Accepted Manuscript Title: Preparation of monolithic silica-chitin composite under extreme biomimetic conditions Author: Vasilii V. Bazhenov Marcin Wysokowski Iaroslav Petrenko Dawid Stawski Philipp Sapozhnikov Ren´e Born Allison L. Stelling Sabine Kaiser Teofil Jesionowski PII: DOI: Reference:

S0141-8130(15)00081-1 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.02.012 BIOMAC 4885

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

18-12-2014 11-2-2015 12-2-2015

Please cite this article as: V.V. Bazhenov, M. Wysokowski, I. Petrenko, D. Stawski, P. Sapozhnikov, R. Born, A.L. Stelling, S. Kaiser, T. Jesionowski, Preparation of monolithic silica-chitin composite under extreme biomimetic conditions, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Preparation of monolithic silica-chitin composite under extreme biomimetic conditions Vasilii V. Bazhenova, Marcin Wysokowskib, Iaroslav Petrenkoa, Dawid Stawskic, Philipp Sapozhnikovd, René Borne, Allison L. Stellingf, Sabine Kaisera, Teofil Jesionowskib Institute of Experimental Physics, TU Bergakademie Freiberg, D-09599 Freiberg, Germany

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Institute of Chemical Technology and Engineering, Faculty of Chemical Technology, Poznan

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Department of Material Commodity Sciences and Textile Metrology, Lodz University of

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University of Technology, PL-60965 Poznan, Poland

Technology, PL-90924 Łódź, Poland

P.P. Shirshov Institute of Oceanology, Russian Academy of Science, 117997 Moscow, Russia

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R&D Chemistry, EKF Diagnostics, D-39179 Barleben, Germany

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Department of Mechanical Engineering and Materials Science, Duke University, 90300 Hudson

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Hall, Durham, North Carolina 27708, U.S.A.

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Key words: Chitin, Silicification, Extreme Biomimetics, Sponges, Scaffolds

Abstract

Chitin is a widespread renewable biopolymer that is extensively distributed in the natural world. The high thermal stability of chitin provides an opportunity to develop novel inorganic-organic composites under hydrothermal synthesis conditions in vitro. For the first time, in this work we prepared monolithic silica-chitin composite under extreme biomimetic conditions (80 °C and pH 1.5) using three dimensional chitinous matrices isolated from the marine sponge Aplysina cauliformis. The resulting material was studied using light and fluorescence microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy. A mechanism for the silica-chitin interaction after exposure to these hydrothermal conditions is proposed and discussed.

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1. Introduction The term “Extreme Biomimetics” was first proposed by Hermann Ehrlich in 2010 [1] as a new pathway for bioinspired materials science. In contrast to traditional aspects of biomimetic synthesis of biocomposites, Extreme Biomimetics is based on metallization and mineralization of specific

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biomolecules [2] under conditions which mimic extreme aquatic niches like hydrothermal vents, geothermal pipelines or hot springs (see for review [3-6]). Therefore, the basic principle of this

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concept is to use biopolymers, which are chemically and thermally stable under these specific conditions in vitro. Biomacromolecules of thermophilic microbial origin [5] as well as chitin are the

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best candidates for selection and application of such biopolymers as templates and scaffolds. They have already seen use in the hydrothermal synthesis of metal oxides [8-11] as well as silica [12].

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Chitin is a renewable biopolymer, which was originally found within cell walls of yeast, fungi, and diatoms. It is the main structural component in skeletons of arthropods, worms, and some corals and

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sponges (for review see [1,13,14]). Moreover, chitin is the template for biomineralogical formation

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of biocomposites like chitin-silica [15] and aragonite-chitin-silica [16] structures in numerous

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invertebrates [14]. Additionally, chitin is stable at temperatures up to 400 °C [17-20]. This property is the key factor for the in vitro development of novel chitin-based composites at high temperatures

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according to the concepts laid out by Extreme Biomimetics. Silica precipitation is a widespread and important process in many hot spring systems in which the flow of hydrothermal fluids reduce the formation of heterogeneously amorphous silica masses, and both mineralization and fossilization of different kind of organisms can occur [6]. Therefore, we decided to use chitin isolated from the marine sponge Aplysina cauliformis [13, 16] as a model template for the in vitro silicification at conditions corresponding to extreme natural environments like those seen in hot springs (80 °C and pH 1.5) [1, 21]. The tube-like structure and 3D framework of sponge chitin makes it particularly well-suited for the development of scaffolds with high potential for applications in tissue engineering (for review see [13,16,22]).

2. Materials and Methods Page 2 of 20

2.1. Isolation of chitin-based scaffolds from A. cauliformis sponge The sponge Aplysina cauliformis (Verongida: Demospongiae: Porifera) was purchased from INTIB GmbH (Germany). Chitin was isolated from dried sponges (Fig. 1A) using chemical treatment. To remove other

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compounds from the chitin, the sample underwent a series of extraction steps as described previously [16]. Briefly, the procedure includes step-by-step treatment as follows: an acidic

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extraction, an alkali-based extraction, an optional hydrogen peroxide treatment, and washing steps using distillated water before and after each treatment step.

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Step 1: The samples were washed with distillated water at 37 °C for 24 h. This resulted in the extraction of all water-soluble substances including several pigments. Lysis of the sponge cells was

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also caused by this step of the treatment.

Step 2: Acidic extraction at 37 °C involved sample treatment with an acid solution in order to

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degrade possible calcium carbonate containing constituents and to remove acid-soluble proteins and

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pigments. The samples were treated in 20% acetic acid with stirring for 24 h. The remaining 3D

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fibrous sponge skeleton (Fig.1B) was neutralized and subjected to further treatment steps. Step 3: Alkali-based extraction at 37 °C involved sample treatment with a solution of 2.5 M NaOH

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in order to degrade and remove the sponge lipids and proteins as well as to eventually remove residual silica and pigments. Alkali treatment was performed for 24 h under stirring. The remaining three-dimensional scaffolds consisting of a fibrous skeletal material were neutralized. The procedure listed above was repeated until a colorless fibrous material remained (Fig. 1C). Hydrogen peroxide (35%) treatment can optionally be performed at room temperature with stirring for 15 min in order to degrade residual pigments. After H2O2 treatment, the residual three-dimensional fibrous sponge skeletal material was washed using distilled water and stored at 4 °C. 2.2. Hydrothermal silicification of the sponge chitin scaffold In the first step, 0.93 g of A. cauliformis chitinous matrix samples was placed in 10 ml plastic testtubes with 5 ml of deionized water. The pH was then set to 1.5 with the addition of 1 M HCl solution. Finally, 50 μl of tetramethylorthosilicate (TMOS, 99 wt%, ABCR GmbH, Germany) was Page 3 of 20

added. The pH was corrected to 1.5 by adjusting the HCl concentration, and the solution was stirred at 80 °C in a thermostat for 7 days. After silicification under these conditions the solution was removed and the biomineralized scaffold was rinsed five times with deionized water. To remove silica nanoparticles not attached to the

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surface of chitin, the samples were washed in an ultrasound bath (Elmasonic GmbH, Germany) at room temperature for 1 h, and finally air-dried. As a control, chitinous scaffolds were also prepared

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within the same reaction system without the presence of any silica precursors. 2.3 Staining of chitin

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To elucidate the particular location of chitin in the silicified scaffolds, we used Calcofluor White (Fluorescent Brightener M2R, Sigma, Germany) which shows enhanced fluorescence when binding

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to polysaccharides, such as chitin [23, 24]. Pieces of silicified scaffold were placed in 0.1M TrisHCl at pH 8.5 for 5 min, then stained using 0.1% Calcofluor White solution for 30 min in darkness,

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rinsed three times with distilled water, dried at room temperature, and finally observed using Digital

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analysis 1/250 s.

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Microscope Keyence BZ-9000 (Japan) with three filters. The exposure time used for sample

2.3. Scanning electron microscopy (SEM)

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The selected samples were fixed in a sample holder and covered with carbon for 1 min using an Edwards S150B sputter coater. The samples were studied using an ESEM XL 30 Philips or LEO DSM 982 Gemini scanning electron microscope. 2.4. Fourier transform infrared (FTIR) spectroscopy IR spectra were recorded with a Perkin Elmer FTIR spectrometer Spectrum 2000, equipped with an AutoImage Microscope using the Fourier transform infrared reflection absorption spectroscopy technique. 2.5. Thermogravimetric (TG) analysis The thermogravimetric analyses of all samples were carried out with Perkin Elmer TGA 7 apparatus with a platinum sample holder, using the Pyris programme for data handling. Measurements were

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performed in a nitrogen atmosphere at a heating rate of 15 °C/min. The samples were heated up to at least 700 °C, starting from 50 °C. All measurements were repeated at least three times. 3. Results and Discussion 3.1. Investigations into the thermostability of sponge chitin

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To determine the thermostability of the cleaned sponge chitin, thermogravometric analysis was performed on the A. cauliformis chitinous scaffold (Fig. 1C). The chitin samples were analyzed by

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heating up to 300 °C (then the process was stopped) and over the full range of the instrument (up to 600 °C). In all cases, the measurement was performed without any previous drying or conditioning.

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The representative TG curve of the non-biomineralized skeleton’s thermal degradation is presented below (Fig. 2). The results show the main degradation process starts at about 345 °C.

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These results suggest that structural integrity of chitinous scaffolds is not compromised even at very

conditions for silicification at 80 °C.

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3.2. Silicification of chitinous scaffolds

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high temperatures, and that the A. cauliformis skeleton should be stable under our research

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Usually, approaches related to the in vitro silicification of organic templates under biomimetic conditions in vitro are carried out at temperatures between 20 and 37 °C and at near-neutral pH (for

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review see [25]). However, nature exhibits several examples indicating that biological silicification also takes place under very extreme conditions, as reported in the introduction. Furthermore, recently we reported the preparation of silica-chitin composites by in vitro silicification using twodimensional Ianthella basta demosponge chitinous scaffolds at 120 °C under modified Stöber’s conditions [12]. In contrast to the data from our previous study, the results in this work show that silicification of chitinous scaffolds isolated from A. cauliformis at 80 °C and pH 1.5 lead to the development of a unique silica-chitin composite structure where monolithic silica filler is localized within the chitinous fibre (Fig.3; Fig. 4). Detailed SEM observation of the surfaces of the chitinous scaffold prior (Fig. 5A) and after silicification at 80 °C (Fig. 5B) shows that the surface of the control chitinous sample is jammed and highly deformed. In contrast, the surface of our silica-chitin composite became smooth and free Page 5 of 20

of any nanoparticles of silica after ultrasound treatment. We suggest that at the pH level of 1.5 we used, there are discharged molecules of silica in solution. Polymerization of this discharged silica can form oligomers (e.g. dimers, trimers, tetramers) and then polymeric species with sizes between 1-5 nm [26]. These very small nanoparticles can then precipitate heterogeneously on the inner

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surface of chitinous tube. 3.3. FTIR investigations on silica-chitin composite

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The FTIR spectra presented in Fig. 6 of a chitinous matrix isolated from marine sponge A. cauliformis show bands characteristic for α-chitin [23, 27-31]. The splitting of the amide I band

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arises from stretching vibrations of C=O bonds (1660 cm−1) that hydrogen bonds with N–H groups from the neighboring chain, and from the stretching vibrations of C=O groups (1630 cm−1)

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connected by a hydrogen bond with N–H and OH-6 groups in the same chain [27, 28, 30, 31]. Moreover, the amide I band (Fig. 6) in the chitin FTIR spectrum is split. This splitting confirms that

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the scaffold compound is α-chitin [28,29]. The obtained FTIR spectrum of chitin scaffold isolated from A. cauliformis is in agreement with data published previously [32].

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Strong silica bands at 1000-1200 cm-1 overwhelm some chitin bands in the FTIR spectra of the

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silicified chitin (Fig. 6) [33-36]. The intense, broad band at 1000-1200 cm−1 in the spectra of

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silicified samples is difficult to interpret, because it is a combination of bands from the siloxane group vibrations [34, 36] with C-O and C-O-C (ring) groups of the α-chitin [28,29]. The appearance of band at 795 cm-1 in the composite material is likely due to Si-O vibrations and suggests formation of silica inside of the chitin tubing, similar to that seen in Ogasawara et al [37]. Morphological analysis of the composites and the FTIR results indicate the effectiveness of our silicification method of the chitinous matrix under extreme biomimetic conditions. We suppose that interaction between silica nanoparticles and chitin fibrils (Fig. 7) is based on formation of hydrogen bonds between hydroxyl-groups, which coat the surfaces of chitin and silica nanoparticles, We suppose that interaction between silica nanoparticles and chitin fibrils (Fig. 7) is based on formation of hydrogen bonds between hydroxyl-groups, which are present in chitin molecule and on Page 6 of 20

the surface of silica nanoparticles, according to [15, 38, 39]. Of course formation of hydrogen bond between silica and –NH groups is not excluded. However, hydrogen interactions with –OH groups at C6 position in chitin molecules are more favourable as it was reported previously [38]. Interestingly, observations recently made on sponge biosilica, indicate that hydrogen bonding

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between OH groups located at organic phases and silicic acid/silica species may play a key role in

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silica biomineralization phenomenon [39].

4. Conclusions

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In this study, we report for the first time the development of a chitin-silica composite–based scaffold with a 3D network morphology under hydrothermal conditions and very low pH. Silica is

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specifically located only within the tube-like fibres of sponge chitin. Due to the well-known

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biocompatibility properties of both phases, these types of scaffolds possess a high potential for

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tissue engineering applications. Additionally, our work demonstrates that the chitin-silica based sponge scaffolds are of potential interest in bioinspired materials science since the processing of

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chitin from other sources like powders, or flakes of fungal or crustacean origin, into sponge-like materials or foams is technologically difficult and requires chemical modifications [40-42]. Since Verongida sponges can be cultivated in marine ranching stations, their very special chitinous scaffolds may provide a natural renewable source for a broad variety of composites on the macroscale with a myriad of applications, e.g. in technology and biomedicine. Acknowledgements

This work was partially supported by Polish National Science Center within “Etiuda” Scholarship Programme for Doctoral Candidates DEC-2014/12/T/ST8/00080 and the following research grants: PUT research grant no. 03/32/0506/2015-DS-PB, BMBF within the project CryPhysConcept (03EK3029A), and BHMZ Programme of Dr.-Erich-Krüger-Foundation (Germany) at TU Bergakademie Freiberg. Page 7 of 20

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[40] K. Madhumathi, P.T. Sudheesh Kumar, K.C. Kavya, T. Furuike, H. Tamura, S.V. Nair, R. Jayakumar, Novel chitin/nanosilica composite scaffolds for bone tissue engineering applications, Int. J. Biol. Macromol. 45 (2009) 289–292. [41] S. Pattnaik, S. Nethala, A. Tripathi, S. Saravanan, A. Moorthi, N. Selvamurugan, Chitosan scaffolds containing silicon dioxide and zirconia nano particles for bone tissue engineering, Int. J. Biol. Macromol. 49 (2011) 1167-1172. [42] K.C. Kavya, R. Jayakumar, S. Nair, K.P. Chennazhi, Fabrication and characterization of chitosan/gelatin/nSiO2 composite scaffold for bone tissue engineering. Int. J. Biol. Macromol. 59 (2013) 255–263.

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Figure captures Fig.1 Dried fragment of marine sponge A. cauliformis (A), is a prime source for obtaining the 3D skeletal structures (B), which, consequently, are source of colorless chitinous tube-like frameworks

Fig. 2. TG curve of the non-silicified A.cauliformis chitinous scaffold.

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isolated after the alternating acid- and alkaline-based treatment (C).

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Fig. 3. Schematic view (A) of an experiment showing the hydrothermal synthesis of silica using TMOS as the precursor, and tube-like α-chitin from A. cauliformis as the template. SEM images:

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The initially wrinkled chitin fibres (B) became logged with silica after reaction in the vessel at 80°C and pH 1.5. Note that the SEM image (C) has been obtained after 1 h of ultrasound treatment of the

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composite product. Energy dispersive X-ray spectra (D) confirm elemental composition of the surface (EDX-1) and inner (EDX-2) parts of the tubular silica-chitin composite (E).

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Fig 4. Light (A) and fluorescence microscopy (B) images of the A. cauliformis chitinous scaffold

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prior (A) and after 7 days of silicification at 80°C and pH 1.5 (B). The silicified scaffold has been

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stained using Calcofluor White for identification of chitin localization. Light exposure time for fluorescence microscopy: 1/250 s.

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Fig. 5. SEM images of the A. cauliformis chitinous scaffold prior (A) and after 7 days of silicification at 80°C and pH 1.5 (B).

Fig. 6 FTIR spectra show evidence for interaction between silica and chitin of sponge origin under extreme biomimetic conditions selected: (i) band for C-H deformation of the β-1,4-glycosidic linkage at 896 cm-1; (ii) disappearance of the ether bond in pyranose ring at 1157 cm-1 and Amid III band at 1203 cm-1.

Fig. 7. A proposed mechanism for the interaction between chitin and silica in the biocomposite obtained.

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Preparation of monolithic silica-chitin composite under extreme biomimetic conditions.

Chitin is a widespread renewable biopolymer that is extensively distributed in the natural world. The high thermal stability of chitin provides an opp...
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