Letter pubs.acs.org/NanoLett
Biomimetic Self-Templated Hierarchical Structures of Collagen-Like Peptide Amphiphiles Hyo-Eon Jin,†,‡ Jaein Jang,§ Jinhyo Chung,§ Hee Jung Lee,† Eddie Wang,† Seung-Wuk Lee,*,†,‡ and Woo-Jae Chung*,†,‡,§ †
Department of Bioengineering, University of California, Berkeley, California 94720, United States Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States § Department of Genetic Engineering, College of Biotechnology & Bioengineering, Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, Korea ‡
S Supporting Information *
ABSTRACT: Developing hierarchically structured biomaterials with tunable chemical and physical properties like those found in nature is critically important to regenerative medicine and studies on tissue morphogenesis. Despite advances in materials synthesis and assembly processes, our ability to control hierarchical assembly using fibrillar biomolecules remains limited. Here, we developed a bioinspired approach to create collagen-like materials through directed evolutionary screening and directed selfassembly. We first synthesized peptide amphiphiles by coupling phage display-identified collagen-like peptides to long-chain fatty acids. We then assembled the amphiphiles into diverse, hierarchically organized, nanofibrous structures using directed selfassembly based on liquid crystal flow and its controlled deposition. The resulting structures sustained and directed the growth of bone cells and hydroxyapatite biominerals. We believe these self-assembling collagen-like amphiphiles could prove useful in the structural design of tissue regenerating materials. KEYWORDS: self-templating, biomimetic hierarchical structure, collagen-like peptide amphiphile, nanofiber, liquid crystalline assembly, tissue regeneration
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amphiphiles is one of the most promising strategies to develop biomimetic materials that can elicit desired cellular behavior23−25 and templated biomineralization.7,26 These selfassembled nanofibrous surfaces are reminiscent of natural extracellular matrices in that they can display high densities of biochemical cues and provide physical cues due to the unique geometry of the nanofibrous structures. However, creating biomimetic structures with controllable structural complexity and hierarchy over large areas from such self-assembling peptides is still challenging. In nature, highly structured biological tissues often employ liquid crystalline self-assembly processes that are frozen in time27−29 using helical nanofibrous building blocks. Collagen, the main structural protein in the body, plays a critical role in
he formation of hierarchically ordered structures from nanofibrous building blocks is essential for defining the physicochemical features of many biological tissues.1,2 The organization of these biomaterials has inspired scientists to develop a variety of strategies for guiding molecular selfassembly to form biomimetic materials.3−5 For example, amphiphilic molecules made of peptides, proteins or their conjugates with alkyl chains, fatty acids, and hydrophobic polymers have been demonstrated to undergo supramolecular self-assembly into nanofibrous networks.5−13 The resulting structures are tunable in their biochemical and physical properties, which allows for guidance of cell growth and tissue remodeling.14−16 In particular, studies of the supramolecular design, assembly, and bioactivity of peptide amphiphiles have demonstrated their potential for biomedical applications including biomineralization,7 tissue regeneration,5,17−20 diagnosis/targeted drug delivery,21 and biosensors.22 Utilizing nanoscopic fibrous surfaces or coating made from peptide © XXXX American Chemical Society
Received: August 18, 2015 Revised: September 14, 2015
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DOI: 10.1021/acs.nanolett.5b03313 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 1. Design of collagen-like peptide amphiphile (CLPA) into biomimetic structures based on the discovery of collagen-like motifs and their application for tissue regeneration: (a) Evolutionary screening of hydroxyapatite-binding peptide using phage display. (b) Molecular model of a collagen-like peptide amphiphile and nanofiber: a 12-mer peptide identified through phage display library screening is coupled to a hydrophobic fatty acid chain (C16), which undergoes self-assembly into nanofibers. The self-assembly of peptide amphiphiles (CLPA) is triggered by their local accumulation at meniscus area during self-templating assembly. (c) Nanofibers are self-templated into hierarchically organized matrices through the interplay of liquid crystal phase transitions and interfacial forces at the deposition site. (d) Resulting materials can serve as soft- and hard-tissue guiding scaffolds.
flow and the subsequent deposition of such building blocks to create biomimetic liquid crystal flow patterns27,28 over large areas. In this context, the recent discovery of directed selfassembly process termed “self-templating assembly”, which emulates nature’s self-assembly of incoming chiral molecules through the control of thermodynamic, kinetic, and environmental factors, has opened new avenues to control liquid crystalline behaviors and the deposition of nanofibrous building blocks for the creation of various hierarchical structures, as demonstrated using the M13 bacteriophage as a model molecule.39,40 Therefore, expanding the self-templating assembly to other supramolecular and self-assembly system will be a great logical next step to develop hierarchically organized functional biomaterials. Here, we report the synthesis and assembly of collagen-like peptide amphiphiles into functional hierarchical, supramolecular structures (Figure 1). We designed a collagen-like peptide amphiphile (CLPA) through the conjugation between the previously identified collagen-like hydroxyapatite binding peptide38 together with hydrophobic alkyl chain (Figure 1). The resulting CLPA forms nanofibers of uniform diameter through molecular self-assembly. This nanofiber-forming peptide amphiphile exhibits lyotropic liquid crystalline behavior in solution. We exploited the selftemplating approach39 to create diverse hierarchical architectures analogous to structures often found in collagenous tissues (i.e., bones, cartilage, and tendons).41 The structural evolution of the self-templated architectures depends on initial CLPA
creating hierarchical structures such as twisted plywood structures from decalcified bone tissues and crimp patterns from tendon tissues, which are considered to be associated with liquid crystal-like development processes. The liquid crystalline properties of collagen have been demonstrated in studies showing the formation of nematic or cholesteric liquid crystal structures either in solution30,31 or in gel and thin film structures.32−34 Although natural collagen has been highly utilized as a structural building block material in tissue engineering and many biomedical applications, emulating hierarchically organized functional collagen structures such as corneal or bone tissue is still challenging. For the last two decades, numerous collagen-mimetic and collagen-like peptides have been extensively studied for elucidation of molecular assembly mechanism and development of collagen substitutes.35−37 Collagen-mimetic or collagen-like peptide has commonly employed a repeating triplet sequence of Gly− Pro−Hyp (Hyp: hydroxyproline). Recently, we identified a new class of 12-residue peptide that could bind to single crystal (100) hydroxyapatite (HAP) surfaces.38 Interestingly, the sequence responsible for HAP binding resembled the tripeptide repeat (Gly−Pro−Hyp) of type I collagen (see the sequence in Figure 1). This peptide was able to template the nucleation and growth of crystalline HAP minerals in a sequence- and composition-dependent manner.38 Therefore, there is increasing interest for developing collagen-mimetic building blocks and their self-assembly through controlling the liquid crystal B
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Figure 2. Structure of self-assembling peptide (CLPA) and its molecular self-assembly behavior. (a) Structures of collagen-like peptide amphiphile (CLPA) and control peptide amphiphile (Control-PA). (b) Photos of CLPA solution, Control-PA solution, and their mixtures (1 wt % in DI water at various molar ratios of CLPA/Control-PA) before and after pH adjustment. Solutions are placed upside down to show their fluidity. (1) CLPA (pH 3), (2) Control-PA (pH 3), (3) Control-PA (pH 7.5), (4) CLPA:Control-PA = 2:8 (pH 7.5), (5) CLPA:Control-PA = 4:6 (pH 7.5), (6) CLPA:Control-PA = 6:4 (pH 7.5), (7) CLPA:Control-PA = 8:2 (pH 7.5), and (8) CLPA (pH 7.5). (c) TEM image of precipitated nanofibers of CLPA. (d) AFM image of nanofibers formed from a drop-cast CLPA solution (10 μg/mL).
protonated in acidic conditions (calculated isoelectric point of CLPA: 8.29). At around pH 7.5, the solution became turbid and white precipitates formed in the solution. The peptide motif in CLPA is relatively hydrophobic compared to the motifs in conventional gel-forming peptide amphiphiles reported by other research groups and resulted in precipitation rather than gelation at controlled pH. We found that gelation occurs when mixed with an amphiphile (Control-PA: GGGCCK-Palmitoyl) without the collagen-like peptide motif when the CLPA/Control-PA molar ratio is greater than 6/4 (Figure 2b). The TEM analysis showed that CLPA assembles into nanofibrous bundle consisting of individual nanofiber morphologies in gel (Figure 2c). At concentrations as low as 1−10 μg/mL, individual nanofibers of uniform diameter were clearly observed in drop-cast samples (Figure 2d and Figures S1 and S2 in Supporting Information). In the AFM image, the individual nanofiber seemed to have the radius of ∼17 nm (Figure S2 in Supporting Information), which is larger than we expected considering that the stretched length of the CLPA is around ∼7 nm. We believe that this result is due to the AFM tip geometry and evaporation-driven compression. The dropcast nanofibers showed no preferred orientations or any additional levels of structural order. Liquid Crystalline Behavior of CLPA Solution. We examined whether CLPA solutions possess lyotropic LC behavior, which is critical to self-templated assembly. At concentrations lower than 40 mg/mL, CLPA exhibited an
concentration and the rate of deposition, which impose both thermodynamic and kinetic factors during the self-templating process. The resulting CLPA-based materials direct cellularoriented growth and serve as scaffolds for biomineralization, showing their potential for soft and hard tissue engineering. To our knowledge, this work represents the first report describing the self-assembly of peptide amphiphiles into hierarchical collagen-like liquid crystalline surface coatings that are effective over large areas and which may contribute to developing biomimetic materials and understanding biomacromolecular assembly in in vivo systems.
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RESULTS AND DISCUSSION Preparation and Self-Assembling Property of CLPA. The collagen-like peptide amphiphile, CLPA (NPYHPTIPQSVHGGGCCK-Palmitoyl), is composed of a previously discovered hydroxyapatite-binding/nucleating peptide and a cross-linkable spacer peptide (GGGCC), and lysine linked to a long fatty acid (C16). The CLPA was synthesized (Figure 2a) using standard Fmoc-based solid-phase peptide synthesis (details in Methods). CLPA solutions were prepared at concentrations ranging from 1 μg/mL to 100 mg/mL in DI water. Unless otherwise specified, the pH of the solution was adjusted to pH 3 to solubilize CLPA. We initially triggered selfassembly of CLPA by gradually increasing the pH to 7.5 using 1 N NaOH. This reduced the electrostatic repulsion between the N-terminal amino and imidazole groups of peptide amphiphiles C
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Figure 3. Liquid crystalline properties of CLPA solution. Polarized optical microscopy images of CLPA solution incubated for 24 h at room temperature in capillary tubes at different concentrations. We observed (a) isotropic (20 mg/mL), (b) nematic (60 mg/mL), and (c) cholesteric (80 mg/mL) phases. Crossed arrows indicate the direction of polarizer and analyzer. Scale bar indicates 100 μm.
Figure 4. Biomimetic hierarchically organized structures created from self-templating assembly of CLPA. (a) Structural color patterns appearing on self-templated structures pulled at 20−50 μm/min from 1 and 3 mg/mL of CLPA solution. (b) Twisted plywood-like phase in self-templated structures showing vertical periodic spacing (top). Periodic arc structures appear between the vertical stripes (bottom). The film was pulled at 30 μm/min from 1 mg/mL of CLPA solution. (c) Crimp pattern phase in self-templated structures showing periodic horizontal patterns (top). Periodic crimp patterns appear within the horizontal stripes. The film was pulled at 50 μm/min from 5 mg/mL of CLPA solution. (d) Structural periodicity in crimp pattern phase (5 mg/mL) evolving in response to different pulling speeds. (e) Crimp pattern phase clearly showing periodic microstructural undulating topography in AFM analysis. (f) Self-templated structures pulled at 50 μm/min from 5 mg/mL of Control-PA solution show no hierarchical patterns. Red bidirectional arrows indicate the microstructural periodicity of self-templated structures.
isotropic phase without LC ordering (Figure 3a). At concentrations greater than 60 mg/mL, CLPA solutions exhibited nematic LC phases as indicated by birefringent textures under a polarized optical microscope (Figure 3b). At 80 mg/mL (Figure 3c), multiple aligned domains were observed, whereas a relatively uniformly aligned phase was observed at 60 mg/mL indicating lyotropic LC behavior of CLPA solutions. During LC phase characterization, we did not observe any evidence of long-range ordered hierarchical arrangements. Interestingly, when we allowed CLPA solutions with concentrations as low as 5 mg/mL (isotropic phase) to evaporate in capillary tubes, areas near the meniscus exhibited birefringence due to localized concentrations of CLPA (Figure
S3 in Supporting Information). As the meniscus receded, organized CLPA nanofiber structures were deposited on the wall of the capillary tube. These results suggest that CLPA is amenable to our self-templating approach based on liquid crystal flow and deposition. Self-Templating Assembly of CLPA into Hierarchical Architectures. We investigated the effects of self-templating assembly conditions on the formation of CLPA hierarchical architectures. To perform assembly, we immersed solid substrates in CLPA solutions (1−5 mg/mL) and pulled them out at controlled rates. During this process, competing interfacial forces (e.g., surface tension and friction forces) are exerted at the air−liquid−solid interface, where evaporation of D
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Figure 5. Hierarchical CLPA biomaterials guiding directional cell growth cells and templating biomineralization of calcium phosphate. (a) Bright field and fluorescence images of MC3T3-E1 cells cultured for 5 days on the drop-cast CLPA film. (b) Bright field and fluorescence images of MC3T3-E1 cells cultured for 5 days on the self-templated CLPA (actin: green and nuclei: blue). (c) Profile of orientation order parameter for cells cultured on aminated glass, drop cast, and self-templated CLPA film (5 days). (d)−(g) SEM images of self-templated CLPA films before biomineralization (e) and after biomineralization (d), (f), and (g) using three different methods. Biomineralization was carried out by treating films with (d) [Ca2+] = 3 mM and [PO43−] = 1.5 mM in HEPES buffer (pH 7.5) at 37 °C for 7 days, (f) [Ca2+] = 3 mM, [PO43−] = 1.5 mM and [poly(aspartate)] = 20 μg/mL in HEPES buffer (pH 7.5) at 37 °C for 7 days, or (g) alternating dipping in [Ca2+] = 100 mM and [PO43−] = 100 mM (50 cycles). White bidirectional arrows indicate the orientations of groove structures from the underlying self-templated CLPA.
the solution takes place on the solid substrate. We attempted to impose thermodynamic and kinetic controls on the assembly process by manipulating two variables. The first is the concentration of CLPA solution, a thermodynamic factor controlling liquid crystal phase transitions, and the second is the pulling speed, a kinetic factor inducing liquid crystal flow and subsequent deposition that results in the formation of nonequilibrium structures with different periodicity. The most evident characteristic that emerged from the different assembly conditions was variety in structural colors (Figure 4a). Similar structurally colored matrices were previously observed from phage-based self-templating processes.39,40 Two distinct, concentration-dependent, hierarchical phases were observed at the microscale (Figure 4b and c): The first is a twisted plywood-like phase (1 mg/mL) and the second is a crimped filament phase (3 and 5 mg/mL). In the twisted plywood-like films, striped patterns normal to the meniscus line were observed, with upward arcing fibrous structures filling the gaps between stripes (Figure 4b). Structures deposited earlier on the substrate guided the subsequent deposition of CLPA, resulting in continuity of the cholesteric phase along the pulling direction (Figure S4, Supporting Information). The morphologies bear similarities with the characteristic half-turn pitch (P1/2) on the oblique side of cholesteric phase collagen matrices found in decalcified bone.42 As the substrate was pulled faster (20−80 μm/min), the interspacing between the vertical domains of the pattern decreased from 85 to 30 μm (Figure S5 in Supporting Information). The crimped nanofilament phase films exhibited periodic zigzag morphologies composed of ridge-groove
structures perpendicular to the pulling direction (Figure 4c and e) over a long length scale (up to a few cm). The crimp pitches increased from 14 to 42 μm as a function of the pulling speed (20−80 μm) (Figure 4d), indicating that kinetic factors influence the periodicity of the liquid crystalline phases. Such crimped patterns are found in assembled structures of collagen fibrils (e.g., tendon) that have a helical arrangement and follow a wavy, undulating course.41,43 These results demonstrate that the CLPA not only forms one-dimensional nanofibers but also can be organized into multilevel hierarchical supramolecular structures using a self-templating approach. In contrast, Control-PA does not form hierarchically ordered supramolecular structures (Figure 4f and Figure S6 in Supporting Information), indicating that the CLP peptide in the peptide amphiphile plays a critical role in hierarchical assembly. The self-templating assembly of peptide amphiphiles and phages34 showed similarity in that they were both assembled into hierarchical structures in the concentration-dependent manner, they created both cholesteric phase and filamentous crimp phase in long-range order over large area and the periodic pitches of the LC phase structures were controllable by tuning pulling speed. The distinctive differences between the two systems are the molecular design features of the building blocks and building block production (Table S1 in the Supporting Information). Directed Growth of Preosteoblast Cultures on Anisotropic Collagen-Mimetic Patterns. Then, we studied the potential use of self-templated collagen-mimetic patterns as tissue-regenerative materials. We assessed the capability of E
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successfully functioned as templates for hydroxyapatite crystal growth (theoretical value =1.67).
hierarchical patterns to support cell viability and guide directional cell growth. A live/dead assay was performed to assess cell viability on drop-cast samples of CLPA, resulting in an over 95% survival rate of MC3T3-E1 preosteoblast cells after at least 5 days (Figure S7 in Supporting Information). On dropcast CLPA and glass substrates without peptide amphiphile deposition, the preosteoblast cells grew without a preferred orientation (Figure 5a and Figure S8 in Supporting Information). In contrast, cells cultured on the self-templated CLPA (crimp pattern) showed growth oriented parallel to the ridge/groove direction (Figure 5b). The stained actin filaments and nuclei in the fluorescence images clearly showed preferred orientation (Figure 5b). On the aligned patterns, most of the cultured cells grew in a uniform direction, demonstrating significant effects of the anisotropic microstructure patterns. Meanwhile, a significantly lower proportion of the cultured cells were oriented on the drop-cast samples and glass substrates due to the lack of anisotropic topography. The cell alignment on the substrates was further evaluated and compared by measuring the orientation order parameter (OOP) (Figure 5c). First, cell alignment was determined according to the angles of cell nuclei which had their long axis, with respect to the direction of ridge/ groove topography (for self-templated pattern) and the average angle of orientation (for drop-cast samples and glass substrates). Second, the OOP was calculated based on the orientation angle of the nuclei (OOP = 1/2 ⟨3cos2θ − 1⟩, θ = orientation angle of cell). The measurements of OOP on the self-templated pattern, drop-casted sample, and glass substrate also provide evidence of oriented growth (OOP = 0.83, 0.37, and 0.35, respectively) (Figure 5c). Morphologies of the selftemplated (Figure 4c and e) versus drop-cast CLPA (Figure 4f) strongly support the correlation between surface topography and the orientation of cell growth on the surfaces (Figure 5a− c). Biomineralization of calcium phosphate by collagenmimetic patterns. We studied the biomineralization of calcium phosphate by the self-templated collagen-mimetic patterns. When treated with a mixture of Ca2+ and PO43− ions in a supersaturated condition, the pattern structure supported calcium phosphate cluster formation due to nucleation on the surface (Figure 5d), similar to crystal growth in the presence of CLP12 short peptide as previously reported.38 Self-templated CLPA showed little structural features before biomineralization other than periodic lines in SEM analysis due to the lack of electrical conductivity of organic matrices (Figure 5e). To promote mineralization within organic matrices not only on the surface, as occurs in bone tissue, we added polyaspartate to the ion mixtures (Ca2+ and PO43−) as previously reported.44−46 This additive has proven to play a critical role in templated mineral growth. The presence of additive significantly influenced the way biomineralization proceeds in self-templated CLPA and resulted in the retention of hierarchical morphologies of the structures (Figure 5f). Repetitive alternate dipping of the film into Ca2+ and PO43− solutions led to layered inorganic crystal deposition on the pattern structure, resulting in the formation of CLPA-templated organic/inorganic composites. SEM and AFM images show that the aligned ridge/groove microstructures were preserved even after the deposition of layered minerals (Figure 5g and Figure S9a in Supporting Information). XPS analysis revealed that the inorganic crystals were composed of calcium and phosphorus at an atomic ratio of 1.64 (Figure S9b and c in Supporting Information), which suggests that the CLPA films
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CONCLUSION We demonstrated that diverse biomimetic hierarchical structures can be created by the self-templating assembly of CLPA, a peptide amphiphile derived from directed evolutionary screening. Like natural biomolecular structural building blocks, CLPA forms nanofibers that display active molecules and themselves form lyotropic liquid crystalline structures. Our peptide amphiphile-based self-templating system generated various long-range ordered liquid crystal structures, including twisted plywood-like and crimped nanofilament structures, under different environmental conditions. Bioactive functions of collagen were mimicked by the self-templated structures, as demonstrated by preosteoblast culture and biomineralization experiments. Our combined strategy of directed evolutionary screening and self-templating assembly provides opportunities for developing multifunctional biomimetic peptide-based materials and represents a means to study the dynamics of biomolecule-based supramolecular assemblies, areas that will play essential roles in tissue engineering and regenerative medicine.
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MATERIALS Rink resins, Fmoc-amino acids, N,N′-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and organic solvents N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), methanol, ethanol, and diethyl ether were purchased from Advanced ChemTech (Louisville, KY, U.S.A.). Palmitic acid, N,N′-diisopropylethylamine (DIEA) trifluoroacetic acid (TFA), thioanisole, phenol, 1,2-ethanedithiol, triisopropyl silane (TIPS), (3-aminopropyl) triethoxysilane (APTS), glutaraldehyde (25%), 2-aminoethanol, CaCl2, and Na2HPO4 were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Slide glass was purchased from Fisher Scientific (Pittsburgh, PA, U.S.A.). MC3T3-E1 fibroblasts were obtained from the University of California, Berkeley Cell Culture facility. αMEM media, fetal bovine serum (FBS), phosphate buffered saline (PBS, pH 7.5), trypsin, Alexa Fluor 488-phalloidin and DAPI were purchased from Invitrogen (Carlsbad, CA, USA). Viability was assessed with the Invitrogen Live/Dead Viability test (Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions.
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METHODS Synthesis of Self-Assembling CLPA. Collagen-like peptide amphiphile (CLPA) was synthesized using standard Fmoc chemistry based solid-phase peptide synthesis using NFmoc-amino acids and Rink resins. For all coupling reactions, building blocks, DIC and HOBt (5 equiv each) were mixed in NMP (∼0.1 M for each component) and reacted with the resins. Complete coupling was confirmed by Kaiser ninhydrin tests. If necessary, the coupling reaction was repeated. After deprotection of Fmoc groups on the resin, Fmoc-Lys(Mtt) was coupled to the resin and dried under vacuum. Mtt protecting groups were cleaved with 4% TFA and 2.5% TIS in dichloromethane for 10 min. The resins were neutralized with 5% DIEA in DCM for 10 min. Next, palmitic acid (5 equiv) was coupled to the ε-amino group of the lysine that was loaded on the resin. Subsequently, cross-linkable residues (CC), spacer (GGG) and target sequence (NPYHPTIPQSVH) F
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in PBS), and the nuclei were counterstained with DAPI (300 nM) for 30 min. Fluorescence images were collected using an IX71 fluorescence microscope (Olympus, Tokyo, Japan). Biomineralization. The CLPA crimp patterns were mineralized in two different ways. The first method was to treat the crimp pattern with a mixture of CaCl2 (6 mM), Na2HPO4 (3 mM), and polyaspartate (20 μg/mL) for 7 days at 37 °C. The mineralized crimp pattern was washed with DI water by dipping (10 times, 1 min each). The second method was to use a home-built programmable alternate dipping machine. The pattern was immersed in 100 mM CaCl2 solution for 30 min, washed with DI water for 1 min, and then immersed in 100 mM Na2HPO4 solution (pH 9) for 30 min. The cycle was repeated 50 times and the mineralized patterns were washed with DI water and dried in a desiccator prior to analysis. Microscopy. Optical and fluorescence images were collected using an IX71 Inverted Fluorescence Microscope (Olympus, Tokyo, Japan) equipped with a digital CCD camera (QImaging, Surrey, Canada). SEM images were collected at 50 kV using a scanning electron microscope (Hitachi America, Pleasanton, CA, U.S.A.). Atomic force microscopy images were collected using an MFP3D AFM (Asylum Research, Santa Barbara, CA, U.S.A.) and analyzed using Igor software 6.0 (WaveMetrics, Inc., Lake Oswego, OR, U.S.A.) and Asylum software package (Asylum Research). AFM images were taken in air using the tapping mode (cantilever: AC240TS). TEM images were taken using JEOL-200 and a Philips CM200/FEG at a 200 kV acceleration voltage at the National Center of Electron Microscopy, Lawrence Berkeley National Laboratories (Berkeley, CA, U.S.A.). X-ray Photoelectron Spectroscopy (XPS) Analysis. XPS was performed with a PHI VersaProbe Scanning XPS Microprobe with an Al Kα X-ray source (1486.6 eV). The spectra were collected with a pass energy of 23.5 eV and a spot size of 200 μm. An electron gun was used to neutralize excess charge on the substrate surface. The binding energies were calibrated with reference to the carbon 1s orbital.
were coupled to the N-terminal group of palmitic acidderivatized lysine on the resin. After complete synthesis and deprotection of the N-terminal Fmoc-group, CLPA was cleaved from the resins for 2 h using a cocktail of 82.5% TFA, 5% thioanisole, 2.5% water, 5% phenol, 2.5% ethanedithiol, and 2.5% TIPS. The solvent was removed under vacuum. Crude products were collected by precipitating in cold diethyl ether (20-fold volume) and purified by HPLC to >95% purity. Transformation of CLPA into Nanofibers. Two different methods were exploited to form nanofibers using CLPA. (1) pH-driven assembly was performed as follows. First, 10 mg/mL of CLPA solution was prepared in DI water (adjusted to pH 3). The pH of the solution was raised by adding 1N NaOH aqueous solution until the solution became turbid. For TEM analysis, 5 μL of CLPA was placed on a holey carbon-coated TEM grid for 10 min. The peptide amphiphile loaded TEM grid was exposed to ammonia gas by placing it in the closed chamber with a container of 28% NH4OH solution inside until the solution turned white (pH ∼ 7.5). The TEM grid was washed with DI water three times and dried in a stream of nitrogen. (2) Evaporation-driven assembly was performed by placing CLPA solution (10 μL) on a silicon wafer and drying in air. Observation of Liquid Crystalline Phase Behaviors of CLPA. CLPA solutions with different concentrations were placed in capillary tubes (ID 400 μm) using capillary force. Both ends of the capillary tubes were sealed with epoxy. The solutions trapped in the capillary tubes were incubated for 1 day at room temperature. Each sample was observed under a cross-polarized optical microscope. Self-Templating of CLPA into Long-Range Hierarchically Ordered Patterns. Glass slides were placed in piranha solution for 10 min (H2O2:H2SO4 = 1:4), thoroughly rinsed with DI water, and dried under a nitrogen stream. Then, the substrates were treated with 1% (v/v) APTS solution in EtOH (10 min, rt). Subsequently, the substrates were rinsed with EtOH to remove excess silane and annealed at 100 °C for 10 min. APTS-treated glass or gold-coated silicon wafers (5 mm × 15 mm) were immersed in CLPA solution (1, 3, and 5 mg/mL) and pulled vertically to assemble building blocks and deposit hierarchical pattern structures using a home-built pulling machine (pulling speed: 20−100 μm/min). For cell culture experiments, as-prepared structures were treated with iodine solutions (10 mM) in DI water for 30 min to induce disulfide bond formation between the nanofibrils in the matrix. Crosslinked structures were washed with DI water (×5) and dried with a stream of N2. Successful cross-linking was confirmed by observing the preserved structures under optical microscope (×200). MC3T3-E1 Preosteoblast Cell Culture on Self-Templated CLPA Structures. Preosteoblast (MC3T3-E1) cells were used at passage number 12 and seeded at a density of ∼2 × 103 cells/cm2 on top of the CLPA structures (self-templated and drop cast) and APTS-treated glass in six-well culture plates (BD Biosciences, San Jose, CA, U.S.A.). The cells were grown in α-MEM media supplemented with 10% FBS at 37 °C with 5% CO2. Cells cultured on the films and APTS-treated glass were fixed in 4% paraformaldehyde for 30 min and then blocked with a solution of 0.5% (v/v) Triton X-100 and 4% (v/ v) normal goat serum in PBS for 45 min. To visualize the MC3T3-E1 cells, actin filaments were stained with Alexa Fluor 488-phalloidin (1:500 in PBS) for 30 min at room temperature. The cells were then rinsed with PBS-T (0.05% (v/v) Tween 20
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.5b03313. AFM images of drop-casted or self-templated CLPA and Control-PA, polarized optical microscope image of the meniscus area of CLPA, optical microscope images of twisted plywood-like structures, analysis of structural periodicity in twisted plywood-like structures, fluorescence images of MC3T3-E1 cultured on CLPA, ControlPA and aminated glass substrate, and AFM image and XPS analysis of mineralized CLPA crimp pattern. (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. H.-E.J. and J.J. contributed equally. G
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Nano Letters Notes
(25) Gouveia, R. M.; Castelletto, V.; Alcock, S. G.; Hamley, I. W.; Connon, C. J. J. Mater. Chem. B 2013, 1 (44), 6157−6169. (26) Huang, Z.; Jin, H.; Che, S. Dalton Trans 2014, 43 (43), 16169− 16172. (27) Cowin, S. C. J. Non-Newtonian Fluid Mech. 2004, 119 (1−3), 155−162. (28) Smalyukh, I. I. Nature 2011, 478 (7369), 330−331. (29) Hamley, I. W. Soft Matter 2010, 6 (9), 1863−1871. (30) Giraud Guille, M. M.; Mosser, G.; Helary, C.; Eglin, D. Micron 2005, 36 (7−8), 602−608. (31) De Sa Peixoto, P.; Deniset-Besseau, A.; Schmutz, M.; Anglo, A.; Illoul, C.; Schanne-Klein, M.-C.; Mosser, G. Soft Matter 2013, 9 (47), 11241−11248. (32) Giraud-Guille, M.-M.; Besseau, L.; Martin, R. J. Biomech 2003, 36 (10), 1571−1579. (33) Kirkwood, J. E.; Fuller, G. G. Langmuir 2009, 25 (5), 3200− 3206. (34) De Sa Peixoto, P.; Deniset-Besseau, A.; Schanne-Klein, M.-C.; Mosser, G. Soft Matter 2011, 7 (23), 11203−11210. (35) Holmgren, S. K.; Bretscher, L. E.; Taylor, K. M.; Raines, R. T. Chem. Biol. 1999, 6 (2), 63−70. (36) Yu, S. M.; Li, Y.; Kim, D. Soft Matter 2011, 7 (18), 7927−7938. (37) O’Leary, L. E. R.; Fallas, J. A.; Bakota, E. L.; Kang, M. K.; Hartgerink, J. D. Nat. Chem. 2011, 3 (10), 821−828. (38) Chung, W.-J.; Kwon, K.-Y.; Song, J.; Lee, S.-W. Langmuir 2011, 27 (12), 7620−7628. (39) Chung, W.-J.; Oh, J.-W.; Kwak, K.; Lee, B. Y.; Meyer, J.; Wang, E.; Hexemer, A.; Lee, S.-W. Nature 2011, 478 (7369), 364−368. (40) Oh, J.-W.; Chung, W.-J.; Heo, K.; Jin, H.-E.; Lee, B. Y.; Wang, E.; Zueger, C.; Wong, W.; Meyer, J.; Kim, C.; Lee, S.-Y.; Kim, W.-G.; Zemla, M.; Auer, M.; Hexemer, A.; Lee, S.-W. Nat. Commun. 2014, 5, 3043. (41) Giraud-Guille, M. M.; Mosser, G.; Belamie, E. Curr. Opin. Colloid Interface Sci. 2008, 13 (4), 303−313. (42) Giraud-Guille, M. Calcif. Tissue Int. 1988, 42 (3), 167−180. (43) Silver, F. H.; Freeman, J. W.; Seehra, G. P. J. Biomech 2003, 36 (10), 1529−1553. (44) Olszta, M. J.; Cheng, X.; Jee, S. S.; Kumar, R.; Kim, Y.-Y.; Kaufman, M. J.; Douglas, E. P.; Gower, L. B. Mater. Sci. Eng., R 2007, 58 (3−5), 77−116. (45) Nudelman, F.; Pieterse, K.; George, A.; Bomans, P. H. H.; Friedrich, H.; Brylka, L. J.; Hilbers, P. A. J.; de With, G.; Sommerdijk, N. A. J. M. Nat. Mater. 2010, 9 (12), 1004−1009. (46) Wang, Y.; Azaïs, T.; Robin, M.; Vallée, A.; Catania, C.; Legriel, P.; Pehau-Arnaudet, G.; Babonneau, F.; Giraud-Guille, M.-M.; Nassif, N. Nat. Mater. 2012, 11 (8), 724−733.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the National Science Foundation Early Career Development Award (DMR-0747713) (S.W.L.), U.S. Army Engineering Research Development Center (W912HZ-11-2-0047) (S.W.L.), and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2015037593). We also thank K. Braam for the XPS analysis.
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REFERENCES
(1) Neville, A. C. Biology of Fibrous Composites: Development beyond the Cell Membrane; Cambridge University Press: New York, 1993. (2) Lakes, R. Nature 1993, 361 (6412), 511−515. (3) Whitesides, G. M.; Grzybowski, B. Science 2002, 295 (5564), 2418−2421. (4) Mann, S. Nat. Mater. 2009, 8 (10), 781−792. (5) Zhang, S. Nat. Biotechnol. 2003, 21 (10), 1171−1178. (6) Cui, H.; Webber, M. J.; Stupp, S. I. Peptide Sci. 2010, 94 (1), 1− 18. (7) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Science 2001, 294 (5547), 1684−1688. (8) Lim, Y.-b.; Lee, E.; Lee, M. Angew. Chem. 2007, 119 (47), 9169− 9172. (9) Berndt, P.; Fields, G. B.; Tirrell, M. J. Am. Chem. Soc. 1995, 117 (37), 9515−9522. (10) Maeda, K.; Kamiya, N.; Yashima, E. Chem. - Eur. J. 2004, 10 (16), 4000−4010. (11) Zhang, Q.; Ariga, K.; Okabe, A.; Aida, T. J. Am. Chem. Soc. 2004, 126 (4), 988−989. (12) Croisier, E.; Liang, S.; Schweizer, T.; Balog, S.; Mionić, M.; Snellings, R.; Cugnoni, J.; Michaud, V.; Frauenrath, H. Nat. Commun. 2014, 5, 4728. (13) Li, Y.; Yu, S. M. Curr. Opin. Chem. Biol. 2013, 17 (6), 968−975. (14) Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington, D. A.; Kessler, J. A.; Stupp, S. I. Science 2004, 303 (5662), 1352−1355. (15) Ellis-Behnke, R. G.; Liang, Y.-X.; You, S.-W.; Tay, D. K. C.; Zhang, S.; So, K.-F.; Schneider, G. E. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (13), 5054−5059. (16) Arslan, E.; Garip, I. C.; Gulseren, G.; Tekinay, A. B.; Guler, M. O. Adv. Healthcare Mater. 2014, 3 (9), 1357−1376. (17) Shah, R. N.; Shah, N. A.; Del Rosario Lim, M. M.; Hsieh, C.; Nuber, G.; Stupp, S. I. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (8), 3293−3298. (18) Webber, M. J.; Tongers, J.; Newcomb, C. J.; Marquardt, K.-T.; Bauersachs, J.; Losordo, D. W.; Stupp, S. I. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (33), 13438−13443. (19) Mammadov, R.; Mammadov, B.; Toksoz, S.; Aydin, B.; Yagci, R.; Tekinay, A. B.; Guler, M. O. Biomacromolecules 2011, 12 (10), 3508−3519. (20) Rexeisen, E. L.; Fan, W.; Pangburn, T. O.; Taribagil, R. R.; Bates, F. S.; Lodge, T. P.; Tsapatsis, M.; Kokkoli, E. Langmuir 2010, 26 (3), 1953−1959. (21) Mlinar, L. B.; Chung, E. J.; Wonder, E. A.; Tirrell, M. Biomaterials 2014, 35 (30), 8678−8686. (22) Wu, J.; Zawistowski, A.; Ehrmann, M.; Yi, T.; Schmuck, C. J. Am. Chem. Soc. 2011, 133 (25), 9720−9723. (23) Harrington, D. A.; Cheng, E. Y.; Guler, M. O.; Lee, L. K.; Donovan, J. L.; Claussen, R. C.; Stupp, S. I. J. Biomed. Mater. Res., Part A 2006, 78A (1), 157−167. (24) Andukuri, A.; Kushwaha, M.; Tambralli, A.; Anderson, J. M.; Dean, D. R.; Berry, J. L.; Sohn, Y. D.; Yoon, Y.-S.; Brott, B. C.; Jun, H.W. Acta Biomater. 2011, 7 (1), 225−233. H
DOI: 10.1021/acs.nanolett.5b03313 Nano Lett. XXXX, XXX, XXX−XXX
