U.S. patent application number 14/480778 was filed with the patent office on 2014-12-25 for scaffolds formed from polymer-protein conjugates, methods of generating same and uses thereof.
The applicant listed for this patent is Regentis Biomaterials Ltd.. Invention is credited to Dror SELIKTAR, Yonatan Shachaf.
Application Number | 20140377209 14/480778 |
Document ID | / |
Family ID | 43632644 |
Filed Date | 2014-12-25 |
United States Patent
Application |
20140377209 |
Kind Code |
A1 |
SELIKTAR; Dror ; et
al. |
December 25, 2014 |
SCAFFOLDS FORMED FROM POLYMER-PROTEIN CONJUGATES, METHODS OF
GENERATING SAME AND USES THEREOF
Abstract
Conjugates are provided herein which comprise a protein attached
to at least two polymeric moieties, at least one of which exhibits
reverse thermal gelation. The conjugates are suitable for being
cross-linked by non-covalent and/or covalent cross-linking.
Compositions-of-matter comprising cross-linked conjugates are
provided herein, as well as processes for producing same. Methods
of controlling a physical property of compositions-of-matter are
also provided herein. The conjugates and compositions-of-matter may
be used for various applications, such as cell growth, tissue
formation, and treatment of disorders characterized by tissue
damage or loss, as described herein.
Inventors: |
SELIKTAR; Dror; (Haifa,
IL) ; Shachaf; Yonatan; (Haifa, IL) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Regentis Biomaterials Ltd. |
Or-Akiva |
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IL |
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|
Family ID: |
43632644 |
Appl. No.: |
14/480778 |
Filed: |
September 9, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13515298 |
Jun 12, 2012 |
8846020 |
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PCT/IL2010/001072 |
Dec 16, 2010 |
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14480778 |
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61282104 |
Dec 16, 2009 |
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Current U.S.
Class: |
424/78.3 ;
424/93.7; 514/7.6; 524/498; 525/54.1 |
Current CPC
Class: |
C12N 2533/52 20130101;
A61K 35/12 20130101; A61K 47/6903 20170801; A61K 47/60 20170801;
A61K 47/50 20170801; A61L 27/48 20130101; A61L 27/227 20130101;
A61K 38/18 20130101; C12N 5/0068 20130101; A61K 38/363 20130101;
A61K 47/6435 20170801; C12N 2533/30 20130101; A61L 27/222 20130101;
A61L 27/225 20130101 |
Class at
Publication: |
424/78.3 ;
525/54.1; 524/498; 424/93.7; 514/7.6 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 38/18 20060101 A61K038/18; A61K 38/36 20060101
A61K038/36; A61K 35/12 20060101 A61K035/12 |
Claims
1. A conjugate comprising a polypeptide having attached thereto at
least two polymeric moieties, at least one of said polymeric
moieties exhibiting a reverse thermal gelation.
2. The conjugate of claim 1, wherein each of said polymeric
moieties exhibits a reverse thermal gelation.
3. The conjugate of claim 1, wherein said polypeptide comprises a
fibrinogen or a fragment thereof.
4. The conjugate of claim 1, wherein at least one of said polymeric
moieties comprises a poloxamer (poly(ethylene oxide-propylene
oxide) copolymer).
5. A composition-of-matter comprising a cross-linked form of the
conjugate of claim 1, said cross-linked form comprising a plurality
of molecules of the conjugate cross-linked to one another.
6. The composition-of-matter of claim 5, being a hydrogel.
7. The composition-of-matter of claim 5, generated by a reverse
thermal gelation of said plurality of molecules of the conjugate in
an aqueous solution.
8. The composition-of-matter of claim 5, wherein said plurality of
molecules of the conjugate are non-covalently cross-linked to one
another.
9. The composition-of-matter of claim 5, wherein at least one of
said polymeric moieties further comprises a cross-linking moiety,
and said plurality of molecules of the conjugate are covalently
cross-linked to one another.
10. The composition-of-matter of claim 5, further comprising cells
therein.
11. The composition-of-matter of claim 5, further comprising growth
factors.
12. A process of producing the composition-of-matter of claim 5,
the process comprising heating a solution of a plurality of
molecules of a conjugate comprising a polypeptide having attached
thereto at least two polymeric moieties, at least one of said
polymeric moieties exhibiting a reverse thermal gelation, from a
first temperature to a second temperature, said second temperature
being such that a reverse thermal gelation of the conjugate in said
solution is effected, thereby producing the
composition-of-matter.
13. The process of claim 12, wherein said composition-of-matter is
produced in vivo.
14. The process of claim 13, wherein said solution further
comprises cells derived from an autologous source.
15. A process of producing the composition-of-matter of claim 9,
the process comprising subjecting a solution comprising a plurality
of molecules of a conjugate comprising a polypeptide having
attached thereto at least two polymeric moieties, at least one of
said polymeric moieties exhibiting a reverse thermal gelation, at
least one of said polymeric moieties further comprising at least
one cross-linking moiety for covalently cross-linking a plurality
of molecules of the conjugate to one another, to conditions that
effect covalent cross-linking of said cross-linking moieties,
thereby producing the composition-of-matter.
16. The process of claim 15, wherein said covalent cross-linking is
effected in vivo.
17. The process of claim 16, wherein said solution further
comprises cells derived from an autologous source.
18. A process of producing the composition-of-matter of claim 9 in
vivo, the process comprising: (a) subjecting a solution comprising
a plurality of molecules of a conjugate comprising a polypeptide
having attached thereto at least two polymeric moieties, at least
one of said polymeric moieties exhibiting a reverse thermal
gelation, at least one of said polymeric moieties further
comprising at least one cross-linking moiety for covalently
cross-linking a plurality of molecules of the conjugate to one
another, to conditions that effect covalent cross-linking ex vivo,
to thereby produce a covalently cross-linked scaffold; and (b)
subjecting said covalently cross-linked scaffold to a physiological
temperature in vivo, such that a reverse thermal gelation of said
scaffold is effected in vivo, thereby producing the
composition-of-matter.
19. The process of claim 18, wherein said solution further
comprises cells derived from an autologous source.
20. A method of inducing formation of a tissue in vivo, the method
comprising implanting the composition-of-matter of claim 5 in a
subject, to thereby induce the formation of the tissue.
21. The method of claim 20, wherein said composition-of-matter
further comprises cells derived from said subject.
22. The method of claim 20, wherein said composition-of-matter
further comprises growth factors.
23. The method of claim 20, wherein said tissue is cartilage.
24. A method of inducing formation of a tissue in vivo, the method
comprising implanting a plurality of molecules of the conjugate of
claim 1 in a subject, to thereby induce the formation of the
tissue.
25. The method of claim 24, further comprising implanting in said
subject cells derived from said subject in combination with said
plurality of molecules of the conjugate.
26. The method of claim 24, wherein said tissue is cartilage.
27. A method of treating a subject having a disorder characterized
by tissue damage or loss, the method comprising implanting the
composition-of-matter of claim 5 in a subject, to thereby induce
formation of said tissue, thereby treating the disorder
characterized by tissue damage or loss.
28. The method of claim 27, wherein said composition-of-matter
further comprises cells derived from said subject.
29. The method of claim 27, wherein said composition-of-matter
further comprises growth factors.
30. The method of claim 27, wherein said disorder is an articular
cartilage defect.
31. A method of treating a subject having a disorder characterized
by tissue damage or loss, the method comprising implanting a
plurality of molecules of the conjugate of claim 1 in a subject, to
thereby induce formation of said tissue, thereby treating the
disorder characterized by tissue damage or loss.
32. The method of claim 31, further comprising implanting in said
subject cells derived from said subject in combination with said
plurality of molecules of the conjugate.
33. The method of claim 31, wherein said disorder is an articular
cartilage defect.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/515,298 filed on Jun. 12, 2012, which is a
National Phase of PCT Patent Application No. PCT/IL2010/001072
having International filing date of Dec. 16, 2010, which claims the
benefit of priority under 35 USC .sctn.119(e) of U.S. Provisional
Patent Application No. 61/282,104 filed on Dec. 16, 2009. The
contents of the above applications are all incorporated by
reference as if fully set forth herein in their entirety.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention, in some embodiments thereof, relates
to polymer-protein conjugates and, more particularly, but not
exclusively, to polymer-protein conjugates which form a scaffold,
to processes of generating same and to uses thereof in, for
example, tissue engineering.
[0003] As the field of tissue engineering evolves, there is a need
for new biomaterial scaffolds that can provide more than just
architectural and mechanical support. New "hybrid" materials are
being developed as sophisticated scaffolds wherein biological
polymers such as alginate, collagen or fibrinogen are combined with
synthetic polymers to provide added versatility and bioactivity at
the material/cell interface. From the perspective of cellular
interactions, the biological domains of the hybrid material may
actively participate in stimulating cells towards the formation of
functional tissues. Bioactive signals are controlled via biological
macromolecules such as protein segments [Cutler and Garcia,
Biomaterials 2003, 24:1759-1770], growth factors [Seliktar et al.,
J Biomed Mater Res A 2004, 68:704-716; Zisch et al., FASEB J 2003;
17:2260-2262; DeLong et al., Biomaterials 2005, 26:3227-3234] or
short bioactive peptides [Mann et al., Biomaterials 2001,
22:3045-3051; Lutolf et al., Proc Natl Acad Sci USA 2003,
100:5413-5418; Stile and Healy, Biomacromolecules 2001, 2:185-194].
These elements are capable of influencing cell migration,
proliferation, and guided differentiation [Dikovsky et al.,
Biomaterials 2006, 27:1496-1506]. From the perspective of
biomaterial properties, "smart" polymers may also be used to
provide better control over bulk features of the scaffold in
response to changes in temperature, pH, or light [Furth et al.,
Biomaterials 2007, 28:5068-5073; Galaev and Mattiasson, Trends
Biotechnol 1999, 17:335-340]. Hybrid materials made with smart
polymers have additional degrees of freedom, including control over
bulk density, degradability, porosity and compliance, all of which
can be regulated by the synthetic polymer component [Peppas et al.,
Annu Rev Biomed Eng 2000, 2:9-29; Tsang and Bhatia, Adv Drug Deliv
Rev 2004, 56:1635-1647; 3] Baier Leach et al., Biotechnol Bioeng
2003, 82:578-589].
[0004] Hybrid materials have been prepared based on conjugation of
endogenous proteins with versatile synthetic polymers [Almany and
Seliktar, Biomaterials 2005, 26:2467-2477; Gonen-Wadmany et al.,
Biomaterials 2007, 28:3876-3886; Peled et al., Biomed Mater Res A
2007, 80:874-884; Seliktar, Ann NY Acad Sci 2005, 1047:386-394].
The effect of alternating structural properties of hydrogels made
from poly(ethylene glycol) (PEG) conjugated to fibrinogen on the
morphology and remodeling of encapsulated smooth muscle cells has
been investigated [Dikovsky et al., Biomaterials 2006,
27:1496-1506; Dikovsky et al., Biophys J 2008, 94:2914-2925]. These
materials exhibited an ability to control cellular behavior by
changing factors such as density, stiffness, and proteolytic
degradability through the versatile synthetic component. The
fibrinogen is a natural substrate for tissue remodeling which
contains several cell signaling domains, including a protease
degradation substrate and cell adhesion motifs [Herrick et al., Int
J Biochem Cell Biol 1999, 31:741-746; Werb, Cell 1997,
91:439-442].
[0005] International Patent Application PCT/IL2004/001136
(published as WO2005/061018) and U.S. patent application Ser. No.
11/472,437 describe a biodegradable scaffold composed of a protein
(e.g., fibrinogen) backbone cross-linked by a synthetic polymer
such as poly(ethylene glycol), and methods of generating such
scaffolds from polymer-protein conjugates.
[0006] International Patent Application PCT/IL2008/000521
(published as WO 2008/126092) describes scaffolds composed of
albumin or thiolated collagen cross-linked by a synthetic polymer
such as poly(ethylene glycol).
[0007] Reverse thermo-responsive polymers are capable of producing
low viscosity aqueous solutions at ambient temperature, and forming
a gel at a higher temperature. This property may be used to
generate implants in situ [Cohn et al., Biomacromolecules 2005,
6:1168-1175].
[0008] Stile and Healy [Biomacromolecules 2001, 2:185-194] modified
a smart polymer, N-isopropylacrylamide, with RGD (Arg-Gly-Asp)
containing peptides to form a reversible thermo-sensitive hydrogel
with bioactive segments for cell culture studies. They reported
that the conjugation of RGD peptides to the thermo-responsive smart
polymer does not compromise the temperature-induced sol-gel
transition of the hydrogels. They further reported that the
conjugated RGD peptide enhanced the biological interactions of the
otherwise inert N-isopropylacrylamide polymer network.
[0009] Reverse thermo-responsive polymers having a poly(ethylene
oxide) (PEO)-poly(propylene oxide) (PPO)-PEO tri-block structure,
referred to as "poloxamers", have also been reported. The
endothermic sol-gel transition takes place due to an increase in
entropy caused by release of water molecules bound to the PPO
segments as temperature increases [Alexandridis, Colloid Surface A
1995, 96:1-46].
[0010] Pluronic.RTM. F127 poloxamer is a well known synthetic
triblock copolymer (PEO.sub.99-PPO.sub.67-PEO.sub.99) [Nagarajan
and Ganesh, J Colloid Interface Sci 1996, 184:489-499; Sharma and
Bhatia, Int J Pharm 2004, 278:361-377; Cohn et al., Biomaterials
2003, 24:3707-3714], that exhibits a reverse thermal gelation (RTG)
property above a critical temperature in aqueous solutions. Cohn et
al. [Polym Adv Tech 2007; 18:731-736] reported that polymerized
F127 displays reverse thermal gelation at lower concentrations and
with enhanced mechanical properties, as compared with F127.
[0011] Additional background art includes Halstenberg et al.
[Biomacromolecules 2002, 3:710-723], Cohn et al. [Polym Adv Tech
2007; 18:731-736], and U.S. Pat. No. 7,842,667.
SUMMARY OF THE INVENTION
[0012] According to an aspect of some embodiments of the present
invention there is provided a conjugate comprising a polypeptide
having attached thereto at least two polymeric moieties, at least
one of the polymeric moieties exhibiting a reverse thermal
gelation.
[0013] According to an aspect of some embodiments of the present
invention there is provided a composition-of-matter comprising a
cross-linked form of a conjugate described herein, the cross-linked
form comprising a plurality of molecules of the conjugate
cross-linked to one another.
[0014] According to an aspect of some embodiments of the present
invention there is provided a process of producing a
composition-of-matter described herein, the process comprising
heating a solution of a plurality of molecules of a conjugate
described herein from a first temperature to a second temperature,
the second temperature being such that a reverse thermal gelation
of the conjugate in the solution is effected, thereby producing the
composition-of-matter.
[0015] According to an aspect of some embodiments of the present
invention there is provided a process of producing a
composition-of-matter described herein, the process comprising
subjecting a solution comprising a plurality of molecules of a
conjugate described herein, the conjugate comprising at least one
cross-linking moiety, to conditions that effect covalent
cross-linking of the cross-linking moieties, thereby producing the
composition-of-matter.
[0016] According to an aspect of some embodiments of the present
invention there is provided a process of producing a
composition-of-matter described herein in vivo, the process
comprising:
[0017] (a) subjecting a solution comprising a plurality of
molecules of a conjugate described herein, the conjugate comprising
at least one cross-linking moiety, to conditions that effect
covalent cross-linking ex vivo, to thereby produce a covalently
cross-linked scaffold; and
[0018] (b) subjecting the covalently cross-linked scaffold to a
physiological temperature in vivo, such that a reverse thermal
gelation of the scaffold is effected in vivo, thereby producing the
composition-of-matter.
[0019] According to an aspect of some embodiments of the present
invention there is provided a method of controlling a physical
property of a composition-of-matter described herein, the method
comprising controlling a parameter selected from the group
consisting of a concentration of a conjugate described herein in
solution, an ambient temperature, a presence or absence of an
initiator, a dose of irradiation during covalent cross-linking, and
a cross-linking temperature.
[0020] According to an aspect of some embodiments of the present
invention there is provided a process of producing the conjugate
described herein, the process comprising covalently attaching a
polymer to a polypeptide, the polymer and the polypeptide being
such that at least two polymer molecules covalently attach to a
molecule of the polypeptide, wherein at least one of the two
polymer molecules exhibits a reverse thermal gelation, thereby
producing the conjugate.
[0021] According to an aspect of some embodiments of the present
invention there is provided a use of a conjugate described herein
or of a composition-of-matter described herein in the manufacture
of a medicament for repairing tissue damage.
[0022] According to an aspect of some embodiments of the present
invention there is provided a use of a conjugate described herein
or of a composition-of-matter described herein in the manufacture
of a medicament for treating a subject having a disorder
characterized by tissue damage or loss.
[0023] According to an aspect of some embodiments of the present
invention there is provided a method of inducing formation of a
tissue in vivo, the method comprising implanting a
composition-of-matter described herein in a subject, to thereby
induce the formation of the tissue.
[0024] According to an aspect of some embodiments of the present
invention there is provided a method of inducing formation of a
tissue in vivo, the method comprising implanting a plurality of
molecules of a conjugate described herein in a subject, to thereby
induce the formation of the tissue.
[0025] According to an aspect of some embodiments of the present
invention there is provided a method of inducing formation of a
tissue ex vivo, the method comprising subjecting a
composition-of-matter which comprises cells, as described herein,
to conditions conductive to growth of the cells, to thereby induce
tissue formation.
[0026] According to an aspect of some embodiments of the present
invention there is provided a method of treating a subject having a
disorder characterized by tissue damage or loss, the method
comprising implanting a composition-of-matter described herein in a
subject, to thereby induce formation of the tissue, thereby
treating the disorder characterized by tissue damage or loss.
[0027] According to an aspect of some embodiments of the present
invention there is provided a method of treating a subject having a
disorder characterized by tissue damage or loss, the method
comprising implanting a plurality of molecules of a conjugate
described herein in a subject, to thereby induce formation of the
tissue, thereby treating the disorder characterized by tissue
damage or loss.
[0028] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical, cosmetic or
cosmeceutical composition comprising a plurality of molecules of a
conjugate described herein, the composition being identified for
use in inducing formation of a tissue upon being contacted with a
tissue and further upon subjecting the composition to a
physiological temperature.
[0029] According to an aspect of some embodiments of the present
invention there is provided a kit for inducing formation of a
tissue, the kit comprising:
[0030] (a) a conjugate described herein;
[0031] (b) an aqueous solvent; and
[0032] (c) instructions for cross-linking an aqueous solution the
conjugate in order to form a scaffold for inducing formation of the
tissue.
[0033] According to some embodiments of the invention, each of the
polymeric moieties exhibits a reverse thermal gelation.
[0034] According to some embodiments of the invention, at least one
of the polymeric moieties further comprises at least one
cross-linking moiety for covalently cross-linking a plurality of
molecules of the conjugate to one another.
[0035] According to some embodiments of the invention, the
conjugate is of the general formula:
X(--Y--Zm)n
[0036] wherein:
[0037] X is a polypeptide described herein;
[0038] Y is a polymeric moiety described herein;
[0039] Z is a cross-linking moiety described herein;
[0040] n is an integer greater than 1; and
[0041] m is 0, 1 or an integer greater than 1.
[0042] According to some embodiments of the invention, the
polypeptide comprises a protein or a fragment thereof.
[0043] According to some embodiments of the invention, the protein
is selected from the group consisting of a cell signaling protein,
an extracellular matrix protein, a cell adhesion protein, a growth
factor, protein A, a protease, and a protease substrate.
[0044] According to some embodiments of the invention, the
extracellular matrix protein is selected from the group consisting
of fibrinogen, collagen, fibronectin, elastin, fibrillin, fibulin,
vimentin, laminin and gelatin.
[0045] According to some embodiments of the invention, the
polypeptide comprises a fibrinogen or a fragment thereof.
[0046] According to some embodiments of the invention, the protein
is denatured.
[0047] According to some embodiments of the invention, the
polypeptide is a denatured fibrinogen.
[0048] According to some embodiments of the invention, the
polymeric moiety comprises a synthetic polymer.
[0049] According to some embodiments of the invention, at least one
of the polymeric moieties comprises a poloxamer (poly(ethylene
oxide-propylene oxide) copolymer).
[0050] According to some embodiments of the invention, each of the
polymeric moieties comprises a poloxamer.
[0051] According to some embodiments of the invention, the
poloxamer is F127 poloxamer.
[0052] According to some embodiments of the invention, at least one
of the polymeric moieties comprises T1307 polymer.
[0053] According to some embodiments of the invention, the
polymeric moieties are selected from the group consisting of a
Pluronic.RTM. polymer and a Tetronic.RTM. polymer.
[0054] According to some embodiments of the invention, each of the
polymeric moieties comprises from 1 to 10 of the cross linking
moieties.
[0055] According to some embodiments of the invention, the
cross-linking moiety comprises a polymerizable group.
[0056] According to some embodiments of the invention, the
polymerizable group is polymerizable by free radical
polymerization.
[0057] According to some embodiments of the invention, the
polymerizable group is selected from the group consisting of an
acrylate, a methacrylate, an acrylamide, a methacrylamide, and a
vinyl sulfone.
[0058] According to some embodiments of the invention, the
polypeptide is denaturated fibrinogen and the polymeric moieties
comprise F127 poloxamer.
[0059] According to some embodiments of the invention, the
conjugate comprises F127 poloxamer diacrylate moieties, wherein an
acrylate group of each of the F127 poloxamer diacrylate moieties is
attached to a cysteine residue of the fibrinogen.
[0060] According to some embodiments of the invention, the
polypeptide is denaturated fibrinogen and the polymeric moieties
comprise T1307 polymer.
[0061] According to some embodiments of the invention, the
conjugate comprises T1307 tetraacrylate moieties, wherein an
acrylate group of each of the T1307 tetraacrylate moieties is
attached to a cysteine residue of the fibrinogen.
[0062] According to some embodiments of the invention, the
conjugate is characterized by an ability to undergo reverse thermal
gelation in an aqueous solution.
[0063] According to some embodiments of the invention, the reverse
thermal gelation is effected at a concentration of less than 10
weight percents of the conjugate in the aqueous solution.
[0064] According to some embodiments of the invention, the reverse
thermal gelation of the conjugate increases a shear storage modulus
of the aqueous solution by at least ten-folds.
[0065] According to some embodiments of the invention, the reverse
thermal gelation increases a shear storage modulus of the aqueous
solution to at least 20 Pa.
[0066] According to some embodiments of the invention, the reverse
thermal gelation increases a shear storage modulus of the aqueous
solution from less than 2 Pa to at least 20 Pa.
[0067] According to some embodiments of the invention, the reverse
thermal gelation occurs upon an increase of temperature from
10.degree. C. to 55.degree. C.
[0068] According to some embodiments of the invention, the reverse
thermal gelation is reversible.
[0069] According to some embodiments of the invention, the reverse
thermal gelation forms a biodegradable gel.
[0070] According to some embodiments of the invention, the
conjugate is identified for use in generating a scaffold.
[0071] According to some embodiments of the invention, the
conjugate is identified for use in reversibly generating a
scaffold.
[0072] According to some embodiments of the invention, the scaffold
is a hydrogel.
[0073] According to some embodiments of the invention, the hydrogel
is characterized by a shear storage modulus of at least 15 Pa at a
temperature of 37.degree. C.
[0074] According to some embodiments of the invention, the hydrogel
is capable of undergoing a reverse thermal gelation.
[0075] According to some embodiments of the invention, the
composition-of-matter is a hydrogel.
[0076] According to some embodiments of the invention, the
composition-of-matter is generated by a reverse thermal gelation of
the plurality of molecules of the conjugate in an aqueous
solution.
[0077] According to some embodiments of the invention, the
plurality of molecules of the conjugate are non-covalently
cross-linked to one another.
[0078] According to some embodiments of the invention, the
cross-linked form of the conjugate is reversible.
[0079] According to some embodiments of the invention, at least one
of the polymeric moieties comprises a cross-linking moiety, and the
plurality of molecules of the conjugate are covalently cross-linked
to one another.
[0080] According to some embodiments of the invention, the
composition-of-matter is generated by subjecting a plurality of
molecules of the conjugate to conditions for effecting
cross-linking of the cross-linking moieties.
[0081] According to some embodiments of the invention, the
composition-of-matter is characterized by a shear storage modulus
of at least 20 Pa at a temperature of 37.degree. C.
[0082] According to some embodiments of the invention, the
composition-of-matter is capable of undergoing a reverse thermal
gelation.
[0083] According to some embodiments of the invention, the reverse
thermal gelation of the composition-of-matter increases a shear
storage modulus of the composition-of-matter by at least 200%.
[0084] According to some embodiments of the invention, the reverse
thermal gelation of the composition-of-matter increases a shear
storage modulus of the composition-of-matter to at least 15 Pa.
[0085] According to some embodiments of the invention, the reverse
thermal gelation of the composition-of-matter increases a shear
storage modulus of the composition-of-matter from a first value in
a range of from 0.5 Pa to 200 Pa to a second value which is at
least 20% higher than the first value.
[0086] According to some embodiments of the invention, the reverse
thermal gelation of the composition-of-matter increases a shear
storage modulus of the composition-of-matter from a first value to
a second value in a range of from 20 Pa to 5000 Pa, the second
value being at least 20% higher than the first value.
[0087] According to some embodiments of the invention, the reverse
thermal gelation of the composition-of-matter occurs upon an
increase of temperature from 10.degree. C. to 55.degree. C.
[0088] According to some embodiments of the invention, the reverse
thermal gelation of the composition-of-matter is reversible.
[0089] According to some embodiments of the invention, the
composition-of-matter is characterized by a shear storage modulus
of one portion of the composition-of-matter that is different from
a shear storage modulus of at least one other portion of the
composition-of-matter.
[0090] According to some embodiments of the invention, the
composition-of-matter is biodegradable.
[0091] According to some embodiments of the invention, the
composition-of-matter further comprises cells therein.
[0092] According to some embodiments of the invention, the
composition-of-matter is identified for use in inducing a formation
of a tissue.
[0093] According to some embodiments of the invention, the
composition-of-matter is identified for use in repairing tissue
damage.
[0094] According to some embodiments of the invention, the
composition-of-matter is produced in vivo.
[0095] According to some embodiments of the invention, the
abovementioned second temperature is a physiological
temperature.
[0096] According to some embodiments of the invention, the
conjugate comprises at least one polymeric moiety that further
comprises at least one cross-linking moiety, and the process
further comprises subjecting the solution to conditions that effect
cross-linking of the cross-linking moieties.
[0097] According to some embodiments of the invention, subjecting
the solution to the conditions that effect cross-linking is
effected prior to the heating.
[0098] According to some embodiments of the invention, subjecting
the solution to the conditions that effect cross-linking is
effected subsequent to the heating.
[0099] According to some embodiments of the invention, the covalent
cross-linking is effected in vivo.
[0100] According to some embodiments of the invention, the covalent
cross-linking is effected ex vivo, to thereby produce a covalently
cross-linked scaffold, and the process further comprises subjecting
the covalently cross-linked scaffold to a physiological temperature
in vivo, such that a reverse thermal gelation of the scaffold is
effected in vivo, thereby producing a composition-of-matter
described herein.
[0101] According to some embodiments of the invention, the
conditions comprise irradiation.
[0102] According to some embodiments of the invention, the
conditions comprise a presence of a free radical initiator.
[0103] According to some embodiments of the invention, the solution
further comprises cells, and the process is for producing a
composition-of-matter comprising cells embedded therein.
[0104] According to some embodiments of the invention, the
conjugate comprises at least one cross-linking moiety, and the
method further comprises covalently cross-linking the plurality of
molecules of the conjugate.
[0105] According to some embodiments of the invention, the
cross-linking is effected by subjecting the plurality of molecules
of the conjugate to conditions that effect covalent cross-linking
of the cross-linking moiety.
[0106] According to some embodiments of the invention, the
conjugate comprises at least one cross-linking moiety, and the
composition described herein is identified for use in inducing
formation of a tissue upon further subjecting the plurality of
molecules of the conjugate to conditions that effect covalent
cross-linking of the cross-linking moiety.
[0107] According to some embodiments of the invention, a
pharmaceutical, cosmetic or cosmeceutical composition described
herein further comprises an initiator for inducing covalent
cross-linking of the cross-linking moiety.
[0108] According to some embodiments of the invention, a
pharmaceutical, cosmetic or cosmeceutical composition described
herein is packaged in a packaging material and identified in print,
in or on the packaging material, for use in inducing formation of
the tissue.
[0109] According to some embodiments of the invention, the
conjugate comprises at least one cross-linking moiety, and the kit
further comprises an initiator for inducing covalent cross-linking
of the cross-linking moiety.
[0110] According to some embodiments of the invention, the kit
further comprises cells for embedding in the scaffold described
herein.
[0111] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0112] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0113] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0114] In the drawings:
[0115] FIGS. 1A and 1B are schemes showing the synthesis of F127
poloxamer diacrylate (FIG. 1A) from F127 poloxamer and acryloyl
chloride in a 1:2 mixture of dichloromethane (DCM) and toluene with
triethylamine ((ET).sub.3N) at room temperature (R.T.), and the
synthesis of an F127 poloxamer-fibrinogen conjugate (FF127) using
the poloxamer diacrylate (FIG. 1B) in phosphate buffer saline (PBS)
with 8 M urea, according to some embodiments of the invention;
[0116] FIG. 2 presents comparative plots showing the storage
modulus (G') of FF127 solutions at fibrinogen concentrations of 4,
6 and 8 mg/ml, as a function of temperature; the inset graph shows
the storage modulus (G') and loss modulus (G'') of an FF127
solution with 8 mg/ml fibrinogen;
[0117] FIGS. 3A and 3B present graphs showing the storage moduli of
FF127 solutions with (FIG. 3B) and without (FIG. 3A) chemical
(covalent) cross-linking of the FF127, as a function of time with
cyclic temperature changes between 15.degree. C. and 37.degree. C.,
in the presence of 0.1 or 0.01 mg/ml collagenase, and in the
absence of collagenase;
[0118] FIG. 4 is a schematic illustration of fibrinogen
polypeptides (red, green and blue) conjugated to a polymer (black)
and hydrogel assembly according to some embodiments of the
invention by reversible (non-covalent) cross-linking of the polymer
in a temperature-dependent manner or irreversible UV-induced
(covalent) cross-linking;
[0119] FIG. 5 is a graph showing a reversible increase in storage
modulus (G') of an FF127 solution by increasing the ambient
temperature (T.sub.amb) and a subsequent irreversible UV-induced
increase of the storage modulus;
[0120] FIG. 6 presents comparative plots showing the storage
modulus (G') of a chemically (covalently) cross-linked FF127 at
fibrinogen concentrations of 4, 6 and 8 mg/ml, as a function of
temperature; the inset graph shows the storage modulus (G') and
loss modulus (G'') of a chemically (covalently) cross-linked FF127
with 8 mg/ml fibrinogen;
[0121] FIGS. 7A and 7B present graphs showing the effect of
oscillatory stress and temperature changes on the storage modulus
(G'; FIG. 7A) and loss modulus (G''; FIG. 7B) of hydrogels of 8
mg/ml FF127 with (black line) and without (dotted line) chemical
(covalent) cross-linking (temperatures were cycled between
37.degree. C. (red lines) and 15.degree. C. (blue lines) at a rate
of 1.degree. C./second; oscillation frequency was 1 Hz; strain was
2%);
[0122] FIG. 8 presents a graph showing the storage modulus (G') of
FF127 hydrogels (8 mg/ml fibrinogen) cross-linked (covalently) by
application of UV light at different cross-linking temperatures
(T.sub.cl), following exposure to ambient temperatures (T.sub.amb)
(before T.sub.amb=37.degree. C., T.sub.amb=T.sub.cl);
[0123] FIG. 9 is a bar graph showing the swelling ratio of FF127
hydrogels (6 mg/ml fibrinogen) formed with cross-linking
temperatures (T.sub.cl) of 21.degree. C. or 37.degree. C. and a
hydrogel formed from cross-linked 12 kDa PEG-fibrinogen conjugates
(PF12 kDa), at ambient temperatures (T.sub.amb) of 4.degree. C. and
37.degree. C.;
[0124] FIGS. 10A and 10B are images showing the diameters (marked
by black circles) of FF127 hydrogels (6 mg/ml fibrinogen)
chemically (covalently) cross-linked at a temperature of 21.degree.
C. (FIG. 10A) or 37.degree. C. (FIG. 10B), and then subjected to
ambient temperatures of 37.degree. C.; images on left show the
hydrogels at the cross-linking temperature immediately after
chemical cross-linking, and images on right show the chemically
(covalently) cross-linked hydrogels after incubation at 37.degree.
C.;
[0125] FIG. 11 presents comparative plots showing the degradation
in trypsin solution of hydrogels formed by cross-linking FF127 or
12 kDa PEG-fibrinogen conjugate (PF12) at a cross-linking
temperature (T.sub.cl) of 21.degree. C. or 37.degree. C. (storage
moduli (G') and degradation half-lives (t50) of the hydrogels are
indicated);
[0126] FIG. 12 is a bar graph showing the storage modulus (G') of
hydrogels formed by cross-linking FF127 (at a cross-linking
temperature (T.sub.cl) of 21.degree. C. or 37.degree. C.), 12 kDa
PEG-fibrinogen conjugate (PF12 kDa), or F127 diacrylate (F127-DA),
at an ambient temperature (T.sub.amb) of 37.degree. C.;
[0127] FIGS. 13A and 13B are schemes illustrating the synthesis
(FIG. 13A) of a T1307-fibrinopeptide conjugate (FT-1307) in
phosphate buffer saline (PBS) with 8 M urea at room temperature
(R.T.), and the structure of the conjugate (FIG. 13B), according to
some embodiments of the invention;
[0128] FIGS. 14A and 14B present comparative plots (FIG. 14A) and a
bar graph (FIG. 14B) showing the storage modulus (G') of FT-1307 (6
mg/ml fibrinogen) hydrogels cross-linked at a temperature
(T.sub.cl) of 4.degree. C., 21.degree. C. or 37.degree. C., as a
function of ambient temperature (FIG. 14A), and as a mean.+-.SEM of
4 samples at an ambient temperature of 37.degree. C. (FIG.
14B);
[0129] FIG. 15 is a bar graph showing the swelling ratio (Q.sub.M)
of FT1307 hydrogels (6 mg/ml fibrinogen) cross-linked at a
temperature (T.sub.cl) of 4.degree. C., 21.degree. C. or 37.degree.
C., at an ambient temperature (T.sub.amb) of 4.degree. C. and
37.degree. C.;
[0130] FIG. 16 is a bar graph showing the biodegradation half-life
(T.sub.112) in trypsin solution of FT1307 hydrogels (6 mg/ml
fibrinogen) cross-linked at a temperature (T.sub.cl) of 4.degree.
C., 21.degree. C. or 37.degree. C.;
[0131] FIG. 17 presents images showing human foreskin fibroblasts
seeded in hydrogels formed by cross-linking FF127 (at a
cross-linking temperature (T.sub.cl) of 21.degree. C. or 37.degree.
C.), 12 kDa PEG-fibrinogen conjugate (PEG-Fib 12 kDa), or F127
diacrylate (F127-DA), 3 and 6 days after seeding (scale bar=100
.mu.m);
[0132] FIG. 18 is an image showing human foreskin fibroblasts
seeded in FF127 hydrogels with (Physical+Chemical) and without
(Physical) chemical cross-linking of the FF127 (at a cross-linking
temperature of 37.degree. C.), 3 and 6 days after seeding (scale
bar=100 .mu.m);
[0133] FIG. 19 is a graph showing the viability of human foreskin
fibroblasts seeded for 0 or 3 days in hydrogels formed by
cross-linking FF127 at a cross-linking temperature (T.sub.cl) of
21.degree. C. or 37.degree. C. (storage moduli (G') and degradation
half-lives (t50) of the hydrogels are indicated);
[0134] FIGS. 20A and 20B are an image (FIG. 20A) and a graph (FIG.
20B) showing the cellular invasion from smooth muscle tissue into
hydrogels formed by cross-linking FF127 (at a cross-linking
temperature (T.sub.cl) of 21.degree. C. or 37.degree. C.) or 12 kDa
PEG-fibrinogen conjugate (PF12 kDa), on days 1, 3 and 5 after
encapsulation of the tissue in the hydrogel; FIG. 20B shows the
invasion distance as a function of time (scale bar=100 .mu.m);
[0135] FIG. 21 is an image showing human foreskin fibroblasts 3
hours, 3 days or 6 days after being seeded in FT1307 hydrogels with
storage moduli of 52, 244 or 373 Pa (viable cells are stained with
calcein (green) and non viable cells are stained with ethidium
(orange); scale bar=100 .mu.m);
[0136] FIG. 22 is an image showing HeLa cells 3 hours, 3 days or 6
days after being seeded in FT1307 hydrogels with storage moduli of
52, 244 or 373 Pa (viable cells are stained with calcein (green)
and non viable cells are stained with ethidium (orange); scale
bar=100 .mu.m);
[0137] FIGS. 23A and 23B depict the preparation of a cell-seeded
FF127 capsule embedded in an FT1307 hydrogel, according to some
embodiments of the invention;
[0138] FIGS. 24A and 24B are photographs showing an FF127 capsule
(6 mg/ml fibrinogen) seeded with human foreskin fibroblasts (green)
embedded for 6 days in an FT1307 hydrogel (6 mg/ml fibrinogen)
having a storage modulus of 373 Pa (FIG. 24A) or 52 Pa (FIG. 24B)
(scale bar=200 .mu.m);
[0139] FIGS. 25A and 25B are photographs showing an FF127 capsule
(6 mg/ml fibrinogen) seeded with Hela cells (green) embedded for 6
days in an FT1307 hydrogel (6 mg/ml fibrinogen) having a storage
modulus of 373 Pa (FIG. 25A) or 52 Pa (FIG. 25B) (scale bar=200
.mu.m);
[0140] FIGS. 26A and 26B are photographs showing FF127 capsules (6
mg/ml fibrinogen) seeded with a co-culture of human foreskin
fibroblasts (stained green) and Hela cells (stained red) on day 0
(FIG. 26A) and on day 5 (FIG. 26B) of being embedded in an FT1307
hydrogel (6 mg/ml fibrinogen) (dashed circles in FIG. 26B show the
diameter of the cell culture on day 0, scale bar=200 .mu.m);
and
[0141] FIGS. 27A and 27B are photographs showing FF127 capsules (6
mg/ml fibrinogen) seeded with a co-culture of human foreskin
fibroblasts (stained green) and Hela cells (stained red) on day 0
(FIG. 26A) and on day 5 (FIG. 26B) of being embedded in an FT1307
hydrogel (6 mg/ml fibrinogen) (scale bar=200 .mu.m).
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0142] The present invention, in some embodiments thereof, relates
to polymer-protein conjugates and, more particularly, but not
exclusively, to polymer-protein conjugates which form a scaffold,
to processes of generating same and to uses thereof in, for
example, tissue engineering.
[0143] The conjugation of a synthetic polymer to a natural protein
such as fibrinogen provides a means of creating biocompatible
hydrogels while controlling their physical properties. The
conjugation reaction is intended to endow the protein constituent
with additional structural versatility, without compromising its
biocompatibility.
[0144] The present inventors have previously disclosed a
methodology of generating hydrogels made from a synthetic polymer
such as poly(ethylene glycol) (PEG) conjugated to fibrinogen, which
enables to control cellular behavior of the formed hydrogels by
manipulating factors such as density, stiffness, and proteolytic
degradability through the versatile synthetic component.
[0145] In a search for methodologies for generating hydrogels with
improved control of the hydrogel's characteristics, the present
inventors have designed and successfully practiced a methodology of
generating "smart" hydrogels, by conjugating to proteins a
synthetic polymer that exhibits a reverse thermal gelation (RTG)
property above a critical temperature in aqueous solutions.
[0146] This methodology was found to produce hydrogels with an
exceptional control of physical characteristics of the hydrogels,
since it allows manipulating these characteristics by selecting,
for example, the degree and nature of the cross-linking reactions
that lead to gel formation. Since it was uncovered that the
protein-polymer conjugates exhibit a reverse thermal gelation
property, the degree and occurrence of non-covalent (physical)
cross-linking can be controlled, whereby chemical conditions can be
selected for effecting covalent cross-linking if desired.
[0147] Thus, using a combination of photo-polymerization
cross-linking and temperature, an exceptional control over physical
properties of the generated hydrogels was demonstrated. The ability
of the generated hydrogels to act as a matrix for cell and tissue
growth and survival (e.g., as a scaffold) has also been
demonstrated.
[0148] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways.
[0149] The present inventors have demonstrated the novel
methodology while utilizing Pluronic.RTM. F127 poloxamer and
Tetronic.RTM. T1307 copolymer (a poloxamer derivative) which are
end-functionalized with acryl groups and are reacted with denatured
fibrinogen via a Michael-type addition reaction to form a
protein-copolymer conjugate. These exemplary polymeric conjugates
could cross-link to form a structure comprising multiple units
("unimers") of the conjugate. Rheological measurements were
conducted on the functionalized unimers and the hydrogels generated
therefrom in order to characterize the physical response of these
conjugates to environmental stimuli (e.g., temperature
responsiveness).
[0150] The present inventors have thus further uncovered that the
generated hydrogels retain the biocompatibility of their fibrinogen
constituent with the added advantage of enhanced precision in
controlling the physical properties of the polymeric network using
the synthetic F127 constituent.
[0151] It was shown that the conjugation reaction does not
eliminate the self-assembly properties of the F127, but rather
enhances it, thus endowing the obtained protein-polymer conjugates
with reverse thermal gelation (RTG) properties. Thus, it was
uncovered that the poloxamer-fibrinogen conjugate surprisingly
undergoes gelation at low concentrations (e.g., below 20 mg/ml
conjugate), which are considerably lower than the concentrations
necessary for reverse thermal gelation of the poloxamer alone. This
indicates that the protein acts as a chain extender that allows the
poloxamer-protein conjugate to undergo gelation at these
exceptionally lower concentrations.
[0152] The ability to obtain hydrogels at low conjugate
concentrations is advantageous for applications such as tissue
regeneration, because such hydrogels are better suited for allowing
cell growth and migration within a hydrogel.
[0153] Using a combination of photo-polymerization cross-linking
and temperature, an exceptional control over physical properties of
the generated hydrogels was demonstrated. The ability of the
generated hydrogels to act as a matrix for cell and tissue growth
and survival has also been demonstrated.
[0154] Referring now to the drawings, FIGS. 1A and 1B illustrate
the synthesis of an exemplary F127 poloxamer-fibrinogen
conjugate.
[0155] FIG. 2 shows the gelation of the conjugate by an increase of
temperature (i.e., reverse thermal gelation) at various
concentrations, including at conjugate concentrations below 20
mg/ml. Such concentrations are lower than the concentrations that
allow reverse thermal gelation of F127 poloxamer alone, indicating
that conjugation to fibrinogen enhanced the RTG properties of the
poloxamer by acting as a chain extender.
[0156] FIGS. 3A and 3B show that the reverse thermal gelation of
the conjugate is reversible, such that gelation can be repeatedly
induced and reversed, even after the conjugate has been covalently
cross-linked (FIG. 3B). FIG. 6 shows the reverse thermal gelation
of covalently cross-linked conjugate at various concentrations.
[0157] FIG. 4 illustrates two types of cross-linking which
molecules of the conjugate can undergo to form a hydrogel; a
reversible temperature-dependent cross-linking of conjugate
molecules (by reverse thermal gelation), and an irreversible
cross-linking induced by UV light. FIG. 5 shows increases in shear
storage modulus resulting from both reversible and irreversible
cross-linking of conjugate molecules.
[0158] FIGS. 7A and 7B show the different behaviors of exemplary
covalently cross-linked and non-covalently cross-linked hydrogels
in response to stress. FIGS. 7A and 7B also show that after
collapsing in response to stress, both types of hydrogel recover
completely after lowering and increasing the temperature so as to
undo and restore the reverse thermal gelation.
[0159] FIGS. 8 and 12 show that the shear storage modulus of
exemplary covalently cross-linked hydrogels depends strongly on the
temperature at which the conjugate is covalently cross-linked. FIG.
11 shows that the effect of cross-linking temperature on
biodegradability is considerably weaker, and that biodegradability
is affected more by the type of polymer conjugated to the
protein.
[0160] FIGS. 9-10B show that the swelling properties of covalently
cross-linked poloxamer-fibrinogen hydrogels are
temperature-dependent (in contrast to cross-linked PEG-fibrinogen
hydrogels), and that the degree of temperature dependency is
affected by the cross-linking temperature.
[0161] FIGS. 13A and 13B illustrate the synthesis of an exemplary
T1307-fibrinogen conjugate, wherein each T1307 moiety in the
conjugate comprises three acrylate cross-linking moieties.
[0162] FIGS. 14A and 14B show that the shear storage modulus of
covalently cross-linked T1307-fibrinogen hydrogels depends strongly
on the temperature at which the conjugate is covalently
cross-linked. FIG. 15 shows that the swelling properties of
covalently cross-linked T1307-fibrinogen hydrogels are
temperature-dependent, and that the degree of temperature
dependency is affected by the cross-linking temperature. FIG. 16
shows that that biodegradability is not clearly correlated with the
cross-linking temperature.
[0163] The results presented in FIGS. 14A-16 indicate that the
properties of T1307-containing hydrogels are similar to those of
F127 poloxamer-containing hydrogels.
[0164] FIGS. 17-22 and 24A-27B show that exemplary hydrogels can
serve as matrices for cell growth and invasion, and that the rate
and type of cellular growth and invasion depends on the covalent
cross-linking temperature of the hydrogels. FIGS. 26A-27B show the
effects of different hydrogel properties on cell growth in a
co-culture of different cell types.
[0165] FIGS. 23A and 23B illustrate an exemplary process for
preparing a hydrogel capsule with one set of physical properties,
embedded within a hydrogel with a different set of physical
properties.
[0166] Thus, it has been demonstrated that polymer-fibrinogen
conjugates according to exemplary embodiments of the invention can
be readily cross-linked so as to form hydrogel scaffolds. In
addition, non-covalent and covalent cross-linking can be readily
combined. The hydrogels exhibit high flexibility, biodegradability,
good biofunctionality and support for cell spreading and invasion,
and a shear storage modulus which can be readily controlled by
various parameters. The temperature at which covalent cross-linking
is performed was particularly useful for controlling the shear
storage modulus, as it has relatively little effect on other
properties, such as biodegradability.
[0167] According to one aspect of the present invention, there is
provided a conjugate comprising a polypeptide having attached
thereto at least two polymeric moieties, at least one of the
polymeric moieties exhibiting a reverse thermal gelation. In some
embodiments, each of the polymeric moieties exhibits a reverse
thermal gelation.
[0168] As used herein, the phrase "reverse thermal gelation"
describes a property whereby a substance (e.g., an aqueous solution
of a compound) increases in viscosity upon an increase in
temperature. The increase in viscosity may be, for example,
conversion from a liquid state to a semisolid state (e.g., gel),
conversion from a liquid state to a more viscous liquid state, or
conversion from a semisolid state to a more rigid semisolid state.
Herein, all such conversions are encompassed by the term
"gelation". The increase in temperature which effects gelation may
be between any two temperatures. Optionally, the gelation is
effected at a temperature within the range of 0.degree. C. to
55.degree. C.
[0169] Herein, a polymeric moiety is considered to exhibit a
reverse thermal gelation when an aqueous solution of a polymer
which corresponds to the polymeric moiety (e.g., a polymer not
attached to the abovementioned polypeptide) exhibits a reverse
thermal gelation, as described herein.
[0170] A variety of polymers exhibit a reverse thermal gelation.
Each polymer may be characterized by a critical gelation
temperature, wherein gelation is effected at the critical gelation
temperature or at temperatures above the critical gelation
temperature.
[0171] Herein, "critical gelation temperature" refers to the lowest
temperature at which some gelation of a material is observed (e.g.,
by increase in shear storage modulus).
[0172] The polymeric moiety may be selected so as to impart to the
conjugate containing same a reverse thermal gelation that is
characterized by a critical gelation temperature within a
temperature range (e.g., in a range of 0.degree. C. to 55.degree.
C.) which allows for convenient manipulation of the properties of
the conjugate by exposure to an ambient temperature above and/or
below the critical gelation temperature.
[0173] The critical gelation temperature of the polymer may be
selected, for example, based on the intended use or desired
properties of a conjugate. For example, the critical gelation
temperature may be selected such that the conjugate is in a gelled
state at a physiological temperature but not at room temperature,
such that gelation may be effected in vivo. In another example, the
critical gelation temperature may be selected such that the
conjugate is in a gelled state at room temperature but not at a
moderately lower temperature, such that gelation may be effected,
for example, by removal from refrigeration.
[0174] The polymeric moiety optionally comprises a synthetic
polymer. Poloxamers (e.g., F127 poloxamer) are exemplary polymers
which exhibit a reverse thermal gelation at temperatures suitable
for embodiments of the present invention.
[0175] As used herein and in the art, a "poloxamer" refers to
poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO) block
copolymer having a PEO-PPO-PEO structure. Suitable poloxamers are
commercially available, for example, as Pluronic.RTM. polymers.
[0176] Typically, reverse thermal gelation is mediated by the
formation of non-covalent cross-linking (e.g., via hydrophobic
interactions, ionic interactions, and/or hydrogen bonding) between
molecules, wherein the degree of non-covalent cross-linking
increases in response to an increase of temperature.
[0177] Herein, "non-covalent" cross-linking (formed as a result of
a reverse thermal gelation) is also referred to as "physical"
cross-linking or as "non-chemical cross-linking". The non-covalent
cross-linking can therefore be understood as a
temperature-dependent cross-linking.
[0178] The polymeric moiety may comprise one or more moieties which
effect non-covalent cross-linking (e.g., hydrophobic moieties). The
degree of gelation and the conditions (e.g., temperature) under
which gelation is effected may optionally be controlled by the
nature and the number of moieties which participate in non-covalent
cross-linking.
[0179] The polymeric moiety may comprise from 1 and up to 100 and
even 1000 moieties which participate in non-covalent cross-linking.
In many embodiments, the higher the number of such moieties, and
the larger the moieties are (e.g., the higher the molecular weights
are), the lower the temperature under which gelation is
effected.
[0180] The polymeric moiety may comprise one or more types of
moieties which effect cross-linking. These moieties may effect
non-covalent cross-linking via the same intermolecular interactions
(e.g., hydrophobic interactions) or via different intermolecular
interactions (e.g., hydrophobic and ionic interactions). Polymers
that exhibit reverse thermal gelation (also referred to in the art
as RTG polymers) include, but are not limited to,
poly(N-isopropylacrylamide), which undergoes reverse thermal
gelation at temperatures above about 32-33.degree. C., as well as
copolymers thereof (e.g.,
poly(N-isopropylacrylamide-co-dimethyl-.gamma.-butyrolactone),
poly(ethylene glycol)-poly(amino urethane) (PEG-PAU) block
copolymers, poly(.epsilon.-caprolactone)-poly(ethylene glycol)
(PCL-PEG) block copolymers (e.g., PCL-PEG-PCL), and poly(methyl
2-propionamidoacrylate). In addition, polyorganophosphazenes with
PEG and hydrophobic oligopeptide side groups (which provide
intermolecular hydrophobic interactions) have been described, which
are gelled at temperatures of 35-43.degree. C. [Seong et al.,
Polymer 2005, 46:5075-5081].
[0181] For example, a poloxamer moiety comprises a hydrophobic PPO
moiety which mediates gelation. A polymeric moiety may optionally
comprise one such PPO moiety, or alternatively, a plurality (e.g.,
2, 3, 4, etc., up to 100 and even 1000 such moieties) of such
moieties.
[0182] Similarly PCL-PEG copolymers comprise hydrophilic PEG and a
relatively hydrophobic poly(.epsilon.-caprolactone) (PCL) moiety,
and PEG-PAU copolymers comprise hydrophilic PEG and a hydrophobic
poly(amino urethane) (PAU) moiety (e.g., a
bis-1,4-(hydroxyethyl)piperazine-1,6-diisocyanato hexamethylene
condensation polymer moiety).
[0183] Thus, in general, many block polymers exhibiting reverse
thermal gelation may be prepared from a combination of hydrophilic
and hydrophobic building blocks.
[0184] In some embodiments, each polymeric moiety comprises a
poloxamer (e.g., F127 poloxamer).
[0185] Optionally, a polymeric moiety comprises one poloxamer.
[0186] Alternatively or additionally, at least one polymeric moiety
comprises a plurality of poloxamer moieties. Polymers comprising a
plurality of poloxamer moieties are commercially available, for
example, as Tetronic.RTM. polymers. T1307 (e.g.,
Tetronic.RTM.T1307) is an exemplary polymer which comprises four
poloxamer moieties.
[0187] According to optional embodiments, at least one of the
polymeric moieties further comprises at least one cross-linking
moiety for covalently cross-linking a plurality of molecules of the
conjugate to one another. Optionally, the polymeric moiety
comprises from 1 to 10, optionally from 1 to 5, and optionally from
1 to 3 cross-linking moieties.
[0188] It to be noted that the expression "cross-linking moiety" is
used herein to describe moieties that are attached to the polymeric
moiety (e.g., as an end group or as pendant groups), or which form
an integral part of the polymeric moiety, yet it differs from those
moieties in the polymeric moiety that effect non-covalent
cross-linking, as described hereinabove.
[0189] A "cross-linking moiety" as used herein thus describes
moieties on the polymeric moiety that effect covalent
cross-linking, as defined herein, between molecules of the
conjugate.
[0190] Herein, "covalent cross-linking" (also referred to herein as
"chemical cross-linking") refers to a formation of a covalent bond
("cross-link") between two or more molecules (e.g., two conjugate
molecules described herein). A molecule may be attached to a
plurality of other molecules, each other molecule being attached by
a different covalent bond. Thus, a plurality of molecules (e.g., at
least 5, at least 10, at least 20, at least 50, at least 100) may
be linked together.
[0191] A conjugate as described may optionally be represented by
the general formula:
X(--Y--Zm)n
[0192] wherein X is a polypeptide as described herein, Y is a
polymeric moiety as described herein, Z is a cross-linking moiety
as described herein, n is an integer greater than 1 (e.g., 2, 3, 4
and up to 20), and m represents the number of cross-linking
moieties per polymeric moiety. Thus, m is 0 in embodiments lacking
the optional cross-linking moiety, and m is 1 or an integer greater
than 1, in embodiments which comprise the optional cross-linking
moiety.
[0193] It is to be understood that as the above formula includes
more than one --Y--Zm moiety, different --Y--Zm moieties in a
conjugate may optionally have a different values for m.
[0194] As used herein, the phrase "cross-linking moiety" refers to
a moiety (e.g., a functional group) characterized by an ability to
effect covalent cross-linking with a functional group of another
molecule (e.g., another conjugate).
[0195] According to optional embodiments, the cross-linking moiety
is able to effect cross-linking with a conjugate similar to and/or
identical to the conjugate described herein (e.g., a conjugate
comprising a cross-linking moiety chemically related to and/or
identical to the cross-linking moiety of the conjugate described
herein).
[0196] Thus, the cross-linking moiety described herein provides a
conjugate with an ability to undergo covalent cross-linking,
whereas a polymeric moiety which exhibits reverse thermal gelation,
as described herein, provides a conjugate with an ability to
undergo non-covalent cross-linking (self-assembly). Hence, in
embodiments without a cross-linking moiety (e.g., wherein m in the
general formula is 0), cross-linking of the conjugate may be
effected solely by non-covalent cross-linking by the polymeric
moiety, whereas in embodiments with a cross-linking moiety (e.g.,
wherein m in the general formula is 1 or more), cross-linking of
the conjugate may be effected by non-covalent cross-linking and/or
by covalent cross-linking, as discussed in more detail herein.
[0197] Exemplary cross-linking moieties that are suitable for use
in the context of embodiments of the invention include, but are not
limited to, polymerizable groups, as further detailed
hereinbelow.
[0198] Thus, in some embodiments, the cross-linking moiety
comprises a polymerizable group, such that cross-linking may be
effected by polymerization of the polymerizable group. In the
context of embodiments of the present invention, the polymerizable
groups may act as monomers, whereby polymerization of the
polymerizable groups cross-links the conjugates comprising the
polymerizable groups.
[0199] Many polymerizable groups are known in the art, including
groups (e.g., unsaturated groups) which readily undergo free
radical polymerization, and cyclic groups (e.g., lactones) which
readily undergo polymerization via ring-opening. Polymerization can
be effected, for example, via photoinitiation (in the presence of
an appropriate light, e.g., 365 nm), via chemical cross-linking (in
the presence of a free-radical donor) and/or heating (at the
appropriate temperatures).
[0200] In some embodiments, a polymerizable group is selected such
that polymerization thereof may be effected under relatively mild
conditions which are non-harmful to living cells. For example, the
polymerization conditions are optionally sufficiently non-toxic and
non-hazardous so as to be suitable for effecting polymerization in
vivo, as described herein.
[0201] It is to be noted that covalent cross-linking can be
effected also in presence of a cross-linking agent. Such an agent
is typically a bifunctional chemical moiety that is capable of
reacting with the cross-linking group. Examples include, but are
not limited to, PEGs terminated at both ends with a reactive group
that can readily react with the cross-linking group.
[0202] In some embodiments, the polymerizable group is
polymerizable by free radical polymerization. Examples of such
groups include, without limitation, an acrylate, a methacrylate, an
acrylamide, a methacrylamide, and a vinyl sulfone.
[0203] According to optional embodiments, the conjugate comprises
polymeric moiety which comprise a plurality cross-linking moieties
which can attach to a polypeptide. For example, acrylate,
methacrylate, acrylamide, methacrylamide, and vinyl sulfone, in
addition to being polymerizable groups, are suitable for attachment
to a thiol group (e.g., in a cysteine residue) via Michael-type
addition.
[0204] Thus, as exemplified in the Examples section herein, a
polymeric moiety may comprise a plurality of such moieties (e.g.,
acrylate), one of which attached the polymeric moiety to the
polypeptide, the remaining moieties being cross-linking moieties as
described herein.
[0205] Thus, in exemplary embodiments, the conjugate comprises
poloxamer diacrylate (e.g., F127 poloxamer diacrylate) moieties,
wherein one acrylate group in each moiety is attached to a cysteine
residue of a polypeptide (e.g., denatured fibrinogen), and one
acrylate group serves as a cross-linking moiety.
[0206] In additional exemplary embodiments, the conjugate comprises
a polymeric tetraacrylate (e.g., T1307 tetraacrylate) moieties,
wherein one acrylate group in each moiety is attached to a cysteine
residue of a polypeptide (e.g., denatured fibrinogen), and three
acrylate groups serve as cross-linking moieties.
[0207] The polypeptide of the conjugate is at least 10 amino acids
in length, optionally at least 20 amino acids in length, and
optionally at least 50 amino acids in length.
[0208] The term "polypeptide" as used herein encompasses native
polypeptides (either degradation products, synthetically
synthesized polypeptides or recombinant polypeptides) and
peptidomimetics (typically, synthetically synthesized
polypeptides), as well as peptoids and semipeptoids which are
polypeptide analogs, which may have, for example, modifications
rendering the polypeptides more stable while in a body or more
capable of penetrating into cells. Such modifications include, but
are not limited to, N-terminus modification, C-terminus
modification, peptide bond modification, including, but not limited
to, CH.sub.2--NH, CH.sub.2--S, CH.sub.2--S.dbd.O, O.dbd.C--NH,
CH.sub.2--O, CH.sub.2--CH.sub.2, S.dbd.C--NH, CH.dbd.CH or
CF.dbd.CH, backbone modifications, and residue modification.
Methods for preparing peptidomimetic compounds are well known in
the art and are specified, for example, in Quantitative Drug
Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press
(1992), which is incorporated by reference as if fully set forth
herein. Further details in this respect are provided
hereinunder.
[0209] Peptide bonds (--CO--NH--) within the peptide may be
substituted, for example, by N-methylated bonds
(--N(CH.sub.3)--CO--), ester bonds (--C(R)H--C--O--O--C(R)--N--),
ketomethylen bonds (--CO--CH.sub.2--), _-aza bonds
(--NH--N(R)--CO--), wherein R is any alkyl, e.g., methyl, carba
bonds (--CH.sub.2--NH--), hydroxyethylene bonds
(--CH(OH)--CH.sub.2--), thioamide bonds (--CS--NH--), olefinic
double bonds (--CH.dbd.CH--), retro amide bonds (--NH--CO--),
peptide derivatives (--N(R)--CH.sub.2--CO--), wherein R is the
"normal" side chain, naturally presented on the carbon atom. These
modifications can occur at any of the bonds along the polypeptide
chain and even at several (2-3) at the same time.
[0210] As used herein throughout, the term "amino acid" or "amino
acids" is understood to include the 20 naturally occurring amino
acids; those amino acids often modified post-translationally in
vivo, including, for example, hydroxyproline, phosphoserine and
phosphothreonine; and other unusual amino acids including, but not
limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine,
nor-valine, nor-leucine and ornithine. Furthermore, the term "amino
acid" includes both D- and L-amino acids.
[0211] According to optional embodiments, the polypeptide comprises
a protein or a fragment thereof.
[0212] The protein may be a naturally occurring protein (e.g., a
protein existing in eukaryotic and/or prokaryotic organisms, cells,
cellular material, non-cellular material, and the like) or a
polypeptide homologous (e.g., at least 90% homologous, optionally
at least 95% homologous, and optionally at least 99% homologous) to
a naturally occurring protein.
[0213] In some embodiments, the protein (or protein fragment) is
denatured.
[0214] It is to be understood that the protein described herein may
optionally comprise more than one polypeptide chain.
[0215] In embodiments comprising a protein characterized by more
than one polypeptide chain, the conjugate described herein
optionally comprises one polypeptide of the protein.
[0216] Alternatively, the conjugate described herein comprises a
plurality of polypeptides of the protein (e.g., all of the
polypeptides of the protein). Optionally, the plurality of
polypeptides are linked together (e.g., by non-covalent and/or
covalent bonds) so as to form a multimer (e.g., a dimer, a trimer,
a tetramer, a hexamer, etc.), the multimer having attached thereto
at least two polymeric moieties, as described herein. Optionally,
the polypeptides of the protein are separate (e.g., separated by
denaturation of the protein), such that the conjugate described
herein is a mixture of different conjugate species, wherein each of
the conjugate species comprises a different polypeptide.
[0217] Optionally, the polypeptide (e.g., protein or protein
fragment) is selected so as to exhibit a biological activity.
Optionally, the biological activity comprises support for cell
growth and/or invasion.
[0218] Examples of proteins exhibiting a biological activity which
is advantageous in the context of embodiments of the present
invention include, without limitation, a cell signaling protein, an
extracellular matrix protein, a cell adhesion protein, a growth
factor, protein A, a protease and a protease substrate. Optionally,
the protein is an extracellular matrix protein.
[0219] According to optional embodiments, the polypeptide comprises
a fibrinogen polypeptide (.alpha., .beta. and/or .gamma. chains of
fibrinogen) or a fragment thereof. Optionally, the conjugate
described herein comprises the .alpha., .beta. and .gamma. chains
of fibrinogen. In exemplary embodiments, the polypeptide is a
denatured fibrinogen (e.g., a mixture of denatured .alpha., .beta.
and .gamma. chains of fibrinogen).
[0220] Examples of extracellular matrix proteins include, but are
not limited to, fibrinogen (e.g., .alpha.-chain--GenBank Accession
No. NP.sub.--068657; (.beta.-chain--GenBank Accession No. P02675;
.gamma.-chain--GenBank Accession No. P02679), collagen (e.g.,
GenBank Accession No. NP.sub.--000079), fibronectin (e.g., GenBank
Accession No. NP.sub.--002017), vimentin (e.g., GenBank Accession
No. NP.sub.--003371), elastin, fibrillin, fibulin, laminin (e.g.,
GenBank Accession No. NP.sub.--000218) and gelatin.
[0221] Examples of cell signaling proteins include, but are not
limited to, p38 mitogen-activated protein kinase (e.g., GenBank
Accession No. NP.sub.--002736), nuclear factor kappaB (e.g.,
GenBank Accession No. NP.sub.--003989), Raf kinase inhibitor
protein (RKIP) (e.g., GenBank Accession No. XP.sub.--497846), Raf-1
(e.g., GenBank Accession No. NP.sub.--002871), MEK (e.g., GenBank
Accession No. NP.sub.--002746), protein kinase C (PKC) (e.g.,
GenBank Accession No. NP.sub.--002728), phosphoinositide-3-kinase
gamma (e.g., GenBank Accession No. NP.sub.--002640), receptor
tyrosine kinases such as insulin receptor (e.g., GenBank Accession
No. NP.sub.--000199), heterotrimeric G-proteins (e.g.,
Galpha(i)--GenBank Accession No. NP.sub.--002060;
Galpha(s)--GenBank Accession No. NP.sub.--000507;
Galpha(q)--GenBank Accession No. NP.sub.--002063), caveolin-3
(e.g., GenBank Accession No. NP.sub.--001225), microtubule
associated protein 1B, and 14-3-3 proteins (e.g., GenBank Accession
No. NP.sub.--003397).
[0222] Examples of cell adhesion proteins include, but are not
limited to, integrin (e.g., GenBank Accession No. NP.sub.--002202),
intercellular adhesion molecule (ICAM) 1 (e.g., GenBank Accession
No. NP.sub.--000192), N-CAM (e.g., GenBank Accession No.
NP.sub.--000606), cadherin (e.g., GenBank Accession No.
NP.sub.--004351), tenascin (e.g., GenBank Accession No.
NP.sub.--061978), gicerin (e.g., GenBank Accession No.
NP.sub.--006491), and nerve injury induced protein 2 (ninjurin2)
(e.g., GenBank Accession No. NP.sub.--067606).
[0223] Examples of growth factors include, but are not limited to,
epidermal growth factor (e.g., GenBank Accession No.
NP.sub.--001954), transforming growth factor-.beta. (e.g., GenBank
Accession No. NP.sub.--000651), fibroblast growth factor-acidic
(e.g., GenBank Accession No. NP.sub.--000791), fibroblast growth
factor-basic (e.g., GenBank Accession No. NP.sub.--001997),
erythropoietin (e.g., GenBank Accession No. NP.sub.--000790),
thrombopoietin (e.g., GenBank Accession No. NP.sub.--000451),
neurite outgrowth factor, hepatocyte growth factor (e.g., GenBank
Accession No. NP.sub.--000592), insulin-like growth factor-I (e.g.,
GenBank Accession No. NP.sub.--000609), insulin-like growth
factor-II (e.g., GenBank Accession No. NP.sub.--000603),
interferon-.gamma. (e.g., GenBank Accession No. NP.sub.--000610),
and platelet-derived growth factor (e.g., GenBank Accession No.
NP.sub.--079484).
[0224] Examples of proteases include, but are not limited to,
pepsin (e.g., GenBank Accession No. NP.sub.--055039), low
specificity chymotrypsin, high specificity chymotrypsin, trypsin
(e.g., GenBank Accession No. NP.sub.--002760), carboxypeptidases
(e.g., GenBank Accession No. NP.sub.--001859), aminopeptidases
(e.g., GenBank Accession No. NP.sub.--001141),
proline-endopeptidase (e.g. GenBank Accession No. NP.sub.--002717),
Staphylococcus aureus V8 protease (e.g., GenBank Accession No.
NP.sub.--374168), proteinase K (PK) (e.g., GenBank Accession No.
P06873), aspartic protease (e.g., GenBank Accession No.
NP.sub.--004842), serine proteases (e.g., GenBank Accession No.
NP.sub.--624302), metalloproteases (e.g., GenBank Accession No.
NP.sub.--787047), ADAMTS17 (e.g., GenBank Accession No.
NP.sub.--620688), tryptase-.gamma. (e.g., GenBank Accession No.
NP.sub.--036599), matriptase-2 (e.g., GenBank Accession No.
NP.sub.--694564).
[0225] Examples of protease substrates include the peptide or
peptide sequences being the target of the protease protein. For
example, lysine and arginine are the target for trypsin; tyrosine,
phenylalanine and tryptophan are the target for chymotrypsin.
[0226] Such naturally occurring proteins can be obtained from any
known supplier of molecular biology reagents.
[0227] As exemplified in the Examples section below, it has been
surprisingly uncovered that a conjugate comprising a polypeptide as
described herein and at least one polymeric moiety exhibiting
thermal gelation may provide the conjugate with an ability to
undergo reverse thermal gelation.
[0228] Hence, according to optional embodiments, the conjugate is
characterized by an ability to undergo reverse thermal gelation in
an aqueous solution, as described herein.
[0229] Optionally, the reverse thermal gelation of the conjugate
occurs at a temperature below 55.degree. C., optionally below
50.degree. C., optionally below 40.degree. C., and optionally below
30.degree. C. Optionally, the reverse thermal gelation occurs at a
temperature below about 37.degree. C., such that at a physiological
temperature of about 37.degree. C., the conjugate is in a gelled
state.
[0230] Optionally, the reverse thermal gelation of the conjugate
occurs at a temperature above 0.degree. C., optionally above
10.degree. C., optionally above 20.degree. C. and optionally above
30.degree. C.
[0231] In some embodiments, the reverse thermal gelation of the
conjugate occurs upon an increase of temperature from 0.degree. C.
to 55.degree. C., optionally from 10.degree. C. to 55.degree. C.,
optionally from 10.degree. C. to 40.degree. C., optionally from
15.degree. C. to 37.degree. C., and optionally from 20.degree. C.
to 37.degree. C. Reverse thermal gelation which occurs upon an
increase of temperature from a room temperature (e.g., about
20.degree. C., about 25.degree. C.) to a physiological temperature
(e.g., about 37.degree. C.) are particularly useful for some
applications (e.g., medical applications), as gelation can be
induced by transferring the conjugate from a room temperature
environment to a physiological temperature, for example, by placing
the conjugate in a body.
[0232] As exemplified herein, the temperature at which gelation of
a conjugate solution occurs may be controlled by varying the
concentration of the conjugate.
[0233] Furthermore, the gelation temperature may be controlled by
selecting a polymer with an appropriate gelation temperature for
inclusion in the polymeric moiety, and/or by varying the
concentration of polymeric moieties which exhibit reverse thermal
gelation (e.g., by varying the number of polymeric moieties
attached to a polypeptide and/or by varying the size of the
polymeric moieties).
[0234] As further exemplified in the Examples section, aqueous
solutions comprising conjugates described herein may undergo
reverse thermal gelation at relatively low concentrations, for
example, less than 20 weight percents conjugate, optionally less
than 10 weight percents, optionally less than 5 weight percents,
and optionally less than 2 weight percents.
[0235] Without being bound by any particular theory, it is believed
that conjugation of a polypeptide to a polymer exhibiting reverse
thermal gelation acts as chain extension of the polymer, which
lowers the minimal concentration necessary for gelation.
[0236] It is to be noted that a phenomenon of a chain extender of a
biological nature or origin (e.g., a polypeptide) has never been
reported heretofore.
[0237] The reverse thermal gelation of the conjugate as described
herein can be determined by measuring a shear storage modulus of an
aqueous solution containing same. An temperature-dependent increase
in the storage modulus is indicative of a gel formation via a
reverse thermal gelation.
[0238] As used herein and in the art, a "shear modulus" is defined
as the ratio of shear stress to the shear strain. The shear modulus
may be a complex variable, in which case the "storage modulus" is
the real component and the "loss modulus" is the imaginary
component. The storage modulus and loss modulus in viscoelastic
solids measure the stored energy, representing the elastic portion,
and the energy dissipated as heat, representing the viscous
portion.
[0239] In some embodiments, the reverse thermal gelation described
herein increases a shear storage modulus (also referred to herein
as "storage modulus", or as "G'") of the aqueous solution of the
conjugate by at least ten-folds, optionally at least 30-folds,
optionally at least 100-folds, and optionally at least
300-folds.
[0240] In some embodiments, the reverse thermal gelation described
herein increases a shear storage modulus of the aqueous solution to
at least 5 Pa, optionally at least 15 Pa, optionally at least 20
Pa, optionally at least 50 Pa, optionally at least 100 Pa, and
optionally at least 200 Pa.
[0241] In some embodiments, the shear storage modulus of the
aqueous solution containing the conjugate before reverse thermal
gelation (e.g., at a temperature below a temperature at which
gelation occurs) is less than 2 Pa, optionally less than 1 Pa,
optionally less than 0.5 Pa, and optionally less than 0.2 Pa.
[0242] According to optional embodiments, the reverse thermal
gelation is reversible, i.e., a gelled state obtained by increasing
a temperature can revert to the non-gelled state by lowering the
temperature, the non-gelled state having substantially the same
properties as existed prior to the reverse thermal gelation.
Reversible gelation is advantageous in that a gelled state can be
modified and/or reconstructed by causing at least a portion of the
gelled state to revert to a non-gelled state (by decreasing a
temperature), followed by formation of a gelled state (by
increasing a temperature) in a desired form. In addition,
reversible gelation does not create spoilage of a product by
gelation before a product is used (e.g., a product in storage), as
any such gelation prior to use of the product may be eliminated (by
cooling).
[0243] Optionally, the gelation is reversible over many cycles
(e.g., at least 10 cycles, at least 50 cycles) of increasing and
decreasing a temperature.
[0244] Optionally, a gel formed by reverse thermal gelation of an
aqueous solution of the conjugate is a biodegradable gel, i.e., the
gel degrades in contact with a tissue and/or a cell (e.g., by
proteolysis and/or hydrolysis). Biodegradable materials are useful
in various medical applications, for example as temporary implants.
In addition, biodegradable materials are highly suitable as
matrices for supporting cell growth and/or migration, as cell
growth and/or migration is associated with degradation of a
surrounding matrix.
[0245] As exemplified in the Examples section below, a gel formed
by reverse thermal gelation of a solution of a conjugate described
herein may serve as a suitable matrix for cell growth, spreading,
expansion and/or invasion.
[0246] Hence, the conjugate described herein is optionally
identified for use in generating a scaffold, as defined herein. The
scaffold may be generated by reverse thermal gelation of the
conjugate (e.g., by non-covalent cross-linking of the conjugate)
and/or by covalent cross-linking of the conjugate.
[0247] The conjugate described herein can therefore be referred to
also as a precursor molecule for generating a scaffold. Thus, the
scaffold is formed by cross-linking (covalently and/or
non-covalently) a plurality of precursor molecules to one
another.
[0248] As used herein, the term "scaffold" describes a
two-dimensional or a three-dimensional supporting framework. The
scaffold according to embodiments of the present invention is
composed of precursor units (comprising the conjugates as described
herein) which are cross-linked therebetween. In some embodiments, a
scaffold can be used as a support for cell growth, attachment
and/or spreading and thus facilitates tissue generation and/or
tissue repair. In some embodiments, a scaffold maintains a desired
shape of a tissue and/or cell colony supported thereby.
[0249] In exemplary embodiments, the scaffold is a hydrogel, i.e.,
the gel formed from the conjugate comprises water absorbed therein,
for example, water from an aqueous solution of the conjugate which
underwent gelation.
[0250] As used herein and is well-known in the art, the term
"hydrogel" refers to a material that comprises solid networks
formed of water-soluble natural or synthetic polymer chains,
typically containing more than 99% water.
[0251] Optionally the hydrogel is characterized by a shear storage
modulus of at least 15 Pa (optionally at least 50 Pa, optionally at
least 100 Pa, and optionally at least 200 Pa) at 37.degree. C.
[0252] Optionally the generation of the scaffold is reversible.
Reversible scaffold generation is optionally obtained in
embodiments wherein scaffold generation is by reverse thermal
gelation, as discussed hereinabove.
[0253] Optionally, the scaffold is generated by means other than
reverse thermal gelation, for example, by covalent cross-linking.
The obtained scaffold (e.g., a hydrogel) is optionally capable of
further undergoing a reverse thermal gelation. Further optionally,
the scaffold is generated by a reverse thermal gelation and is
thereafter further subjected to covalent cross-linking, as
described herein.
[0254] As discussed herein, conjugates described herein may be
cross-linked by non-covalent (physical) cross-linking and/or by
covalent (chemical) cross-linking.
[0255] Hence, according to another aspect of embodiments of the
invention, there is provided a composition-of-matter (e.g., a
scaffold or a hydrogel) comprising a cross-linked form of a
conjugate described herein. The composition-of-matter thus
comprises a plurality of molecules of the conjugate cross-linked to
one another.
[0256] It is to be understood that although the
composition-of-matter is described herein for the sake of
simplicity as comprising a conjugate, compositions-of-matter
comprising a plurality of conjugate species (e.g., a mixture of
different conjugates) are encompassed by the term
"composition-of-matter".
[0257] In some embodiments, the conjugate molecules are
cross-linked non-covalently.
[0258] Optionally the molecules are cross-linked only
non-covalently (i.e., no substantial covalent cross-linking is
present).
[0259] Compositions-of-matter described herein may optionally be
generated by non-covalent and/or covalent cross-linking of the
conjugate molecules in a solution, preferably an aqueous solution.
Optionally, the solution remains absorbed to the cross-linked
conjugate, for example, in the form of a gel (e.g., a
hydrogel).
[0260] The solution may be selected suitable for effecting the
abovementioned covalent and/or non-covalent cross-linking.
[0261] In some embodiments, the solution is an aqueous
solution.
[0262] Compositions-of-matter comprising only non-covalent
cross-linking may optionally be generated by reverse thermal
gelation of the conjugate molecules in an aqueous solution (e.g.,
as described herein). Optionally, the non-covalently cross-linked
form is reversible, as described herein.
[0263] In some embodiments, the conjugate molecules are
cross-linked covalently. In such embodiments, the conjugate
comprises a cross-linking moiety (as described herein). The
composition-of-matter is optionally generated by subjecting a
plurality of conjugate molecules to conditions for effecting
covalent cross-linking of the cross-linking moieties of the
conjugate molecules.
[0264] Optionally the covalently cross-linked composition-of-matter
is characterized by a shear storage modulus of at least 20 Pa at
37.degree. C., and optionally at least 50 Pa, optionally at least
100 Pa, optionally at least 200 Pa, and optionally at least 300
Pa.
[0265] In some embodiments a composition-of-matter comprises
non-covalent cross-linking, in addition to the covalent
cross-linking.
[0266] For example, a composition-of-matter comprising covalent
cross-linking may be capable of undergoing reverse thermal gelation
(e.g., a reversible reverse thermal gelation).
[0267] Such a reverse thermal gelation of a covalently cross-linked
composition-of-matter may optionally increase a shear storage
modulus of the composition-of-matter by at least 20%, optionally at
least 50%, optionally at least 200%, optionally at least 400%, and
optionally at least 900%.
[0268] The shear storage modulus prior to reverse thermal gelation
is optionally in a range of from 0.5 Pa to 200 Pa, optionally in a
range of from 0.5 Pa to 100 Pa, and optionally in a range of from
10 Pa to 100 Pa.
[0269] The shear storage modulus following reverse thermal gelation
is optionally at least 15 Pa, and optionally in a range of from 20
Pa to 5000 Pa, optionally from 20 Pa to 1000 Pa, optionally from 20
Pa to 500 Pa, and optionally from 50 Pa to 500 Pa.
[0270] Optionally, the reverse thermal gelation of a covalently
cross-linked composition-of-matter is at a temperature described
herein for gelation of a conjugate.
[0271] As exemplified in the Examples section below, a
composition-of-matter may be characterized by a shear storage
modulus of one portion of the composition-of-matter that is
different from a shear storage modulus of at least one other
portion of the composition-of-matter. Each portion may
independently be characterized by non-covalent cross-linking,
covalent cross-linking or a combination of non-covalent and
covalent cross-linking (e.g., as described hereinabove).
[0272] Such a composition-of-matter may be prepared, for example,
using two solutions of a conjugate (e.g., solutions of different
conjugates and/or solutions with different concentrations of
conjugate). Optionally, one solution is cross-linked to obtain a
first composition-of-matter (e.g., as described herein), whereupon
the first composition-of-matter is added to the second solution.
Upon cross-linking of the second solution (e.g., under conditions
which do not significantly affect the first composition-of-matter),
a composition-of-matter having portions with different properties
may be obtained.
[0273] Regardless of the type (non-covalent and/or covalent) of
cross-linking, compositions-of-matter described herein are
optionally biodegradable. In some embodiments, the incorporation of
a polypeptide in a network of cross-linked conjugates within the
composition-of-matter causes the composition-of-matter to
biodegrade upon biodegradation of the polypeptide.
[0274] According to optional embodiments, the composition-of-matter
further comprises cells (preferably live cells) therein. The cells
may comprise one cell type or a two or more cell types.
[0275] Compositions-of-matter described herein may be useful for
inducing formation of a tissue, for example, by serving as a matrix
for supporting cellular growth and/or invasion, and/or by providing
cells (e.g., embedded in the composition-of-matter) which induce
tissue formation. Such properties may be useful for repairing
tissue damage.
[0276] Hence, in some embodiments, the composition-of-matter is
identified for use in inducing formation of a tissue, as discussed
in further detail hereinbelow.
[0277] In some embodiments, the composition-of-matter is identified
for use in repairing tissue damage, as discussed in further detail
hereinbelow.
[0278] The compositions-of-matter described herein may be prepared
by various processes, depending on the type of
composition-of-matter, and particularly, on the type of
cross-linking (i.e., non-covalent and/or covalent) in the
composition-of-matter.
[0279] Thus, according to another aspect of embodiments of the
invention, there is provided a process of producing a
composition-of-matter which comprises non-covalent cross-linking
(e.g., as described herein). The process comprises heating a
solution (e.g., an aqueous solution) which comprises a plurality of
molecules of a conjugate as described herein, from a first
temperature to a second temperature. The second temperature is such
that a reverse thermal gelation of the conjugate in solution is
effected, thereby producing a composition-of-matter with
non-covalent cross-linking.
[0280] The second temperature is a temperature at or above the
critical temperature of the precursor conjugate, as detailed
hereinabove.
[0281] Optionally, the composition-of-matter is produced in vivo,
for example, by heating to a physiological temperature (e.g., about
37.degree. C.). Such heating may be effected simply by contacting a
solution of the conjugate with a body.
[0282] In some embodiments, the conjugate is a conjugate comprising
at least one cross-linking moiety described herein, and the process
further comprises subjecting the conjugate solution to conditions
that effect cross-linking of the cross-linking moieties (e.g.,
prior to the aforementioned heating, subsequent to the heating or
concomitant with the heating). Cross-linking of the cross-linking
moieties may optionally be performed so as to obtain a
composition-of-matter comprising both non-covalent and covalent
cross-linking.
[0283] According to another aspect of embodiments of the invention,
there is provided a process of producing a composition-of-matter
which comprises covalent cross-linking (e.g., as described herein).
The process comprises subjecting a solution comprising a plurality
of molecules of a conjugate described herein, wherein the conjugate
comprises at least one cross-linking moiety (as described herein),
to conditions that effect covalent cross-linking of the
cross-linking moieties, thereby producing a composition-of-matter
with covalent cross-linking.
[0284] Optionally, the covalent cross-linking is effected in
vivo.
[0285] Alternatively, the covalent cross-linking is effected ex
vivo.
[0286] Optionally, the process further comprises forming
non-covalent cross-links, for example, by exposure to a temperature
at which reverse thermal gelation occurs.
[0287] In some embodiments, covalent cross-linking is effected ex
vivo, to thereby produce a covalently cross-linked scaffold, and
the process further comprises subjecting the covalently
cross-linked scaffold to a physiological temperature in vivo (e.g.,
by contacting the scaffold with a body), such that reverse thermal
gelation of the scaffold is effected in vivo, thereby producing a
composition-of-matter in vivo which comprises non-covalent and
covalent cross-linking.
[0288] In some embodiments, the solution of the conjugate further
comprises cells. Consequently, the process produces a
composition-of-matter comprising cells embedded therein (as
described herein).
[0289] The conditions which effect cross-linking of cross-linking
moieties will depend on the chemical properties of the
cross-linking moieties.
[0290] Various conditions for effecting cross-linking are known in
the art. For example, cross-linking may be effected by irradiation
(e.g., by UV light, by visible light, by ionizing radiation), by an
initiator (e.g., free radical donors) and/or heat.
[0291] Preferably, the conditions for effecting covalent
cross-linking are biocompatible, namely, use agents or conditions
which are not considered as hazardous in in vivo applications.
[0292] According to an optional embodiment of the present
invention, the cross-linking is by illumination with UV (e.g., at a
wavelength of about 365 nm).
[0293] As used herein the term "about" refers to .+-.10%.
[0294] When cross-linking in vivo, it is preferable to avoid
irradiation doses that are harmful. The maximal dose which is
non-harmful will depend, for example, on the type (e.g.,
wavelength) of irradiation, and on the part of the body exposed to
the irradiation. One skilled in the art will readily be capable of
determining whether a dose is harmful or non-harmful.
[0295] In some embodiment, the conditions comprise a presence of an
initiator which is added to facilitate cross-linking.
[0296] Optionally, the initiator is capable of effecting
cross-linking without irradiation.
[0297] Alternatively, the initiator is a photoinitiator which
effects cross-linking in the presence of irradiation (e.g., UV
light, visible light). Addition of a photoinitiator will typically
enable one to use lower doses of UV light for cross-linking.
[0298] As used herein, the term "photoinitiator" describes a
compound which initiates a chemical reaction (e.g., cross-linking
reaction, chain polymerization) when exposed to UV or visible
illumination. Many suitable photoinitiators will be known to one
skilled in the art. Exemplary photoinitiators include, without
limitation, bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide
(BAPO), 2,2-dimethoxy-2-phenylacetophenone (DMPA), camphorquinone
(CQ), 1-phenyl-1,2-propanedione (PPD), the organometallic complex
Cp'Pt(CH(3))(3) (Cp'=eta(5)-C(5)H(4)CH(3)),
2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (e.g.,
Irgacure.TM. 2959), dimethylaminoethyl methacrylate (DMAEMA),
2,2-dimethoxy-2-phenylacetophenone, benzophenone (BP), and
flavins.
[0299] As exemplified in the Examples section below, physical
properties (e.g., shear storage modulus) of compositions-of-matter
depend on certain parameters which may be readily controlled. Thus,
a composition-of-matter having a desired physical property may be
prepared by selecting a suitable value of one or more of such
parameters.
[0300] Hence, according to another aspect of embodiments of the
invention, there is provided a method of controlling a physical
property (e.g., a shear storage modulus) of a composition-of-matter
such as described herein. The method comprises controlling a
parameter which characterizes the composition-of-matter. Such a
parameter can be, for example, a concentration of a conjugate
described herein in the solution (aqueous solution), an ambient
temperature, a cross-linking temperature. In addition, the
parameter can be the presence or absence of covalent cross-linking,
a concentration of initiator (e.g., a presence or absence of
initiator) during covalent cross-linking, and/or a dose of
irradiation used for covalent cross-linking.
[0301] The concentration of a conjugate in a composition-of-matter
may be readily controlled by preparing a solution of the conjugate
at a selected concentration, and cross-linking the conjugate by
covalent and/or non-covalent cross-linking, as described herein,
such that the solution of the conjugate is converted into a
composition-of-matter described herein, having the selected
concentration of conjugate.
[0302] In some embodiments, the concentration of conjugate is
positively correlated with the shear storage modulus, as
exemplified in the Examples herein.
[0303] In some embodiments, the concentration of conjugate is
negatively correlated with a temperature at which reverse thermal
gelation is effected (e.g., a critical gelation temperature), as
exemplified in the Examples herein.
[0304] In some embodiments, the ambient temperature controls a
physical property of a composition-of-matter by affecting reverse
thermal gelation of a composition-of-matter, as described
herein.
[0305] The ambient temperature may be selected, for example, such
that gelation is not effected (e.g., at a relatively low
temperature) and the shear storage modulus is relatively low, such
that gelation is effected (e.g., at a relatively high temperature)
and the shear storage modulus is relatively high. In addition, an
ambient temperature may be selected (e.g., at an intermediate
temperature) such that gelation is partially effected to any
desired degree, such that the shear storage can be at any
intermediate level which is desired.
[0306] Typically, the composition-of-matter will be characterized
by a relatively narrow temperature range (e.g., a 5.degree. C.
range, a 10.degree. C. range, a 15.degree. C. range) in which a
physical property (e.g., a shear storage modulus) exhibits a
particularly strong temperature dependence. Optionally, an ambient
temperature is selected from within this temperature range, such
that the physical property may be conveniently controlled by
relatively small changes in ambient temperature.
[0307] The cross-linking temperature (i.e., a temperature at which
conjugates in the composition-of-matter are covalently
cross-linked) may be used to control a physical property of a
composition-of-matter which comprises covalent cross-linking (e.g.,
as described herein).
[0308] In some embodiments, the cross-linking temperature is
negatively correlated with a shear storage modulus of the
composition-of-matter, as exemplified in the Examples herein.
[0309] In some embodiments, a correlation between a physical
property (e.g., shear storage modulus) and cross-linking
temperature is particularly strong when the cross-linking
temperature is in a temperature range in which a physical property
exhibits a particularly strong temperature-dependence, as described
hereinabove. Optionally a cross-linking temperature is selected
from within this temperature range, such that the physical property
may be conveniently controlled by relatively small changes in
cross-linking temperature.
[0310] In some embodiments, the presence of covalent cross-linking
is associated with a higher shear storage modulus, as exemplified
herein.
[0311] In some embodiments, a degree of covalent cross-linking by
modulating the conditions for effecting covalent cross-linking.
[0312] Thus, for example, low degree of covalent cross-linking may
be obtained by effecting covalent cross-linking without an
initiator or with a smaller amount of initiator, and/or without
irradiation or with a small dose of irradiation (e.g., using a
short irradiation time and/or a low intensity of irradiation).
[0313] In some embodiments, the parameter (e.g., ambient
temperature, cross-linking temperature) is relatively independent
of some physical properties (e.g., biodegradation rate). This
advantageously allows for controlling two or more physical
properties of interest (e.g., degradation rate and shear storage
modulus) without creating a need for experimentation to determine
how such physical properties are interdependent. For example, a
shear storage modulus may optionally be controlled by selecting a
suitable cross-linking temperature, while a degradation rate may be
controlled by selecting an appropriate polymer for the polymeric
moieties described herein.
[0314] Thus, in some embodiments, changing a parameter described
herein (e.g., ambient temperature, cross-linking temperature) will
change a biodegradation rate by a factor of less than 4, optionally
by a factor of less than 3, optionally by a factor of less than 2,
and optionally by a factor of less than 1.5.
[0315] The biodegradation rate is optionally quantified by
measuring a half-life of the composition-of-matter in a trypsin
solution (e.g., using procedures described herein).
[0316] Conjugates according to embodiments of the invention may be
produced in a relatively simple and inexpensive manner.
[0317] Thus, according to another aspect of embodiments of the
invention, there is provided a process of producing a conjugate as
described herein, the process comprising covalently attaching a
polymer to a polypeptide, the polymer and polypeptide being such
that at least two polymer molecules attach to a molecule of the
polypeptide, wherein at least one of the two polymer molecules
exhibits a reverse thermal gelation.
[0318] The polymer may optionally comprise at least one
cross-linking moiety (e.g., as described herein), so as to produce
a conjugate comprising at least one cross-linking moiety, as
described herein.
[0319] Optionally, the polymer comprises at least one first moiety
(optionally a single first moiety) which is capable of reacting so
as to attach the polymer to the polypeptide, and optionally at
least one second moiety which is a cross-linking moiety described
herein.
[0320] In some embodiments, the first moiety and the second moiety
are different, such that the first moiety may be reacted so as to
attach the polymer to the polypeptide, without causing the second
moiety (cross-linking moiety) to react prematurely (e.g., before
cross-linking of conjugate molecules is desired).
[0321] In some embodiments, the first moiety and second moiety are
the same, the moiety being suitable for attaching the polymer to
the polypeptide and for cross-linking the conjugate.
[0322] Optionally, such a cross-linking moiety is selected as being
capable of undergoing two different reactions, each under different
conditions, such that the moiety may be reacted under one set of
conditions so as to attach the polymer to the polypeptide, and then
reacted under different conditions so as to cross-link conjugate
molecules. For example, as described herein, some unsaturated
moieties (e.g., acrylates) may undergo Michael-type addition by a
thiol (e.g., under basic conditions) so as to attach the polymer to
a polypeptide, and also undergo polymerization (e.g., under
conditions for initiating free radical polymerization) so as to
cross-link conjugates.
[0323] In some embodiments wherein the first and second moieties
described herein are the same (or otherwise capable of undergoing
similar reactions under the same conditions), the polypeptide is
reacted with a molar excess (e.g., at least 20:1, at least 50:1, at
least 100:1, at least 200:1) of the polymer, so as to prevent each
polymer molecule from attaching to more than one site on the
polypeptide.
[0324] Apart from being inexpensive to produce, the
compositions-of-matter of embodiments of the present invention are
highly reproducible, flexible (can be stressed or stretched
easily), exhibit controllable structural properties, and are
amenable to controllable biodegradation; characteristics which make
it highly suitable for in vivo or ex vivo regeneration of tissues
such as bone, cartilage, heart muscle, skin tissue, blood vessels,
and other tissues (soft and hard) in the body. For example, such a
scaffold hydrogel can be easily placed into gaps within a tissue or
an organ, following which it can fill the void and initiate the
process of regeneration as the scaffold degrades away.
[0325] Hence, according to another aspect of embodiments of the
invention, there is provided a use of a conjugate described herein
or of a composition-of-matter described herein in the manufacture
of a medicament for repairing tissue damage.
[0326] The medicament is optionally for inducing formation of a
tissue (in vivo and/or ex vivo).
[0327] Optionally, the medicament is for treating a disorder
characterized by tissue damage or loss (e.g., as described herein).
Herein, the phrase "tissue" refers to part of an organism
consisting of an aggregate of cells having a similar structure and
function. Examples include, but are not limited to, brain tissue,
retina, skin tissue, hepatic tissue, pancreatic tissue, bone,
cartilage, connective tissue, blood tissue, muscle tissue, cardiac
tissue brain tissue, vascular tissue, renal tissue, pulmonary
tissue, gonadal tissue, hematopoietic tissue and fat tissue.
Preferably, the phrase "tissue" as used herein also encompasses the
phrase "organ" which refers to a fully differentiated structural
and functional unit in an animal that is specialized for some
particular function. Non-limiting examples of organs include head,
brain, eye, leg, hand, heart, liver kidney, lung, pancreas, ovary,
testis, and stomach.
[0328] According to another aspect of embodiments of the invention,
there is provided a use of a conjugate described herein or of a
composition-of-matter described herein in the manufacture of a
medicament for treating a subject having a disorder characterized
by tissue damage or loss.
[0329] As used herein the phrase "disorder characterized by tissue
damage or loss" refers to any disorder, disease or condition
exhibiting a tissue damage (e.g., non-functioning tissue, cancerous
or pre-cancerous tissue, broken tissue, fractured tissue, fibrotic
tissue, or ischemic tissue) or a tissue loss (e.g., following a
trauma, an infectious disease, a genetic disease, and the like)
which require tissue regeneration. Examples for disorders or
conditions requiring tissue regeneration include, but are not
limited to, liver cirrhosis such as in hepatitis C patients (liver
tissue), type-1 diabetes (pancreatic tissue), cystic fibrosis
(lung, liver, pancreatic tissue), bone cancer (bone tissue), burn
and wound repair (skin tissue), age related macular degeneration
(retinal tissue), myocardial infarction, myocardial repair, CNS
lesions (myelin), articular cartilage defects (chondrocytes),
bladder degeneration, intestinal degeneration, and the like. In
addition, cosmetic tissue damage or loss is encompassed by the term
"disorder".
[0330] As used herein, the term "cosmetic" refers to apparent
(e.g., visible) tissue, including, but not limited to, skin tissue.
Cosmetic tissue damage or loss is typically detrimental
aesthetically, and may be detrimental for additional reasons (e.g.
psychological factors).
[0331] Herein, the phrase "treating" refers to inhibiting or
arresting the development of a disease, disorder or condition
and/or causing the reduction, remission, or regression of a
disease, disorder or condition in an individual suffering from, or
diagnosed with, the disease, disorder or condition. Those of skill
in the art will be aware of various methodologies and assays which
can be used to assess the development of a disease, disorder or
condition, and similarly, various methodologies and assays which
can be used to assess the reduction, remission or regression of a
disease, disorder or condition.
[0332] In some embodiments, a medicament comprising a conjugate as
described herein is identified for being cross-linking the
conjugate (in vivo and/or ex vivo), as described herein.
[0333] In some embodiments, a medicament comprising a
composition-of-matter described herein is identified for being
implanted in a subject.
[0334] As used herein, the term "subject" refers to a vertebrate,
preferably a mammal, more preferably a human being (male or female)
at any age.
[0335] Implantation is optionally effected using a surgical tool
such as a scalpel, spoon, spatula, or other surgical devices.
Optionally, implantation is effected via injection (e.g. via
syringe, catheter, and the like)
[0336] Herein, the terms "implant" and "implantation" encompass
placing a substance (e.g., a conjugate or composition-of-matter
described herein) in a body or on a body surface (e.g., on a skin
surface). According to another aspect of embodiments of the
invention, there is provided a method of inducing formation of a
tissue in vivo, the method comprising implanting a
composition-of-matter described herein in a subject (e.g., as
described herein), to thereby induce the formation of the
tissue.
[0337] In some embodiments, the composition-of-matter is a
composition-of-matter which comprises covalently cross-linked
conjugate as described herein, and is non-covalently cross-linked
in vivo following implantation (e.g., to provide the
composition-of-matter with a desired rigidity). Optionally, the
non-covalent cross-linking is effected by exposure to a
physiological temperature (e.g., as described herein), the exposure
to the physiological temperature being a direct result of
implantation.
[0338] According to another aspect of embodiments of the invention,
there is provided a method of inducing formation of a tissue in
vivo, the method comprising implanting a plurality of molecules of
a conjugate described herein in a subject, to thereby induce the
formation of the tissue.
[0339] In some embodiments, the conjugate is non-covalently
cross-linked in vivo following implantation (e.g., to form a
scaffold). Optionally, the non-covalent cross-linking is effected
by exposure to a physiological temperature (e.g., as described
herein), the exposure to the physiological temperature being a
direct result of implantation.
[0340] In some embodiments, the conjugate is covalently
cross-linked in vivo following implantation (e.g., to form a
scaffold). Cross-linking can be performed as described herein,
using non-toxic, non-hazardous agents and/or conditions (e.g.,
application of UV irradiation).
[0341] According to another aspect of embodiments of the invention,
there is provided a method of inducing formation of a tissue ex
vivo, the method comprising subjecting a composition-of-matter
having cells therein (as described herein) to conditions conductive
to growth of the cells, to thereby induce tissue formation.
[0342] As used herein, the phrase "ex vivo" refers to living cells
which are derived from an organism and are growing (or cultured)
outside of the living organism, for example, outside the body of a
vertebrate, a mammal, or human being. For example, cells which are
derived from a human being such as human muscle cells or human
aortic endothelial cells and cultured outside of the body are
referred to as cells which are cultured ex vivo.
[0343] The cells in a composition-of-matter described herein are
optionally selected so as to be capable of forming a tissue. Such
cells can be, for example, stem cells such as embryonic stem cells,
bone marrow stem cells, cord blood cells, mesenchymal stem cells,
adult tissue stem cells, or differentiated cells such as neural
cells, retinal cells, epidermal cells, hepatocytes, pancreatic
cells, osseous cells, cartilaginous cells, elastic cells, fibrous
cells, myocytes, myocardial cells, endothelial cells, smooth muscle
cells, and hematopoietic cells.
[0344] The composition-of-matter comprising cells may comprise
cells embedded within and/or on the surface of the
composition-of-matter. Cells may optionally be embedded within the
composition-of-matter by cross-linking a conjugate described herein
in the presence of cells (e.g., as described herein). Incorporation
of cells onto a surface of the composition-of-matter may optionally
be effected by contacting a prepared composition-of-matter with the
cells.
[0345] The concentration of cells in and/or on the
composition-of-matter depends on the cell type and the scaffold
properties. Those of skill in the art are capable of determining
the concentration of cells used in each case.
[0346] The composition-of-matter is optionally contacted with
tissue culture medium and growth factors.
[0347] Alternatively or additionally, the composition-of-matter
comprises tissue culture medium and growth factors, for example, in
an aqueous phase of a hydrogel.
[0348] Optionally, the cells are routinely examined (e.g., using an
inverted microscope) for evaluation of cell growth, spreading and
tissue formation, in order to facilitate control over the tissue
formation, and/or to determine when a process of tissue formation
has been completed.
[0349] Following ex vivo tissue formation, the obtained tissue
and/or composition-of-matter comprising the formed tissue is
optionally implanted in the subject (e.g., to induce further tissue
formation, to repair tissue damage, and/or to treat a disorder as
described herein). Those of skills in the art are capable of
determining when and how to implant the tissue and/or
composition-of matter to thereby induce tissue formation and/or
repair, and/or to treat a disease described herein.
[0350] It will be appreciated that the cells to be implanted in a
subject (e.g., for inducing in vivo tissue formation and/or
following ex vivo formation of a tissue), as described herein, may
optionally be derived from the treated subject (autologous source),
and optionally from allogeneic sources such as embryonic stem cells
which are not expected to induce an immunogenic reaction.
[0351] According to another aspect of embodiments of the invention,
there is provided a method of treating a subject having a disorder
characterized by tissue damage or loss (e.g., as described herein),
the method comprising implanting a composition-of-matter described
herein in a subject, as described herein, to thereby induce
formation of the tissue, thereby treating the disorder
characterized by tissue damage or loss.
[0352] According to another aspect of embodiments of the invention,
there is provided a method of treating a subject having a disorder
characterized by tissue damage or loss (e.g., as described herein),
the method comprising implanting a plurality of molecules of a
conjugate described herein in a subject, as described herein, to
thereby induce formation of the tissue, thereby treating the
disorder characterized by tissue damage or loss.
[0353] In some embodiments of the methods described herein which
are effected by implanting a conjugate, the conjugate optionally
comprises at least one cross-linking moiety (e.g., as described
herein). In such embodiments, the method optionally further
comprising covalently cross-linking the plurality of molecules of
the conjugate, for example, by subjecting the plurality of
molecules to conditions (e.g., as described herein) that effect
covalent cross-linking of the cross-linking moieties of the
molecules.
[0354] A conjugate described herein may be provided as a
composition, for example a composition for effecting a method or
use described herein. The composition may be for effecting a
pharmaceutical (e.g., medicinal) treatment and/or a cosmetic
treatment (e.g., as described herein).
[0355] Hence, according to another aspect of embodiments of the
invention, there is provided a pharmaceutical, cosmetic or
cosmeceutical composition comprising a plurality of molecules of a
conjugate described herein, the composition being identified for
use in inducing formation of a tissue upon being contacted with a
tissue and further upon subjecting the composition to a
physiological temperature.
[0356] Herein, the phrase "cosmeceutical composition" refers to a
composition characterized by both pharmaceutical and cosmetic
uses.
[0357] Optionally, the conjugate comprises at least one
cross-linking moiety (as described herein), and the composition is
identified for use in inducing formation of a tissue upon further
subjecting the plurality of molecules of the conjugate to
conditions (e.g., as described herein) that effect covalent
cross-linking of the cross-linking moieties of the molecules.
[0358] Optionally, the composition further comprises an initiator
(e.g., as described herein) for inducing the covalent cross-linking
of the cross-linking moieties.
[0359] Optionally, the composition described herein is packaged in
a packaging material and identified in print, in or on the
packaging material, for use in inducing formation of tissue and/or
for treating a disorder, as described herein.
[0360] The composition may further comprise a pharmaceutically
acceptable carrier, and be formulated for facilitating its
administration (e.g., implantation).
[0361] Herein, the term "pharmaceutically acceptable carrier"
refers to a carrier or a diluent that does not cause significant
irritation to an organism and does not abrogate the biological
activity and properties of the administered compound. Examples,
without limitations, of carriers are: propylene glycol, saline,
emulsions and mixtures of organic solvents with water, as well as
solid (e.g., powdered) and gaseous carriers.
[0362] Optionally, the carrier is an aqueous carrier, for example,
an aqueous solution (e.g., saline).
[0363] The conjugate may also be provided as part of a kit.
[0364] Thus, according to another aspect of embodiments of the
invention, there is provided a kit for inducing formation of a
tissue, the kit comprising a conjugate described herein, an aqueous
solvent, and instructions for cross-linking an aqueous solution of
the conjugate in order to form a scaffold for inducing formation of
tissue.
[0365] Optionally, the conjugate and solvent are stored separately
within the kit (e.g., in separate packaging units), such that the
conjugate is stored in a dry state until being contacted with the
solvent for formation of a solution of the conjugate (e.g., a
solution described herein). Such storage of the conjugate prior to
use may increase an effective life span of the conjugate (and
kit).
[0366] Optionally, the conjugate comprises at least one
cross-linking moiety (e.g., as described herein), and the kit
further comprises an initiator (e.g. as described herein) for
inducing covalent cross-linking of the cross-linking moiety.
[0367] Optionally, the kit further comprises cells for embedding in
the scaffold (e.g., as described herein).
[0368] The cells may form a part of the solvent or may be packaged
separately.
[0369] In some embodiments, the kit comprises instructions as a
package insert.
[0370] Instructions for cross-linking the conjugate in the solvent
can be, for example, mixing the conjugate and solvent and
subjecting the obtained solution to a certain temperature (e.g.,
for effecting reverse thermal gelation).
[0371] For example, if gelation of the conjugate is effected at
ambient temperature, instructions may be to store the kit under
refrigeration (e.g., below 10.degree. C. or at 4.degree. C.), mix
the components at room temperature and wait until gel formation is
observed.
[0372] If gelation is effected at higher temperatures, instructions
may be to mix the components and then heat the solution for an
indicated time period.
[0373] If covalent cross-linking is to be effected by irradiation,
instructions may be to mix the components (optionally including a
photoinitiator as described herein), irradiate the solution, and
optionally heating the solution to effected thermal gelation as
described hereinabove. The irradiation can be prior to, concomitant
with or after irradiation.
[0374] If covalent cross-linking is to be effected by free radical
polymerization, instructions may be to mix the components
(including a polymerization initiator as described herein), and
optionally heating the solution to effect thermal gelation as
described hereinabove and/or to effect polymerization (if heating
is desired). The heating to effect thermal gelation and to effect
polymerization can be to the same temperature or to different
temperatures.
[0375] In some embodiments, the conjugate and the solution are
packaged within the kit at a ratio suitable for obtaining a
composition-of-matter with the desired properties. Such a ratio can
be pre-determined as detailed hereinabove.
[0376] Optionally, the instructions further include guidance for
selecting a suitable ratio for obtaining a suitable property of the
composition-of-matter, in accordance with the description provided
hereinabove.
[0377] The instructions may further include guidance with regard to
selecting the cross-linking conditions (e.g., with or without
irradiation; with or without heating; with or without adding a
polymerization initiator) for obtaining a composition-of-matter
with desired properties.
[0378] It is expected that during the life of a patent maturing
from this application many relevant polymers exhibiting reverse
thermal gelation will be developed and the scope of the phrase
"polymeric moieties exhibiting a reverse thermal gelation" is
intended to include all such new technologies a priori.
[0379] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0380] The term "consisting of" means "including and limited
to".
[0381] The term "consisting essentially of" means that the
composition, method or structure may include additional
ingredients, steps and/or parts, but only if the additional
ingredients, steps and/or parts do not materially alter the basic
and novel characteristics of the claimed composition, method or
structure.
[0382] The word "exemplary" is used herein to mean "serving as an
example, instance or illustration". Any embodiment described as
"exemplary" is not necessarily to be construed as preferred or
advantageous over other embodiments and/or to exclude the
incorporation of features from other embodiments.
[0383] The word "optionally" is used herein to mean "is provided in
some embodiments and not provided in other embodiments". Any
particular embodiment of the invention may include a plurality of
"optional" features unless such features conflict.
[0384] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0385] Throughout this application, various embodiments of this
invention may be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0386] Whenever a numerical range is indicated herein, it is meant
to include any cited numeral (fractional or integral) within the
indicated range. The phrases "ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are
used herein interchangeably and are meant to include the first and
second indicated numbers and all the fractional and integral
numerals therebetween.
As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0387] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0388] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0389] Reference is now made to the following examples, which
together with the above descriptions illustrate some embodiments of
the invention in a non-limiting fashion.
Materials and Methods
[0390] Materials:
[0391] Acetone was obtained from Bio-Lab (Israel);
[0392] acryloyl-chloride was obtained from Merck;
[0393] calcein AM was obtained from Sigma-Aldrich;
[0394] collagen type-I was obtained from BD Biosciences;
[0395] collagenase 1A was obtained from Sigma-Aldrich;
[0396] dichloromethane was obtained from Aldrich;
[0397] diethyl ether was obtained from Frutarom (Israel);
[0398] Dulbecco's modified Eagle medium was obtained from
Gibco;
[0399] ethidium homodimer-1 was obtained from Sigma-Aldrich;
[0400] fetal bovine serum was obtained from Biological Industries
(Israel);
[0401] formalin was obtained from Sigma-Aldrich;
[0402] Hoechst 33342 was obtained from Sigma Aldrich;
[0403] Irgacure.RTM. 2959 initiator was obtained from Ciba;
[0404] mercaptoethanol was obtained from Gibco;
[0405] N-hydroxysuccinimide-fluorescein was obtained from Thermo
Scientific;
[0406] non-essential amino acids wee obtained from Biological
industries (Israel);
[0407] penicillin-streptomycin was obtained from Biological
Industries (Israel);
[0408] petroleum ether 40-60 was obtained from Bio-Lab
(Israel);
[0409] Pluronic.RTM. F127 (12.6 kDa) was obtained from Sigma;
[0410] poly(ethylene glycol) (12 kDa) was obtained from Fluka;
[0411] sodium azide was obtained from Riedel-deHaen;
[0412] Tetronic.RTM. tetraol T1307 was obtained from BASF;
[0413] toluene was obtained from Bio-Lab (Israel);
[0414] triethylamine was obtained from Fluka;
[0415] tris(2-carboxyethyl)phosphine hydrochloride was obtained
from Sigma;
[0416] trypsin was obtained from MP Biomedicals.
[0417] F127 Poloxamer-Diacrylate, Tetronic.RTM. T1307-Tetraacrylate
and PEG-Diacrylate Synthesis:
[0418] F127 poloxamer-diacrylate (F127-DA), Tetronic.RTM.
T1307-tetraacrylate (T1307-TA) and poly(ethylene glycol)-diacrylate
(PEG-DA) were prepared from Pluronic.RTM. F127 (12.6 kDa),
Tetronic.RTM. tetraol T1307 (18 kDa) and poly(ethylene glycol)
(PEG) diol (12 kDa), respectively, according to the procedures
described in Halstenberg et al. [Biomacromolecules 2002,
3:710-723]. As depicted in FIG. 1A, acrylation of the polymers was
carried out under argon by reacting the hydroxyl-terminated
polymers in a solution of dichloromethane and toluene with acryloyl
chloride (Merck, Darmstadt, Germany) and triethylamine at a molar
ratio of 1.5:1 relative to the hydroxyl groups. The final product
was precipitated in ice-cold diethyl ether (for PEG-DA) or
petroleum ether 40-60 (for F127-DA and T1307-TA). The solid polymer
was dried under vacuum for 48 hours.
[0419] Using proton NMR, the average number of acryl groups per
F127-DA molecule was determined to be 2.15, the average number of
acryl groups per T1307-TA molecule was determined to be 4.38, and
the average number of acryl groups per PEG-DA was determined to be
1.74.
[0420] Rheological Characterization:
[0421] Rheological measurements were carried out using an AR-G2
rheometer (TA Instruments) equipped with a Peltier plate
temperature-controlled base. A 40 mm quartz plate geometry was used
in all experiments. Each measurement was carried out with 0.4 ml of
the polymer solution containing 0.1% (weight/volume) Irgacure.RTM.
2959 initiator. UV light (365 nm) was applied by a circular
multi-diode array (Moritex, Japan). The testing conditions for all
measurements were 2% strain at an oscillation frequency of 1
Hz.
[0422] Water Uptake Measurements:
[0423] Hydrogel constructs were made from a volume of 100 .mu.l
polymer-fibrinogen conjugate solution with 0.1% (weight/volume)
Irgacure.RTM. 2959 initiator in a 5 mm diameter silicon tube. The
constructs were cross-linked under UV light (365 nm, 4-5
mW/cm.sup.2) to form a 5 mm tall cylinder. FF127 was cross-linked
at a temperature of 4.degree. C., 21.degree. C. or 37.degree. C.
The water uptake was evaluated by calculating the swelling ratio
(Q.sub.M), i.e., the ratio of the wet weight (mass after swelling)
divided by the dry weight (weight after lyophilization).
[0424] Biodegradation Measurements:
[0425] Biodegradation of the hydrogels was characterized by
fluorometrically labeling the biological component in the
bio-synthetic hydrogel with amine-reactive
N-hydroxysuccinimide-fluorescein (NHS-fluorescein). The rate of
degradation was quantified by measuring the release of the protein
during the enzymatic dissolution of the hydrogel. 100 .mu.l
hydrogel plugs were stained overnight in a PBS solution containing
0.05 mg/ml NHS-fluorescein, and washed extensively to remove
unbound fluorescein. The plugs were then transferred into 3 ml of
PBS with 0.01 mg/ml trypsin and 0.1% sodium azide (Riedel-deHaen,
India), and incubated at 37.degree. C. with continuous agitation.
Fluorescence measurements were carried out in a Thermo Varioskan
Spectrophotometer (excitation wavelength 494 nm, emission
wavelength 518 nm) with Skanit2.2.RTM. Software. After the last
time point, each hydrogel was hydrolytically dissociated by adding
0.1 M NaOH. After 30 minutes, the emission values were recorded at
100% degradation. Labeled hydrogel plugs without enzyme and
unstained plugs with enzyme solution were used as negative
controls.
[0426] Preparation of Cell-Seeded Constructs:
[0427] Cell-seeded hydrogel constructs were prepared by UV-induced
cross-linking of FF127 or FT1307 conjugates in solution in the
presence of dispersed human foreskin fibroblasts or HeLa cells. The
passaged cells were trypsinized and suspended in 100 .mu.l of a
solution of the conjugate at a concentration of 10.sup.6 cells/ml,
along with a photoinitiator (0.1% w/v). The disc-shaped constructs
were exposed to UV light for 5 minutes at 4.degree. C., 21.degree.
C. or 37.degree. C. Control cell-seeded constructs were prepared
from PEG (12 kDa)-fibrinogen, F127 poloxamer diacrylate or T1307
tetraacrylate (3% w/w in PBS). The cell-seeded constructs were
cultured for up to 6 days in Dulbecco's modified Eagle medium
(DMEM) containing 10% fetal bovine serum (FBS), 1%
penicillin-streptomycin, 1% non essential amino acids, and 0.2%
2-mercaptoethanol.
[0428] Light Microscopy and Fluorescent Microscopy:
[0429] Light microscopy and fluorescent microscopy were performed
using an Eclipse TE2000-S microscope (Nikon) or an Eclipse TS100
microscope (Nikon), and a digital camera.
[0430] Statistical Analysis:
[0431] Statistical analysis was performed using Microsoft Excel
statistical analysis software. Data from independent experiments
were quantified and analyzed for each variable. Comparisons between
two treatments were made using student's T-test (two tail, equal
variance) and comparisons between multiple treatments were made
with analysis of variance (ANOVA). A p-value of <0.05 was
considered to be statistically significant.
Example 1
F127 Poloxamer-Fibrinogen Conjugate
[0432] Fibrinogen was conjugated to F127 poloxamer-diacrylate
(prepared as described hereinabove, and as depicted in FIG. 1A) by
a Michael-type addition reaction, as depicted in FIG. 1B. In order
to compare the properties of poloxamer-protein conjugates with
those of poly(ethylene glycol) (PEG)-protein conjugates, fibrinogen
was conjugated with 12 kDa PEG-diacrylate (prepared as described
hereinabove), using the same reaction.
[0433] A 3.5 mg/ml solution of fibrinogen in 150 mM phosphate
buffer saline (PBS) with 8 M urea was supplemented with
tris(2-carboxyethyl)phosphine hydrochloride (TCEP) at a molar ratio
of 1.5:1 TCEP to fibrinogen cysteine residues. PBS with 8 M urea
and 280 mg/ml of the functionalized polymer (F127-DA or PEG-DA) was
then added at a molar ratio of 4:1 polymer molecules to fibrinogen
cysteine residues. The mixture was allowed to react for 3 hours at
room temperature. The conjugated protein was then precipitated by
adding 4 volumes of acetone. The precipitate was redissolved in PBS
containing 8 M urea at a protein concentration of 10 mg/ml and then
dialyzed against PBS for 2 days at 4.degree. C., with the PBS being
replaced twice per day. The dialysis tubing had a cutoff of
12-14-kDa (Spectrum).
[0434] In order to establish total concentration of the
F127-fibrinogen and PEG-fibrinogen conjugates, 0.5 ml of the
conjugate solution was lyophilized for 24 hours and weighed. The
net fibrinogen concentration was determined using a standard
BCA.TM. Protein Assay (Pierce Biotechnology) and the concentrations
of the conjugates (dry weight) and fibrinogen were compared in
order to determine the concentration of synthetic polymer in the
conjugates. The efficiency of the conjugation reaction
(.epsilon..sub.conjugation) was calculated based on the
concentrations and molecular weights of the synthetic polymer and
fibrinogen, assuming a theoretical maximum of 29 synthetic polymer
molecules per fibrinogen molecule (as fibrinogen comprises 29 thiol
groups), using the following formula:
conjugation = [ S . Polymer ] [ Fibrinogen ] .times. theortical {
MW fibrinogen 29 .times. MW S . Polymer } ##EQU00001##
[0435] The mean fibrinogen concentration and conjugation efficiency
obtained for 4 batches of each of F127-fibrinogen and
PEG-fibrinogen conjugates are summarized in Table 1.
TABLE-US-00001 TABLE 1 Mean fibrinogen concentration and
conjugation efficiency of synthetic polymer-fibrinogen conjugates
(mean .+-. standard error of the mean) Synthetic Fibrinogen
Conjugate polymer Conjugation concentration concentration
concentration efficiency Synthetic MW (measured) (measured)
(calculated) (.epsilon..sub.conjugation) polymer (kDa) (mg/ml)
(mg/ml) (mg/ml) (%) F127-DA 12.6 7.7 .+-. 0.5 21 .+-. 2.3 13 .+-.
1.9 79 .+-. 8.4 PEG-DA 12 8.9 .+-. 2 24.7 .+-. 6.7 15.8 .+-. 4.8
83.8 .+-. 10.5
[0436] As shown in Table 1, both F127 poloxamer and PEG were
conjugated to fibrinogen with a relatively high conjugation
efficiency. There was no statistically significant difference
between the conjugation efficiency or fibrinogen concentration
obtained with F127 poloxamer and PEG.
Example 2
Rheological Properties of F127 Poloxamer-Fibrinogen Conjugate
(FF127) and Hydrogels Formed by Cross-Linking FF127
[0437] The rheological properties of the F127 poloxamer-fibrinogen
conjugate (FF127) described in Example 1 was studied, as described
in the Materials and Methods section hereinabove.
[0438] As shown in FIG. 2, the shear storage modulus (G') of FF127
increased considerably at temperatures above about 20.degree. C.
The transition was dependent on the concentration of FF127, as the
storage modulus of 8 mg/ml FF127 increased at a slightly lower
temperature than did the storage modulus of 4 mg/ml FF127.
[0439] As further shown in FIG. 2, the increase in the shear
storage modulus was accompanied by a peak in the shear loss modulus
(G'') of the FF127.
[0440] As shown in FIG. 3A, the shear storage modulus was
repeatedly increased (up to about 185 Pa) and decreased by raising
the temperature to 37.degree. C. and lowering the temperature to
15.degree. C., indicating a reversible transition.
[0441] These results indicates that FF127 undergoes a reverse
thermal gelation (RTG) phase transition at such temperatures, as a
result of the formation of a continuous polymeric matrix due to
physical (i.e., non-covalent) cross-linking of FF127 molecules, as
depicted in FIG. 4.
[0442] It is notable that the reverse thermal gelation occurred at
concentrations of less than 20 mg/ml of conjugate (corresponding to
a fibrinogen concentration of approximately 8 mg/ml), as F127 does
not exhibit reverse thermal gelation at concentrations less than
14.6% (w/w) [Cohn et al., Biomacromolecules 2005, 6:1168-1175].
[0443] Chemical (i.e., covalent) cross-linking of the FF127
molecules was performed by adding 0.1% (weight/volume)
Irgacure.RTM. 2959 initiator to FF127 solutions, and irradiating
the solution with UV light (365 nm, 4-5 mW/cm.sup.2).
[0444] As shown in FIG. 5, chemical cross-linking of FF127 resulted
in an irreversible increase in the storage modulus.
[0445] This result indicates that a hydrogel is formed due to
UV-initiated free radical polymerization of the acryl functional
groups on the FF127 molecules.
[0446] As shown in FIG. 6, the chemically cross-linked hydrogel
exhibited temperature-dependent increases in the storage modulus
and loss modulus.
[0447] As shown in FIG. 3B, the storage modulus of the chemically
cross-linked hydrogel was repeatedly increased (up to about 300 Pa)
and decreased by raising the temperature to 37.degree. C. and
lowering the temperature to 15.degree. C., indicating a reversible
transition.
[0448] This result indicates that the chemically cross-linked
hydrogel further exhibits RTG phase transitions due to physical
cross-linking of FF127 unimers, as observed in FF127 without
chemical cross-linking.
[0449] As further shown in FIGS. 3A and 3B, the gelation of FF127
and chemically cross-linked FF127 at 37.degree. C. was gradually
eliminated in the presence of collagenase (which degrades
fibrinogen), in a dose-dependent manner.
[0450] These results indicate that the reverse thermal gelation of
both FF127 and chemically cross-linked FF127 is associated with the
molecular weight of the fibrinogen which forms the backbone of the
FF127. As the fibrinogen was proteolytically degraded by the
collagenase, the FF127 unimers become smaller and the ability to
form a physical polymeric matrix was thereby affected.
[0451] In order to explore the stability of the hydrogel network
properties under applied loading conditions, hydrogels were
prepared from FF127 (8 mg/ml) with or without chemical
cross-linking and exposed to time-sweep rheological measurements as
the applied shear stress levels were increased incrementally.
[0452] As shown in FIG. 7, the chemically cross-linked hydrogel was
more responsive to temperature changes compared to the physical
hydrogel, exhibiting a higher storage modulus at 37.degree. C., but
it collapsed under less oscillatory stress (70 Pa) than did the
physical hydrogel (200 Pa).
[0453] As further shown therein, when the applied stress was
removed at 37.degree. C., the chemically cross-linked hydrogel was
restored almost completely, whereas the physically cross-linked
hydrogel recovered only slightly from the applied stress. However,
lowering the temperature to 15.degree. C. and raising it back to
37.degree. C. completely restored the mechanical properties of both
hydrogels.
[0454] These results indicate that the properties of the gels can
be "reset" by lowering and raising the temperature.
Example 3
Effect of Cross-Linking Temperature on Physical Properties of F127
Poloxamer-Fibrinogen Conjugate (FF127) Hydrogels
[0455] As the interactions between molecules of the FF127 conjugate
are temperature-dependent, it was hypothesized that the temperature
during the chemical cross-linking reaction (T.sub.cl) influences
the chemical cross-linking reaction. The chemical cross-linking of
a hydrogel network in the presence of free radicals may depend upon
the mobility of the molecular precursors and their likelihood to
form chemical cross-links when undergoing a temperature-dependent
physical transition.
[0456] Hydrogels were formed by UV-activated cross-linking, as
described in Example 2, at different temperatures.
[0457] As shown in FIG. 8, the G' value of the hydrogels at
37.degree. C. was inversely proportional to the temperature at
which the UV-induced cross-linking was performed. As further shown
therein, the G' values of hydrogels chemically cross-linked at
different temperatures were nearly identical at 15.degree. C.
[0458] These results indicate that physical cross-linking has a
highly significant effect on the physical properties which
characterize chemically cross-linked networks, as the properties of
the various hydrogels varied considerably at 37.degree. C., when
physical cross-linking is present, but not at 15.degree. C., when
physical cross-linking is absent.
Example 4
Water Uptake by F127 Poloxamer Fibrinogen Conjugate (FF127)
Hydrogels
[0459] Water uptake of cross-linked FF127 hydrogel constructs was
determined as described in the Materials and Methods section
hereinabove. FF127 was cross-linked at a temperature of 21.degree.
C. or at a temperature of 37.degree. C. As a control, water uptake
of cross-linked PEG (12 kDa)-fibrinogen hydrogels was determined as
described hereinabove.
[0460] The water uptake in each hydrogel represents a
characteristic measure of its equilibrium state between water and
polymeric matrix, and gives an indication of the structural forces
involved in forming and sustaining the hydrogel network. The
swelling ratio (Q.sub.M) was measured for the three hydrogels at
two separate ambient temperatures, 4.degree. C. and 37.degree.
C.
[0461] As shown in FIG. 9, there was no significant difference in
swelling ratio between the different hydrogels at 4.degree. C.,
whereas at 37.degree. C., FF127 and PEG-fibrinogen exhibit
significantly different properties. FF127 hydrogels expelled water
when warmed to 37.degree. C., whereas PEG-fibrinogen hydrogels did
not.
[0462] As shown in FIGS. 9, 10A and 10B, FF127 cross-linked at
21.degree. C. expelled more water than did FF127 cross-linked at
37.degree. C.
[0463] These results indicate that at a temperature at which
reverse thermal gelation effects are negligible (e.g., 4.degree.
C.), the different cross-linked polymers exhibit similar
properties, whereas at a temperature at which reverse thermal
gelation effects are significant (e.g., 37.degree. C.), the degree
of reverse thermal gelation affects the swelling properties of the
polymer networks.
Example 5
Comparison of Biodegradation and Rheological Properties of F127
Poloxamer-Fibrinogen Conjugate (FF127) Hydrogels
[0464] The biodegradation kinetics of chemically cross-linked FF127
and PEG (12 kDa)-fibrinogen hydrogels were determined in a 0.01
mg/ml trypsin solution at 37.degree. C., as described hereinabove.
FF127 hydrogels were cross-linked at temperatures of 21.degree. C.
and 37.degree. C. were compared. The hydrogels were cross-linked by
exposure to UV, as described hereinabove.
[0465] The storage moduli of the hydrogels were determined as
described hereinabove. For comparison, a hydrogel was prepared by
cross-linking F127 diacrylate at 37.degree. C. the storage modulus
was determined
[0466] As shown in FIG. 11, there was a statistically significant
difference between the three materials in terms of their
biodegradation rate (p<0.05). The hydrogels made of
PEG-fibrinogen degraded the fastest, with a half-life of 105.+-.5 4
minutes, and were fully degraded after 24 hours in 0.01 mg/ml
trypsin. The hydrogels made of FF127 reached only .about.60%
degradation after 24 hours. The half-life of the FF127 hydrogels
was 420.+-.66 minutes when cross-linked at 37.degree. C., and
580.+-.90 minutes when cross-linked at 21.degree. C.
[0467] As shown in FIG. 12, the storage modulus of FF127
cross-linked at 37.degree. C. was similar to that of the
PEG-fibrinogen, and considerably lower than that of the FF127
cross-linked at 21.degree. C. As further shown therein, the storage
modulus of FF127 cross-linked at 21.degree. C. was similar to that
of F127 diacrylate cross-linked at 37.degree. C.
[0468] Thus, although the biodegradation rate of cross-linked FF127
was lower than that of cross-linked PEG-fibrinogen, and was only
moderately affected by the cross-linking temperature, the storage
modulus of cross-linked FF127 was strongly affected by the
cross-linking temperature.
[0469] These results indicate that factors determining
biodegradation rate (e.g., type of polymer) can be selected
relatively independently of the factors determining rheological
properties (e.g., cross-linking temperature).
Example 6
Tetronic.RTM. T1307-Fibrinogen Conjugate
[0470] Fibrinogen was conjugated to Tetronic.RTM. T1307
tetraacrylate (prepared as described hereinabove) by a Michael-type
addition reaction, using essentially the same procedures as
described in Example 1. As depicted in FIGS. 13A and 13B,
conjugation of a tetraacrylate polymer to fibrinogen results in 3
free acrylate groups per conjugated polymer (1 acrylate group
attaches the fibrinogen to the polymer), providing increased
cross-linking ability.
[0471] The mean fibrinogen concentration and conjugation efficiency
was determined for 4 batches of T1307-fibrinogen, as described in
Example 1.
[0472] The obtained solution of T1307-fibrinogen conjugate
comprised 20.4.+-.1.4 mg/ml of the conjugate, 6.7.+-.1 mg/ml
fibrinogen, and 13.7.+-.0.5 mg/ml synthetic polymer. The
conjugation efficiency was 66.3.+-.8.5%.
Example 7
Physical Properties of T1307-Fibrinogen (FT1307) Hydrogels
[0473] The T1307-fibrinogen conjugate (FT1307) described in Example
6 was chemically cross-linked by UV light at a concentration of 6
mg/ml, at temperatures of 4.degree. C., 21.degree. C. or 37.degree.
C. Rheological properties, water uptake and biodegradation of the
obtained hydrogels were determined, as described hereinabove.
[0474] As shown in FIGS. 14A and 14B, the cross-linking temperature
of FT1307 was inversely correlated to the storage modulus at
37.degree. C.
[0475] As shown in FIG. 15, the cross-linking temperature of FT1307
was inversely correlated to the amount of water expelled from the
hydrogel when the hydrogel was warmed to 37.degree. C. In contrast,
the cross-linking temperature had little effect on the water uptake
of the polymers at 4.degree. C.
[0476] In contrast, as shown in FIG. 16, the cross-linking
temperature of FT1307 did not exhibit any clear correlation with
the degradation rates of the FT1307.
[0477] These results are similar to those presented in Examples 3
and 4, and indicate that the cross-linking temperature can be used
to determine the properties of polymer-protein hydrogels formed
using a variety of reverse thermal gelation polymers, and that the
rheological properties of the hydrogels can be determined
independently of the degradation rates.
Example 8
Cell-Seeded F127-Fibrinogen (FF127) Hydrogels
[0478] Cell-seeded hydrogel constructs were prepared by UV-induced
cross-linking of a FF127 conjugate solution in the presence of
dispersed human foreskin fibroblasts (Lonza, Walkersville, Md.,
USA), as described in the Materials and Methods section. Control
cell-seeded constructs were prepared from PEG (12 kDa)-fibrinogen
and F127 poloxamer diacrylate. Samples for histology were fixed in
4% formalin on day 3 and on day 6 of each experiment.
Cross-sections were stained with hematoxylin and eosin (H & E)
for imaging.
[0479] As shown in FIG. 17, the formation of lamellipodia and a
spindled cellular morphology proceeded more rapidly in FF127
cross-linked at 37.degree. C. than in FF127 cross-linked at
21.degree. C. On day 3, the cells in FF127 cross-linked at
21.degree. C. were relatively rounded and had only begun to form
lamellipodia, whereas in the FF127 cross-linked at 37.degree. C.,
the cells were highly spindled with many cellular lamellipodia.
Accordingly, on day 6, the cells in FF127 cross-linked at
21.degree. C. had begun to invade the matrix through cellular
lamellipodia, but only a few were fully spindled, whereas most of
the cells in FF127 cross-linked at 37.degree. C. were fully
spindled and exhibited many lamellipodia.
[0480] As further shown therein, in the cross-linked
PEG-fibrinogen, which is characterized both by a relatively high
biodegradability and low storage modulus (as shown hereinabove),
cells were highly spindled by day 3.
[0481] As further shown therein, in cross-linked F127 diacrylate,
which lacks fibrinogen, cells remained completely rounded and did
not form cellular extensions.
[0482] Cell-seeded FF127 hydrogel constructs were also prepared by
physical cross-linking at 37.degree. C. without chemical
cross-linking by UV light. The cells in such hydrogels were
compared to those in FF127 hydrogel constructs chemically
cross-linked at 37.degree. C.
[0483] As shown in FIG. 18, cells in FF127 hydrogels with only
physical cross-linking and cells in FF127 hydrogels with both
physical and chemical cross-linking both displayed a similar
morphology. On day 3 in both materials, the cells exhibited
spindled morphology with protrusions invading the matrix, and on
day 6 in both materials, the cells were fully spread and highly
spindled.
[0484] The viability of the encapsulated cells was determined on
day 0 and on day 3 of each experiment. The cells were removed from
the construct by dissolving the fibrinogen in 0.4 mg/ml collagenase
1A solution for 4 hours followed by 5 minutes centrifugation (1000
rotations per minute). The pellet was redissolved in 100 .mu.l of
staining solution containing 0.004 mM ethidium homodimer-1 and 2
mg/ml Hoechst 33342 in PBS. The cells were stained for 30 minutes
on an orbital shaker in the dark and then centrifuged for 5 minutes
(1000 rotations per minute). The cell pellet was dissolved in 25
.mu.l of PBS, and imaged on a glass microscope slide overlaid with
a cover slip. The stained cells were imaged by fluorescent
microscopy. The live and dead cells were counted and normalized by
a control suspension that was not exposed to UV light.
[0485] As shown in FIG. 19, the viability of cells in chemically
cross-linked FF127 was at least 88% on day 0 and at least 85% on
day 3. The cell viability on both days was higher in FF127
cross-linked at 37.degree. C. than in FF127 cross-linked at
21.degree. C., although the differences were not statistically
significant.
[0486] The above results indicate that hydrogels formed from
poloxamer-fibrinogen conjugates, including hydrogels with and
without chemical cross-linking of the conjugates, can serve as
matrices for cell growth and invasion. The results further indicate
that the rate of cell invasion can be modulated by selecting the
physical properties of the gel, for example, by selecting a
suitable cross-linking temperature.
Example 9
Cellular Outgrowth into F127-Fibrinogen (FF127) Hydrogels
[0487] Outgrowth experiments were performed using a dense tissue
construct made from compacted bovine aortic smooth muscle cells
(Genlantis) seeded in collagen gels. Each compacted cell-seeded
collagen gel was encapsulated inside an FF127 hydrogel. As a
control, a compacted cell-seeded collagen gel was encapsulated
inside a PEG-fibrinogen hydrogel.
[0488] The collagen-based tissue was made from a solution of
5.times.DMEM, 10% fetal bovine serum, reconstituted collagen type-I
solution in 0.02 N acetic acid (2 mg/ml), and 1 M NaOH with smooth
muscle cells dispersed at a concentration of 3.times.10.sup.6
cells/ml. The cell-seeded collagen gels were cultured for 2 days in
culture medium before the compacted tissue was placed in 300 .mu.l
of FF127 (or PEG-fibrinogen) conjugate solution and photoinitiator
in a 48-well plate. After exposure to 5 minutes of UV light at
37.degree. C. or 21.degree. C., the encapsulated tissue was
cultured inside the hydrogel with 500 .mu.l of culture medium. The
cellular outgrowth from the collagen gel into the FF127 (or
PEG-fibrinogen) encapsulating matrix was monitored daily for up to
5 days. The outgrowth results were quantified by measuring the
average travel distance of the cells from the margins of the dense
collagen tissue into the FF127 (or PEG-fibrinogen) hydrogel using
phase contrast micrographs of the samples taken at set time
intervals.
[0489] As shown in FIGS. 20A and 20B, in each of the three tested
materials (FF127 cross-linked at 21.degree. C. and at 37.degree.
C., and cross-linked PEG-fibrinogen), the cells began to invade the
matrix surrounding the tissue mass after 1 day and continued to
invade the matrix for the duration of the experiment.
[0490] As shown in FIG. 20B, the rate of invasion in the FF127
cross-linked at 37.degree. C. remained constant for the duration of
the experiment, whereas the rate of invasion decreased in the FF127
cross-linked at 21.degree. C. and in the PEG-fibrinogen, starting
on the third day of the experiment. Beginning from day 3, there was
a statistically significant difference between the cell migration
distance in FF127 cross-linked at 21.degree. C. and in FF127
cross-linked at 37.degree. C. On day 4, the distance the cells
traveled was 21% lower in FF127 cross-linked at 21.degree. C. than
in FF127 cross-linked at 37.degree. C., and on day 5, the distance
was 11% lower in FF127 cross-linked at 21.degree. C. The invading
cells did not exhibit a morphological difference among the three
materials tested.
[0491] These results further indicate that the rate of cell
invasion can be modulated by selecting the physical properties of
the gel.
Example 10
Cell-Seeded T1307-Fibrinofen (FT1307) Hydrogels
[0492] Cell-seeded hydrogel constructs were prepared by UV-induced
cross-linking of a FT1307 conjugate solution in the presence of
human foreskin fibroblasts and HeLa human adenocarcinoma cells, as
described in the Materials and Methods section. Control cell-seeded
constructs were prepared from T1307 tetraacrylate.
[0493] In order to view the seeded cells and determine their
viability, the cell-seeded constructs were placed in a well holding
2 ml of 4 mM calcein AM and 2 mM ethidium homodimer-1 in DMSO, and
incubated for 45 minutes. Viable cells are stained by calcein and
non-viable cells are stained by ethidium. Each construct was then
washed twice for 15 minutes in PBS in order to remove excess dye
molecules. The cells were then imaged by fluorescent
microscopy.
[0494] As shown in FIG. 21, cell spreading of fibroblasts proceeded
relatively rapidly in FT1307 cross-linked at 37.degree. C., and
more slowly in FT1307 cross-linked at 21.degree. C., and was almost
completely halted in FT1307 cross-linked at 4.degree. C. The rate
of cell spreading was inversely correlated to the storage modulus,
which was 52 Pa, 244 Pa and 373 Pa following cross-linking
temperatures of 37.degree. C., 21.degree. C. and 4.degree. C.,
respectively.
[0495] As further shown in FIG. 21, cell viability was high in all
three types of FT1307 matrices, as evidenced by the paucity of
ethidium (orange-colored) staining.
[0496] As shown in FIG. 22, HeLa cell colonies were relatively
dense and confined in FT1307 cross-linked at 4.degree. C., somewhat
less dense and confined in FT1307 cross-linked at 21.degree. C.,
and relatively disperse in FT1307 cross-linked at 37.degree. C.
[0497] The above results indicate that the rate of cell spreading
and the structure of cell colonies is affected by the physical
properties of the matrix, which can be determined by cross-linking
temperature.
Example 11
Cellular Outgrowth in F127-Fibrinogen (FF127) Hydrogels
Encapsulated within T1307-Fibrinofen (FT1307) Hydrogels
[0498] Outgrowth experiments were performed using FF127 physically
cross-linked capsules containing cultures or co-cultures of human
dermal fibroblasts and HeLa cells, which were entrapped in FT1307
chemically cross-linked hydrogels. Trypsinized cells were suspended
in 500 .mu.l of FF127 conjugate solution at a concentration of
10.sup.7 cells/ml, and loaded into a Micro-Fine.TM. 30 G syringe
(BD, New Jersey, USA).
[0499] As shown in FIG. 23A, while keeping the temperature below
20.degree. C., drops 20 of the suspension of cells in FF127 were
added from syringe 10 into a gently stirred phosphate buffered
saline (PBS) medium 30 kept at a temperature of 37.degree. C. The
drops 20 gelled upon exposure to a temperature of 37.degree. C. in
PBS medium 30, forming cell-seeded capsules 40. The cell-seeded
capsules 40 were isolated and incubated in DMEM cell culture medium
for 2 days at 37.degree. C., and then seeded in 300 .mu.l of FT1307
conjugate solution with a photoinitiator (0.1% w/v), and exposed to
UV light for 5 minutes at temperatures of 37.degree. C., 21.degree.
C. or 4.degree. C.
[0500] As shown in FIG. 23B, this procedure resulted in a
co-polymeric construct--so as to entrap the relatively soft
physically cross-linked FF127 capsules 50 within a harder
chemically cross-linked FT1307 milieu 60.
[0501] As described hereinabove, cross-linking temperatures of
37.degree. C., 21.degree. C. or 4.degree. C. resulted in FT1307
storage moduli of 52 Pa, 244 Pa and 373 Pa, respectively.
[0502] As shown in FIGS. 24A and 24B, fibroblasts exhibited
outgrowths in a hydrogel with a low storage modulus (52 Pa), but
not in a hydrogel with a high storage modulus (373 Pa).
[0503] In comparison, as shown in FIGS. 25A and 25B, HeLa cells
exhibited different migration/invasion strategies in hydrogels with
different moduli; the cells exhibited individual amoeboid migration
in a hydrogel with a low storage modulus (52 Pa), and collective
multicellular migration in a hydrogel with a high storage modulus
(373 Pa).
[0504] Co-cultures of HeLa cells and fibroblasts were seeded in
FF127 capsules within FT1307 hydrogels in order to assess how the
hydrogel modulus affects the development of heterogenic cultures.
In order to differentiate between the fibroblasts and HeLa cells,
GFP (green fluorescent protein)-labeled fibroblasts and DiI
(1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine
perchlorate)-stained HeLa cells were co-cultured.
[0505] As shown in FIGS. 26A and 26B, in an FT1307 hydrogel with a
high storage modulus (373 Pa), HeLa cells pushed into the FT1307
hydrogel, increasing the diameter of the capsule, whereas
fibroblast outgrowth was halted.
[0506] As shown in FIGS. 27A and 27B, in an FT1307 hydrogel with a
low storage modulus (52 Pa), the capsule front was dominated by
fibroblasts, which effectively performed mesenchymal migration into
the FT1307 hydrogel.
[0507] The above results indicate that the outgrowth of cells from
homogeneous and heterogeneous cultures can be modulated according
to the physical properties of a surrounding hydrogel.
[0508] The above results further indicate that heterogeneous
hydrogels can be prepared from more than one type of
polymer-protein conjugate.
[0509] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0510] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.
* * * * *