Structure and characteristics of milled silk particles

Powder Technology 254 (2014) 488–493

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Structure and characteristics of milled silk particles Mehdi Kazemimostaghim a, Rangam Rajkhowa a, Kiran Patil a, Takuya Tsuzuki a,b, Xungai Wang a,c,⁎ a b c

Australian Future Fibres Research and Innovation Centre, Deakin University, Geelong, VIC 3217, Australia Research School of Engineering, Australian National University, Canberra, ACT 0200, Australia School of Textile Science and Engineering, Wuhan Textile University, Wuhan, China

a r t i c l e

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Article history: Received 25 September 2013 Received in revised form 16 January 2014 Accepted 19 January 2014 Available online 4 February 2014 Keywords: Silk nanoparticle Bead mill Ion adsorption Silk particle characterization

a b s t r a c t This study examined the structure, thermal property, and ion adsorption of silk particles. The particles were prepared by attritor–bead mill combination, using alkaline (pH 10) charge repulsion and surfactant steric repulsion methods. Both methods produced particles with a dominant β-sheet structure, similar to the silk fibre. There was no significant difference in the decomposition temperatures for either the silk fibre or the micro/nano silk particles. An important finding from this study is clear evidence of reduction of amorphous content during the final stage of powdering using the bead mill. As a result, despite reduction in β-sheet crystallites with the progressive milling, the relative β-sheet content actually increased during this process. However, intermolecular forces between the β-sheets reduced significantly and hence the XRD results showed significant reduction in crystallinity in nano silk particles but crystal forming segments remained with β-sheet conformations after milling. The structural change influenced the ion-adsorption property where particle-size reduction resulted in a significant increase in both the rate and volume of HCrO− 4 adsorption. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Silk fibre is a protein produced to form cocoon by the larva of caterpillar. A cocoon protects the silkworm from microbes, dehydration, and predators during metamorphosis [1]. In a cocoon, twin silk filaments are held together by silk gum called sericin [2,3]. The fibroin fibre itself is a bundle of several fibrils and microfibrils [4]. Fibroin has a β-sheet secondary structure with stabilized inter-chain and inter-sheet hydrogen bonds [5]. Silk fibroin is a block copolymers in which crystalline sections are built up by amino acids with short side chains such as glycine and alanine and spread into amorphous sections consisted of amino acids with bulkier side chains [5]. High wet strength, ability to withstand enzymatic breakdown, and good transfer of oxygen and drugs make silk protein a suitable biomaterial [6]. Silk particle is a useful form of silk which has been used for many applications such as drug delivery [7–11], reinforcing scaffolds [12,13], enzyme immobilization [14], cosmetics [15], and coatings [16]. Silk particles possess high potential in the biomedical field of application due to their ability to bind various ionic species and their controllable release profile [17]. Silk can be dissolved in solvents which have the ability to break down the strong intermolecular bonds [18]. Liquid silk can then be converted to various morphologies such as films, gels, fibres and particles [19]. However, during regeneration, the original crystal structure is lost. Structural stabilization can be achieved by stimulating the

⁎ Corresponding author at: Australian Future Fibres Research and Innovation Centre, Deakin University, Geelong, VIC 3217, Australia. Tel.: +61 3 5227 2894. E-mail address: [email protected] (X. Wang). 0032-5910/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.powtec.2014.01.060

formation of the β-sheet structure by various means such as methanol treatment, which can improve mechanical properties and decrease the water solubility [18]. Different methods have been reported to fabricate silk particles from silk solution and improve their stability [20,21]. An alternative method to fabricate silk particles while preserving the primary structure of silk fibre is ball milling. Although mostly used for inorganic materials, ball milling processes have also been used for breaking solid organic materials. For example, ball milling was applied for reducing the particle size of drugs to increase their bioavailability [22]. In our previous study, a combination of different milling systems were also used to produce a broad size-range of silk particles from micron to nano levels [23,24]. For the production of silk nanoparticles, charge stabilization [23] or steric repulsion [24] was essential to avoid their aggregation. Charge stabilization was achieved by milling at pH 10 where electrostatic repulsion forces prevent particle aggregation [23]. The pH was restricted to 10 to be safe for silk processing as silk is sensitive to alkali hydrolysis and the hydrolysis is enhanced by increasing pH and temperature [25,26]. In addition to pH assisted milling to produce nano particles, similar silk particle can be obtained by milling at neutral pH using Tween 80, a surfactant suitable for biomedical applications [24]. Unlike alkali, Tween 80 does not hydrolyse silk. Despite progress in milling of silk to produce fine particles and understanding of the particle morphology, there is lack of understanding of the change in silk structure during milling. Understanding changes in secondary structure during milling is important as it largely determines the stability of silk against dissolution, and various forms of destabilisation process such as thermal, mechanical and enzymatic degradation. In this study, we try to correlate the secondary structure and milling process which will be helpful to engineer

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silk particles with appropriate structure for achieving desired properties for a specific application. 2. Experimental 2.1. Milling The process flow chart for the production and drying of samples is shown in Fig. 1. Details of the powdering processes were reported in our previous works [23,24]. Briefly, degummed Eri fibres were chopped with a cutter mill (Pulverisette 19 from Fritsch) until the snippets could pass through a 1 mm grid. Wet milling of snippets was carried out in an attritor (1S from Union Process). 200 g of snippets was mixed with 1500 mL of distilled water. The slurry was treated in the attritor mill with yttrium-doped zirconium oxide balls of 5 mm in diameter in deionised water for 7 h. The slurry obtained from the attritor milling was further processed with a laboratory spray dryer (B-290 from Buchi) to produce dry powder (Powder-1). 2 g of the spray-dried powder was further milled in a bead mill (DYNO® Mill Research Lab) using100 mL deionised water. In bead milling, pH 10 buffer or Tween 80 at 30% concentration on the weight of powder (owp) was used to avoid particle aggregation. The grinding medium was cerium-doped zirconium oxide (0.5–0.6 mm in diameter) having a volume of 60 mL. The milling speed was 2000 rpm. Processing time was 10 h for pH 10, and 7 h for Tween 80 assisted milling. After milling, alkali was removed by dialysis followed by washing and centrifuging the dialysed powder with deionised water. Subsequently, washed particles were freeze dried (Powder 2). Similar procedure was followed for removing Tween 80 and drying to obtain dry powder (Powder 3). 2.2. Nano-spray dying and freeze drying of particles bead milled in pH 10 Bead milled slurry with pH 10 without removing alkali was freeze dried (Labconco FreeZone), and nano-spray dried (B-90 from Buchi) to get dry powder. The inlet temperature for nano-spray drier was 120 °C with a flow rate of 20 mL/h.

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2.3. Preparation of silk film Water soluble silk films can be used as standard of amorphous silk while characterising structure of silk powder. Eri silk film was prepared using a method explained elsewhere with some modification [27]. Briefly, Eri fibre was dissolved in a 10 M lithium thiocyanate solution with a material to liquor ratio of 1 (g): 10 (mL) at room temperature. After removing undissolved parts by centrifuging at 6500 rpm for 20 min, the supernatant was dialyzed for 4 days at 4 °C against deionised water by a dialysis tube with molecular weight cut off 12,000 Da (from Sigma Aldrich). 10 mL 1% (w/v) silk solution was cast on a 10 cm diameter polyethylene disc at room temperature.

2.4. Characterisations Mastersizer 2000 (Malvern, UK) was used for determining particle sizes and specific surface areas using deionised water as a dispersion media. The reflective index of 1.542 was used for Eri silk for the calculation of particle size distribution [28]. Measurements were repeated three times. The shapes of the snippets and particles were observed under a scanning electron microscope (SEM, Zeiss Supra 55VP) using 3–10 kV and working distance of 5–10 mm. Gold coating was applied to the samples prior to the observation. Fourier transform infrared (FTIR) spectra were obtained using a Bruker® VERTEX 70 spectrometer under the resolution of 4 cm−1 and 128 scans per sample. Differential scanning calorimetric (DSC) analysis was done using thermal analysis instrument DSCQ200. Scanning rate of 10 °C min−1 between (− 30 and 400 °C), and nitrogen gas flow rate of 50 mL/min were used. Thermogravimetric analysis (TGA) was performed under the flow of nitrogen gas at a scanning speed of 10 °C min−1 using a Netzch STA409PC DSC/TGA instrument. XRD experiments were carried out with PANalytical X'Pert PRO instrument. CuKα radiation with a wavelength of 1.54 °A was used. The scanning speed was 0.1° with time per step of 1 s, and measurement angle 2θ from 5 to 40° under 40 kV and 50 mA.

Fig. 1. Production process of dry silk particles.

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2.5. Metal ion adsorption To measure metal ion adsorption property, the bead milled silk particles prepared at pH 10 with a volume median particle size, d(0.5), of about 200 nm were used after dialysis. The aqueous dispersion of the particles was exposed to a chromium(VI) aqueous solution at pH 2 and examined for its adsorption yield at different time points in comparison to the attritor milled silk particles (Powder 1). All the chemicals used were laboratory grade. The % solid content was determined by evaporating water from the dispersion in an oven at 90 °C for 2 h. The particle dispersions were separately pipetted according to their % solid content and poured in a reaction tube containing pH 2 buffer solution. A stock solution of pH 2 buffer was prepared by combination of 0.1 M KCl and HCl in deionised water. 0.1 M potassium dichromate was added to the reaction tube to achieve 50 ppm Cr6 + concentration in the solution. Finally the solution was made to 5 mL by adding the pH 2 buffer solution so that the particles were exposed to 50 ppm Cr6+ solution at the sorbent concentration of 5 mg/ml at pH 2. The reaction tubes were gently agitated with a rotating disc under ambient conditions. At set time intervals, the reaction tubes were centrifuged at 5000 rpm for 5 min and the aliquots were sampled. The sample solutions were analysed for its Cr6+ concentration using a UV–Vis spectrophotometer (CARY 300) by measuring its optical density at 350 nm wavelength. The spectrophotometer was previously calibrated with the standard Cr6+ solutions in the pH 2 buffer solutions. The adsorption yield was calculated as the percentage of adsorbed Cr6 + on the basis of initial Cr6+ concentration as below:

% Adsorption Yield ¼

A0 −At  100 A0

where A0 is the initial Cr6+ concentration in the reaction solution and At is the Cr6+ concentration in the reaction solution at time t after exposure to the silk particle dispersion. 3. Results and discussion 3.1. FTIR analysis Based on the preparation methods of silk materials, three different conformations of silk fibroin (silk I, silk II, and silk III) have been reported [29,30]. Conformation of silk I is related to the silk in the silkworm gland and silk III is related to the water–air interface in thin films. These two conformations have not been fully understood. In contrast, silk II is the conformation of silk after spinning by silkworm which consists of dominant β-sheet conformation. Silk fibre is a polymorphic compound made of blocks of amorphous and β crystal groups [31]. Highly repetitive units of (alanine)n blocks with a very short side chains are the base of β-sheet structure in Eri silk [32]. The conformations can be easily identified by FTIR spectra and particularly the amide I–V vibrations are useful for analysing the secondary structure of polypeptides and proteins [33]. It is reported that peak absorbance at 1628 cm−1 (amide I), 1520 cm−1 (amide II), and 1240 cm−1 with shoulder at 1222 cm−1 (amide III) are due to β-sheet structure; whereas peaks at 1653 cm−1 (amide I), 1541(amide II), and 1269 cm−1 (amide III) are due to α-helix structure for Eri silk [27]. Moreover, the amide III region (1200 to 1300 cm−1) has been suggested as a highly sensitive area for detecting structural changes of Eri silk [27,34]. Fig. 2 shows the FTIR spectra of silk fibre and powders in amide III, amide I and II areas. It is seen in Fig. 2(a) that Eri fibre (scan a) has a strong peak at 1240 cm−1 with a shoulder at 1220 cm−1. The relative height of the peak at 1220 cm−1 is more than the one at 1240 cm−1. The shoulder at 1220 cm−1 disappeared for Eri film (scan e), and a peak maximum at 1269 cm−1 was observed corresponding to α-helix absorbance. In amide II and I areas (Fig. 2(b)) the peak maxima for Eri

Fig. 2. FTIR spectra of Eri Silk (a) fibre, (b) Powder 1, (c) Powder 2, (d) Powder 3, and (e) film.

fibre was observed at 1513 cm−1 and 1626 cm−1 while peaks for Eri film were seen at 1538 cm−1 and 1646 cm−1, respectively. For the attritor milled powder (Powder 1), the amide III shoulder (Fig. 2(a)) bent slightly at 1220 cm−1, and the peaks in amide II and III regions (Fig. 2(b)) moved towards higher wavenumber, which suggests some reduction in β-sheet conformation due to attritor milling in comparison with silk fibre. The reduction in intermolecular β-sheet during attritor milling was reported in our previous study on mulberry silk [35]. This was expected due to energy input during high energy attritor milling. Surprisingly, after bead milling, the peaks appeared in the same position as that of Eri fibre for both alkali assisted and surfactant assisted milling (Powders 2 and 3). The proportional increase in β-sheet after bead milling could be the result of reduction in the amorphous region during bead milling. Particles became very small and frictional forces were very high due to high speed of milling and the use of finer media. SEM image of bead milled particles in our previous report [24] showed a smoother surface than attritor milled particles, confirming

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the removal of most of the low strength areas related to amorphous parts. Xu et al. also observed an increase of crystallinity during treatment of micron sized silk particles with enzyme as result of reduction in amorphous content and associated reduction in mass [36]. The rise in β structure from submicron particles to nano particles may also be due to rupture of particles at amorphous areas which were more susceptible because of open structures. Particularly the presence of water in wet milling could further open up the structure in the amorphous regions as they contain polar and water soluble parts [32]. In addition, the scans (c) and (d) in Fig. 2 show that the FTIR spectra for submicron particles prepared by alkali method and surfactant (Tween 80) are similar, suggesting that the two milling media had similar effects on the structure of milled particles. 3.2. DSC analysis Fig. 3 shows DSC curves of silk powders. Eri snippets and Powder 1 showed similar thermal behaviours. Two endotherms for these materials at ~ 75 °C and ~ 370 °C are related to the evaporation of water and thermal decomposition of silk, respectively [37]. The small endotherms at ~ 220 °C is attributed to molecular motions related to glass transition temperature [38]. The other small endotherm at ~ 300 °C is assigned to the decomposition of fibroin in the amorphous region which occurs prior to crystal decomposition [39]. The main decomposition temperature (366.7 °C) for Powder 1 is lower than silk snippets (371.3 °C), confirming that a lower quantity of β-sheets was present compared to snippets. This result is in good agreement with the FTIR results. Plots (c) and (d) in Fig. 3 show the DSC graphs of Powder 2 and Powder 3, respectively. Endotherms related to glass transition and decomposition of amorphous region reduced significantly in bead milled silk (Powder 2 and Powder 3), indicating that they contained less amorphous phase than Powder 1. This supports the FTIR results showing higher relative content of β-sheets after bead milling. Thus the DSC results confirm the hypothesis that bead milling removed some amorphous regions from silk. Such changes, however, did not enhance the final decomposition temperature compared to Powder 1, as a relative increase in β-sheet proportions does not change the decomposition temperature if the size of β-sheet crystallites remains the same. The decomposition temperatures of Powder 2 and Powder 3 were nearly the same, which further suggests that the alkali and surfactant used during milling induced similar effects on the structure of silk particles. To investigate the effect of alkali on silk particles during milling, the bead milled slurry at pH 10 was nano-spray dried and freeze dried without removing alkali. The DSC graph of these two samples is shown in Fig. 4.

Fig. 3. DSC thermograms of (a) snippets, (b) Powder 1, (c) Powder 2, (d) Powder 3.

Fig. 4. DSC thermograms of bead milled slurry without removing alkali (a) freeze dried, and (b) nano-spray dried.

Comparing Fig. 4 with Fig. 3, a significant difference can be seen between thermal decompositions if the alkali is not removed during drying. The thermal decomposition temperatures of nano-spray dried and freeze dried sample with alkali were 314.6 and 322.4 °C, respectively. These temperatures are about 50 °C lower than that of the same materials dried after removing the alkali. This indicates that, if the alkali is not removed, the resulting silk particles have more structural disorder. As mentioned earlier, silk is sensitive to alkali. Alkali hydrolysis the peptide bonds and the extent of reaction depends on the concentration of the alkali and temperature. In case of nano-spray drying, the particles were subjected to the nozzle temperature of 120 °C. The alkali concentration also increased during the drying process. These conditions along with a high surface area of silk nanoparticles helped the alkali to degrade some parts of the silk structure. Similar trend happened to freeze dried particles, but due to lower drying temperature, the decomposition temperature was slightly higher than nano-spray dried particles. To avoid high alkali concentrations and high temperatures in the production of bead milled particles, the molarity of pH 10 buffer was adjusted to less than 0.05, and the temperature during the bead milling process was below 40 °C [23]. 3.3. TG and DTG The TG graphs of the Eri silk snippets and particles are shown in Fig. 5. The first weight loss at around 100 °C is associated with the evaporation of moisture. The second weight loss starting at about 300– 370 °C is attributed to the cleavage of side chain groups of amino acids and the breaking of peptide bonds [40]. The total weight loss of Powder 1 in this temperature range was more than Powder 2 and Powder 3, indicating that Powder 1 had a higher amount of amorphous regions than Powder 2 and Powder 3 [41]. Amorphous regions have amino acids with bulky side chains that produce more volatile decomposed products than crystalline regions [5,32,40]. When derivatives of the TG graphs are plotted, the weight loss due to the decomposition of amorphous regions during heating becomes clearer. Fig. 6 shows such DTG curves of silk snippets and powders. Eri snippets and Powder 1 showed a minor peak at around 320 °C, attributed to the breaking of side chain group of amino acid molecules and breaking of peptide bonds in amorphous regions [40]. This minor peak disappeared in Power 2 and Powder 3, indicating a smaller amorphous region in Powder 2 and Powder 3 compared to Powder 1. Thus the results of mass loss support the results of DSC and FTIR, confirming the higher proportion of β-sheet structure in Powder 2 and Powder 3 compared to Powder 1.

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Fig. 5. TG graph of (a) silk snippets, (b) Powder 1, (C) Powder 3, and (d) Powder 2. Fig. 7. X-ray diffractograms of (a) silk snippets, (b) Powder 1, (C) Powder 2, and (d) Powder 3.

3.4. XRD Fig. 7 shows the XRD patterns of Eri silk materials. Eri fibre showed a characteristic peak of Eri fibre at 16.45°, and 20.15° corresponding to β-sheet crystalline spacing of 5.38 and 4.40 Å, respectively [42]. After attritor milling (Powder 1), the intensity of 16.45° peak dropped sharply. This peak is related to the index (200) and associated with intra-sheet ordering in the β-sheet crystallites [35]. Hence the significant reduction in the intensity of this peak indicates increased disorder between β-sheets. After further milling using the bead mill, the peaks at 16.45°, and 20.15° disappeared completely. This indicates that the regular stacking of β-sheets was largely destroyed. However, the FTIR and DSC studies indicated the presence of β-sheets after milling. The results therefore suggest that, with the progress in milling along with the reduction in particle size, long range orders between β-sheets were destroyed but the short range orders and internal structures of β-sheets are largely preserved.

Silk is an amphoteric protein with the isoelectric pH 3.8–4.5 [43,44]. Silk particles have positive charge below isoelectric point due to adsorption of H+ by electron donor groups such as amines. As Cr6 + is primarily available in the form of HCrO− 4 anionic species at pH 2 aqueous solution [45], the metal anion would have electrostatically bound with positively charged groups in silk particles. The bead milled particles with d(0.5) = ~ 200 nm in diameter exhibited faster and higher amount of HCrO− 4 absorption in comparison to the attritor milled particles with d(0.5) = ~ 7 μm. The difference may be attributable to the difference in specific surface areas (28.4 m2/g in comparison with 1.18 m2 /g) and the amount of crystal defects which make the reaction sites in the submicron particles more accessible to HCrO− 4 ions. The bead milled particle had a higher number of ion-adsorption sites due to the increase in the surface area and crystal defects. 4. Conclusion

3.5. Metal ion adsorption Fig. 8 shows adsorption of Cr6+ as a function of time. As shown in Fig. 8, silk particles demonstrated good adsorption kinetics for Cr6 +.

In milling silk fibres into fine powders, the crystalline regions of silk progressively opened up but the β-sheet conformations of the crystal forming domains remained essentially unchanged. However, when bead milling was used to produce nano silk particles in the final stage

Fig. 6. DTG graph of (a) silk snippets, (b) Powder 1, (C) Powder 2, and (d) Powder 3.

Fig. 8. Adsorption yield of chromium(VI) for silk particles.

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of particle production, the milling energy could permanently remove some amorphous portions from the material thereby the relative content of β-sheet forming domains increased after bead milling. These findings were confirmed by shifts in FTIR peaks, reduction in crystalline peaks in XRD scatterings and drop in endotherms in DSC thermograms. The residual mass of particles after thermal degradation was also higher than attritor milled particles after bead milling due to reduction in amorphous material. The rate and the amount of HCrO− 4 adsorption were higher for silk nanoparticles in comparison with micron-size silk particles due to increase in surface area, and the reduction in crystallinity.

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