Regulation of silica morphology by proteins serving as a template for mineralization

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Colloids and Surfaces B: Biointerfaces 63 (2008) 7–11

Regulation of silica morphology by proteins serving as a template for mineralization Yury Shchipunov ∗ , Nadya Shipunova Institute of Chemistry, Far East Department, Russian Academy of Sciences, 690022 Vladivostok, Russia Received 19 June 2007; received in revised form 30 October 2007; accepted 30 October 2007 Available online 9 November 2007

Abstract The fabrication of inorganic materials under the control of biopolymers is a research field of tremendous interest because in diatoms and sponges the specific proteins direct the formation of silica at ambient conditions that have highly organized structure at multiple length scales and properties that the material scientists can only aspire to achieve. Here it is demonstrated by using a novel biocompatible precursor that common proteins – albumin, caseins and gelatin – can mediate formation of hybrid nanocomposite materials through catalyzing the sol–gel processes and templating silica generated in situ. Serving as the template, the proteins provide a unique opportunity to regulate the silica morphology and properties by means of a change of their secondary and tertiary structure through pH and temperature. © 2007 Elsevier B.V. All rights reserved. Keywords: Albumin; Gelatin; Casein; Sol–gel; Nanocomposite

1. Introduction It is thought that the structure-driven synthesis of silica nanomaterials by means of sol–gel processing can be performed on organic templates. As usual, surfactants and copolymers are used in labs [1–3]. Biological systems present an example of synthesis of nanostructured silica under regulation by proteins [4,5]. The latter are responsible for the highly controllable formation of hierarchically structured materials with precisely defined size, structure, shape, spatial orientation and organization as well as with mechanical properties exceeding those of materials from inorganic world [6–12]. This is the reason for tremendous interest to proteins. As demonstrated in the case of sponges and diatoms [8,13], the biomineralization is related to specific proteins called as silicatein and silaffin, respectively. They induced precipitation of silica in solutions with silicic acid or silanes at neutral pH at which the sol–gel processes were not observed in their absence. Identification of amino acids in these proteins has inspired the use of synthetic polypeptides consisting of certain amino acids. They could manipulate the sol–gel processes and provide the formation of materials with controlled

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nanostructure varied from spherical to fibrillar (see, e.g. Refs. [14–18]). Here we demonstrate by the example of gelatin, albumin and caseins that any protein can catalyze the sol–gel processing and serve as template for silica if a novel silica precursor, tetrakis(2-hydroxyethyl) orthosilicate (THEOS), with improved biocompatibility, as shown in Ref. [19], is used instead of common tetramethoxy- and tetraethoxysilanes. THEOS was previously applied for fabrication of hybrid polysaccharide–silica nanocomposites [20–22]. Relative to polysaccharides, the proteins offer an additional opportunity for manipulating the silica morphology and architecture at the nanoscale level through its secondary and tertiary structure changed by simple shift of pH of aqueous solution or temperature [23,24]. 2. Materials and methods Sodium azide and ethylene glycol were from Fluka. Bovine serum albumin (BSA) was obtained from Serva and gelatin, from Fluka. Caseins were prepared by defatting of milk powder that was made by acetone treatment. The treated powder contained about 30 wt.% caseins and 5 wt.% whey proteins and minor proteins. There was also ca. 55 wt.% lactose and 5 wt.% salts. Tetrakis(2-hydroxyethyl) orthosilicate was synthe-

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sized from tetraethoxysilane (ABCR company) as previously described [20,25]. Proteins were dissolved in distilled water with gentle agitation using a magnetic stirrer. 0.2 mg/ml of sodium azide was added to prevent bacterial growth. The desired pH value was adjusted with HCl or NaOH. Hydrogels were synthesized by admixing THEOS into aqueous solutions of proteins, thoroughly stirred for 1–2 min and left at ambient temperature at least for a week for further experiments. Aerogels for observations under a scanning electron microscope (SEM) were prepared by drying the initially synthesized hydrogels at supercritical conditions: To make this procedure, the water from the hydrogels was exchanged first to acetone and then to liquid CO2 , followed by the increasing of pressure and temperature until the supercritical state (about 50 atm. and 38 ◦ C), whereupon CO2 was removed by slow depressurizing. Fresh surface of aerogels obtained by cutting was covered by carbon layer. SEM pictures were taken by using a LEO 430 microscope. Rheological measurements were performed by means of a Rotovisco RT20 (Haake) stress-controlled rheometer, using a cell with cone-and-plate geometry as detailed in Ref. [20]. The diameter and angle of the cone were 60 mm and of 1◦ , respectively. The rheometer was run in oscillation regime. The oscillatory frequency was varied from 0.001 to 10 Hz. To examine a dependency of rheological parameters on the temperature, measurements were made at 1 Hz within a certain time interval, while a sample was heated or cooled. The evaporation of solvents during the measurements was decreased by using a special chamber. 3. Results and discussion The effects of BSA, gelatin and caseins on the sol–gel processing were studied. These proteins were chosen because of difference in their composition, structure and structural organization in solution. The BSA is a globular protein (Mw 66.3 kDa) [24], whereas gelatin, fibrillar one, liable to form a helix from three macromolecules and experiencing a reversible phase transition from helix to coil at a certain temperature [26–28]. Caseins are a family of phosphorylated proteins (␣S1 -, ␣S2 -, ␤- and ␬-caseins) of milk that self-assemble into aggregates called micelles [29,30]. Their shell is formed by ␬-casein that protects three other caseins locating in the micellar core. The ␬-casein differs from albumin and gelatin by a carbohydrate chain bearing by its macromolecules. These chains are located on the surface of micelles, forming a “hair” shell around them [31]. The silica precursor, tetrakis(2-hydroxyethyl) orthosilicate, was previously used to fabricate hybrid polysaccharide–silica nanocomposite materials in Refs. [20,21]. When being added into solutions of the studied proteins, THEOS resulted in their transition into the gel state owing to the sol–gel processes. The prepared materials can be assigned to hydrogels because of water that is their main constituent. Its content was as much as 90 wt.%. It is worth also mentioning that THEOS was first used by Hoffmann with collaborators to synthesize hybrid nanomateri-

als on the basis of surfactants [32,33]. They demonstrated that this precursor did not cause the precipitation of surfactants and restructuring of their lyotropic mesophase. These experimental evidences were indicative of improved compatibility of THEOS with organic substances. This was then shown in set of articles published by Husing et al. [34,35]. The jellification of BSA- and gelatin-containing solutions was observed in a wide pH range lying between 3.3 and 10.6. The THEOS admixing did not induce a phase separation or precipitation. It was observed only at pH around the isoelectric point (pI) of BSA (ca. 4.8 [24]). When the pH was shifted into the acidic or alkaline region in relation to this point even for 0.3 units, the precipitation was absent. There was a white, turbid monolith. It became translucent and then optically transparent with moving away from the pI. The formed hydrogels remained monolithic during an observation continuing for more than 9 months. Fabrication of hydrogels with caseins had some features. Their micelles aggregate and then precipitate when pH of aqueous solution becomes lower than 5.5 that is at the basis of production of fermented diary product [36,37]. Because of the casein precipitation, it is unreasonable to carry out the sol–gel processes in the acidic media as for gelatin and BSA. The smallest pH value at which the synthesis could be made was 5.5. The monolithic hydrogels with caseins were formed from this pH up to pH 9.4. The performed experiments revealed two features by which the formation of hybrid protein–silica hydrogels can be distinguished from the common sol–gel processing. (i) The jellification took place at conditions at which it was not observed in the absence of proteins. For example, it was found in the neutral pH region. A related effect was observed previously for silicatein and silaffin separated from sponges and diatoms [8,13]. It was also mentioned in experiments with polysaccharides where THEOS was used as the precursor [20,21,38]. The similarity to processes of fabrication of hybrid polysaccharide–silica nanocomposites allows us suggesting that the proteins have a catalytic effect on the sol–gel processing. (ii) A hydrogel, which is generated after mixing of proteins and THEOS, was monolithic. The precipitation or phase separation was not observed. This differed even from processing performed with silicatein and silaffin that precipitate generated silica [8,13]. In the case of THEOS monolithic nanocomposites were fabricated everywhere over the studied pH range extended from 3 up to 10. The sole exception, as was mentioned above, was the system with BSA in which a precipitation was observed at pH equal to the protein pI. Morphological peculiarities of aerogels prepared from the starting water-containing gels synthesized at various pHs are obvious from Fig. 1. There are three extended sets of images of samples with gelatin, BSA and caseins taken with a scanning electron microscopy. One may see porous nanocomposites consisting of clusters arranged in various manners that results

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Fig. 1. SEM micrographs of aerogels that were prepared from initial gels synthesized by adding 10 wt.% THEOS into an aqueous solution containing 1 wt.% of protein. The pH value and protein being in a solution are shown close to pictures.

in substantial difference in the morphology. The differences can be resolved among samples fabricated with various proteins as well as at various pH. The largest clusters and pores are seen in the albumincontaining sample prepared at pH 4.8 and gelatin-containing ones, at pH 4.3 and 5.5. These aerogels were fabricated either from precipitated or turbid gels. When the synthesis was performed by shifting pH into the acidic or alkaline region, the hydrogels became translucent and then optically transparent that causes a decrease of nanoparticles and pores. In the acidic region there are extended strings from nanoparticles (BSA-containing sample, pH 3.3). They can be seen also in the case of gelatin, but these strings are less pronounced, shorter and curved. With shift pH into the alkaline region, aerogels with gelatin remain mainly amorphous, whereas in the BSA-containing samples one may observe a transition to plate-like clusters. The planar morphology looking like stacked plates appear at pH 6.4. With increasing the pH, it seems finer (pH 8.1). Spherical particles with diameter slightly exceeding 1 ␮m are revealed in case of sample synthesized at pH 9.6. Preliminary observation demonstrated that these spheres are hollow. It seems that they were formed in the course

of aerogel preparation from the synthesized gel, but it calls for further study. The morphological changes with the pH shift in aerogelscontaining caseins (bottom set of SEM images in Fig. 1) are not so diverse as in the foregoing samples. The morphology is amorphous, consisting of cross-linked clusters. Their dimension is decreased with the increase of pH. It is worth mentioning that this is of the same order as the diameter of micelles in milk (see, e.g. Ref. [29]). There is a reason to believe that these nanoparticles present casein micelles covered by silica. Fig. 2 presents SEM pictures taken at higher magnification. This allows emerging additional morphological details. The clusters seen in Fig. 1 are made up of cross-linked or merged nanoparticles. Their dimensions vary, being under 100 nm. One may reveal differences in the morphology between samples prepared with various proteins. Fibrillar-like nanoparticles can be found in the gelatin-containing aerogel, whereas they are spherical for the most part in samples synthesized on the basis of BSA and caseins. It is worthy mentioning that this morphological distinction follows difference in the tertiary structure of examined proteins. Gelatin is in the fibrillar state at the experimental

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Fig. 2. SEM micrographs of some aerogels shown in Fig. 1 that were taken at higher magnification.

conditions, while BSA is a globular protein and caseins are selfassembled into micelles of which shape is close to spherical. It seems (Fig. 2) that casein micelles are aggregated that is typical of them at this pH (see, e.g. Ref. [29]). The morphological features of hybrid protein–silica nanocomposites suggest a mineralization of biopolymer macromolecules that is provided by THEOS. This conclusion was previously drawn in detailed study of processing with polysaccharides [20–22,25]. Experimental evidences obtained with the help of atomic force microscopy demonstrated that carbohydrate macromolecules served as a template for silica generated in situ that resulted in their encasement in inorganic matrix [25]. Preliminary experiments demonstrated that this mechanism can be extended to the studied proteins. Further evidence of the influence of protein secondary and ternary structure on the properties of hybrid nanocomposites was found when rheological measurements were performed at various temperatures. Results are presented in Fig. 3. There are temperature dependences of shear (storage G and loss G ) moduli for a 5 wt.% gelatin solution (Fig. 3A) and a hydrogel fabricated by admixing 10 wt.% of THEOS into a 5 wt.% gelatin solution (Fig. 3B). The former demonstrates rheological behavior typical of this protein in water. One may see a step-like change of shear moduli with the temperature. It occurs in a certain temperature range when the solution was heated or cooled. An intersection of curves 1 with 2 and 3 with 4 corresponding, respectively, to the storage G and loss G moduli means that there is a phase transition in the system. This type of behavior is caused by the melting of hydrogel that leads to its transition into

the liquid state when it is heated and jellifying of solution when it is cooled. The reversible solution–hydrogel transition is due to the thermotropic coil–triple helix transformations experienced by gelatin in water [26–28]. A similar step-like change in the shear moduli is observed for the hybrid gelatin-silica hydrogel, but the curves 1 and 2 as well as 3 and 4 in Fig. 3B do not intersect each other as it was found in the previous system. Because the storage modulus is higher than the loss one in the measured temperature range, the transition of hybrid gel into a liquid state with heating is absent. This signifies that the silica fixes the gel structure. Nevertheless, the observed changes in the shear moduli with the temperature, which correlate with those in Fig. 3A, suggest restructuring of hybrid matrix owing to the conformational changes of protein macromolecules. The foregoing proves that the mineralization of various types of proteins is feasible in wide pH range including the neutral region and at ambient conditions if THEOS is used as the silica precursor. This is something akin to the biomineralization in living organisms and biomimetic sol–gel processes catalyzed by separated proteins in vitro [8,13] or bioinspired synthetic polypeptides [14–18]. The possibility of any protein being involved in the synthesis of hybrid silica nanomaterials is of great importance from fundamental point of view and for various applications. An important point is that the structuredriven effect is based on conformational changes of protein macromolecules. It can be regulated, as demonstrated here, by a simple variation of pH of aqueous media, but this is only one of many opportunities. The protein secondary and tertiary structure

Fig. 3. The storage (1 and 3) and loss (2 and 4) moduli vs. the temperature for hydrogels containing 5 wt.% gelatin (A) and 5 wt.% gelatin and 10 wt.% THEOS (B). The shear moduli were measured at 1 Hz when the temperature, as shown by arrows, was increased (solid symbols) or decreased (open symbols). pH value was 8.

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is very liable to various influences. The conformation changes can be brought about by salts, solvents, organic additives and temperature [23,39]. This means that the spectrum of means to regulate the protein structure and thus the nanocomposite morphology is highly diverse. Therefore, we believe that the suggested approach could open new possibilities in the rational structure-driven synthesis of nanomaterials under mild conditions. Next in importance for the successful fabrication of protein–silica hybrids with regulated structure and properties is the precursor. Its effect on the protein secondary and tertiary structure should be as minimal as possible. The common silica precursors, such as tetramethoxy- and tetraethoxysilanes, generate, respectively, methanol and ethanol in the course of their hydrolysis. The alcohols have the strong denaturating effect on proteins; that is, they cause an uncontrollable change of protein conformation. This disadvantage is absent in the case of THEOS. Ethylene glycol generated after its hydrolysis is compatible with biopolymers. It was found in our experiments where hybrid polysaccharide–silica nanocomposites were fabricated [20–22,38] and highly labile enzymes were entrapped into the sol–gel derived silica matrix [40,41]. The absence of perceptible effect of THEOS on the protein conformation let us reveal the effect of pH change on the morphology of protein–silica hybrids caused by the pH-tuned protein denaturation (Fig. 1), and in its turn can be applied for manipulation on structure of sol–gel derived nanocomposite materials. Acknowledgements The study was supported by grants from the Russian Foundation for Basic Research (No. 06-03-96007-p east a) as well as from the Presidiums of Russian Academy of Sciences (No. 06I-OXHM-138) and Far East Department of Russian Academy of Sciences (No. 06-II-CO-04-018). We appreciate the help of Denis Fomin with the SEM observations. References [1] A.C. Pierre, Introduction to Sol–Gel Processing, Kluwer, Boston, 1998. [2] S. Forster, M. Antonietti, Adv. Mater. 10 (1998) 195. [3] G.J.A.A. Soler-Illia, C. Sanchez, B. Lebeau, J. Patarin, Chem. Rev. 102 (2002) 4093. [4] T.L. In, B.E. Simpson, Volkani (Eds.), Silicon and Siliceous Structures in Biological Systems, Springer-Verlag, New York, 1981. [5] J.J.R. Frausto da Silva, R.J.P. Williams, The Biological Chemistry of the Elements. The Inorganic Chemistry of Life, Oxford University Press, Oxford, 2001.

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