Nanocrystal Width Controls Fibrinogen Orientation and As-sembly

1 Chair of Materials Science (CMS), Otto Schott Institute of Materials Research (OSIM), Friedrich Schiller University Jena,. Löbdergraben 32, 07743 J...
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Nanocrystal Width Controls Fibrinogen Orientation and As-sembly Kinetics on Poly(butene-1) Surfaces Xiaoyuan Zhang, Christian Helbing, Matthias Michael Lothar Arras, Klaus D. Jandt, and Izabela Firkowska-Boden Langmuir, Just Accepted Manuscript • Publication Date (Web): 09 Jun 2017 Downloaded from http://pubs.acs.org on June 10, 2017

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Langmuir

Nanocrystal Width Controls Fibrinogen Orientation and Assembly Kinetics on Poly(butene-1) Surfaces Xiaoyuan Zhang,1 Christian Helbing,1 Matthias M. L. Arras,1,2 Klaus D. Jandt*,1,3 and Izabela Firkowska-Boden1 1

Chair of Materials Science (CMS), Otto Schott Institute of Materials Research (OSIM), Friedrich Schiller University Jena, Löbdergraben 32, 07743 Jena, Germany 2 Oak Ridge National Lab, Biology and Soft Matter Division, Oak Ridge, TN 37831, USA 3 Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany Supporting Information Placeholder ABSTRACT: From the view of biomedical relevance, it is known that a specific arrangement of surface-immobilized human plasma fibrinogen (HPF) molecules is required to retain their biological functionality. Here, we demonstrate a topographical effect of chemically identical isotactic poly(butene-1) (iPB-1) semicrystalline nanostructures on the adsorption behavior, i.e., conformation change and orientation of HPF molecules. Using the distinct crystallization of iPB-1 under different shear conditions, polymer thin films consisting of needlelike crystals (NLCs) or shish-kebab crystals (SKCs) having lateral dimension, i.e., width, smaller than or comparable to the HPF major axis, respectively, were fabricated. The protein adsorption kinetic studies by quartz crystal microbalance with dissipation (QCM-D) revealed surface-dependent packing density and assembly configuration of HPF. High-resolution imaging disclosed a “side-on” protein adsorption and anisotropic network formation on the NLCs. With a two-fold orientation analysis performed at both “single” protein and multiprotein levels, we quantitatively proved the preferential alignment of adsorbed HPF molecules with respect to the axial direction of the NLCs. Remarkably, the iPB-1 surface with SKCs perturbed the “end-to-end” protein-protein interactions, and thus hindered the network formation. The distinguished adsorption behavior of HPF molecules on iPB- 1 surfaces is explained by the physical effect of crystal width, which is additionally supported by the synergistic effect of crystal curvature and aspect ratio. Our studies provide fundamental insight into purely topography-controlled self-assembly of HPF molecules, which might be further exploited in creating topographically defined implant surfaces for preventing protein aggregation related disorders.

INTRODUCTION Protein adsorption on implant surfaces is a key biological response to mediate cell attachment and subsequent extracellular matrix (ECM) formation.1 Among all the proteins contacting implants, human plasma fibrinogen (HPF), the most abundant adhesive plasma protein, participates in the initiation of immune reactions by activating inflammatory cells.2 HPF is also essential for blood clotting,3 wound healing,4 cellular responses,5 as well as an indicator for tumor cell circulation6. Conformational changes in HPF induced by adsorption on the material surface may lead to changes in protein activity, which could dramatically affect biocompatibility of implant material. Accordingly, interactions of HPF with a wide range of surfaces have been studied both experimentally and theoretically.7-14 In general, on hydrophobic atomically flat surfaces, HPF adsorbs with high affinity and undergoes significant structural alterations.14 For instance, HPF molecules flatten over time on highly ordered pyrolytic graphite (HOPG)9 and form a network structure.15 In contrast, on hydrophilic mica, HPF shows little if any adsorption and preserves its native-state secondary structure.9 Similar behavior has been reported for topographically flat polymeric surfaces, where HPF rather adsorbed preferentially on hydrophobic polystyrene (PS), than on a hydrophilic poly(methyl methacrylate) (PMMA) surface.10 According to experimental studies reported in the literature, the HPF conformation but also orientation can be additionally mediated by nanotopographies present on the semicrystalline polymeric surfaces. Recent findings by Helbing et al.16 established that the surface of a polyethylene single crystal (PE-SC), with topographical features one order of magnitude smaller than the smallest dimension of the protein, can trigger a preferential alignment of HPF molecules. On the supramolecular scale, the influence of the crystal lamellae arrangement, on aggregation behavior of HPF but also on anisotropic network formation was exemplified on melt-

drawn (MD) ultrahigh molecular weight polyethylene (UHMWPE).12 In addition to protein conformational change, the nanostructure topography of the polymeric surface was found to influence the diffusion direction of the protein. Single-molecule tracking studies by Kastantin et al. 13 revealed lateral HPF diffusion mediated by well-oriented crystalline lamellae present on nanostructured melt-drawn high-density polyethylene (HDPE) films. The overall picture that has emerged from these studies is that semicrystalline polymer films can be used as versatile model surfaces to provide unique information on protein interactions with nanoscale building blocks. Such knowledge is of great importance as the polymers are increasingly employed as promising coating layers of existing biomaterials to improve the biocompatibility of metals and ceramics.17,18 So far, it is known that adsorption and assembly nature of the proteins may be driven by the nanoscale crystal arrangement in the polymeric surface. However, no attempts were made to investigate the effect of crystal width on HPF adsorption and assembly configuration. Controlled alterations in the lateral dimension of oriented polymer crystals may enable fine-tuning of the protein-surface interaction, which may be further exploited to prevent, for instance, unwanted blood clot formation on implant materials. In view of these considerations, we have chosen to examine the effect of the lateral dimension of crystalline polymeric nanostructures on the adsorption behavior, e.g., conformation and orientation of HPF molecules. Our approach to create topographically different, yet chemically identical, polymeric surfaces employs polymer crystallization to induces crystalline phases that impart nanoscale topography to the film.19 The polymer used in this current work is isotactic poly(butene-1) (iPB-1). This semicrystalline polymer, on which the nature of protein adsorption and assembly has not yet been reported, shows a distinct crystallization behavior under shear force conditions. Namely, depending on the

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fabrication technique, i.e., melt drawing or spin coating, thin films with (i) highly-oriented needle-like crystals (NLC)20 having a lateral repeating unit smaller than the length of the HPF major axis and (ii) shish-kebab crystals (SKC)21 with a lateral size comparable to the HPF major axis length can be fabricated. Apart from identical chemistry, both nanotopographies have an equivalent anisotropy on the molecular and supramolecular levels. Hence, we hypothesize that the difference in surface adsorption behavior of HPF will be purely induced by the alterations in the lateral physical dimension of the polymer crystals. Performing adsorption kinetic measurements with quartz crystal microbalance with dissipation (QCM-D), we find the surface-dependent HPF packing density and indications towards its preferred assembly configuration and geometry. The protein concentration-dependent atomic force microscopy (AFM) investigations complement the information on conformation and network geometry of HPF molecules with respect to laterally different iPB-1 crystals. In addition, image and corresponding statistical analysis of the HPF network structure unambiguously confirm balanced proteinprotein and protein-NLCs surface interactions, which facilitate the rearrangement of HPF molecules into a network assembly with a preferred orientation. The results of this study imply that the physical dimensions of the underlying nanoscale polymeric surface can be used to control the protein surface density, adsorption configuration and network/protein orientation. Thus, this work is not only important for the design of new types of materials for implant applications, but also for an improved understanding of the HPF adsorption mechanisms and to establish new strategies for preventing protein aggregation related disorders. EXPERIMENTAL SECTION Preparation of Nanostructured Polymer Thin Films. Ultraflat, highly-oriented melt-drawn iPB-1 thin films with NLCs were produced by the melt-drawing technique as follows: iPB-1 pellets (molecular weight Mw = 570 kg/mol, Sigma-Aldrich, Schnelldorf, Germany, semicrystalline isostatic thermoplastic) were dissolved in p-xylene (synthesis grade, Merck KGaA, Darmstadt, Germany) at a concentration of 1 wt% and heated to approximately 120 °C. The iPB-1 solution was poured onto a glass slide mounted on a heating plate kept at a temperature of approximately 125 °C. The drawing temperature was slightly higher than the usual crystallization temperature of iPB-1. Solvent remaining in the melt depresses the crystallization temperature, yielding a slightly undercooled melt. The solvent depressed crystallization temperature together with the reduction in viscosity allows to homogenously spread the polymer melts on the hot plate, a prerequisite to subsequently obtain a thin film.22 After evaporation of most of the solvent, a thin iPB-1 film was drawn off the plate at a drawing rate of approximately 6 cm/s by an electromechanical roller. The originally self-supporting film was fixed on a metal ring to temporarily store it for further use. Subsequently, the MD thin films were deposited on 1 cm × 1 cm glass cover slips and silicon wafer. In addition, the MD iPB-1 films were attached to QCM sensors (Q-Sense, Goteborg, Sweden) by using solvent vapor to ensure the tight bonding of the film. To obtain the SKCnanostructured surface, iPB-1 solution (1.0 wt % in p-xylene) was spin-coated on silicon wafers and QCM sensors at 2000 rpm and fully dried in a vacuum oven at 50 °C for 3 days. HPF Adsorption on Nanostructured iPB-1 Surfaces. A stock solution of HPF (Calbiochem, Merck KGaA, Darmstadt, Germany) in Dulbecco’s phosphate-buffered saline solution (PBS, with-

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out calcium chloride and magnesium chloride, pH~7.3 - 7.7) (Biochrom, Germany) was prepared with an HPF concentration of 1 mg/mL. For concentration-dependent adsorption studies, the stock solution was diluted with PBS to concentrations ranging from 10-4 to 100 mg/L. For each HPF concentration, 1 mL PBS solution of the respective HPF concentration was pipetted on the iPB-1 films and left for 30 min adsorption under quasiphysiological conditions at 37 °C.8, 9 Subsequently the samples were gently rinsed with PBS solution to remove non-adsorbed proteins. Afterwards, PBS residues were removed by rinsing the samples with Millipore water. Finally, the samples were dried in ambient atmosphere. Characterization. The iPB-1 film surfaces before and after protein adsorption were characterized by performing AFM imaging. Accordingly, the height and phase scans were recorded with a MultimodeTM III AFM interfaced with a Nanoscope IV controller (Digital Instruments, Veeco, Santa Barbara, CA, USA), operating in tapping mode at a scan rate 0.5 Hz using silicon cantilevers with a typical resonance frequency of 341 kHz and a spring constant of 42 N/m (model RTESP, Vecco, Bruker, Santa Barbara, CA, USA). X-ray photoelectron spectroscopy (XPS) analysis of pure iPB-1 films was conducted on a EA200-ESCA-system (SPECS) using nonmonochromatic Mg Kα radiation (hν = 1253.6 eV). Dynamic HPF Adsorption Measurement by QCM-D. The adsorption kinetics of HPF on the nanostructured surfaces were measured with QCM-D (Q-Sense, Goteborg, Sweden). Piezoelectric quartz crystals with a fundamental frequency of 5 MHz were purchased from Q-Sense (Goteborg, Sweden) with gold electrodes and used as purchased or coated with iPB-1 films. Before use/coating, they were immersed in piranha solution for 10 min and subsequently rinsed thoroughly with Millipore water. UVtreatment was applied as a final stage of cleaning the gold surface of the QCM sensors. The QCM sensor was installed into the QCM-D chamber (KSV instruments, Helsinki, Finland) connected to a temperature controller (Oven Industries, Inc. Mechanicsburg, PA) set to 37.0±0.1 °C. During rinsing and exchange of PBS buffers, the liquids were pumped through the chamber with a flow rate of 100 µL/min. Twenty minutes were required to obtain a stable frequency baseline. For protein adsorption, the HPF solution was pumped into the sensor with a flow rate of 20 µL/min.23 During the QCM-D measurement, the frequency shift and dissipation change were simultaneously recorded at first five overtones (n=3, 5, 7, 9, 11).24 The QCM-D frequency shift was converted to adsorbed mass using the Sauerbrey equation:25 ∆m = - Cf ∆f/n, (1) where Cf is the sensitivity factor for the crystal (56.6 Hz·µg1 ·cm2 for a 5 MHz AT-cut quartz crystal at room temperature26), ∆f is the frequency shift and n is the overtone. RESULTS AND DISCUSSION Topographically well-defined and chemically homogenous surfaces prepared from iPB-1 were first characterized by AFM. The adsorption kinetic of HPF on iPB-1 surfaces as a function of protein concentration was investigated with QCM - D. Finally, the effect of supramolecular features of the iPB-1 surfaces on the orientation and packaging geometry of HPF assemblies was visualized by AFM and quantitatively analyzed.

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Figure 1. AFM analysis of pristine iPB-1 thin film surfaces used in the HPF adsorption study. The micrographs display the characteristic polymer crystal morphologies and alignments specific to (A) iPB-1 with the NLCs and (B) iPB-1 with the SKCs. In the paired set of AFM panels shown, the left and right images correspond to the height and phase scans, respectively, of the same sample area. Enlarged phase scans shown under the paired AFM panels depict the NLC and SKC structure (dashed white grid on SKC phase scan highlights the segmented structure of the SKC). Height versus distance profiles were measured along the white lines inserted in the height scans. The line analysis shows the difference in iPB-1 surfaces᾽ nanotopography, namely height and lateral periodicity. The C(1s) XPS spectra placed above the topography line profiles show no measurable differences in the surface chemistry between NLC and SKC samples. The lateral dimensions of the NLCs and SKCs, the arrangement of the polymer chains in the crystals and their relation to the size of the single HPF molecule, with its typical trinodular shape and the hydrophobic centered E and D domains, is schematically illustrated on the bottom of the figure (in scale).

Nanostructures on iPB-1 Film Surfaces. The surface morphologies of the iPB-1 thin films used in this study are shown in Fig. 1. The characteristic crystal size and alignment specific to NLC and SKC iPB-1 surfaces can be seen in the paired set of AFM panels as well as in the height versus distance profile of each surface. The native NLCs in the iPB-1 film surface (Fig. 1A) exhibit a regular arrangement of close-packed longitudinal needles, which are aligned parallel to the drawing direction,20 indicated by the white arrow on the phase image. The NLCs protrude out of the plane with an arithmetic average roughness of 2 ± 1 nm (average local values) and with a lateral peak-to-peak distance of 27 ± 10 nm. The diameter of the tubular needles is 22 ± 8 nm. Thus, the lateral dimension of the NLCs is smaller than the major axis of an HPF molecule (the length of the dried molecule is 47.5 ± 0.2 nm),1 which is schematically illustrated in Fig. 1 (see bottom part). The simplified structure of the elongated HPF protein consists of rod-like coils connecting three spherical domains denoted D (at the two ends, red balls) and E (at the center, black

ball). The protein has a net negative surface charge (isoelectric point, pI = 5.2) in a physiological pH of 7.4.27 The surface morphology of the iPB-1 SKC reveals long striped lamellae, which are typical for the SKC nanostructure.28 It is known that shear forces lead to the formation of highly extended chain crystals (shishs) which may serve as nucleation sites for chain folded crystals (kebabs)29 (see the schematic illustration of the polymer chain arrangement in the SKC on the bottom of the Fig. 1). The SKCs protrude out of the plane with an arithmetic average roughness of 4 ± 1 nm and have a lateral peak to peak distance of 62 ± 12 nm. The half peak width of the SKCs is 50 ± 11 nm. The length of the kebab crystal segments of the SKC lamellae, highlighted by the dashed white grid on the enlarged SKC phase image in Fig. 1B, is 40 ± 10 nm. Contrary to the NLCs structure, the SKCs lateral dimension corresponds to the size of the HPF molecule’s major axis. 3

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Figure 2. HPF adsorption kinetic on nanostructured polymeric surfaces. (A) Frequency shift ∆f upon adsorption of HPF on NLCs (solid line) and SKCs (dashed line) surfaces at different protein concentrations. (B) The change in adsorbed mass as a function of HPF concentration after 120 min. The size of the symbols reflects the error bars.

HPF Adsorption Kinetics on iPB-1 Film Surfaces. To attain a better understanding of how the nanostructured surfaces modulate protein-surface interaction, the adsorption kinetic of HPF on nanostructured iPB-1 films was investigated using QCM-D. For this, the NLCs and SKCs films were directly attached to gold QCM electrode (see Experimental). Figure 2A displays the changes of the resonance frequency (∆f) upon adsorption of HPF on NLC and SKC surfaces. For all concentration levels, the addition of HPF led to a decrease in the ∆f over time, which reflects the increase in adsorbed protein mass on the surfaces. Thus, the appearance of a significant difference in ∆f between NLC and SKC structure is attributed to a different amount of adsorbed proteins. As the HPF molecules fill the surface, the adsorption process slows down, reaching the adsorption saturation (plateau). The time needed to reach surface saturation

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depended mainly on protein concentration and it occurred between 40 min and 2 h. Note, at a concentration of 1 mg/L, HPF adsorbed slowly on both NLCs and SKCs and reached no saturation within the measurement time. Interestingly, ∆f for HPF on NLCs reached a plateau value (10 mg/L and 100 mg/L) and stayed constant after approximately 50 min of exposure to the protein solution. Conversely, on SKCs, HPF seems to adsorb prolonged, which appears in a late stage small linear decrease of ∆f (see data for 100 mg/L). As common in protein adsorption, the formation of the HPF layer is mainly driven by hydrophobic and electrostatic interactions. As there is neither a difference in NLC and SKC surface hydrophobicity (difference in the measured water contact angle below 3%) nor in surface chemistry (XPS spectra revealed no measurable changes), the increase in weight as indicated by the frequency shift must be due to the distinct iPB – 1 surface nanotopographies. Possible explanations for the observed ∆f values being higher for SKCs could be (i) surface diffusion of the molecules allowing rearrangement of the randomly adsorbed proteins into a more tightly packed layer,30 (ii) increased hydration of the layer or (iii) formation of a multilayer. The latter can be validated by comparing the experimental mass with the mass of a theoretical protein complete monolayer. The mass of the adsorbed proteins can be obtained by converting the observed frequency shift to a mass-uptake by the Sauerbrey equation (see Eq. 1), while the validity of the latter depends on whether the adsorbed layer can be considered rigidly coupled to the iPB-1 modified QCM electrode (viscoelastic adsorbed layer will dampen the crystal’s oscillation). To determine the nature of the adsorbed layer, the QCM dissipation signal (∆D) has been used to calculate the ∆D/(−∆f/n) dimensional ratio, and compared to the 4×10-7 Hz-1 threshold suggested by Reviakine 31 to distinguish between viscoelastic or rigid adsorbed protein layers. For both nanostructured films, the dimensional ratio fell in the range of 3.2 – 7.6×10-9 Hz-1, and thus, the layer formed by the adsorbed HPF can be considered rigidly coupled and the Sauerbrey model was considered valid for the samples under investigation. The calculated mass-uptakes (∆m) of the HPF protein on SKCs and NLCs as a function of protein concentration are shown in Fig. 2B. While for the lowest HPF concentration, the absorbed mass is close to zero, an increase of ∆m from 200 to 1300 ng/cm2 is observed for the protein concentration in the 0.1 – 100 mg/L range. The adsorbed mass varies substantially between the different iPB1 nanotopographies. As an analogy to ∆f data, the overall adsorbed mass for the SKCs is higher compared to that of NLCs. The ∆m difference between NLCs and SKCs varies between 12 % and 25 % for 10 mg/L and 100 mg/L, respectively. These results indicate that the lateral dimensions of the iPB-1 nanostructures (nanocrystals) affect the adsorption behavior and arrangement of HPF molecules. It is worth to note that the influence of a possible variation in iPB-1 film thickness on the adsorption of protein is negligible.32 To elucidate the effect of the studied nanotopographies on HPF orientation and to distinguish between the mono- and multilayer

Table 1. Comparison between the experimentally determined mass of iPB-1 absorbed HPF and theoretical protein surface coverage based on assumed HPF orientation and size (47.5 nm).1 iPB-1 film

Experimental mass*

RSA (ng/cm2)

RSA (ng/cm2)

Total coverage in mixed mode

∆D/∆f

nanotopography

∆m (ng/cm²)

“side-on”

“end-on”

incl. 20% hydration (ng/cm2)

(10−9 cm2/ng)

NLCs

1009 ± 30 210

1570

570

SKCs

3.63

1250 ± 28

3.05

* The adsorbed mass refers to samples with the HPF buffer concentration of 100 mg/L.

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Figure 3. Analysis of HPF network assembly on NLC topography. (A) AFM height and phase scan images depict HPF adsorption behavior and network structure upon interaction with NLCs. The scale bar corresponds to 300 nm. The arrow indicates the film’s drawing direction, which is the same for all depicted images. The zoomed-in magnifications with false colored HPF assemblies (green) are shown on the right side of the left image panel (scale bar 30 nm). (B) Quantification of HPF network orientation. (Top) Overlaid height and phase image with HPF network (green) and “eyelet” structure represented by solid ellipses. (Middle) Polar plot with the orientation angle, θ, distribution of (i) “eyelets” major axis as a function of axis length (half-filled green circles) and (ii) “single” HPF molecule (black dots). (Bottom) Extracted HPF network and its color-coded orientation obtained with ImageJ.

formation, the experimental ∆m was compared to the theoretical complete HPF monolayer mass calculated by the random sequential adsorption (RSA) model.33,34 The theoretical values and experimental data from this current work are summarized in Table 1. The orientation of an ellipsoidal protein on the surface can be characterized as “side-on” or “end-on” depending on which axis, long or short, is predominantly interacting with the surface.28 Obviously, a higher protein area density in an adsorbed monolayer is possible with an “end-on” orientation. Within the RSA model “side-on” and “end-on” adsorbed HPF form a monolayer area density from 210 to 1570 ng/cm2, respectively 25,33,34 The experimentally determined amount of adsorbed proteins on iPB-1 nanostructures lies within this range (see Table 1). According to Roach et al.,34 the “end-on” orientation of HPF molecules after the initial adsorption stage would favor the adsorption of additional molecules because of increased hydrophobic interaction between HPF molecules aligned parallel to each other. Considering the data for protein concentration of 100 mg/L, the ∆m indicates that the SKCs on the iPB-1 surface, with a lateral dimension comparable to the major axis of HPF, mediate the “end-on” packing configuration of the adsorbed proteins. Assuming a combination of both orientations, the total maximum coverage would be 570 ng/cm², including 20% hydration of the protein.35 One should keep in mind that the mass value obtained via the measured fre-

quency shift of the QCM also includes water in the protein adlayer, coupled via direct hydration or water entrapped in cavities formed between and near the adsorbed proteins.36 Accordingly, little or no water is expected for a densely packed monolayer. The information about water content can be extracted from ∆D/∆f, measured at saturation.37 As shown in Tab. 1, the ratio comparison between NLCs and SKCs demonstrates that noticeably larger amount of water is entrapped in HPF adsorbed onto NLCs rather than on SKCs. This, once again, suggests that HPF molecules undergo a different assembly configuration upon interaction with topographically dissimilar iPB-1 surfaces. To obtain more detailed information about how the studied nanostructures influence the protein-surface interaction, the iPB-1 samples exposed to HPF buffer solution with concentrations lower than 1 mg/L were further characterized by AFM. Based on the QCM-D results, at mentioned low HPF concentrations, the adsorption behavior of isolated proteins as well as assembly characteristic of several HPF molecules should be visible. Adsorption and Orientation Characteristics of HPF Molecules on NLC Nanotopography. The height and phase images of a series of NLCs surfaces after HPF adsorption are shown in Fig. 3A. For the HPF concentration of 0.0001 mg/L only star-like structures of HPF molecules which are resulting from the connec5

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tion between several adjacent molecules can be seen. The appearance of such HPF assemblies indicates “end-to-end” proteinprotein interaction, which is dominant on surfaces, which are smooth on the nanoscale.12 With increasing concentration, HPF proteins progressively interact with each other, which leads to network structure formation on the entire NLC surface. Assembly of HPF molecules into a network structure, however, requires a sufficiently high protein concentration and/or enhanced proteinprotein interaction at simultaneously hindered protein-surface interaction. Similar assembly patterns to those obtained in this study were observed previously on HOPG15 and nanostructured MD UHMWPE12 surfaces. It is noteworthy that the HPF network shows a very regular structure, with no overlaps. The extracted height of the HPF molecules within the network, 1.94 ± 0.84 nm, corresponds well to the reported dimensions of the HPF molecule D and E domains, namely 2.9 ± 0.3 and 1.8 ± 0.3 nm, respectively.38 It also confirms a single layer of the “side-on” adsorbed molecules. Another characteristic feature of the widespread HPF assembly is, as termed by Sit and Marchant,39 an “eyelet” structure. This morphology is exemplified in the zoomed-AFM image on the top of Fig. 3B, which displays the overlaid height and phase scans and false-colored HPF network on the NLC surface to show the HPF network in relation to the underlying nanotopography. A closer look at this AFM data reveals that the “eyelets” shape resembles ellipses, whose major axes are predominantly aligned parallel to the NLCs orientation, i.e., the drawing direction. This would imply that protein-NLCs interaction is indeed present. Since the HPF molecules surround the “eyelet”, one can assume that their orientation is a result of the arrangement of the protein’s major axis parallel to the axis of the NLCs. To validate this hypothesis, we performed a statistical analysis of the orientation angle, θ, between the “eyelet’s” major axis and the NLCs long axis, as schematically illustrated in the top part of Fig. 3B. Accordingly, the statistical analysis of the angle distribution as a function of the “eyelet’s” major axis is presented on the polar plot, where θ = 0° and θ = 90° corresponds to the extreme cases, namely parallel and perpendicular “eyelet” orientation, respectively (see half-filled green dots in Fig. 3B middle). It can be clearly seen that the “eyelets” are mostly aligned parallel with respect to the axial direction of the NLCs, having an orientation factor f = 0.82 (calculated with Herman’s orientation function.39 f = 1 corresponds to a system with a perfect uniaxial orientation and f = 0 to random orientation). In addition to the orientation, the performed analysis provides insight into the “eyelet” length distribution, and the assembly structure of several molecules. Fig. 3B (middle) clearly shows that the length of the “eyelet’s” major axis oriented parallel to the NLC nanostructure varies between 25 nm and 60 nm. This lays in the range of reported HPF molecule length, 53 ± 3.4 nm, in the “eyelet” network on MD-UHMWPE film.12 Further, an “eyelet” formed by two HPF proteins touching each other at the D domains at each end and having a 90° bend in the E domain is 24 nm long. The described would be the smallest “eyelet” with touching D domains. Thus, seeing the “eyelet’s” length distribution fade away below 25 nm one can conclude that on average more than two HPF molecules form the “eyelet”. In combination with the high aspect ratio of the eyelets’ major to minor axis, 1.6 ± 0.1, this substantiates the HPF end-to-end interaction and associated alignment. Shifting the focus from studying the “eyelet” orientation, to examining the orientation of individual network segments, we can make statements on the “single” protein orientation. We aimed to extract this information from the local orientation of the protein network by means of 2D image processing analysis with the ImageJ software. Accordingly, we started by extracting a clear image of the HPF network from the AFM height scan using the

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“Tubeness” plug-in. Images processed in this manner (see Fig. 3B, bottom) were then analysed by the OrientationJ plug-in developed by Razakhaniha et al.40 This plug-in allows to evaluate the local pixel-wise orientation of the image. The local orientation of the network structure is visualized on the color-coded image presented in Fig. 3B (bottom right). Here, the network segments/HPF molecules having alike orientation represent one color, and thus molecules oriented parallel to the NLCs appear red. The distribution of the molecules’ orientation angle, θm, is presented on the polar plot (Fig. 3B, middle). As shown by the black filled circles, the distribution peaks at θm≈ 0°, which points toward preferred alignment of the HPF molecules along the NLCs axis. Interestingly, the calculated Herman’s orientation factor, f = 0.61 indicates a lower degree of orientation of “single” HPF molecules in comparison to the assembly structure of several molecules, i.e., “eyelets”. This discrepancy in orientation (factors) may be explained by our simplistic assumption that the locally oriented network segment corresponds to a single HPF. Likely, a different local orientation is ascribed to the molecules shared by adjacent “eyelets”. The overall findings of the orientation analysis unambiguously show that the iPB-1 surface with NLCs induces the arrangement of HPF molecules into a highly anisotropic network structure. The alignment mechanism can be explained by (i) a preferred adsorption of HPF molecules on crystalline regions compared to amorphous ones12 and (ii) the high aspect ratio (above 10) in combination with the low width of the nanocrystals. Both factors induce an alignment along the NLC axis, whereas the small amorphous regions between the NLCs allow for interaction between the adjacent HPF molecules. At coverage close to a complete monolayer, the protein-protein interactions outweigh the topographical factor, and the network anisotropy disappears. The close-up in Fig. 3A, 1 mg/L, shows that the “eyelet” network structure is still present, although with significantly smaller voids. Another insight into the surface structure-mediated width and orientation of HPF has already been provided in the work of Rasmussen et al.41 They showed that ordered poly(tetrafluoroethylene), PTFE, fibers having width below 100 nm induce perpendicular orientation of the HPF long axis with respect to the fiber direction. Thicker PTFE fibers, on the other hand, lead to network formation. An important distinction to our work is that the fiber width was higher than the width of NLC. Moreover, different surface chemistry and crystallinity exclude the direct comparison to our system. Adsorption and Assembly Characteristics of HPF Molecules on SKC Nanotopography. By comparing the protein adsorption results on the SKC surface presented in Fig. 4, it is seen that the structure of isolated proteins as well as overall structure of the HPF assembly is dramatically different from that shown on NLCs. For the SKC nanotopography, individual HPF molecules can be seen in mainly globular and fewer trinodal shaped (see Fig.4 middle). This is indicative for strong protein-surface interaction and hindered “end-to-end” protein-protein interaction. The latter can be explained by the low aspect ratio of the crystalline SKC phase, ~ 1 (see amorphous regions between kebabs of individual shish-kebab on phase scan in Fig. 1B), which contrary to the NLCs, may act as interaction barrier. Another relevant topographical aspect that distinguishes the iPB-1 surfaces under investigation is the surface curvature, K. In comparison to NLCs (tubular diameter of 22 nm, K = 0.09 nm-1), the SKCs with a diameter of 50 nm have a two times smaller surface curvature (K = 0.04 nm-1). Theoretical studies on fibrinogen adsorption onto tubular structures, such as carbon nanofibers, with different width have shown that protein adsorption becomes more prominent as the local curvature decreases.42 Another investigation examining 6

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Langmuir hand, might present much better biocompatibility. Consequently, the future direction of this work will be to include in-vitro cell adhesion studies to (i) determine whether adsorbed HPF retained their biofunctionality and (ii) to investigate the feasibility of the nanostructured iPB-1 surfaces as competitive alternatives to conventional polymers used for biomedical purposes.

Figure 4. AFM analysis of HPF adsorption behavior on the SKC topography. At the lowest concentration (top), a single protein cannot be clearly seen. The amorphous regions hinder the protein-protein interaction, which together with large lateral dimensions of SKCs favors rearrangement of the adsorbed molecules into tightly packed monolayer (bottom). The scale bar corresponds to 300 nm. The zoomed areas of smaller length scale with a false colored HPFs are shown on the right side (scale bar 30 nm).

fibrinogen and albumin adsorption, strongly suggests that surface curvature modifies protein conformation upon binding.7 Namely, surfaces with low surface curvature, such as particles with a diameter above 30 nm, favor the “end-on” orientation (below this diameter “side-on” orientation is preferred, which is in agreement with the HPF orientation on the NLCs). Thus, one can expect that the HPF molecules upon binding to SKC will not assemble into an ordered network structure but will create a densely packed layer. The assumed high surface coverage is indeed well visible on the phase scan (1 mg/L), where the underlying SKC structure is no more distinguishable. Based on our AFM observations we suggest that the SKC iPB -1 nanotopography having a lateral size comparable to the HPF major axis, promotes an “end-on” arrangement of the protein molecules. This is in agreement with the conclusions obtained from the QCM data presented in an earlier paragraph of this study. According to a considerable body of experimental evidence, an immunological response is observed for a high density of adsorbed HPF molecules (note, increased protein density is commensurate with “end-on” orientation).43-46 A recent theoretical work30 suggested that under a condition that favors an “end-on” approach of the protein to the surface, the segments implicated in the immune response will be unfolded, and thus the immunological reaction to foreign material will be triggered. Hence, we suggest, tentatively, that SKC nanotopography might be proinflammatory, and thus is not suitable for blood contacting surfaces. The iPB-1 thin film having NLCs topography, on the other

CONCLUSIONS The ability to endow a material with biocompatibility based on a topographical structure and interactions with blood components would greatly benefit the design of implant materials. This study demonstrates a path to direct protein adsorption by nanoscale features of chemically homogenous semicrystalline thin film surfaces. By exploiting topographically distinct polymeric nanostructures with lateral dimensions smaller than or comparable to the HPF major axis, we discuss resulting differences in protein orientation and assembly configuration. iPB-1 crystalline structure, NLCs, with a lateral dimension lower than the length of the HPF major axis, supports “side-on” adsorption and trinodular conformation. A two-fold orientation analysis performed on a “single” and multiprotein (“eyelets”) level clearly shows preferential alignment of HPF molecules with respect to the axial direction of the NLCs, and high anisotropy of the HPF network assembly, respectively. As the NLCs modulated the affinity of HPF molecules for “end-to-end” protein-protein and protein-surface interactions in a balanced manner, the SKC structure with lateral features comparable to the HPF length blocked “end-to-end” proteinprotein interaction, and thus hindered the network formation. The observed globular conformation and densely-packed monolayer assembly on SKCs indicates an “end-on” orientation, which is in line with the QCM - D data. The observed changes in HPF adsorption on NLCs and SCKs unambiguously show that surface topographical factors such as structure width, curvature, aspect ratio, and crystallinity, concomitantly influence protein interaction preferences. The correlation between these physical factors and protein adsorption, which has emerged from this study, may provide a basis for directing the surface-induced protein arrangement to mediate biological reactions at the blood proteins-implant material interface and subsequent cellular behavior.

AUTHOR INFORMATION Corresponding Author *E-mail [email protected]; Tel +49 (0) 3641 94 77 30; Fax +49 (0) 3641 94 77 32.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors acknowledge the China Scholarship Council (CSC) for a PhD fellowship as well as the Chair of Material Science, Friedrich Schiller University Jena for the financial support. KDJ gratefully acknowledges the partial financial support of the Deutsche Forschungsgemeinschaft (DFG) project: “Antimicrobial Effect of Nano-Rough Titanium Surfaces: Reduction of Microbial Adhesion and Mechanisms of Reduction”, AOBJ: 622946. The author thanks Mr. Ralf Wagner (CMS) for technical support and Dr. Bernd Schröter (Institute of Solid State Physics, Friedrich 7

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Schiller University Jena) for XPS measurements. The research at Oak Ridge National Laboratory's Spallation Neutron Source was sponsored by the Scientific User Facilities Division, Office of Basic Energy Science, U.S. Department of Energy.

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