U.S. patent application number 13/863294 was filed with the patent office on 2013-12-19 for hydrogel compositions.
This patent application is currently assigned to The University of Strathclyde. The applicant listed for this patent is Julie Elizabeth Gough, Vineetha Jayawarna, Andrew Smith, Rein Vincent Ulijn. Invention is credited to Julie Elizabeth Gough, Vineetha Jayawarna, Andrew Smith, Rein Vincent Ulijn.
Application Number | 20130338084 13/863294 |
Document ID | / |
Family ID | 37997230 |
Filed Date | 2013-12-19 |
United States Patent
Application |
20130338084 |
Kind Code |
A1 |
Ulijn; Rein Vincent ; et
al. |
December 19, 2013 |
HYDROGEL COMPOSITIONS
Abstract
Hydrogel compositions comprise an aqueous dispersion phase and a
plurality of peptides, or derivatives, or analogues thereof. Each
peptide comprises at least two amino acid residues and an aromatic
stacking ligand and the hydrogel is formed by self-assembly of said
peptides in said aqueous dispersion medium. The aqueous dispersion
phase is physiologically acceptable and may have a pH of 6 to 8, as
may the hydrogel itself. The hydrogel may be used for cell culture
or for treatment of medical conditions characterised by tissue
loss/damage.
Inventors: |
Ulijn; Rein Vincent;
(Rainhill, GB) ; Jayawarna; Vineetha; (Cheadle
Hulme, GB) ; Smith; Andrew; (Leeds, GB) ;
Gough; Julie Elizabeth; (Ramsbottom, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ulijn; Rein Vincent
Jayawarna; Vineetha
Smith; Andrew
Gough; Julie Elizabeth |
Rainhill
Cheadle Hulme
Leeds
Ramsbottom |
|
GB
GB
GB
GB |
|
|
Assignee: |
The University of
Strathclyde
Galsgow
GB
|
Family ID: |
37997230 |
Appl. No.: |
13/863294 |
Filed: |
April 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11470962 |
Sep 7, 2006 |
8420605 |
|
|
13863294 |
|
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|
60714583 |
Sep 7, 2005 |
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Current U.S.
Class: |
514/21.8 ;
435/29; 435/375; 435/404; 514/21.9; 514/21.91 |
Current CPC
Class: |
A61K 38/05 20130101;
A61K 9/0014 20130101; A61K 38/06 20130101; A61K 38/07 20130101;
A61K 47/42 20130101; A61K 38/08 20130101 |
Class at
Publication: |
514/21.8 ;
514/21.9; 514/21.91; 435/404; 435/375; 435/29 |
International
Class: |
A61K 38/07 20060101
A61K038/07; A61K 38/05 20060101 A61K038/05; A61K 38/08 20060101
A61K038/08; A61K 38/06 20060101 A61K038/06 |
Claims
1. A hydrogel composition comprising an aqueous dispersion phase
and a plurality of peptides, or derivatives, or analogues thereof,
wherein each peptide comprises at least two amino acid residues and
an aromatic stacking ligand, and wherein the hydrogel is formed by
self-assembly of said peptides in said aqueous dispersion
medium.
2. A hydrogel composition as claimed in claim 1 wherein the aqueous
dispersion phase is physiologically acceptable.
3. (canceled)
4. A hydrogel composition as claimed in claim 1 having a pH of
6-8.
5. (canceled)
6. A hydrogel composition as claimed in claim 1 wherein the peptide
comprises a dipeptide and the aromatic stacking ligand.
7. (canceled)
8. A hydrogel composition as claimed in claim 6 wherein the
dipeptide is Leu-Leu.
9. A hydrogel composition as claimed in claim 6 wherein the
hydrogel comprises first and second peptides each incorporating a
dipeptide and an aromatic stacking ligand wherein the dipeptide of
the first peptide is Phe-Phe and the dipeptide of the second
peptide is Gly-Gly.
10. A hydrogel composition as claimed in claim 1 wherein the
peptide comprises a tripeptide and the aromatic stacking
ligand.
11. (canceled)
12. A hydrogel composition as claimed in claim 10 wherein the
tripeptide is Leu-Leu-Leu.
13. A hydrogel composition as claimed in claim 1 wherein the
peptide has the structure A.S.L.-AA.sub.1-AA.sub.2-X, where A.S.L.
denotes the Aromatic Stacking Ligand, where AA.sub.n denotes amino
acid residues in the peptide where n is the number of the amino
acid residue, and where X is an amino acid residue selected from
the group consisting of Phe, Leu, IKVAV, RGD and KPV.
14. A hydrogel composition as claimed in claim 1 wherein the
peptide comprises an Arginine-Glycine-Aspartate (RGD) peptide
motif.
15. (canceled)
16. A hydrogel composition as claimed in claim 1 wherein at least
one peptide in the hydrogel comprises an
Isoleucine-Lysine-Valine-Alanine-Valine (IKVAV) peptide motif.
17. (canceled)
18. A hydrogel composition as claimed in claim 1 wherein at least
one peptide in the hydrogel comprises a Lysine-Proline-Valine (KPV)
motif.
19. (canceled)
20. A hydrogel composition as claimed in claim 1 wherein the
hydrogel incorporates a bioadditive which comprises an aromatic
stacking ligand and has the structure A.S.L.-K, where A.S.L.
denotes a Aromatic Stacking Ligand, and where K denotes a Lysine
residue.
21.-22. (canceled)
23. A hydrogel composition as claimed in claim 20 wherein the
peptide comprises a mixture of Fmoc-Phe-Phe and Fmoc-Lys.
24. A liquid hydrogel precursor composition comprising an aqueous
dispersion phase and a plurality of peptides, or derivatives, or
analogues thereof, wherein each peptide comprises at least two
amino acid residues and an aromatic stacking ligand, said
composition being capable of being induced to form a hydrogel by
self-assembly of said peptides.
25. A method of treating an individual suffering from a medical
condition characterised by tissue loss/damage, the method
comprising providing at a treatment site of an individual in need
of such treatment, a hydrogel comprised of gel-forming peptides, or
derivatives, or analogues thereof, wherein each peptide comprises
at least two amino acid residues and an aromatic stacking
ligand.
26.-27. (canceled)
28. A cell-supporting medium comprising a hydrogel composition as
claimed in claim 1 and at least one cell.
29. A method of preparing a cell supporting medium according to
claim 28, the method comprising the steps of:-- (i) contacting
either a hydrogel of the first aspect, or a liquid hydrogel
precursor composition according to the second aspect, or a hydrogel
composition according to the second with at least one cell; and
(ii) exposing the hydrogel or composition to conditions such that
the at least one cell is supported on and/or in a hydrogel, thereby
forming a cell-supporting medium.
30. A method of culturing cells wherein the cells are cultured on
or in a hydrogel composition as claimed in claim 1.
31. A method as claimed in claim 30 for use in vitro testing,
pharmaceutical screening or as extracellular matrix models.
Description
FIELD OF INVENTION
[0001] The present invention relates to hydrogels and particularly,
although not exclusively, to hydrogels formed from self-assembling
peptides. More specifically, the invention relates to the use of
such hydrogels as cell supporting media and cell scaffolds, and to
methods of preparing such scaffolds. The invention further extends
to uses of the cell supporting media and scaffolds, for example, in
medicine, including methods of treatment.
BACKGROUND
[0002] All publications herein are incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference. The following description includes information that may
be useful in understanding the present invention. It is not an
admission that any of the information provided herein is prior art
or relevant to the presently claimed invention, or that any
publication specifically or implicitly referenced is prior art.
[0003] Pre-designed self-assembling scaffolds are highly
advantageous in areas such as tissue regeneration/engineering, 3D
cell culture, in vitro toxicity testing, understanding
cell/extracellular matrix interactions, controlled stem cell
differentiation, studies of mechanical loading effects on cells,
and the study of metastasis models. In particular, developments in
the use of self-assembling peptides provide potential for the use
of such novel bionanomaterials in tissue engineering. The various
properties of the amino acids in peptides, their biological
compatibility, and the inherent properties of their bonded
structure make peptides a very powerful building block for the
fabrication of self-assembling scaffolds.
[0004] Advances have been made in creating synthetic mimics of the
Extracellular Matrix for in vivo and in vitro applications. Some
researchers have described the use of peptides with alternating
charged, hydrophobic and hydrophilic amino acids to culture nerve
cells, endothelial cells and chondrocytes. Other researchers have
demonstrated the use of synthetic amphiphile peptide-containing
molecules that can self-assemble into fibrous scaffolds that
support cell growth and stem cell differentiation. These successes
illustrate that man-made hydrogels could be useful for forming
scaffold materials for 3D cell culture and tissue engineering
applications.
[0005] Xu et. al. (J. Am. Chem. Soc. 2003, 125, 13680) described
that Fmoc (fluorenylmethoxycarbonyl) protected di-peptides could
form fibrous scaffolds at low pH values by taking advantage of
.pi.-stacking of the highly conjugated Fmoc group. Examples of
Fmoc-dipeptides disclosed by Xu et al as being capable of forming
such gels are Fmoc-D-Ala-D-Ala (3), Fmoc-L-Ala-L-Ala (3),
Fmoc-Gly-Gly (3), Fmoc-Gly-D-Ala (5) and Fmoc-Gly-L-SER (5), the
numbers in parentheses being the pH value for gelation. Fmoc is
widely used as a protecting group in peptide chemistry and when
coupled to amino acids, is known to have anti-inflammatory
properties, as demonstrated in animal studies. The Fmoc group acts
as a "stacking ligand", thought to offer order and directionality
to the self-assembly process. However, Xu et al carried out all of
their investigations at substantially acidic pH's (i.e. pH 3-5),
and did not investigate whether the compounds could be used in
biologically acceptable (ie. physiologically agreeable)
conditions.
[0006] Although considerable efforts have been made towards
understanding the behaviour of hydrogel scaffolds, the present
knowledge on the subject is very limited as much of these studies
have been based on trial and error. Furthermore, little has been
reported on the rules that govern self-assembly or the functioning
of the peptide scaffolds under different conditions. For use of
such scaffolds in biological or medical conditions, it is important
to understand the scaffold behaviour, especially under
environmental conditions similar to those experienced in vivo.
Furthermore, ultimately, researchers would like to design scaffolds
rationally for use in vivo.
[0007] Furthermore, up until now, it has not been possible to make
scaffolds from small molecule building blocks that are: (i) stable
under tissue culture conditions (i.e. high ionic strength, and pH
7); (ii) of similar dimensions to fibrous components of the
extracellular matrix; (iii) capable of supporting cell culture in
3D; (iv) optically transparent; and (v) capable of liquid to gel
transitions on demand by biocompatible means.
[0008] Therefore, it is an aim of the present invention to obviate
or mitigate one or more of the problems of the prior art, whether
identified herein or elsewhere, and to provide improved hydrogels,
which may be used in vitro or in vivo to support cell cultures, and
to provide methods of treatment, which use such hydrogels.
BRIEF DESCRIPTION OF THE FIGURES
[0009] Exemplary embodiments are illustrated in referenced figures.
It is intended that the embodiments and figures disclosed herein
are to be considered illustrative rather than restrictive.
[0010] FIG. 1 depicts spectroscopic data and putative structures in
relation to hydrogels comprised of Fmoc-Phe-Phe in accordance with
various embodiments of the present invention.
[0011] FIG. 2 shows chondrocyte cell culture in self-assembled
Fmoc-dipeptide hydrogel scaffolds in accordance with various
embodiments of the present invention. (a): scheme representing
formation of gel in the presence of cells (b): cell morphological
phenotype is retained on surface of gel Fmoc-Phe-Phe-OH (c): two
photon fluorescence microscopy reveals the presence of DAPI stained
cells throughout the gel Fmoc-Phe-Phe-OH (d): ESEM shows the
structure of gel Fmoc-Phe-Phe-OH+Fmoc-Gly-Gly-OH with chondrocytes
attached (arrows).
[0012] FIG. 3 shows a colorimetric assay (MTT Assay) which shows
that the number of surviving cells observed at three different time
up to 7 days has a continuous cell growth in accordance with
various embodiments of the present invention. Black=hydrogel 1
(Phe-Phe); Light grey=hydrogel 2 (Gly-Gly+Phe-Phe); Dark
grey=hydrogel 3 (Phe-Phe plus Lys); White=tissue culture plastic
control. Error bars represent standard deviations of mean values
where n=3.
[0013] FIG. 4 shows the structure of Fmoc used in accordance with
various embodiments of the present the invention.
[0014] FIG. 5 illustrates the appearances of human adult dermal
fibroblasts cultured on top of self-assembled peptide hydrogels of
Fmoc-Phe-PheOH at time points of 4 hours (spreaded), 6 hours
(spreaded) and 24 hours (rounded) in accordance with various
embodiments of the present invention.
[0015] FIG. 6 illustrates the live/dead staining of cells inside
Fmoc-Phe-Phe-OH hydrogel in accordance with various embodiments of
the present invention. A: magnified from part of figure B, showing
partially spreaded cells (3 hours after culture); B: 3 hours after
culture; C: 72 hours after culture.
[0016] FIG. 7 illustrates the results of the LDH assay (as
described in Example 6) of cell viability (3D culture of human
adult dermal fibroblasts in Fmoc-Phe-Phe-OH) in accordance with
various embodiments of the present invention.
[0017] FIG. 8 illustrates the cell phenotype and size comparison
(as described in Example 7) in accordance with various embodiments
of the present invention. (A) Fmoc-GGRGD+Fmoc-Phe-Phe-OH; (C)
Fmoc-GGRGE+Fmoc-Phe-Phe-OH. The arrows point to the cells.
[0018] FIG. 9 illustrates the expression of GAPDH and type I
collagen by MSCs in Fmoc-F-F gels after 14 days in accordance with
various embodiments of the present invention. Agarose gel showing
results of PCR on Fmoc-Phe-Phe gels seeded with human MSCs
following 14 days in culture is depicted. Mw=molecular weight
ladder. Lane 1=GAPDH, lane 2=SOX-9, lane 3=type I collagen, lane
4=type II collagen, lane 5=aggrecan.
[0019] FIG. 10 illustrates a comparison of Fmoc and CBz as aromatic
stacking ligands in accordance with various embodiments of the
present invention, A: the structure of Fmoc-Phe-Phe-OH; B: the
structure of Cbz-Phe-Phe-OH; C: Cyro-SEM image of Fmoc-Phe-Phe-OH;
D: Cryo-SEM image of Cbz-Phe-Phe-OH.
DESCRIPTION OF THE INVENTION
[0020] All references cited herein are incorporated by reference in
their entirety as though fully set forth. Unless defined otherwise,
technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which
this invention belongs. One skilled in the art will recognize many
methods and materials similar or equivalent to those described
herein, which could be used in the practice of the present
invention. Indeed, the present invention is in no way limited to
the methods and materials described.
[0021] The inventors of the present invention investigated the
design and preparation of hydrogels consisting of self-assembling
peptides, as they believed that these could be used to form a
scaffold that mimics the extracellular matrix (ECM) of certain
tissues. The inventors wanted to investigate if these hydrogels
would be capable of supporting individual cells and cell cultures
under biologically acceptable conditions, i.e. stable under in vivo
tissue culture conditions of high ionic strength, and a neutral pH.
As a model cell culture, the inventors focussed their research on
supporting cultures of chondrocytes (cartilage cells) on the
hydrogel scaffold.
[0022] The inventors therefore produced a dipeptide as discussed in
Example 1, which consists of the amino acid, Phenylalanine (Phe),
the structure of which will be known to the skilled technician. The
dipeptide was attached to, and protected with, Fmoc
(fluorenylmethoxycarbonyl), the structure of which is shown in FIG.
4, and will also be known to the skilled technician. The inventors
wished to investigate whether or not an Fmoc cap could be useful in
the formation of a hydrogel scaffold, Hence, the peptide produced
was Fmoc-Phe-Phe.
[0023] The inventors attempted to prepare self-assembled hydrogels
by suspending the Fmoc-Phe-Phe dipeptide in purified water, and
then varying the pH. The inventors were surprised to find that the
Fmoc-Phe-Phe (diphenylalanine) was able to self-assemble into a
hydrogel in a physiological buffer under biologically acceptable
conditions (pH=7.0). To date, this had not been possible. The
inventors also investigated the stability of the hydrogel by adding
the amino acid lysine thereto, and again found that the hydrogel
(Phe-Phe+Lysine) was stable at pH 7. Finally, the inventors
investigated the stability of the hydrogel by adding a further
dipeptide, Fmoc-Gly-Gly, to the mixture, and again found that the
hydrogel (formed from a mixture of Phe-Phe+Gly-Gly) formed was
stable at pH 7.
[0024] Surprised to find that these dipeptides and mixtures were
able to produce stable hydrogels under physiological conditions,
the inventors decided to carry out further experiments. They also
found that the hydrogels formed by the self-assembled peptides as
described herein are surprisingly adapted to support cell cultures
therein. Following on from the promising results produced with
Fmoc-Phe-Phe dipeptides and mixtures thereof, the inventors wanted
to investigate further how the design of the self-assembling
peptides could be modified to produce other stable hydrogels under
physiological conditions. The inventors therefore produced four
tripeptides each of which consisted of Fmoc-X-Phe-Phe, where
X=Alanine, Valine, Leucine, Phenylalanine. In addition, the
inventors also made the tripeptide: Fmoc-Leu-Leu-Leu The inventors
were surprised to see that each of these five tripeptides formed
stable hydrogels, and were also able to support cell cultures.
[0025] Hence, in summary, the inventors have surprisingly
demonstrated that the hydrogels formed from self-assembling
peptides are: --(i) stable under biologically acceptable, tissue
culture conditions; (ii) are of similar dimensions to fibrous
components of the extracellular matrix (i.e. nano-sized fibres);
and (iii) are capable of supporting cell culture in both 21) and in
3D. Hence, advantageously, the inventors believe that the hydrogels
formed by such self-assembling Fmoc-dipeptides may be used in a
wide range of medical applications, for example, in tissue
engineering and regeneration scenarios, and in methods of
treatment.
[0026] In its broadest, first aspect the present invention provides
a hydrogel composition comprising an aqueous dispersion phase and a
plurality of peptides, or derivatives, or analogues thereof,
wherein each peptide comprises at least two amino acid residues and
an aromatic stacking ligand, and wherein the hydrogel is formed by
self-assembly of said peptides in said aqueous dispersion
medium.
[0027] Preferred embodiments of hydrogel composition in accordance
with the invention are formulated with a physiologically acceptable
aqueous dispersion phase, preferably having a pH of 6 to 8. The
hydrogel composition itself may have a pH of 6 to 8.
[0028] Preferred embodiments of hydrogel compositions in accordance
with the invention are disclosed below in conjunction with proposed
uses of the compositions.
[0029] The invention is able to provide hydrogels in the form of
nanofibrous dense networks that are stable under physiological
conditions. The hydrogels are comprised of di- or higher-peptides
modified with aromatic stacking ligands and are stabilised by a
combination of pi-pi interactions, hydrogen bonding and/or other
non-covalent interactions (such as electrostatics). The gels have
uses in maintaining and/or directing cell phenotype and cell
behaviour such as motility, morphology, proliferation rate,
adhesion, differentiation, or matrix production.
[0030] Furthermore, advantageously, by choosing specific amino acid
residues, which make up the plurality of peptides, it is possible
to vary the structural and functional properties of the hydrogel
formed. Therefore, the peptides and hence the hydrogel may be
specifically `tailored`, depending on the final use of the
hydrogel.
[0031] According to a second aspect of the present invention, there
is provided a method of treating an individual suffering from a
medical condition characterised by tissue loss/damage, the method
comprising providing at a treatment site of an individual in need
of such treatment, a hydrogel comprised of gel-forming peptides, or
derivatives, or analogues thereof, wherein each peptide comprises
at least two amino acid residues and an aromatic stacking
ligand.
[0032] The inventors have surprisingly found that the use of such
peptides to form a hydrogel at the treatment site enables the
formation of a hydrogel scaffold structure, which is adapted to
support cell growth. The inventors observed that the cells are able
to infiltrate the hydrogel at the treatment site, thereby forming a
3D cell culture. This cell culture therefore can replace and/or
repair the tissue lost or damaged at the treatment site.
[0033] By the term "hydrogel", we mean a gel in which water is the
major dispersion medium. Preferably, the water disperses the
components of the hydrogel, ie. the peptides, derivatives or
analogues thereof. Preferably, the hydrogel comprises at least 80%
(w/w) water, more preferably, at least 85% (w/w) water, and more
preferably, at least 90% (w/w), even more preferably, at least 95%
(w/w) water.
[0034] The self-assembling subunits of the hydrogel (ie. the
gel-forming peptides, derivatives or analogues thereof) may have a
molecular weight of between 100 and 20,000Da, more preferably,
between 200 and 15,000Da, and even preferably, between 300 and
12,000Da.
[0035] In one embodiment, the hydrogel may be provided as a liquid
precursor composition, which may then be induced in situ to form
the hydrogel. Hence, the hydrogel may be prepared in situ in the
treatment site. In another embodiment, the hydrogel may be formed
remote from the treatment site, for example, in a mould, which may
then be administered to the treatment site. The choice of how to
administer the hydrogel to the treatment site will depend on the
medical condition being treated. In either case, the hydrogel may
be used as a scaffold structure to support cells therein, to
thereby repair the site of tissue loss or damage.
[0036] Hence, the inventors believe that the method according to
the second aspect, may be used in wide variety of different medical
treatments for treating a medical condition characterised by tissue
loss/damage. Examples of conditions that may be treated include the
treatment of wounds, and related injuries, and tissue degenerative
disorders. For example, the wound may be chronic, and may be
abrasive, for example, burns. The wound may be formed by pressure,
such as decubitus ulcers, and bed-sores. The wound may be acute,
and may be penetrative such as a cut, or a stab wound, or the
result of a crush to the body of the individual requiring
treatment.
[0037] Tissue degenerative disorders that may be treated using the
method include neurodegenerative, intervertebral disc disorders,
cartilage or bone degeneration such as osteoarthritis,
osteoporosis, liver degenerative disorders, kidney degenerative
disorders, muscle atrophy.
[0038] Preferably, and advantageously, the peptides, or
derivatives, or analogues thereof used in the method according to
the invention may be induced to form a hydrogel. The hydrogel is
preferably optically transparent, which is an advantage for medical
practitioners to clearly see the treatment site when using the
hydrogel in the method. It is preferred that the hydrogel is
provided in a physiologically acceptable excipient (or aqueous
dispersion medium). By the term "physiologically acceptable
excipient", we mean any suitable solution, which is capable of
conferring biologically acceptable conditions on the peptides such
that they self-assemble (i.e. with each other) resulting in
gelation to form the hydrogel. Examples of suitable excipients will
be known to the skilled technician, and may comprise a
physiological buffer, such as saline. Preferably, the excipient is
provided at a biologically acceptable pH.
[0039] Hence, the inventors have demonstrated for the first time
that peptides, derivatives or analogues thereof may be contained
within a physiologically acceptable excipient, such that the
peptides which are attached to an aromatic stacking ligand,
self-assemble to form the hydrogel. Hence, preferably, the
excipient confers biologically acceptable conditions on the
peptides, derivatives or analogues thereof, such that interactions
between the stacking ligands cause the peptides, derivatives or
analogues thereof to form a hydrogel either in the treatment site,
or prior to administration thereto.
[0040] Previous researchers have only demonstrated preparation of
hydrogels under non-physiological (i.e. biologically unacceptable)
conditions, for example, where the pH is substantially low and
therefore acidic. Hence, to date, it has not been possible to form
hydrogels at biologically acceptable pH's. Hence, the prior art
does not contemplate the use of such hydrogels in medical contexts,
as it will be appreciated that acidic conditions will be wholly
unsuitable for biological applications of the hydrogel used in the
method according to the invention. Therefore, the inventors believe
that use of the hydrogel in the method of the invention is a
significant advance over current technology.
[0041] It is preferred that the biologically acceptable excipient
is at a pH of between 5 and 9, more preferably between 6 and 8,
even more preferably, between about 6.5 and about 7.5. It will be
appreciated that the pH of most cells is about 7.4. Hence, a most
preferred excipient has a pH of between about 7 and about 7.5. It
will be appreciated that such pHs are referred to as being
biologically acceptable conditions.
[0042] By the term "biologically acceptable conditions", we mean
the hydrogel used in the method of the invention is substantially
stable under in vivo conditions, i.e. conditions of pH, ionic
strength and temperature, which would be found in vivo. The
inventors envisage primarily using the method according to the
invention, and hence, the hydrogel, to treat disorders charaterised
by tissue damage/loss in mammals and, in particular, man,
Therefore, it is preferred that the hydrogel is formed and is
stable under biologically acceptable conditions in mammals, and
preferably, in man.
[0043] Hence, the inventors investigated the stability of the
hydrogel at a biologically acceptable pH. Since the inventors
envisage primarily using the hydrogel in mammals, they considered a
biologically acceptable pH at which the hydrogel should be stable
to be between about 5.0 to about 9.0. The inventors believe that
the treatment site in the disorders being treated would be within
this pH range. However, it is preferred that the hydrogel is formed
at a pH of between about 6.0 to about 8.0. As described herein, the
method may be used to treat wounds, In chronic wounds, the pH may
be between a 6.0 and 8.0. Hence, when treating chronic wounds, it
is preferred that the hydrogel is stable between a pH of about 6.0
and 8.0.
[0044] However, when treating other disorders, the hydrogel may be
formed at a pH of between about 6.5 to about 7.5. It is more
preferred that the hydrogel is formed at a pH of between about 6.7
to about 7.3, and still more preferably, between about 6.9 to about
7.1. It will be appreciated that it is most preferred that the
hydrogel is formed at about pH 7.0. It is preferred that the
hydrogel is substantially stable at these biologically acceptable
pH's.
[0045] The inventors also investigated the stability of the
hydrogel under biologically acceptable ionic conditions. The
inventors believe that the treatment site of the individual being
treated would be at a high ionic strength. Hence, it is preferred
that the hydrogel is formed in conditions of substantially high
ionic strength. Hence, the ionic strength may be between about
0.01M to about 1M, preferably, between about 0.05M to about 0.5M,
more preferably, between about 0.1 to about 0.2, and even more
preferably, between about 0.12M and about 0.17M.
[0046] Furthermore, the inventors investigated the stability of the
hydrogel at biologically acceptable temperatures. Since the
inventors envisage primarily using the hydrogel in the method to
treat mammals and in particular man, they considered biologically
acceptable temperatures to be between about 32.degree. C. to about
40.degree. C. Hence, it is preferred that the hydrogel used in the
method is substantially liquid at temperatures above about
40.degree. C.
[0047] The inventors were surprised to find that it was possible to
tightly control the gelation of the hydrogel at temperatures below
40.degree. C. In fact, they found that the critical gelation
temperature for the hydrogel was at about body temperature (i.e.
37.degree. C. and below), and that the gel liquifies at
temperatures greater than body temperature. This is a major
advantage for use of the hydrogel in medicine, as it is therefore
possible to induce transition of the peptides from liquid form
(sol) to hydrogel (gel) on demand when in situ in the treatment
site. Hence, preferably, the hydrogel used in the method is formed
below about 40.degree. C., more preferably below about 39.degree.
C., and even more preferably, below about 38.degree. C. Therefore,
preferably, the hydrogel is formed at a temperature of between
about 36.degree. C. to about 38.degree. C., and most preferably, at
about 37.degree. C.
[0048] However, it should be appreciated that in chronic wounds,
and also in surface organs (such as the skin, the eye etc.) the
temperature may be a few degrees lower, for example, about
32.degree. C. to 34.degree. C. Hence, in embodiments of the method
where the composition is used to treat chronic wounds or surface
organs, it is preferred that the hydrogel forms at a temperature of
between about 32.degree. C. to 34.degree. C.
[0049] Therefore, in preferred embodiments of the invention, it is
preferred that the hydrogel forms at a pH of between about 6.8 to
about 7.5, a high ionic strength, and at a temperature of between
about 32.degree. C. to about 38.degree. C.
[0050] It will be appreciated that the hydrogel used in the method
according to the second aspect of the invention may comprise a
plurality of identical peptides, or a plurality of peptides that
are different. Nevertheless, in either case, each peptide in the
hydrogel comprises at least two amino acid residues or derivatives
or analogues thereof attached to an aromatic stacking ligand, such
that interactions therebetween causes the hydrogel to form. The
inventors have found that surprisingly, at least two amino acid
residues are required in each peptide. This is because if a peptide
comprises less than two amino acid residues, it results in either
no hydrogel forming at all, or an inferior hydrogel being formed,
at biologically acceptable conditions.
[0051] Peptides of the hydrogel used in the method may comprise at
least three, four, five, six, or more amino acids or derivatives or
analogues thereof, or any combination thereof. However, it is
preferred that the peptides may comprise less than 10 amino acids
or derivatives or analogues thereof, more preferably less than 8
amino acids or derivatives or analogues thereof, and even more
preferably, less than 6 amino acids or derivatives or analogues
thereof. Hence, peptides of the hydrogel may comprise at least 2
amino acids and less than 7 amino acids, or derivatives or
analogues thereof. For example, the hydrogel used in the method
according to the invention may comprise a dipeptide, a tripeptide,
a tetrapeptide, a pentapeptide, hexapeptide, and/or a heptapeptide
etc., or derivatives or analogues thereof, or any combination
thereof.
[0052] The hydrogel used in the method according to the invention
may comprise a number of identical peptides, a number of peptides
that are different from each other, or any combination thereof.
Therefore, in one embodiment, the hydrogel may comprise all
dipeptides, or all tripeptides, or all tetrapeptides etc. In
another embodiment, the hydrogel may comprise a combination of
dipeptides and tripeptides, or a combination or tripeptides and
tetrapeptides. In yet another embodiment, the hydrogel may comprise
a combination of dipeptides, tripeptides, and tetrapeptides, and so
on.
[0053] Advantageously, smaller peptides such as dipeptides and
tripeptides are conveniently small molecules compared to longer
peptides (greater than 10 amino acid residues), and are therefore
relatively simple and cheap to synthesise. Moreover, due to their
small size, dipeptides and tripeptides also exhibit excellent
stacking characteristics to thereby form the scaffold under the
biologically acceptable conditions.
[0054] The inventors have found that the physical properties of the
hydrogel formed by the hydrogel under biologically acceptable
conditions in the treatment site may be altered or `tuned` by
choosing different combinations of amino acid residues in the
plurality of peptides. As described in the Examples, the
characteristics of the resultant hydrogels may then be analysed by
Circular Dichroism, and imaged by a CryoScanning Electron
Microscope, examples of which are shown in FIG. 1.
[0055] Hence, the amino acids in the plurality of peptides in the
hydrogel used in the method according to the second aspect of the
invention may be selected from the repertoire of twenty amino acids
commonly found in proteins, or any non-naturally occurring amino
acids, and the specific amino acids chosen will depend on the final
use of the hydrogel, and the condition being treated. For example,
the hydrogel may comprise an acidic amino acid, such as aspartic
acid, glutamic acid, asparagines, or glutamine; or a basic amino
acid, such as histidine, lysine, or arginine. Variation of such
amino acids in the peptide will influence the pH of the peptide,
and hence, the hydrogel formed. The pH of the hydrogel may
therefore be varied depending on the pH of the treatment site.
[0056] The gel-forming peptides may comprise a dipeptide and the
aromatic stacking ligand. Alternatively the gel-forming peptides
may comprise a tripeptide and the aromatic stacking ligand.
[0057] The hydrogel may comprise a hydrophobic amino acid, such as
alanine, cysteine, isoleucine, leucine, methionine, phenylalanine,
proline, tryptophan, valine or tyrosine; or a hydrophilic amino
acid, such as arginine, asparagine, aspartate, glutamine,
glutamate, histidine, lysine, serine, or threonine.
[0058] The inventors found that if the peptide comprises two
consecutive or adjacent phenylalanine residues, that stable and
effective hydrogels are formed. Hence, preferably the peptide
comprises at least two consecutive phenylalanine residues.
[0059] Therefore, a preferred peptide used in accordance with the
invention is Phe-Phe, which is described in the Example. The
inventors carried out further investigations as described in
Example 2, and found that introduction of a further amino acid
immediately before the Phe-Phe also formed stable hydrogels. Hence,
the inventors produced four tripeptides each of which consisted of
Fmoc-X-Phe-Phe, where X=Ala, Val, Leu, Phe. Hence, further
preferred peptides include Ala-Val-Phe; Val-Phe-Phe; Leu-Phe-Phe;
and Phe-Phe-Phe.
[0060] In addition, the inventors also made the tripeptide:
Fmoc-Leu-Leu-Leu, which also formed stable hydrogels and is also
considered a preferred peptide for use in the method according to
the invention.
[0061] The hydrogel may comprise first and second peptides each
incorporating a dipeptide and an aromatic stacking ligand. The
dipeptide of the first peptide may be Phe-Phe and the dipeptide of
the second peptide may be Gly-Gly.
[0062] The inventors investigated modifying the peptides in the
hydrogel used in the method according to the invention by choosing
specific amino acids and combinations thereof. They found that it
was possible to tailor the structural and functional
characteristics of the resultant hydrogel formed under biologically
acceptable conditions. For example, at least one peptide in the
hydrogel may comprise at least one amino acid, which is adapted to
initiate or promote cell-cell adhesion. For example, the or each
peptide may comprise at least one tryptophan residue, which may
mimic cadherin-mediated cell-cell interactions. It is preferred
that the tryptophan residue is the amino acid residue distal from
the aromatic stacking ligand.
[0063] As discussed herein, the inventors have surprisingly found
that the hydrogel used in the method according to the invention is
formed due to the presence of the aromatic stacking ligand
(A.S.L.). Hence, the peptide may preferably have the following
structure: A.S.L.-AA.sub.1-AA.sub.2-X, where A.S.L. denotes the
Aromatic Stacking Ligand, where AA.sub.n denotes amino acid
residues in the peptide (n=the number of the amino acid residue,
e.g. n=1 or 2), and where X is a amino acid residue selected from
the group consisting of Phe, Leu, IKVAV, RGD and KPV.
[0064] At least one peptide in the hydrogel may comprise an
Arginine-Glycine-Aspartate (RGD) peptide motif. The inventors
believe that incorporation of the RGD motif (which is a known cell
adhesive) will improve the efficacy of the hydrogel to adhere to
cells, which would be useful in the method of the second aspect as
cell proliferation in the hydrogel will be promoted. Hence, the or
each peptide may preferably have the following structure:
A.S.L.-AA.sub.1-AA.sub.2-RGD, where A.S.L. denotes the Aromatic
Stacking Ligand, where AA.sub.n denotes amino acid residues in the
peptide, and where RGD denotes the RGD motif. It will be
appreciated that the above structure is a pentapeptide.
[0065] At least one peptide in the hydrogel may comprise an
Isoleucine-Lysine-Valine-Alanine-Valine (IKVAV) peptide motif. The
inventors believe that incorporation of the IKVAV motif (which is
known to directionally guide nerve cells) will improve the efficacy
of the hydrogel to guide nerve cells, which would be useful in the
method of the second aspect when involving nerve growth, wound
repair or nerve tissue regeneration. Hence, the or each peptide may
preferably have the following structure:
A.S.L.-AA.sub.1-AA.sub.2-IKVAV, where A.S.L. denotes the Aromatic
Stacking Ligand, where AA.sub.n denotes amino acid residues in the
peptide, and where IKVAV denotes the IKVAV motif. It will be
appreciated that the above structure is a heptapeptide.
[0066] At least one peptide in the hydrogel may comprise
Lysine-Proline-Valine (KPV) motif. The inventors believe that
incorporation of the KPV motif (which has anti-inflammatory
properties) will improve the efficacy of the hydrogel the method of
the second aspect as inflammation may occur in the treatment site.
Hence, the or each peptide may preferably have the following
structure: A.S.L.-AA.sub.1-AA.sub.2-KPV, where A.S.L. denotes the
Aromatic Stacking Ligand, where AA.sub.n denotes amino acid
residues in the peptide, and where KPV denotes the KPV motif. It
will be appreciated that the above structure is a pentapeptide.
[0067] The inventors were surprised to observe that if a peptide in
the hydrogel includes an aromatic amino acid, such as
phenylalanine, then this resulted in the formation of effective
hydrogels under biologically acceptable conditions. This is
illustrated by the efficacy of the Phe-Phe dipeptide investigated.
Hence, preferably, at least one peptide of the composition used in
the method according to the invention comprises at least one
aromatic amino acid. By the term "aromatic amino acid", we mean an
amino acid comprising a benzene (or other aromatic group) ring in
its side chain.
[0068] Preferably, more than one of the peptides of the hydrogel
comprises at least one aromatic amino acid. Preferably, the or each
peptide comprises a plurality of aromatic amino acids. In preferred
embodiments, each amino acid of each peptide in the composition is
an aromatic amino acid. Therefore, by way of example, in
embodiments where the hydrogel comprises a dipeptide, the dipeptide
preferably comprises two aromatic amino acids, and where the
hydrogel comprises a tripeptide, the tripeptide preferably
comprises three aromatic amino acids.
[0069] Examples of suitable aromatic amino acids, which could be
included in each peptide in the hydrogel include tyrosine,
tryptophan, or phenylalanine. However, it is most preferred that
the aromatic amino acid in the peptide comprises phenylalanine.
While the inventors do not wish to be bound by any hypothesis, they
believe that aromatic amino acids comprising an aromatic side chain
contribute to side branching between the peptides in the hydrogel.
The inventors believe that such side branching considerably
enhances the generation of the hydrogel under biologically
acceptable conditions, and this produces an improved scaffold for
supporting cell tissues.
[0070] Accordingly, it is preferred that the hydrogel used in the
method of the second aspect comprises a plurality of peptides, or
derivatives, or analogues thereof, wherein each peptide comprises
at least two amino acid residues, and an aromatic stacking ligand,
wherein at least one amino acid comprises an aromatic side chain,
and wherein under biologically acceptable conditions, interactions
between the stacking ligands cause the hydrogel to form a hydrogel.
It is preferred that the amino acid comprising an aromatic side
chain is phenylalanine.
[0071] Surprisingly also it has been found that peptides containing
amino acids with acidic and basic side chains (for example those
containing the sequences RGD or RGE) form gels that are less shear
sensitive and of higher mechanical strength than those not
incorporating such side chains.
[0072] Derivatives or analogues of the peptide hydrogel used in the
method according to the invention may include derivatives or
analogues that increase or decrease the peptide's half-life in
vivo. Examples of derivatives or analogues capable of increasing
the half-life of the peptide according to the invention include
peptoid derivatives, D-amino acid derivatives of the peptides, and
peptide-peptoid hybrids.
[0073] The peptide used in the invention may be subject to
degradation by a number of means (such as protease activity in
biological systems). Such degradation may limit the bioavailability
of the peptide, and hence the ability of the peptide to achieve its
biological function. There are wide ranges of well-established
techniques by which peptide derivatives or analogues that have
enhanced stability in biological contexts can be designed and
produced. Such peptide derivatives may have improved
bioavailability as a result of increased resistance to
protease-mediated degradation.
[0074] Preferably, a peptide derivative or analogue suitable for
use according to the invention is more protease-resistant than the
peptide from which it is derived. Protease-resistance of a peptide
derivative and the peptide from which it is derived may be
evaluated by means of well-known protein degradation assays. The
relative values of protease resistance for the peptide and the
peptide derivative or analogue may then be compared.
[0075] Peptoid derivatives of the peptide hydrogel used in the
invention may be readily designed from knowledge of the structure
of the peptide. Peptoid compounds have two properties that make
them suitable for use as peptide derivatives/analogues according to
the invention:--
[0076] (i) In peptoid residues, no hydrogen bond involving the NH
would be possible.
[0077] (ii) The peptoids are resistant to enzymatic
degradation.
[0078] Commercially available software may be used to develop
peptoid derivatives according to well-established protocols.
[0079] Retropeptoids, (in which all amino acids are replaced by
peptoid residues in reversed order) are also able to mimic
peptides. A retropeptoid is expected to bind in the opposite
direction in the ligand-binding groove, as compared to a peptide or
peptoid-peptide hybrid containing one peptoid residue. As a result,
the side chains of the peptoid residues are able to point in the
same direction as the side chains in the original peptide.
[0080] As discussed herein, the inventors have surprisingly found
that the hydrogel used in the method according to the invention
forms a stable hydrogel due to the presence of the aromatic
stacking ligand.
[0081] By the term "aromatic stacking ligand", we mean an aromatic
molecule comprising at least one benzene ring, or a related planar,
cyclic structure with a delocalised .pi. electron structure, such
as pyridine, furan or thiophene or, more generally, ligands that
can be covalently attached either to the N or C terminus or side
chain of amino acids in a peptide sequence and, which preferably
adhere to the 4n+2 (Huckel) rule. It is preferred that the stacking
ligand is adapted to interact with at least one other aromatic
stacking ligand. Hence, the molecules are able to self-assemble
with each other, Surprisingly, such self-assembly of the stacking
ligands results in the self-assembly of the peptides to which they
are attached. As the peptides assemble together, the hydrogel is
formed under biologically acceptable conditions.
[0082] Examples of a suitable aromatic stacking ligand, which may
be attached to the peptide in the hydrogel used in the method of
the invention include any aromatic compound, which comprises at
least one benzene ring. The skilled technician will appreciate that
there are many different types of aromatic compounds available that
could be attached to the peptide in the hydrogel, and which would
interact with each other to form a hydrogel. However, examples of
suitable aromatic stacking ligand to which the peptide may be
attached include benzoyl (Bz) or carboxybenzoyl (Cbz), both of
which are common protecting groups used in peptide synthesis, and
which will be known to the skilled technician.
[0083] However, a preferred aromatic stacking ligand comprises Fmoc
(fluorenylmethoxycarbonyl), which is another type of protecting
group used in peptide synthesis, the structure of which is shown in
FIG. 4. As shown in FIG. 1a, which is a Circular Dichroism (CD)
spectrum, so-called .pi.-stacking (or .pi.-.pi. interactions)
between the fluorenyl groups on an Fmoc aromatic group gives rise
to a peak at approximately 308 nm. While the inventors do not wish
to be bound by any hypothesis, they believe that such .pi.-stacking
between the Fmoc groups enables and encourages hydrogen bonding to
occur between the peptides in the hydrogel used in the method
according to the invention, The inventors believe that such
hydrogen bonding between the peptides causes the formation of
structures, which resemble .beta.-sheets between the plurality of
peptides in the hydrogel. The inventors believe that these
.beta.3-sheet-type structures cause the formation of the hydrogel.
Another advantage of Fmoc is that it is thought to have
anti-inflammatory properties, which will have significant
advantages as the hydrogel is used in medical applications.
[0084] Hence, it is preferred that the method comprises
administering to the treatment site, a hydrogel which comprises
comprise a plurality of peptides, or derivatives, or analogues
thereof, wherein each peptide comprises at least two amino acid
residues attached to Fmoc.
[0085] Preferably, under biologically acceptable conditions,
interactions between the Fmoc structures cause the formation of the
hydrogel. A preferred peptide is Fmoc-Phe-Phe. Another preferred
peptide comprises a mixture of Fmoc-Phe-Phe and Fmoc-Gly-Gly.
[0086] Another preferred aromatic stacking ligand, which may be
attached to the peptide in the hydrogel used, comprises an aromatic
amino acid, i.e. an amino acid residue comprising an aromatic side
group (i.e. at least one benzene ring). Accordingly, in this
embodiment, because the aromatic stacking ligand is itself an
aromatic amino acid, and because it is attached to at least two
other amino acid residues, the hydrogel comprises at least three
amino acid residues. Where the ligand is an aromatic amino acid
attached to a tripeptide, the hydrogel comprises a tetrapeptide,
and so on.
[0087] Examples of suitable aromatic amino acids may include
tyrosine, tryptophan, or phenylalanine, or less common aromatic
amino acids such as di-hydroxy-phenylalanine (DOPA), or other
natural or non-natural amino acids with aromatic side chains.
Hence, the hydrogel used in the method according to the invention
may comprise a plurality of peptides, or derivatives, or analogues
thereof, wherein each peptide comprises at least two amino acid
residues attached to an aromatic amino acid residue.
[0088] In addition to the peptides, which comprise at least two
amino acid residues, in the hydrogel used in the method, the
inventors also investigated modifying the hydrogel used in the
method according to the invention by adding further components
thereto, They added various additives to the hydrogel components,
and found that it was possible to further tailor the structural and
functional characteristics of the resultant hydrogel formed under
biologically acceptable conditions, such characteristics depending
on the intended use of the hydrogel. Therefore, the hydrogel may
further comprise a bioadditive.
[0089] By the term "bioadditive", we mean a compound exhibiting
biologically active functionality.
[0090] By way of example, the bioadditive may be adapted to promote
or improve cell adhesion. It is known that cells respond favourably
to positive charges. Hence, it is preferred that the bioadditive is
positively charged. The bioadditive may comprise at least one
further amino acid, or a peptide. Therefore, the bioadditive may
comprise a positively charge amino acid residue, for example,
arginine, histidine, or lysine. The inventors have demonstrated in
the Examples that the addition of lysine (K) significantly improves
cell adhesion.
[0091] It is preferred that the bioadditive itself comprises an
aromatic stacking ligand, which may be provided so that the
bioadditive is able to form hydrogen bonds with the peptides of the
hydrogel used according to the invention. Suitable aromatic
stacking ligands, are as described hereinbefore. Hence, a preferred
aromatic stacking ligand comprises Fmoc. As mentioned herein, Fmoc
is thought to have anti-inflammatory properties. In another
embodiment, the bioadditive may be Fmoc.
[0092] Hence, the bioadditive may preferably have the following
structure: A.S.L.-K, where A.S.L. denotes the Aromatic Stacking
Ligand, and where K denotes the Lysine residue. It will be
appreciated that the above structure is a single amino acid
attached to the aromatic stacking ligand. Preferably, the aromatic
stacking ligands comprise Fmoc.
[0093] Hence, a preferred peptide used in the method in accordance
with the invention comprises a mixture of Fmoc-Phe-Phe with
Fmoc-Lys.
[0094] With all of the above considerations in mind, particularly
suitable di- or higher-peptides (incorporating an aromatic stacking
ligand) for producing hydrogels in accordance with the invention
are as follows:
[0095] (i) Fmoc-Phe-Phe either alone or in combination with one or
more of Fmoc-Lys, Fmoc-Gly, Fmoc-Gly-Gly, Fmoc-Gly-Gly-Arg-Gly-Asp,
Fmoc-Gly-Gly-Arg-Gly-Glu or Fmoc-Trp;
[0096] (ii) Fmoc-Phe-Phe-Phe;
[0097] (iii) Fmoc-Leu-Leu-Leu;
[0098] (iv) Cbz-Phe-Phe;
[0099] (v) Cbz-Phe-Phe-Phe; and
[0100] (vi) Cbz-Leu-Leu-Leu.
[0101] Generally the amount of each di- or higher-peptide
(incorporating the aromatic stacking ligand) and (if present) amino
acid incorporating an aromatic stacking ligand will each be in the
range of 1 to 50 mM/L, more preferably 5-30 mM/L.
[0102] The hydrogels may be formed by increasing the pH of a
solution of the gel-forming components to 9-11 (more preferably
about 10) and then reducing the pH into the range 6-8 (such that
gel formation occurs. More preferably gel formation occurs about pH
7. Gel formation may be effected at ambient temperature or on
incubation, e.g. at a temperature of up to 40.degree. C. (for
example 35.degree.-40.degree. C.).
[0103] It will be appreciated that the hydrogel used in the method
of the second aspect may be either used effectively in a number of
different physical forms. For example, in one embodiment, the
method may comprise administering to the treatment site a liquid
hydrogel precursor composition in the form of a solution, which may
then be induced to form the hydrogel. Alternatively, in another
embodiment, the method may comprise administering to the treatment
site the already formed hydrogel composition. The inventors believe
that each of these embodiments is an important aspect of the
invention, which may be used with the method of the second
aspect.
[0104] Hence, in a third aspect, there is provided a liquid
hydrogel precursor composition comprising a plurality of peptides,
or derivatives, or analogues thereof, wherein each peptide
comprises at least two amino acid residues and an aromatic stacking
ligand, and a physiologically acceptable excipient.
[0105] Preferably, the hydrogel precursor composition may be
induced to form a hydrogel, for example, by reducing the
temperature to below the critical gelation temperature.
[0106] As mentioned herein, prior art hydrogels have only been made
at acidic pH, and it will be appreciated that low pHs are
unsuitable for medical applications. Therefore, because the
hydrogel according to the invention forms in a physiological
excipient under biologically acceptable conditions, the inventors
wanted to assess whether functional cues or moieties could be
incorporated into the hydrogel's structure so that they could be
adapted for medical uses. The inventors therefore tested the
hydrogel formed from Fmoc-Phe-Phe (and mixtures therewith) for its
stability in cell culture conditions, and its ability to support
cell cultures or tissues. As discussed in the Examples, microscopic
images shown in FIG. 2 confirmed that the hydrogels tested had the
surprising ability to organise cells into a three-dimensional
architecture. The inventors have therefore observed that the
hydrogels according to the invention are surprisingly suitable for
culturing and supporting cells therein. The inventors then
conducted statistical analysis of data used in an MTT Assay, which
further confirmed the surprising finding the cell growth actually
continued for the entire time measured, i.e. up to 7 days.
[0107] Therefore, it is preferred that the hydrogel of the first
aspect or that used in the method according to the second aspect,
or the precursor composition of the third aspect is adapted to
support at least one cell, to thereby form a physiologically stable
cell-supporting medium or cell scaffold. Hence, the hydrogel used
in the method of the first aspect, or the composition of the second
or third aspect may be seeded with at least one cell.
[0108] Hence, according to a fourth aspect of the present
invention, there is provided a cell-supporting medium comprising
the hydrogel of the first aspect, or that used in the method
according to the second aspect or the precursor composition of the
third aspect, and at least one cell.
[0109] The cell-supporting medium of the fourth aspect may be
referred to as a cell-hydrogel scaffold. Preferably, the
cell-supporting medium is adapted to support a plurality of cells.
Preferably, the or each cell is biochemically functional in vivo.
Accordingly, the plurality of cells may form a cell culture or a
tissue.
[0110] As the hydrogel precursor composition in the third aspect is
a liquid, at least one cell may be suspended therein. As the
hydrogel composition in the first aspect is a gel, at least one
cell may be supported on and/or in the structure of the hydrogel,
which therefore acts as a supporting scaffold structure.
[0111] The inventors investigated various methods for preparing the
cell-supporting medium according to the fourth aspect.
[0112] Hence, in a fifth aspect, there is provided a method of
preparing a cell supporting medium according to the fourth aspect,
the method comprising the steps of:-- [0113] (i) contacting either
a hydrogel of the first aspect, or that used in the method of the
second aspect, or the precursor composition of the third aspect
with at least one cell; and [0114] (ii) exposing the hydrogel or
composition to conditions such that the at least one cell is
supported on and/or in a hydrogel, thereby forming a
cell-supporting medium.
[0115] It will be appreciated that the method according to the
fifth aspect may be carried out in situ in the treatment site, or
remote from the treatment site, and then transferred thereto.
[0116] The skilled technician will appreciate how to culture
various cell types with the hydrogel or precursor. Hence, it will
be appreciated that the specific details of the methodologies
(culture time, temperatures, growth media etc) used will depend on
the type of cell involved, and the final use of the cell-supporting
medium (ie. the scaffold). By way of the example, the Example
provides details of how to culture chondrocytes and to produce a
chondrocyte cell scaffold.
[0117] In one embodiment, step (i) of the method according to the
fifth aspect may comprise contacting the liquid hydrogel precursor
composition according to the third aspect with the at least one
cell. In another embodiment, step (i) of the method according to
the fifth aspect may comprise contacting the hydrogel composition
according to the first aspect with the at least one cell. The
nature of step (ii) of the method will be determined by whether the
composition in step (i) is in liquid form or a hydrogel.
[0118] Hence, in one embodiment, the method may comprise exposing
the composition of the first aspect to conditions such that a
hydrogel is formed in step (i) prior to contacting the at least one
cell therewith. Such conditions may comprise lowering the
temperature of the composition to below the critical gelation
temperature, e.g. less than 40.degree. C. The inventors
investigated this embodiment of the method, and surprisingly found
that cells in a culture media were rapidly taken up by the hydrogel
in step (ii) of the method to form the cell-supporting medium. They
found that the cell culture distributed itself on and throughout
the hydrogel in step (ii). The inventors envisage that this
embodiment will have great utility in the method of the second
aspect.
[0119] In an alternative embodiment, the composition may be
initially maintained under conditions in which it is in the form of
the liquid precursor in step (i) of the method, to which the at
least one cell is added in step (ii). Hence, the method may
comprise initially exposing the composition in step (i) to
conditions in which it is substantially liquid (i.e. not a
hydrogel). For example, the composition may be exposed to a pH or
temperature or ionic strength at which the compound is liquid. For
example, the composition may be exposed to a temperature above the
critical gelation temperature of about 40.degree. C. or more, such
that it liquifies. The method may then comprise the step of
contacting the at least one cell with the liquid precursor in step
(i). After step (i), step (ii) preferably comprises exposing the
liquid precursor composition to conditions in which it forms a
hydrogel. For example, the temperature may be cooled to about
37.degree. C., or the pH may be adjusted such that the hydrogel is
preferably formed with cells distributed throughout. The hydrogel
which forms, in which the at least one cell is supported is
referred to as the cell-supporting medium or cell scaffold. Again,
the inventors believe that this embodiment will have great utility
in the method of the second aspect.
[0120] The composition according to the first or third aspect, or
the medium according to the fourth aspect may be used in a number
of ways. A common problem with many wounds or tissue degenerative
disorders is that a cavity or space may be formed in the body of
the individual being treated, and this cavity or space will need to
be repaired using the composition of cell support medium. Hence,
the composition or medium may be prepared either in vitro or in
vivo. Furthermore, the composition or medium may be prepared
either: (i) in situ (in the wound itself); or (ii) remote from the
wound, and then transferred to the area to be treated after it has
been prepared.
[0121] Preferably, the method according to the fifth aspect is used
to prepare the cell-supporting medium. Therefore, in one
embodiment, the hydrogel or composition according to the third
aspect is preferably administered to the area to be treated (wound,
cavity, or degenerated area). It will be appreciated that the
composition according to the third aspect is in liquid form and the
composition according to the first aspect is in the form of a
hydrogel. Once the composition is in position in the area to be
treated, at least one cell is then contacted therewith as in step
(i) of the method according to the fifth aspect. If the composition
is a hydrogel, then at least one cell can be contacted therewith to
allow the cell scaffold to form. If the compound is in the form of
the liquid hydrogel precursor, then as it cools to body
temperature, it will form the hydrogel.
[0122] In another embodiment, the cell-supporting medium may be
prepared remote from the wound (eg. in the lab), and is then
preferably administered to the area to be treated. In this
approach, the gel would be formed in a pre-determined
three-dimensional shape for example, by using a mould, and cells
may either be added prior to the gelation process or after the gel
has formed. The pre-formed gel may then be implanted in the body
where the patient's cells migrate into the gel scaffold. Examples
of this use would be in tissues, which have a migratory capacity
and/or those, which are responsible for tissue remodelling.
Examples are skin, bone, and peripheral nerves. The implant may
also be supplemented with further cells externally by the medical
practitioner. In addition, other factors, which may simulate cell
and preferably tissue growth, may be added to the implant, for
example, growth factors.
[0123] Preferably, the cell supporting medium according to the
fourth aspect, whether prepared in situ in the area to be treated,
or remote from it, is suitably maintained to allow the at least one
cell to divide to form a culture or tissue therein. Accordingly, it
will be appreciated that the hydrogel acts as a supporting scaffold
for the tissue and thereby allows repair of the wound, or
regeneration of the damaged tissue.
[0124] The inventors believe that the method according to the first
aspect, may be used in wide variety of different, medical treatment
methods, such as tissue regeneration/engineering applications,
controlled stem cell differentiation, and in wound healing. The
types of tissues and wound which could be treated are varied, and
hence, it will be appreciated that the invention is not limited to
any specific type of cell, which could be supported and cultured on
the hydrogel administered to the treatment site. However, by way of
example, suitable cells, which may be supported in the hydrogel
include epithelial cells (e.g., hepatocytes), neurons, endothelial
cells, osteoblasts (bone cells), chondrocytes (cartilage cells),
fibroblasts, smooth muscle cells, osteoclasts, keratinocytes, nerve
progenitor cells, Schwann cells, stem cells, macrophages, islet
cells, and tumour cells, etc.
[0125] The cell type contacted with the composition or
cell-supporting medium will depend on the type of wound being
repaired, or the type of tissue being regenerated. Therefore, by
way of example, if the wound is in skin, then at least one skin
cell may be contacted with the hydrogel, composition or
cell-supporting medium. If the wound is in bone, then at least one
bone cell or osteoblast is preferably contacted with the hydrogel,
composition or cell-supporting medium. If the wound is in
cartilage, then at least one chondrocyte is preferably contacted
with the hydrogel, composition or cell-supporting medium. If the
eye tissue has been damaged, it may be required to contact the
hydrogel, composition or cell-supporting medium with eye stem
cells. It will be appreciated that different types of cell type may
be contacted with the hydrogel, composition, or cell supporting
medium, if necessary.
[0126] As discussed in the Examples, the inventors focussed their
research on investigating the efficacy of the hydrogel
cell-supporting medium (or scaffold) according to the fourth aspect
to support cartilage cells. Hence, it is preferred that the at
least one cell is a chondrocyte. This would be advantageous, if the
treatment site is a site in which cartilage has been damaged or
lost. However, the at least one cell may be an osteoblast or bone
cell. This would be useful if the site being treated is bone. The
osteoblast may be autologous or autogenous.
[0127] Alternatively, the at least one cell may be a stem cell,
which may be either mesenchymal, or haematopoeic, or embryonic, or
cloned. The inventors believe that the ability to culture and
support a wide variety of cells such as chondrocytes, osteoblasts
and stem cells, will be of significant importance in many aspects
of medicine.
[0128] The method according to the second aspect may comprise use
of the composition according to either the first or third aspect,
or of the cell-supporting medium according to the fourth aspect.
The composition or cell supporting medium may be combined in
formulations having a number of different forms depending, in
particular on the manner in which the formulation is to be used. It
will be appreciated that the vehicle of the composition of the
invention should be one which is well-tolerated by the subject to
whom it is given, and preferably enables efficient delivery of the
composition to a target site. Thus, for example, the composition
may be in the form of a liquid (composition according to the third
aspect), or gel or hydrogel (composition according to the first
aspect), or any other suitable form that may be administered to a
person or animal.
[0129] The inventors believe that the Fmoc peptides described
herein may be formulated with a physiologically acceptable
excipient to form a medicament. The inventors believe that the
prior art does not hint at or even suggest that hydrogels according
to the invention may be used as a medicament.
[0130] Therefore, according to a sixth aspect of the invention,
there is provided a composition according to the first or third
aspect, or a cell-supporting medium according to the fourth aspect,
for use as a medicament.
[0131] In particular, the inventors envisage the composition of the
first or third aspect or cell-supporting medium according to the
fourth aspect will have major uses in a wide variety of tissue
engineering and regeneration applications, and also in wound
healing. Such disorders are commonly linked in that they a
characterised by tissue damage or loss.
[0132] Therefore, according to a seventh aspect, there is provided
use of a composition according to the first or third aspect, or a
cell supporting medium according to the fourth aspect, for the
preparation of a medicament for the treatment of a medical
condition characterised by tissue loss/damage.
[0133] It will be appreciated that the medicament may be used to
treat individuals suffering from a wide variety of disease
conditions characterised by tissue loss or damage. Examples include
wounds and/or tissue degenerative disorders.
[0134] The wound may be chronic or acute. Tissue degenerative
disorders that may be treated include neurodegenerative,
intervertebral disc disorders, cartilage or bone degeneration such
as osteoarthritis, osteoporosis, liver degenerative disorders,
kidney degenerative disorders, muscle atrophy.
[0135] It will be appreciated that in chronic wounds, it has been
described that modulating the pH of the wound may help improve
wound healing. The pH in chronic wounds varies between 6 and 8, and
the inventors believe that wound healing appears to work best at
reduced pH values. Hence, the composition may comprise acidic or
basic amino acids (His, Arg, Lys, Glu, Asp), which may help
maintain the pH of the hydrogel in the treatment site.
[0136] Furthermore, in chronic wounds, the temperature may be a few
degrees lower than normal body temperature, i.e. about 32.degree.
C. to 34.degree. C. Furthermore, for treating surface organs such
as the eye, skin, and so on, etc the preferred temperature will be
lower than normal body temperature. However, the composition will
need to gel at this temperature range to form the scaffold.
[0137] It will be appreciated that the hydrogel of the first
aspect, that used in the second aspect, the composition according
to the third aspect, or the cell-supporting medium according to the
fourth aspect may be used to formulate the medicament of the sixth
or seventh aspect. Furthermore, the medicament may be used in the
method of treatment according to the second aspect.
[0138] The hydrogel, compositions, cell-supporting medium, or
medicament according to the invention may be used in a monotherapy
(i.e. use of the hydrogel, composition, cell supporting medium, or
medicament, alone). Alternatively, the hydrogel, compositions,
cell-supporting medium, or medicament according to the invention
may be used as an adjunct, or in combination with other known
therapies.
[0139] In some circumstances, the composition, compound or scaffold
according to the invention may be administered by injection into
the wound areas. Injections may be intravenous (bolus or infusion)
or subcutaneous (bolus or infusion).
[0140] The hydrogel, compositions, cell-supporting medium, or
medicament may also be incorporated within a slow or delayed
release device. Such devices may, for example, be positioned on or
adjacent the area to be treated, for example by implantation, and
the hydrogel, compositions, cell-supporting medium, or medicament
may be released over weeks or even months. Such devices may be
particularly advantageous when long-term treatment with the
medicament is required and which would normally require frequent
administration (e.g. at least daily injection or implant).
[0141] It will be appreciated that the amount of hydrogel,
compositions, cell-supporting medium, or medicament according to
the invention required will be determined by its biological
activity and bioavailability, which in turn depends on the mode of
administration, the physicochemical properties of the medicament
employed, and whether the hydrogel, compositions, cell-supporting
medium, or medicament is being used as a monotherapy or in a
combined therapy. The frequency of administration will also be
influenced by the above-mentioned factors and particularly the
half-life of the medicament within the subject being treated.
[0142] Optimal dosages to be administered may be determined by
those skilled in the art, and will vary with the particular
medicament in use, the strength of the preparation, the mode of
administration, and the advancement of the disease condition.
Additional factors depending on the particular subject being
treated will result in a need to adjust dosages, including subject
age, weight, gender, diet, and time of administration.
[0143] Known procedures, such as those conventionally employed by
the pharmaceutical industry (e.g. in vivo experimentation, clinical
trials, etc.), may be used to establish specific formulations of
the medicament according to the invention, and precise therapeutic
regimes (such as daily doses and the frequency of
administration).
[0144] Generally, a daily dose of between 0.01 .mu.g/kg of body
weight and 1.0 g/kg of body weight of the hydrogel according to the
invention may be used for the prevention and/or treatment of the
specific medical condition. More preferably, the daily dose is
between 0.01 mg/kg of body weight and 100 mg/kg of body weight.
Daily doses may be given as a single administration (e.g. a single
daily tablet). Alternatively, the medicament may require
administration twice or more times during a day. As an example, the
medicament according to the invention may be administered as two
(or more depending upon the severity of the condition) daily doses
of between 25 mg and 5000 mg. A patient receiving treatment may
take a first dose upon waking and then a second dose in the evening
(if on a two dose regime) or at 3 or 4 hourly intervals thereafter.
Alternatively, a slow release device may be used to provide optimal
doses to a patient without the need to administer repeated
doses.
[0145] The invention further provides a pharmaceutical composition
comprising a therapeutically effective amount of a hydrogel,
compositions, cell-supporting medium, or medicament according to
the invention. In one embodiment, the amount of the hydrogel is an
amount from about 0.01 mg to about 800 mg. In another embodiment,
the amount of the hydrogel is an amount from about 0.01 mg to about
500 mg. In another embodiment, the amount of the hydrogel is an
amount from about 0.01 mg to about 250 mg. In another embodiment,
the amount of the hydrogel is an amount from about 0.1 mg to about
60 mg. In another embodiment, the amount of the hydrogel is an
amount from about 0.1 mg to about 20 mg.
[0146] The invention also provides a process for making a
pharmaceutical composition, the process comprising combining a
therapeutically effective amount of a hydrogel, compositions, or
cell-supporting medium according to the present invention, and a
pharmaceutically acceptable vehicle. A "therapeutically effective
amount" is any amount which, when administered to a subject
provides prevention and/or treatment of a specific medical
condition. A "subject" may be a vertebrate, mammal, domestic animal
or human being.
[0147] A "pharmaceutically acceptable vehicle" as referred to
herein is any physiological vehicle known to those of ordinary
skill in the art useful in formulating pharmaceutical compositions.
The pharmaceutically acceptable vehicle may be a liquid, and the
pharmaceutical composition is in the form of a solution. In a
further preferred embodiment, the pharmaceutical vehicle is a gel
or hydrogel, and the composition is in the form of a cream or the
like. In both cases, the composition may be applied to the
treatment site.
[0148] The composition may comprise one or more substances, which
may also act as lubricants, solubilisers, suspending agents,
fillers, glidants, compression aids, or binders. It can also be an
encapsulating material. Liquid vehicles are used in preparing
solutions, suspensions, emulsions, syrups, elixirs and pressurized
compositions. The hydrogel, compositions, cell-supporting medium,
or medicament may be dissolved or suspended in a pharmaceutically
acceptable liquid vehicle such as water, an organic solvent, a
mixture of both or pharmaceutically acceptable oils or fats. The
liquid vehicle may contain other suitable pharmaceutical additives
such as solubilisers, emulsifiers, buffers, preservatives,
sweeteners, flavouring agents, suspending agents, thickening
agents, colours, viscosity regulators, stabilizers or
osmo-regulators. Suitable examples of liquid vehicles for oral and
parenteral administration and implants include water (partially
containing additives as above, e.g. cellulose derivatives,
preferably sodium carboxymethyl cellulose solution), alcohols
(including monohydric alcohols and polyhydric alcohols, e.g.
glycols) and their derivatives, and oils (e.g. fractionated coconut
oil and arachis oil). For parenteral administration, the vehicle
can also be an oily ester such as ethyl oleate and isopropyl
myristate. Sterile liquid vehicles are useful in sterile liquid
form compositions for parenteral administration. The liquid vehicle
for pressurized compositions can be halogenated hydrocarbon or
other pharmaceutically acceptable propellent.
[0149] In cases where it is desired to inject or implant the
hydrogel, compositions, cell-supporting medium, or medicament
directly to the treatment site, liquid pharmaceutical compositions
which are sterile solutions or suspensions can be utilized by for
example, intramuscular, intrathecal, epidural, intraperitoneal,
intravenous and particularly subcutaneous, intracerebral or
intracerebroventricular injection. The hydrogel may be prepared as
a sterile hydrogel composition that may be dissolved or suspended
at the time of administration using sterile water, saline, or other
appropriate sterile injectable medium. Vehicles are intended to
include necessary and inert binders, suspending agents, lubricants,
sweeteners, preservatives, dyes, and coatings.
[0150] It is preferred that the hydrogel, compositions,
cell-supporting medium, or medicament according to the invention
may be implanted in the form of a sterile solution or suspension or
gel or hydrogel containing other solutes or suspending agents (for
example, enough saline or glucose to make the solution isotonic),
bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80
(oleate esters of sorbitol and its anhydrides copolymerized with
ethylene oxide) and the like. Preferably, the hydrogel is implanted
either in liquid or solid (hydrogel) composition form. Compositions
suitable for implants include liquid forms, such as solutions,
syrups, elixirs, and suspensions.
[0151] It will be appreciated that the self-assembling hydrogels
according to the invention have a wide range of medical
applications, for use in the method of the second aspect. In
addition, the inventors also explored the use of the
self-assembling hydrogel in a range of non-medical applications,
for example, in 3D cell culturing, in vitro toxicity testing,
understanding cell/extracellular matrix interactions, studies of
mechanical loading effects on cells, and cell study or metastasis
models.
[0152] Therefore, the inventors made a comparison of current
materials, which are available for in vitro 3D cell studies, with
the hydrogel used in the method according to the first aspect of
the invention. The currently available materials that the inventors
tested included: Puramatrix; Bovine Collagen; Agarose; and
chitosan.
[0153] Hence, according to a further aspect, there is provided use
of a composition according to the cell-supporting medium according
to the fourth aspect for studying a cell culture in vitro.
[0154] The comparison showed that Puramatrix can be somewhat
difficult to handle, and initially somewhat toxic to cells (pH
3-4). Furthermore, bovine collagen, agarose and chitosan are
unsatisfactory model systems due to batch-to batch variations of
the material, difficulty in handling and/or significantly different
properties to the in vivo extracellular matrix. It is preferred
that the use comprises initially preparing a hydrogel from the
self-assembling peptides, and then adding a cell culture thereto,
so that the cell behaviour under conditions that mimic in vivo
growth environment can be studied. Hence, preferably the cell
culture grown on the cell-supporting medium is substantially 3D.
The growth experiments may be carried out in 20 or 96 well plate
format and may have applications in 3D cell culture, in vitro
toxicity testing, understanding cell/extracellular matrix
interactions, controlled stem cell differentiation, studies of
mechanical loading effects on cells, and the study of metastasis
models. Currently, Puramatrix, Bovine Collagen, Agarose or chitosan
are used, which the inventors have found to be significantly
inferior cell supporting medium according to the fourth aspect.
[0155] All of the features described herein (including any
accompanying claims, abstract and drawings), and/or all of the
steps of any method or process so disclosed, may be combined with
any of the above aspects in any combination, except combinations
where at least some of such features and/or steps are mutually
exclusive.
[0156] For a better understanding of the invention, and to show how
embodiments of the same may be carried into effect, reference will
now be made, by way of example, to the accompanying diagrammatic
drawings.
Example 1
[0157] The inventors conducted a series of experiments to
investigate the design and preparation of hydrogel scaffolds, and
their use in supporting tissue cell cultures. The inventors
believed that such hydrogel scaffolds could have significant uses
in biological tissue regeneration and engineering.
Materials and Methods
(1) Preparation of Peptide Hydrogels
[0158] Fmoc peptides (from Bachem Ltd) were weighed out into 4 ml
glass vials. 2 ml purified water was added followed by sonication
for 30-60 seconds. 0.5M NaOH solution was added in 50 .mu.l
droplets until a clear solution was formed. 0.1M HCl was added to
the solution by stepwise addition of 50 .mu.l followed by mixing
using a vortex. pH values were estimated by transferring a spatula
tip of hydrogel onto universal indicator paper.
(2) Cell Cultures
[0159] Bovine chondrocytes were isolated from cartilage of the
proximal side of the metacarpalphalangeal joint, washed in PBS and
incubated overnight in Dulbeccos Modified Eagles Medium (DMEM)
supplemented with 10% foetal calf serum, 100 units ml
penicillin/streptomycin and 0.85 mM ascorbic acid. The cartilage
was finely chopped and incubated with pronase type E (700 units
ml.sup.-1) (BDH Ltd., Poole, UK) in medium for 2 h followed by
collagenase type 1a (300 units ml.sup.-1) (Sigma-Aldrich Co. Ltd.,
Poole, UK) in medium for 2 h. The cell suspension was centrifuged
at 1500 rpm for 5 min to pellet the cells. The cells were washed
twice in medium and seeded directly onto the peptide scaffolds (and
tissue culture plastic control) or encapsulated within the
scaffolds during the gelation stage at a cell density of
5.times.10.sup.5 ml.sup.-1 in medium. The cultures were maintained
in an incubator at 37.degree. C. with a humidified atmosphere of 5%
CO.sub.2 for up to 4 weeks. Chondrocytes were used up to passage
5.
(3) MTT Assay for Cell Proliferation
[0160] Cells were cultured for 1, 3 and 7 days. A 5 mg/ml of MTT
reagent in acidified isopropanol was used at a dilution factor of
1:10 in culture medium. This was added to cells and incubated for 4
hours. Culture medium was then removed and the reduced MTT reagent
removed by dissolution with acidified isopropanol. Optical density
of the reagent was then measured using a LabSystems Ascent
colourimetric plate reader at 570 nm. By preparing a standard curve
of MTT reduction against cell number, optical density values can be
converted into actual cell numbers.
(4) Microscopy CryoSEM
[0161] Microscopy CryoSEM was performed using a Philips XL30
ESEM-FG equipped with an Oxford Instrument Alto 25000 Cryotransfer
system, ESEM was performed using a Philips/FEI XL30 FEG-ESEM
equipped with a Peltier effect cooling stage. The
Confocal/multiphoton is a Biorad 1024MRC connected to an inverted
Nikon TE300 microscope. The laser for FITC is an American Laser
Corp Krypton Argon multiline 25 mW laser with 488 nm, 568 nm and
648 nm laser lines. The emission filter used was a 522df35, the
DAPI laser was a Ti-Sapphire Spectra-Physics Tsunami multiphoton
laser at 740 nm. The emission filter is 450/40, the lens was a
Nikon 40X 1.3NA oil Plan Fluor objective,
(5) Preparation of Fmoc-Dipeptides
[0162] The inventors prepared using standard techniques an Fmoc
capped dipeptide, Fmoc-Phe-Phe and prepared hydrogels in accordance
with Table 1 Referring to FIG. 1e, there is shown the standard
formula of the dipeptide, which shows the position of R1 and R2, as
used in Table 1.
TABLE-US-00001 TABLE 1 Properties of a self-assembled
Fmoc-dipeptides Fmoc- Concentration Entry Peptide R1 R2 pH (mM) 1
Phe-Phe CH.sub.2--C.sub.5H.sub.6 CH.sub.2--C.sub.5H.sub.6 4-8
2.9-30.0 2 Gly-Gly + 4-8 2.2-30.0 Phe-Phe 3 Phe-Phe + 4-8 3.7-30.0
Lys
(6) Preparation of Hydrogel Scaffolds
[0163] Self-assembled scaffolds were prepared by first suspending
the Fmoc-dipeptide in purified water. Upon increase of the pH by
addition of concentrated NaOH (thus de-protonating the carboxylic
acid group) to a value of >8, a clear solution was obtained. To
this solution, concentrated hydrochloric acid was added drop wise
until the pH of the solution became 7, at which point a clear,
self-supporting gel was formed.
[0164] The circular dichroism spectrum of Fmoc-Phe-Phe-OH was
collected on a Jasco J-810 spectrometer, using a 0.5 mm cuvette,
190-350 nm, with a 1 nm slit width and 4 second accumulation and 3
acquisitions. The gel was formed at 10 mM/L, as previously
described.
[0165] The results are shown in FIG. 1.
[0166] The minima at 218 nm indicates the formation of a
.beta.-sheet structure and the minima at 305 nm is due to the Fmoc
group (1A). Fluorescence spectrum of Fmoc-Phe-OH as a solution
indicates that the monomer fluoresces at 320 nm after excitation at
290 nm, when this is a precipitate the emission peak shifts to 330
nm. The gel formed by Fmoc-Phe-Phe-OH has also has an emission peak
at 330 nm, this shift is an indicator of excimer formation where
Fmoc groups are close enough to form dimers. The additional broad
peak observed in the Fmoc-Phe-Phe-OH gel spectrum indicates that
higher order aggregates have been formed (1B). A putative model of
the structure formed is presented in 1C and D, in this model Fmoc
groups stack over one another creating .pi.-.pi. interactions,
while the .beta.3-sheets form anti-parallel to one another with
hydrogen bonding (1D). This can form a tubular structure due to the
inherent twist present in .beta.-sheets and the arrangement of
several .beta.-sheets next to one another (1C).
(7) Stability of Hydrogel Scaffolds In Vivo
[0167] For these hydrogels to have applications in biomedicine, it
is essential that they are able to withstand near neutral pH values
and also high ion concentrations. Fmoc-Phe-Phe, which is a very
hydrophobic peptide formed a stable gel at pH 7 as summarised in
Table 1. It was then assessed whether the gel properties could be
tuned using mixtures of different peptides. As a starting point,
Phe-Phe was mixed with the dipeptide Gly-Gly in different ratios.
This is shown as entry 2 in Table 1. It was found that stable gels
were obtained at pH 7 down to 25 mol % of 7. Interestingly, the
50:50 mixture of Phe-Phe with Gly-Gly formed a more stable gel
compared to that of pure peptide material (Phe-Phe). Furthermore,
this mixture liquefied into a clear solution by slightly increasing
the temperature to 40.degree. C. Upon cooling to 37.degree. C., the
hydrogel was reformed, which is a useful transition temperature for
applications involving cells (as shown in FIG. 2a).
[0168] It was then assessed whether functional cues or moieties
could be incorporated into the hydrogel scaffold structures. It is
known that cells respond favourably to positive charges. Hence, the
inventors tested the addition of a positively charged Fmoc amino
acid (i.e. lysine). This is shown as entry 3 in Table 1. The
inventors hypothesised that the lysine residues would be
incorporated into the Fmoc .pi.-stack thus giving rise to a
distribution of charged groups throughout the structure. As
summarised in Table 1, the mixture of Phe-Phe composition with
Fmoc-Lys was subsequently tested for its stability in cell culture
conditions, and it was found to retain its gel like structures when
placed in culture media and incubated at 37.degree. C.
(8) Preparation of a 3D Culture of Chondrocytes on the Hydrogel
Scaffold
[0169] Finally, all three gels prepared (ie, entries 1, 2 and 3)
were tested for their ability to support proliferation and
retention of phenotype of bovine chondrocytes. For entries 1 and 2,
cells in culture media were seeded on top of the preformed gel, and
the culture media was rapidly taken up by the hydrogel. For entry
3, cells were incorporated into the gel by mixing with the
appropriate Fmoc-peptide solution that was liquefied by slightly
increasing the temperature to 40.degree. C. Upon cooling to
37.degree. C., the hydrogel was reformed with cells distributed
throughout (as shown by the arrows in FIG. 2d).
[0170] During cell culture of up to 7 days, chondrocytes were found
to retain morphological phenotype and to proliferate on all three
gels tested. FIG. 2b shows chondrocyte cells on the surface of gel
entry 1. The rounded cell shape is the typical phenotype for
chondrocytes. This observation suggests that this hydrogel scaffold
would be suitable for 3D tissue culture of these cells in vitro or
for cartilage regeneration in vivo. Two-photon fluorescence
microscopy was used to observe samples stained with DAPI, a
fluorescent nucleic acid stain that enables visualisation of cell
nuclei. This experiment confirmed the presence of cells throughout
the gel matrix (as shown in FIG. 2b), ESEM allows for interrogation
of the hydrogel structures while hydrated, and revealed a number of
rounded features of 10-20 micron in diameter, thought to be
chondrocyte cells (as shown in FIG. 2c).
[0171] The number of metabolically active cells in the scaffold was
then determined by using a simple colorimetric assay (MIT).
Continuous cell growth was measured at three different time points
up to 7 days (as shown in FIG. 3). These experiments revealed that
the three gels 1-3 shown in Table 1 support cell proliferation,
with gels 1 and 2 showing similar growth profiles. However, gel 2
(i.e. Fmoc-Phe-Phe mixed with Fmoc-Lys) showed significantly more
cells after 7 days (p<0.05). While the inventors do not wish to
be bound by any hypothesis, they believe that this observation may
be related to the incorporated cationic Fmoc-Lys residues into the
structure of gel 3.
Example 2
[0172] Starting with the promising results produced in Example 1,
i.e. Phe-Phe dipeptides were shown to self-assemble into stable
hydrogels, the inventors wanted to investigate further how the
design of the self-assembling peptides could be modified to produce
other stable hydrogels under physiological conditions.
[0173] The inventors thought it would be sensible to retain the two
consecutive phenylalanine residues in the peptide, but introduce a
third amino acid immediately after the Fmoc cap and before the
Phe-Phe. Hence, the inventors produced four tripeptides each of
which consisted of Fmoc-X-Phe-Phe, where X=Ala, Val, Leu, Phe. In
addition, the inventors also made the tripeptide
Fmoc-Leu-Leu-Leu.
[0174] The five tripeptides all formed stable hydrogels as shown in
Table 2 below.
TABLE-US-00002 TABLE 2 A number of further Fmoc-amino
acid/di-peptide combinations that formed hydrogels Entry Fmoc-AA
Di-peptide Gel formed? 1 Ala Phe-Phe 2 Val Phe-Phe 3 Leu Phe-Phe 4
Phe Phe-Phe 5 Leu Leu-Leu .sup.a mixture of Fmoc-peptides formed
.sup.b 60 .mu.mol starting materials was used
Example 3
[0175] Table 3 below identifies various peptide derivatives
(incorporating an aromatic stacking ligand) and mixtures thereof
that formed gels at a pH of 6-8 at the indicated
concentrations:
TABLE-US-00003 TABLE 3 Conc. Cone. Component 1 mM/L Component 2
mM/L pH Gel? Fmoc-Gly-Gly-OH 10-40 -- 3-5 Fmoc-Gly-Gly-OH 10-20
Fmoc-Lys-OH 10-20 5 Fmoc-Ala-Ala-OH 40 -- 3 Fmoc-Phe-Phe-OH 10 -- 7
Fmoc-Phe-Phe-OH 4-16 Fmoc-Lys-OH 16-4 7 Fmoc-Phe-Phe-OH 10
Fmoc-Gly-Gly-OH 10 7 Fmoc-Phe-Phe-OH 7.5 Fmoc-Gly-Gly-Arg- 7.5 7
Gly-Asp-OH Fmoc-Phe-Phe-OH 7.5 Fmoc-Gly-Gly-Arg- 7.5 7 Gly-Glu-OH
Fmoc-Phe-Phe-OH 10-20 Fmoc-Gly-Gly-OH 10-20 7 Fmoc-Phe-Phe-OH 10
Fmoc-Trp-OH 10 7 Fmoc-Phe-Phe-Phe-OH 5-20 -- 7 Fmoc-Phe-Gly-OH 20
-- 3-5 Cbz-Phe-Phe-OH 10-30 -- 6-8 Fmoc-Phe_OH 10-20 Fmoc-Lys-OH
10-20 3-5 Fmoc-Leu-Gly-OH 20 -- 2 Cbz-Leu-Leu-Leu-OH 5-20 -- 7
Table abbreviations Fmoc--fluorenylmethyoxycarbonyl,
Cbz--carboxybenzyl
Example 4
[0176] An initial 2D culture of human adult dermal fibroblasts was
carried out on the surface of 10 mM/L Fmoc-Phe-Phe-OH
self-assembled peptide hydrogels and cell phenotype was
investigated under inverted light microscope.
[0177] Generally, 0.0107 gram of Fmoc-Phe-Phe-OH was weighed in a
glass vial and sterilized for 30 minutes by an ultraviolet light
with bottles of distilled water, filtered NaOH (Sodium Hydroxide,
0.5M/L), filtered HCl (Hydrochloric Acid, 0.5M/L), and relevant
apparatus (spatulas, pipettes, Vortex). 2 mL of the sterile
distilled water was then added into the glass vial of Fmoc-Phe-Phe,
and the mixture was vortexed for a few seconds to create a
suspension. Afterwards, approximate 100 .mu.L of NaOH was gradually
pipetted into the suspension (20 .mu.L each pipetting) and the
mixture was vortexed after every addition of the alkaline. The
whole mixture was shaken continually until a homogeneous
transparent solution was obtained. The basic peptide solution (pH
around 10) was finally neutralized to pH 7 by dropwise addition of
HCl and pH values were monitored by a pH meter with a
micro-probe.
[0178] The above peptide solutions of physiological pH were
aliquoted into a 24 well-plate with 500 .mu.L in each well and the
well plate was maintained in a 37.degree. C./5% CO.sub.2 incubator
overnight. The solution underwent a self-assembling to become
hydrogels. Human adult dermal fibroblasts in suspension were then
poured on top of the self-assembled hydrogels with 1mL cell
suspension for each well. Cell suspensions of 8.times.10.sup.4/mL
were used with a serum-free DMEM (Dulbecco's modified Eagle's
medium) supplemented by 1% antibiotics/antimicotics. The cell
culture was maintained for up to 72 hours in the 37.degree. C./5%
CO.sub.2 incubator and cell phenotype was observed at different
time points. The results are shown in FIG. 5 which shows the
appearances of human adult dermal fibroblasts cultured on top of
self-assembled peptide hydrogels of Fmoc-Phe-PheOH at time points
of 4 hours (spreaded), 6 hours (spreaded) and 24 hours (rounded),
Within the first 8 hours of culturing, cell attachment and
spreading was observed as cells were flattened on the surfaces and
possessing a spindle-like to polygonal phenotype, regardless of the
hydrogel types. These originally spreaded cells, however, became
rounded with a diameter of around 1.0 microns between 24 and 72
hours. The phenomenon indicated of a dynamic solid (gel)-liquid
(culture medium) interface which formed a malleable, unstable gel
surface to wrap and sink cells into the interacted nano-filaments
of the gel structure resulting the cells' rounding up.
[0179] With 10 mM/L Fmoc-Phe-Phe-OH self-assembled hydrogels, human
adult dermal fibroblast were also seeded 3 dimensionally (31)
culture) inside the peptide gels. When trapped in a 3D aqueous
hydrogel, oval to round cell shape remained for a long term; cells
falsely sensed themselves in a cell-cell contact environment
therefore proliferation phase for increasing cell population was
shut off, Despite proliferation, whether ECM components (proteins
and saccharides) were synthesized and secreted by these cells is
another way to justify cells' reaction to the material, However,
viability of the cells is of necessity to maintain a steady ECM
secretion or to restore proliferation after possible cell spreading
at later time points.
Example 5
[0180] In order to coarsely test cell viability, a Live/Dead assay
was chosen in which two reagents of EthD-1 and Calcine AM were
involved. EthD-1 is able to enter intact cell membranes of living
cells and to selectively react with the cells metabolically to form
a bright green fluorescence dye, while Calcine AM can only enter
broken nucleus membranes of dead cells to stain the nucleus red.
With the help of this staining and fluorescence microscope, living
and dead cells could be easily visualized therefore viability
inside the culture is measured.
[0181] For making Fmoc-Phe-Phe-OH peptide solution, 0.0107 gram of
Fmoc-Phe-Phe-OH was weighed in a glass vial and sterilized for 30
minutes by an ultraviolet light with bottles of distilled water,
filtered NaOH (Sodium Hydroxide, 0.5M/L), filtered HCl
(Hydrochloric Acid, 0.5M/L), and relevant apparatus (spatulas,
pipettes, Vortex). 2 mL of the sterile distilled water was then
added into the glass vial of Fmoc-Phe-Phe-OH, and the mixture was
vortexed for a few seconds to create a suspension, Afterwards,
approximate 100 .mu.L of NaOH was gradually pipetted into the
suspension (20 .mu.L each pipetting) and the mixture was vortexed
after every addition of the alkaline. The whole mixture was shaken
continually until a homogeneous transparent solution was obtained.
The basic peptide solution (pH around 10) was finally neutralized
to pH 7 by dropwise addition of HCl and pH values were monitored by
a pH meter with a micro-probe. The solution was left in a 4.degree.
C. refrigerator overnight until usage.
[0182] The solution was warmed at room temperature on the day of
cell culture for around 1 hour. Human dermal fibroblasts were
trypsinised and centrifuged into a loose pellet of 2 million in a
centrifuge tube. About 200 .mu.l of complete culture medium (DMEM
with 10% bovine fetal serum and 1% antibiotics/antimicotics) was
added to the pellet and pipetted to obtain a condensed cell
suspension; after which 1800 .mu.l of the Fmoc-Phe-Phe-OH solution
was poured into the tube and the whole thing was vortexed gently to
get a homogeneous pale-pink viscous solution with 1 million/ml cell
density. The cell-containing solution was then transferred to a 24
well-plate with 500 .mu.l in each well. A further 1 ml of complete
culture medium was poured onto each cell-peptide solution drop by
drop. The self-assembling mechanism was rapidly triggered by medium
components and stable hydrogels was formed in seconds.
[0183] The 3D culture inside Fmoc-Phe-Phe-OH was maintained for 3
days and live/dead staining was done at various time points. The
majority of cells were shown to be living in the Fmoc-Phe-Phe-OH
hydrogel (stained green) after 72 hours, although cells did not
complete spreading in the gels during the first 3 days. A few
pictures at 3 hour time-point showed tiny stretched-out filophodia
suggesting there might be partial spreading cells. The results are
shown in FIG. 6 which shows live/dead staining of cells inside
Fmoc-Phe-Phe-OH hydrogel: A: magnified from part of photo B showing
partially spreaded cells (3 hours after culture); B: 3 hours after
culture; C. 72 hours after culture.
Example 6
[0184] In order to quantitatively test cell viability in the 3D
culture, LDH assay was adopted which tested the amount of lactate
dehydrogenase inside cell mitochondria membrane (reflecting cell
numbers).
[0185] 0.0107 gram of Fmoc-Phe-Phe-OH was weighed in a glass vial
and sterilized for 30 minutes by an ultraviolet light with bottles
of distilled water, filtered NaOH (Sodium Hydroxide, 0.5M/L),
filtered HCl (Hydrochloric Acid, 0.5M/L), and relevant apparatus
(spatulas, pipettes, Vortex). 2 mL of the sterile distilled water
was then added into the glass vial of Fmoc-Phe-Phe-OH, and the
mixture was vortexed for a few seconds to create a suspension.
Afterwards, approximate 100 .mu.L of NaOH was gradually pipetted
into the suspension (20 .mu.L each pipetting) and the mixture was
vortexed after every addition of the alkaline. The whole mixture
was shaken continually until a homogeneous transparent solution was
obtained. The basic peptide solution (pH around 10) was finally
neutralized to pH 7 by dropwise addition of HCl and pH values were
monitored by a pH meter with a micro-probe. The solution was left
in a 4.degree. C. refrigerator for overnight until usage.
[0186] The solution was warmed at room temperature on the day of
cell culture for around 1 hour. Human dermal fibroblasts were
trypsinised and centrifuged into a loose pellet of 2 million in a
centrifuge tube. About 200 .mu.l of complete culture medium (DMEM
with 10% bovine fetal serum and 1% antibiotics/antimicotics) was
added to the pellet and pipetted to obtain a condensed cell
suspension; after which 1800 .mu.l of the Fmoc-Phe-Phe-OH solution
was poured into the tube and the whole thing was vortexed gently to
get a homogeneous pale-pink viscous solution with 1 million/ml cell
density. The cell-containing solution was then transferred to a 24
well-plate with 500 .mu.l in each well. A further 1 ml of complete
culture medium was poured onto each cell-peptide solution drop by
drop to get hydrogel formed in seconds.
[0187] The culture was maintained in a 37.degree. C./5% CO.sub.2
incubator for a 10 day period and LDH assay was carried out after 1
hour, 1 day, 3 days, 7 days, and 10 days. The gels were scooped out
and vortexed to a viscous liquid mixture and freeze-thawed to
release lactate dehydrogenase (LDH) from cells. Assay reagent was
then added, the mixture was incubated, and light absorbance at 490
nm was then measured. Absorbance was converted to cell number
according to a standard. The results of this LDH assay of cell
viability (3D culture of human adult dermal fibroblasts in
Fmoc-Phe-Phe-OH) are shown in FIG. 7. The results showed that there
was initially a decreasing of cell numbers, but with the remaining
living cells, the proliferation happened after 1 week with rapid
cell number increasing of 5 folds from day 7 to day 10.
Example 7
[0188] Fmoc-Gly-Gly-Arg-Gly-Asp-OH (Fmoc-GGRGD) adhesion motifs
were introduced to tackle the issue of un-spreaded cells in
Fmoc-Phe-Phe-OH gels therefore to induce spreading and focal
adhesion of dermal fibroblasts in these self-assembled peptide
hydrogels. Fmoc-Phe-Phe-OH and
Fmoc-Phe-Phe-OH+Fmoc-Gly-Gly-Arg-Gly-Glu-OH (Fmoc-GGRGE)
combination were set as comparisons.
[0189] 0.0014 grams of Fmoc-GGRGD or Fmoc-GGRGE (1 mM/L in later
hydrogels), were weighed and mixed with 0. 0107 grams
Fmoc-Phe-Phe-OH respectively into glass vials, and 0.0107 grams
Fmoc-Phe-Phe-OH alone was weighed as well. The above peptide and
peptide mixtures were sterilized for 30 minutes by an ultraviolet
light exposure with bottles of distilled water, filtered NaOH
(Sodium Hydroxide, 0.5M/L), filtered HCl (Hydrochloric Acid,
0.5M/L), and relevant apparatus (spatulas, pipettes, Vortex). 2mL
of the sterile distilled water was then added into each glass vial
of Fmoc-Phe-Phe-OH, Fmoc-Phe-Phe-OH+Fmoc-GGRGD, and
Fmoc-Phe-Phe-OH+Fmoc-GGRGE, and the mixtures were vortexed for a
few seconds to create suspensions, Afterwards, approximately 100
.mu.L of NaOH was gradually pipetted into every suspension (20
.mu.L each pipetting) and the mixtures were further vortexed after
every addition of the alkali. The whole mixtures were shaken
continually until homogeneous transparent solutions were obtained.
The basic peptide solutions (pH around 10) were finally neutralized
to pH 7 by dropwise addition of HCl and pH values were monitored by
a pH meter with a micro-probe. The solutions were left in a
4.degree. C. refrigerator for overnight until usage.
[0190] The solutions were warmed at room temperature on the day of
cell culture for about an hour. Human dermal fibroblasts were
trypsinised and centrifuged into a loose pellet of 2 million in a
centrifuge tube. About 200 .mu.L of complete culture medium (DMEM
with 10% bovine fetal serum and 1% antibiotics/antimicotics) was
added to the pellet and pipetted to obtain a condensed cell
suspension; after which 1800 .mu.L of the Fmoc-Phe-Phe-OH,
Fmoc-Phe-Phe-OH+Fmoc-GGRGD, or Fmoc-Phe-Phe-OH+Fmoc-GGRGE solution
was poured into the tube and the whole thing was vortexed gently to
get a homogeneous pale-pink viscous solution with 1 million/mL cell
density. The cell-containing solutions were then transferred to 24
well-plates separately with 500 .mu.L in each well. A further 1 mL
of complete culture medium was poured onto each cell-peptide
solution drop by drop to get hydrogel formed in seconds.
[0191] The cell culture in the 3 different gel types were kept in
the 37.degree. C./5% CO.sub.2 incubator and cell phenotype was
observed after 24 hours by an inverted optic microscope.
[0192] The results are resulted are illustrated in FIG. 8 which
shows cell phenotype and size comparison in
Fmoc-GGRGD+Fmoc-Phe-Phe-OH(A), Fmoc-GGRGE+Fmoc-Phe-Phe-OH(C) arrows
pointing to cells).
[0193] Different from the other two, Fmoc-Phe-Phe-OH+Fmoc-GGRGD
hydrogels made cells larger with obvious oval to round nucleus and
flattened membranes. The average cell size in the RGD-containing
hydrogels was 35 microns compared to around 20 microns in the
RGE-containing hydrogels or gels without RGE/RGD.
[0194] Higher concentrations of RGD/RGE-containing Fmoc-Phe-Phe-OH
were prepared with 50% (7.5 mM/L) of Fmoc-GGRGD (or Fmoc-GGRGE) and
50% of Fmoc-Phe-Phe-OH (7.5 mM/L). Stronger transparent hydrogels
were formed as they were easily lifted up with a thin spatula
without broken pieces after even 10 days. Same cell-size phenomenon
were observed as in 50%:50% (Fmoc-GGRGD: Fmoc-Phe-Phe-OH) hydrogels
cells were larger with flat membranes compared to those of 50%:50%
(Fmoc-GGRGE: Fmoc-Phe-Phe-OH) and Fmoc-Phe-Phe-OH hydrogels.
Example 8
[0195] Human mesenchymal stem cells (MSCs) were isolated from bone
marrow taken from patients (with both patient and ethical consent)
undergoing hip replacement surgery. A Histopaque (Sigma) gradient
was used to isolate mononuclear cells and these cells were cultured
in monolayer with .alpha.-MEM (with 10% heat-inactivated foetal
calf serum, 100 U/ml streptomycin/penicillin and 0.85 mM ascorbic
acid) under standard culture conditions (humidified atmosphere,
37.degree. C., 5% CO.sub.2). After 5 days non-adherent cells were
removed by washing with media.
[0196] At 80% confluence in passage 3 the MSCs were trypsinised and
a cell count was performed. A suitable number of cells were
centrifuged at 400 g for 5 minutes and then resuspended in a 10 mM
Fmoc-Phe-Phe solution to a final concentration of 4.times.10.sup.6
cells/ml. The cell suspension was mixed to ensure even cell
distribution and 200 .mu.l layers were pipetted into high pore
density (0.4 .mu.m pore size) cell culture inserts in 24-well
plates. Media (.alpha.-MEM as previously described) was added
gently to both the well and the insert and gels were allowed to
polymerise.
[0197] The cell-seeded gels were then cultured under standard
conditions for 14 days with media changed every 2 days. Following
culture 1 ml of TRIzol was added to each insert and the gels
disrupted by pipetting. A modified RNA extraction procedure was
performed combining both the TRIzol and Purelink (Invitrogen)
extraction procedures. RNA was reverse transcribed to cDNA using
Superscript II (Invitrogen) and PCR performed using a standard
HotStarTaq (Qiagen) procedure for the house-keeping gene GAPDH as
well as the transcription factor SOX-9, aggrecan and collagen types
I and II.
[0198] PCR products were run on a 1.5% agarose gel containing
ethidium bromide and visualised on a UV-transilluminator.
[0199] The results are shown in FIG. 9. As shown in FIG. 9,
expression of GAPDH and type I collagen by MSCs in Fmoc-F-F gels
after 14 days showed that there were viable cells present and the
lack of expression of SOX-9, type II collagen or aggrecan suggests
these cells may be in an undifferentiated state.
Example 9
[0200] This Example provides a comparison of Fmoc and CBz as
aromatic stacking ligands.
[0201] In this Example Cryo-SEM was performed using a Philips XL30
ESEM-FG equipped with an Oxford Instrument Alto CT2500 for
cryo-transfer and cryo image purposes.
[0202] Fmoc-Phe-Phe-OH and Cbz-Phe-Phe-OH where prepared as
previously described. Approximately 100 mg of each of the hydrogels
prepared at a concentration of 40 mM/L were frozen using liquid
nitrogen and then placed in the SEM chamber for sublimation and
fracture. The sample specimens were then analysed at various
magnifications to observe different characteristics of interest.
The images were recorded digitally.
[0203] The results in the Cryo-SEM images of FIGS. 10C
(Fmoc-Phe-Phe-OH) and 10D (Cbz-Phe-Phe-OH), FIG. 10 shows that gels
formed by Fmoc-Phe-Phe-OH and Cbz-Phe-Phe-OH shows slightly
different macro-structure but within the dimensions of 5-300 nm
similar to that of the extracellular matrix. For the sake of
completeness, FIG. 10A shows the structure of Fmoc-Phe-Phe-OH and
FIG. 10B shows that of Cbz-Phe-Phe-OH.
CONCLUSIONS
[0204] As can be seen from the results, the research carried out by
the inventors has provided some very promising data. Preliminary
analysis of the test samples revealed the possibility of designing
stable gels that can withstand cell culture conditions (neutral pH
and high ion concentration and 37.degree. C.). These were found to
support the proliferation and retention of the phenotype of bovine
chondrocytes, human mesenchymal stem cells and human adult dermal
fibroblasts.
[0205] Hence, the inventors have demonstrated for the first time
that short Fmoc-dipeptides and tripeptides cause the self-assembly
of a range of fibrous hydrogel scaffolds with different structural
and functional properties. These hydrogels are: --(i) stable under
tissue culture conditions (high ionic strength, pH 7); (ii) of
similar dimensions to fibrous components of the extra cellular
matrix (nano-sized fibres); and (iii) capable of supporting cell
culture of chondrocytes in 2D and 3D.
[0206] The inventors believe that the peptides and the hydrogel
cell scaffolds they form may be used in a wide variety of medical
applications, such as in wound healing and in tissue
regeneration.
* * * * *