Biomimetic Designing of Functional Silk Nanotopography Using Self

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Biomimetic designing of functional silk nano-topography using self-assembly Banani Kundu, Mohamed Eltohamy, Vamsi K Yadavalli, Subhas C. Kundu, and Hae-Won Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07872 • Publication Date (Web): 30 Sep 2016 Downloaded from http://pubs.acs.org on October 6, 2016

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ACS Applied Materials & Interfaces

Biomimetic Designing of Functional Silk NanoTopography Using Self-Assembly

Banani Kundua, ‡, Mohamed Eltohamya,b, ‡, Vamsi K. Yadavallic, Subhas C. Kundua,d, Hae-Won Kima,e,f,* a

Institute of Tissue Regeneration Engineering (ITREN), Dankook University, South Korea

b

c

Glass Research Department, National Research Centre, Dokki, Cairo, Egypt

Department of Chemical and Life Science Engineering, Virginia Commonwealth University,

Richmond, VA, USA d

Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal-721302,

India e

Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for

Regenerative Medicine, Dankook University, Cheonan 330-714, South Korea f

Department of Biomaterials Science, College of Dentistry, Dankook University, Cheonan 330-

714, South Korea

ACS Paragon Plus Environment

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ABSTRACT

In nature inorganic-organic building units create multifunctional hierarchical architectures. Organic silk protein is particularly attractive in this respect because of its micro-nano scale structural blocks that attribute to sophisticated hierarchical assembly imparting flexibility and compressibility to designed bio-hybrid materials. In the present study, aqueous silk fibroin is assembled to form nano-/micro-topography on inorganic silica surface via a facile diffusion limited aggregation process. This process is driven by electrostatic interaction and only possible at a specified aminated surface chemistry. The self-assembled topography depends on the age and concentration of protein solution as well as on the surface charge distribution of the template. The self-assembled silk trails closely resemble natural cypress leaf architecture, which is considered structural analogue of neuronal cortex. This assembled surface significantly enhances anchorage of neuronal cell and cytoskeletal extensions, providing an effective nano/micro-topographical cue for cellular recognition and guidance. KEYWORDS: Silk, silica, self-assembly, topographical cue, cellular guidance


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INTRODUCTION Supramolecular assembly is ubiquitous in nature, occurring in egg shells, pearls, corals, enamel, nacre and bone, which use protein templates to induce the nucleation and growth of inorganic materials.1 Over billions of years ~20 amino acids, a dozen lipids, polysaccharides and few nucleotides, have evolved into building blocks that can self-assemble into flexible, functional and self-sustaining architectures. Bio-macromolecules, especially proteins can self-assemble into nanotubes, nanofibrils or other nano-shapes.2 Unfolding the mystery of molecular intercalation at nanoscale provides the possibility to mimic zero- to three-dimensional structures in nature, incorporating engineered products with extraordinary properties. However, regular fabrication methods are typically unsuccessful at recreating natural, bottom-up designs, particularly in a laboratory setting. To address such issue, we draw inspiration from nature, wherein molecular self-assembly is guided using protein templates that induce the assembly or growth of inorganic materials.1 Here, we ask the question whether the reverse strategy may be proposed, that employs inorganic materials to serve as a template for the induction of protein assembly. Toward this goal, we study combination of silk and silica as the self-assembling protein and inorganic template, respectively. Specifically, we modulate silica to control the assembly of the silk protein. Silk is widely considered one of nature’s unique biopolymers, abundantly available and low-cost, while also featuring mechanical robustness, flexibility,3-4 optical transperancy5-6 and electrical conductivity.7 Previous studies on self-assembly of silk fibroin into nano-structures are summarized in Table 1.8-14,1 On the other hand, silica is widespread in nature and performs significant roles in biological systems, primarily offering structural support and protection in animals. This ranges from single-


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celled diatoms to multi-cellular plants and animals,15 bone repair16 and as source material for various low-molecular weight proteins; termed as silaffins.15 Structurally stable silk-silica composite has also been studied as potential biomedical materials in space.17 Table 1. Self-assembly of mulberry Bombyx mori silk protein fibroin on different surfaces

Surfaces Aminated glass cover slips

Protein Conc. (mg/ml) 4 - 0.005 10 ± 0.5 1.5 – 0.15

1.5 – 0.015 Mica 0.3-0.06 Poly-styrene surface Aqueous solution of protein Graphene oxide

200 - 60 70 1.5 – 0.015

Self-assembled topologies At 4: rod-like particles At 0.5: globular particles > 0.5: dispersed globular particles Beads on a string at nano-meter scale At 1.5: cuboid At 0.3: bead At 0.1: proto-filament to fibrils at 0.15: proto-filament At 0.015 & 0.06: Increasing temperature results in β-sheet proto-fibrils; decreasing temperature results in random coil protofilaments/ globule-like molecules At 0.3 & 1.5: Increasing temperature results in β-sheet proto-fibrils and globule-like features Nanofibrils-nanoparticles-aggregates > 0.3: no self-assembly Micelles

Ref. 8 9 10

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12 13

Nanofibrils

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Nano-composites tunable in the plane of conductivity

1

Inorganic surfaces like mica,18 graphene1 and silicon19 were previously reported for their use in stimulating protein nano-assembly. For instance, surface mediated nano-assembly of oligopeptides occured by physisorption on mica and graphite,18 or by chemisorption on amino (NH2) or carboxyl (-COOH)-terminated surfaces.20 Conversely, none of these reports clearly demonstrated the prerequisites of surface induced self-assembly. The study of Hwang et al.,21


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using silk-elastin-like proteins revealed the significance of electro-static interactions on nanofibrillar protein growth, indicating that charged hydrophilic surfaces were more suitable for nano-assembly. However, the extent of hydrophilicity was not taken into consideration in the study. Therefore, the present report focuses on three aspects of directed assembly of natural silk protein using silica as an inorganic template: (i) nanoscale structural assembly of silk protein fibroin through the control of surface charge distribution, (ii) recognition of surface topologies resulting from this self-assembly process, and (iii) observation of the impact of surface hydrophobicity/ hydrophilicity on fibroin assembly. The resulting protein assembly is then briefly investigated if the topology can help the cellular recognition and cytoskeletal guidance using rat pheochromocytoma (PC12) cells.

EXPERIMENTAL SECTION Materials Tetraethyl orthosilicate (TEOS), (3-aminopropyl)triethoxysilane; (APTS) (Sigma-Aldrich, USA), microscopic slides (Marienfeld, Germany), dialysis membrane (MWCO 3500, Orange Scientific, Belgium), sodium carbonate (Na2CO3), lithium bromide (LiBr), hydrochloric acid (0.1 N), ethanol (Daejung Chemicals and Metals Co. Ltd., South Korea), deionized water (Synergy Millipore, Billerica, MA; resistivity 18 MΩ•cm at 25 °C), Dokdo Mark 240 (DokDo-MARKTM Broad-range, dual color, Cat. No. EBM-1032), PC12 (rat pheochromocytoma cells), American Type Culture Collection (Rockville, MD, USA), DMEM, fetal bovine serum (FBS), horse serum


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(HS), 100 U/mL penicillin-streptomycin (Gibco, USA), trypsin – EDTA (Invitrogen, Basel, Switzerland), MTS assay kit [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; AMRESCO, Solon, OH) were used as received. Mulberry Bombyx mori silk cocoons were purchased from Kyoung-buk Ulsin Gunnam myun Meogil 146, Ulsin Silk Farm, South Korea Aminated silica coating on glass slides Glass slides were cut into rectangular pieces with a dimension of 2.5 cm x 1.5 cm using a glass cutting machine (Accutom-50, Struers, Denmark) and cleaned with acid-acetone solution, followed by deionized water. Immediately before coating, both APTS and TEOS were prepared in ethanol and mixed in a ratio of 0/100, 25/75, 50/50, 75/25 and 100/0 (APTS/TEOS) by volume. Slides were coated using a spin coater (SPIN-3000A-S, Midas system Co. Ltd, South Korea) at 5866 g for 30 sec, dried at room temperature overnight; then heat treated at 70oC for one hour. Immediately before protein coating processes, the slide surfaces were subjected to brief hydrochloric acid (0.1 N) treatment to activate the surface pendant amino groups (as illustrated in Scheme 1).


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Scheme 1: (A) Schematic illustration of step-wise preparation of silk fibroin assembled on aminated silica surface. (B) Systematic mechanism of self-assembly of silk fibroin molecule in aqueous solution. (C) Dendrite structure of Cypress leaf, the structural analogous of the assembled silk topology. (D) Electrostatic attraction between negative site of protein with positive site of surface and vice-versa during protein deposition and self-assembly.

Silk protein preparation and characterization Silk fibroin solution from the mulberry silkworm Bombyx mori was obtained following standard protocols of Na2CO3-LiBr.22 After dissolution, aqueous silk solution was dialyzed against deionized water for 72 h at room temperature to remove the traces of salt and centrifuged at 10,000 rpm for 20 min before freezing for overnight. The frozen silk solution was then lyophilized to obtain silk powder. Molecular weight of regenerated protein was estimated by discontinuous 6% SDS-PAGE under both reducing and non-reducing conditions. Protein band was visualized after staining with Coomassie-Brilliant Blue R-250 (Sigma, St. Louis., USA).


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Silk assembly into nano-micro-structures A series of fibroin concentrations (0.02 to 7.0 mg/ml) were prepared by dissolving predetermined amount of silk powder in deionized water and filtering through 0.22 µm syringe filter. For self-assembly, an aliquot (5 µl) of transparent protein solution was dropped onto the silica-coated surface followed by drying at room temperature under sterile condition overnight.23 Characterization of assembled protein The morphology of protein assembled structures was observed by scanning electron microscopy (SEM). For SEM analysis, dry samples were sputter-coated with platinum and imaged (JEOLSEM 3000, FE-SEM S-4300, Hitachi Japan and MIRA2 LM, TESCON, Czech Republic). The images were further processed using Image J software (version 1.47, NIH, USA) for fractal morphology and estimating the coating thickness of silica. Digimizer software (version 4.0.0.0) was used to calculate the particle size. Both particle size distribution (PSD) and zeta (ζ) potential measurements were carried out using aqueous protein and a Zetasizer (Malvern, Worcestershire, UK) at 25 °C based on dynamic light scattering principle. Infrared spectra of coated surface and protein fibroin were obtained using a Varian 640-IR spectrometer (Varian, Australia) based on attenuated total reflectance infrared (ATR-IR) mode with a resolution of 4 cm-1 in the range of 4000 to 400 cm-1. Wettability of surfaces was determined by placing protein droplet (~20 µl) on dried surface. The contact angle at the intersection of the liquid and solid phase was measured using a Phoenix 300 device (S.E.O., Gyunggido, South Korea).


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Sterilization with ethylene oxide (EO) treatment The slides (both protein coated and regenerated Si-surface) were subjected to EO treatment for 4 h at 55 oC followed by overnight aeration to eradicate the effect of residual EO gas. Stability of assembled structure in solution Protein-coated slides after EO treatment were incubated in sterile phosphate buffer saline (PBS, pH 7.4) up to seven days (separate set for each time point) at 37oC in humidified atmosphere. At each pre-determined time interval (day 1, 2, 3, 5 and 7), PBS extracts were assessed for leached protein using Bradford assay and Microplate reader (Biorad 550, Japan). The quantity of released protein was estimated from corresponding standard curve. Cell culture PC12 cells initially were maintained in DMEM (Gibco) containing 10% horse serum - 5% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) till reached confluence.24 Then the cells were cultured in DMEM supplemented with only 10 % fetal bovine serum and 1% penicillin-streptomycin in order to evaluate the direct effect of substrate on cells at 37oC - 5% CO2 humidified atmosphere. Prior to cell seeding, EO treated slides were rinsed thoroughly with sterile PBS (pH 7.4) and exposed to UV radiation for 30 min. The slides were then partially dehydrated to facilitate cell attachment. Cells were treated with trypsin/EDTA to get into solution and 5 µl of suspensions containing 5 × 104 cells25 was added dropwise on top of each slide. They were left undisturbed for 30 min in a humidified atmosphere (37oC - 5% CO2) to promote cellular adhesion and thereafter maintained in adequate media. The aminated silica surface (75Si:25NH2) without the


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protein assembly was compared, and tissue culture plate without any treatment was also used as a control. Cell adherence, viability and morphology Adherence of cells on protein surfaces was investigated as a function of time by both qualitative and quantitative ways at regular interval (1, 3 and 6 h) after seeding.26 At each time point, floating cells in culture media were counted using hemocytometer (separate set for each time point). The number of cells adhered to each surface was determined by deducting the number of washed out cells from initial cell seeding density. For imaging, the cells were incubated with 25 µM CellTracker™ Green CMFDA (C7025, Molecular probes, Life technologies) dye at 37 °C for 45 min before seeding followed by washing with complete media. The stained cells were imaged using Olympus fluorescence microscope equipped with DP72 microscope digital camera. The imaged cells at 24 h were also counted to compare the cell adhesion level between groups. Cells were cultured for up to 72 hr. After the culture, the cells were fixed in 4% paraformaldehyde and stained with Alexa Fluor 546 conjugated Phalloidin (Molecular Probes, USA) for F-actin, and counter-stained with DAPI for nuclei. The arrangement of actin filaments was visualized using Olympus fluorescence microscope equipped with a DP72 microscope digital camera. Statistical analysis


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Data were expressed as mean ± standard deviation (n = 3 or as otherwise mentioned). The analysis of variance was performed using one way ANNOVA. P values of ≤ 0.05 were considered as statistically significant.

RESULTS AND DISCUSSION Aminated surface and regenerated protein Sol-gel spin coating process is used to prepare amino-functionalized surfaces with formulations of TEOS/APTS, on which the silk fibroin aqueous solution is self-assembled, analogue to Cypress leaf dendritic structure (Scheme 1). The electrostatic attraction between negative sites of protein with positive site of surface is engaged in the silk self-assembly. APTS serves as a base catalyst in the hydrolysis and condensation process of the porous silica.27 The influence of surface amino group (APTS) content on the assembly of silk fibroin (0.1 mg/ml) was shown by SEM images (Figure 1A). The self-assembly was only possible when APTS was used (on pure TEOS the silk coating was severely cracked). The contact angle value (ϴ) measured (Figure 1B) with a protein solution (0.1 mg/ml) was relatively low on the aminogroup containing surfaces (about 45-50o), with the lowest value observed on 25% APTS surface. However, the contact angle was significantly higher on the pure TEOS surface (about 105o), implying the surface was very hydrophobic. Spin coating at 4000 rpm resulted in homogeneous coating layer with a thickness of ~50 nm as measured by AFM (Figure 1C). Root mean square roughness (Rq) value of the resultant surface was 12 nm, which was observed to be directly proportional to the APTS/TEOS ratio.27 Thermogravimetric analysis of the surfaces revealed a


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two-stage thermal decomposition (Figure 1D). Weight loss in the region of < 300oC was assigned to evaporation of chemically and physically attached water. As the heating proceeded, an intensive weight loss (14.3 wt%) in the temperature range of 300-500oC was attained due to functionalized organic groups on surface, indicating the quantitative ratio of (CH2)3NH2 to SiO2 groups in APTS/TEOS (25/75). With further increase in temperature over 500oC, the weight loss curve reached a stable plateau. Investigation using FT-IR spectroscopy exhibited distinctive peaks at 1077 and 794 cm-1 corresponding to asymmetric and symmetric stretching vibrations of Si–O–Si framework (Figure 2A).28 Characteristic peaks at 673 and 1514 cm-1 were assigned to bending of the N-H bond and symmetrical -NH3+, respectively. In addition, peaks at 3000 to 2878 cm-1 were representative of sp2 and sp3 hybridized C–H bond stretching within the alkane group of APTS.29 A broad band appeared around 3664 cm-1 denoting the hydrophobic nature of APTS/TEOS (25/75)21 and the surface zeta-potential value of APTS/TEOS (25/75) was + 21.1 (± 3) mV (Figure 2B).


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Figure 1. (A) Influence of amino groups (APTS percentage) of surface on the assembled topology of silk fibroin (0.1 mg/ml).

Percentage of APTS (NH2) with respect to TEOS

designated in each image (0, 25, 50, 75 and 100%). Scale bar = 20 µm; (B) Wettability of silk protein on the aminated silica surfaces measured by means of contact angle using aqueous silk fibroin solution (0.1 mg/ml). Contact angle was ~45-50o on the aminated surfaces (APTS 0%, 25%, 50%, 75%), but ~105o on the pure silica surface (APTS 0%); (C) Field emission-scanning electron micrograph of the sample assembled on 25% APTS (cross-section view, Scale bar = 200 nm) and the AFM image showing nano-topology of the assembled silk; (D) Thermogravimetric analysis of the 25% APTS surface, confirming the TEOS/APTS ratio of 75/25 by a thermal degradation.


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Figure 2. (A) FTIR of 75% TEOS/ 25% APTS surface confirming the presence of functional groups on the surface; (B) zeta potential value of 75% TEOS/ 25% APTS and pure silk fibroin. The zeta potential value of freshly prepared regenerated silk fibroin solution is measured to ensure the electrostatic interaction between the aminated surface and protein biomolecules.

Extracted fibroin from cocoon exhibited a molecular weight band greater than 250 kDa, corresponding to a heavy chain (Mw ~390 kDa) under both reducing and non-reducing conditions (Figure S1). Broad molecular weight band around 26 kDa was observed under non-


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reducing condition compared to narrow molecular weight band under reducing condition, which corresponding to the fibroin light chain of 25 kDa and P25, the glycoprotein (~30 kDa).30 Smearing observed in the gel is due to the damage associated with fibroin peptide chain during Na2CO3-LiBr treatment.31 Native globular silk fibroin heavy chains possess hydrophilic and hydrophobic blocks, which form micelles in regenerated protein (RSF) solution.9 The presence of different molecular weight fraction in RSF is necessary for the integrated assembly of silk, as recommended by Khire et al.,.30 As observed earlier, silk protein sericin fractions from 24 to ~ 210 kDa imaged individually revealed lack of any assembled structure, in comparison to the corresponding mixed blend. This confirms the co-ordination between different fragments to form integrated assemblies.30 The zeta potential of fibroin was -10.5 mV in neutral aqueous solution (Figure 2B), which is far less than that reported elsewhere (-43 mV).32 This indicates the negative charge distribution within metastable silk nanostructures that are involved in repulsive charge–charge interactions, providing stability to meta-phase.14 The optimal host surface as confirmed from SEM observation and contact angle measurement is considered to be 25%APTS/ 75%TEOS, which is thus used for the rest of our study. Nano- to macro-scale assemblies of biomacromolecules are chiefly driven by diffusion limited aggregation (DLA), which is characteristic phenomenon of globular protein subunits such as silk fibroin.33 For DLA-mediated self-assembly of negatively charged silk fibroin, an electrostatically attractive surface is required; which is fulfilled by the underlying amino-functionalized silica. Facile, one-step fabrication approach was demonstrated by dispersing protein solution on silicasurface and allowed the slow evaporation of aqueous solvent over 24 hr at room temperature under atmospheric humidity. Solvent evaporation resulted in concentration gradient with time, determining the start and end of deposition. A wide range of silk fibroin solution was studied


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(0.02-7 mg/ml), which in turn indicated concentration dependent fluctuations of fractal morphology, as shown in Figure 3. Random aggregation in square lattice33 was revealed in 0.020.1 mg/ml concentrations with branches radiating from center. Linear growth of the fractal structures indicated persistent and robust assemblies. Within the range of 0.02–0.1 mg/ml, the protein micelles self-assembled into microstructures; wherein each micelle could serve as a nucleation site.34 At the concentration < 0.1 mg/ml, the resultant self-assembled dendritic structures showed discontinuous fragments. Saffman Taylor finger-like morphology35 was observed with an increase in concentration of protein from 0.1 to 0.4 mg/ml. At a concentration of 0.1 mg/ml, completely connected assembles with denser subunits were revealed, leading to branched dendrite structures with length several microns. The periodicity and complexity of such self-assemblies was observed up to ~0.4 mg/ml. Further increase in protein concentration from 0.8 to 3.5 mg/ml revealed individual star-like aggregates with irregular shape of each sub-unit. At concentrations greater than 3.5 mg/ml, long filament- like structures with 20 ± 6 µm diameters were obtained. Fractal dimensions were estimated using the box-counting algorithm as discussed earlier by our group.18,34,36


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Figure 3. Concentration dependent assembly of aqueous protein solution generated after dryingdissolving process on APTES/TEOS (25/75) surface; 0.02 to 7.0 mg/ml of silk protein fibroin. High resolution pictographs of building subunits of dendrite branches reveal concentration dependent change in size and diffusion limited aggregation; resulted in diverse length selfassembled dendrite structures. Enlarged images are shown in inset.

The ImageJ program was used for these calculations (Rasband, W.S., ImageJ, NIH, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2015). At lower concentrations 0.01-0.1 mg/ml, the fractal dimensions ranged from 1.25-1.5 (Figure S2). However, at concentrations of 0.1-0.8 mg/ml, the fractal dimensions of the filamentous structures ranged from 1.6-1.8. The fractal architecture observed at 0.1 mg/ml concentration resembles the DLA of colloids proposed by Witten and Sander33 and also corroborate with our group’s earlier observation using silk protein sericin.36 In this model, a random particle seeds at the origin of a lattice, followed by an encounter of a second particle travelling from infinity via Brownian diffusion. This particle may either add to the pre-existing structure facilitating its growth, or just escape. The probability of a particle to adhere to the encountered structure is termed “stickiness” and governs the growth and morphology of DLA structure. In solution, repulsive forces among negatively charged protein micelles tend to prevent aggregation or stickiness, whereas the partially aminated surfaces provide the sticky points to adhere and aggregate. By comparing Figure 3 (silk concentration of 0.1 mg/ml and higher) with Scheme 1C, it is observed that the morphologies begin to approximate natural dendritic architectures similar to that of a cypress leaf (Chamaecyparis obtusa or Hinoki). The cypress leaf is considered to be structurally analogous to the neuronal cell network in neocortex.37


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A neuron in brain contains a cell body, hundreds of tree like branches called dendrites and a long axon; together forming a dendritic living architecture. Quantitatively, the fractal dimensions at concentrations of 0.1 mg/ml to 0.8 mg/ml range from 1.6 to 1.8, which are very similar to the values for the cypress leaf (1.65-1.75, calculated using the same approach as above). Therefore, we can conclude that it is possible to mimic such natural architectures using this simple selfassembly technique. By careful control of surface properties and silk fibroin concentration, precise dendritic architectures may be fabricated. Such topographical cues can favor neural cells, prompting us to hypothesize that the observed biomimetic silk nanotopographies may be used for similar applications. Although here we tested the fractal structures only with the APTES/TEOS (25/75) surface at varying protein concentrations of 0.02-7.0 mg/ml, further studies on other surface compositions might gain other intriguing assembled morphologies due to the different surface charge, wettability, and chemical interactions. Measurement of the size of protein nanostructures revealed a range from 182 to 750 nm (Figure 4); size increased with increasing protein concentration up to 0.8 mg/ml (or 1.75 mg/ml) and then decreased with further increase in protein concentration. Dynamic light scattering (DLS) of the aqueous protein solutions also showed the hydrodynamic size of the protein assemblies (Figure S3). Due to the aggregation and charge repulsion behavior of protein fibroin an equilibrium statue with the smallest size is achieved at certain concentration; below or above of this, the nanoscale subunits aggregate and exhibit larger particle sizes. Though DLS method has a limitation in characterizing real-time particle size during the self-assembly on the surface (specially the case in 7 mg protein concentration), the current findings can explain the thermodynamic self-assembly process of various silk fibroin solutions, which is primarily corroborated with the SEM images.


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Figure 4. Particle size distributions of silk fibrioin building blocks generated from different fibroin concentrations. Size of the particles calculated from scanning electron micrographs using Digimizer software (version 4.0.0.0). Scale bar = 2 µm.

Further analysis of contact angle indicated an increase in hydrophilicity with increased protein concentration; 61.8o (0.02 mg/ml), 40.8o (0.1 mg/ml) and 28.7o (3.5 mg/ml), respectively (Figure 5A). ATR-FTIR absorption spectrum of the protein assembly was also analyzed. The band at 1700-1600 cm−1 corresponds to amide I, 1600-1500 cm−1 corresponds to amide II, and 12101280 cm−1 corresponds to amide III, respectively (Figure 5B). The peaks at 1610-1630 cm−1 (i.e. 1628 cm−1) (amide I) and 1510-1520 cm−1 (i.e. 1512 cm−1) (amide II) are characteristic of β-


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sheet confirmation and silk II secondary structure. The peak at 1628 cm−1 is precisely indicative of intramolecular anti-parallel β-sheet.38 The adsorption band at 1234 cm−1 (i.e. 1227 cm−1 here) (amide III) is suggestive of silk I structural conformation.39 In addition, peak between 1697-1703 cm−1 i.e. at 1700 cm−1 is responsible for weak intermolecular β-sheet.38 In general, intra and intermolecular β-sheet bands increase during silk crystallization38 and impart stability to the assembled structure. Increased crystallinity restricts the molecular mobility of polyalanine (A)n and polyalanine glycine (AG)n repeats in silk fibroin chain, leading to an increase in contact angle value;40 which is corroborated with our contact angle observation.

Figure 5. (A) Wettability of silk fibroin-coated 75 TEOS/ 25 APTS surfaces, measured by using a water droplet (~30 µl); (B) FTIR spectra of regenerated assembled silk fibroin; (C) Protein assembled on the surface slowly degrades, remaining ~ 96 % after day 7 in phosphate buffer solution (pH 7.4); (D) SEM images of silk building blocks at different degradation times (day 5,


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20 and 40), showing a time-dependent size distribution and topographical changes (0.1 mg/ml silk protein solution).

At the initial stages in RSF (up to 8 hr), bulk silk fibroin remains in random coil configuration and a small quantity of β-sheet aggregates.41 However, over time aggregations of β-sheet reach a critical concentration and become insoluble. The transition from random coil to β-sheet is a thermodynamically favorable process mediated by a multi-step ordered nucleus formation. In the resultant silk II structure, small hydrophilic blocks move out of micellar structures and interact with surrounding water to reduce the free energy of fibroin-water system.13 The transition of hydrophilic/hydrophobic interaction and molecular mobility is the rate determining step in selfassembly to form a stable structure. From FTIR spectra (Figure 5B), the presence of the intermolecular β-sheet bands indicates stable silk crystallization in the fractal structures, which is in agreement with the findings of protein leaching. Overall, the size of each building block (nano-crystals) regulates final DLA (diffusion-limited aggregation) cluster morphology. Large blocks have less homogeneous aggregation than small blocks.42 The durability of protein coated surface in aqueous medium was investigated in order to evaluate the possibility to use these surfaces for stable coating. The degradation of protein from silica surface was examined using the protein concentration 3.5 mg/ml as a representative sample (Figure 5C). Two other concentrations, 0.1 and 0.02 mg/ml were too low to carry out the release study. The amount of effectively coated protein on per slide used for the study was 262.5 µg. The release of protein was gradual with time as measured up to 7 days and the release rate decreased with time. At day 7, ~96% of the protein still remained on the surface. Analysis of aqueous silk fibroin in fresh and aged/old solutions indicated the absence of any specific


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nanostructures in fresh solution compared to homogenous structures after 40 days when stored at 4°C (Figure 5D).43 The sizes of the building blocks/nanocrystals reduced with prolonged storage time.

Cell behaviors After EO gas sterilization, a brief exposure to UV radiation was carried out to ensure complete aseptic surface. The surfaces with protein concentrations of 0.02 and 0.1 mg/ml along with protein-free aminated Si surface (75Si:25NH2) were used to investigate the responses of cells. A drop-wise seeding yielded cell clusters following Eden’s model of lattice formation, which is independent of distance travelled by sub-units (here cells) during lattice formation.32 Initially after dropping, a single cell situated at a random site of protein assembly, which was the peripheral site as indicated in fluorescence images (Figure 6A). The next cell adhered adjacent to this occupied site. The phase-contrast pictograph at 3 h showed the PC12 cells largely gathered on the silk-assembled area, suggesting the silk topological cue should provide adhesion sites for cells. The initial cellular adhesion quantified up to 6 hr showed significantly higher levels on the protein assembled surfaces than on the aminated Si surface without protein (75Si:25NH2), suggesting favorable interactions between cells and silk protein assembly units (Figure 6B). Moreover, the cell adhesion on the protein assembled surfaces was comparable to that on TCP. The favorable response of cell adhesion to the protein coating on Si substrate was also reported by Lee et al.45 The current silk fibroin assembled surface was clearly shown to provide anchorage sites for PC12 cells, which was considered in part due to the role of positively charged


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silk protein residues such as arginine, situated near the C-terminus of non-repetitive (hydrophobic) regions of fibroin, attracting negatively charged PC12 cells44. However, along with the positive charge density, the other surface parameters such as wettability can also be important in cell adhesion. It was observed that the adhesive proteins (fibronectin and vitronectin) in serum favorably adsorbed on the moderately hydrophobic surfaces45, which could allow more rapid anchorage of PC12 cells on the 0.02 mg/ml surface (less hydrophilic with a contact angle ~61o) than on 0.1 mg/ml surface (more hydrophilic with a contact angle ~40.8o). After 24 hr of culture, the adhered cells were densely observed on the protein assembled surfaces, but those on the Si surface without protein were sparsely distributed (Figure 6C). The quantification of cells at 24 hr revealed significant higher level on the protein assembled surfaces than on the Si surface. The adhesion might be through the non-specific interactions (e.g., electrostatic, van der Waals, hydrophobic and steric hindrance) between cells and the substrate during the initial stages of cell adhesion. Subsequent observation of cells over 72 hr revealed that the cellular spreading was pronounced on the silk-assembled surfaces, which however, was absent on the aminated Si substrate without the silk assembly. Moreover, the spreading morphology on the silk-assembled surfaces was directional (elongated); however, the cell spreading was primarily polygonal on the randomly coated silk surface, suggesting the possible cytoskeletal arrangement of cells along the self-assembled topological trail (Figure 6D).


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Figure 6. (A) Anchorage of PC12 cells on the fibroin assembled sites (0.1 mg/ml protein representatively shown) pictured in a time-sequence manner (1, 3, and 6 h). Cells initially adhered at the periphery of the protein and gradually approached to the center with time. The culture surface was treated with Alexa fluor 546 before cell seeding to visualize the selfassembled protein structure while cells were live-imaged with 25 µM CellTracker™ Green CMFDA. Phase-contrast images of cells at low and high magnification revealing a unique topology of silk-assembly and cells gathered on the assembled sites (indicated as arrows). (B) Cell adhesion level quantified at 1, 3 and 6 hr (*P < 0.05; statistical significance was noticed between groups; aminated silica surface w/o protein vs. protein assembled surfaces). (C) Distribution of cells on different surfaces at 24 hr of culture; fluorescence images of cells and the quantified cell number. (*P < 0.05; statistical significance was noticed between groups; aminated silica surface w/o protein vs. protein assembled surfaces, n = 3, analyzed image field size = 200


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µm x 100 µm). Scale bar = 50 µm. (D) Cytoskeleton extension of cells on different surfaces at 72 hr of culture; stained with Alexa fluor 546 conjugated Phalloidin for F-actin and with DAPI for nucleus. Well-developed elongated F-actins were revealed on the silk-assembled surfaces, whereas no such cytoskeletal development was observed on the aminated silica surface. Of note, the cell spreading on the randomly coated silk surface (used for comparison purpose) was not directionally elongated but primarily polygonal. Scale bar = 50 µm.

Taken all, the enhanced cell anchorage and cytoskeletal extensions of PC12 cells upon the silk fibroin assembled surfaces demonstrate their possible roles as neural matrices, by providing cellular recognition sites and further guidance cues (as illustrated in Scheme 2). In fact, neuronal differentiation of cells is governed by electrical and topological cues along with biochemical signals, and here the silk fibroin assembled topology is thought to offer some of the physicochemical cues to cells. Silk fibroin, as a self-assembling protein, has been shown to support a wide range of cells including stem cells, osteoblasts, fibroblasts, muscle cells, vascular endothelial cells, and neural cells46-48, facilitating the adhesion and migration through the up-regulation of MEK, JNK and PI3K signalling pathways49. Although here we did not observe the neuronal differentiation upon the silk assembled surfaces, the favorable actions in the cell anchorage and cytoskeletal extensions encourage further studies on this area. It is hoped that the silk-assembled trail, with its nature mimic topology, can provide appropriate stimulating and guidance cues for neural cell differentiation. One merit of the current silk assembled surface is that the method relies on its simplicity and mild processing conditions, without introducing sophisticated techniques like lithography, which are traditionally used to obtain micro- and nano topographies for nerve


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regeneration.50 Therefore, the current method may be used to tailor the surface of complex 3D structures of existing nerve conduits with nature mimic topology and composition of silk for functional nerve generation, which warrants further exploration.

Scheme 2. Illustration of the neuronal cell recognition to the surface of silk protein and the possible guidance of cellular elongation along the self-assembled trail.

Conclusions The work demonstrated the applicability of nano/microscale self-assembled silk fibroin topographical cue for cellular guidance. Improved adhesion and cytoskeleton arrangement of PC12 cells on the assembled surface suggest the ability to serve as a guidance platform for neural cells, an alternative to other substrates. The fabrication method is simple and yields stable biomimetic topography that closely resembles the neuro-cortex, as demonstrated by a fractal


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dimension calculation. Factors such as source, aging and concentration of silk fibroin protein along with the threshold ratio of pendant hydrophilic - hydrophobic groups on surface are considered to regulate the assembled structure, which remain as an intriguing study to follow.

Supporting Information Molecular weight estimation of aqueous regenerated silk fibroin using 6% SDS-PAGE; Particle size obtained by DLS measurement; Fractal structures and dimensions of different silk assembled structures This material is available free of charge via the Internet at http://pubs.acs.org

Corresponding Author † Corresponding author: Tel: +82 41 550 3081; Fax: +82 41 550 3085; E-mail: [email protected] Author Contributions B.K. and M.E. designed and performed the work, analyzed data, wrote the paper; V.K.Y analyzed data, S.C.K, V.K.Y and H-W.K. revised the write up. ‡

B.K. and M.E. contributed equally

Funding Sources Global Research Laboratory (GRL) Program (Grant no. 2015032163), Priority Research Centers Program (Grant no. 2009-0093829), and KRF R-2015-01277 through the National Research Foundation (NRF), Republic of Korea


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ACKNOWLEDGMENT This work was supported by a grant of Global Research Laboratory (GRL) Program (Grant no. 2015032163), Priority Research Centers Program (Grant no. 2009-0093829) and KRF R-201501277 through the National Research Foundation (NRF), Republic of Korea.


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Biomimetic Designing of Functional Silk Nanotopography Using Self

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