Deakin Research Online
This is the published version:
Byrne, Nolene, DeSilva, Rasike, Whitby, Catherine P. and Wang, Xungai 2013, Silk
scaffolds achieved using pickering high internal phase emulsion templating and ionic liquids,
RSC advances, vol. 3, no. 46, pp. 24025-24027.
Available from Deakin Research Online:
http://hdl.handle.net/10536/DRO/DU:30058918
Reproduced with the kind permission of the copyright owner.
Copyright : 2013, RSC Publications
RSC Advances
Published on 01 October 2013. Downloaded by Deakin University on 04/12/2013 00:07:57.
COMMUNICATION
View Article Online
View Journal | View Issue
Silk scaffolds achieved using Pickering high internal
phase emulsion templating and ionic liquids†
Cite this: RSC Adv., 2013, 3, 24025
Nolene Byrne,*a Rasike DeSilva,a Catherine P. Whitbyb and Xungai Wangac
Received 29th August 2013
Accepted 27th September 2013
DOI: 10.1039/c3ra44749a
www.rsc.org/advances
We describe a convenient route to the preparation of silk scaffolds
that does not require silk fiber dissolution and regeneration. We
prepare the silk scaffolds via a single step Pickering-high internal
phase emulsion (HIPE) method. Additionally, we find that the use of
biocompatible ionic liquids significantly improves the compressive
properties of the HIPEs.
Silk as a biomaterial has gained signicant interest due to its
biocompatibility and excellent mechanical properties.1
However, normally silk to be produced into a scaffold needs to
be dissolved and regenerated.1,2 The dissolution of silk involves
the use of harsh solvents and multiple processing steps.3 The
typical procedure for native silk ber dissolution is to boil in
alkaline pH to remove the sericin, dissolve the silk ber in
9MLiBr, dialyse to remove the salt and solubilize the amorphous silk in either formic acid4 or hexauoroisopropanol,5
although ionic liquids (ILs) have been shown to directly dissolve
silk cocoons.6 The regeneration of silk is also a complex process,
with many different solvents and processing techniques being
investigated currently.7–11 The major disadvantage with the
dissolution and regeneration of silk, apart from the complexity
and time involved, is the loss of the native silk ber structure
which impacts the material properties of the regenerated silk.12
In this communication, we describe the preparation of novel
silk scaffolds using a Pickering emulsion – HIPE process.
High internal phase emulsions (HIPEs) are dened as having
an internal phase volume (f) of 0.74 or greater.13 HIPEs nd use
in numerous applications including food preparation, fuels, oil
recovery, cosmetics and recently as templates in material
science.14,15 Oen HIPEs require surfactants14,16,17 or particles18
to enhance stabilization and prevent coalescence or sedimentation. Recently ionic liquids have been successfully used as
surfactant materials19 as well as to form emulsions.20 In addition, ionic liquids, ILs, have been shown to enhance protein
stability against aggregation21,22 as well as being biocompatible
crosslinking agents for collagen proteins.23 For these reasons,
we have explored the impact of using biocompatible ionic
liquids in the preparation of the silk particle stabilized HIPEs.
We studied 3 different biocompatible ILs, cholineTa, chloineLa
and EOALa. The choline salts were selected as they have recently
been shown to cross link collagen23 as well as stabilize
proteins,22 while EOALa is a simple protic ionic liquid known to
possess amphiphilic properties.24
High internal phase emulsions were achieved via a single
step process at 10 wt% silk (Fig. 1). Briey the ball milled silk
particles25 were dispersed in the aqueous phase, the required
amount of oil was added and the solution homogenised for
1 minute. The oil used was the biocompatible dodecane. We
found that by using the 2 step process,26 lower silk wt% could be
used to achieve a HIPE (see Table S1 in ESI†). We achieved
HIPEs with internal volumes of up to 87% without the use of a
surfactant. The inuence of ionic liquid addition on the HIPEs
was investigated at concentrations of 0.5 M, 1 M and 2.5 M. The
IL was dissolved in the aqueous phase prior to the silk particles
being added. Addition of IL resulted in a decrease in the
internal volume fraction of the HIPEs to 80%. Fig. 2a–d shows
a
Institute for Frontier Materials, Deakin University, Geelong, Vic 3217, Australia.
E-mail: [email protected]
b
Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA 5095,
Australia. Fax: +61 8 8302 3683; Tel: +61 8 8302 6866
c
School of Textile Science & Engineering, Wuhan Textile University, Wuhan, China
† Electronic supplementary information (ESI) available: Tables containing HIPE
conditions are provided plus additional graphs. See DOI: 10.1039/c3ra44749a
This journal is ª The Royal Society of Chemistry 2013
Fig. 1 (a) Images showing samples at 1 wt, 5 wt% and 10 wt% silk loadings. (b)
HIPE formation was achieved at 10 wt% silk particle loading.
RSC Adv., 2013, 3, 24025–24027 | 24025
View Article Online
Published on 01 October 2013. Downloaded by Deakin University on 04/12/2013 00:07:57.
RSC Advances
Communication
Fig. 3 (a) Storage and loss modulus of the HIPEs as a function of frequency. (b)
Storage and loss modulus of the HIPE as a function of strain: solid lines: storage
modulus G0 , broken line: loss modulus G0 0 . Black lines: 0.0 M, blue: 1.0 M EOALa,
red: 1.0 M cholineLa and green line 1.0 M cholineTA.
Fig. 2 (a–d) Optical images of HIPEs formed using the ionic liquid cholineTA at
0 M, 0.5 M, 1.0 M and 2.5 M. scale 100 mm (e–h) corresponding fluorescent
microscope images, scale 100 mm.
the optical images of the HIPEs formed at 0 M, 0.5 M, 1.0 M and
2.5 M cholineTA. As the IL concentration is increased the
droplet size increases, and at IL concentrations of 2.5 M no
HIPE formation was achieved in any of the ILs studied here (see
Table S2 in ESI†). This was a surprising result and suggests that
the IL is likely absorbed onto the surface of the silk particle
changing the surface properties, resulting in the lower internal
volumes. Fig. 2e–h shows the corresponding uorescent
microscope images.
Next, we explored the rheological properties of the HIPEs.
Fig. 3a shows the storage (G0 ) and loss modulus (G00 ) of the
HIPEs as a function of frequency at 1.0 M IL. It can clearly be
seen that all HIPEs are in the “gel” form, even in the absence of
IL. However, the addition of the IL has improved the gel properties. Creating HIPEs with 1.0 M cholineTa resulted in a 3 fold
improvement in the gel properties. Fig. 3b shows the impact of
varying the strain rate, again the use of IL improves the HIPEs
response to strain. The compressive strength of the HIPEs
without IL was measured to be 32 0.012 kPa and compressive
strain at failure of 52%. The addition of the IL further improves
both the compressive strength and the compressive strain of the
HIPEs. HIPEs prepared with 1.0 M cholineTA had a compressive
strength of 48 0.026 kPa and a compressive strain at failure of
24026 | RSC Adv., 2013, 3, 24025–24027
62% (Stress–strain curves in ESI†). This suggests that the IL, in
particular the cholineTA, is enhancing the inter-particle
binding, accounting for the signicant improvement in material properties of the scaffolds. Interestingly, cholineTa also
showed the best crosslinking capabilities for collagen when
compared to other choline salts23 its likely the hydrogen bond
capability of this anion, both acceptor and donor sites available,
is responsible for the improvement.
Finally, we explored the morphology of the HIPEs. Fig. 4a–d
shows the SEM images of the HIPEs without IL and at 1.0 M of
Fig. 4 SEM images of freeze-dried HIPEs at (a) 0.0 M, (b) 1 M EOALa, (c) 1.0 M
cholineLa and (d) 1.0 M cholineTa. Scale 50 mm.
This journal is ª The Royal Society of Chemistry 2013
View Article Online
Published on 01 October 2013. Downloaded by Deakin University on 04/12/2013 00:07:57.
Communication
EOALa, cholineLa and cholineTa respectively. Signicant
differences in the matrix can be observed between the different
HIPEs. Clear binding and/or partial dissolution of the silk
particles can be observed for HIPEs prepared with 1.0 M cholineTa and cholineLa. The improved binding of the silk particles
in the presence of the IL further suggests the IL is modifying the
silk particle surface, this likely explains the lower internal
volume fraction obtained with the IL. However, it would be
interesting to study the impact of a more traditional surfactant
like ionic liquid such as a long chain imidazolium. The inability
for crosslinking to occur in the presence of the protic IL EOALa
suggests that the unique hydrogen bond network of the protic
ionic liquid, which is different to the aprotic ionic liquid,27
limits this ILs ability to bind the silk particles.
Conclusions
We have shown that silk scaffolds can be prepared using a
Pickering – HIPE process, thus eliminating the need to dissolve
and regenerate the silk ber. Additionally, using biocompatible
ionic liquids a signicant improvement in the scaffold material
properties was measured. The improved compressibility is
likely due to improved binding between the silk particles due
either increased hydrogen bonding or particle silk particle
dissolution. The improved interface properties was observed by
SEM. CholineTa was shown to improve the material properties
the most, this anion has multiple hydrogen sites. Future works
will explore the mechanism of ionic liquid improvement as well
as other methods to improved interfacial contact.
Acknowledgements
NB and XW acknowledge funding from the Australian Research
Council through a discovery project. CPW acknowledges receipt
of an Australian Research Council Future Fellowship. The
authors thank Dr Rajkhowa for the silk particles.
Notes and references
1 F. G. Omenetto and D. L. Kaplan, Science, 2010, 329, 528.
2 G. H. Altman, F. Diaz, C. Jakuba, T. Calabro, R. L. Horan,
J. Chen, H. Lu, J. Richmond and D. L. Kaplan, Biomaterials,
2003, 24, 401.
3 H. Yamada, H. Nakao, Y. Takasu and K. Tsubouchi, Mater.
Sci. Eng., C, 2001, 14, 41.
4 I. C. Um, H. Kweon, Y. H. Park and S. Hudson, Int. J. Biol.
Macromol., 2001, 29, 91.
This journal is ª The Royal Society of Chemistry 2013
RSC Advances
5 J. Yao, H. Masuda, C. Zhao and T. Asakura, Macromolecules,
2001, 35, 6.
6 D. M. Phillips, L. F. Drummy, D. G. Conrady, D. M. Fox,
R. R. Naik, M. O. Stone, P. C. Trulove, H. C. De Long and
R. A. Mantz, J. Am. Chem. Soc., 2004, 126, 14350.
7 J. Yan, G. Zhou, D. P. Knight, Z. Shao and X. Chen,
Biomacromolecules, 2009, 11, 1.
8 J. Yin, E. Chen, D. Porter and Z. Shao, Biomacromolecules,
2010, 11, 2890.
9 N. Goujon, X. Wang, R. Rajkowa and N. Byrne, Chem.
Commun., 2012, 48, 1278.
10 R. Nazarov, H.-J. Jin and D. L. Kaplan, Biomacromolecules,
2004, 5, 718.
11 S.-W. Ha, A. E. Tonelli and S. M. Hudson, Biomacromolecules,
2005, 6, 1722.
12 M. E. Kinahan, E. Filippidi, S. Köster, X. Hu, H. M. Evans,
T. Pfohl, D. L. Kaplan and J. Wong, Biomacromolecules,
2011, 12, 1504.
13 N. R. Cameron and D. C. Sherrington, in Biopolymers Liquid
Crystalline Polymers Phase Emulsion, Springer, Berlin
Heidelberg, 1996, vol. 126, p. 163.
14 N. R. Cameron, Polymer, 2005, 46, 1439.
15 B. P. Binks, Adv. Mater., 2002, 14, 1824.
16 H. Zhang and A. I. Cooper, So Matter, 2005, 1, 107.
17 S. D. Kimmins and N. R. Cameron, Adv. Funct. Mater., 2011,
21, 211.
18 S. Fujii, M. Okada, T. Nishimura, H. Maeda, T. Sugimoto,
H. Hamasaki, T. Furuzono and Y. Nakamura, J. Colloid
Interface Sci., 2012, 374, 1.
19 F. Yan and J. Texter, Chem. Commun., 2006, 2696.
20 B. P. Binks, A. K. F. Dyab and P. D. I. Fletcher, Chem.
Commun., 2003, 2540.
21 N. Byrne, L.-M. Wang, J.-P. Belieres and C. A. Angell, Chem.
Commun., 2007, 2714.
22 K. Fujita, D. R. MacFarlane and M. Forsyth, Chem. Commun.,
2005, 4804.
23 R. Vijayaraghavan, B. C. Thompson, D. R. MacFarlane,
R. Kumar, M. Surianarayanan, S. Aishwarya and
P. K. Sehgal, Chem. Commun., 2010, 46, 294.
24 T. L. Greaves, A. Weerawardena, C. Fong and
C. J. Drummond, J. Phys. Chem. C, 2007, 111, 4082.
25 R. Rajkhowa, L. Wang and X. Wang, Powder Technol., 2008,
185, 87.
26 I. Capron and B. Cathala, Biomacromolecules, 2013, 14, 291.
27 C. A. Angell, N. Byrne and J.-P. Belieres, Acc. Chem. Res., 2007,
40, 1228.
RSC Adv., 2013, 3, 24025–24027 | 24027