Distinctive Stress-Stiffening Responses of Regenerated Silk Fibroin

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Distinctive Stress-Stiffening Responses of Regenerated Silk Fibroin Protein Polymers under Nanoscale Gap Geometries: Effect of Shear on Silk Fibroin-based Materials Yuanzhong Zhang, Yuchen Zuo, Shihao Wen, Yupeng Hu, and Younjin Min Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00070 • Publication Date (Web): 26 Feb 2018 Downloaded from http://pubs.acs.org on February 27, 2018

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Biomacromolecules

Distinctive

Stress-Stiffening

Responses

of

Regenerated Silk Fibroin Protein Polymers under Nanoscale Gap Geometries: Effect of Shear on Silk Fibroin-based Materials Yuanzhong Zhang§, Yuchen Zuo§, Shihao Wen§, Yupeng Hu,§ and Younjin Min*§ §

Department of Polymer Engineering, University of Akron, 250 South Forge Street, Akron, Ohio

44325, United States KEYWORDS Bombyx mori silk fibroin; nanoconfinement; compression; shear; hierarchical assembly; elastic stiffening

1 ACS Paragon Plus Environment

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ABSTRACT Interfacial dynamics, assembly processes, and changes in nanostructures and mechanical properties of Bombyx mori silk fibroin (SF) proteins under varying degrees of nanoconfinement without and with lateral shear are investigated. When only compressive confinement forces were applied, SF proteins adsorbed on the surfaces experienced conformational changes following the Alexander–de theory of polymer brushes. By contrast, when SF proteins were exposed to a simultaneous nanoconfinement and shear, remarkable changes in interaction forces were observed, displaying the second order phase-transitions, which are attributed to the formation of SF micelles and globular superstructural aggregates via hierarchical assembly processes. The resultant nanostructured SF aggregates show several folds greater elastic moduli than those of SF films prepared by drop-casting and compression-only, and even degummed SF fibers. Such a striking improvement in mechanical strength is ascribed to a directional organization of -sheet nanocrystals, effectively driven by nanoconfinement and shear stress-induced stiffing and ordering mechanisms.


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INTRODUCTION Regenerated and processed silk fibroin (SF) protein polymers have received a great attention as adaptable material candidates for a wide range of practical applications. In particular, SF protein polymers in aqueous medium have been processed into high performance functional materials in various biomedical fields such as drug delivery vehicles,1,2 regenerative engineering for vascular,3,4 neural,5,6 skin,7,8 bone,9–11 cartilage,12,13 and ligament and tendon tissues,14,15 artificial implants as resorbable screws,16 and coatings for cell culture or antifouling purposes, some of which are shown in Figure 1a. All of these biomedical applications entail desirable materials properties such as biocompatibility,17–19 easy processibility in aqueous medium,20–22 as well as superior mechanical properties23–25 and degradability24,26 that SF can offer in a tunable manner. Notably, the controllability on mechanical strength featuring a rare combination of high tensile strength and extensibility (thus, high toughness) accompanied with a flexible degradation rate is one of the key advantages of using SF in biomedical applications aforementioned. Thus, an adequate knowledge on how to precisely adjust macroscopic material properties of SF has become essential for further successful technological advances. This need can be properly addressed only when a detailed understanding on material responses and behaviors of SF under explicit stresses such as normal compression and lateral shear is provided. Raw silk cocoons of silkworm (e.g. Bombyx mori, B. mori) are made from double-stranded fibers of fibroins (i.e. structural proteins consisting of the fibroin heavy (~390 kDa) and light (~26 kDa) chains connected by a disulfide linkage27,28). The most of SF is highly periodic with repeating amino acid sequences containing glycine (45%), alanine (30%), and serine (12%).25,29 The periodic alternation of glycine with alanine or serine forms the hydrophobic crystalline regions with β-sheet conformation, which are connected via small hydrophilic, amorphous linking segments composed



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of bulky aromatic amino acids (see Figure 1b), including phenylalanine and tyrosine, acidic residues, and proline.30 While covalent linkage and hydrogen bonding (intra- and interchain) within the crystalline domains of SF promote an anti-parallel configuration of β-sheets, attractive van der Waals interactions maximize and effectively induce the stacking of β-sheets due to the tiny size of side groups of amino acids predominantly found in the SF (i.e. glycine, alanine, and serine aforementioned).31,32 The tunability in SF material properties can be achieved by controlling the extents of β-sheet structures and assembly pathways of SF protein polymers (primary building unit) into hierarchical structures (secondary and tertiary building units) during a course of desirable processes and post-treatments (See Figure 1b). Various processing approaches including chemical (e.g. methanol33,34 and acidic35,36 treatments) and physical (e.g. centrifugation and vacuum assisted molding,37 electrospinning,38–40 and layer-by-layer assembly,23,41) methods have been adopted in order to suit SF-based materials for a wide range of practical purposes, especially for biomedical applications. While a lot of technological advances have been made on practical processing steps in order to control and optimize SF-based material properties depending on specific needs in applications, a fundamental understanding on how to achieve certain macromolecular constructs with molecular precision at the nanoscale is still lacking with limited studies accessible,42 especially for the cases where the restricted spaces and surfaces are involved in a course of processes into desired material formats. To date, the SF conformations have been studied mainly in solution which can be summarized as following: (i) random coil, which tends to occur at low concentrations of SF; (ii) silk I, which is the meta stable conformation with repeated β-turns forming at high concentrations in the absence of shear; (iii) silk II, which corresponds to the antiparallel β-sheet structure forming in the presence of shear;43–45 and (iv) silk III, which tends to form at the water-air interface.46 Prior


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mechanistic reports have proposed that SF protein suspensions lead to the formation of irregularlysized micellar structures in the size range of 100-200 nm to minimize unfavorable energy associated with the contact of hydrophobic groups and water and to maximize favorable energy due to hydration of hydrophilic groups.21 At higher SF concentrations and lower water content in solution, it has been suggested that the spacing between micelles decreases, thereby amplifying the inter-micellar interactions to result in coalescence and the formation of globules.47 The present study aims to provide answers on many fundamental questions on the intra- and inter-molecular interactions of SF protein polymers confined between two negatively charged mica surfaces under a couple of experimental conditions that can distinctly mimic processing steps involving normal compression in the absence and presence of lateral shear. For this purpose, we used the surface force apparatus (SFA) to determine adsorption dynamics and configurations (nanolayer structuring) of SF protein polymers on surfaces and discriminate their assembly steps responding to different degrees of confinement. Mechanical aspects of research have been carried out by Atomic Force Microscopy (AFM) on the SF assembled materials that were produced by a couple of simulated processing steps. We anticipate that the insights obtained from this study will provide comprehensive understanding on how the interactions arising from SF protein polymers by themselves as well as against surfaces in contact govern structural organizations and mechanical properties of SF assembled architectures, of which relation has not been well explored. EXPERIMENTAL SECTION Materials. Bombyx mori silkworm cocoons were purchased from Aurora Silk (Portland, OR). Salts utilized for the disassembly of silk fibroin fibers were sodium carbonate (99.5%, Sigma Aldrich, St Louis, MO), lithium bromide (99%, Sigma Aldrich, St Louis, MO), and silver nitrate (99%, Sigma Aldrich, St Louis, MO), all of which were used as received. Muscovite ruby mica



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sheets were obtained from Axim Mica (Grade 1, V1, Robbinsville, NJ). Unless otherwise stated, throughout the text, water refers to milli-Q water with a resistivity of 18.2 MΩ.cm. Preparation and Characterization of Silk Protein Solution. Silk fibroin protein was extracted from Bombyx mori silkworm cocoons according to previously published protocol48. Briefly, each 5 g of cocoon was boiled (degummed) in 2 L of 0.02 M sodium carbonate water solution for 30 minutes to remove Sericin gum coating. After removing excess sodium carbonate and complete drying, every 1 g of degummed silk was dissolved in 4 mL of 9.3 M lithium bromide solution at 60 oC within a typical duration of 4 hours. The solution was then transferred into 15,000 cut-off dialysis bags (Spectrum, Rancho Dominguez, CA) and dialyzed for 48 hours to remove lithium bromide salts from silk protein polymer solution. Dialysis medium was changed six times and the final medium was checked with 0.1 M silver nitrate solution to ensure a complete removal of lithium bromide salts (Ksp = 7.7 ×10-13 mol2L-6, no precipitation indicates c[Br-] < 7.7 × 10-12 M). Sample Preparation prior to Force Measurements by Surface Forces Apparatus (SFA). Molecularly-smooth mica surfaces were chosen for SFA studies and analysis because of atomically smooth nature, optical transparency, chemical inertness, and physical robustness thereof. The mica surfaces were cleaved from mica blocks (Ruby Muscovite, Robbinsville, NJ) to the thickness of 1 to 3 μm. 50 nm silver mirrors were then deposited on the back side of cleaved mica pieces while protecting the front side (actual experimental surface) by mica backing-sheets. The back-silvered mica pieces were peeled from the backing-sheets in laminar flow hood and then glued to a quartz cylindrical lens (radius of curvature, RoC = 2 cm) using thermosetting glue Epon 1004F (Hexion, Columbus, OH). The surfaces were then installed into the SFA chamber in a crosscylindrical configuration and dried with dehydrated N2 gas flow for at least 30 minutes before injecting silk protein polymer solution.


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Surface Forces Apparatus Studies. The surface forces apparatus (SFA)49 was used to measure the interactions between an aqueous suspension of silk fibroin (SF) proteins confined between atomically smooth mica substrates as a function of concentrations and surface separation (level of confinement). The fringes of equal chromatic order (FECO) was generated by the silver coating layer on the back side of mica substrates50,51 and continuously monitored and post analyzed to determine the thickness, the mean refractive index, and the contact area of the confined thin film of silk fibroin protein polymers in solution in situ. For each individual experiment, a droplet of silk fibroin solution of 0.1, 1, and 10 % was injected between two surfaces and water was subsequently injected into beakers pre-placed into the SFA chamber with syringe needle to maintain concentration of silk fibroin during measurements. The SFA chamber was completely sealed and allowed to thermally equilibrate until thermal drift of surfaces stopped. The bottom surface was then driven to approach or separate from the top surface at a constant velocity of 2 nm/s, which is slow enough to neglect hydrodynamic force. The bottom surface was attached to a double cantilever spring (spring constant, k = 200 N/m), the deflection of which upon onset of interfacial force, Δx, was recorded through surface separation change from FECO with an angstrom resolution. Interfacial force, F, was then calculated through Hook’s law (F = k × Δx), with a corresponding force resolution of 10-8 N with respect to surface separation, D. For force measurements during simultaneous approach/separation (compression/decompression) and lateral shearing, the bottom surface was attached to a piezoelectric bimorph slider, which drove the lateral movement

of

bottom

surface

at

Vlateral

(V‫)׀׀‬

=

1.2

μm/s

while

simultaneously

approaching/separating from top surface in the normal direction at Vnormal (V┴) = 2 nm/s. The normal force was measured with same mechanism described above. The lateral shearing force was



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sensed by a pair of piezoelectric spring attached to the top surface, which converted lateral spring deformation to the electric signal. Determination of Morphology and Mechanical Properties of Silk Fibroin Protein Polymer Aggregates. Both imaging and mechanical test were carried out using a Multimode Atomic Force Microscope (AFM, Bruker, Billerica, MA). After force measurement experiments, surfaces were carefully taken out from the SFA chamber and rinsed with Milli Q water drop-wise to remove unbound SF protein polymers from the surface. The surfaces were subsequently dried under mild N2 gas stream overnight. Surface morphology and nanostructures of bulk film drop-casted from SF protein polymer solution and SF assembled structures after SFA experiments were first imaged with tapping mode using a FESP probe (Bruker, tip radius = 8 nm, cantilever spring constant = 2.8 N/m) at a scan rate of 2 m/s with 512×512 resolution. For the case of degummed Bombyx mori silk fibers, microscopic imaging was carried out using a polarized light microscopy (ECLIPSE LV100POL, Nikon, Tokyo, Japan). AFM nanoindentation was performed in dry condition on degummed silk fibers, bulk film drop-casted from SF protein polymer solution and SF assembled structures after SFA experiments. The indentation experiments were performed in force volume mode using a diamond like carbon coated probe (TAP190DLC, NanoandMore USA, tip radius = 20 ± 3 nm, cantilever spring constant = 34.7 ± 3.8 N/m). Indentation depths were controlled indirectly through cantilever deflection threshold to be roughly around 10% of total film thickness to avoid substrate effects.52 For each type of sample, a minimum of 60 force curves were collected and fitted by a couple of contact models to estimate respective elastic moduli. For degummed silk fibers and drop casted silk films, the Sneddon model was applied, given that the surface roughness was much rougher than the tip radius and the samples were several micrometers (up to 10-20 micrometers) in thickness:53


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F 2



EY tan( ), 2 (1  v 2 )

(1)

where, F is the loading force, EY is the Young’s modulus of the sample, ν is the Poisson’s ratio (νsilk = 0.3), α is the opening angle of the cone, and δ is the indentation depth. Considering the raw output of force volume mode is the relation between absolute cantilever deflection, d, and absolute vertical position of sample surface (driven by piezoelectric crystal), z, the above equation can be modified into the format:54 z  zo  d  do 

k d  do , E   tan( )   Y 2  1  v 2 

 

(2)

where zo and do are initial absolute values of cantilever deflection and vertical position of sample surface, respectively. do can be read directly from baseline of force curves and zo (initial contact position) was set as fitting parameter along with EY. For the case of SF assembled structures obtained from the SFA experiments, the sample thickness ranged from a few nanometers to several micrometers and thus, the Hertzian contact model was used for force curve fitting. The classic Hertzian contact model addresses the situation where an elastic sphere indents an elastic half substrate, where the relationship between applied force and indentation depth can be expressed as:55 F

4EY R 3/2  . 3(1  v 2 )

(3)

Equation 3 can be further linearized to the following more convenient form:56 F

2/3

 4E R  C Y 2   3(1  v ) 

2/3

D,

(4)



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where, D is tip-sample separation (distance from tip to maximum indentation depth) which can be directly provided from force volume mode measurement. C is the constant generated during the conversion process of  to D. RESULTS AND DISCUSSION Interactions of silk fibroin proteins under nanoconfinement. Figure 3 shows the normal force F(D) as a function of the separation distance D between two mica surfaces in SF protein polymer solutions in the absence of shearing motions of which experimental set-up is shown in Figure 2a. As shown in Figure 3a (Csilk = 10%), purely repulsive normal forces (no adhesion) were measured without hysteresis between approach and separation and no to little changes were observed in force curves while repeating approach-separation cycles (For Csilk = 0.1 and 1.0%, see Figure S1). In addition, almost no increase on hard-wall distance, dHW, was observed as repeating approachseparation cycles for all concentrations of SF protein solutions (Figures 3a, S1, and S3). The follow-up analysis further indicates that the reflective index of SF protein polymer covered surfaces at dHW, n(dHW), stays almost constant for the following three consecutive force runs (1.426±0.003, 1.428±0.0030.006, 1.459±0.007 for 0.1, 1.0, and 10 % solutions respectively) as does surface concentration (which is proportional to n(dHW)2 and dHW). This set of observation suggests that the adsorption process of SF protein polymers on the mica surface was quickly terminated and almost no further adsorption occurred during consecutive force runs once the surface area was saturated (fully covered) by SF protein polymers. While the onsets of long-range repulsion were found to start at different separation distances of D 100, 125, and 175 nm in 0.1 %, 1.0 %, and 10% SF protein polymer solutions in pure water (Csilk) as shown in Figures 3a-d and S1, the measured exponential decay lengths in a long-range distance regime were similarly ranged between 20-35 nm irrespective of concentration, Csilk. Such a similar decay length of the long-


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range interaction force profiles are well below the hydrodynamic radius of the SF micelles (~100 nm),21 leading to the conclusions that the SF micelles are not present under given experimental conditions and/or at least not in the midway of confining two mica surfaces across the SF protein polymer solution. If these forces were entirely of electrostatic origin, such experimental decay length should be equivalent to the Debye length which indicates how diffused or compact the electrostatic double layer is. For the aqueous system containing charged polymers, the effective Debye length (eff-1) should be computed considering both ionic concentrations in solution and electrostatic charges from polymers.57 The eff-1 values were estimated to be about 414 nm, 131 nm, and 42 nm for 0.1 %, 1.0 %, and 10% SF protein polymer solutions in pure water, respectively assuming that the maximum charge density per SF protein polymer chain was achieved at the pH range of 6-8 in pure water. These eff-1 values are distinctively different from exponential decay lengths experimentally observed (20-35 nm), confirming that the long-range repulsive interactions are of entropic (steric) origin rather than being electrostatic. Given the chemical structure and amino acid sequences of SF protein polymer,25 the origin of such long-range repulsive forces can be dictated by the Alexander-de Gennes (AdG) model considering osmotic repulsion and elastic stretching effects:58,59 F 16 kTB Li  R 35 i3

  2 L 5/4  D 7/4  7  i   5    12 ,   D    2Li 

(5)

where F/R is the normalized force with the units of energy per unit area according to the Derjaguin approximation,60,61 Li is the equilibrium brush length, i is the average spacing between close neighbor chains (grafting density), D is the separation distance, kB is the Boltzmann constant, and T is temperature. The subscript i can be either l, x or s, x that represents long- or short-range interaction force regimes, respectively where “x” stands for % concentration of SF protein polymer



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solution investigated. The analysis of long- and short-range regimes with the AdG model allowed us to estimate Li and i at different concentrations as fitted values that are shown in Figures 3b-d and summarized in Table 1. All the Li values were below the effective chain length of SF protein polymer with possible intramolecular interactions (Leff), which is about 160 nm.47 For 0.1 and 1.0 % concentrations, no significant difference was observed on the Li at the long-range regime (Ll,0.1 and Ll,1.0), which was in the range of 72-74 nm. The equilibrium length of 72-74 nm is about half of Leff, indicating that SF protein polymers are adsorbed on mica surfaces with a similar loop configuration for 0.1 and 1.0 % concentrations but with different surface coverages (l, 1.0 > l, 0.1). For the 10% SF protein polymer solution, the Ll,10 was 109 nm, which is closer to Leff, suggesting that the polymer chains are mostly extended with a tail configuration. In the case of short-range regime (i.e. D < 40 nm with further compression and confinement), both fitted Ls, x and s, x values became much smaller compared to the values in long-range regime and Ls, x showed a stronger dependence on concentrations. This result indicates that freely floating (non-adsorbed) polymer chains in solution would be mostly depleted from the gap between SF polymer-covered two mica surfaces rather than further participating on adsorption events as the confinement continues, giving rise to two distinct conformations of SF protein polymers according to different levels of confinements (in the long- or short-range regime). We envision that the SF protein polymer chains at the surface undergo a conformational transition from loop/tail configurations with less anchoring points to multiple loops/train configurations with more anchoring points as depicted in Figure 4a. The transition points where the SF protein polymer chains make such conformational changes are likely to coincide with the transection regions where the two AdG fitted lines meet displaying deviations from experimental data, i.e. upper bound in short-range distance regime and lower bound in long-range distance regime and thus, we have


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limited access to accurately deduce molecular information. Near the hard-walls (within the shortrange regime), the fitted Ls, 0.1 and s, 0.1 values based on Equation 1 appear to be minimum for the Csilk = 0.1%, likely possessing the most number of train conformation. The equilibrium brush length in 10 % silk protein polymer solution, Ls,

10,

in short-range regime increased by

approximately 26% compared to the Csilk = 1.0% case (Ls, 1.0 = 17.3 ± 0.8 nm) while their respective grafting densities are quite similar (s, 1.0 ≈ s, 10 ≈ 2 nm). This finding suggests that the surface coverages of silk protein polymers on two negatively charged mica surfaces should increase with a strong dependence on bulk concentration (Csilk) upon nanoconfinement, especially near the hardwall (See Figure 4b). The FECO optical technique51,62 was used to determine the refractive index n (D) of the aqueous medium in the gap, which can be subsequently used to estimate the surface coverage of polymer chains adsorbed on mica surfaces. Given that n2(D) is a linear function of the volume fraction y of each component y,63 n2(D) may be given by wnw2 + SnS2 where w + S=1 and subscripts w and S denote water and silk protein polymer, respectively. Therefore, the volume fraction of silk, S, can be readily obtained from the following expression by experimentally measuring n2(D) and adopting that the refractive indexes of water and silk protein polymers are 1.33 and 1.4764 respectively: S 

n2 ( D)  nw2 . nS2  nw2

(6)

Finally, the surface coverage of SF protein polymers confined between two mica surfaces, silk,x, can be estimated from an expression of 1/2silkDS65 where “x” again stands for % concentration of SF protein polymer solution and S the density of silk protein polymer (1.31 g/cm3).32 The surface concentration of silk,x and the corresponding surface number density of silk,x indeed increased according to bulk concentration (x or Csilk) as shown in Figure 4b. All of these



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quantitative analysis on Li, i, silk,x, and silk,x suggest the followings: (i) the kinetic adsorption process was quite fast and majority of adsorption events were complete soon after negatively charged mica surfaces were exposed to amphoteric SF protein polymers in pure water, determining ultimate values of silk, x and silk, x within a short time period; (ii) the free SF protein polymers were most likely all squeezed out upon compression (confinement) in the absence of shearing motion; and (iii) the adsorbed silk protein polymers undergo conformational changes to accommodate increased normal pressure (up to ~ 47 atm) on approach as increasing the levels of nanoconfinement. Interactions of silk fibroin proteins under simultaneous shear and nanoconfinement. To investigate if the addition of shear action aside from the compressive normal force can also alter the dynamics and aggregation processes of SF fibroin protein polymers in pure water, we designed and performed dynamic force measurements using the SFA (Figure 2b). In this experimental setup, the normal forces were obtained while two surfaces were being sheared laterally one against each other with the velocity of Vlateral = 1.2 m/s. Figure 5 shows an experimental data obtained by bringing the mica surfaces across the solution of SF protein polymers (Csilk = 10%) under simultaneous nanoconfinement and shear. The force-distance profiles remained still repulsive but displayed very unusual shape in the force-distance profile curve, showing the instabilities that can cause the confining surfaces to be separated from each other while the driving force is compressing them together. This behavior can be attributed to the fact that the formation rate of assembled structures (association rate to form secondary and tertiary building units by consuming up primary and secondary building units, respectively) is faster than the confinement speed maintained by double cantilever springs with a finite spring constant. As shown in Figure 1, SF protein polymer consists of alternative hydrophilic (orange colored solid line) and hydrophobic (green colored


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cylinder) segments of which both ends are terminated by large hydrophilic blocks with amino and carboxy termini. This amphoteric character of SF protein polymer suggests the formation of micellar structures (secondary building units) as may be described as a primary assembly process (Figure 1b). Initially, such intervening hydrophilic internal blocks make the polymer chain more extended (less unfolded) in water, keeping it hydrated. As the confinement proceeds with shearing, the system gradually reaches at the critical micellar concentration (CMC) and the sliding motion may serve as an additional energetic trigger to accelerate a primary assembly process (Region I in Figures 5a and b). Under further compression and simultaneous shearing, the concentration of SF micelles increases further, leading to the formation of globular shaped aggregations through a secondary assembly process (Figure 1b) at a critical distance D* (Figures 5a and b). Once the secondary assembly process begins, the reaction rate to form assembled structures appears to be fast enough to resist mechanical compression (at a rate of 2 nm/min), giving rise to separation distance increased in Region II as shown in Figures 5a and b. Once such a reaction rate reaches at the equilibrium or slows down by consuming out all the available constituent building units (e.g. SF micelles), the compression energy comes back, effectively confining globular shaped aggregations, and thereby causing a sharp increase in F/R as decreasing in D (Region III in Figures 5a and b). The size of aggregations grew up as repeating approach-separation cycles probably by aggregations being fused each other and/or by replenishing the gap confined by two mica surfaces with SF protein polymers, spherical micelles, and globular aggregates (Figures 5c and S3). We found that the unique features on force-distance profiles under compression and simultaneous shear were more strongly depended on concentrations. First, the hard-wall distances under shear and simultaneous compression were significantly larger (Figures 6 and S4) than those under compression only (Figures 3 and S1); such an increase continues as increasing approach-



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separation cycles and becomes much dramatic at higher concentrations. The primary and secondary building units are replenished upon separation for the following consecutive approach process, leading to the formation of higher levels of assembled structures (spherical micelles and/or globular shaped aggregations) and, thus increasing hard-wall distances. Second, the hysteresis between approach and separation became much significant at higher concentrations (Figures 6 and S4), due to the continued formation of assembled/aggregated structures upon successive confinements. Third, the repelling distance of the instability (D*) increased as increasing concentrations; D* was about 65 nm, 120 nm, and 200 nm for 0.1 %, 1%, and 10 %, respectively (Figures 6b-d). This increase in D* as increasing Csilk confirms that the hierarchical assembly process strongly depends on the concentration of SF protein polymers in solution trapped between two mica surfaces (Csilk, confined) that is presumably proportional to but higher than the original bulk concentration (Csilk). The fundamental mechanism behind such an assembly process may be described by the equation of K = Ka/Kd = exp[-N(oN – o1)/kBT],66 where K is the equilibrium constant (ratio of two reaction rates of association (Ka, e.g. Kp and Ks in Figure 1b) and dissociation (Kd, e.g. K-p and K-s in Figure 1b), oN and o1 the mean interaction free energies per molecule in aggregates of aggregation number N and in isolated molecules or primary building units (e.g. SF protein polymers) respectively.66,67 Using the law of mass action (e.g. rate of association = KaX1N), the definition of K can be written in the following form of XN = N {X1exp[(o1 – oN)/ kBT]}N, where XN and X1 are the concentrations of molecules in aggregates of aggregation number N and in isolated molecules or primary building units (SF protein polymers), respectively. The larger Csilk (interchangeably written as X1 in solution), therefore, will favor the association (assembly) processes, leading to the formations of spherical micelles as well as globular shaped aggregations at larger separations as experimentally observed in Figures 6b-d. Equivalently, this


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analysis explains the increasing trend in D* as increasing Csilk, suggesting that the formation of higher levels of aggregations (SF spherical micelles and/or globular shaped aggregations) can be triggered at the critical value of Csilk, confined. The larger D* observed at higher Csilk, therefore, indicates the more number of SF micelles (larger Cmicelles and therefore, XN(micelles)) are formed in a period of Region I. Since the SF micelles can be consumed as secondary building units to generate globular shaped aggregates (tertiary building units) as shown in Figures 1b and 5a, the larger Cmicelles can produce the more number of globular shaped aggregates (larger Cglobules), leading to the longer reverse pathway (increasing in D) from the start (D*) and end points of Region II as observed at higher Csilk (Figure 6d). Subsequently, the critical compressive energy (E = F/2R by Derjaguin approximation) to cause a turnaround in force curves from Region II to Region III has been also increased from about 4.0 to 4.8 mJ/m2 as increasing Csilk. Mechanical Properties of Assembled Structures of Silk Protein Polymers Processed under Different Conditions. In order to further establish experimental evidences on how in situ processing conditions such as compression only and compression with simultaneous shear can maneuver the mechanical properties of SF assembled structures, the respective SFA disks were collected after force measurements for the follow-up AFM nanoindentation experiments. The Figure S5 shows sets of applied force (F) vs. indentation depth (δ) curves obtained from sufficiently large numbers of indentation locations (at least 60), and the corresponding elastic modulus EY for degummed B. mori silk fibers, drop casted silk films and SF assembled structures obtained from the SFA experiments was calculated from Equations 1-4. The degummed B. mori silk fibers and drop casted SF films were prepared and their mechanical properties were compared with those of SF aggregates processed under normal compression without and with lateral shear through SFA force measurements. The degummed B. mori silk fibers can be obtained by removal



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of sericin (hydrophilic type of “glue-like” protein) of which structural characteristics can be defined as semi-crystalline fibrils (more orderly -sheet structures by hydrogen bonds) longitudinally embedded in an amorphous matrix (predominantly -helix and random coil structures). While such degummed B. mori silk fibers can be also used in some practical purposes such as sutures, the regenerated form of SF protein solution has been much more commonly used for a variety of practical applications covering from biomedical to electrical to optical demands.17,68–70 In that, the drop casted SF films were also fabricated from SF protein polymer solutions by further disassembling degummed B. mori silk fibers in highly concentrated ionic solution (e.g. 9.3 M LiBr) at elevated temperature. Figure 7 delivers several important notes: First, an averaged elastic modulus Eav of degummed B. mori silk fibers (Eav, fiber) was approximately three times higher than that of drop casted SF film samples (Eav, film), stressing an importance of hierarchical structures that nature has been offering in silk (Figure 7a). While degummed B. mori silk fibers can still preserve -sheet nanocrystals reinforced structures, the drop casted SF films lose such features after being disassembled in high salts, leading to decrease Eav, film to one-third of Eav,

fiber.

Second, the averaged elastic modulus Eav of assembled SF nanostructures appears

insensitive to the concentration of silk in solution, Csilk (Figures 7b and c) but quite susceptible to the ways of how to process SF protein polymers in solution (Figure 7d). The elastic modulus values of SF assembled structures processed under normal compressions (Eav, SF(compression)) was quantified about six times less than those produced by both normal compression and lateral shear (Eav, SF(compression and shear)).

In the absence of shearing motion, SF protein polymers first adsorb on the

surfaces and change their conformations according to the levels of confinement which are well fitted according to the AdG theory with comparable effective chain lengths and respective hardwall distances (11.9 nm for Csilk= 0.1%, 17.4 nm for Csilk= 1.0%, and 22.8 nm for Csilk= 10.0%) as


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shown in Figures 3 and S1. The spherical micellar structures with a diameter of approximately 5070 nm shown in the inset of Figure 7b, therefore, are somewhat less expected, especially given that the decay length (25-30 nm) of the long-range force profiles does not scale with the size of spherical micelles observed here. Thus, these spherical micelles were likely to be formed inevitably during AFM sample preparation steps due to the amphiphilic nature of SF protein polymers (See Figure 1) when the SF protein polymers covered mica surfaces were secured after SFA experiments and dried prior to AFM experiments. Interestingly, the elastic modulus values obtained from the areas with depleted micelles are quite close to those of SF spherical micelles, all of which are well below the elastic modulus values of the SF aggregates assembled under simultaneous compression and shear (Figure 7d). Once two surfaces begin to be slid against each other across respective SF protein polymer solutions, shear motions force spherical micelles to get closer, finally leading to the formation of globular shaped aggregates with diameters in the micrometer range up to several tens of m (See insets of Figure 7c). This set of data clearly indicates that the elastic modulus of surface-mediated SF nanostructures is subject to be changed during a course of assembly stage (step) that can be predominantly influenced by different processing pathways but has little to no correlation with the number of constituent molecules present in solution (Csilk). The shearing motion under normal compression could rearrange -sheet nanocrystals in a way of orienting them according to the shearing direction analogously to a twodimensional nematic phase, giving rise to the substantial increase in Eav of SF assembled nanostructures compared to no shear case. The existence of nematic liquid crystal textures in the fibroin was indeed directly proven by disclosing changes in optical properties such as birefringence and specific retardation in a region of the spinning duct,71 suggesting a parallel with our experimental observation and interpretation pertaining to shear-induced molecular alignment



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which was confirmed by the increase of mechanical properties although both studies deal with different length scales experimentally emulated. Once a certain number of  -sheet nanocrystals has been accumulated in the SF aggregates and aligned upon compression and shear, their crystallinity could remain almost constant with no significant changes in the percolation structure (aligned -sheet nanocrystals embedded in amorphous matrix), resulting in similar mechanical properties of SF aggregates independently of the concentration of silk in solution as observed. CONCLUSIONS In this study, we directly measured surface forces to characterize the structural and dynamic properties of SF protein polymers confined between two mica surfaces. The surface force measurements on SF protein polymers with different concentrations suggest that the adsorption behaviors, interaction dynamics, and assembled nanostructures of SF protein polymers have been dramatically influenced by the action of shear under normal confinement (compression). Under normal compression only, both long-range and short-range repulsive forces were confirmed to be of steric origin where the architecture of the SF protein polymer chains at the solid-liquid interface was varying with the concentration of SF protein polymers in solution as well as the relative length scale of the confined gap. The chain conformations of SF protein polymers underwent drastic changes from tail to train configurations with loops of the adsorbed chains as the degrees of confinement became comparable with relevant characteristic length scales of SF protein polymers. The refractive index analysis complementing the AdG fitting results demonstrates that the surface coverages of SF protein polymers on mica are determined in the early stage of adsorption process in long-range distance regime and the unbound (unabsorbed) SF protein polymers remained in the confined gap are depleted upon further compression in short-range distance regime. When the SF protein polymers were exposed to the confined geometry under the action of shear, hierarchical


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assembly processes of SF protein polymers started, completely altering corresponding interfacial dynamics. Peculiar features observed in interaction force profiles under simultaneous normal compression and lateral shear could be resolved into sequenced assembly steps of SF protein polymers to form spherical micelles and then globular shaped aggregates, displaying a clear dependence on the concentration of SF protein polymers and/or micelles in a confined space (contact zone). We postulate that different stress-stiffening responses of SF protein polymers to external compressive and shear forces alter the elastic properties of SF-based materials, which have not been well understood by now. These findings could be also related to the molecular extension mechanism of B. mori silkworm silk, accompanied by increased shearing forces along the anterior division and the spinneret. For instance, SF solution is processed through the spinneret into air, which intrinsically involves a narrow duct of decreasing diameters from millimeters to micrometer i.e. introducing confinement effects and shearing forces. This tapering has a crucial role to induce the alignment of  -sheet nanocrystals in a way to enhance mechanical strengths of silk fibers, which has been similarly identified from our work despite the different length scales, i.e. SFA is operated from nanometer to micrometer scales. From a practical perspective, converting SF protein polymers into functional materials and devices often requires multiple processing steps at different length scales as in the case of producing silk microneedles and micro/nanoporous scaffolds. If one designs a processing system with the different dimensions (analogues to different levels of confinement), shear stresses can be generated in a positive way to produce the stronger and more robust SF functional materials based on our observations. We believe that our experimental observation and fundamental interpretation at the molecular scale can also answer critical questions pertaining to the dynamics and assembly behaviors of other protein-based materials (e.g. collagen, mucin) and membrane proteins (e.g. myelin basic protein, lung surfactant



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protein) in aqueous medium. Overall, this work can bring new insights not only to the fabrication point of view in biomaterials but also to the mechanistic principles of hierarchical structures in nature. We anticipate that these insights enable a better control on the properties of SF proteinbased materials, which have been used as multifunctional material platforms in numerous biomedical applications and devices. ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Consecutive interaction force profiles and changes in hard-wall distances with corresponding FECO and top-view microscopic images in the absence and presence of shear and AFM nanoindentation force profiles (PDF); Videos on the dynamic formation of SF assembled aggregations under simultaneous confinement and shear (AVI). AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ORCID Younjin Min: 0000-0002-1156-3373 Author Contributions Y.Z., Y.Z. and Y.M. conceived the concept. Y.M. initiated the SFA effort and supervised the study. Y.Z. and S.W. prepared the mica disks and silk fibroin protein solutions. Y.Z., Y.Z. S.W, and Y.H. implemented SFA measurements. Y.Z. performed AFM nanoindentation. Y.Z. and Y.M. analyzed the data. All authors were involved in manuscript writing. All authors have given approval to the final version of the manuscript.


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Science Foundation (CBET Award # 1511626) and the American Chemistry Society Petroleum Research Fund. We thank Dr. Yu Zhu for allowing us to use the thermal evaporator for mica silvering. REFERENCES (1)

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FIGURES

a

b

Figure 1. (a) Pictorial description showing steps to produce SF protein polymers in aqueous medium with different concentrations that have been used for various purposes ranging from microneedles, fibers/mats, and films. (b) Illustrations of SF protein polymers with hydrophilic and hydrophobic blocks which can serve as primary building units to form SF spherical micelles as secondary building units. The spherical micelles can be subsequently consumed for the next hierarchical structures such as globular aggregations (tertiary building units). The cartoons are adapted by permission from Springer Nature: ref. 21. Copyright 2003 Nature Publishing Group. http://www.nature.com/nature/ Kp and Ks represent the reaction constants of association while K-p and K-s are reaction constants of dissociation through (dis)assembly processes using primary and secondary building units, respectively.



a

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b

Figure 2. Shematic illubstrations of the SFA setups used to measure the interactions across the SF protein polymer solutions confined between back-silvered mica surfaces (a) in the absence of and (b) in the presence of lateral sliding motions. Both experimental set-ups are designed for simulating confined situations without and with shear which can be directly correlated to some of representative post processing steps to fabricate various types of SF-based materials as shown in Figure 1b.


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a

b

c

d

Figure 3. (a) Consecutive force runs measured across SF protein polymers in solution (Csilk = 10%) confined between two mica surfaces by repeating approach-separation (compression and decompression) cycles. Normal interaction forces between two mica surfaces across SF protein polymer solutions at the concentrations of (b) 0.1%, (c) 1.0%, and (d) 10% in the absence of lateral sliding motion. All the interaction forces are long ranged with the steric origin at a fixed confinement speed of V┴ = 2 nm/s. Solid symbols indicate approaches and grey solid lines represent the best fits of the data (solid symbols) to the Alexander-de Gennes model, Equation 5, where the values of the polymer layer thicknesses Ll,x and Ls,x and the distances between polymer anchoring points l,x and s,x were obtained from long-range and short-range interaction forces, respectively where x refers to the % concentration of SF protein polymer solution in bulk, Csilk. The short-range distance regimes in (b), (c), and (d) are magnified and given in Figure S2 in Supporting Information.



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a

b

Figure 4. (a) Schematic representations describing the conformational changes of the SF protein polymers at the long- and short-range regimes. (b) Hard wall distances, refractive indexes, surface concentrations, and corresponding surface density values of SF protein polymers confined between two mica surfaces at hard-wall distances, dHW.


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a

b

c

Figure 5. (a) Illustration of four different regimes emerging during the processing of SF suspension under simultaneous confinement and shear. In Region I, the degree of confinement is small enough that the existence of shear is likely decoupled from interfacial effects. The point of D* is the onset of the instability causing surfaces to recede away from each other although there is an inward driving force by the DC motor. In Region II, the micellization process further progresses and multilayers of micelles (globular shaped aggregations) are trapped between the confining surfaces. In Region III, the micellar multilayers are compressed, causing elastic repulsion. The corresponding regimes are shown in the force curve, (b) with matching labels. (c) In situ top view images showing the growth of globular shaped aggregations during repeated approach-separation cycles for Csilk = 10%. The size of globular shaped aggregations is clearly evolved as repeating approach-separation cycles. The Supporting Information, Video S1 shows a representative movie that visualizes the evolution in aggregation size of SF protein polymers responding to simultaneous confinement and shear that were captured by top view microscopy and corresponding FECO images for Csilk = 10%.



a

c

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b

d

Figure 6. (a) Consecutive force runs measured across SF protein polymers in solution (Csilk = 10%) confined between two mica surfaces by repeating approach-separation (compression and decompression) cycles in the presence of shearing motion. Normal interaction forces measured during the first approach/separation cycle (●/○) between two mica surfaces across silk protein polymer solutions at the concentrations of (b) 0.1%, (c) 1.0%, and (d) 10% in the presence of lateral sliding motions with the speed of Vlateral = 1.2 m/s.


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b

c

d

Figure 7. Elastic modulus distribution of SF protein polymer structures prepared by (a) drop casting (silk films) and degumming raw cocoons (degummed B. mori silk fibers) and SF assembled structures processed (b) under nanoconfinement only and (c) under simultaneous nanoconfinement and shear. The averaged-out elastic moduli shown in b and c display a clear contrast in the mechanical properties of SF assembled structures depending on their processing histories as replotted in (d).



Page 36 of 37

TABLE

Table 1 Equilibrium brush length Li and average spacing between close neighbor chains i from the AdG model, Equation 5 used to fit the normal force experimental data obtained from silk protein polymers in solution confined between two mica surfaces. Silk Concentration (Csilk, %)

Long Range

Short Range

Ll,x (nm)

σl,x (nm)

Ls,x (nm)

σs,x (nm)

0.1

74.4 ± 2.7

16.3 ± 0.5

9.9 ± 0.5

1.5 ± 0.1

1.0

72.5 ± 0.7

12.0 ± 0.1

17.3 ± 0.8

2.2 ± 0.1

10

109. 1 ± 1.6

14.3 ± 0.2

21.8 ± 0.6

2.3 ± 0.1


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