Special Technical Feature
PHOTOCHEMICAL CROSSLINKING OF PROTEINS TO
MAKE NOVEL BIOMEDICAL MATERIALS
Chris Elvin* and Tony Vuocolo
CSIRO Livestock Industries, Queensland Bioscience Precinct, St Lucia, QLD 4067
*Corresponding author: [email protected]
Introduction
We have adopted a biomimetics approach to address
unmet medical needs for better adhesives, tissue sealants and
cross-linked protein biomaterials for clinical applications.
We examined an original concept for its potential as a new
and facile practical crosslinking technology to produce
new protein-based biomaterials. Further, we have shown
the biological safety of the technology and the suitability
of this original approach to be exploited in a range of
medical applications. We have termed these new proteinbased biomaterials PhotoSeal™ (a tissue sealant) and
PhotoMatrix™ (a tissue engineering scaffold).
We exploited an original observation by Kodadek and
colleagues in 1999 (1) that uses a photochemical method
to generate protein dimers in order to understand
arrangements in protein complexes. The possibilities of
protein polymer formation and tissue stabilisation were
not envisaged, nor its application to provide unmet
medical needs in the form of new biomaterials. The method
entails the photochemical crosslinking of self-associating
proteins through the production of dityrosine cross-links.
In the presence of visible light (~450 nm) and an electron
acceptor (e.g., persulfate), the RuII in [RuII(bpy)3]2+ is
converted to a RuIII state. This in turn reacts with an
exposed tyrosine residue in a protein to give a tyrosyl
radical intermediate that reacts with another, nearby
tyrosyl radical residue to form a highly stable dityrosine
crosslink (Fig. 1).
Leveraging the interdisciplinary expertise of protein
and peptide chemists, cell and molecular biologists,
biochemists, mechanical engineers, veterinarians and
clinicians, the team has, in a very short time, been able to
develop a next-generation platform technology that has
potential for significant impacts on the medical device
industry and promotes the well-being of all Australians.
The technology delivers new biomedical materials that are
cheaper, more flexible, easier to use, and more effective
than current medical devices and can be tailored to target
unmet clinical needs as sealants, haemostats and scaffolds
for regenerative medicine.
Protein -based Biomaterials For med via
Dityrosine For mation
Fig. 1. Formation of dityrosine via RuII(Bpy) 3 catalysed photochemical oxidation.
Reprinted from Chemistry & Biology 7(9), Fancy, D.A.,
Denison, C., Kim, K., Xie, Y., Holdeman, T., Amini, F.,
and Kodadek, T., Scope, limitations and mechanistic
aspects of the photo-induced cross-linking of proteins
by water-soluble metal complexes, 697-708, Copyright
2000, with permission from Elsevier.
Vol 42 No 3 December 2011
It has been known for more than 40 years that fleas
are able to perform their extraordinary jumping feats
due to the presence of dityrosine-crosslinked resilin
pads in the joints of their legs. They use muscles to store
energy in these tiny protein-filled pads and, in less than
a millisecond, release nearly all of the stored energy,
thus launching into the air and onto a passing animal (2).
Dragonflies and cicadas also use resilin for improving
flying efficiency and for sound production, respectively
– in fact, probably all insects use resilin to perform
a variety of repetitive movements. This crosslinked
invertebrate protein is the vertebrate equivalent of elastin.
Elvin et al. (3) showed that a recombinant form of this
elastic insect protein, resilin (with a high tyrosine content
~7%), could be crosslinked by the Ru-based photochemical
method (Fig. 2). The biomaterial formed displayed many
of the properties of the native biomaterial, namely,
extremely high resilience (return of stored energy) and
elasticity (extension to break). We hypothesised that other
self-assembling, tyrosine-containing proteins might also
be susceptible to this facile photochemical crosslinking
method. The Ru-based method was found to be very rapid
and efficient, and so formed the core technology that has, in
the last five years, been used to produce a number of new
biomedical materials for a range of clinical applications.
AUSTRALIAN BIOCHEMIST
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Special Technical Feature
A
B
HN
HO
C CH
H2 NH
O
NH
O
C
O
OH
HC C
H2
HN
O
+
dityrosine + 2H + 2e–
2 tyrosine
D
soluble
pro-resilin
A 214
1.0 mm
resilin
tyrosine
A 214
crosslinked
resilin
air-filled core
cuticular
tendon
0.1 mm
dityrosine
1.0 mm
0
1
2
3
4
Retention time (min)
5
Fig. 2. Dityrosine formation in crosslinked recombinant (rec1) resilin.
(A) Structure of the dityrosine adduct.
(B) Fluorescence of resilin in the wing tendon from adult dragonfly (Zyxomma sp.). The lower panels show
photomicrographs of the tendon in phosphate-buffered saline under white light and ultraviolet light.
(C) Fluorescence of a moulded rod. A solution of rec1-resilin containing 2 mM [Ru(bpy)3]2+ and 10 mM
persulphate was drawn into a siliconised glass capillary tube (1 mm diameter) and irradiated with white light.
The rod was removed and washed extensively in buffer before photomicrography under white (upper panel) or
ultraviolet (lower panel) light.
(D) HPLC analysis of acid-hydrolysed uncrosslinked rec1-resilin and crosslinked rec1-resilin. Samples were
analysed by reverse-phase HPLC for dityrosine. A214 is the absorbance at 214 nm.
A Rapidly For med and Adhesive Surgical
Tissue Sealant – PhotoSeal™
One area that has been a particular focus has been
examining the potential of this novel technology for use as
a medical adhesive and sealant. Presently, this is an area
of significant unmet medical need (4). In general, synthetic
adhesives such as cyanoacrylates perform very well as
adhesives but are not particularly suitable in the medical
area as they are associated with cytotoxicity, poor resorption
and brittleness. Some are approved for topical use, but
they are unsuitable for internal applications. A number of
synthetic tissue sealants have also been developed in recent
years, but they also have a number of limitations, including
excessive swelling, low to moderate adhesive strength
and poor elasticity for use in sealing highly elastic tissues
such as lung and colon. A commonly used tissue adhesive
is based on fibrinogen, activated by added thrombin,
mimicking the natural coagulation pathway. This has been
broadly approved for use, but it is difficult to use and the
adhesive is not strong, taking more than 15 minutes to gain
even moderate strength.
Soon after demonstrating the facile, rapid and
quantitative photochemical crosslinking of recombinant
resilin, a CSIRO team showed that other self-assembling
proteins that contain tyrosine residues could also be
Page 16
photochemically crosslinked into hydrogel materials. This
was initially shown for fibrinogen, a blood protein involved
in clot formation and haemostasis. Fibrinogen is a 340 kDa
glycoprotein comprised of a disulphide-crosslinked dimer
of trimers (a, b and g subunits). During the normal bloodclotting cascade, thrombin cleaves two small peptides
(fibrinopeptides) from fibrinogen, causing the now-formed
fibrin to self-assemble into a clot. The clot is then stabilised
via enzymatic (transglutaminase) crosslinking of glutamine
and lysine residues, both within the fibrin clot and between
the fibrin clot and the tissue. A current commercial product
(Tisseel™) is based on the final stages of this clotting
cascade and is widely used in surgical applications. This
product has a number of limitations, however, including
poor adhesive strength, limited elasticity, minutes to hours
of curing to achieve full strength bonding, a requirement to
be shipped frozen and significant expense.
A fibrinogen-based form of PhotoSeal™ was evaluated
and shown to be rapidly (within 15 seconds of white light
illumination) crosslinked via dityrosine formation, forming
a hydrogel that fluoresces on illumination with UV light (5)
(Fig. 3). This biomaterial showed high adhesive strength
(about 10-fold higher than Tisseel™) when used to bond
two collagen membranes together.
AUSTRALIAN BIOCHEMIST
Vol 42 No 3 December 2011
Special Technical Feature
Biocompatibility of PhotoSeal™ Technology
For a photo-crosslinked protein to be useful in biomedical
applications, it is essential that it is biocompatible. Thus,
we have shown by various methods that neither the
RuII(Bpy)3 nor the persulfate are toxic to cells at the
concentrations present, with a 20- to 50-fold safety margin
being present (6). A key aspect of this is that we have
shown that the persulphate is rapidly consumed during
the photochemical reaction, so cells are not in contact
with high concentrations except for a few seconds. We
have further shown that cells can readily grow on the
photo-crosslinked protein, and that implants in rodents
are readily accepted. Furthermore, when C2C12 cells are
implanted in nude mice using photo-crosslinked gelatin/
fibrinogen scaffolds, they differentiate into multinucleate
muscle cells with formation of microvasculature
throughout the scaffold (7). Soluble keratins can also be
used to form photo-crosslinked scaffolds that support cell
growth and development (8).
Effectiveness in use is also a critical issue in biomedical
applications. Photo-crosslinked fibrinogen was shown to
easily match commercial thrombin/fibrinogen products
in strength and scarless repair in a rat dermal incision
model (9), but curing was >10-fold more rapid. The new
biomaterial showed strong adhesion, with no local toxicity
or organ toxicity, with neovascularisation and sound
healing. The process was also shown to be highly effective
in the repair of pig vascular puncture wounds, and readily
sealed and prevented blood loss from the wound.
A Generic Peptide/Protein Crosslinking
Approach
This new approach has evolved into a platform
technology that can be used for proteins and peptides
that have tyrosine functionality available. Electrophoresis
has also shown, for example, that many other proteins,
including collagen, gelatin, fibronectin and keratin, are
all efficiently and effectively crosslinked by the new
method (10). In addition, molecular biology and peptide
chemistry have been used to develop new tyrosinerich polypeptides based on novel repeating structures
which are also readily crosslinked. For example, in one
approach, designer oligonucleotides have been made
to allow the construction of peptide structures with one
tyrosine residue for every 7, 11 or 16 residues. Constructs
with polymers of either 4, 8, 16, 32 or 64 copies were
made and expressed in E. coli (11). Photo-crosslinking
has led to a highly crosslinked polymeric material. In a
complementary approach, short peptide monomers that
include termini for ligation have been made by solid phase
synthesis. Olefin methathesis using a second-generation
Grubbs’ catalyst is then employed to achieve complete
N-to-C ligation and peptide polymers of up to 90 kDa (12).
The process is generic and can be used to ligate short or
long monomeric units.
Subsequent photochemical crosslinking of these
assembled peptide sequences then leads to formation
of higher molecular weight polymeric materials. Of the
readily obtainable industrial proteins, gelatin was of
particular interest as it is readily available as a sterile,
approved medical material and is very cost effective
(>1,000 times cheaper than fibrinogen). Gelatin is currently
used as an adhesive/haemostat, for example as GRF™
glue (gelatin/resorcinol/formaldehyde), but this product
is well known to be brittle and for residual formaldehyde
to cause significant inflammation (13,14).
A Highly Elastic and Adhesive Tissue Sealant –
Gelatin PhotoSeal™
In addition to its low cost and currently accepted use
in medical devices, photo-crosslinked gelatin was found
to be highly elastic, showing an extension to break of
> 700%, highly adhesive with >110kPa adhesive strength
and caused no inflammation in vivo. The crosslinked
gelatin was also able to swell. In cases where swelling is
not required, this can be controlled by addition of extra
Fig. 3. Photochemical crosslinking of native bovine fibrinogen.
Fibrinogen solution was prepared from
frozen citrated bovine plasma.
(A) Lane 1, 50 mg of bovine plasma
cryoprecipitate; lane 2, following
photochemical crosslinking. Lane 3,
protein molecular weight markers
(molecular weight shown in kDa).
(B) Fluorescence of a moulded rod. The
rod was removed from the mould and
washed extensively in buffer. The rod
appeared straw-coloured under white
light (left panel) or blue under ultraviolet
light (right panel).
(C) HPLC analysis of acid-hydrolysed
uncrosslinked fibrinogen and crosslinked
fibrinogen. Samples were analysed by
reverse-phase HPLC for dityrosine.
Vol 42 No 3 December 2011
AUSTRALIAN BIOCHEMIST
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Special Technical Feature
Fig. 4. Applications of Gelatin-PhotoSeal™ to seal defects in various tissues.
(A) Photocrosslinking of Gelatin-PhotoSeal™ with a 300W xenon endoscope lamp. (B) Porcine vascular defect.
(C) Sheep lung lobe defect. (D) Sheep dura repair.
phenolic side chains by either tyramine or Bolton-Hunter
modification. These materials fully retain their excellent
adhesive properties, but the swelling is reduced with
the degree of modification, with a commensurate loss in
elasticity (5). Excessive swelling of some surgical sealants
has led to complications in their use and concerns have
been raised about their safety (15,16).
Gelatin formulations have been used very successfully
in animal models (Fig. 4), especially for tissues that are
elastic and need an elastic adhesive/sealant, such as
lung and colon. A number of surgical models have been
carried out using gelatin PhotoSeal™ to repair wounds
in colon, lung and dura in sheep. Most recently, further
applications of the technology have been investigated.
These include the manufacture of protein sheets that allow
delivery of certain antibiotics and proteins, the delivery of
particles, for example for orthopaedic applications, and
the crosslinking of native tissues for wound repair and
cardiovascular applications (10,17). Of particular interest
has been the success with this approach for the delivery
of cells for tissue engineering applications (7,8). The
implantable scaffolds that have been developed can use
the same composition and biochemistry as the sealant, and
can be used with or without cells and biological factors,
and for clinical use could be delivered arthroscopically
and cured rapidly in situ.
Conclusions
We continue to develop biomaterials based on this facile
and simple photochemical crosslinking technology that
may find applications in both medicine and industry. These
materials may be fabricated from synthetic polymers or be
based on native proteins. We predict that new generation
materials based on fabrication of non-degradable, synthetic
peptide mimics will yield new materials for use in medicine
(as spinal disc prostheses or heart valves) and industry (as
actuators, running shoes, golf balls or green energy storage
materials).
Page 18
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Vol 42 No 3 December 2011