle was also used. The dope delivery needle
and the gas delivery needle were mounted onto a steel "L"-bracket
using miniature "C"-clamps and pads of neoprene rubber such that a
gap of 1 mm separated their tips. The tips were displaced in the
vertical direction by 0.5 mm such that the gas stream passed
slightly over the flanged end of the hypodermic needle. The gas
stream was supplied either with compressed air or high purity
(extra dry) nitrogen gas. Compressed air was supplied by an oiless
compressor using a diaphragm pump. The air in the reservoir was a
ca. 8 atm pressure and was regulated down to ca. 2-6 atm before
being fed to the spray apparatus. When nitrogen was used, it was
delivered at 20 psi. The relative humidity was less than 47%.
[0216] Fine filaments were formed on and around the edges of a
rectangular, {fraction (1/16)} inch polypropylene mesh that was
used as a target approximately 7-12 inches from needle tips.
Filaments streamed off the edges of the target and when they were
approximately 5 cm in length, they were collected on a circular,
metal wire loop of 38 mm in diameter. Filaments were collected
across the loop forming a web of suspended filaments in the center.
The web was removed from the loop by compressing the web between
two 35 mm polystyrene discs and pressing the web through the wire
frame. Fibrous meshes were built up by compressing 5-8 webs between
the same discs.
[0217] The meshes were stabilized by flooding them with 1 ml of
either 100% methanol or 100% ethanol and allowing them to dry under
ambient conditions. The meshes were sterilized by electron beam
irradiation at a dose of 2.5 MRads. Under microscopic observation,
the meshes consisted of fine filaments which varied in diameter
from 0.1 to 10.mu.m. The meshes were stable when placed in saline
for more than 24 hrs.
[0218] The meshes were applied to 2.times.2 cm partial and full
thickness dermal wounds in pigs in order to investigate their
biocompatibility and their ability to incorporate within the
healing tissue. The meshes were removed from the polystyrene discs
with forceps and applied to the wound bed. The edges of the meshes
could be pulled across the tissue allowing the mesh to be spread
and/or rearranged over the wound. The wounds were covered and
examined every two days for signs of bioincompatibility. No adverse
effects were observed in wounds containing SELP fibrous meshes.
After 14 days, the wounds were completely epithelialized.
Histological examination of tissues from wounds to which SELPF
fibrous webs had been applied showed that foreign material in the
form of filaments had been incorporated into the healing
tissue.
[0219] These data indicate that SELP fibrous meshes are well
tolerated in healing tissue. Their presence does not interfere with
normal healing. In one case, SELP filaments were clearly shown to
reside within the healed tissue.
[0220] SELP films, meshes, and sponges can serve as resorbable
packing materials that can be used to augment the loss of soft
tissue that occurs during traumatic injury or surgical dissection.
Their application at the time of injury can encourage infiltration,
overgrowth, and eventual replacement of the materials with healthy
tissue. The mass of the implanted material can provide enough
stability to maintain the geometric contours of the body site at
which the tissue was lost. Their presence can also mechanically
reinforce the wound site such that delicate, healing tissues can
form while protected from further physical injury.
G. EXAMPLE 6
SELP Gel Characterization
[0221] The gelling behavior of three SELP polymers were
investigated using a Brookfield cone and plate viscometer. The
studies demonstrated that gelation was a function of time,
temperature, polymer composition, and that certain additives
inhibited or accelerated the onset or rate of gelation.
[0222] SELP0K batch 96042, SELP8K batch 96072 and SELP5 batch 5VA1
were dissolved in phosphate buffered saline solution (1.times.PBS,
Irvine Scientific) at 20% (w/w). At the time of these studies, each
of the polymer samples had the following moisture contents: 5VA-1,
5.55% moisture; 96072, 6.84% moisture; and 96042, 11.03% moisture.
The polymer solutions were mixed by hand until dissolved (3-4 min)
and centrifuged for 2-3 min at high speed in a clinical centrifuge
(International Equipment Co.) in order to clear air bubbles from
the solution. The polymer solution was transferred to the cup of a
Brookfield Syncro-Lectric rotational viscometer (Model RVTCP, CP-52
cone and cup configuration, Brookfield Engineering Laboratories)
using a 1.0 cc polypropylene syringe (Becton Dickinson). Generally,
the elapsed time from which the 1.times.PBS was added to the
protein to the time in which the viscometer was started, was about
6 to 10 min.
[0223] The CP-52 cone, which had a cone angle of 3.0.degree.,
permitted the use of a 0.5 mL sample and the measurement of
viscosities up to 196.6 Pa.multidot.sec. The cup, which allowed the
circulation of liquid to control sample temperature, was connected
to either a Cannon constant temperature bath (Cannon Instrument
Company, Model M-1) or a VWR thermostated bath (VWR Scientific,
Model 1150). Generally, the VWR thermostated bath was used for
ambient and sub-ambient temperatures. For supra-ambient
temperatures, either one of the temperature control systems was
used interchangeably. Both temperature control systems were capable
of controlling temperature to within +0.1.degree. C. Temperature
was monitored using a type J thermocouple (OMEGA Engineering,
catalog number SA1-J) surface-mounted on the exterior of the cup.
The temperatures used in this study were 4, 23, and 37.degree.
C.
[0224] Prior to use, the temperature of the cone and cup on the
Brookfield viscometer was allowed to equilibrate, and then the gap
between the cone and cup was set according to the manufacturers
instructions. The viscometer was started and allowed to rotate
continuously at 20 rpm. The initial viscosity and subsequent
viscosities were measured. The experiments were generally allowed
to continue until the viscometer approached its maximum
capability.
[0225] The gelation of SELP8K batch 96072 was also studied in
solutions containing additives that affect gelation. 20% (w/w)
SELP8K batch 96072 was studied in 1.times.PBS containing 6 molar
urea (UltraPURE.TM. Urea, Life Technologies). Urea is known to
inhibit the crystallization of proteins by disrupting hydrogen
bonding. The gelation of SELP8K batch 96072 solution to which
pregelled SELP8K powder was added was also studied. Previously
gelled SELP8K batch 96072 solution was frozen at -20.degree. C. and
lyophilized. The lyophilized SELP8K protein was comminuted to a
powder by milling in methanol against 240 grit carborundum paper.
The crude protein powder was transferred to a vial and chloroform
was added to float the protein powder away from the carborundum
residues. Residual chloroform was allowed to evaporate overnight
under ambient conditions. 2.19 mg of pregelled protein powder was
added to 1.18 g of 20% (by weight) SELP8K batch 96072 solution. The
actual weight-fraction protein for this solution was 20% (w/w),
thus 0.24 g of protein was present. The addition of pregelled
protein on a weight basis was calculated to be about 0.9% (by
weight, based on protein content).
[0226] The behavior of SELP8K solutions was measured using
modulated differential scanning calorimetry (MDSC) TA Instruments
Modulated DSC, Model MDSC 2920. 20.4780 milligrams of a 33% (w/w)
solution of SELP8K batch 95092 in 1.times.PBS was hermetically
sealed inside of a 40 microliter aluminum pan. The amplitude and
period of the sinusoidally applied heating function was 1.0.degree.
C. and 60 seconds, respectively. The experiment showed that, after
an induction time, an exothermic peak occurred (FIG. 1). The
exothermic peak (the lower trace in FIG. 1) was integrated using a
software-adjusted baseline based on the change in heat capacity of
the sample. The 18.90 J.multidot.g.sup.-1 peak integration
corresponds to the entire mass of the sample which was 33% by
weight protein polymer. Thus, the integration value was multiplied
by a factor of 3 to adjust for that portion of the sample which was
water. The corrected integration of the peak which takes into
consideration only the mass of the polymer which caused the
exotherm, was 56.70 J.multidot.g.sup.-1. This peak was consistent
with the occurrence of a non-reversible crystallization event. Data
gathered to date suggests that the viscosity build-up observed in
solutions of various SELP polymers is due to crystallization of the
protein chains, most likely, the silk-like blocks through a
mechanism involving, at least in part, hydrogen bond formation.
[0227] The results of Brookfield viscometry experiments also add
further validation that gelation of SELP solutions occurs and that
the process might be controlled by adjusting conditions that affect
crystallization. Three SELPs with varying amounts of silk-like
blocks in their repeat units were chosen for this study. The
experiments were performed isothermally at 37.degree. C. The
protein solutions were 20% (w/w) in 1.times.PBS and subjected to a
steady shear rate of 40 s.sup.-1. FIG. 2 shows that the viscosity
build-up as a function of time ranked the polymers according to the
number of silk-like blocks contained in their repeat units. SELP0K,
which has only two silk blocks per repeat unit, demonstrated no
rise in viscosity over the time period studied. SELP8K, which has 4
silk-like blocks per repeat unit, showed a delayed viscosity rise
with time when compared to SELP5 which has 8 silk-like blocks per
repeat unit.
[0228] Further evidence of crystallization was obtained from
examining the viscosity buildup of SELP solutions at various
temperatures. FIGS. 3A and 3B show the viscosity results for 20%
(w/w) solutions of SELP8K and SELP5, respectively. SELP5, which has
more silk units than SELP8K, gelled faster than SELP8K at all
temperatures.
[0229] Both graphs show that temperature dramatically affects the
rate of gelation. Higher temperatures resulted in faster rates of
gelation.
[0230] Additives which control the rate of crystallization of SELP
polymer solutions were identified. Urea is known to denature
proteins by disrupting and preventing the formation of hydrogen
bonds. If crystallization is the mechanism by which SELP solutions
gel, urea should retard or inhibit gelation. Additionally,
crystallization is a process that can be accelerated by additives
which serve to nucleate crystal formation. Precrystallized SELP
incorporated into a fresh SELP solution is expected to act as a
nucleating agent. FIG. 4 shows that the gelation of 20% (w/w)
solutions of SELP8K at 37.degree. C. was effectively accelerated by
the addition of pregelled SELP8K protein, an expected nucleating
agent, and virtually eliminated by the addition of urea, compared
to the gelation rate of the control, SELP8K in 1.times.PBS. The
slow increase in viscosity of the 6 M urea-containing solution was
due to moisture loss during the experiment.
H. EXAMPLE 7
Release of Compounds From SELP Gels
[0231] The release of compounds incorporated into SELP solutions
that had been converted to gels at 37.degree. C. was studied. SELP
powder was dissolved in phosphate buffered saline (1.times.PBS, pH
7.4) at various concentrations and fluorescently labeled amino
acids and dextrans of various molecular weights were added. The
solutions were mixed and loaded into 0.5 and 1.0 cc plastic
syringes and incubated at 37.degree. C. Gel discs were excised from
the syringes and placed in elution tubes with 5 ml of phosphate
buffered saline containing 0.01 % sodium azide (1.times.PBSA) and
incubated at 37.degree. C. At various times, the tubes were removed
from the incubator, agitated by inversion and the eluate measured
for fluorescence using a Sequoia and Turner fluorometer, model 450.
Dansyl derivatives were read using excitation and emission filters
NB360 and SC475, respectively. Fluorescein derivatives were read
using excitation and emission filters NB490 and SC515. The tubes
were replaced at 37.degree. C. for further analysis. The loading
amounts of each compound was adjusted such that 100% release would
allow a fluorescent reading within the linear range of the
instrument. The fluorescence remaining in the gel at termination
was determined by dissolving the gel in 88% formic acid,
neutralizing with sodium hydroxide, and diluting to a total volume
of 5 ml with PBS containing 2 M Urea. Dansyl-L-glutamine (free
acid, FW 379.4) and N(epsilon)-dansyl-L-lysine (free acid, FW
379.5) were obtained from Sigma Chemical, Co., St. Louis, Mo.
Dextran, dansyl, 40,000 MW and 70,000 MW and dextran, fluorescein,
10,000 MW and 500,000 MW were obtained from Molecular Probes, Inc.,
Eugene, Oreg.
[0232] FIG. 5 shows that compounds ranging in molecular weight from
several hundred to 500,000 kD can be released from SELP gels at
37.degree. C. in PBS. The amount of compound eluted is a linear
function of the square root of time, indicitive of Fickian
diffusion. The release of SELP protein from SELP gels was also
monitored to determine the stability of the gelled matrix.
Approximately 28% of the SELP protein in the gel was released in 24
hrs. After that time, the gel was stable in PBS at 37.degree.
C.
[0233] The consistency of the gel as observed by the release
characteristics of added compounds is dependent on the
concentration of SELP in solution prior to gelation. The time
required to achieve 50% release of dansyl-glutamine (FW 379.4),
dansyl-dextran (40 kDa), and fluorescein-dextran (500 kDa) from
SELP gels containing 20%, 30% and 40% (w/w) SELP is shown in Table
10. Increasing the SELP concentration from 20% to 30% had no effect
on the release of dansyl-glutamine and had little effect on the
release of 40 kDa dansyl-dextran. However, an increase from 30% to
40% tripled the time required to achieve 50% release of 40 kDa
dansyl-dextran. Increasing the SELP concentration from 20% to 30%
almost tripled the time required to achieve 50% release of 500 kDa
fluorescein-dextran, and increasing the SELP concentration from 30%
to 40% increased its 50% release time more than 8-fold.
9TABLE 10 Time Required For 50% Release of Fluorescent Compounds
from SELP Gels SELP8K Concentration Dansyl-glutamine Dansyl-dextran
Fluorescein- (w/w) (MW 379.4) (40kDa) dextran (500kDa) 20% 2 hours
5 hours 12 hours 30% 1.5 hours 6 hours 34 hours 40% Not Determined
18 hours >288 hours
[0234] This effect is further illustrated in FIG. 6 where the
release of 70 kda dansyl-dextran was studied as a function of SELP
composition and concentration. The results show that at 20% (w/w)
SELP8 did not gel and, therefore, it released its entire content of
dansyl-dextran immediately into the elution medium. 20% SELP9K,
SELP5 and SELP8K released 70 kda dansyl-dextran over 300 hrs with
similar, first order release rates. At 40% (w/w), the release rate
of dansyl-dextran from SELP9K gel was not changed over that of the
20% gel. 40% SELP8 and SELP5 gels gave identical release profiles
and the release from the SELP5 gel at 40% was reduced by about 15%
over that of the 20% gel. The release rate of 40% SELP8K gel was
reduced by 40% over that of the SELP8K gel at 20%.
[0235] The timing of SELP gel formation and its effect on release
rate was investigated in a mode that would approximate the use of a
SELP solution containing a releasing compound injected into the
body at 37.degree. C. 0.2 grams of lyophilized SELP8K batch 96072
was dissolved in 0.675 ml 1.times.PBSA (50 mM sodium phosphate, pH
7.4, 100 mM sodium chloride, 0.01% sodium azide) and 0.125 ml of 20
mg/ml 70 kda dansyl-dextran in 1.times.PBS to make a 20% (w/w)
solution of SELP8K. The solution was mixed several minutes until
homogeneous and centrifuged for 3 min in a table top clinical
centrifuge to clear entrapped bubbles. 0.1 ml of solution was
dispensed into the bottom of glass 12.times.75 mm test tubes that
had been prewarmed to 37.degree. C. using a syringe and a 26 gauge
needle. 5 ml of prewarmed (37.degree. C.) 1.times.PBSA was
immediately added to one tube and mixed by inversion. The SELP
solution dispersed with no visible gel remaining on the tube. The
remaining tubes were preincubated at 37.degree. C. for various
periods of time before 5 ml of prewarmed (37.degree. C.)
1.times.PBSA was added. Preincubation times included 5, 45 min,
4.25 and 21 hrs. The release of fluorescent 70 kda dansyl-dextran
into the elution medium was monitored as a function of time at
37.degree. C. with time zero corresponding to the time that
1.times.PBSA was added to the tube.
[0236] The SELP8K solution was observed to have formed a solid gel
that adhered to the bottom walls of the glass tube even after
repeated mixing by inversion at even the shortest preincubation
time (5 min). Preincubation time at 37.degree. C. had no effect on
release characteristics of 70 kDa dansyl-dextran. Prepared and
dispensed in this manner, SELP8K solutions formed solid gels in
less than 5 min at 37.degree. C.
[0237] I. EXAMPLE 8
Compatibility and Release of a Protein Drug From SELP Gel
[0238] rFGF-SAP is a genetically engineered mitotoxin produced by
Prizm Pharmaceuticals, Inc., San Diego, Calif., called Pantarin
(Casscells et al., Proc. Natl. Acad. Sci. USA 89:7159-7163 (1992)
and Lappi and Baird, Progress in Growth Factor Research 2:223-236
(1990)). The protein is a recombinantly produced fusion of human
basic fibroblast growth factor, bFGF, and the plant toxin, saporin.
The product has been shown to significantly inhibit the
proliferation of numerous FGF receptor expressing cell types
including tumor cells and vascular smooth muscle cells in in vitro
and in vivo systems. It would be of particular interest to deliver
this product in vivo in a sustained, localized fashion to tissues
that undergo pathological hyperproliferation.
[0239] SELP8K gels were measured for controlled delivery of
Pantarin. .sup.125I Pantarin was incorporated into 33% (w/w) SELP8K
gel at an approximate loading concentration of 0.2 mg/ml using a
buffer system previously shown to provide optimal stability to the
structure and activity of Pantarin. The buffer composition was 50
mM sodium citrate, 80 mM NaCl, 0.1 mM EDTA, pH 6.0 (CBS). The gel
was cast in a 0.5 cc hypodermic syringe at 37.degree. C.
Cylindrical sections of the gel were cut from the syringe and
placed in elution tubes containing CBSGT buffer (CBS buffer
containing 0.1% gelatin, 0.05% Tween-20) at 37.degree. C. The
radioactivity remaining in the gel specimens were monitored using a
gamma counter. An initial rapid release of Pantarin in the first 24
hrs was followed by a slow, steady release of approximately 1% per
day for at least 8 days (FIG. 7). The time allowed for gelation
(preincubation time) of the SELP/Pantarin solution at 37.degree. C.
prior to initiation of elution affected the amount of Pantarin
released in the initial phase. 56% and 26% Pantarin release was
observed after 6 hrs of elution from 4 and 24 hr preincubated gels,
respectively. At 24 hrs of elution, 74% and 37% of Pantarin was
released, respectively.
[0240] The bioactivity of Pantarin released from 33% (w/w) SELP8K
gels after 24 hrs at 37.degree. C. was investigated using an in
vitro bioactivity assay (McDonald et al., Protein Expression and
Purification 8:97-108 (1996)). Control doses of fresh Pantarin or
Pantarin eluted from SELP gel for 6 and 24 hrs at 37.degree. C.
were added to growing cells. The amount of Pantarin in the test
sample was quantified by comparison to a dose response curve
generated with a Pantarin reference standard. Control samples of
Pantarin incubated in elution buffer for 6 and 24 hrs at 37.degree.
C. with no SELP gel were also included as controls. The results
shown in Table 11 demonstrate that Pantarin was stable after being
mixed with SELP, allowed to gel, and eluted from the gel at
37.degree. C. for at least 24 hrs. Recovery of 70 to 82% of the
bioactivity of the Pantarin contained in the SELP gel samples after
6 and 24 hrs of elution time is consistent with the release
experiments which indicate that up to 70% of Pantarin could be
released at 24 hrs.
10TABLE 11 Bioactivity of Pantarin Released from SELP Gels
Incubation Pantarin Expected Time or Concentration Concentra- % of
Elution Time Assayed tion if 100% Expected Sample at 37 C.
(Average) Recovered Recovery 33% SELP8K 6 hours 18.9 ug/ml 27 ug/ml
70% gel (n = 6) Control 6 hours 136 ug/ml 133 ug/ml 102% Pantarin*
(n = 3) 33% SELP8K 24 hours 22.1 ug/ml 27 ug/ml 82% gel (n = 6)
Control 24 hours 133 ug/ml 133 ug/ml 100% Pantarin* (n = 3)
Pantarin** None 124 ug/ml 133 ug/ml 93% *Incubated in elution
buffer for 6 or 24 hours as indicated. **Not incubated in elution
buffer prior to assay.
J. EXAMPLE 9
In Vivo Biocompatibility and Resorption of Injected SELP Gels
[0241] SELP8K batch 96072 solutions were produced at 20% (w/w) in
cell culture grade PBS, filter sterilized through 0.22 .mu.m
syringe filters and loaded into 5 cc sterile plastic syringes. A
syringe was mounted onto a programmable syringe pump (Cole Parmer).
The tip of the syringe was adapted with approximately 8 inches of
{fraction (1/16)} inch i.d. PTFE extension tubing to which Luer
press fit syringe-to-tubing adaptors were attached. The other end
of the tubing was fit with a disposable 30 gauge stainless steel
hypodermic needle. 0.1 cc of SELP8K solution was injected into
guinea pigs subcutaneously and intradermally (six injections per
animal). Each 5 cc syringe of solution was used over the course of
several hours at surgical room temperature through which time it
remained fluid and injectable.
[0242] Under the skin of the injection sites, injected material
converted to a firm mass momentarily after injection. The material
did not migrate during the several hours of postsurgical
inspection. No evidence of material migration was observed in any
of the injection sites through one week of daily observations
followed by weekly examinations.
[0243] Animals were sacrificed at 3, 7, and 28 days. The injection
sites were excised, fixed, sectioned and stained with
Hematoxylin/Eosin and Masson's Trichrome. The in vivo
biocompatibility of SELP solutions was investigated by histological
examination. No gross clinical signs of tissue reaction due to
toxicity, allergy or irritancy were observed. Histologically, the
injected material could be seen throughout the observation period
in the tissues either interpenetrating the tissue collagen or
isolated in subcutaneous pockets. In both cases, there was minimal
evidence of acute inflammation. At 28 days, cells infiltrated the
periphery of the gel and could be seen apparently resorbing the
material. There were no signs of immunological reactivity.
Occasionally, isolated portions of gel material located
subcutaneously were observed at 28 days to be mildly
encapsulated.
K. EXAMPLE 10
Release of DNA From SELP Gels
[0244] 20% (w/w) solutions of SELP8K and SELP9K were produced in
TSAE buffer (50 mM Tris-HCl, pH 7.5, 0.9% sodium chloride, 0.02%
sodium aside, 1 mM EDTA) in which DNA was dissolved. The DNA was a
BanI REN digest of a purified E. Coli plasmid that generated the
following size fragments: 21, 114, 520, 1097, and 1374 bp. 0.5 mg
of DNA was added to 100 mg of SELP solution to give a loading
concentration of 0.5% (w/w). After mixing, the solutions were
centrifuged for 2 min in a microfuge to collect them bubble-free in
the bottom of 1.5 ml capped microfuge tubes. The solutions were
incubated at 37.degree. C. for either 1.5 hrs or 4 hrs prior to
commencing elution experiments. The gels were overlayed with 0.1 ml
of TSAE and incubated at 37.degree. C. Periodically, the gels were
centrifuged for 2 min in the microfuge and the elution buffer was
collected and replaced with fresh buffer. Elution samples were
applied to a 6% polyacrylamide gel cast and run in 1.times.TBE
buffer and analyzed by electrophoresis and ethidium bromide
staining to visualize the eluted DNA.
[0245] DNA fragments were released from the SELP9K gel at a much
greater rate than from the SELP8K. For the first 2 days, DNA bands
of all sizes eluted according to their initial concentration in the
DNA digest from the SELP9K gel (FIG. 8). After 6 days, elution of
the 114 bp DNA fragment was rapidly diminishing while that of the
larger fragments (520 bp and up) continued.
[0246] In order to examine the release of larger (gene size)
fragments of DNA from SELP gels, an .sup.35S-end-labeled lambda
phage DNA digest containing 17 fragments ranging in size from 60 bp
to 22,000 bp (Amersham, cat. No. SJ5000) was added to SELP5, SELP8K
and SELP9K gels prepared as described above. In this case, 100 mg
of each SELP solution was loaded with 3.9 .mu.Ci of labeled DNA.
The amount in weight of DNA was not known. The gels were allowed to
set for 24 hrs at 37.degree. C. before elution commenced. 0.1 cc of
TSAE elution buffer was added to each gel, withdrawn and replaced
with fresh elution buffer after specific incubation periods at
37.degree. C. The samples were analyzed by PAGE using 3-10%
gradient acrylamide gels, dried, and autoradiographed. The results
indicate that throughout the 7 days of analysis DNA fragments up to
22,000 bp were released from the three SELP gels.
[0247] It is evident from the above results, that the subject
compositions have particularly desirable properties for uses in
implants, for tissue augmentation and for sustained release of
bioactive compounds in vivo. By varying compositional ratios, the
rate of resorption can be varied greatly, without significant
changes in the tensile properties of polymer films. The
compositions can be formed in a wide variety of devices or objects,
to find extensive use for a variety of purposes and context as
implants.
[0248] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0249] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
* * * * *