U.S. patent application number 15/398379 was filed with the patent office on 2017-06-29 for crosslinked peptide hydrogels.
The applicant listed for this patent is Agency for Science, Technology and Research. Invention is credited to Charlotte Hauser, Wei Yang Seow.
Application Number | 20170182113 15/398379 |
Document ID | / |
Family ID | 49292792 |
Filed Date | 2017-06-29 |
United States Patent
Application |
20170182113 |
Kind Code |
A1 |
Hauser; Charlotte ; et
al. |
June 29, 2017 |
Crosslinked Peptide Hydrogels
Abstract
The present invention relates to hydrogels comprising a
plurality of amphiphilic peptides and/or peptoids capable of
self-assembling into three-dimensional macromolecular nanofibrous
networks, which entrap water and form said hydrogels, wherein at
least a portion of said plurality of amphiphilic peptides and/or
peptoids is chemically cross-linked. The present invention further
relates to methods for preparing such hydrogels and to various uses
of such hydrogels, e.g. as cell culture substrates, for drug and
gene delivery, as wound dressing, as an implant, as an injectable
agent that gels in situ, in pharmaceutical or cosmetic
compositions, in regenerative medicine, in tissue engineering and
tissue regeneration, or in electronic devices. It also relates to a
method of tissue regeneration or tissue replacement using a
hydrogel in accordance with the present invention.
Inventors: |
Hauser; Charlotte;
(Singapore, SG) ; Seow; Wei Yang; (Singapore,
SG) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Agency for Science, Technology and Research |
Singapore |
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SG |
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|
Family ID: |
49292792 |
Appl. No.: |
15/398379 |
Filed: |
January 4, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14634559 |
Feb 27, 2015 |
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15398379 |
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13751295 |
Jan 28, 2013 |
8999916 |
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14634559 |
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13638152 |
Sep 28, 2012 |
9067084 |
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PCT/SG10/00469 |
Dec 15, 2010 |
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13751295 |
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61319838 |
Mar 31, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 2400/06 20130101;
C12N 5/0018 20130101; A61L 27/38 20130101; A61L 27/50 20130101;
A61L 27/52 20130101; A61P 43/00 20180101; A61L 2300/426 20130101;
A61L 2430/38 20130101; A61L 26/0061 20130101; C12N 5/0068 20130101;
A61L 26/0028 20130101; A61K 35/33 20130101; C12N 2533/50 20130101;
A61K 47/42 20130101; C12N 5/0062 20130101; A61L 2300/412 20130101;
C12N 5/0671 20130101; C12N 5/0691 20130101; A61L 26/008 20130101;
A61K 38/02 20130101; A61L 27/22 20130101; A61L 27/3804 20130101;
C12N 2513/00 20130101; C12N 2537/10 20130101; A61K 9/06 20130101;
A61L 2430/00 20130101; A61L 2430/06 20130101; C12N 2533/54
20130101 |
International
Class: |
A61K 38/02 20060101
A61K038/02; A61L 27/52 20060101 A61L027/52; C12N 5/071 20060101
C12N005/071; A61L 27/38 20060101 A61L027/38; A61L 26/00 20060101
A61L026/00; A61K 9/06 20060101 A61K009/06; A61L 27/22 20060101
A61L027/22 |
Claims
1.-61. (canceled)
62. A method of treatment of wounds, treatment of degenerative
diseases, comprising administration of a hydrogel prepared by a
method comprising steps of: dissolving amphiphilic peptides and/or
peptoids having the general formula:
Z.sub.p--(X).sub.n--(Y).sub.m-AA.sub.thiol-Z'.sub.q, wherein Z is
an N-terminal protecting group, X is, at each occurrence,
independently selected from an aliphatic amino acid, Y is, at each
occurrence, independently selected from a hydrophilic amino acid,
AA.sub.thiol is an amino acid comprising a thiol group, Z' is a
C-terminal protecting group, n is an integer selected from 2 to 6,
m is selected from 0, 1 and 2, and p and q are independently
selected from 0 and 1, in an aqueous solution, wherein the aqueous
solution comprises an oxidizing agent or wherein the method further
comprises the step of exposing the ready-made hydrogel to a
solution of an oxidizing agent.
63.-85. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 14/634,559, entitled "CROSSLINKED PEPTIDE
HYDROGELS" and filed on Feb. 27, 2015, which is a divisional
application of U.S. patent application Ser. No. 13/751,295,
entitled "CROSSLINKED PEPTIDE HYDROGELS", and filed on Jan. 28,
2013, now U.S. Pat. No. 8,999,916, which is a continuation in part
application of U.S. patent application Ser. No. 13/638,152,
entitled "AMPHIPHILIC LINEAR PEPTIDE/PEPTOID AND HYDROGEL
COMPRISING THE SAME", and filed on Sep. 28, 2012, now U.S. Pat. No.
9,067,084, which is a 35 U.S.C. .sctn.371 National Stage of
International Application No. PCT/SG2010/000469, entitled
"AMPHIPHILIC LINEAR PEPTIDE/PEPTOID AND HYDROGEL COMPRISING THE
SAME" and filed on Dec. 15, 2010 and claims the benefit of priority
to U.S. Provisional Patent Application Ser. No. 61/319,838, filed
Mar. 31, 2010, the entire contents of each of which are hereby
incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to hydrogels comprising a
plurality of amphiphilic peptides and/or peptoids capable of
self-assembling into three-dimensional macromolecular nanofibrous
networks, which entrap water and form said hydrogels, wherein at
least a portion of said plurality of amphiphilic peptides and/or
peptoids is chemically cross-linked. The present invention further
relates to methods for preparing such hydro gels and to various
uses of such hydrogels, e.g. as cell culture substrates, for drug
and gene delivery, as wound dressing, as an implant, as an
injectable agent that gels in situ, in pharmaceutical or cosmetic
compositions, in regenerative medicine, in tissue engineering and
tissue regeneration, or in electronic devices. It also relates to a
method of tissue regeneration or tissue replacement using a
hydrogel in accordance with the present invention.
SEQUENCE LISTING
[0003] In accordance with 37 CFR 1.52(e)(5), the present
specification makes reference to a Sequence Listing submitted
electronically in the form of an ASCII text file (entitled
"Sequence_Listing.txt", created on May 18, 2015 and 11 KB in size).
The entire contents of the Sequence Listing are herein incorporated
by reference, with the intention that, upon publication (including
issuance), this incorporated sequence listing will be inserted into
the published document immediately before the claims.
BACKGROUND OF THE INVENTION
[0004] Supramolecular structures are held together by
intermolecular bondings that are responsible for the organization
of polymolecular systems. The non-covalent, intermolecular forces
which are required for the assembly of the defined supramolecular
structures are mainly electrostatic interactions, hydrogen
bondings, van der Waals forces, etc. Supramolecular chemistry or
biology gathers a vast body of two or three dimensional complex
structures and entities formed by association of chemical or
biological species. These associations are governed by the
principles of molecular complementarity or molecular recognition
and self-assembly. The knowledge of the rules of intermolecular
association can be used to design polymolecular assemblies in form
of membranes, films, layers, micelles, tubules, gels for a variety
of biomedical or technological applications (J.-M. Lehn, Science,
295, 2400-2403, 2002).
[0005] Peptides have been used for the fabrication of
supramolecular structures through molecular self-assembly (S.
Zhang, Nature Biotechnology, 21, 1171-1178, 2003). Peptides are for
instance able to assemble into nanotubes (US 7, 79, 84) or into
supramolecular hydrogels consisting of three dimensional scaffolds
with a large amount of around 98-99% immobilized water or aqueous
solution. The peptide-based biomaterials are powerful tools for
potential applications in biotechnology, medicine and even
technical applications. Depending on the individual properties
these peptide-based hydrogels are thought to serve in the
development of new materials for tissue engineering, regenerative
medicine, as drug and vaccine delivery vehicles or as peptide chips
for pharmaceutical research and diagnosis (E. Place et al., Nature
Materials, 8, 457-470, 2009). There is also a strong interest to
use peptide-based self-assembled biomaterial such as gels for the
development of molecular electronic devices (A. R. Hirst et al.,
Angew. Chem. Int. Ed., 47, 8002-8018, 2008).
[0006] A variety of "smart peptide hydro gels" have been generated
that react on external manipulations such as temperature, pH,
mechanical influences or other stimuli with a dynamic behavior of
swelling, shrinking or decomposing. Nevertheless, these
biomaterials are still not "advanced" enough to mimic the
biological variability of natural tissues as for example the
extracellular matrix (ECM) or cartilage tissue or others. The
challenge for a meaningful use of peptide hydrogels is to mimic the
replacing natural tissues not only as "space filler" or mechanical
scaffold, but to understand and cope with the biochemical signals
and physiological requirements that keep the containing cells in
the right place and under "in vivo" conditions (R. Fairman and K.
Akerfeldt, Current Opinion in Structural Biology, 15, 453-463,
2005). Much effort has been undertaken to understand and control
the relationship between peptide sequence and structure for a
rational design of suitable hydrogels. In general hydrogels contain
macroscopic structures such as fibers that entangle and form
meshes. Most of the peptide based hydrogels utilize as their
building blocks 0-pleated sheets which assemble to fibers. Later it
was shown that it is possible to design hydrogelating
self-assembling fibers purely from a-helices. Besides 0-sheet
structure-based materials (S. Zhang et al., PNAS, 90, 3334-3338,
1993; A. Aggeli et al., Nature, 386, 259-262, 1997, etc.) a variety
of a-helical hydrogels has been developed (W. A. Petka et al.,
Science, 281, 389-392, 1998; C. Wang et al., Nature, 397, 417-420,
1999; C. Gribbon et al., Biochemistry, 47, 10365-10371, 2008; E.
Banwell et al., Nature Materials, 8, 596-600, 2009, etc.).
[0007] Nevertheless, the currently known peptide hydrogels are in
most of the cases associated with low rigidity, sometimes
unfavourable physiological properties and/or complexity and the
requirement of substantial processing thereof which leads to high
production costs. There is therefore a widely recognized need for
peptide hydrogels that are easily formed, non-toxic and have a
sufficiently high rigidity for standard applications. The hydrogels
should also be suitable for the delivery of bioactive moieties
(such as nucleic acids, small molecule therapeutics, cosmetic and
anti-microbial agents) and/or for use as biomimetic scaffolds that
support the in vivo and in vitro growth of cells and facilitate the
regeneration of native tissue.
SUMMARY OF THE INVENTION
[0008] It is therefore desirable to provide a biocompatible
compound that is capable of forming a hydrogel that meets at least
some of the above requirements to a higher extent than currently
available hydro gels and that is not restricted by the above
mentioned limitations.
[0009] Disclosed is an amphiphilic peptide and/or peptoid capable
of forming a hydrogel, the amphiphilic peptide and/or peptoid
comprising an amphiphilic sequence consisting of:
a hydrophobic sequence stretch of n aliphatic amino acids, wherein
n is an integer from 2 to 15, and a hydrophilic sequence stretch
linked to said hydrophobic sequence stretch and having a polar
moiety which is acidic, neutral or basic, said polar moiety
comprising m adjacent hydrophilic amino acids, wherein m is an
integer from 1 to 5.
[0010] In one embodiment the amphiphilic peptide and/or peptoid has
a C-terminus and an N-terminus wherein the N-terminus is protected
by a protecting group, wherein said protecting group preferably is
an acetyl group.
[0011] In one embodiment, the amphiphilic peptide and/or peptoid
has a C-terminus, which, if a basic polar amino acid is located at
the C-terminus, is preferably amidated.
[0012] In one embodiment, n is an integer from 2 to 6.
[0013] In one embodiment, m is an integer from 1 to 2.
[0014] In one embodiment, the amphiphilic peptide and/or peptoid
consists of o amphiphilic sequences, as defined above, which
amphiphilic sequences are linked to each other, o being an integer
from 1 to 50.
[0015] In one embodiment, for a given amphiphilic peptide and/or
peptoid, said aliphatic amino acids and said hydrophilic amino
acids are either D-amino acids or L-amino acids.
[0016] In one embodiment, each of the hydrophilic amino acids has a
polar group which is independently selected from a hydroxyl, an
ether, a carboxyl, an imido, an amido, an ester, an amino, a
guanidine, a thio, a thioether, a seleno, and a telluro group.
[0017] In one embodiment, said polar moiety of said hydrophilic
sequence stretch comprises m adjacent hydrophilic amino acids, m
being defined as defined above, said hydrophilic amino acids being
selected from the group comprising aspartic acid, asparagine,
glutamic acid, glutamine, 5-N-ethyl-glutamine (theanine),
citrulline, thio-citrulline, cysteine, homocysteine, methionine,
ethionine, selenomethionine, telluromethionine, threonine,
allo-threonine, serine, homoserine, arginine, homoarginine,
ornithine, lysine and N(6)-carboxymethyllysine, histidine, and
wherein said hydrophobic sequence stretch comprises n aliphatic
amino acids, n being as defined above, said aliphatic amino acids
being selected from the group comprising isoleucine, norleucine,
leucine, valine, alanine, glycine, homoallylglycine and
homopropargylglycine. In one embodiment, m is 1 to 2.
[0018] In one embodiment, m is 2 and said polar moiety comprises
two identical amino acids, or m is 1 and said polar moiety
comprises any one of aspartic acid, asparagine, glutamic acid,
glutamine, serine, threonine, cysteine, methionine, lysine and
histidine.
[0019] In one embodiment, said polar moiety is adjacent to the
hydrophobic sequence stretch of n aliphatic amino acids.
[0020] In one embodiment, said polar moiety has a sequence selected
from Asp, Asn, Glu, Gin, Ser, Thr, Cys, Met, Lys, His, Asn-Asn,
Asp-Asp, Glu-Glu, Gln-Gln, Asn-Gln, Gln-Asn, Asp-Gln, Gin-Asp,
Asn-Glu, Glu-Asn, Asp-Glu, Glu-Asp, Gln-Glu, Glu-Gln, Asp-Asn,
Asn-Asp Thr-Thr, Ser-Ser, Thr-Ser, Ser-Thr, Asp-Ser, Ser-Asp,
Ser-Asn, Asn-Ser, Gln-Ser, Ser-Gln, Glu Ser, Ser-Glu, Asp-Thr,
Thr-Asp, Thr-Asn, Asn-Thr, Gin-Thr, Thr-Gln, Glu-Thr, Thr-Glu.
[0021] In one embodiment, said polar moiety comprises the
C-terminus of the amphiphilic peptide and/or peptoid, or wherein
said polar moiety comprises the N-terminus of the amphiphilic
peptide and/or peptoid.
[0022] In one embodiment, both said C-terminus and said N-terminus
do not carry any protecting groups attached to them.
[0023] In one embodiment, said polar moiety comprises the
C-terminus of the amphiphilic peptide and/or peptoid, wherein both
said C-terminus and said N-terminus do not carry any protecting
groups attached to them.
[0024] In one embodiment, said polar moiety comprises the
C-terminus of the amphiphilic peptide and/or peptoid, wherein said
C-terminus does not carry any protecting group, and wherein said
N-terminus carries a protecting group.
[0025] In one embodiment, said protecting group is an acetyl group
attached to the amino-group of said N-terminus.
[0026] In one embodiment, said polar moiety comprises the
C-terminus of the amphiphilic peptide and/or peptoid, wherein said
C-terminus carries a protecting group, and wherein said N-terminus
does not carry any protecting group.
[0027] In one embodiment, said protecting group is an amido-group
attached to the carboxyl group of said C-terminus.
[0028] In one embodiment, said polar moiety comprises the
C-terminus of the amphiphilic peptide and/or peptoid, wherein both
said C-terminus and N-terminus carry a protecting group.
[0029] In one embodiment, said C-terminus protecting group is an
amido-group attached to the carboxyl group of said C-terminus, and
wherein said N-terminus protecting group is an acetyl group
attached to the amino-group of said N-terminus.
[0030] In one embodiment, said polar moiety consists of at least
one amino acid positioned at the C-terminus of the amphiphilic
peptide and/or peptoid.
[0031] In one embodiment, said hydrophobic sequence stretch
comprises and/or forms the N-terminus of the amphiphilic peptide
and/or peptoid.
[0032] In one embodiment, all or a portion of the aliphatic amino
acids of the hydrophobic sequence stretch are arranged in an order
of decreasing amino acid size in the direction from N- to
C-terminus of the amphiphilic peptide and/or peptoid, wherein the
size of the aliphatic amino acids is defined as
I=L>V>A>G.
[0033] In one embodiment, said aliphatic amino acids arranged in an
order of decreasing amino acid size have a sequence which is a
repetitive or non-repetitive sequence.
[0034] In one embodiment, said aliphatic amino acids arranged in
order of decreasing amino acid size have a sequence with a length
of 2 to 7, preferably 2 to 6, more preferably 2 to 5 amino acids.
In one embodiment, said aliphatic amino acids arranged in an order
of decreasing amino acid size have a sequence selected from LIVAG,
ILVAG, LIVAA, LAVAG, IVAG, LIVA, LIVG, IVA and IV, wherein,
optionally, there is an A preceding such sequence at the
N-terminus.
[0035] In one embodiment, all or a portion of the aliphatic amino
acids of the hydrophobic sequence stretch are arranged in an order
of identical amino acid size in the amphiphilic peptide and/or
peptoid.
[0036] In one embodiment, said aliphatic amino acids arranged in
order of identical amino acid size have a sequence with a length of
2 to 4 amino acids. In one embodiment, said aliphatic amino acids
arranged in an order of identical size have a sequence selected
from LLLL, LLL, LL, IIII, III, II, VVVV, VVV, VV, AAAA, AAA, AA,
GGGG, GGG, and GG.
[0037] In one embodiment, the amphiphilic sequence undergoes a
conformational change during self-assembly, preferably a
conformational change from a random coil conformation to a helical
intermediate structure to a final beta conformation. In one
embodiment, the conformational change is concentration
dependent.
[0038] In one embodiment, the amphiphilic linear sequence comprises
a single hydrophilic and at least two aliphatic amino acids.
[0039] In one embodiment, the amphiphilic sequence is one of SEQ ID
NO: 1-42. It should be noted that any of the amphiphilic sequences
may carry a protecting group at the N-terminus or the C-terminus or
both. For example, SEQ ID NO:1-42 may all carry an acetyl group as
protecting group at the N-terminus. As a further example, SEQ ID
NO: 19 (LIVAGK) may carry an amido-group as protecting group at the
C-terminus, and additionally it may have an acetyl group at the
N-terminus as protecting group.
[0040] In one embodiment, said amphiphilic peptide and/or peptoid
is stable in aqueous solution at physiological conditions at
ambient temperature for a period of time in the range from 1 day to
at least 6 months, preferably to at least 8 months more preferably
to at least 12 months.
[0041] In one embodiment, the amphiphilic peptide and/or peptoid is
stable in aqueous solution at physiological conditions, at a
temperature up to 90.degree. C., for at least 1 hour.
[0042] Also disclosed is a hydrogel comprising the amphiphilic
peptide and/or peptoid as defined above.
[0043] In one embodiment, the hydrogel is stable in aqueous
solution at ambient temperature for a period of at least 7 days,
preferably at least 2 to 4 weeks, more preferably at least 1 to 6
months.
[0044] In one embodiment, the hydrogel is characterized by a
storage modulus G' to loss modulus G'' ratio that is greater than
2.
[0045] In one embodiment, the hydrogel is characterized by a
storage modulus G' from 100 Pa to 80, 00 Pa at a frequency in the
range of from 0.02 Hz to 16 Hz.
[0046] In one embodiment, the hydrogel has a higher mechanical
strength than collagen or its hydrolyzed form (gelatin).
[0047] In one embodiment, the hydrogel as defined above comprises
fibers of the amphiphilic peptide and/or peptoid as defined above,
said fibers defining a network that is capable of entrap-ping at
least one of a microorganism, a virus particle, a peptide, a
peptoid, a protein, a nucleic acid, an oligosaccharide, a
polysaccharide, a vitamin, an inorganic molecule, a synthetic
polymer, a small organic molecule or a pharmaceutically active
compound.
[0048] In one embodiment, the hydrogel comprises at least one of a
microorganism, a virus particle, a peptide, a peptoid, a protein, a
nucleic acid, an oligosaccharide, a polysaccharide, a vitamin, an
inorganic molecule, a synthetic polymer, a small organic molecule
or a pharmaceutically active compound entrapped by the network of
fibers of the amphiphilic polymer.
[0049] In one embodiment, the fibers of the amphiphilic polymer are
coupled to the at least one of a microorganism, a virus particle, a
peptide, a peptoid, a protein, a nucleic acid, an oligosaccharide,
a polysaccharide, a vitamin, an inorganic molecule, a synthetic
polymer, a small organic molecule or a pharmaceutically active
compound entrapped by the network of fibers of the amphiphilic
polymer.
[0050] In one embodiment, the hydrogel is comprised in at least one
of a fuel cell, a solar cell, an electronic cell, a biosensing
device, a medical device, an implant, a pharmaceutical composition
and a cosmetic composition. In one embodiment, the hydrogel as
defined above is for use in at least one of the following:
release of a pharmaceutically active compound, medical tool kit, a
fuel cell, a solar cell, an electronic cell, tissue regeneration,
stem cell therapy and gene therapy.
[0051] In one embodiment, the hydrogel as defined above is
injectable.
[0052] Also disclosed is a method of preparing a hydrogel, the
method comprising dissolving an amphiphilic peptide and/or peptoid
as defined above in an aqueous solution.
[0053] In one embodiment, the dissolved amphiphilic peptide and/or
peptoid in aqueous solution is further exposed to temperature,
wherein the temperature is in the range from 20.degree. C. to
90.degree. C., preferably from 20.degree. C. to 70.degree. C.
[0054] In one embodiment, the amphiphilic peptide and/or peptoid is
dissolved at a concentration from 0.01 .mu.g/ml to 100 mg/ml,
preferably at a concentration from 1 mg/ml to 50 mg/ml, more
preferably at a concentration from about 1 mg/ml to about 20
mg/ml.
[0055] Also disclosed is a surgical implant, or stent, the surgical
implant or stent comprising a peptide and/or peptoid scaffold,
wherein the peptide and/or peptoid scaffold is formed by a hydrogel
as defined above.
[0056] Also disclosed is a pharmaceutical and/or cosmetic
composition and/or a biomedical device and/or electronic device
comprising the amphiphilic peptide and/or peptoid as defined
above.
[0057] In one embodiment, the pharmaceutical and/or cosmetic
composition and/or the biomedical device, and/or the electronic
devices as defined above, further comprises a pharmaceutically
active compound.
[0058] In one embodiment, the pharmaceutical and/or cosmetic
composition as defined above, further comprises a pharmaceutically
acceptable carrier.
[0059] Also disclosed is a kit of parts, the kit comprising a first
container with an amphiphilic peptide and/or peptoid as defined
above and a second container with an aqueous solution.
[0060] In one embodiment, the aqueous solution of the second
container further comprises a pharmaceutically active compound.
[0061] In one embodiment, the first container with an amphiphilic
peptide and/or peptoid further comprises a pharmaceutically active
compound.
[0062] Also disclosed is a method of tissue regeneration comprising
the steps:
providing a hydrogel as defined above, exposing said hydrogel to
cells which are to form regenerated tissue, allowing said cells to
grow on said hydrogel.
[0063] In one embodiment, the method as defined above is performed
in-vitro or in-vivo.
[0064] In one embodiment, the method as defined above is performed
in vivo, wherein, in step a), aid hydrogel is provided at a place
in a body where tissue regeneration is intended.
[0065] In one embodiment, said step a) is performed by injecting
said hydrogel at a place in the body where tissue regeneration is
intended.
[0066] In a first aspect the present disclosure provides an
amphiphilic peptide and/or peptoid capable of forming a hydrogel.
The amphiphilic peptide and/or peptoid includes a hydrophobic and a
hydrophilic sequence. This hydrophobic sequence has a length of n
L- or D-amino acids. n is an integer, which may typically range
from 2 to about 15. The hydrophilic sequence has a polar and/or
charged moiety comprising m L- or D-amino acids. m is an integer
from 1 to 5. Each of the m aliphatic amino acids carries an
independently selected polar group. The amphiphilic linear sequence
has a net charge at physiological pH and a N-terminus carrying a
protecting group. The protecting group can be an acetyl group. The
amphiphilic peptide and/or peptoid may comprise o linked
amphiphilic peptide and/or peptoid sequences of n hydrophobic and m
hydrophilic L- and D-amino acids, wherein o is an integer from 1 to
about 50. The amphiphilic peptide and/or peptoid may consist of o
linked amphiphilic peptide and/or peptoid sequences of n
hydrophobic and m hydrophilic L- and D-amino acids. The value of n
may be an integer from 2 to about 15. The value of m may be 1 to 5.
The charged and/or polar group of each of the m hydrophilic L- and
D-amino acids may be independently selected from a hydroxyl, an
ether, a carboxyl, an amido, an ester, an amino, a guanidino,
athio, a thioether, a seleno, and a telluro group. The charged or
polar moiety of the hydrophilic sequence may comprise m L- or
D-amino acids selected from the group consisting of aspartic acid,
asparagine, glutamic acid, glutamine, 5-N-ethyl-glutamine
(theanine), citrulline, thiocitrulline, cysteine, homocysteine,
methionine, ethionine, selenomethionine, telluromethionine,
threonine, allo-threonine, serine, homoserine, arginine,
homoarginine, ornithine, lysin and N(6)-carboxymethyllysine. The
charged and/or polar moiety of the hydrophilic sequence may
comprise two identical amino acids. The two identical amino acids
may be adjacent to the non-polar hydrophobic moiety. The charged
and/or polar moiety may consist of two amino acids with a sequence
selected from Asn-Asn, Asp-Asp, Glu-Glu, Gln-Gln, Asn-Gln, Gln-Asn,
Asp-Gin, Gin-Asp, Asn-Glu, Glu-Asn, Asp-Glu, Glu-Asp, Gln-Glu,
Glu-Gln, Asp-Asn, Asn-Asp, Thr-Thr, Ser-Ser, Thr-Ser, Ser-Thr,
Asp-Ser, Ser-Asp, Ser-Asn, Asn-Ser, Gln-Ser, Ser-Gln, Glu-Ser,
Ser-Glu, Asp-Thr, Thr-Asp, Thr-Asn, Asn-Thr, Gin-Thr, Thr-Gln,
Glu-Thr, Thr-Glu. The charged and/or polar moiety may comprise the
C-terminus of the amphiphilic peptide and/or peptoid. The charged
and/or polar moiety may comprise (i) the C-terminus, the C-terminus
carrying an unprotected C-terminal carboxyl group or (ii) the
N-terminus, the N-terminus carrying an unprotected N-terminal amino
group. The charged and/or polar moiety may comprise the C-terminus
of the amphiphilic peptide and/or peptoid, the C-terminus carrying
an unprotected C-terminal carboxyl group and wherein the N-terminus
carries a protecting group preferably the acetyl group. The
protecting group may be an amido protecting group. The charged
and/or polar moiety may consist of at least one amino acid
positioned at the C-terminus of the amphiphilic peptide and/or
peptoid. The hydrophobic sequence may comprise at least two
aliphatic amino acids that is defined by a main chain comprising 1
to about 20 carbon atoms. A portion of the amino acids of the
non-polar moiety may be arranged in a general sequence of
decreasing size in the direction from N- to C-terminus of the
amphiphilic peptide and/or peptoid, and the size of adjacent amino
acids of the non-polar moiety may be identical or smaller in the
direction of the general sequence of decreasing size. The general
sequence of decreasing size may be preferably a non-repetitive
sequence. The direction of the general sequence of decreasing size
in which adjacent amino acids may be of identical or smaller size
may be the direction toward the charged and/or polar moiety of the
sequence. The portion of the amino acids arranged in a general
sequence of decreasing size may have a length of 2-7, preferably
2-6, more preferably 2, 3, 4, 5 or 6 amino acids. The portion of
the amino acids arranged in a general sequence of decreasing size
may also have a length of n-m-1 acids and wherein the portion of
the amino acids arranged in the general sequence of decreasing size
may be positioned between the remaining non-polar amino acid of the
non-polar moiety of n-m amino acids and the polar moiety. The
remaining nonpolar amino acid of the non-polar moiety of n-m amino
acids may define the N-terminus or the C-terminus of the
amphiphilic peptide and/or peptoid. The remaining non-polar amino
acid of the non-polar moiety of n-m amino acids may be one of
alanine, valine and glycine. The amphiphilic linear sequence may
undergo a conformational change from a random coil conformation to
a helical conformation during self-assembly. The conformational
change may be concentration dependent. The non-polar moiety of the
amphiphilic linear sequence may comprise at least one L- or D-amino
acid selected from the group consisting of glycine,
homoallylglycine, homopropargylglycine, alanine, valine, leucine,
norleucine and isoleucine. The amphiphilic linear sequence may
comprise a single polar and/or charge and a single nonpolar moiety.
The amphiphilic linear sequence may have a positive or a negative
net charge. The net charge may be from about -1 to about -4 or from
about +5 to about +1. The net charge may be from about -1 to about
-2. The net charge may be -2. The net charge may be +1 or +2 or +5.
The amphiphilic peptide and/or peptoid may be stable in aqueous
solution at physiological conditions at ambient temperature for a
period of time in the range from 1 day to at least 6 months,
preferably at least 8 months, more preferably at least 12 months.
The amphiphilic peptide and/or peptoid may be stable in aqueous
solution at physiological conditions at a temperature to 90.degree.
C. for at least 1 hour.
[0067] In a second aspect the disclosure provides a hydrogel. The
hydrogel includes an amphiphilic peptide and/or peptoid according
to the first aspect. The hydrogel may be stable in aqueous solution
at ambient temperature for a period of at least 7 days. The
hydrogel may be stable in aqueous solution at ambient temperature
for a period of at least 2 to 4 weeks. The hydrogel may be stable
in aqueous solution at ambient temperature for a period of at least
1 to 6 months. The hydrogel mechanical property may be
characterized by a loss modulus G'' to storage modulus G' ratio
that is less than 1. The hydrogel may be characterized by magnitude
of storage modulus G' greater than loss modulus G'' by minimum
factor of 1.5. The hydrogel may be characterized by a storage
modulus G' of from 100 Pa to 80, 00 Pa at a frequency in the range
of from 0.02 Hz to 16 Hz. The hydrogel may be characterized by
higher storage modulus G' with increase in the concentration of
peptide. The hydrogel may have a higher mechanical strength than
collagen or hydrolyzed form (gelatin). The hydrogel may comprise
fibers of an amphiphilic peptide and/or peptoid described herein.
The fibers may define a network that is capable of entrapping at
least one of a microorganism, a virus particle, a peptide, a
peptoid, a protein, a nucleic acid, an oligosaccharide, a
polysaccharide, a vitamin, an inorganic molecule, a synthetic
polymer, a small organic molecule or a pharmaceutically active
compound. The hydrogel may comprise at least one of a
microorganism, a virus particle, a peptide, a peptoid, a protein, a
nucleic acid, an oligosaccharide, a polysaccharide, a vitamin, an
inorganic molecule, a synthetic polymer, a small organic molecule
or a pharmaceutically active compound entrapped by the network of
fibers of the amphiphilic polymer. The fibers of the amphiphilic
polymer may be coupled to the at least one of a microorganism, a
virus particle, a peptide, a peptoid, a protein, a nucleic acid, an
oligosaccharide, a polysaccharide, a vitamin, an inorganic
molecule, a synthetic polymer, a small organic molecule or a
pharmaceutically active compound entrapped by the network of fibers
of the amphiphilic polymer. The hydrogel may be comprised in at
least one of a fuel cell, a solar cell, a electronic cell, a
biosensing device, a medical device, an implant, a pharmaceutical
composition, drug delivery system, tissue culture medium, biosensor
devices and a cosmetic composition. The hydrogel may be for at
least one of release of a pharmaceutically active compound, medical
tool kit, a fuel cell, a solar cell, an electronic cell, tissue
regeneration, stem cell therapy and gene therapy. In some
embodiments the hydrogel may be used for tissue regeneration, drug
release or gene therapy.
[0068] In a third aspect the disclosure provides a method of
preparing a hydrogel. The method includes providing an amphiphilic
peptide and/or peptoid according to the first aspect. The method
further includes dissolving and/or dispersing the amphiphilic
peptide and/or peptoid in an aqueous solution. The
dissolved/dispersed amphiphilic peptide and/or peptoid in aqueous
solution may be further exposed to a temperature. The temperature
may be selected in the range from about 20.degree. C. to about 90,
preferably from 20.degree. C. to 70.degree. C. The amphiphilic
peptide and/or peptoid may be dissolved at a concentration from
about 0.01 .mu.g/ml to about 100 mg/ml. The amphiphilic peptide
and/or peptoid may be dissolved at a concentration from about 1
mg/ml to about 50 mg/ml. The amphiphilic peptide and/or peptoid may
be dissolved and/or dispersed at a concentration from about 1 mg/ml
to about 30 mg/ml.
[0069] In a fourth aspect the disclosure provides a surgical
implant or stent. The surgical implant or stent includes a peptide
and/or peptoid scaffold. The peptide and/or peptoid scaffold is
defined by a hydrogel according to the second aspect.
[0070] In a fifth aspect the disclosure provides a pharmaceutical
and/or cosmetic composition. The pharmaceutical and/or cosmetic
composition includes the amphiphilic peptide and/or peptoid
according to the first aspect. The pharmaceutical and/or cosmetic
composition may comprise a pharmaceutically active compound. The
pharmaceutical and/or cosmetic composition may comprise a
pharmaceutically acceptable carrier.
[0071] In a sixth aspect the disclosure provides a kit of parts.
The kit includes a first container and a second container. The
first container includes a peptide and/or peptoid according to the
first aspect. The second container includes an aqueous solution.
The aqueous solution of the second container may further comprise a
pharmaceutically active compound. The first container with an
amphiphilic peptide and/or peptoid may further comprise a
pharmaceutically active compound.
[0072] It was an object of the present invention to further improve
the above disclosed hydrogels in terms of their material
properties, such as stiffness, elasticity and resistance to
degradation. It was a further object of the present invention to
facilitate the conjugation of bioactive agents or other compounds
of interest (e.g. nanoparticles) to the hydrogel. Yet another
object was to reduce the amount of amphiphilic peptides and/or
peptoids required for preparing hydrogels as disclosed above.
[0073] The objects of the present invention are solved by a
hydrogel comprising a plurality of amphiphilic peptides and/or
peptoids capable of self-assembling into three-dimensional
macromolecular nanofibrous networks, which entrap water and form
said hydrogel, the amphiphilic peptides and/or peptoids having the
general formula:
Z.sub.p--(X).sub.n--(Y).sub.m-AA.sub.thiol-Z'q,
wherein
[0074] Z is an N-terminal protecting group, X is, at each
occurrence, independently selected from an aliphatic amino acid, Y
is, at each occurrence, independently selected from a hydrophilic
amino acid, AA.sub.thiol is an amino acid comprising a thiol group,
Z' is a C-terminal protecting group, n is an integer selected from
2 to 6, preferably 2 to 5, m is selected from 0, 1 and 2,
preferably 0 and 1, and p and q are independently selected from 0
and 1, wherein, preferably, p is 1, wherein at least a portion of
said plurality of amphiphilic peptides and/or peptoids is
chemically (e.g. covalently) cross-linked.
[0075] In one embodiment, said amino acid comprising a thiol group
is selected from cysteine and homocysteine.
[0076] In one embodiment, said at least a portion of said plurality
of amphiphilic peptides and/or peptoids is chemically cross-linked
via sulfhydryl-to-sulfhydryl cross-linking (i.e. via disulfide
bridges), via sulfhydryl-to-hydroxyl cross-linking, via
sulfhydryl-to-aldehyde cross-linking, via sulfhydryl-to-amine
cross-linking, via peptidoglycans or via photo-induced
cross-linking, preferably via sulfhydryl-to-sulfhydryl
cross-linking. In one embodiment, said N-terminal protecting group
has the general formula --C(O)--R, wherein R is selected from the
group consisting of H, unsubstituted or substituted alkyls, and
unsubstituted or substituted aryls. Preferred alkyls are methyl,
ethyl, butyl, isobutyl, propyl and isopropyl.
[0077] In one embodiment, said N-terminal protecting group is an
acetyl group (R=methyl).
[0078] In one embodiment, said N-terminal protecting group is a
peptidomimetic molecule, including natural and synthetic amino acid
derivatives, wherein the N-terminus of said peptidomimetic molecule
may be modified with a functional group selected from the group
consisting of carboxylic acid, amide, alcohol, aldehyde, amine,
imine, nitrile, an urea analog, thiol, phosphate, carbonate,
sulfate, nitrate, maleimide, vinyl sulfone, azide, alkyne, alkene,
carbohydrate, imide, peroxide, ester, thioester; aryl, ketone,
sulphite, nitrite, phosphonate and silane.
[0079] In one embodiment, said C-terminal protecting group is an
amide group.
[0080] In one embodiment, the C-terminus of said amphiphilic
peptides and/or peptoids has the formula --CONHR or --CONRR', with
R and R' being selected from the group consisting of H,
unsubstituted or substituted alkyls, and unsubstituted or
substituted aryls. Preferred alkyls are methyl, ethyl, butyl,
isobutyl, propyl and isopropyl.
[0081] In one embodiment, said C-terminal protecting group is an
ester group.
[0082] In one embodiment, the C-terminus of said amphiphilic
peptide and/or peptoid has the formula --CO.sub.2R, with R being
selected from the group consisting of H, unsubstituted or
substituted alkyls, and unsubstituted or substituted aryls.
Preferred alkyls are methyl, ethyl, butyl, isobutyl, propyl and
isopropyl.
[0083] In one embodiment, said C-terminal protecting group is a
peptidomimetic molecule, including natural and synthetic amino acid
derivatives, wherein the C-terminus of said peptidomimetic molecule
may be modified with a functional group selected from the group
consisting of carboxylic acid, amide, alcohol, aldehyde, amine,
imine, nitrile, an urea analog, thiol, phosphate, carbonate,
sulfate, nitrate, maleimide, vinyl sulfone, azide, alkyne, alkene,
carbohydrate, imide, peroxide, ester, thioester, aryl, ketone,
sulphite, nitrite, phosphonate and silane.
[0084] In one embodiment, for a given amphiphilic peptide and/or
peptoid, said aliphatic amino acid, said hydrophilic amino acid and
said amino acid comprising a thiol group are either D-amino acids
or L-amino acids.
[0085] In one embodiment, said hydrophilic amino acid has a polar
group which is independently selected from a hydroxyl, an ether, a
carboxyl, an imido, an amido, an ester, an amino, a guanidino, a
thio, a thioether, a seleno, and a telluro group.
[0086] In one embodiment, said hydrophilic amino acid is selected
from the group consisting of aspartic acid, asparagine, glutamic
acid, glutamine, 5-N-ethyl-glutamine (theanine), citrulline,
thio-citrulline, cysteine, homocysteine, methionine, ethionine,
selenomethionine, telluromethionine, threonine, allo-threonine,
serine, homoserine, arginine, homoarginine, ornithine (Om),
2,-diaminobutyric acid (Dab), 2,-diaminopropionic acid (Dap),
lysine and N(6)carboxymethyllysine and histidine.
[0087] In one embodiment, said hydrophilic amino acid is selected
from the group consisting of aspartic acid, asparagine, glutamic
acid, glutamine, serine, threonine, cysteine, methionine, lysine,
ornithine (Om), 2,-diaminobutyric acid (Dab), 2,-diaminopropionic
acid (Dap) and histidine.
[0088] In one embodiment, said aliphatic amino acid is selected
from the group consisting of isoleucine, norleucine, leucine,
valine, alanine, glycine, homoallylglycine and
homopropargylglycine. Preferably, said aliphatic amino acid is
selected from the group consisting of isoleucine, leucine, valine,
alanine and glycine.
[0089] In one embodiment, all or a portion of the aliphatic amino
acids of the amphiphilic peptides and/or peptoids, i.e. (X)n, are
arranged in an order of decreasing amino acid size in the direction
from N- to C-terminus of the amphiphilic peptides and/or peptoids,
wherein the size of the aliphatic amino acids is defined as
I=L>V>A>G.
[0090] In one embodiment, said aliphatic amino acids arranged in an
order of decreasing amino acid size have a sequence which is a
repetitive or non-repetitive sequence. In one embodiment, said
aliphatic amino acids arranged in an order of decreasing amino acid
size have a sequence selected from LIVAG, ILVAG, LIVAA, LAVAG,
LAVAG, LIVA, LIVG, IVA and IV, wherein, optionally, there is an A
preceding such sequence at the N-terminus.
[0091] In one embodiment, said amphiphilic peptides and/or peptoids
undergo a conformational change during self-assembly, preferably a
conformational change from a random coil conformation to a helical
intermediate structure to a final beta conformation.
[0092] In one embodiment, the conformational change is dependent on
the concentration of the amphiphilic peptides and/or peptoids,
dependent on the ionic environment, pH dependent and/or temperature
dependent. In one embodiment, said amphiphilic peptides and/or
peptoids are the same or different. In one embodiment,
(X).sub.u--(Y).sub.m is selected from the group consisting of SEQ
ID NO: 1 to 42. In one embodiment,
(X).sub.u--(Y).sub.m-AA.sub.thiol is selected from the group
consisting of LIVAGKC (SEQ ID NO:43), LIVAGSC (SEQ ID NO: 44),
LIVAGDC (SEQ ID NO: 45), ILVAGKC (SEQ ID NO: 46), ILVAGDC (SEQ ID
NO: 47), LIVAGC (SEQ ID NO: 48), AIVAGC (SEQ ID NO: 49), ILVAGC
(SEQ ID NO: 50), IVKC (SEQ ID NO: 51), IVDC (SEQ ID NO: 52) and
IVSC (SEQ ID NO: 53).
[0093] In one embodiment, the hydrogel is stable in aqueous
solution at ambient temperature for a period of at least 7 days,
preferably at least 2 to 4 weeks, more preferably at least 1 to 6
months.
[0094] In one embodiment, at least 5%, preferably at least 10%,
more preferably at least 15%, more preferably at least 20%, more
preferably at least 25%, more preferably at least 30%, more
preferably at least 35%, more preferably at least 40%, more
preferably at least 45%, even more preferably at least 50% of said
plurality of amphiphilic peptides and/or peptoids are chemically
cross-linked.
[0095] In one embodiment, at least 60% of said plurality of
amphiphilic peptides and/or peptoids are chemically cross-linked.
In one embodiment, the hydrogel is characterized by a storage
modulus G' to loss modulus G'' ratio that is greater than 2.
[0096] In one embodiment, the hydrogel is characterized by a
storage modulus G' from 100 Pa to 400, 00 Pa, preferably 500 Pa to
400, 00 Pa, even more preferably 1000 Pa to 400, 00 Pa, at a
frequency in the range of from 0.02 Hz to 16 Hz.
[0097] In one embodiment, the hydrogel has a higher mechanical
strength than collagen or its hydrolyzed form (gelatine).
[0098] In one embodiment, the hydrogel has an elasticity defined as
% strain at linear viscoelasticity (LVE) limit above 0.01% strain,
preferably above 0.5% strain, more preferably above 1% strain, more
preferably above 2% strain.
[0099] In one embodiment, the hydrogel further comprises a
non-peptidic polymer.
[0100] In one embodiment, the hydrogel further comprises at least
one of a microorganism, a cell, a virus particle, a peptide, a
peptoid, a protein, a nucleic acid, an oligosaccharide, a
polysaccharide, a vitamin, an inorganic molecule, a nano- or
microparticle, a synthetic polymer, a small organic molecule, a
cosmetic agent or a pharmaceutically active compound.
[0101] In one embodiment, said at least one of a microorganism, a
cell, a virus particle, a peptide, a peptoid, a protein, a nucleic
acid, an oligosaccharide, a polysaccharide, a vitamin, an inorganic
molecule, a nano- or microparticle, a synthetic polymer, a small
organic molecule, a cosmetic agent or a pharmaceutically active
compound is entrapped by said three-dimensional macromolecular
nanofibrous networks.
[0102] In one embodiment, said at least one of a microorganism, a
cell, a virus particle, a peptide, a peptoid, a protein, a nucleic
acid, an oligosaccharide, a polysaccharide, a vitamin, an inorganic
molecule, a nano- or microparticle, a synthetic polymer, a small
organic molecule, a cosmetic agent or a pharmaceutically active
compound is coupled to said amphiphilic peptides and/or peptoids,
preferably via a disulfide bridge.
[0103] In one embodiment, said at least one of a microorganism, a
cell, a virus particle, a peptide, a peptoid, a protein, a nucleic
acid, an oligosaccharide, a polysaccharide, a vitamin, an inorganic
molecule, a nano- or microparticle, a synthetic polymer, a small
organic molecule, a cosmetic agent or a pharmaceutically active
compound is coupled to said non-peptidic polymer. In one
embodiment, said pharmaceutically active compound is selected from
the group consisting of haemostatic agents, antibiotics,
anti-microbial agents, anti-fungal agents, anti-inflammatory
agents, analgesics, anti-coagulants, antibodies, antigens, growth
factors and cytokines. In one embodiment, said nano- or
microparticle is a metal nano- or microparticle, preferably a gold
nano- or microparticle.
[0104] In one embodiment, said peptide comprises a signal sequence,
wherein, preferably, said peptide is coupled to said amphiphilic
peptides and/or peptoids via a disulfide bridge.
[0105] In one embodiment, said signal sequence comprises an
adhesion or growth signal, such as an integrin binding sequence
(e.g. CRGD).
[0106] In one embodiment, said hydrogel is provided in an
injectable form and gels in situ. The objects of the present
invention are also solved by a method of preparing a hydrogel,
preferably a hydrogel according to the present invention, the
method comprising the step of dissolving amphiphilic peptides
and/or peptoids, as defined above in connection with the hydrogel
according to the present invention, in an aqueous solution, wherein
said aqueous solution comprises an oxidizing agent or wherein said
method further comprises the step of exposing the ready-made
hydrogel to a solution of an oxidizing agent.
[0107] In one embodiment, said oxidizing agent is H.sub.2O.sub.2,
wherein, preferably, H.sub.2O.sub.2 is used at a concentration from
0.02 to 0.1% (w/w), preferably 0.04 to 0.08% (w/w), more preferably
0.05 to 0.07% (w/w).
[0108] In one embodiment, said amphiphilic peptides and/or peptoids
are dissolved at a concentration from 0.01 .mu.g/ml to 50 mg/ml,
preferably at a concentration from 1 mg/ml to 25 mg/ml, more
preferably at a concentration from 1 mg/ml to 15 mg/ml, even more
preferably at a concentration from 5 mg/ml to 12 mg/ml.
[0109] In one embodiment, the dissolved amphiphilic peptides and/or
peptoids in aqueous solution are further exposed to a temperature
in the range of from 20.degree. C. to 90.degree. C., preferably
20.degree. C. to 70 C, more preferably 20.degree. C. to 40.degree.
C.
[0110] In one embodiment, the dissolved amphiphilic peptides and/or
peptoids in aqueous solution are exposed to said temperature for at
least 1 hour, preferably at least 2 hours, more preferably at least
4 hours, more preferably at least 6 hours, more preferably at least
8 hours, more preferably at least 10 hours, more preferably at
least 12 hours, even more preferably at least 24 hours.
[0111] In one embodiment, the method further comprises the step of
exposing the ready-made hydrogel to an aqueous solution not
comprising said oxidizing agent, wherein, if said method comprises
the step of exposing the ready-made hydrogel to a solution of said
oxidizing agent, said step of exposing the ready-made hydrogel to
an aqueous solution not comprising said oxidizing agent is
performed after said step of exposing the ready-made hydrogel to a
solution of said oxidizing agent.
[0112] In one embodiment, said step of exposing the ready-made
hydrogel to an aqueous solution not comprising said oxidizing agent
is repeated at least once.
[0113] In one embodiment, said step of exposing the ready-made
hydrogel to an aqueous solution not comprising said oxidizing agent
occurs for at least 1 hour, preferably at least 2 hours, more
preferably at least 4 hours, even more preferably at least 6
hours.
[0114] In one embodiment, said step of exposing the ready-made
hydrogel to an aqueous solution not comprising said oxidizing agent
occurs at a temperature in the range of from 30.degree. C. to
45.degree. C., preferably 35.degree. C. to 40.degree. C.
[0115] The step of exposing the ready-made hydrogel to an aqueous
solution not comprising said oxidizing agent is used to remove
unreacted oxidizing agent and/or residual acid from solid-phase
peptide synthesis. In one embodiment, more than 90%, preferably
more than 95%, more preferably more than 97%, even more preferably
more than 99% of the unreacted oxidizing agent (e.g.
H.sub.2O.sub.2) are removed.
[0116] In one embodiment, said aqueous solution not comprising said
oxidizing agent is water or a buffered aqueous solution (e.g.
PBS).
[0117] In one embodiment, said aqueous solution not comprising said
oxidizing agent is a cell culture medium. In one embodiment, the
method further comprises at least one of the steps of: [0118]
adding at least one of a microorganism, a cell, a virus particle, a
peptide, a peptoid, a protein, a nucleic acid, an oligosaccharide,
a polysaccharide, a vitamin, an inorganic molecule, a nano- or
microparticle, a synthetic polymer, a small organic molecule, a
cosmetic agent or a pharmaceutically active compound; [0119] adding
at least one non-peptidic polymer; [0120] adding at least one
gelation enhancer; [0121] adding at least one buffer, preferably at
least one physiologically acceptable buffer.
[0122] In one embodiment, said gelation enhancer is a salt or a
solution of a salt. The objects of the present invention are also
solved by a hydrogel prepared by the method according to the
present invention.
[0123] The objects of the present invention are also solved by the
use of a hydrogel according to the present invention as a cell
culture substrate, preferably a cell culture substrate for 3-D cell
culture.
[0124] The objects of the present invention are also solved by the
use of a hydrogel according to the present invention as a device
for drug or gene delivery, preferably for sustained or controlled
release drug delivery, or as a wound dressing or as an implant or
as an injectable agent that gels in situ.
[0125] The objects of the present invention are also solved by a
cell culture substrate, preferably a cell culture substrate for 3-D
cell culture, comprising a hydrogel according to the present
invention.
[0126] The objects of the present invention are also solved by a
device for drug or gene delivery, preferably sustained or
controlled release drug delivery, comprising a hydrogel according
to the present invention.
[0127] The objects of the present invention are also solved by an
implant or injectable agent or wound dressing comprising a hydrogel
according to the present invention.
[0128] The objects of the present invention are also solved by a
pharmaceutical or cosmetic composition comprising a hydrogel
according to the present invention.
[0129] In one embodiment, the pharmaceutical or cosmetic
composition is provided in the form of a topical gel or cream, a
spray, a powder, or a sheet, patch or membrane.
[0130] In one embodiment, the pharmaceutical or cosmetic
composition is provided in the form of an injectable solution. In
one embodiment, the pharmaceutical or cosmetic composition further
comprises a pharmaceutically active compound.
[0131] In one embodiment, the pharmaceutical or cosmetic
composition further comprises a pharmaceutically acceptable
carrier.
[0132] The objects of the present invention are also solved by a
hydrogel according to the present invention for use in regenerative
medicine or for use in tissue engineering and tissue regeneration,
e.g. regeneration of adipose and cartilage tissue.
[0133] The objects of the present invention are also solved by a
hydrogel according to the present invention for use in the
treatment of wounds.
[0134] The objects of the present invention are also solved by a
hydrogel according to the present invention for use in the
treatment of degenerative diseases of the skeletal system, e.g.
degenerative disc disease, or urinary incontinence.
[0135] The objects of the present invention are also solved by a
hydrogel according to the present invention for cosmetic use.
[0136] The objects of the present invention are also solved by an
electronic device comprising a hydrogel according to the present
invention.
[0137] In one embodiment, the electronic device is selected from a
fuel cell, a solar cell, an electronic cell or a biosensing
device.
[0138] The objects of the present invention are also solved by a
method of tissue regeneration or tissue replacement comprising the
steps: [0139] a) providing a hydrogel according to the present
invention; [0140] b) exposing said hydrogel to cells which are to
form regenerated tissue; [0141] c) allowing said cells to grow on
or in said hydrogel.
[0142] In one embodiment, the method is performed in vitro or in
vivo or ex vivo.
[0143] In one embodiment, the method is performed in vivo, wherein,
in step a), said hydrogel is provided at a place in the body of a
patient where tissue regeneration or tissue replacement is
intended. In one embodiment, said tissue is selected from the group
comprising skin tissue, nucleus pulposus in the intervertebral
disc, cartilage tissue, synovial fluid and submucosal connective
tissue in the bladder neck. In one embodiment, said step a) is
performed by injecting said hydrogel or a solution of amphiphilic
peptides and/or peptoids as defined above in connection with the
hydrogel according to the present invention at a place in the body
of a patient where tissue regeneration or tissue replacement is
intended. In one embodiment, said step a) further comprises the
co-injection of a gelation enhancer, preferably of a solution of a
salt, and/or the co-injection of an oxidizing agent.
[0144] In one embodiment, the method is performed ex vivo, wherein,
in step a) or b), cells from a patient or from a donor are mixed
with said hydrogel, and the resulting mixture is provided at a
place in the body of a patient where tissue regeneration or tissue
replacement is intended.
[0145] In one embodiment, said hydrogel comprises one or more
bioactive therapeutics that stimulate regenerative processes and/or
modulate the immune response.
[0146] Other aspects and features of the present invention will
become apparent to those skilled in the art upon review of the
following description of specific embodiments of the invention in
conjunction with the accompanying FIGS. 1K-M and 17 to 40.
BRIEF DESCRIPTION OF THE DRAWINGS
[0147] Reference is now made to the figures, wherein:
[0148] FIGS. 1A to 1M represent a sorted list of some exemplary
peptides capable of forming hydrogels. These peptides are
embodiments in which the entire peptide consists of a single linear
amphiphilic sequence. Peptides which are forming hydrogels are
named with a short code, but their individual sequence is
disclosed. The peptides of these examples consist of a sequence of
natural amino acids containing 3 to 7 amino acids. The N-terminus
is acetylated which removes the charge that would otherwise
restrain the amphiphilic character of the peptides.
[0149] FIG. 2 depicts gelation pictures for peptide based hydro
gels at lowest concentrations.
[0150] FIG. 3 depicts gelation pictures for Ac-AS-6 (Ac-AIVAGS) (L)
at concentrations of 5 mg/ml, 10 mg/ml, 15 mg/ml.
[0151] FIG. 4 depicts a hypothesis of self-assembly from peptide
monomers to supramolecular network of condensed fibers. (A)
Assembly is believed to initiate with antiparallel pairing of two
peptide monomers by changing to a-helical conformations.
Subsequently, peptide pairs assemble to fibers and nanostructures.
Condensation of peptide fibers to fiber aggregates results in
hydrogel formation.
[0152] FIGS. 5A-C depict environmental scanning electron microscopy
(ESEM) images of hydrogels of Ac-LD6 (Ac-LIVAGD) (L) (10 mg/ml),
where FIG. 5A, FIG. 5B and FIG. 5C are images obtained at
magnification of 260.times., 1000.times., 2000.times., 2400.times.,
4000.times. at a temperature of 4.degree. C. with HV at 10 KV. The
images indicate the formation of fibrous structures.
[0153] FIGS. 6A-D show field emission scanning electron microscopy
(FESEM) images of hydrogels of Ac-LD6 (Ac-LIVAGD) (L) (15 mg/ml),
where FIGS. 6A-D are images obtained at magnifications of
6000.times., 45000.times., 45000.times. and 40000.times. with HV at
10 KV.
[0154] FIGS. 7A-B depict field emission scanning electron
microscopy (FESEM) images of Ac-AD6 (Ac-AIVAGD) (D) hydrogels (20
mg/ml) at a magnification of 50.times. (FIG. 7A) and 20000.times.
(FIG. 7B) at 12 KV.
[0155] FIGS. 8A-B show field emission scanning electron microscopy
(FESEM) images of hydrogels of Ac-AD6 (Ac-AIVAGD) (D) (20 mg/ml)
obtained at 120.times. (FIG. 8A), and 450.times. (FIG. 8B).
[0156] FIG. 9A shows the morphology and structure evaluation of the
peptide scaffolds as determined by field emission scanning electron
microscopy (a-f). (a) A honeycomb porous structure is observed
following lyophilization of 20 mg/mL Ac-AD.sub.6 (Ac-AIVAGD) (D)
hydrogel. The pores are bounded by membranes of condensed fibers as
shown in close-up views of 15 mg/mL (b) and 20 mg/mL (c)
Ac-ID.sub.3 (Ac-IVD) (L) hydrogels. Further magnification of 20
mg/mL Ac-AD.sub.6 (L) hydrogel revealed single fibers (d, e). At
lower concentrations, 0.1 mg/mL Ac-LD.sub.6 (Ac-LIVAGD) (L),
nanostructures are observed (f).
[0157] FIG. 9B shows an image obtained at a magnification of
1000.times., HV of 12 KV, FIG. 9C obtained at a magnification of
2500.times., HV of 12 KV, FIG. 9D obtained at a magnification of
4000.times., HV of 10 KV, FIG. 9E obtained at a magnification of
35000.times., HV of 10 KV, FIG. 9F at a magnification of
80000.times., HV of 5 KV, FIG. 9G obtained at a magnification of
120000.times., HV of 10 KV, and FIG. 911 at a magnification of
200000.times., HV of 10 KV.
[0158] FIG. 10A shows Far-UV CD spectra demonstrating that with
increasing concentration there is the transition of Ac-LD.sub.6
(Ac-LIVAGD) peptide conformation from random coil (below threshold
concentration) to .alpha.-helical (222 and 208 nm peaks) and
further (3-type (negative band at 218 nm) structures. Heating the
samples to facilitate gelation increased the (3 type aggregation.
FIG. 10B Below threshold concentration, the random coil
conformations of 0.2 mg/mL Ac-LD.sub.6 were reversibly affected by
step-wise temperature increases (solid lines) from 25.degree. C. to
90.degree. C. and cooling (dotted lines). FIGS. 10C and 10D Above
the threshold concentration in 1 mg/mL Ac-LD.sub.6 gel, stepwise
temperature increases FIG. 10C stabilized the (.beta.-type
structures irreversibly, such that subsequent cooling FIG. 10D did
not alter the CD spectra. FIG. 10E Far-UV CD spectra of AcID.sub.3
(Ac-IVD) at different concentrations. All curves were done at
25.degree. C.
[0159] FIGS. 11A and 11B show rheological measurements. The high
mechanical strengths of different peptide hydrogels at 20 mg/mL
concentration was determined by measuring storage moduli (G') as a
function of angular frequency under 0.1% strain, at 25.degree. C.
and 50.degree. C. respectively. The gels demonstrate good thermal
stability compared to gelatin, which liquidified at 50.degree. C.
(hence excluded in 4B). FIG. 11C Mechanical strength is a function
of concentration, as determined from oscillatory frequency sweep
studies using Ac-LD.sub.6 (Ac-LIVAGD) (L) under 0.1% strain at
25.degree. C. FIG. 11D Increasing salt concentration (NaCl)
decreases G', reducing the rigidity of 10 mg/mL Ac-LD.sub.6 (L)
hydrogels, demonstrating the tuneability and reversibility of
gelation.
[0160] FIGS. 12A-D show further examples of a rheology measurements
for peptide based hydrogels. FIG. 12A and FIG. 12B depict
oscillatory amplitude sweep studies at temperatures of 25.degree.
C. and 50.degree. C. for Ac-AD6 (Ac-AIVAGD) (L) and Ac-AD6 (D) at a
concentration of 20 mg/ml with a constant frequency of [1 rad-s]
and a gap of 0.8 mm. The graphs indicate the plot of moduli [Pa]
versus strain (%) at temperatures of 25.degree. C. and 50.degree.
C. The linear viscoelastic range was observed at 0.07% to 0.2
strain % at temperatures of 25.degree. C. and 50.degree. C. FIG.
12C and FIG. 12D depict oscillatory frequency sweep Studies at
temperatures of 25.degree. C. and 50.degree. C. for Ac-AD6(L) and
Ac-AD6(D) at a concentration of 20 mg/ml with varying frequency
ranges from 0.1 to 100 [Rad/s] with a constant strain [%] of 0.1%
linear viscoelastic range and a gap of 0.8 mm.
[0161] FIG. 13 shows a further example of a rheology measurement
for peptide based hydrogels. Depicted is a frequency sweep study of
a UV cross-linked peptide at a temperature of 25.degree. C. with
0.1% strain.
[0162] FIG. 14 depicts rheology measurements for gelatin-1890 (type
A, porcine skin). This figure shows moduli data obtained at
25.degree. C. when applying different frequencies.
[0163] FIGS. 15A-D illustrate the biocompatibility of peptide-based
hydrogels of the invention using further cell lines. FIG. 15A shows
a microscopy image of human primary renal tubule cells (HPRTC)
after 72 hours after seeding on a hydrogel of Ac-LD.sub.6
(Ac-LIVAGD) (L) in DMEM medium, grown at optimum conditions. FIG.
15B shows microscopy images of human primary renal tubule cells
(HPRTC) after 72 hrs after seeding on tissue culture plastic, grown
at optimum conditions. FIG. 15C shows microscopy images of human
umbilical vein endothelial cells (HUVEC) after 72 hrs after seeding
on gels of Ac-LD.sub.6 (L) in DMEM medium, grown at optimum
conditions. FIG. 15D shows microscopy images of human umbilical
vein endothelial cells (HUVEC) after 72 hrs after seeding on tissue
culture plastic, grown at optimum conditions.
[0164] FIGS. 16A-B are further illustrations on the viability of
cells in presence of a hydrogel of the invention. Human fibroblast
cells were cultured in the presence (FIG. 16A) and absence (FIG.
16B) of Ac-LD.sub.6 (Ac-LIVAGD) (L) (5 mg/ml). Fluorescein
isothiocyanate (FITC) stained cells (left panels), Texas red
stained cells (center panels) and cells stained with both FITC and
Texas red (right panels) are shown.
[0165] Embodiments of the invention will now be described by way of
example with reference to the following figures, in which:
[0166] FIG. 17 shows the crosslinking strategy of the present
invention using (A) disulfide bridges between two thiol-containing
amino acids (here: two cysteines) which introduce (B) chemical
intra- and inter-fiber crosslinks among LK.sub.6C peptide fibers.
The thiol groups further facilitate the conjugation of bioactive
agents, such as the integrin binding sequence CRGD (C).
[0167] FIG. 18 at A illustrates the determination of a suitable
gelation concentration with the LK.sub.6 control peptide sequence.
A working concentration was established that was suitably low so as
to save on material during testing but yet, capable of forming gels
strong enough to be manipulated. LK.sub.6 gels were therefore
casted at different concentrations and their stiffness was measured
(average.+-.s.d. of triplicates). As expected, G' values increased
with peptide concentration and it was determined that a working
concentration of 12 mM afforded gels with sufficient mechanical
integrity to withstand handling. FIG. 18 at B shows that gels can
be formed with 12 mM of LK.sub.6C (.about.10 mg/mL after accounting
for an amino acid content of 89.4%) and that gel formation is
compatible with various aqueous media.
[0168] FIG. 19 shows the kinetics of disulfide formation when
LK.sub.6C was subjected to oxidation by air, as compared to
H.sub.2O.sub.2-assisted oxidation+/-HRP (average.+-.s.d. of
duplicates). For oxidation by air, LK.sub.6C dissolved in water at
12 mM was left in capped micro-centrifuge tubes at room
temperature. As can be seen, kinetics of disulfide formation was
sluggish and .about.80% of thiols still remained after 5 days, as
determined using an Ellman's assay. H.sub.2O.sub.2-assissted
oxidation was much more efficient in the formation of disulfide
bridges. Interestingly, horse-radish peroxidase (HRP, 0.6 U/mL), an
enzyme commonly used with H.sub.2O.sub.2 to boost oxidation
efficiency, did not significantly increase the rate of disulfide
formation.
[0169] FIG. 20 shows representative UPLC chromatograms where the
area under the peaks were used to follow the rate of disulfide
formation in H.sub.2O.sub.2-assisted oxidation. 12 mM LK.sub.6C
dissolved in water containing 0.06% H.sub.2O.sub.2 was used, and
the disulfide peak at .about.3.6 min gradually increased. Only
.about.50% of thiols remained after 1 day. The R.sup.2 value of the
thiol calibration curve was 1.000.
[0170] FIG. 21 shows mass spectra corresponding to the a) LK.sub.6C
monomer-thiol and b) (LK.sub.6C).sub.2 dimer-disulfide peaks in the
UPLC elution profile. In both cases, the expected masses were
detected and verified the assignment of peaks.
[0171] FIG. 22 shows the effect of the H.sub.2O.sub.2 concentration
on the rate of disulfide formation. LK.sub.6C was incubated with
different concentrations of H.sub.2O.sub.2 for eight hours at
25.degree. C. before UPLC analysis. The area under the thiol peaks
were then normalised to that at 0 hour to give the % of thiol
remaining. Before that, a calibration curve had been generated with
pure LK.sub.6C as standard (R.sup.2=0.999). As expected, a higher
concentration of H.sub.2O.sub.2 increased the kinetics of disulfide
formation. However, as a compromise between oxidation efficiency
and H.sub.2O.sub.2-associated toxicity, 0.06% H.sub.2O.sub.2 was
chosen for subsequent experiments.
[0172] FIG. 23 illustrates two different methods to effect
cross-linking: 1) cast-and-soak: gel is first formed in water and
then soaked in a H.sub.2O.sub.2 solution for the desired amount of
time. 2) in situ oxidation: LK.sub.6C peptide powder is dissolved
directly in water containing H.sub.2O.sub.2 to form the gel.
[0173] FIG. 24 shows that oxidation dramatically improved the
ability of the gels to retain their shapes, as seen in panels A to
D: (A) LK.sub.6C gel casted at the start of experiment which was
either (B) not oxidised and soaked in water for 24 hours, or (C)
oxidised for two hours and soaked in water for 96 hours. (D) The
control peptide sequence, LK.sub.6, did not survive the 24-hour
water soak.
[0174] FIG. 25 and FIG. 26 show that the hydrogel was uniformly
oxidised in the cast-and-soak method. In the cast-and-soak method,
LK.sub.6C was dissolved in water, casted overnight in a ring mould
before being soaked in a solution containing H.sub.2O.sub.2 for the
desired amount of time (FIG. 25A). To investigate if oxidation was
uniform throughout the bulk or enriched only in the surface layers,
the gel was removed from the H.sub.2O.sub.2 solution after two
hours and carefully separated into its surface (top, bottom and
circumference) and core fractions using a surgical blade (FIG. 25B
and FIG. 26A). The amount of H.sub.2O.sub.2 in the respective
fractions was quantified using the PeroXOquant H.sub.2O.sub.2 assay
kit (Pierce, Ill., USA), according to the manufacture's
recommendations (average.+-.s.d. of triplicates). Samples were
always diluted such that absorbance readings fell within the linear
portion (R.sup.2=0.999) of the calibration curve obtained with
H.sub.2O.sub.2 standards (FIG. 25C). The gel fractions were also
analysed using UPLC (FIG. 26B; average.+-.s.d. of duplicates). To
normalise for any differences in fiber density between the surface
and core layers, the area ratio between the disulfide and thiol
(D/T) peaks was taken to indicate the extent of oxidation that had
occurred in the fractions. As can be seen, the amount of
H.sub.2O.sub.2 detected in both the fractions was comparable,
suggesting that the diffusion of small H.sub.2O.sub.2 molecules was
extremely efficient. Compared to without oxidation, the D/T ratio
of both layers was, as expected, significantly elevated after
oxidation. However, the D/T ratio of the surface and core layer was
insignificantly different after oxidation, indicating that the gel
was uniformly oxidised in the cast-and-soak method.
[0175] FIG. 27 illustrates the rheological properties and the
effects of oxidation on the microstructure and ability of LK.sub.6C
gels to retention their shape. The (A) stiffness and (B) elasticity
of LK.sub.6C gels were measured after various oxidation regimes
(average.+-.s.d. of triplicates). Generally, elasticity increased
whereas stiffness was either maintained or increased after the
introduction of S--S bonds.
[0176] FIG. 28 illustrates the rheological properties of oxidized
hydrogels. 12 mM LK.sub.6C gels were subjected to various oxidation
regimes with 0.06% H.sub.2O.sub.2 and various durations of water
soak before their (A) stiffness and (B) elasticity were measured
(average.+-.s.d. of triplicates). Without oxidation, LK.sub.6C gels
could maintain their stiffness after two hours of water soak but
broke down and lost their stiffness after 24 hours of water soak.
On the other hand, gels subjected to 2 or 24 hours of cast-and-soak
oxidation, or 22 hours of in situ oxidation maintained their shapes
and in fact, became stiffer after up to four days of water soak.
Presumably, the introduction of chemical S--S bonds was important
in helping to keep the peptide fibers together and increase the
ability of the gels to maintain their shape and stiffness. Compared
to gels without oxidation, the increase in elasticity of the
oxidised gels was also maintained after up to four days of water
soak.
[0177] FIG. 29 shows the effects of concentration and LK6-doping on
the rheological properties of LK.sub.6C. LK.sub.6C was dissolved in
water at 12 or 13. 5 mM and casted overnight in a ring mould. The
(A) stiffness and (B) elasticity of the gels were then measured
(average.+-.s.d. of triplicates). Increasing the concentration of
LK.sub.6C increased the gel stiffness significantly but only
resulted in a modest increase in elasticity. (C-D) LK.sub.6C was
also mixed with varying amount of LK6 to a final concentration of
12 mM in water and casted overnight in a ring mould. The "%" values
presented above refer to the proportion of LK.sub.6C in the
formulation. The gels were then soaked in a 0.06% H.sub.2O.sub.2
solution for two hours (i.e., cast-and-soak) before their (C)
stiffness and (D) elasticity were measured (average.+-.s.d. of
triplicates). As can be seen, pure LK.sub.6C (100%), pure LK6 (0%)
and LK.sub.6C/LK6 75/25 (75%) gels were relatively stiff. Gels
containing .ltoreq.50% LK.sub.6C, however, broke down before/during
handling, resulting in low G' values. These suggest that the window
to dope LK.sub.6C with LK.sub.6 (at a final concentration of 12 mM)
while still maintaining gel stiffness, lies between 75-50% of
LK.sub.6C. Gels containing oxidised LK.sub.6C were more elastic
than pure LK.sub.6 gels. However, the difference in elasticity
between gels containing 75% and 100% of LK.sub.6C was only
modest.
[0178] FIG. 30 depicts FESEM images showing that the fibrous
microstructure of LK.sub.6C was maintained after oxidation.
Freeze-dried gels were deposited onto carbon tapes, sputtered with
platinum and observed under a JSM-7400F electron microscope (Jeol,
Tokyo, Japan).
[0179] FIG. 31 illustrates the purification of hydro gels before
their use in cell culture. LK.sub.6C was dissolved in 200 .mu.L of
water containing 0.06% H.sub.2O.sub.2 and casted directly into a
48-well plate for 22 hours at 25.degree. C. 1 mL of water was then
added on top of the gel and replaced at regular time intervals to
leach out (A) unreacted H.sub.2O.sub.2 and (B) residual H.sup.+
from solid-phase peptide synthesis (average.+-.s.d. of duplicates
at least). The amount of H.sub.2O.sub.2 in the supernatant was
quantified as before and plotted with respect to time. The total
amount of H.sub.2O.sub.2 at the start of experiment was obtained
by: sum of all removed H.sub.2O.sub.2 in the supernatants+whatever
amount of H.sub.2O.sub.2 remaining in the gel at the end of
experiment. The amount of H.sup.+ removed was determined by
measuring the pH of the supernatant with respect to control wells
with no gel (only water). Since pH is proportional to the negative
logarithm of the activity of H.sup.+ ions, the amount of H.sup.+
removed from the gel can be calculated with respect to the control
wells, which account for natural air acidification. Experiment was
stopped when pH of supernatant matched that of the control wells
and cumulative activity of H.sup.+ removed was plotted as a
function of time. As can be seen, >96% of H.sub.2O.sub.2 was
removed after 4 hrs and >99% after 7 hrs. Similarly, >85% of
residual acid was removed after 8 hrs. We however note that as the
purification efficiency is diffusion controlled, the plots above
are only valid for the reported frequency of water change. A more
frequent regime of water change or using a buffered media (e.g.,
growth media or PBS) instead of water is expected to give even
better results.
[0180] FIG. 32 illustrates the gradual and tunable release kinetics
of the hydro gels according to the present invention. LK.sub.6C
containing 1 mg/mL of dextran-dye (10 kDa) was first casted in
48-well plates and incubated overnight. The hydrogel was either not
oxidized or oxidized for 2 hrs before water was introduced at room
temperature. Water was then extracted to assay for dextran release
from the hydrogel (R.sup.2 of calibration curve was 0.999). The
release profile showed no burst release, but a gradual release up
till 28 days. The release rate of the oxidized hydrogel was lower,
presumable due to its greater stability in water.
[0181] FIG. 33 shows the chemical conjugation of CRGD onto
LK.sub.6C fibers via cysteine-mediated disulfide bonds. CRGD was
mixed with LK.sub.6C in the presence of 0.06% H.sub.2O.sub.2 and
casted in a ring mould at 25.degree. C. for 22 hours. (A) UPLC
later revealed that there were three main peaks in the chromatogram
which could be assigned, based on their respective MS, to: (B)
unreacted LK.sub.6C monomers, (C) (LK.sub.6Ch dimers and (D)
disulfide-linked LK.sub.6C-CRGD conjugates. All peaks have been
colour-coded to facilitate interpretation. Earlier experiments also
determined that unreacted CRGD monomers or (CRGD).sub.2 dimers
eluted at .about.1.1 min (data not shown), both of which were not
observed here (see magnified inset of A). The integrin-recognition
motif, CRGD was therefore successfully conjugated onto LK.sub.6C
fibers. The conjugation of the CRGD motif is only intended to
illustrate the versatility of this platform and the simplicity of
reaction conditions. Future conjugations need not be limited to
this sequence.
[0182] FIG. 34 shows the effects of 3D cell culture and the
incorporation of RGD on the viability and spreading of HepG2 cells.
(A) En face views of viable cells stained with calcein cultured on
regular 2D surfaces for four days, compared to cells in
LK.sub.6C+/-RGD gel culture. All images, except for the 2D culture,
were mergers of several z-stacks. (B) Cells were viable, as evident
from the positive calcein signals, after four days of culture on
the RGD functionalised, crosslinked and purified LK.sub.6C gels. 3D
distribution was also achieved, as con-31 firmed by cross-sectional
slices which revealed multi-layered cell growth. The presence of
RGD increased the viability of cells, as visually suggested in (A)
and corroborated by (C) the MTT assay. (D) In the case of HepG2
cells, LK.sub.6C gel culture in the presence or absence of RGD had
insignificant effects (p>0.05) on cell spreading compared to
those in regular 2D culture. (C-D) Average.+-.s.d. of triplicates
at least.
[0183] FIG. 35 illustrates the 3D primary cell culture and their
cell-spread area. (A) Fibroblasts isolated from the cornea/sclera
of New Zealand White rabbits were maintained in regular completed
DMEM and grown in 2D culture or purified LK.sub.6C gels+/-various
concentrations of CRGD. Cells were incubated for four days before
they were stained with calcein AM for confocal imaging. Images from
gel cultures were mergers of several z-stacks. As observed, viable
fibroblasts were seen in all cases. In the case of 2D culture, it
was repeatedly observed that the cells were heterogeneously
distributed throughout the well area, i.e., the middle of the well
was sparsely populated while the edges were confluent. Cells in gel
culture, on the contrary, were evenly distributed across the gel.
We note that seeding of cells was done on the same day using the
same methods and most probably did not account for the in
homogeneous distribution of cells. (B) Images taken at a higher
magnification were then analysed using the ImageJ software to
quantify the average cell-spread area of the fibroblasts
(Average.+-.s.d. of triplicates at least). The rabbit fibroblasts
spreaded more in 2D culture compared to those in the various gel
cultures (ANOV A, p<0.05). There were no significant differences
in terms of cell-spread area between gels with or without CRGD in
them.
[0184] FIG. 36 shows confocal images (40.times. magnification) of
HepG2 cells either cultured on LK.sub.6C/CRGD gels or on regular 2D
wells. In the former, the image presented was a merger of several
z-stacks. Cells were stained with calcein AM and Hoechst
(Invitrogen, Singapore) prior to confocal observations. The images
from at least two independent locations were then processed with
the ImageJ software to quantify the average spreading area of
cells.
[0185] FIG. 37 shows the rheological properties of gels of
LK.sub.6C (10 mg/mL) with or without CRGD (1 mg/mL) were casted
overnight in the presence of 0.06% H.sub.2O.sub.2 before their (A)
stiffness and (B) elasticity were quantified with oscillation
rheometry (Average.+-.s.d. of triplicates). Upon CRGD conjugation,
the gel maintained their stiffness but became less elastic.
[0186] FIG. 38 shows that 3T3 murine fibroblasts cultured in 3D in
LK.sub.6C+CRGD hydrogels remained viable for at least 21 days.
Cells were stained with calcein AM prior to confocal
microscopy.
[0187] FIG. 39 illustrates the surgical procedure used for testing
LK.sub.6C+CRGD hydrogels in the treatment of wounds. (1) Hair was
removed, and the area for surgical removal was marked. Both the
epidermis and dermis were removed to simulate injury. The removed
skin flap was killed by a repeated cycle of freezing and thawing.
(2) Mouse with open wound. (3) LK.sub.6C+CRGD hydrogels with or
without 3T3 fibroblast cells were placed onto wound and the killed
skin flap was sutured back to act as a bandage dressing. (4) Mouse
after skin flap dressing was sutured back.
[0188] FIG. 40 shows that hydrogel treatment promotes
vascularisation in wounds (see red arrows). Preliminary data
obtained with H&E stained slides revealed that the
gel+3T3+dressing group has the most significant re-epithelisation
and regeneration of a thicker dermis (data not shown).
DETAILED DESCRIPTION OF THE INVENTION
[0189] The inventors have previously described short peptide
sequences (3-7 residues) capable of self-assembly into helical
fibers that ultimately result in hydrogel formation. While these
peptides have good mechanical properties and can entrap large
amount of water, they rely purely on physical forces to keep the
interwoven fiber network together. Consequently, some peptide
sequences--one example of which is LIVAGK, or abbreviated to
LK6--are typically used at rather high concentrations to ensure the
integrity and stability of the gel in aqueous solution. This has
contributed significantly to cost.
[0190] The present invention as defined above offers significant
improvements in material properties such as stiffness, elasticity
and resistance of the gel to degradation compared to previous
disclosures-ultra-small peptide-based and peptide/polymer-based
hydrogels. Reactive chemical groups are also introduced to the
peptide fibers which allow the facile in-situ chemical conjugation
of biological signals. Furthermore, compared to previous
formulations, less material is now needed for gelation (e.g. 10
mg/ml LK.sub.6C instead of 25 mg/ml for LK6), which translates into
significant cost savings and higher water contents. This class of
material is suitable for biomedical applications, for example but
not limited to, three-dimensional cell culture, tissue engineering,
sensing and drug and gene delivery.
[0191] The present invention is based on the introduction of
chemical bonds to crosslink the peptide fibers, resulting in gels
with increased stiffness, elasticity and resistance to degradation.
According to the present invention chemical crosslinks are
introduced by the modification of the peptide sequence with a
cysteine residue at the C-terminus, e.g., LIVAGKC (or LK.sub.6C).
This way, the pure amino acid composition of the gel is still
conserved, which makes it, in general, a biocompatible material.
Three-dimensional gels have been casted using these cross-linkable
peptides in water or cell culture medium, such as growth medium
completed with FBS/Penicillin/Streptomycin, and cargoes have been
encapsulated within the matrix. Crucially, on top of physical self
assembly forces, inter- and intra-fiber chemical bonds (in
particular, disulfide bonds between cysteine or homocysteine
residues which each has a thiol group) are now introduced. These
chemical bonds can be encouraged in the presence of an oxidising
agent like hydrogen peroxide (H.sub.2O.sub.2). Alternatively,
chemical bond (e.g. disulfide) formation can also be encouraged in
the oxidative blood environment when used in vivo. The formation of
crosslinks significantly increases the elasticity of the gel due to
the presence of additional chemical bonds. Another advantage of
introducing crosslinks includes the increased resistance of the gel
to degradation due to stronger interactions within and between
fibers. This also means that less material is needed to obtain gels
with mechanical properties comparable or even superior to earlier
formulations, which in turn translates into significant cost
savings and higher water contents. The present invention also
allows the tuning of the physical properties of the hydrogels
(stiffness, elasticity etc.) by modifying the oxidation
cross-linking regime. The presence of reactive chemical groups also
enables the functionalization of the gel with cargos or adhesive
signals (e.g., CRGD) that further increase the biocompatibility of
the gel. Therefore, cargos can now be covalently attached to the
gel matrix besides simple physical encapsulation. The conjugation
procedure can furthermore be conveniently done in situ, while the
gel is being formed. According to the present invention, only mild
conditions for the hydrogen peroxide-assisted oxidation process are
used. Importantly, the use of horse-radishperoxidase (HRP)
prescribed in many other oxidation protocols can be avoided. This
further reduces cost and facilitates regulatory approval in the
future. Moreover, the inventors have devised a method to remove
most of the unreacted hydrogen peroxide (>99%) and residual acid
from solid-phase peptide synthesis before the introduction of e.g.
cells onto the gels. The release profile of cargoes encapsulated
within the gel can also be tuned by modifying the oxidation
strategy.
[0192] The hydrogels according to the present invention are
non-allergenic and non-toxic. The inventors also showed the
biocompatibility of the hydrogels by successfully culturing several
cell types on purified and crosslinked LK.sub.6C-CRGD gels. They
also managed to show the 3D distribution of cells within
LK.sub.6C-CRGD hydrogels. More particularly, cells were seeded on
the gel and stained with the fluorescent live cell marker calcein.
By obtaining a vertical cross sectional slice of the hydrogel and
directly imaging the penetration depth profile of the cells, they
observed multi-layered cell growth. This confirmed the infiltration
of cells into the gel and convincingly demonstrated the 3D cell
growth environment.
[0193] Potential applications of the hydrogels according to the
present invention include: [0194] (Injectable) application for
tissue engineering, particularly in orthopaedic and aesthetic
surgery applications. H.sub.2O.sub.2 may even be avoided altogether
as the overall redox potential of the blood is oxidative (while the
intracellular overall redox potential is reductive). Thus,
disulfide bonds will be naturally formed and in the process and,
thus, increase the long-term stability of gel, which is already an
improvement over current formulations. [0195] 3D cell culture
substrate. Adhesion or growth signals can be covalently attached to
increase biocompatibility. Such 3D cell culture substrate may, for
example, be used as 3D cancer model. [0196] Skin Grafting. Using
e.g. an LK.sub.6C-based hydrogel as a scaffold, first fibroblasts
are embedded within the bulk of the gel, followed by seeding of
keratinocytes onto the surface of the gel to mimic the dermis and
epidermis, respectively, of the human skin. This artificial skin
layer can then be explored for grafting applications. [0197]
Scaffold for corneal endothelial transplantation. The endothelium
of a human cornea is a monolayer and is critical to the maintenance
of vision. Handling of the endothelium is therefore technically
challenging and can influence the success of a corneal endothelial
transplant. Hydrogels according to the present invention, such as
those based on LK.sub.6C, could be used as a matrix support, on
which corneal endothelial cells are cultured, and which can then be
used as an endothelium replacement for transplants. [0198] Cargo
delivery. Drugs and nanoparticles, e.g., EI/DNA complexes can be
encapsulated for gradual release. Physical concentration of cargo
in the vicinity of cells. Less cargo may be needed to achieve
similar effects (compared to dilution of particles by dripping
directly into growth medium), reducing on cost and toxicity. [0199]
Surface adhesion of cargoes, e.g., DNA solution can first be
layered on top of gel. Electrostatic interactions can hold DNA on
surface of gel before DNA solution removed and cells cultured over
gel. In this case, localised transfection is achieved. [0200]
Attachment of particles, e.g., gold particles for application in
sensing or other biodevices. [0201] Wound treatment. Advantages of
using hydro gels according to the present invention for the
treatment of wounds include: the hydrogel keeps the wound moist; it
provides a scaffold for the regenerating skin layers; therapeutics
can be loaded into the gel to aid recovery.
[0202] Disclosed herein is a novel class of hydrogels comprising
hydrogel-forming peptides/peptoids derived from inter alia natural
amino acids. These peptides/peptoids are small amphiphilic peptides
with a hydrophobic portion of aliphatic amino acids and,
preferably, one or two polar amino acids. The peptides/peptoids
(typically 3-7-mers) are typically in the L- or D-form and can self
assemble into supramolecular fibers which are organized into
mesh-like structures. The hydrogels are generally characterized by
a remarkable rigidity and are biocompatible and non-toxic.
Depending on the peptide/peptoid sequence these hydrogels can show
thermoresponsive or thixotropic character. By selecting the peptide
assembling conditions the thickness and length of the fibers can be
controlled. The rigid hydro gels can be used for cultivation of a
variety of primary human cells, providing peptide scaffolds that
can be useful in the repair and replacement of various tissues.
Also disclosed is the procedure of preparing these hydrogels.
Disclosed is further the use of respective hydro gels in
applications such as cell culture, tissue engineering, plastic
surgery, drug delivery, oral applications, cosmetics, packaging and
the like as well as for technical applications, as for example for
use in electronic devices which may include solar or fuel
cells.
[0203] Also disclosed herein is an amphiphilic peptide and/or
peptoid capable of forming a hydrogel, i.e. a polymer network in
which water is the dispersion medium. The amphiphilic peptide
and/or peptoid includes one or more linear amphiphilic sequences,
each having a polar and anon-polar portion. For sake of simplicity
explanations are in the following to a large extent focused on
amphiphilic peptides and/or peptoids that consist of a single
respective linear sequence. In these explanations a respective
peptide and/or peptoid is denominated a "linear peptide and/or
peptoid". Respective explanations apply to any linear sequence,
which may also be included in an amphiphilic peptide and/or peptoid
with a plurality of these linear sequences. Each of these linear
sequences is individually selected. In some embodiments an
amphiphilic peptide and/or peptoid disclosed herein includes
several linear amphiphilic sequences, each of them differing from
any other of the linear amphiphilic sequences. In some embodiments
an amphiphilic peptide and/or peptoid disclosed herein includes
several identical linear amphiphilic sequences. In one embodiment
an amphiphilic peptide and/or peptoid disclosed herein includes a
plurality of linear amphiphilic sequences, each linear amphiphilic
sequence being identical to each other linear amphiphilic
sequence.
[0204] Also disclosed is a peptide and/or peptoid that includes o
amphiphilic linear sequences. The symbol o represents an integer
selected in the range from 1 to about 25, such as from 1 to about
20, from 1 to about 18, from 1 to about 15, from 1 to about 12,
from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to
about 5 from 1 to about 4 or from 1 to about 3.
[0205] In some embodiments these amphiphilic linear sequences are
linked in a consecutive manner, thereby defining a linear portion
of the peptide and/or peptoid.
[0206] In some embodiments the peptide and/or peptoid has a
backbone with one or more branches. In such an embodiment these
amphiphilic linear sequences may be included on different branches.
As mentioned above, each of the o amphiphilic linear sequences is
independently selected. A respective amphiphilic linear sequence
has a length of n aliphatic amino acids. The symbol n represents an
integer selected in the range from 3 to about 18, such as from 3 to
about 15, from 3 to about 14, from 3 to about 13, from 3 to about
12, from 3 to about 11, from 3 to about 10, from 3 to about 9, from
3 to about 8 or from 3 to about 7, such as 3, 4, 5, 6, 7, 8, 9 or
10 aliphatic amino acids.
[0207] In some embodiments an amphiphilic linear sequence of a
peptide and/or peptoid described herein is chiral, rendering the
entire amphiphilic peptide and/or peptoid chiral. A corresponding
linear peptide and/or peptoid, i.e. an embodiment that consists of
a single respective linear sequence, is accordingly a chiral
peptide or peptoid. A respective amphiphilic linear sequence may
include any linear non-aromatic amino acid. The term "amino acid"
as used herein refers to an alpha-amino carboxylic acid, i.e. a
carboxylic acid with an amino group in the .alpha.-position. The
respective amino group may be an --NH.sup.2 group or an --NHR.sup.1
group. The moiety R.sup.1 may be any aliphatic group, whether
alkyl, alkenyl or alkynyl, with a main chain that includes 1 to 5,
to 10, to 15 or to 20 carbon atoms. Examples of alkenyl radicals
are straight chain or branched hydrocarbon radicals which contain
one or more double bonds. Alkenyl radicals generally contain about
two to about twenty carbon atoms and one or more, for instance two,
double bonds, such as about two to about ten carbon atoms, and one
double bond. Alkynyl radicals normally contain about two to about
twenty carbon atoms and one or more, for example two, triple bonds,
preferably such as two to ten carbon atoms, and one triple bond.
Examples of alkynyl radicals are straight-chain or branched
hydrocarbon radicals which contain one or more triple bonds.
Examples of alkyl groups are methyl, ethyl, propyl, butyl, pentyl,
hexyl, heptyl, octyl, nonyl, decyl, the n isomers of these
radicals, isopropyl, isobutyl, isopentyl, sec-butyl, tert-butyl,
neopentyl, 3, dimethylbutyl.
[0208] A peptoid is an oligo(N-alkyl) glycine that, similar to the
side chain connected to the a carbon atom (see below) of a peptide,
at the amide nitrogen carries a moiety that is an aliphatic moiety.
Accordingly, in embodiments where an --NHR.sup.1 group (supra) is
included in the amino acid and the a carbon atom is included in a
--CH.sub.2-- group, the reaction product of coupling a plurality of
such amino acids may be called a peptoid. A peptoid can also be
taken to differ from a peptide in that it carries its side chain at
the amide nitrogen rather than at the a carbon atom. Peptoids are
typically resistant to proteases and other modifying enzymes and
can have a much higher cell permeability than peptides (see e.g.
Kwon, Y.-U., and Kodadek, T., J. Am. Chem. Soc. (2007) 129,
1508-1509).
[0209] The term "amino acid" includes compounds in which the
carboxylic acid group is shielded by a protecting group in the form
of an ester (including an ortho ester), a silyl ester, an amide, a
hydrazide, an oxazole, an 1,3-oxazoline or a 5-oxo-1,3,
oxazolidine. The term "amino acid" also includes compounds in which
an amino group of the form --NH.sup.2 or --NHR.sup.1 (supra) is
shielded by a protecting group. Suitable amino protecting groups
include, but are not limited to, a carbamate, an amide, a
sulfonamide, an imine, an imide, histidine, a
N-2,5,-dimethylpyrrole, an
N-1,1,4,4-tetramethyldisilylazacyclopentane adduct, an
N-1,1,3,3-tetramethyl-1,-disilisoindoline, an
N-diphenylsilyldiethylene, an 1,3,5-dioxazine, a N-[2-(trimethyl
silyl)ethoxy]methylamine, a N-(5,
5-dimethyl-3-oxo-1-cyclohexenyl)amine, a
N-4,4,4-trifluoro-3-oxo-1-butenylamine, a N-9-borabicyclononane and
a nitroamine. A protecting group may also be present that shields
both the amino and the carboxylic group such as e.g. in the form of
a 2,2-dimethyl-4-alkyl-2-sila-5-oxo-1,3-oxazolidine. The alpha
carbon atom of the amino acid typically further carries a hydrogen
atom. The so called "side chain" attached to the alpha carbon atom,
which is in fact the continuing main chain of the carboxylic acid,
is an aliphatic moiety that may be linear or branched. The term
"side chain" refers to the presence of the amino acid in a peptide
(supra), where a backbone is formed by coupling a plurality of
amino acids. An aliphatic moiety bonded to the a carbon atom of an
amino acid included in such a peptide then defines a side chain
relative to the backbone. As explained above, the same applies to
an aliphatic moiety bonded to the amino group of the amino acid,
which likewise defines a side chain relative to the backbone of a
peptoid.
[0210] The term "aliphatic" means, unless otherwise stated, a
straight or branched hydrocarbon chain, which may be saturated or
mono- or poly-unsaturated and include heteroatoms. The term
"heteroatom" as used herein means an atom of any element other than
carbon or hydrogen. An unsaturated aliphatic group contains one or
more double and/or triple bonds (alkenyl or alkynyl moieties). The
branches of the hydrocarbon chain may include linear chains as well
as non-aromatic cyclic elements. The hydrocarbon chain, which may,
unless otherwise stated, be of any length, and contain any number
of branches. Typically, the hydrocarbon (main) chain includes 1 to
5, to 10, to 15 or to 20 carbon atoms. Examples of alkenyl radicals
are straight-chain or branched hydrocarbon radicals which contain
one or more double bonds. Alkenyl radicals generally contain about
two to about twenty carbon atoms and one or more, for instance two,
double bonds, such as about two to about ten carbon atoms, and one
double bond. Alkynyl radicals normally contain about two to about
twenty carbon atoms and one or more, for example two, triple bonds,
preferably such as two to ten carbon atoms, and one triple bond.
Examples of alkynyl radicals are straight-chain or branched
hydrocarbon radicals which contain one or more triple bonds.
Examples of alkyl groups are methyl, ethyl, propyl, butyl, pentyl,
hexyl, heptyl, octyl, nonyl, decyl, the n isomers of these
radicals, isopropyl, isobutyl, isopentyl, sec-butyl, tert-butyl,
neopentyl, 3, dimethylbutyl. Both the main chain as well as the
branches may furthermore contain heteroatoms as for instance N, 0,
S, Se or Si or carbon atoms may be replaced by these
heteroatoms.
[0211] An aliphatic moiety may be substituted or unsubstituted with
one or more functional groups. Substituents may be any functional
group, as for example, but not limited to, amino, amido, azido,
carbonyl, carboxyl, keto, cyano, isocyano, dithiane, halogen,
hydroxyl, nitro, organametal, organoboron, seleno, silyl, silano,
sulfonyl, thio, thiocyano, trifluoromethyl sulfonyl,
p-toluenesulfonyl, bromobenzenesulfonyl, nitrobenzenesulfonyl, and
methanesulfonyl. As should be apparent from the above, the side
chain of an amino acid in a peptide/peptoid described herein may be
of a length of 0 to about 5, to about 10, to about 15 or to about
20 carbon atoms. It may be branched and include unsaturated
carbon-carbon bonds. In some embodiments one or more natural amino
acids are included in the peptide or peptoid. Such a natural amino
acid may be one of the 20 building blocks of naturally occurring
proteins.
[0212] In a peptide or peptoid, including a peptide/peptoid
disclosed herein individual amino acids are covalently coupled via
amide bonds between a carboxylic group of a first and an amino
group of a second amino acid. A peptide and/or peptoid disclosed
herein is non-repetitive, such that two amino acids coupled to each
other are always different from one another.
[0213] The term amphiphilic refers to a compound that is soluble in
both polar and non-polar fluids. It also encompasses multiphase
compounds. The amphiphilic properties of the peptide and/or peptoid
are due to the presence of both polar and non-polar moieties within
the same peptide and/or peptoid. In this regard the peptide and/or
peptoid may be of surfactant nature. Accordingly, the polar
properties of a peptide and/or peptoid disclosed herein are based
on a polar moiety. Two such moiety are a --COOH side group, in
particular in the form of a charged COO-- group and an amino group.
A further such moiety is a C-terminal --COOH group if it is present
in free, unprotected form. Generally, a surfactant molecule
includes a polar, typically hydrophilic, head group attached to a
non-polar, typically hydrocarbon, moiety. Non-polar moieties of a
peptide or peptoid include a hydrocarbon chain that does not carry
a functional group.
[0214] An amphiphilic linear sequence included in a peptide and/or
peptoid disclosed herein thus includes a polar moiety and a
non-polar moiety. The polar moiety includes an aliphatic amino acid
that carries a polar group such as a hydroxyl group, a thiol group,
a seleno group, an amino group, an amide group, an ether group, a
thioether group or a seleno ether group. Accordingly, the polar
moiety may include an amino acid that carries a functional polar
group with a proton such as hydroxyl, thiol, selenol, amine or
amide. The polar moiety may also include the C-terminus or the
N-terminus of the peptide and/or peptoid. The C-terminus or the
N-terminus may in such a case be present in the form of the free
carboxyl or amino group, respectively, i.e. free of a protecting
group.
[0215] Generally the polar moiety of a linear amphiphilic sequence
of an amphiphilic peptide and/or peptoid disclosed herein is
defined by a single amino acid, by two consecutive amino acids or
by three consecutive amino acids that is/are coupled to the
non-polar moiety of the peptide/peptoid. Accordingly, in some
embodiments the polar moiety of the peptide/peptoid consists of two
amino acids that are covalently coupled via an amide bond, both
amino acids carrying a polar peptide/peptoid side chain. One of
these two amino acids may be a terminal amino aid of the
peptide/peptoid, defining its N- or C-terminus. In some embodiments
the amphiphilic peptide/peptoid has a single amino acid with a
polar side chain with the residual portion of the peptide/peptoid
defining the non-polar moiety. In some embodiments the amphiphilic
peptide/peptoid has two amino acids with a polar side chain while
the residual portion of the peptide/peptoid defines the non-polar
moiety. As three illustrative examples of a respective polar side
chain may serve 4-methyl-4-thio-pentyl,
6-ethoxycarbonyl-4,5-dimethylhexyl and
6-hydroxy-4-(1-hydroxyethyl)-hexyl groups. As used herein, the
numbering of corresponding peptide/peptoid side chains is started
with "1" at the carbon atom that is covalently bonded to the
a-carbon atom of the amino acid or to the amino group of the amino
acid, respectively. Amino acids included in the polar moiety may be
or include, but are not limited to, aspartic acid, asparagine,
glutamic acid, 4-fluoro-glutamic acid, 2-aminoadipic acid,
.gamma.-carboxy-glutamic acid, 4-tert-butyl aspartic acid,
glutamine, 5-N-ethyl-glutamine (theanine), itrulline,
thio-citrulline, cysteine, homocysteine, methionine, ethionine,
selenomethionine, telluromethionine, threonine, allo-threonine,
serine, homoserine, arginine, homoarginine, ornithine, lysine,
5-hydroxy-lysine and N(6)-carboxymethyllysine. Any such amino acid
may be present in the L- or D-form.
[0216] The amphiphilic linear sequence of the amphiphilic
peptide/peptoid disclosed herein can be defined as having n amino
acids. Where a single amino acid with a polar side chain is
included in the amphiphilic linear sequence, the non-polar moiety
may then be taken to have n-1 amino acids. In this case the polar
moiety consists of exactly one amino acid, such amino acid being
selected from any amino acids of the foregoing paragraph. Where two
consecutive amino acids with a polar side chain are included in the
amphiphilic linear sequence of the peptide/peptoid, the non-polar
moiety may then be taken to have n-2 amino acids. In this case the
polar moiety consists of exactly two amino acids. Where three
consecutive amino acids with a polar side chain are included in the
amphiphilic linear sequence, the non-polar moiety may then be taken
to have n-3 amino acids. In this case the polar moiety consists of
exactly three amino acids. In embodiments where the polar moiety
consists of two amino acids, the polar moiety may have a sequence
selected from Asn-Asn, Asp-Asp, Glu-Glu, Gln-Gln, Asn-Gln, Gln-Asn,
Asp-Gin, Gin-Asp, Asn-Glu, Glu-Asn, Asp-Glu, Glu-Asp, Gln-Glu,
Glu-Gln, Asp-Asn, Asn-Asp, Thr-Thr, Ser-Ser, Thr-Ser, Ser-Thr,
Asp-Ser, Ser-Asp, Ser-Asn, Asn-Ser, Gln-Ser, Ser-Gln, Glu-Ser,
Ser-Glu, Asp-Thr, Thr-Asp, Thr-Asn, Asn-Thr, Gin-Thr, Thr-Gln,
Glu-Thr, hr-Glu. In embodiments where the polar moiety consists of
three amino acids, the polar moiety may have a sequence selected
from Asn-Asn-Asn, Asn-Asn-Asp, Asn-Asp-Asn, Asp-Asn-Asn,
Asp-Asp-Asn, Asp-Asn-Asp, Asp-Asp-Asp, Asn-Asn-Glu, Asn-Asn-Gln,
Asn-Glu-Asn, Asn-Gln-Asn, Glu-Glu-Glu, Gln-Gln-Gln, Asn-Gln-Gln,
Asn-Glu-Gln, Asp-Asn-Glu, Gln-Asn-Asn, Gln-Asn-Asn, Glu-Asp-Gln,
Asp-Gin-Asp, Asn-Glu-Asp, Glu-Asn-Gln, Asp-Glu-Gln, Asn-Glu-Gln,
Glu-Asp-Asn, and Gln-Asp-Asn, Thr-Thr-Thr, Ser-Ser-Ser,
Asn-Thr-Thr, Asn-Ser-Ser Asn-Ser-Thr, Asn-Thr-Ser Asp-Asn-Ser,
Ser-Asn-Asn, Thr-Asn-Asn, Ser-Asp-Thr, to name a few.
[0217] The amphiphilic linear sequence of the peptide/peptoid has a
net charge at physiological pH. The term "physiological pH" is
known to those in the art to refer to the pH value of blood, which
has typically a pH value of about 7.4. In embodiments where the
amphiphilic linear sequence is arranged at the C- or N-terminus of
the peptide/peptoid, the respective terminus may provide the
corresponding net charge. In embodiments where the amphiphilic
linear sequence is not arranged at the C- or N-terminus of the
peptide/peptoid, the polar moiety of the amphiphilic linear
sequence includes one or more amino acids that have a side chain
with a functional group that is charged at physiological pH.
Illustrative examples of a respective functional group include an
amino, a nitro-, a guanidino, a esteryl, a sulfonyl or a carboxyl
group. In some embodiments the net charge of the amphiphilic linear
sequence is, as a positive or as a negative charge, equal to or
smaller than the number of amino acids included in the polar moiety
thereof. In some embodiments the net charge of the amphiphilic
linear sequence is one of -3, -2 or -1. In some embodiments the net
charge of the amphiphilic linear sequence is +1, +2 or +3.
[0218] The respective polar side chain of an amino acid of the
polar moiety, coupled to the a-carbon atom of the amino acid
(supra) and/or to the amino group thereof, may typically be defined
by a main chain that includes 1 to about 20, including 1 to about
15, 1 to about 10 or 1 to about 5 carbon atoms. For sake of clarity
it is recited that the term "side chain" is used relative to the
backbone of the peptide and/or peptoid. This peptide and/or peptoid
side chain may be branched and thus be defined by a main chain and
branches. Both the main chain and branches, f present, of the
peptide and/or peptoid side chain may include one or more double or
triple bonds (supra). Examples of side chains include, but are not
limited to, methyl, ethyl, propyl, isopropyl, propenyl, propinyl,
butyl, butenyl, sec-butyl, tert-butyl, isobutyl, pentyl, neopentyl,
sopentyl, pentenyl, hexyl, 3,3 dimethylbutyl, heptyl, octyl, nonyl
or decyl groups. The functional polar group is bonded to this the
peptide and/or peptoid side chain. In some embodiments the polar
moiety of the amphiphilic linear sequence includes two identical
amino acids. Where these amino acids are naturally occurring amino
acids, they may for example define one of the sequences Lys-Lys,
Gln-Gln, Glu-Glu, Asp-Asp, Asn-Asn, Met-Met, Thr-Thr, Arg-Arg or
Ser-Ser. The term "naturally occurring" in this context refers to
the 20 amino acids into which the genetic code is directly being
translated by any organism. Such two identical polar amino acids
may for example be adjacent to the non-polar moiety. In some
embodiments the amphiphilic linear sequence of the peptide/peptoid
has a hydrophobic tail of aliphatic amino acids and at least one
polar, including a charged, amino acid head group. The non-polar
moiety includes an amino acid, generally at least two amino acids,
with a hydrocarbon chain that does not carry a functional group.
The respective side chain, coupled to the a-carbon atom of the
amino acid (supra), may have a main chain that includes 0 to about
20 or 1 to about 20, including 0 to about 15, 1 to about 15, 0 to
about 10, 1 to about 10, 1 to about 5 or 0 to about 5 carbon
atoms.
[0219] The non-polar moiety may thus include an amino acid without
side chain, i.e. glycine. The peptide and/or peptoid side chain may
be branched (supra) and include one or more double or triple bonds
(supra). Examples of peptide and/or peptoid side chains include,
but are not limited to, methyl, ethyl, propyl, isopropyl, propenyl,
ropinyl, butyl, butenyl, sec-butyl, tert-butyl, isobutyl, pentyl,
neopentyl, isopentyl, pentenyl, exyl, 3,3 dimethylbutyl, heptyl,
octyl, nonyl or decyl groups. As a few illustrative examples, the
non-polar moiety may include an amino acid of alanine, valine,
leucine, isoleucine, norleucine, norvaline,
2-(methylamino)-isobutyric acid, 2-amino-5-hexynoic acid. Such an
amino acid may be present in any desired configuration. Bonded to
the non-polar moiety may also be the C-terminus or the N-terminus
of the peptide/peptoid. Typically the C-terminus or the N-terminus
is in such a case shielded by a protecting group (supra).
[0220] In some embodiments the non-polar moiety includes a sequence
of amino acids that is arranged in decreasing or increasing size.
Hence, a portion of the amino acids of the non-polar moiety may be
arranged in a general sequence of decreasing or increasing size.
Relative to the direction from N- to C-terminus or from C- to
N-terminus this general sequence can thus be taken to be of
decreasing size. By the term "general sequence" of decreasing or
increasing size is meant that embodiments are included in which
adjacent amino acids are of about the same size as long as there is
a general decrease or increase in size. Within a general sequence
of decreasing size the size of adjacent amino acids of the
non-polar moiety is accordingly identical or smaller in the
direction of the general sequence of decreasing size. In some
embodiments the general sequence of decreasing or increasing size
is a non-repetitive sequence. As an illustrative example, where a
respective portion of amino acids is a sequence of five amino
acids, the first amino acid may have a 3,-dimethyl-hexyl side
chain. The second amino acid may have a neopentyl side chain. The
third amino acid may have a pentyl side chain. The fourth amino
acid may have a butyl side chain. The fifth amino acid may be
glycine, i.e. have no side chain. Although a neopently and a pentyl
side chain are of the same size, the general sequence of such a
non-polar peptide portion is decreasing in size. As a further
illustrative example of a general sequence of decreasing size in a
non-polar moiety the respective non-polar portion may be a sequence
of three amino acids. The first amino acid may have an n-nonyl side
chain. The second amino acid may have a 3-ethyl-2-methyl-pentyl
side chain. The third amino acid may have a tert-butyl side chain.
As yet a further illustrative example of a general sequence of
decreasing size in a non-polar moiety, the non-polar moiety may be
a sequence of nine amino acids. The first amino acid may have a
4-propyl-nonyl side chain. The second amino acid may have an
n-dodecyl side chain. The third amino acid may have a
6,6-diethyl-3-octenyl side chain. An n-dodecyl side chain and a
6,6-diethyl-3-octenyl sidechain both have 12 carbon atoms and thus
again have a comparable size, Nevertheless, the
6,6-diethyl-3-octenyl group includes an unsaturated carbon-carbon
bond and is thus of slightly smaller size than the dodecyl group.
The fourth amino acid may have a 2-methyl-nonyl sidechain. The
fifth amino acid may have a 3-propyl-hexyl side chain. The sixth
amino acid may have an n-hexyl side chain. The seventh amino acid
may have a 2-butynyl side chain. The 8th amino acid may have an
isopropyl side chain. The ninth amino acid may have a methyl
sidechain.
[0221] Where a portion of the amino acids of the non-polar moiety
arranged in a general sequence of decreasing (or increasing) size
only contains naturally occurring amino acids (whether in the D- or
the L-form), it may for example have a length of five amino acids,
such as the sequence leucine-isoleucine-valine-alanine-glycine or
isoleucine-leucine-valine-alanine-glycine, A general sequence of
decreasing size of only natural amino acids may also have a length
of four amino acids. Illustrative examples include the sequences
isoleucine-leucine-valine-alanine,
leucine-isoleucine-valine-alanine,
isoleucine-valine-alanine-glycine, leucine-valine-alanine-glycine,
eucine-isoleucine-alanine-glycine,
leucine-isoleucine-valine-glycine,
isoleucine-leucine-alanine-glycine or
isoleucine-leucine-valine-glycine. A general sequence of decreasing
size of only natural amino acids may also have a length of three
amino acids. Illustrative examples include the sequences
isoleucine-valine-alanine, leucine-valine-alanine,
isoleucine-valine-glycine, leucine-valine-glycine,
leucine-alanine-glycine, isoleucine-alanine-glycine or
isoleucine-leucine-alanine. A general sequence of decreasing size
of only natural amino acids may also have a length of two amino
acids. Illustrative examples include the sequences
isoleucine-valine, leucine-valine, isoleucine-alanine,
leucine-alanine, leucine-glycine, isoleucine-glycine,
valine-alanine, valine-glycine or alanine-glycine.
[0222] In some embodiments the direction of decreasing size of the
above defined general sequence of decreasing size is the direction
toward the polar moiety of the amphiphilic linear sequence.
Accordingly, in such embodiments the size of adjacent amino acids
within this portion of the non-polar moiety is accordingly
identical or smaller in the direction of the polar moiety. Hence,
as a general trend in such an embodiment, the closer to the polar
moiety of the amphiphilic linear sequence, the smaller is the
overall size of a peptide and/or peptoid side chain throughout the
respective general sequence of decreasing size. In the above
illustrative example of a general sequence of three amino acids
with a n-nonyl, a 3-ethyl-2-methyl-pentyl and a tert-butyl side
chain, the next amino acid may be polar in that it carries a
peptide/peptoid sidechain with a polar functional group. As an
illustrative example, adjacent to the tert-butyl sidechain within
the peptide/peptoid there may be a 3-carboxy-n-butyl side
chain.
[0223] In some embodiments the entire non-polar moiety of the
amphiphilic linear peptide and/or peptoid or the amphiphilic linear
sequence, respectively, consists of the general sequence of
decreasing (or increasing) size. In such an embodiment the general
sequence of decreasing (or increasing) size may have a length of
n-m amino acids (cf. above). In some embodiments the general
sequence of decreasing or increasing size is flanked by further
non-polar side chains of the peptide/peptoid. In one embodiment the
general sequence of decreasing (or increasing) size has a length of
n-m-1 amino acids. In this embodiment there is one further amino
acid included in the peptide/peptoid, providing a non-polar
peptide/peptoid side chain. This amino acid may be positioned
between the general sequence of decreasing (or increasing) size and
the polar amino acid, the polar amino acid may be positioned
between this additional nonpolar amino acid and the general
sequence of decreasing (or increasing) size or the general sequence
of decreasing (or increasing) size may be positioned between the
polar amino acid and this additional non-polar amino acid.
Typically the general sequence of decreasing (or increasing) size
is positioned between the polar amino acid and this additional
non-polar amino acid. The additional non-polar amino acid may for
example define the N-terminus of the peptide/peptoid, which may be
shielded by a protecting group such as an amide, e.g. a propionic
acyl or an acetyl group. Together with the general sequence of
decreasing (or increasing) size as defined above it may define the
non-polar portion of the peptide/peptoid. The polar amino acid may
define the C-terminus of the peptide/peptoid. In this example the
general sequence of decreasing (or increasing) size is thus flanked
by the polar amino acid on one side and by the additional non-polar
amino acid on the other side. In one embodiment where embodiment
the general sequence of decreasing (or increasing) size has a
length of n-m-1 amino acids, the remaining non-polar amino acid of
the non-polar moiety of n-m amino acids is one of alanine and
glycine.
[0224] As explained above, the polar moiety of the amphiphilic
linear sequence may in some embodiments be defined by two or three
consecutive amino acids. The polar moiety includes m aliphatic
amino acids. Each of the m aliphatic amino acids is independently
selected and carries an independently selected polar group. The
symbol m represents an integer selected from 1, 2 and 3. The at
least essentially non-polar moiety (supra) accordingly has a number
of n-m, i.e. n-1, n-2 or n-3 amino acids. In some embodiments n is
equal to or larger than m+2. In such an embodiment m may thus
represent a number of n-2 or smaller.
[0225] In an embodiment where the entire non-polar moiety of the
amphiphilic linear peptide and/or peptoid consists of the general
sequence of decreasing (or increasing) size (supra), this non polar
moiety may thus have a length of n-2 or n-3 amino acids. In an
embodiment where the amphiphilic linear peptide and/or peptoid has
a further non-polar side chain in addition to the non-polar moiety
of decreasing (or increasing) size, this additional non-polar side
chain may be included in an amino acid that is directly bonded to
an amino acid of the general sequence of decreasing (or increasing)
size. The non-polar moiety may thus be defined by the non-polar
moiety of decreasing (or increasing) size and the respective
further amino acid with a non-polar side chain. In one such an
embodiment where m=1, the non-polar moiety may thus have a length
of n-2 amino acids, of which the non-polar moiety of decreasing (or
increasing) size has a length of n-3 amino acids. The general
sequence of decreasing (or increasing) size may be positioned
between the two polar amino acids and this additional non-polar
amino acid, or the additional non-polar amino acid may be
positioned between the general sequence of decreasing (or
increasing) size and the two polar amino acids. Typically the
general sequence of decreasing (or increasing) size is positioned
between the two polar amino acids and this additional non-polar
amino acid. As mentioned above, one of the two polar amino acids
may define the C-terminus of the peptide/peptoid. In this example
the general sequence of decreasing (or increasing) size may thus be
flanked by the two consecutive polar amino acids on one side and by
the additional non-polar amino acid on the other side. Again, in
some embodiments where m=1 the two consecutive polar amino acids
may also be positioned between the general sequence of decreasing
(or increasing) size and the additional non-polar amino acid, in
which case the non-polar moiety has a first portion with a length
of n-3 amino acids and a further portion of one amino acid.
[0226] Electrostatic forces, hydrogen bonding and van der Waals
forces between amphiphilic linear sequences as defined above,
including amphiphilic linear peptides and/or peptoids, result in
these amphiphilic linear sequences to be coupled to each other.
Without being bound by theory, hereby a cross-linking effect occurs
that allows the formation of a hydrogel. In this regard the
inventors have observed the formation of fibers based on helical
structures.
[0227] The fibers formed of amphiphilic linear sequences of
amphiphilic peptides and/or peptoids disclosed herein typically
show high mechanical strength, which renders them particularly
useful in tissue regeneration applications, for instance the
replacement of damaged tissue. Amphiphilic peptides and/or peptoids
disclosed herein have been observed to generally assemble into a
fiber structure that resembles collagen fibers. Collagen, a
component of soft tissue in the animal and human body, is a fibrous
protein that provides most of the tensile strength of tissue. The
mechanical strength of fibers of amphiphilic peptides and/or
peptoids disclosed herein has been found to typically be much
higher than that of collagen (cf. e.g. Figures) of gelatine, the
hydrolysed form of collagen. An amphiphilic peptide and/or peptoid
disclosed herein may thus be included in a hydrogel that is used as
permanent or temporary prosthetic replacement for damaged or
diseased tissue.
[0228] The amphiphilic linear sequence of the peptide/peptoid,
which may represent the entire amphiphilic peptide/peptoid (supra)
has been found to show remarkable stability at physiological
conditions, even at elevated temperatures. It is in some
embodiments stable in aqueous solution at physiological conditions
at ambient temperature for a period of time in the range from 1 day
to 1 month or more. It may in some embodiments be stable in aqueous
solution at physiological conditions at 90.degree. C. for at least
1 hour, at least 2 hours, at least 3 hours, at least 4 hours or at
least 5 hours.
[0229] An amphiphilic linear sequence of an amphiphilic peptide
and/or peptoid disclosed herein, including an amphiphilic linear
peptide and/or peptoid, is capable of providing a self assembling
a-helical fiber in aqueous solution under physiological conditions.
The peptides/peptoids (typically 3-7-mers) in the L- or D-form can
self assemble into supramolecular helical fibers which are
organized into mesh-like structures mimicking biological substances
such as collagen. It has previously been observed in X-ray
crystallography that peptides of a length of 3 to 6 amino acids
with repetitive alanine containing sequences and an acetylated
C-terminus take a helical conformation (Hatakeyama, Y, et al,
Angew. Chem. Int. Ed. (2009) 8695-8698). Using peptides with an
amphiphilic sequence disclosed herein, Ac-LD.sub.6 (L), the
formation of aggregates has for example been observed already at
0.1 mg/ml. As the concentration of peptide is increased to 1 mg/ml,
the peptide monomers were found to align to form fibrous
structures. With a formation of fibers occurring under
physiological conditions at concentrations below 2 mM a
peptide/peptoid disclosed herein is well suited as an injectable
hydrogel material that can form a hydrogel under physiological
conditions. Also disclosed herein is an amphiphilic linear peptide
and/or peptoid as defined above for tissue engineering as well as
to a tissue engineering method that involves applying, including
injecting a respective amphiphilic linear peptide and/or
peptoid.
[0230] A hydrogel as disclosed herein is typically characterized by
a remarkable rigidity and are generally biocompatible and
non-toxic. Depending on the selected peptide/peptoid sequence these
hydrogels can show thermoresponsive or thixotropic character.
Reliant on the peptide/peptoid assembling conditions the fibers
differ in thickness and length. Generally rigid hydro gels are
obtained that are well suited for cultivation of a variety of
primary human cells, providing peptide/peptoid scaffolds that can
be useful in the repair and replacement of various tissues.
Disclosed is also a process of preparing these hydrogels. The
exemplary usage of these hydrogels in applications such as cell
culture, tissue engineering, plastic surgery, drug delivery, oral
applications, cosmetics, packaging and the like is described, as
well as for technical applications, as for example for use in
electronic devices which might include solar or fuel cells.
[0231] As an amphiphilic linear sequence of the peptide/peptoid, a
hydrogel as disclosed herein shows high stability at physiological
conditions, even at elevated temperatures. In some embodiments such
a hydrogel is stable in aqueous solution at ambient temperature for
a period of at least 7 days, at least 14 days, at least a month or
more, such as at least 1 to about 6 months.
[0232] In some embodiments a hydrogel disclosed herein is coupled
to a molecule or a particle, including a quantum dot, with
characteristic spectral or fluorometric properties, such as a
marker, including a fluorescent dye. A respective molecule may for
instance allow monitoring the fate, position and/or the integrity
of the hydrogel.
[0233] In some embodiments a hydrogel disclosed herein is coupled
to a molecule with binding affinity for a selected target molecule,
such as a microorganism, a virus particle, a peptide, a peptoid, a
protein, a nucleic acid, a peptide, an oligosaccharide, a
polysaccharide, an inorganic molecule, a synthetic polymer, a small
organic molecule or a drug.
[0234] The term "nucleic acid molecule" as used herein refers to
any nucleic acid in any possible configuration, such as single
stranded, double stranded or a combination thereof. Nucleic acids
include for instance DNA molecules (e.g., DNA or genomic DNA), RNA
molecules (e.g., RNA), analogues of the DNA or RNA generated using
nucleotide analogues or using nucleic acid chemistry, locked
nucleic acid molecules (LNA), and protein nucleic acids molecules
(PNA). DNA or RNA may be of genomic or synthetic origin and may be
single or double stranded. In the present method of an embodiment
of the invention typically, but not necessarily, an RNA or a DNA
molecule will be used. Such nucleic acid can be e.g. mRNA, cRNA,
synthetic RNA, genomic DNA, eDNA synthetic DNA, a copolymer of DNA
and RNA, oligonucleotides, etc. A respective nucleic acid may
furthermore contain non-natural nucleotide analogues and/or be
linked to an affinity tag or a label. In some embodiments the
nucleic acid molecule may be isolated, enriched, or purified. The
nucleic acid molecule may for instance be isolated from a natural
source by eDNA cloning or by subtractive hybridization. The natural
source may be mammalian, such as human, blood, semen, or tissue.
The nucleic acid may also be synthesized, e.g. by the triester
method or by using an automated DNA synthesizer.
[0235] Many nucleotide analogues are known and can be used in
nucleic acids and oligonucleotides used in the methods of exemplary
embodiments of the invention. A nucleotide analogue is a nucleotide
containing a modification at for instance the base, sugar, or
phosphate moieties. Modifications at the base moiety include
natural and synthetic modifications of A, C, G, and T/U, different
purine or pyrimidine bases, such as uracil-5-yl, hypoxanthin-9-yl,
and 2-aminoadenin-9-yl, as well as non-purine or non-pyrimidine
nucleotide bases. Other nucleotide analogues serve as universal
bases. Universal bases include 3-nitropyrrole and 5-nitroindole.
Universal bases are able to form a base pair with any other base.
Base modifications often can be combined with for example a sugar
modification, such as for instance 2'-O-methoxyethyl, .g. to
achieve unique properties such as increased duplex stability.
[0236] A peptide may be of synthetic origin or isolated from a
natural source by methods well known in the art. The natural source
may be mammalian, such as human, blood, semen, or tissue. A
peptide, including a polypeptide may for instance be synthesized
using an automated polypeptide synthesizer. Illustrative examples
of polypeptides are an antibody, a fragment thereof and a
proteinaceous binding molecule with antibody-like functions.
Examples of (recombinant) antibody fragments are Fab fragments, Fv
fragments, single-chain Fv fragments (scFv), diabodies, triabodies
(Iliades, P., et al., FEBS Lett (1997) 409, 437-441), decabodies
(Stone, E., et al., Journal of Immunological Methods (2007) 318,
88-94) and other domain antibodies (Holt, L. J., et al., Trends
Biotechnol. (2003), 21, 11, 484-490). An example of a proteinaceous
binding molecule with antibody-like functions is a mutein based on
a polypeptide of the lipocalin family (WO 03/029462, Beste et al.,
Proc. Natl. Acad. Sci. U.S.A. (1999)96, 1898-1903). Lipocalins,
such as the bilin binding protein, the human neutrophil
gelatinase-associated lipocalin, human Apolipoprotein D or
glycodelin, posses natural ligand binding sites that can be
modified so that they bind to selected small protein regions known
as haptens. Examples of other proteinaceous binding molecules are
the so-called glubodies (see e.g. international patent application
WO 96/23879), proteins based on the ankyrin scaffold (Mosavi, L.
K., et al., Protein Science (2004) 13, 6, 1435-1448) or crystalline
scaffold (e.g. international patent application WO 01/04144) the
proteins described in Skerra, J. Mol. Recognit. (2000) 13, 167-187,
AdNectins, tetranectins and avimers. Avimers contain so called
A-domains that occur as strings of multiple domains in several cell
surface receptors (Silverman, J., et al., Nature Biotechnology
(2005) 23, 1556-1561). Adnectins, derived from a domain of human
fibronectin, contain three loops that can be engineered for
immunoglobulin-like binding to targets (Gill, D. S. & Damle, N.
K., Current Opinion in Biotechnology (2006) 17, 653-658).
Tetranectins, derived from the respective human homotrimeric
protein, likewise contain loop regions in a C-type lectin domain
that can be engineered for desired binding (ibid.). Where desired,
a modifying agent may be used that further increases the affinity
of the respective moiety for any or a certain form, class etc. of
target matter.
[0237] An example of a nucleic acid molecule with antibody-like
functions is an aptamer. An aptamer folds into a defined
three-dimensional motif and shows high affinity for a given target
structure. Using standard techniques of the art such as solid-phase
synthesis an aptamer with affinity to a certain target can
accordingly be formed and immobilized on a hollow particle of an
embodiment of the invention.
[0238] As a further illustrative example, a linking moiety such as
an affinity tag may be used to immobilise the respective molecule.
Such a linking moiety may be a molecule, e.g. a hydrocarbon-based
(including polymeric) molecule that includes nitrogen-,
phosphorus-, sulphur-, arben-, halogen- or pseudohalogen groups, or
a portion thereof. As an illustrative example, the peptide/peptoid
included in the hydrogel may include functional groups, for
instance on aside chain of the peptide/peptoid, that allow for the
covalent attachment of a biomolecule, for example a molecule such
as a protein, a nucleic acid molecule, a polysaccharide or any
combination thereof. A respective functional group may be provided
in shielded form, protected by a protecting group that can be
released under desired conditions. Examples of a respective
functional group include, but are not limited to, an amino group,
an aldehyde group, a thiol group, a carboxy group, an ester, an
anhydride, a sulphonate, a sulphonate ester, an imidoester, a silyl
halide, an epoxide, an aziridine, a phosphoramidite and a
diazoalkane. Examples of an affinity tag include, but are not
limited to, biotin, dinitrophenol or digoxigenin, ligohistidine,
polyhistidine, an immunoglobulin domain, maltose-binding protein,
glutathione-S-transferase (GST), calmodulin binding peptide (CBP),
FLAG'-peptide, the T7 epitope
(Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly), maltose binding protein
(MBP), the HSV epitope of the sequence
Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp of herpes simplex virus
glycoprotein D, the hemagglutinin (HA) epitope of the sequence
Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala, the "myc" epitope of the
transcription factor c-myc of the sequence
Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, or an oligonucleotide tag.
Such an oligonucleotide tag may for instance be used to hybridise
to an immobilised oligonucleotide with a complementary sequence. A
further example of a linking moiety is an antibody, a fragment
thereof or a proteinaceous binding molecule with antibody-like
functions (see also above). A further example of linking moiety is
a cucurbituril or a moiety capable of forming a complex with a
cucurbituril. A cucurbituril is a macrocyclic compound that
includes glycoluril units, typically self-assembled from an acid
catalyzed condensation reaction of glycoluril and formaldehyde. A
cucurbit[n]uril, (CB[n]), that includes n glycoluril units,
typically has two portals with polar ureido carbonyl groups. Via
these ureido carbonyl groups cucurbiturils can bind ions and
molecules of interest. As an illustrative example cucurbit[7]uril
(CB[7]) can form a strong complex with ferrocenemethylammonium or
adamantylammonium ions. Either the cucurbit[7]uril or e.g.
ferrocenemethylammonium may be attached to a biomolecule, while the
remaining binding partner (e.g. ferrocenemethylammonium or
cucurbit[7]uril respectively) can be bound to a selected surface.
Contacting the biomolecule with the surface will then lead to an
immobilisation of the biomolecule. Functionalised CB[7] units bound
to a gold surface via alkanethiolates have for instance been shown
to cause an immobilisation of a protein carrying a
ferrocenemethylammonium unit (Hwang, I., et al., J. Am. Chem. Soc.
(2007) 129, 4170-4171).
[0239] Further examples of a linking moiety include, but are not
limited to an oligosaccharide, anoligopeptide, biotin,
dinitrophenol, digoxigenin and a metal chelator (cf. also below).
As an illustrative example, a respective metal chelator, such as
ethylenediamine, ethylenediaminetetraaceticacid (EDTA), ethylene
glycol tetraacetic acid (EGTA), diethylenetriaminepentaaceticacid
(DTPA), N,N-bis(carboxymethyl)glycine (also called nitrilotriacetic
acid, NTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA), 2,3-dimercapto-1-propanol (dimercaprol), porphine or
heme may be used in cases where the target molecule is a metal ion.
As an example, EDTA forms a complex with most monovalent, divalent,
trivalent and tetravalent metal ions, such as e.g. silver
(Ag.sup.+), calcium (Ca.sup.2+), manganese (Mn.sup.2+), copper
(Cu.sup.2+), iron (Fe.sup.2+), cobalt (Co.sup.3+) and zirconium
(Zr.sup.4+), while BAPTA is specific for Ca.sup.2+. In some
embodiments a respective metal chelator in a complex with a
respective metal ion or metal ions defines the linking moiety. Such
a complex is for example a receptor molecule for a peptide of a
defined sequence, which may also be included in a protein. As an
illustrative example, a standard method used in the art is the
formation of a complex between an oligo histidine tag and copper
(Cu.sup.2+), nickel (Ni.sup.2+), cobalt (Co.sup.2+), or zink
(Zn.sup.2+) ions, which are presented by means of the chelator
nitrilotriacetic acid (NTA).
[0240] Avidin or streptavidin may for instance be employed to
immobilise a biotinylated nucleic acid, or a biotin containing
monolayer of gold may be employed (Shumaker-Parry, J. S., et al.,
Anal. Chem. (2004) 76, 918). As yet another illustrative example,
the biomolecule may be locally deposited, e.g. by scanning
electrochemical microscopy, for instance via pyrroleoligonucleotide
patterns (e.g. Fortin, E., et al., Electroanalysis (2005) 17, 495).
In other embodiments, in particular where the biomolecule is a
nucleic acid, the biomolecule may be directly synthesised on the
surface of the immobilisation unit, for example using
photoactivation and deactivation. As an illustrative example, the
synthesis of nucleic acids or oligonucleotides on selected surface
areas (so called "solid phase" synthesis) may be carried out using
electrochemical reactions using electrodes. An electrochemical
deblocking step as described by Egeland & Southern (Nucleic
Acids Research (2005) 33, 14, e125) may for instance be employed
for this purpose. A suitable electrochemical synthesis has also
been disclosed in US patent application US 2006/0275927. In some
embodiments light-directed synthesis of a biomolecule, in
particular of a nucleic acid molecule, including UV-linking or
light dependent 5'-deprotection, may be carried out.
[0241] The molecule that has a binding affinity for a selected
target molecule may be immobilised on the nanocrystals by any
means. As an illustrative example, an oligo- or polypeptide,
including a respective moiety, may be covalently linked to the
surface of nanocrystals via a thio-ether bond, or example by using
.omega. functionalized thiols. Any suitable molecule that is
capable of linking a nanocrystal of an embodiment of the invention
to a molecule having a selected binding affinity may be used to
immobilise the same on a nanocrystal. For instance a
(bifunctional.mu.inking agent such as
ethyl-3-dimethylaminocarbodiimide, N-(3-aminopropyl)
3-mercaptobenzamide, 3-aminopropyl-trimethoxysilane,
3-mercaptopropyl-trimethoxysilane, 3-(trimethoxysilyl)
propyl-maleimide, or 3-(trimethoxysilyl) propyl-hydrazide may be
used. Prior to reaction with the linking agent, the surface of the
nanocrystals can be modified, for example by treatment with glacial
mercaptoacetic acid, in order to generate free mercaptoacetic
groups which can then employed for covalently coupling with an
analyte binding partner via linking agents.
[0242] Embodiments of the present invention also include a
hydrogel, which can be taken to be a water-swollen water-insoluble
polymeric material. The hydrogel includes, including contains and
consists of, a peptide and/or peptoid as defined above. Since a
hydrogel maintains a three dimensional structure, a hydrogel of an
embodiment of the invention may be used for a variety of
applications. Since the hydrogel has a high water content and
includes amino acids, it is typically of excellent
biocompatibility.
[0243] A hydrogel according to an embodiment of the invention is
formed by self-assembly. The inventors have observed that the
peptides/peptoids assemble into fibers that form mesh-like
structures. Without being bound by theory hydrophobic interaction
between non-polar portions of peptides/peptoids as disclosed herein
are contemplated to assist such self-assembly process. The method
of forming the hydrogel includes dissolving the peptide/peptoid in
aqueous solution. Agitation, including mixing such as stirring,
and/or sonication may be employed to facilitate dissolving the
peptide/peptoid. In some embodiments the aqueous solution with the
peptide/peptoid therein is exposed to a temperature below ambient
temperature, such as a temperature selected from about 2.degree. C.
to about 15.degree. C. In some embodiments the aqueous solution
with the peptide/peptoid therein is exposed to an elevated
temperature, i.e. a temperature above ambient temperature.
Typically the aqueous solution is allowed to attain the temperature
to which it is exposed. The aqueous solution may for example be
exposed to a temperature from about 25.degree. C. to about
85.degree. C. or higher, such as from about 25.degree. C. to about
75.degree. C., from about 25.degree. C. to about 70.degree. C.,
from about 30.degree. C. to about 70.degree. C., from about
35.degree. C. to about 70.degree. C., from about 25.degree. C. to
about 60.degree. C., from about 30.degree. C. to about 60.degree.
C., from about 25.degree. C. to about 50.degree. C., from about
30.degree. C. to about 50.degree. C. or from about 40.degree. C. to
about 65.degree. C., such as e.g. a temperature of about 40.degree.
C., about 45.degree. C., about 50.degree. C., about 55.degree. C.,
about 60.degree. C. or about 65.degree. C. The aqueous solution
with the peptide/peptoid therein may be maintained at this
temperature for a period of about 5 min to about 10 hours or more,
such as about 10 min to about 6 hours, about 10 min to about 4
hours, about 10 min to about 2.5 hours, about 5 min to about 2.5
hours, about 10 min to about 1.5 hours or about 10 min to about 1
hour, such as about 15 min, about 20 min, about 25 min, about 30
min, about 35 min or about 40 min.
[0244] A hydrogel according to an embodiment of the invention may
be included in a fuel cell, here it may for example provide a
substrate between the anode and the cathode. A liquid electrolyte
may be encompassed by the hydrogel. Likewise, a hydrogel according
to an embodiment of the invention may provide a substrate between
two electrodes in an electrophoresis apparatus. The hydrogel may
also be conducting. The hydrogel may also serve in enhancing the
efficiency of charge-separated states and/or slowing down charge
recombination. The hydrogel may thus be applied in any form
photovoltaics, including a solar cell.
[0245] In some embodiments a hydrogel disclosed herein is a
biocompatible, including a pharmaceutically acceptable hydrogel.
The term "biocompatible" (which also can be referred to as "tissue
compatible"), as used herein, is a hydrogel that produces little if
any adverse biological response when used in vivo. The term thus
generally refers to the inability of a hydrogel to promote a
measurably adverse biological response in a cell, including in the
body of an animal, including a human. A biocompatible hydrogel can
have one or more of the following properties: non-toxic,
non-mutagenic, non-allergenic, non-carcinogenic, and/or
non-irritating. A biocompatible hydrogel, in the least, can be
innocuous and tolerated by the respective cell and/or body. A
biocompatible hydrogel, by itself, may also improve one or more
functions in the body.
[0246] Depending on the amino acids that are included in the
peptide/peptoid that is included in a hydrogel, a respective
hydrogel may be biodegradable. A biodegradable hydrogel gradually
disintegrates or is absorbed in vivo over a period of time, e.g.,
within months or years. Disintegration may for instance occur via
hydrolysis, may be catalysed by an enzyme and may be assisted by
conditions to which the hydrogel is exposed in a human or animal
body, including a tissue, a blood vessel or a cell thereof. Where a
peptide is made up entirely of natural amino acids, a respective
peptide can usually be degraded by enzymes of the human/animal
body.
[0247] A hydrogel according to an embodiment of the invention may
also serve as a depot for a pharmaceutically active compound such
as a drug. A hydrogel according to an embodiment of the invention
may be designed to mimic the natural extracellular matrix of an
organism such as the human or animal body. A fiber formed from the
peptide/peptoid of an embodiment of the invention, including a
respective hydrogel, may serve as a biological scaffold. A hydrogel
of an embodiment of the invention may be included in an implant, in
a contact lens or may be used in tissue engineering. In one
embodiment, the peptides consist typically of 3-7 amino acids and
are able to self-assemble into complex fibrous scaffolds which are
seen as hydrogels, then dissolved in water or aqueous solution.
These hydrogels can retain water up to 99.9% and possess
sufficiently high mechanical strength. Thus, these hydrogels can
act as artificial substitutes for a variety of natural tissues
without the risk of immunogenicity. The hydrogels in accordance
with the present invention may be used for cultivating suitable
primary cells and thus establish an injectable cell-matrix compound
in order to implant or reimplant the newly formed cell-matrix in
vivo. Therefore, the hydrogels in accordance with the present
invention are particularly useful for tissue regeneration or tissue
engineering applications. As used herein, a reference to an
"implant" or "implantation" refers to uses and applications of/for
surgical or arthroscopic implantation of a hydrogel containing
device into a human or animal, e.g. mammalian, body or limb.
Arthroscopic techniques are taken herein as a subset of surgical
techniques, and any reference to surgery, surgical, etc., includes
arthroscopic techniques, methods and devices. A surgical implant
that includes a hydrogel according to an embodiment of the
invention may include a peptide and/or peptoid scaffold. This the
peptide and/or peptoid scaffold may be defined by the respective
hydrogel. A hydrogel of an embodiment of the invention may also be
included in a wound cover such as gauze or a sheet, serving in
maintaining the wound in a moist state to promote healing.
[0248] Depending on the amino acid sequence used in the
peptide/peptoid the hydrogel may be temperature-sensitive. It may
for instance have a lower critical solution temperature or a
temperature range corresponding to such lower critical solution
temperature, beyond which the gel collapses as hydrogen bonds by
water molecules are released as water molecules are released from
the gel.
[0249] The disclosed subject matter also provides improved chiral
amphiphilic natural-based peptides and/or peptoids that assemble to
peptide/peptoid hydrogels with very favorable material properties.
The advantage of these peptide/peptoid hydrogels is that they are
accepted by a variety of different primary human cells, thus
providing peptide scaffolds that can be useful in the repair and
replacement of various tissues. Depending on the chirality of the
peptide monomer the character of the hydro gels can be designed to
be more stable and less prone to degradation though still
biocompatible.
[0250] A hydrogel and/or a peptide/peptoid described herein can be
administered to an organism, including a human patient per se, or
in pharmaceutical compositions where it may include or be mixed
with pharmaceutically active ingredients or suitable carriers or
excipient(s). Techniques for formulation and administration of
respective hydro gels or peptides/peptoids resemble or are
identical to those of low molecular weight compounds well
established in the art. Exemplary routes include, but are not
limited to, oral, transdermal, and parenteral delivery. A hydrogel
or a peptide/peptoid may be used to fill a capsule or tube, or may
be provided in compressed form as a pellet. The peptide/peptoid or
the hydrogel may also be used in injectable or sprayable form, for
instance as a suspension of a respective peptide/peptoid.
[0251] A hydrogel of an embodiment of the invention may for
instance be applied onto the skin or onto a wound. Further suitable
routes of administration may, for example, include depot, oral,
rectal, transmucosal, or intestinal administration; parenteral
delivery, including intramuscular, subcutaneous, intravenous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intraperitoneal, intranasal, or intraocular
injections. It is noted in this regard that for administering
microparticles a surgical procedure is not required. Where the
microparticles include a biodegradable polymer there is no need for
device removal after release of the anti-cancer agent. Nevertheless
the microparticles may be included in or on a scaffold, a coating,
patch, composite material, a gel or a plaster.
[0252] In some embodiments one may administer a hydrogel and/or a
peptide/peptoid in a local rather than systemic manner, for
example, via injection.
[0253] Pharmaceutical compositions that include a hydrogel and/or a
peptide/peptoid of an embodiment of the present invention may be
manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0254] Pharmaceutical compositions for use in accordance with an
embodiment of the present invention thus may be formulated in
conventional manner using one or more physiologically acceptable
carriers including excipients and auxiliaries that facilitate
processing of the hydrogel and/or peptide/peptoid into preparations
that can be used pharmaceutically. Proper formulation is dependent
upon the route of administration chosen.
[0255] For injection, the peptide/peptoid of an embodiment of the
invention may be formulated in aqueous solutions, for instance in
physiologically compatible buffers such as Hanks's solution,
Ringer's solution, or physiological saline buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art.
[0256] For oral administration, the hydrogel and/or peptide/peptoid
can be formulated readily by combining them with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
hydrogel and/or peptide/peptoid, as well as a pharmaceutically
active compound, o be formulated as tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions and the
like, for oral ingestion by a patient to be treated. Pharmaceutical
preparations for oral use can be obtained by adding a solid
excipient, optionally grinding a resulting mixture, and processing
the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. Suitable excipients
are, in particular, fillers such as sugars, including lactose,
sucrose, mannitol, or sorbitol; cellulose preparations such as, for
example, maize starch, wheat starch, rice starch, potato starch,
gelatine, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinylpyrrolidone,
agar, or alginic acid or a salt thereof such as sodium
alginate.
[0257] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0258] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatine, as well as soft, sealed
capsules made of gelatine and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules can contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the peptides/peptoids
may be suspended in suitable liquids, such as fatty oils, liquid
paraffin, or liquid polyethylene glycols. In addition, stabilizers
may be added. All formulations for oral administration should be in
dosages suitable for such administration. For buccal
administration, the compositions may take the form of tablets or
lozenges formulated in conventional manner.
[0259] The hydrogel and/or peptide/peptoid may be formulated for
parenteral administration by injection, .g., y intramuscular
injections or bolus injection or continuous infusion. Formulations
for injection may be presented in unit dosage form, e.g., in
ampules or in multi-dose containers, with an added preservative.
The respective compositions may take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulator agents such as suspending, stabilizing and/or dispersing
agents. The hydrogel and/or peptide/peptoid may be formulated for
other drug delivery systems like implants, or transdermal patches
or stents.
Examples
[0260] Experiments have been performed to illustrate the technical
aspects of exemplary embodiments of the present invention. The
following examples are described in the Experimental Methods and
Results. The skilled artisan will readily recognize that the
examples are intended to be illustrative and are not intended to
limit the scope of the present invention.
Experimental Methods and Results
Peptides
[0261] The peptide sequences were designed to represent an
amphiphilic peptide structure containing a hydrophilic head group
and a hydrophobic tail. The rationale for the peptides design was
to create a peptide monomer of decreasing size resembling a cone
shaped structure. The hydrophobic tail differs by using different
aliphatic amino acids. It is consisting of the following aliphatic
amino acids such as glycine, alanine, valine, leucine and
isoleucine and the hydrophilic head group is consisting of one or
two polar or charged amino acids. The sequence order of the
hydrophobic tail differed by using different aliphatic amino acids.
The peptides were commercially synthesized from GL Biochem,
Shanghai, China. In order to verify the reproducibility of the
peptide hydro gel-forming behavior peptides were also synthesized
from other companies (Biomatik Corp., Anaspec. Inc, USA). The
peptides have a purity of equal or higher than 95% verified by
High-performance liquid chromatography (HPLC) and mass
spectrometry. The peptide stock solutions were dissolved in water
at 5 to 10 mg/ml. Most of the peptides are acetylated at the
N-terminus.
Peptide-Based Hydrogel Preparation
[0262] All peptides (GL Biochem, Shanghai, China, .gtoreq.98%
purity) were freshly prepared in order to avoid premature peptide
aggregation. The peptides were dissolved in water and left at room
temperature to form hydrogels. Depending on the peptide
concentration, the self-assembly process occurred immediately,
within hours or even within days (experimental time frame for
gelation). For higher peptide concentrations peptides were
dissolved in milliQ water by vortexing. If a forced and accelerated
hydrogel preparation was needed, the peptide solution was subjected
to sonication in a water bath (Barnstead Labline 9319
UltrasonicLC60H). No significant structural differences were
observed between hydrogels produced via self-assembly and those
whose assembly was facilitated by sonication. Few peptides formed
hydrogels more easily at elevated temperatures, i.e. at 50.degree.
C.
[0263] To study the effect of concentration variation, both
AcLD.sub.6 (L) and AciD.sub.3 (L) hydrogels were prepared with
varying concentration as specified above. To study the effect of
monovalent and divalent cations, AcLD.sub.6 (L) hydrogels were
prepared by dissolving peptide in 10, 50, 100 and 150 mM NaCl and
CaCl.sub.2 solutions. FESEM and rheology studies were further
performed to characterize the morphology and strength of these
hydro gels.
[0264] Preparation of gelatin and collagen gels: Gelatin (Type A, G
1890; Sigma Aldrich) hydrogels was prepared by first dissolving
gelatin in milli Q water by heating followed by cooling till the
gelation was observed. Collagen (Type I from bovine, Advanced
Biomatrix, USA) was diluted with PBS buffer to a concentration of
1.5 mg/ml and titrated to pH 7.4 using 0.1M NaOH. Gelation was
achieved by incubating the solution at 37.degree. C. for 1
hour.
Circular Dichroism (.omega.) Spectroscopy
[0265] Secondary peptide structures were analyzed by measuring
ellipticity spectra using the Aviv Circular Dichroism Spectrometer,
model 410. .omega. samples were prepared by diluting stock peptides
solutions (5-10 mg/ml) in water. The diluted peptide solutions were
filled in to a cuvette with 1 mm path length and spectra were
acquired. As a blank reference water was used and the reference was
subtracted from the raw data before molar ellipticity was
calculated. The calculation was based on the formula:
[.theta.].lamda.=.theta..sub.obs.times.1/(10 Lcn), where
[.theta.].lamda. the molar ellipticity at .lamda., in deg cm.sup.2
d/mol, is the observed ellipticity at .lamda., in mdeg, L is the
path length in cm, c is the concentration of the peptide in M, and
n is the number of amino acids in the peptide. Secondary structure
analysis was done using CDNN software.
Environmental Scanning Electron Microscopy (ESEM)
[0266] Samples were placed onto a sample holder of FEI Quanta 200
Environmental Scanning Electron Microscopy. The surface of interest
was then examined using accelerating voltage of 10 kV at a
temperature of 4.degree. C.
Field Emission Scanning Electron Microscopy (FESEM)
[0267] Samples were frozen at -20.degree. C. and subsequently to
-80.degree. C. Frozen samples were further freeze dried. Freeze
dried samples were fixed onto a sample holder using conductive tape
and sputtered with platinum from both the top and the sides in a
JEOL JFC-1600 High Resolution Sputter Coater. The coating current
used was 30 mA and the process lasted for 60 sec. The surface of
interest was then examined with a JEOL JSM-7400F Field Emission
Scanning Electron Microscopy system using an accelerating voltage
of 5-10 kV.
Rheological Measurements
[0268] To determine the viscoelastic properties of the
peptide-based hydro gels, hydro gels were subjected to dynamic
time, strain and frequency sweep experiments using the ARES-G2
rheometer (TA Instruments, Piscataway, N.J.) with the 25.0 mm
diameter titanium parallel plate geometry and a 0.8 mm gap
distance. Oscillatory frequency study was performed to compare the
strength of peptide based hydrogel with varying concentration of
peptides, or for peptide in presence of monovalent or divalent
ions. Oscillatory frequency sweep studies were performed at 0.1-100
rad/s frequency and 0.1% strain at 25.degree. C. and 50.degree.
C.
Ac-LD.sub.6 [L]:
[0269] Peptide sequence:
Ac-LIVAGD-COOH
[0270] Molecular weight: 629.56 (1) Temperature sweep study for
Ac-LD.sub.6 (L): [0271] (a) The peptide mixture was then placed on
rheometer lower plate. Following parameters were optimized: [0272]
Gap between two plates: 1 mm [0273] Strain: 10% [0274] Frequency:
6.28 rad/sec [0275] Temperature scan: 4.degree. C. to 60.degree. C.
[0276] Sample volume: 500 .mu.l (2) Frequency sweep study for
Ac-LD.sub.6(L): [0277] Optimized parameter required to perform
frequency sweep study [0278] Gap between two plates: 0.8 mm [0279]
Strain: 0.1% [0280] Temperature: 25 and 50.degree. C. [0281] Sample
volume: 1 ml [0282] Frequency scan: 0.1 rad/sec to 100 rad/sec
[0283] Concentration of Ac-LD-6 (L) in hydrogel: 10 mg/ml (3)
Effect of concentration variation of Ac-LD.sub.6 (L) on gel
strength: [0284] Optimized parameters that are required to perform
frequency sweep studies for measuring gel strength are as follows:
[0285] Gap between two plates: 0.8 mm [0286] Strain: 0.1% [0287]
Temperature: 25 and 50.degree. C. [0288] Sample volume: 1 ml [0289]
Frequency scan: 0.1 rad/sec to 100 rad/sec [0290] Concentrations of
Ac-LD.sub.6 (L) in hydrogels: 5 mg/ml, 10 mg/ml, 15 mg/ml and, 0
mg/ml and 30 mg/ml in water. (4) Effect of sodium chloride (NaCl)
on the gel strength of Ac-LD.sub.6 (L):
[0291] Effect of sodium chloride on Ac-LD.sub.6 (L) based
hydrogels, were studied by performing a frequency sweep study on
hydro gels prepared by dispersing 10 mg of Ac-LD-6 (L) in varying
concentration of NaCl solution for example 10 mM, 50 mM, 100 mM and
150 mM of NaCl solution using optimized procedure to form hydro
gels. Optimized parameter required to perform frequency sweep study
to measure gel strength in presence of NaCl are as follows: [0292]
Gap between two plates: 0.5 mm and 0.8 mm [0293] Strain: 10% and
0.1% respectively [0294] Temperature: 25.degree. C. and 50.degree.
C. [0295] Sample volume: 1 ml [0296] Frequency scan: 0.1 rad/sec to
100 rad/sec [0297] Concentrations of NaCl solutions used to prepare
10 mg/ml of Ac-LD-6 (L) Hydrogels: 10 mM, 50 mM, 100 mM, 150 mM
NaCl solution.
Cell Growth Experiments
[0298] In order to find out whether the peptide hydrogels can serve
as a scaffold for tissue engineering, its biocompatibility was
investigated. Different primary human cells were seeded on top of
the hydrogel after its gelation in tissue culture medium (DMEM
without serum) in 6-well, 4-well or 96-well culture plates, see the
culture conditions below. During the next 2-4 days no change of
medium was necessary, but eventually fresh media was added to the
wells. The cells were analyzed for viability.
[0299] Primary human renal proximal tubule cells (HPTCs) and
primary human umbilical vein endothelial cells (HUVECs) were
obtained from ScienCell Research Laboratories (Carlsbad, Calif.,
SA). HPTCs were cultivated in basal epithelial cell medium
supplemented with 2% fetal bovine serum (FBS) and 1% epithelial
cell growth supplement (all components obtained from ScienCell
Research Laboratories). The culture medium for HUVECs was
endothelial cell medium containing 5% FBS and 1% endothelial cell
growth supplement (ScienCell Research Laboratories). All cell
culture media used were supplemented with 1%
penicillin/streptomycin solution (ScienCell Research Laboratories),
and all cells were cultivated at 37.degree. C. in a 5% CO.sub.2
atmosphere. The seeding density of the cells was about
5.times.10.sup.4 cells/cm.sup.2. However since HUVECs are bigger
than HPTCs the cell number would be slightly lower than one for
HPTC cells (.about.4.5.times.10.sup.4 cells/cm.sup.2). Both cell
types had a confluency of about 80% in the wells after seeding.
Crosslinked Hydrogels
Material & Methods
Peptides
[0300] All peptides were synthesised at American Peptide Company
(CA, USA) using SPPS and purified to >95% (HPLC). Amino acid
content (AA %) analysis was performed and the net weight (gross
weight.times.AA %) was used for calculations.
Kinetics of Disulfide Formation
[0301] For air oxidation, LK.sub.6C was dissolved in MilliQ water
by vigorous vortexing for five minutes and dispensed into 20 .mu.L
aliquots. At appropriate time points, 180 .mu.L of DTNB (Sigma,
Singapore) working solution (4 mg/mL in 0.1M phosphate buffer pH
7.0) was mixed with the peptide solution for 15 minutes. Absorbance
at 412 nm was then measured (InfiniteM200, Tecan, Switzerland).
Using a calibration curve generated with L-cysteine (Sigma) as
standard (R.sup.2=0.999), the background-subtracted value was
normalised to the reading at 0 hour to give the % of thiol
remaining. For H.sub.2O.sub.2-assissted oxidation, LK.sub.6C was
dissolved in water (HPLC grade, J. T. Baker, NJ, USA) containing
0.06% H.sub.2O.sub.2 (Merck, Sinagpore) and dispensed into
aliquots. At appropriate time points, the aliquots were analysed
using an Aquity.RTM. UPLC (Waters, SA) fitted with a
single-quadrupole MS. Using a calibration curve generated with pure
LK.sub.6C as standard (R.sup.2=0.999), the area under the peak
corresponding to LK.sub.6C monomer was normalised to that at 0 hour
to give the % of thiol remaining.
Gel Casting
[0302] Peptide was dissolved in 200 .mu.L of water+/-H.sub.2O.sub.2
and filled into custom-made hollow ring casts (diameter .about.1
cm). The ring ends were sealed with parafilm to minimise
evaporation and the cast was kept at 25.degree. C. for 22 hours
before further manipulations.
[0303] Rheology: The rheological properties of casted gels were
measured with an ARES-G2 (TA Instruments, USA) using the
oscillation method. Frequency-sweep studies were performed with
.omega.=0-100 rad/s at strain, .gamma.=0.1%. The gel stiffness was
represented by plotting G' against .omega.. Amplitude-sweep studies
were performed beforehand at 1 Hz with y=0-100%. The LVE limit of
the gel was defined as the value of .gamma. when G' first dropped
below 90% of the average initial value.
Rheology
[0304] The rheological properties of casted gels were measured with
an ARES-G2 (TA Instruments, SA) using the oscillation method.
Frequency-sweep studies were performed with .omega.=0-100 rad/s at
strain, .gamma.=0.1%. The gel stiffness was represented by plotting
G' against .omega.. Amplitude sweep studies were performed
beforehand at 1 Hz with .gamma.=0-100%. The LVE limit of the gel
was defined as the value of .gamma. when G' first dropped below 90%
of the average initial value.
FESEM
[0305] Freeze-dried gels were deposited onto carbon tapes,
sputtered with platinum and observed under a JSM-7400F electron
microscope (Jeol, Tokyo, Japan).
3D Cell Culture
[0306] LK.sub.6C+/-CRGD gels were casted directly into 8-chamber
Lab-Tek.RTM. wells (Nunc, NY, USA) and purified as reported in the
SI. 0.5 mL of cell suspension in regular completed DMEM
(Invitrogen, Singapore) was either seeded onto the gel or directly
into the well (2D control) and incubated for four days. 0.5 .mu.L
of calcein-AM (Invitrogen) was then added to the media to identify
live cells and images were captured using a confocal microscope
(LSM5 DUO, Carl Zeiss, Germany). Beforehand, calcein-AM was
verified to stain only live cells as ethanol- and
H.sub.2O.sub.2-treated cells excluded the dye. All images, except
for the 2D controls, were presented as mergers of several z-stacks.
To further verify the 3D distribution of cells, the gel was
embedded vertically in Jung Tissue Freezing Medium (Leica
Instruments GmbH, Germany) and 20 .mu.m cross-sectional slices were
obtained with a cryostat (CM3050 S, Leica). Sections were mounted
with coverslips and imaged as above.
Quantification of Cell-Spread Area
[0307] Confocal images after calcein staining were processed using
ImageJ (NIH, USA) to obtain the average cell-spread area under
various culture conditions. For cells in 3D gel culture, images
used for quantification were mergers of several z-stacks to capture
the maximum cell-spread area. In all cases, at least three
independent experiments with at least two locations selected from
each culture were used for averaging.
MTT Viability Assay
[0308] After four days of 3D gel culture, cells were separated from
the substrate by trypsination and centrifugation and re-seeded into
48-well plates (Nunc) overnight for attachment. MTT (Sigma)
dissolved in PBS was then added to the media for four hours, before
the formazan crystals were dissolved using DMSO (Sigma). Absorbance
was read at 560 nm, subtracted with the 680 nm reference and
normalised to the appropriate control to give an indication of
relative cell viability.
Statistical Analysis
[0309] ANOVA testing (OriginLab Corporation, MA, USA) was performed
on sample means with p<0.05 being denoted by * and accepted to
be statistically significant.
Results
[0310] LK.sub.6C Peptides can be Casted into 3-Dimensional
Shapes
[0311] LK.sub.6C peptides can be easily casted into 3-dimensional
gels using the appropriate moulds (see FIG. 18). In this
experiment, a mould in the shape of a ring was used and LK.sub.6C
gels were casted using water or completed growth medium. Dextran
particles, employed here as a model cargo, could also be
encapsulated within the gel matrix.
H.sub.2O.sub.2-Assisted Formation of Disulfide Crosslinks
[0312] The kinetics of disulfide formation under air oxidation is
inefficient (FIG. 19). The addition of H.sub.2O.sub.2 speeds up
disulfide formation (FIG. 20). LC-MS confirms the formation of
disulfide-crosslinked dimers in the presence of H.sub.2O.sub.2
(FIG. 21). The data further suggests that the addition of HRP has
no significant effect on the kinetics of disulfide formation (FIG.
19). The rate of disulfide formation can also be accelerated by
increasing the amount of H.sub.2O.sub.2 used (FIG. 22).
Oxidation Strategy
[0313] Two different methods were devised for the
H.sub.2O.sub.2-assissted cross-linking: 1) The cast-and-soak
method, where the gel is first being casted overnight before being
soaked in an oxidative solution. 2) The in situ oxidation method
where the aqueous solution used to dissolve the peptide powder
already contains the oxidative agent (FIG. 23).
Oxidation Increases the Long-Term Stability of LK.sub.6C in
Water
[0314] LK.sub.6 or LK.sub.6C gels were first casted and then soaked
in water for various durations. After 24 hours of water soak, the
non-crosslinkable LK6 gels were completely degraded while the
non-oxidised LK.sub.6C gels were severely degraded (FIG. 24). In
stark contrast, LK.sub.6C gels oxidised for 2 hours using the
cast-and-soak method and LK.sub.6C gels oxidised in situ remained
intact after 96 hours of water soak. These observations are also
supported by rheological measurements (FIG. 28). Therefore, the
strategy of disulfide cross-linking has increased the resistance of
the gel to degradation and has made it possible to use the gel at a
lower concentration compared to previous formulations. This, in
turn, will translate into significant cost savings.
Gel Oxidised Using the Cast-and-Soak Method is Uniformly
Oxidised
[0315] LK.sub.6C gels were casted overnight and then soaked in
H.sub.2O.sub.2 solution for 2 hours (see scheme in FIG. 25). Then
the surfaces (circumference+top+bottom layers) were separated from
the core and it was shown that the amount of H.sub.2O.sub.2 and
disulfide/thiol ratio of both the layers were comparable (FIGS. 25
and 26). This suggests that oxidation is not restricted to the
surfaces of the gel and that the diffusion of H.sub.2O.sub.2 is
rapid in this experimental set-up. However, if the gel is casted
into a 48-well plate and the H.sub.2O.sub.2 solution is merely
applied over the top of the gel (as per later experiments, see
scheme in FIG. 31), the diffusion rate of H.sub.2O.sub.2 will be
reduced as only the top surface is accessible to the solution in
this configuration.
Effects of Oxidation on G' and Elasticity of the Gel
[0316] The rheological properties of various LK.sub.6C gels were
next quantified. As a measure of stiffness, the elastic modulus, G'
was plotted against angular frequency, m after the gel was
subjected to 2-24 hours of cast-and-soak oxidation with 0.06%
H.sub.2O.sub.2, or 24 hours of in situ oxidation with 0.03-0.1%
H.sub.2O.sub.2 (FIG. 27 A). Compared to non-oxidised gels, the
stiffness was either maintained or increased following different
oxidation regimes. Interestingly, the stiffness achieved after 24
hours of cast-and-soak differed from that after in situ oxidation
with 0.06% H.sub.2O.sub.2. This could be due to the different
availability of H.sub.2O.sub.2 and sequence of oxidation. More
specifically, in cast-and-soak, the gel has access to a sink of
H.sub.2O.sub.2 and cross-linking happens only after self-assembly
of peptide fibers; while during in situ oxidation, H.sub.2O.sub.2
is limited to the gel volume and cross-linking occurs concurrently
with self-assembly. The linear viscoelastic (LVE) limit
(elasticity) of the gel, however, increased 2.4-3.8 fold following
oxidation (FIG. 27B). This is presumably attributed to the
introduction of additional chemical bonds. Other strategies to
modulate the stiffness and elasticity of LK.sub.6C gels include
varying its concentration (FIG. 29A/B) or doping LK.sub.6C with LK6
(FIG. 29C/D). This also allows the tuning of the amount of thiol
groups available in the gel.
Fibrous Microstructures of Gels
[0317] Field emission scanning electron microscope (FESEM) revealed
that the fibrous microstructures of LK.sub.6C gels were maintained
after various oxidation regimes (FIG. 30).
H.sub.2O.sub.2 and Residual Acid Readily Removed
[0318] Using a configuration closer to cell culture experiments,
gels were casted overnight in 48-well plates and water was applied
over the gels to purify it before the introduction of cells. The
majority of H.sub.2O.sub.2 and residual acid can be removed be
simply changing the water regularly (FIG. 31). Growth medium can be
used instead of water to ensure that the gel is essentially free of
H.sub.2O.sub.2 and adjusted to a pH amenable for cell culture.
Gradual and Tunable Release Kinetics
[0319] As a model system, dextran particles were encapsulated
within the gel and their release profile monitored. No burst
release was observed in both cases and release rate was suppressed
in the oxidized sample, presumably due to the increased stability
in water (FIG. 32).
Functionalization of the Hydrogel
[0320] The gel was next functionalized with a bioactive signal.
CRGD (0-1 mg/mL) was simply mixed with LK.sub.6C (fixed at 10
mg/mL, i.e., CRGD ligand density 0-9. 1 gross weight %) in the
presence of H.sub.2O.sub.2 and ring-casted overnight. UPLC-MS
confirmed the formation of LK.sub.6C-CRGD conjugate and the
disappearance of free CRGD (FIG. 33). The mild and simple reaction
conditions are to be noted. A person skilled in the art will also
recognize the versatility of this peptide platform as well as the
fact that future conjugations need not be limited to RGD. The gels
were next purified by a dialysis-inspired method whereby water was
layered on top and changed regularly. Doing so, >96% of
unreacted H.sub.2O.sub.2 was removed after 4 hrs and >99% after
7 hrs (FIG. 31 A). Similarly, >85% of residual acid from SPPS
was removed after 8 hrs (FIG. 31 B).
Biocompatibility and Use for Cell Culture
[0321] To test for biocompatibility, HepG2 cells were either seeded
directly into wells (2D control) or onto purified LK.sub.6C gels
conjugated with different concentrations of RGD. After four days,
calcein staining revealed that cells were viable in all experiments
(FIG. 34 A/B). This was repeated with primary rabbit fibroblasts
(FIG. 35), 3T3 murine fibroblasts (FIG. 38) and NIH-3T3 murine
fibroblasts (data not shown), confirming the biocompatibility of
the gels. With regular media changes, cells remained viable for at
least 3 weeks (FIG. 38). Images obtained from the gel cultures were
presented as mergers of several z-stacks. While that may already
suggest a 3D distribution, the inventors wanted to verify that they
were not artifacts due to, e.g., the meniscus on gel surfaces or
the gel being casted in a non-horizontal position. The gels were
therefore embedded vertically and cross-sectional slices were
obtained for depth profiling. From FIG. 34B, calcein-stained cells
were observed to infiltrate the gel, resulting in multi-layered
growth and validating their 3D spatial distribution. As visually
suggested in FIG. 34A, HepG2 cells appeared to have proliferated
faster within gels with RGD compared to the ones without.
[0322] This was supported by data from the MTT assay (FIG. 34C).
Being an anchorage-dependent cell line, the better adhesion to RGD
conjugated gels presumably provided the cells with a more ideal
growth environment. Higher magnification images (FIG. 36) were
subsequently taken and the average cell-spread area of HepG2 cells
was quantified. Interestingly, 3D gel culture had insignificant
effects (p>0.05) on the average cell-spread area of HepG2 cells
compared to those in regular 2D culture (FIG. 34D). The amount of
RGD ligand present also did not significantly (p>0.05) impact
the cell spreading area of HepG2 cells. The trend was different,
though, in the case of primary rabbit fibroblasts (FIG. 35B) and
NIH-3T3 murine fibroblasts (data not shown), both of which spreaded
more in 2D culture. Cells are known to respond to mechanical
properties such as stiffness and elasticity of their
microenvironment and these observations are consistent with an
earlier report that hydrogel-encapsulated murine fibroblasts
maintained rounder morphologies. However, while a transition from
2D to 3D culture caused the fibroblasts to spread differently, the
amount of RGD ligand present had insignificant effects (p>0.05).
Compared to before CRGD attachment, the gel was as stiff (FIG. 37
A) but less elastic (FIG. 37B) after conjugation.
Non-Allergenicity and Non-Toxicity
[0323] Experiments conducted by the CRO Toxikon further showed that
LK.sub.6C was non-allergenic and non-toxic. More particularly,
LK.sub.6C caused no sensitization on the skins of guinea pigs in
the direct contact Kligman maximization test during a GLP study
conducted by Toxikon according to the ISO 10993-10 guidelines.
Also, LK.sub.6C exhibited no significant toxicity in the direct
contact V79 colony assay in another GLP study performed by
Toxikon.
Use in Wound Treatment
[0324] A first round of wound healing experiments with mice was
conducted. In this model, the epidermis and dermis of mice we
removed to simulate injury (FIG. 39). The following groups were
analyzed:
a) No treatment control (i.e., simply bandage up the wound)
b) Application of LK.sub.6C-CRGD gel
[0325] c) Application of LK.sub.6C-CRGD gel with 3T3 murine
fibroblasts cultured in 3D
[0326] Here, the fibroblasts (the major population of the dermis)
act as a therapeutic/bioactive agent. The hypothesis is that the
3T3 cells secrete factors that encourage the growth of
keratinocytes, which make up the epidermis. This should further aid
healing as compared to the gel-only treatment (group b). After 2
weeks, the extent of vascularisation of the wound (a prerequisite
feature of recovery) was indeed most significant in group c,
followed by group b and then group a (FIG. 40). Subsequent
histology also showed the regeneration of a significant dermal
layer in group c mice. In comparison, group b mice had less
regenerated dermis, and group a mice had the least extent of dermal
regeneration.
SUMMARY
[0327] Cysteine-mediated disulfide-crosslinked ultra small peptide
hydrogel were analyzed. Cross-linking was driven by H.sub.2O.sub.2
and produced only water as a by-product. Moreover, H.sub.2O.sub.2
helped to maintain the sterility of the gel and can be virtually
removed before the introduction of cells. Oxidation increased both
the elasticity of the gel and its ability to keep its shape after
being soaked in water. Due to the cysteine residue, bioactive
signals can be conjugated to the peptide fibers using facile
chemistry and gels can be easily purified. Gels formed were shown
to support the true 3D distribution of cells and influence their
growth and spreading characteristics. Furthermore, their
applicability in wound treatment was demonstrated.
[0328] The listing or discussion of a previously published document
in this specification should not necessarily be taken as an
acknowledgement that the document is part of the state of the art
or is common general knowledge. All documents listed are hereby
incorporated herein by reference in their entirety for all
purposes.
[0329] Exemplary embodiments of the invention illustratively
described herein may suitably be practiced in the absence of any
element or elements, limitation or limitations, not specifically
disclosed herein. Thus, for example, the terms "comprising",
"including", "containing", etc. shall be read expansively and
without limitation. Additionally, the terms and expressions
employed herein have been used as terms of description and not of
limitation, and there is no intention in the use of such terms and
expressions of excluding any equivalents of the features shown and
described or portions thereof, but it is recognized that various
modifications are possible within the scope of the invention
claimed. Thus, it should be understood that although the present
invention has been specifically disclosed by exemplary embodiments
and optional features, modification and variation of the inventions
embodied therein herein disclosed may be resorted to by those
skilled in the art, and that such modifications and variations are
considered to be within the scope of this invention.
[0330] The invention has been described broadly and generically
herein. Each of the narrower species and sub generic groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0331] Other embodiments are within the following claims. In
addition, where features or aspects of the invention are described
in terms of Markush groups, those skilled in the art will recognize
that the invention is also thereby described in terms of any
individual member or subgroup of members of the Markush group.
Sequence CWU 1
1
6017PRTArtificial SequenceChemically synthesized peptide 1Leu Ile
Val Ala Gly Asp Asp 1 5 26PRTArtificial SequenceChemically
synthesized peptide 2Leu Ile Val Ala Gly Asp 1 5 36PRTArtificial
SequenceChemically synthesized peptide 3Leu Ile Val Ala Gly Asp 1 5
46PRTArtificial SequenceChemically synthesized peptide 4Ala Ile Val
Ala Gly Asp 1 5 56PRTArtificial SequenceChemically synthesized
peptide 5Ala Ile Val Ala Gly Asp 1 5 66PRTArtificial
SequenceChemically synthesized peptide 6Ile Leu Val Ala Gly Asp 1 5
76PRTArtificial SequenceChemically synthesized peptide 7Ile Leu Val
Ala Gly Asp 1 5 86PRTArtificial SequenceChemically synthesized
peptide 8Leu Ala Val Ala Gly Asp 1 5 96PRTArtificial
SequenceChemically synthesized peptide 9Leu Ile Val Ala Ala Asp 1 5
106PRTArtificial SequenceChemically synthesized peptide 10Leu Ile
Val Ala Gly Ser 1 5 116PRTArtificial SequenceChemically synthesized
peptide 11Leu Ile Val Ala Gly Ser 1 5 126PRTArtificial
SequenceChemically synthesized peptide 12Ala Ile Val Ala Gly Ser 1
5 136PRTArtificial SequenceChemically synthesized peptide 13Ile Leu
Val Ala Gly Ser 1 5 146PRTArtificial SequenceChemically synthesized
peptide 14Leu Ile Val Ala Gly Thr 1 5 156PRTArtificial
SequenceChemically synthesized peptide 15Ala Ile Val Ala Gly Thr 1
5 167PRTArtificial SequenceChemically synthesized peptide 16Leu Ile
Val Ala Gly Glu Glu 1 5 176PRTArtificial SequenceChemically
synthesized peptide 17Leu Ile Val Ala Gly Glu 1 5 186PRTArtificial
SequenceChemically synthesized peptide 18Leu Ile Val Ala Gly Glu 1
5 196PRTArtificial SequenceChemically synthesized peptide 19Leu Ile
Val Ala Gly Lys 1 5 205PRTArtificial SequenceChemically synthesized
peptide 20Leu Ile Val Ala Asp 1 5 215PRTArtificial
SequenceChemically synthesized peptide 21Leu Ile Val Gly Asp 1 5
224PRTArtificial SequenceChemically synthesized peptide 22Ile Val
Ala Asp 1 234PRTArtificial SequenceChemically synthesized peptide
23Ile Val Ala Asp 1 243PRTArtificial SequenceChemically synthesized
peptide 24Ile Val Asp 1 254PRTArtificial SequenceChemically
synthesized peptide 25Ile Ile Ile Asp 1 264PRTArtificial
SequenceChemically synthesized peptide 26Ile Ile Ile Asp 1
274PRTArtificial SequenceChemically synthesized peptide 27Ile Ile
Ile Lys 1 284PRTArtificial SequenceChemically synthesized peptide
28Ile Ile Ile Lys 1 293PRTArtificial SequenceChemically synthesized
peptide 29Ile Ile Asp 1 303PRTArtificial SequenceChemically
synthesized peptide 30Ile Ile Asp 1 317PRTArtificial
SequenceChemically synthesized peptide 31Leu Ile Val Ala Gly Asp
Asp 1 5 326PRTArtificial SequenceChemically synthesized peptide
32Leu Ala Val Ala Gly Asp 1 5 336PRTArtificial SequenceChemically
synthesized peptide 33Leu Ile Val Ala Ala Asp 1 5 346PRTArtificial
SequenceChemically synthesized peptide 34Ala Ile Val Ala Gly Ser 1
5 356PRTArtificial SequenceChemically synthesized peptide 35Ile Leu
Val Ala Gly Ser 1 5 366PRTArtificial SequenceChemically synthesized
peptide 36Leu Ile Val Ala Gly Thr 1 5 376PRTArtificial
SequenceChemically synthesized peptide 37Ala Ile Val Ala Gly Thr 1
5 387PRTArtificial SequenceChemically synthesized peptide 38Leu Ile
Val Ala Gly Glu Glu 1 5 396PRTArtificial SequenceChemically
synthesized peptide 39Leu Ile Val Ala Gly Lys 1 5 405PRTArtificial
SequenceChemically synthesized peptide 40Leu Ile Val Ala Asp 1 5
415PRTArtificial SequenceChemically synthesized peptide 41Leu Ile
Val Gly Asp 1 5 423PRTArtificial SequenceChemically synthesized
peptide 42Ile Val Asp 1 437PRTArtificial SequenceChemically
synthesized peptide 43Leu Ile Val Ala Gly Lys Cys 1 5
447PRTArtificial SequenceChemically synthesized peptide 44Leu Ile
Val Ala Gly Ser Cys 1 5 457PRTArtificial SequenceChemically
synthesized peptide 45Leu Ile Val Ala Gly Asp Cys 1 5
467PRTArtificial SequenceChemically synthesized peptide 46Ile Leu
Val Ala Gly Lys Cys 1 5 477PRTArtificial SequenceChemically
synthesized peptide 47Ile Leu Val Ala Gly Asp Cys 1 5
486PRTArtificial SequenceChemically synthesized peptide 48Leu Ile
Val Ala Gly Cys 1 5 496PRTArtificial SequenceChemically synthesized
peptide 49Ala Ile Val Ala Gly Cys 1 5 506PRTArtificial
SequenceChemically synthesized peptide 50Ile Leu Val Ala Gly Cys 1
5 514PRTArtificial SequenceChemically synthesized peptide 51Ile Val
Lys Cys 1 524PRTArtificial SequenceChemically synthesized peptide
52Ile Val Asp Cys 1 534PRTArtificial SequenceChemically synthesized
peptide 53Ile Val Ser Cys 1 545PRTArtificial SequenceChemically
synthesized peptide 54Leu Ile Val Ala Gly 1 5 555PRTArtificial
SequenceChemically synthesized peptide 55Ile Leu Val Ala Gly 1 5
565PRTArtificial SequenceChemically synthesized peptide 56Leu Ile
Val Ala Ala 1 5 575PRTArtificial SequenceChemically synthesized
peptide 57Leu Ala Val Ala Gly 1 5 584PRTArtificial
SequenceChemically synthesized peptide 58Ile Val Ala Gly 1
594PRTArtificial SequenceChemically synthesized peptide 59Leu Ile
Val Ala 1 604PRTArtificial SequenceChemically synthesized peptide
60Leu Ile Val Gly 1
* * * * *