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

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aInstitute for Frontier Materials, Deakin U

E-mail: nolene.byrne@deakin.edu.aubIan Wark Research Institute, University of S

Australia. Fax: +61 8 8302 3683; Tel: +61 8cSchool of Textile Science & Engineering, Wu

† Electronic supplementary information (conditions are provided plus additional g

Cite this: RSC Adv., 2013, 3, 24025

Received 29th August 2013Accepted 27th September 2013

DOI: 10.1039/c3ra44749a

www.rsc.org/advances

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Silk scaffolds achieved using Pickering high internalphase emulsion templating and ionic liquids†

Nolene Byrne,*a Rasike DeSilva,a Catherine P. Whitbyb and Xungai Wangac

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 signicant interest due to itsbiocompatibility and excellent mechanical properties.1

However, normally silk to be produced into a scaffold needs tobe dissolved and regenerated.1,2 The dissolution of silk involvesthe use of harsh solvents and multiple processing steps.3 Thetypical procedure for native silk ber dissolution is to boil inalkaline pH to remove the sericin, dissolve the silk ber in9MLiBr, dialyse to remove the salt and solubilize the amor-phous silk in either formic acid4 or hexauoroisopropanol,5

although ionic liquids (ILs) have been shown to directly dissolvesilk cocoons.6 The regeneration of silk is also a complex process,with many different solvents and processing techniques beinginvestigated currently.7–11 The major disadvantage with thedissolution and regeneration of silk, apart from the complexityand time involved, is the loss of the native silk ber structurewhich impacts the material properties of the regenerated silk.12

In this communication, we describe the preparation of novelsilk scaffolds using a Pickering emulsion – HIPE process.

High internal phase emulsions (HIPEs) are dened as havingan internal phase volume (f) of 0.74 or greater.13 HIPEs nd usein numerous applications including food preparation, fuels, oilrecovery, cosmetics and recently as templates in materialscience.14,15 Oen HIPEs require surfactants14,16,17 or particles18

niversity, Geelong, Vic 3217, Australia.

outh Australia, Mawson Lakes, SA 5095,

8302 6866

han Textile University, Wuhan, China

ESI) available: Tables containing HIPEraphs. See DOI: 10.1039/c3ra44749a

Chemistry 2013

to enhance stabilization and prevent coalescence or sedimen-tation. Recently ionic liquids have been successfully used assurfactant materials19 as well as to form emulsions.20 In addi-tion, ionic liquids, ILs, have been shown to enhance proteinstability against aggregation21,22 as well as being biocompatiblecrosslinking agents for collagen proteins.23 For these reasons,we have explored the impact of using biocompatible ionicliquids in the preparation of the silk particle stabilized HIPEs.We studied 3 different biocompatible ILs, cholineTa, chloineLaand EOALa. The choline salts were selected as they have recentlybeen shown to cross link collagen23 as well as stabilizeproteins,22 while EOALa is a simple protic ionic liquid known topossess amphiphilic properties.24

High internal phase emulsions were achieved via a singlestep process at 10 wt% silk (Fig. 1). Briey the ball milled silkparticles25 were dispersed in the aqueous phase, the requiredamount of oil was added and the solution homogenised for1 minute. The oil used was the biocompatible dodecane. Wefound that by using the 2 step process,26 lower silk wt% could beused to achieve a HIPE (see Table S1 in ESI†). We achievedHIPEs with internal volumes of up to 87% without the use of asurfactant. The inuence of ionic liquid addition on the HIPEswas investigated at concentrations of 0.5 M, 1 M and 2.5 M. TheIL was dissolved in the aqueous phase prior to the silk particlesbeing added. Addition of IL resulted in a decrease in theinternal volume fraction of the HIPEs to 80%. Fig. 2a–d shows

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

Fig. 2 (a–d) Optical images of HIPEs formed using the ionic liquid cholineTA at0 M, 0.5 M, 1.0 M and 2.5 M. scale 100 mm (e–h) corresponding fluorescentmicroscope images, scale 100 mm.

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: storagemodulus 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. 4 SEM images of freeze-dried HIPEs at (a) 0.0 M, (b) 1 M EOALa, (c) 1.0 McholineLa and (d) 1.0 M cholineTa. Scale 50 mm.

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the optical images of the HIPEs formed at 0 M, 0.5 M, 1.0 M and2.5 M cholineTA. As the IL concentration is increased thedroplet size increases, and at IL concentrations of 2.5 M noHIPE formation was achieved in any of the ILs studied here (seeTable S2 in ESI†). This was a surprising result and suggests thatthe IL is likely absorbed onto the surface of the silk particlechanging the surface properties, resulting in the lower internalvolumes. Fig. 2e–h shows the corresponding uorescentmicroscope images.

Next, we explored the rheological properties of the HIPEs.Fig. 3a shows the storage (G0) and loss modulus (G00) of theHIPEs as a function of frequency at 1.0 M IL. It can clearly beseen that all HIPEs are in the “gel” form, even in the absence ofIL. However, the addition of the IL has improved the gel prop-erties. Creating HIPEs with 1.0 M cholineTa resulted in a 3 foldimprovement in the gel properties. Fig. 3b shows the impact ofvarying the strain rate, again the use of IL improves the HIPEsresponse to strain. The compressive strength of the HIPEswithout IL was measured to be 32 � 0.012 kPa and compressivestrain at failure of 52%. The addition of the IL further improvesboth the compressive strength and the compressive strain of theHIPEs. HIPEs prepared with 1.0 M cholineTA had a compressivestrength 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, inparticular the cholineTA, is enhancing the inter-particlebinding, accounting for the signicant improvement in mate-rial properties of the scaffolds. Interestingly, cholineTa alsoshowed the best crosslinking capabilities for collagen whencompared to other choline salts23 its likely the hydrogen bondcapability of this anion, both acceptor and donor sites available,is responsible for the improvement.

Finally, we explored the morphology of the HIPEs. Fig. 4a–dshows the SEM images of the HIPEs without IL and at 1.0 M of

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EOALa, cholineLa and cholineTa respectively. Signicantdifferences in the matrix can be observed between the differentHIPEs. Clear binding and/or partial dissolution of the silkparticles can be observed for HIPEs prepared with 1.0 M chol-ineTa and cholineLa. The improved binding of the silk particlesin the presence of the IL further suggests the IL is modifying thesilk particle surface, this likely explains the lower internalvolume fraction obtained with the IL. However, it would beinteresting to study the impact of a more traditional surfactantlike ionic liquid such as a long chain imidazolium. The inabilityfor crosslinking to occur in the presence of the protic IL EOALasuggests that the unique hydrogen bond network of the proticionic 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 aPickering – HIPE process, thus eliminating the need to dissolveand regenerate the silk ber. Additionally, using biocompatibleionic liquids a signicant improvement in the scaffold materialproperties was measured. The improved compressibility islikely due to improved binding between the silk particles dueeither increased hydrogen bonding or particle silk particledissolution. The improved interface properties was observed bySEM. CholineTa was shown to improve the material propertiesthe most, this anion has multiple hydrogen sites. Future workswill explore the mechanism of ionic liquid improvement as wellas other methods to improved interfacial contact.

Acknowledgements

NB and XW acknowledge funding from the Australian ResearchCouncil through a discovery project. CPW acknowledges receiptof an Australian Research Council Future Fellowship. Theauthors thank Dr Rajkhowa for the silk particles.

Notes and references

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13 N. R. Cameron and D. C. Sherrington, in Biopolymers LiquidCrystalline Polymers Phase Emulsion, Springer, BerlinHeidelberg, 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,

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