Article pubs.acs.org/Biomac
Layer-by-Layer Polyelectrolyte Deposition: A Mechanism for Forming Biocomposite Materials YerPeng Tan,†,‡ Umit Hakan Yildiz,§,∥ Wei Wei,⊥ J. Herbert Waite,*,†,‡,⊥ and Ali Miserez*,§,∥,# †
Biomolecular Science and Engineering Program, University of California, Santa Barbara, Santa Barbara, California 93106, United States ‡ Marine Science Institute, University of California, Santa Barbara, Santa Barbara, California 93106, United States § School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore ∥ Center for Biomimetic Sensor Science, Block X Frontier, Research Technological Plaza, Nanyang Technological University, 50 Nanyang Avenue, Singapore ⊥ Materials Research Laboratory, University of California, Santa Barbara, Santa Barbara, California 93106, United States # School of Biological Sciences, Nanyang Technological University, 50 Nanyang Avenue, Singapore S Supporting Information *
ABSTRACT: Complex coacervates prepared from poly(aspartic acid) (polyAsp) and poly-L-histidine (polyHis) were investigated as models of the metastable protein phases used in the formation of biological structures such as squid beak. When mixed, polyHis and polyAsp form coacervates whereas poly-L-glutamic acid (polyGlu) forms precipitates with polyHis. Layer-by-layer (LbL) structures of polyHis−polyAsp on gold substrates were compared with those of precipitate-forming polyHis−polyGlu by monitoring with iSPR and QCM-D. PolyHis−polyAsp LbL was found to be stiffer than polyHis−polyGlu LbL with most water evicted from the structure but with sufficient interfacial water remaining for molecular rearrangement to occur. This thin layer is believed to be fluid and like preformed coacervate films, capable of spreading over both hydrophilic ethylene glycol as well as hydrophobic monolayers. These results suggest that coacervate-forming polyelectrolytes deserve consideration for potential LbL applications and point to LbL as an important process by which biological materials form.
1. INTRODUCTION
The hard tip of the beak (rostrum) is enriched in Gly (27%) and positively charged His residues (11%)12,13 (Figure 1a). When extracting the beak with a denaturing solution of 8 M urea/acetic acid, however, those proteins solubilized exhibit amino acid compositions that are distinct from whole rostrum, with lower His (4%) and Gly (12%) content, but a high aspartic acid (Asp) content of 15%.14 This amino acid composition is similar to that measured in the soft, untanned region,12 suggesting that distinct proteins are processively added to the maturing beak and interact with one another to form the final cured material (Figure 1a). Two models of protein deposition in squid beak have emerged: (a) coacervate and (b) layer-by-layer (LbL). In the first, chitin is impregnated and coated with a fluid−fluid phaseseparated and electrically neutral blend of anionic and cationic proteins (coacervate). In the second, the cationic and anionic proteins are successively applied as layers or films over the
Biomaterials research has two fundamental challenges with respect to better understanding and replicating high performance load-bearing structures such as bone, tendon, silk, and wood: (1) the elaboration of a database of structure−property relationships at multiple length scales, and (2) the elucidation of critical formation processes on which these structures and properties depend. Much progress has been achieved in the first of these, particularly in bone,1−3 tendon,4,5 and silk.6,7 In contrast, processing insights are more elusive: the self-assembly of molecules in silk and tendon formation, for example, is often modeled as a liquid crystalline mesophase,8−11 but these models are difficult to test and involve considerable speculation. Squid beak deserves recognition as a high performance biomaterial: it is the hardest wholly organic material known and consists of chitin, protein, and catechols that are organized to create a functional hydration gradient.12,13 With respect to processing, the tiled chitin scaffold is laid down first, then gradually impregnated with flexible cationic (histidine-rich) and anionic (aspartate-rich) proteins, and cross-linked.14 © 2013 American Chemical Society
Received: November 6, 2012 Revised: April 19, 2013 Published: April 19, 2013 1715
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is phase separated from the bulk solution.16 Complex coacervation has been used for the encapsulation of drugs32 and food flavors.33,34 It has also been proposed as a versatile bioprocessing route by which living organisms produce specific extracellular biological materials.35−37 The capability is exhibited strikingly in the water-resistant adhesive secreted by sandcastle worms,36,38 which they employ to glue together microscale sediments gathered in their surroundings, thereby constructing a protective casing that shields the worm from the external environment. Complex coacervates exhibit two physicochemical characteristics that make them ideally suited for biocomposite processing:37,39 (1) Their relatively low viscosity (compared to polymer melts), low surface tension, as well as shear-thinning characteristics would allow complex coacervates to be injected into and permeate porous networks made of nanoscale fibers. (2) Upon changing their microenvironment such as pH40,41 or salt concentration,41,42 a fluid-to-solid phase change can be triggered by some reactivity such as covalent cross-linking of specific amino acid residues,43 to result, for example, in a hard composite consisting of a nanofibrous network embedded within the coacervate cross-linked matrix. Besides the environmental conditions, formation of soluble complex coacervates is also governed by characteristics of the polyelectrolytes such as mass, charge density, backbone flexibility, and secondary structure.44−46 In order to model the formation of squid beak, we attempted to prepare complex coacervates using polyhistidine and polyaspartic acid polyelectrolytes as synthetic analogs for squid beak’s histidine-rich and aspartate-rich proteins, respectively. Turbidity measurements and light microscopy were used to assess complex coacervate formation. The results show that coacervates (schematics as shown in Figure 2b) can be formed if poly-L,D(αβ)-Asp (polyAsp) is used as a starting material, together with poly-L-histidine (polyHis) (structures shown in Figure 1b,c, respectively). When poly-L-glutamic acid (polyGlu) (Figure 1d) is substituted for poly- L,D (αβ)-Asp, solid precipitation results (schematics as shown in Figure 2b).
Figure 1. Picture of Dosidicus gigas beaks (Top: upper beak, bottom: lower beak) extracted from buccal mass showing the tanning graduation from the stiffest tip to the soft untanned portion. The most abundant amino acids for the tip and the soft untanned region are shown on the right. Structures of polyelectrolytes: (b) polyL,D(αβ)-aspartic acid showing α and β peptide bonds as well as cyclic imide, (c) poly-L-histidine and (d) poly-L-glutamic acid.
chitin. In this study, we compare some of the properties of squid beak-inspired polyelectrolyte films deposited by both processes. A well-known feature of polyelectrolytes is their ability to form complexes with oppositely charged species that are stabilized by electrostatic interactions.15−18 This includes the well-known LbL technique, which yields multilayer films.19−22 Recent advances in multilayer assemblies have shown that the method is flexible and can be expanded to the processing of hollow nanospheres and capsules, which are useful as drug delivery vehicles23−29 and contrast agents for magnetic resonance imaging,30 as well as to create hierarchically organized assemblies that can yield robust nanomaterials.31 Another well-known complex formed by polyelectrolytes is that of complex coacervates which is a dense fluid phase of electrically neutralized cationic and anionic polyelectrolytes that
Figure 2. Schematic representation of (a) individual polyelectrolytes in solution (fully ionized), (b) polyHis−polyAsp and polyHis−polyGlu complexes in solution (soluble and insoluble respectively), (c) polyHis−polyAsp LbL (interface thickness has been exaggerated to highlight the more fluid interfaces) and (d) polyHis−polyGlu LbL. 1716
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Table 1. Characterization of the Polyelectrolytes pKa polyHis polyAsp polyGlu
zeta potential
size (d.nm)
molecular weight
n
free amino acid
polymer (0 mM/70 mM NaCl)
pH 5.0
fully ionized
pH 5.0
fully ionized
16513 5765 34700
108 42 230
6.04 3.9 4.07
5.1/5.6 4.6/4.1 5.8/5.1
19.7 −1.0 −33.8
35.2 −17.0 −21.4
15.2 5.3 41.9
126.0 7.8 15.2
2.3. iSPR. In situ LbL assembly of polyHis and polyAsp on gold substrate (Figure 2c) was monitored by iSPR. The instrument used is the Nanofilm-EP3 from Accurion, Göttingen, Germany, with a xenon lamp as the light source with optical filters. The iSPR cell utilizes the Kretschmann setup for iSPR measurements and consists of a holder for a 60° BK7 prism and a flow cell. Measurements were performed at a wavelength of 740 nm, selected from the spectrum of the xenon lamp by an interference filter. The angle of incidence was 70°. LbL assembly of polyHis and polyGlu was also monitored in the same way as well as preformed coacervates of polyHis and polyAsp. iSPR in ellipsometry mode is an optical method, which is based on the registration of changes in the state of polarization of light after its reflection from the investigated sample. The state of polarization can be described in terms of two parameters Ψ and Δ, representing respectively the ratio of Fresnel reflection amplitudes and phase shift between p- and s-components of polarized light. The method of imaging SPR (in ellipsometry mode) is extremely sensitive to changes in optical parameters of the reflecting substrate, i.e., complex refractive index N = n−ik, where k is the extinction coefficient, as well as to the presence of any coating on its surface. Similar to conventional SPR the values of thicknesses, refractive indices and extinction coefficients of the substrate and coating layers can be found by fitting the ellipsometry data to Fresnel theory. In this work, we elect not to convert the measured parameters into thickness except to obtain the two-dimensional (2D) topology maps, as it is not possible to differentiate between the contribution from polymer adsorption and refractive index change due to density changes as water is removed. For the 2D topology maps, Ψ and Δ signals were fitted with the buildin EP4 software, and the captured images were converted into thickness maps as described elsewhere.48 2.3.1. iSPR Measurements. The iSPR surfaces consisted of 12 mm ×12 mm glass slides coated with a 45 nm thick film of gold, purchased from GE-Healthcare (Biacore), Uppsala, Sweden. Prior to use, iSPR surfaces were modified with a protein resistant surface active molecule; “MeOEG3” (HSCH11(OC2H4)3CH3). MeOEG3 was transferred to the gold surface using μ-contact printing (PDMS stamps with line features). Conditions for PDMS stamp preparation and for μ-contact printing are described elsewhere.46,49−51 For 100 mM acetate buffer, pH 5.0 condition, polyHis (0.1 mg/mL in 100 mM acetate buffer, pH 5.0) was injected into the iSPR flow cell after it had been equilibrated with the same buffer at a flow rate of 0.1 mL/min. After saturation of the iSPR signal, buffer was allowed to flow in to rinse off loosely bound polyHis. Then the polyanion solution (0.1 mg/mL in 100 mM acetate buffer, pH 5.0) was introduced until saturation. This was again followed by buffer rinse. This was repeated four times for the pair of polyHis−polyAsp LbL and three times for polyHis−polyGlu LbL. All flow rates were at 0.1 mL/min. The same set of experiments was repeated for higher salt conditions by replacing the 100 mM acetate buffer, pH 5.0 with 500 mM acetate buffer, pH 5.0 but without MeOEG3 modification of the iSPR surfaces. The structures obtained are as illustrated by the schematics of Figure 2c,d for polyHis−polyAsp and polyHis−polyGlu LbL, respectively. For preformed coacervates, polyHis and polyAsp, were dissolved in 100 mM acetate buffer, pH 5.0 to make 0.1 mg/mL solutions. These were then mixed together at 1:1 ratio just before the experiment and injected into the iSPR flow cell at a rate of 0.1 mL/min for approximately 10 min. Then buffer is passed into the cell to rinse off the loosely bound molecules before flow of preformed coacervates is resumed. This was repeated four times. 2.4. Contact Angle Measurement. Contact angle experiments were performed using the Attension Theta optical tensiometer
We also investigated the feasibility of processing polyHis and polyAsp in an LbL manner (Figure 2c) and compared the results to layered structures made from polyHis and polyGlu (Figure 2d) as well as preformed complex coacervates of polyHis and polyAsp. The process was monitored by imaging surface plasmon resonance (iSPR) and quartz crystal microbalance with dissipation (QCM-D).
2. MATERIALS AND METHODS 2.1. Complex Coacervation from Solution. Poly-L-histidine (polyHis) (Sigma-Aldrich P-9386, Lot 051M5005 V, MW = 16,513), poly-(α,β)-DL-aspartic acid sodium salt (polyAsp) (Sigma-Aldrich P3418, Lot 021M5005, MW = 5,765) and poly-L-glutamic acid sodium salt (polyGlu) (Sigma-Aldrich P4636, Lot 017K5108 V, MW = 34,700) were dissolved separately in acetate buffer of three different concentrations (10 mM, 100 mM and 500 mM) and different pH to obtain 0.5 mg/mL solutions. The following conditions were used: pH 4.5 and 5.0 for 10 mM acetate buffer; pH 4.5, 5.0, 5.5 for 100 mM acetate buffer and pH 5.0 for 500 mM acetate buffer. Different volume ratios of polyHis and polyAsp were then mixed together in plastic disposable cuvettes (Fisher Semimicro Methacrylate), and turbidity measurements taken at 600 nm in a Thermo Scientific NanoDrop 2000c to determine the optimal conditions for complex coacervation of the two polyelectrolytes. Structures of the polyelectrolytes are shown in Figure 1. An alternative to polyGlu which also does not form complex coacervates with polyHis is poly-L(α)-aspartic acid sodium salt (data not shown, MP Biomedical MW = 8905, n = 65). It was not used as it was not available in quantities needed for this project. 2.2. Characterization of Polyelectrolytes. 2.2.1. Circular Dichroism (CD). CD traces of the polyelectrolytes were obtained on a OLIS RSM CD spectrometer. PolyHis, polyAsp, and polyGlu were dissolved in 100 mM acetate buffer, pH 5.0 to obtain 0.5 mg/mL solution and loaded into a clean quartz cuvette individually and spectra obtained from 190 to 250 nm. 2.2.2. Potentiometric Titration. Approximately 5 mg of the polyanions were dissolved separately in 20 mL of Milli-Q water or 70 mM NaCl solution and titrated manually with 0.05 N HCl, with continuous nitrogen gas flow into the solution. Data is collected with a Radiometer Analytical PHM120 pH meter and Mettler Toledo Inlab Micro probe. The same was done for polyHis, but titration was with 0.05 N NaOH. A small amount of 0.05 N NaOH/HCl was added before titration to fully ionize the polyanions/polyHis. Degree of ionization (α) was calculated from the volume of titrant used, taking into account the total charge group residues and initial NaOH/HCl used to fully ionize the polyelectrolytes.47 2.2.3. Zeta Potential. Zeta potential and size of the polyelectrolytes in solution (∼5 mg/mL) in 100 mM acetate buffer at pH 5.0, were obtained using the Malvern Nano ZS, which is calibrated regularly using Malvern Zeta Potential Transfer standard (P/N DTS1230, Batch number 380901). After measurements, 10 μL of 10 N HCl/NaOH were added to the polyanions and polyHis, respectively, to fully ionize all the side chains. Zeta potential and size measurements were then taken of these polyelectrolytes in the fully ionized state. Results are summarized in Table 1. 2.2.4. NMR. About 6 mg of polyAsp was dissolved in 0.8 mL D2O (Cambridge Isotope Laboratories, Andover, MA) by vortex and then transferred to a 5 mm NMR tube. Proton NMR data was collected on a Varian VNMRS 600 MHz with pulse angle of 45°, relaxation delay of 1s, and 32 scans. 1717
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Figure 3. Phase changes associated with complex formation in polyelectrolytes. (a) Turbidity plots at different pH and salt concentration (acetate buffer) for different volume ratios of polyHis and polyAsp mixtures. 100× polarized optical micrographs showing (b) droplets of complex coacervates fluids spread and coalesced on a glass slide as well as spherical droplets floating in solution (white and black dots) and (c) irregularly shaped precipitate solids on a glass slide. (Finland). OneAttension software was used for data collection and analysis. The sessile drop method was used to apply the droplet on the substrate surface: a 3 μL drop was dispensed on the surface, and highresolution images were captured via a 420 fps video camera. Three individual measurements were obtained for each set of data and the mean values calculated. Complex coacervates were prepared by mixing equal amounts of 0.1 mg/mL polyHis and polyAsp solutions. PolyHis and polyAsp were solubilized by dissolving in 100 mM acetate buffer, pH 5.0. The coacervate droplet is allowed to incubate on the surface for about 1 min, then carefully removed and air-dried before water droplet is applied to the same spot for contact angle measurement after surface modification by the coacervate droplet. 2.4.1. Substrate Surface Preparation. Gold-coated (gold layer thickness ∼200 nm) glass slides were used for the substrate (purchased from GE-Healthcare (Biacore), Uppsala, Sweden) in the contact angle experiments. Prior to use, all slides were cleaned with ammonium peroxide mix, then incubated in ethanolic solution of octadecanethiol (ODT) for 24 h to obtain the formation of a selfassembled monolayer on the gold surfaces. 2.5. QCM-D. Gold sensors were purchased from Biolin Scientific, SE (QSX301) and cleaned according to the protocol suggested52 before use. Briefly, the gold surfaces were treated for 10 min to an ultraviolet oxidation (UVO) treatment, followed by 5 min soak in 75 °C ammonium peroxide mix. They were then rinsed with Milli-Q water followed by blow drying with nitrogen gas and finally subjected to another 10 min of UVO treatment. QCM-D experiments were carried out in a Q-Sense E4 system using flow modules in parallel. Samples were flowed into the modules at 0.1 mL/min using a four-channel Ismatec IPC-N4 peristaltic pump. In situ LbL assemblies of polyHis and polyAsp as well as polyHis and polyGlu multilayers were done in a similar manner to that of the iSPR
experiments except that, for QCM-D, the LbL assembly was done in parallel. LbL assemblies were tracked by QCM-D for three different salt conditions at pH 5.0: 10 mM, 100 mM, and 500 mM acetate buffer. In QCM-D, changes in resonance frequency (ΔF) of a quartz crystal are recorded to measure the amount of material deposited onto the sensor and typically includes entrained solvent.53−55 The crystal is excited at its fundamental frequency, approximately 5 MHz, and changes can be observed at the fundamental (n = 1) as well as overtone frequencies (n = 3, 5, 7, 9, 11). The observation from the fundamental frequency is usually not used as it tends to be subjected to artifacts from the sensor clamp.55 A decrease in (ΔF)/n is associated with an increase in hydrodynamic mass (molecules and water) adsorbed onto the crystal surface, which is obtained by the Sauerbrey equation56 for deposited nonviscous films. Energy dissipation due to viscoelastic film adsorption is reflected by the change in dissipation factor, ΔD. A more fluid film will absorb more energy hence higher ΔD values.53−55
3. RESULTS AND DISCUSSION 3.1. Complex Coacervation and Precipitation. Phase changes are well-known for polyelectrolytes and involve transitions between soluble polyions, aggregates, crystallizations, precipitates, and coacervates.57−62 In this work, polyHis and polyAsp are mixed to form complex coacervates. The turbidity plot for aqueous mixtures of polyHis:polyAsp (Figure 3a) shows a maximum at around a volume ratio of 450 μL:550 μL (polyHis:polyAsp) for pH 5.0 at all salt concentrations, indicating that maximum complex coacervation of polyHis and polyAsp occurs at the ratio of 9:11 (0.82). Those polyHis− 1718
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Figure 4. Characterization of the polyelectrolytes (a) CD curves for polyGlu (α-helix), polyAsp (random coil), and polyHis (random coil) at 100 mM acetate buffer, pH 5.0. (b) Potentiometric titration curves of polyHis, polyAsp, and polyGlu at no salt and 70 mM NaCl (equivalent concentration to 100 mM acetate buffer, pH 5.0). The shoulder for polyGlu is indicative of the transition from α-helix to random coil. Degree of ionization (α) was calculated from the volume of titrant used, taking into account the total charge group residues and initial NaOH/HCl used to fully ionize the polyelectrolytes.
Together with the CD spectra (Figure 4a), it is obvious that at pH 5.0, polyGlu adopts a mix of α-helix and random coil secondary structures. This would explain why the zeta potential and size of polyGlu is higher when partially charged (pH 5.0) compared to the fully ionized state (pH 12.5): at pH 5.0, the αhelical structure will spatially presents the ionized groups closer together than a polyelectrolyte adopting a random coil configuration. The α-helical structures will also exhibit a larger hydrodynamic radius. Even though both polyHis and polyGlu are known to adopt an ordered structure at low ionization levels,47,68 polyHis at the condition of 100 mM acetate buffer, pH 5.0, is 75% ionized and beyond the regime of ordered structure. Thus, that polyAsp coacervates with polyHis whereas polyGlu does not is chiefly due to the spatial charge density of the polyelectrolytes. For polyGlu, the charges are so densely spaced that electrostatic interactions with polyHis will be sufficiently extensive to exclude water from the complexes, leading to solid precipitates. This behavior is generally consistent with reports of densely charged polyelectrolytes mixtures.71 A total of three salt concentrations were chosen for the coacervate experiments: 500 mM (seawater ∼ 300 mM), 100 mM (physiological ∼ 150 mM) and 10 mM (low salt). Increasing salt concentration (500 mM vs 100 mM vs 10 mM) results in a broadening of the maximum peak, which is attributed to higher charge screening and ionization of the polyelectrolytes.41 Comparing turbidity as a function of pH at the same salt concentration, we observe that pH increases lead to a shift in the turbidity maximum to the right. This shift suggests that less polyAsp is needed to form charge-neutral complex coacervates as the total charge on polyHis and polyAsp decreases and increases, respectively, with increasing pH. The pH range of 4.5 to 5.5 was selected here as a suitable working pH range as all three polyelectrolytes remain soluble in this range. PolyHis−polyAsp interactions at pH 5.0 in 100 mM acetate buffers were selected for further study by LbL, as this condition is close to the maximum turbidity, and the maximum does not exhibit a sharp decrease with small variations in the ratio of polyelectrolytes.
polyAsp mixtures giving the highest turbidity were scrutinized under light microscopy, and the turbidity was found to be correlated to fluid droplets suspended in the solution. Due to their density, these droplets typically settle and spread on the glass slide (Figure 3b). PolyGlu mixed with polyHis under the same conditions only forms precipitates (Figure 3c). Complex coacervation occurs when two oppositely charged polyelectrolytes assemble to form charge-neutral fluid complexes where the attractive electrostatic force is balanced by the osmotic pressure exerted by the trapped water molecules.37,45,60 This phenomenon implies that in the polyHis−polyAsp system, at pH 5.0, approximately nine polyHis are neutralized by eleven polyAsp within the complexes. NMR spectra (Supporting Figure 1) showed that the polyAsp molecules are not fully hydrolyzed. They are ∼75% hydrolyzed per the ratio of succinimide:bulk methylene peaks as suggested by Wolk et al.63 CD curves (Figure 4a) indicate that polyAsp and polyHis adopt a random coil configuration in 100 mM acetate buffer, pH 5.0 whereas polyGlu exhibits α-helical secondary structure. Figure 4b shows the potentiometric titration curves of the polyanions and polyHis at zero salt and 70 mM NaCl, which represents the equivalence of the salt concentration of 100 mM acetate buffer at pH 5.0. At 70 mM NaCl, the pKa of the polyanions is lower than in zero salt concentration. This trend is expected, since in comparison to single free charge groups, ionization is suppressed among the charged groups in the homopolyelectrolytes due to repulsive interaction between the charged groups.64 Higher salt concentration is then able to screen the repulsive interactions, leading to lower pKa values. For polyHis, the pKa at 70 mM is higher than at 0 mM for the same reason. The measured pKa values at 70 mM NaCl for polyHis (∼5.6), polyAsp (∼4), and polyGlu (∼5) are comparable to previously reported values.47,65−70 At the equivalent of 100 mM acetate buffer, pH 5.0, polyAsp is approximately 60% ionized (Figure 4b). This value is calculated by taking into account that only 75% of the polyelectrolyte is hydrolyzed to carboxylic side chains. PolyGlu is 50% ionized, while polyHis is 75% ionized under this same condition. The shoulder in the titration curve for polyGlu and polyHis (Figure 4b) represents the transition of ordered to random coil secondary structure. These data are summarized in Table 1. 1719
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Figure 5. Polyelectrolytes binding to gold and OEG surfaces as measured by sensograms from iSPR (flow rate: 0.1 mL/min; buffer: 100 mM acetate buffer, pH 5). Orange arrows show the start point of buffer rinse after flow-in of polyelectrolyte solutions. The gray vertical bars show the start of a new injection and the (+) and (−) indicate the charge of the polyelectrolyte solution being injected. There is no charge for preformed complex coacervate solution since it is charge-neutral. Images on the right of the sensograms show the top view of the gold surface before (bottom) and after (top) all layers have been deposited. The thin raised lines in the before images are where the OEG molecules have been deposited using μ-contact printing (a) polyHis−polyGlu LbL: alternating layers of polyHis (+) and polyGlu (−) for a total of six layers. (b) polyHis−polyAsp LbL: alternating layers of polyHis (+) and polyAsp (−) for a total of 8 layers. Dotted box region is expanded to show the S-shape profile when polyHis is laid on polyAsp. (c) Consecutive layers of preformed 0.1 mg/mL complex coacervates interrupted by buffer rinse in between.
When polyHis and polyGlu were mixed together at different ratios under identical conditions (salt and pH) as pHis and pAsp, they did not form coacervates at any of the conditions tested. Instead, the mixture quickly precipitated out of solution, resulting in a clear solution with white particles at the bottom of the cuvettes. Light microscopy showed these white particles to be irregularly shaped solids that can aggregate into 20 μm structures (Figure 3c). Even though precipitation like coacervation also leads to changes in the turbidity of the solution, it is readily distinguished from complex coacervation. During precipitation, the solid particles quickly sediment or form a visible deposit upon microcentrifugation. The difference is particularly striking under light microscopy: precipitates settle onto the glass slide as irregularly shaped objects, whereas coacervates settle as droplets that spread (Figure 3c,b, respectively). 3.2. iSPR Results. Having shown that polyHis−polyAsp form complexes that phase-separate from bulk solution prompted our next question pertaining to polyelectrolyte behavior at solid−liquid interfaces. For example, a succession of soluble polyelectrolytes can be adsorbed to a test surface such as gold, or they can be deposited directly as coacervates. Both would be stabilized electrostatically, but how would the adsorbed quantities, adsorption kinetics, and viscosities of adsorbed films compare? Polyelectrolyte layers made by alternate deposition of soluble polyHis−polyAsp (polyHis− polyAsp LbL) and polyHis−polyGlu (polyHis−polyGlu LbL) on gold were monitored by iSPR in ellipsometry mode. The
measured quantity in an iSPR experiment is the complex reflectance ratio, ρ = Rp/Rs = tan ψeiΔ where Rp and Rs are the complex reflection coefficients for light polarized parallel and perpendicular, respectively, and Δ and ψ are experimentally determined “ellipsometric angles”. The changes in ellipsometric angles Δ and ψ are used to monitor the adsorption process. These changes are related to refractive index changes as polymer molecules are adsorbed, and water molecules are displaced.72 Additionally, molecular rearrangement in the adlayer and adlayer density may change due to water loss, leading to changes in the refractive index and thus ellipsometric angles.64 Figure 5a−c shows that the increase in −Δ upon formation of a new layer is a common feature for both LbL addition of polyelectrolytes and preformed coacervates. The orange arrows on the plots indicate the start of buffer rinse after each successive polyelectrolyte infusion into the SPR cell, whereas the gray vertical lines show the start of polyelectrolyte injections. The topographic images of the surface before and after all layer deposition were obtained by using the modeling software (EP4) to convert the ellipsometric signals into thickness measurements. These two-dimensional images are shown in Figure 5. The images showed a reversal of the surface morphology: OEG-monolayer lines are initially the protruding features, whereas after deposition LbL films are elevated above the printed lines. It should be noted that these images serves only as a representation of the surfaces and do not indicate the true height on the surfaces. 1720
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Figure 6. Total −Δ change versus layers for (a) 0.1 mg/mL preformed coacervates on gold and OEG modified surfaces and (b) polyHis−polyAsp and polyHis−polyGlu LbL at 100 mM and 500 mM acetate buffers.
For both LbL systems, the ellipsometric angle −Δ increases linearly upon alternation of polyelectrolytes (Figure 6b). However the layer formation kinetics for each case differ substantially. PolyHis−polyAsp shows an S-shape kinetic (Figure 5b), whereas polyHis−polyGlu (Figure 5a) displays an instantaneous process. This could be related to the different nature of liquid−liquid and liquid−solid phase separation for polyHis−polyAsp and polyHis−polyGlu, respectively, occurring at the surface. The S-shape kinetics for polyHis−polyAsp LbL is not observed for 500 mM acetate buffer (Supporting Figure 2), which would indicate faster kinetics at this condition due to charge screening. 3.3. Contact Angle Results. The contact angle of water on hydrophobic ODT monolayer was found to be 104 ± 1° (Figure 7), which is in good agreement with previously
A comparison of the sensograms of polyHis−polyGlu LbL (Figure 5a) and polyHis−polyAsp LbL (Figure 5b) in 100 mM acetate buffer, pH 5.0, reveals that both the shape of the curves and the corresponding changes in −Δ are significantly different. PolyHis−polyGlu LbL exhibited a sharp and step-like increase in −Δ for each alternating layer, whereas polyHis−polyAsp LbL showed an S-type of profile when polyHis is laid on top of polyAsp layers. The shape of the sensogram for polyHis− polyGlu LbL (Figure 5a) suggests that LbL formation is an instantaneous process. For polyHis−polyAsp LbL, on the other hand, the S-curve implies a two-step process (with two rate constants) in the complexation of polyHis on polyAsp. We also observed that for polyHis−polyAsp LbL, the OEG modified surface shows trend in −Δ that resemble those on the gold surface, whereas there is almost no change for polyHis− polyGlu LbL. OEG-modified surfaces are known to resist biofouling or nonspecific adhesion,73 and this effect is recapitulated for the pHis−pGlu multilayer. In contrast, pHis−pAsp exhibited significant adhesion to the OEG surface starting from the third injection. For preformed complex coacervates, the slope of the −Δ curve is approximately the same upon addition of each different layer, and the change in −Δ per layer is also similar (Figure 5c), with no major change in −Δ after rinsing. The nonsaturating linear slope of the sensogram during flow of polyHis−polyAsp preformed coacervates suggests that film growth involves adsorbed fluid droplets forming a homogeneous and continuous fluid layer as long as a supply of droplets is present. Figure 6a shows the plot of total −Δ change (after rinsing) versus the layer number for preformed coacervates on gold and OEG surfaces. The values were obtained by averaging the plateau values in Figure 5c. Adhesion by preformed coacervates is observed on both gold and OEG surfaces. However on the gold surface, the increase in −Δ is linear, whereas on the OEG surface, the plot shows signs of saturation. Figure 6b illustrates the total −Δ values after rinsing for the LbLs as each polyHis or polyanion layer is added at 100 mM and 500 mM acetate buffer (pH 5.0) conditions. Overall growth of the LbL is relatively linear for both polyHis−polyGlu and polyHis−polyAsp at these buffer conditions. Acetate buffer (500 mM) results in higher signal for both LbL, which may be due to the higher density of the adsorbed layers as a result of increased charge screening, thus less bound water molecules are likely in the structures.
Figure 7. Contact angle on ODT (hydrophobic) monolayer before (left) and after (right) modification of the surface to be more hydrophilic by polyHis−polyAsp complex coacervate droplet.
published results.74 A slight decrease (by 6 ± 1°) in contact angle was observed for the complex coacervate droplet on the monolayer. After removing the complex coacervate droplet and reapplying a water droplet to the same spot, a large decrease in the contact angle was observed (66 ± 1°), implying decreased surface hydrophobicity after modification by the polyHis− polyAsp complex coacervates. 3.4. QCM-D Results. In QCM-D, hydrodynamic mass (adsorbed polyelectrolyte and water) is sensed by the crystal sensors and reflected by changes in resonance frequency, ΔF. ΔF is thus used to monitor the mass change of adsorbed layers including trapped water molecules on gold QCM-D sensors. 1721
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Figure 8. Polyelectrolyte adsorption to the gold surface of a QCM-D sensor. (a) Sensograms from QCM-D (gold surface sensors). Alternating polyelectrolyte solutions were flowed into the flow cell, interrupted by buffer rinse (orange arrows) in between (flow rate: 0.1 mL/min; buffer: 100 mM acetate buffer, pH 5). (+) and (−) shows the charge of the polyelectrolyte solution being injected. Total mass per unit area for (b) polyHis− polyAsp and (c) polyHis−polyGlu LbL films show an exponential increase for all acetate concentration as an additional layer of polyelectrolytes is deposited onto the gold surface.
the other two curves. This observation suggests that, despite the large range of buffer concentration, approximately the same number of molecules is adsorbed to the surface after each layer, for these polyelectrolytes that coacervates in situ. Overall, the same exponential growth is observed for polyHis−polyGlu LbL (Figure 8c) but a higher mass gain than in polyHis−polyAsp LbL is noticed at all conditions. This is especially obvious at 10 mM acetate, with polyHis−polyGlu showing a much higher rate of mass increase per added layer. (ΔF)/n and ΔD curves for 10 mM and 500 mM acetate buffer, pH 5.0 are shown in Supporting Figure 3. For polyHis− polyAsp LbL, it is observed that the LbL is less rigid (higher ΔD values) during polyAsp adsorption but becomes stiffer during polyHis adsorption (ΔD values fall back to previous level). This trend suggests that during polyAsp adsorption, the layer is more diffuse with somewhat more entrapped water. However, when polyHis is introduced next, these water molecules get forced out quickly (kinetics of ΔD change is approximately the same as (ΔF)/n increase). The trend is particularly apparent for 10 mM acetate buffer. At this condition, charge screening decreases and thus polyAsp layers are more extended and hydrated due to repulsive interactions. Again, the last two layers show a higher increase in ΔD than the earlier layers and are due perhaps to exponential growth of the film that leads to more diffused latter layers. In comparison, polyHis−polyGlu LbL at 10 mM acetate buffer, pH 5.0 shows a
Figure 8a shows the plot of (ΔF)/n and the dissipation factor, ΔD, for overtone number, n = 3,5,7,9,11 versus time for both polyHis−polyGlu LbL and polyHis−polyAsp LbL in 100 mM acetate buffer, pH 5.0. The (ΔF)/n curves for the various overtones are nearly identical, indicating no frequency dependence for ΔF, which in turn indicates a stiff, solid film,75 even for complex coacervates-forming polyHis−polyAsp pair. This is also validated by the small change in dissipation factor (∼2) for the whole duration of the QCM-D experiment (Figure 8a). In fact, most of the increase in ΔD is restricted to the last two layers adsorbed onto the sensor which may be due to the exponential growth of the film (Figure 8b and c), leading to more diffuse latter layers thus more water in the films.76 Figure 8a also shows that the polyelectrolyte layers adsorbed onto the sensor in a stepwise manner, with arrows showing the slight decrease due to buffer rinse. PolyHis−polyGlu LbL displayed a greater change in ΔF for each layer compared to polyHis−polyAsp LbL, suggesting a higher mass increase for the polyHis−polyGlu LbL. Using the Sauerbrey equation,56 ΔF was converted to mass per unit area and replotted in Figure 8b,c to show the total mass adsorbed after each layer. It is observed that for polyHis−polyAsp LbL (Figure 8b), all three buffer conditions displayed an exponential growth in total mass adsorbed as the number of polyelectrolyte layers increase, which implies an exponential-growth-type LbL.77,78 The curves are almost identical with 10 mM showing slight variation from 1722
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to decreased charge screening (Supporting Information, Figure 3). In the 10 mM acetate experiment, polyAsp adsorption is accompanied by an increase in ΔD, which indicates higher fluidity of the layer. When polyHis is adsorbed onto the polyAsp layer, the ΔD signal abruptly drops back to the prepolyAsp level, even though −ΔF is increasing, indicating increased adsorption of material. This observation suggests that water is being extruded from the film as polyHis is adsorbed, and rearrangement occurs due to local complex coacervation of the polyelectrolytes. We thus propose that polyHis−polyAsp LbL contains a liquid phase (coacervate-like) at the nanoscale, which is confined to the interfaces of the polyelectrolyte layers. Even though the mass of polyHis−polyAsp LbL (QCM-D result) adsorbed onto the surface is insensitive to salt concentration (Figure 8b), polyHis−polyAsp LbL in all likelihood depends on electrostatic interactions. The ΔD and Δ signals of the QCM-D and iSPR experiments, respectively, show variations that can be explained by charge screening effect at increased salt concentration. The insensitivity to salt concentration may be due to the use of continuous flow of polyelectrolytes to form the LbL. Debye length (dependence on salt concentration) limits notwithstanding, each layer will be able to reach its maximum saturation of polymers, and any gaps between the same species of polyelectrolytes will be filled in when the next layer is adsorbed and molecular rearrangement occurs. For precipitate-forming polyHis−polyGlu combination, the LbLs display characteristics commonly seen in electrostaticbased LbL:22,29 the viscosity of films is inversely proportional to the salt concentration, and the mass adsorbed shows an inverse relationship due to higher repulsion at low salt. The ability of polyHis−polyAsp LbL but not polyHis− polyGlu LbL to adhere onto OEG-modified surfaces (Figure 5b) provides further important insight into the differences between the complexes. Adhesion of polyHis−polyAsp LbL on OEG surface could be due to surface modification by the polyHis and polyAsp layers during the initial two injections. It is speculated that fluidic complex coacervates formed at the interface of the LbL on the gold surface could spread over the OEG and thus modify its surface.39,82 This assumption is further supported by the behavior of preformed coacervates on gold and OEG surfaces. The build-up of preformed coacervate layers on the gold surface is linear with little change after rinsing, indicating that the fluidic coacervates are coalescing into a homogeneous liquid film on the gold surface. In addition, build-up of preformed coacervates on the raised OEG lines displayed a saturating trend. This suggests that the structure tends to flow down toward the valleys between the OEG lines, again confirming the fluidic nature of the film formed by preformed coacervates. The ability of polyHis−polyAsp complex coacervates to modify surfaces is substantiated by the contact angle experiment, which clearly shows a switch of the monolayer ODT hydrophobic surface into a more hydrophilic one (Figure 7). In spite of the short contact time (∼1 min), the coacervate phase was able to accumulate on the hydrophobic surface and alter its surface properties significantly. The growth pattern of the LbL assembly after addition of successive layer differs significantly when assessed by iSPR or QCM-D experiments. In QCM-D, the assemblies of both LbL display a clear exponential growth pattern (Figure 8b), suggesting either complexation at the interface83 or diffusion of the underlying polyelectrolytes.77,81 This behavior is not as
structure typical of polyelectrolytes at low salt concentration: each layer is diffused (high ΔD and spreading of (ΔF)/n curves) due to repulsive interactions between the same ionized molecules, and interaction between adjacent layers is purely due to electrostatic attraction between the topmost ionized groups of a bottom layer and the bottom-most ionized groups of a top layer. At high salt concentration, the layers are less diffuse with lower ΔD and ΔF values. 3.5. Discussion. Asp and Glu are similar amino acids, differing only in having an extra carbon (β-carbon) in the side chain of Glu. There are, however, more substantial differences between their homopolymers. PolyAsp adopts a random coil secondary structure in solution and may include unhydrolyzed succinimide side groups depending on manufacturing process.79,80 PolyGlu on the other hand, adopts an α-helical structure when uncharged and transitions into random coil with increased ionization (Figure 4a). This structure is also reflected by the unusual zeta potential and size exhibited by polyGlu in solution. When partially ionized (pH 5.0), polyGlu displays a higher zeta potential and size than at the fully ionized pH (12.5, Table 1). The α-helical secondary structure of polyGlu likely results in higher charge density than would be the case in the random coil and lead to a larger hydrodynamic radius as well. Still, polyAsp and polyGlu are the most similar polyanions commercially available, and polyGlu is considered a good homologue of polyAsp. Complex coacervation of polyHis and polyAsp is clearly electrostatic in nature as the ease of complex coacervation increases with increasing salt due to charge screening. This is reflected by the increasing turbidity peak width with increasing salt concentration (Figure 3a). Zeta potential of the complex coacervates also correlates well with the turbidity of the mixture with zero zeta potential when turbidity is highest, showing that complex coacervation is maximum when the complex is fully neutralized (Supporting Information, Figure 4) PolyHis−polyGlu mixtures in solution, however, only result in precipitates under identical conditions (ratios and buffer) used for coacervation of polyHis−polyAsp. Comparing the two polyanions, one would predict that the solution behaviors of the two systems are governed by the spatial charge distribution of the polyanions. Even though polyAsp is slightly more ionized than polyGlu at pH 5.0, polyGlu displays a higher zeta potential, indicating that its charges are spatially more concentrated than polyAsp. As stated earlier, this higher charge density is probably due to the partial α-helical structure adopted by the polyGlu. In polyHis−polyAsp LbL, the S-shape kinetics observed in iSPR for polyHis (Figure 5b) and the stepwise increase for QCM-D data indicate that during polyHis layer deposition, molecular rearrangement occurs upon rinsing and leads to an increase in −Δ (Figure 5b) but not to any increase in −ΔF (Figure 8a). This behavior was observed for each polyHis layer and suggests that the further increase in −Δ during the buffer rinse is probably due to a change in refractive index of the adsorbed film, and not to any real change in the amount of polyelectrolytes adsorbed. A similar behavior was reported by Lavalle et al.81 where diffusion of the polyelectrolytes led to continuous film restructuring, inducing changes in the refractive index of the whole film. For polyHis−polyAsp LbL, the change in refractive index is likely due to a change in the density of adsorbed polyelectrolyte on the surface.64 This is borne out by the QCM-D signals where the trend is most obvious for 10 mM acetate buffer due 1723
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genes into the fluidic interfaces of LbL assemblies or to generate hollow capsules with thin, flexible walls.22,29
obvious for the polyHis−polyAsp iSPR results (Figure 6b), which instead displayed a more linear growth pattern for polyHis--polyAsp LbL. The relative magnitude between polyHis−polyAsp LbL and polyHis−polyGlu LbL is also different depending on monitoring technique: for iSPR, polyHis−polyAsp LbL −Δ values are higher than polyHis− polyGlu LbL, whereas QCM-D shows polyHis−polyGlu LbL having higher −ΔF values. This phenomenon can be explained by polyHis−polyAsp LbL containing a liquid phase sandwiched between two polyelectrolytes layers. The water molecules thus trapped are detected by QCM75 but not iSPR, leading to iSPR underestimating the film growth for polyHis−polyAsp LbL. Conversely, these observations suggest that polyHis−polyGlu LbL is a solid film with even distribution of water molecule trapped within the layers. Our results lead us to conclude that even though both types of polyelectrolyte LbL are driven by electrostatic interactions, their kinetic response can differ widely even for ostensibly similar systems such as polyHis−polyAsp and polyHis− polyGlu, and that this behavior can be predicted from their complexation in solution. The prediction of LbL formation based on the behavior of precursors in solution is an emerging concept.84,85 This study shows that complex coacervateforming precursors (polyHis−polyAsp) form LbLs that are different from solid precipitate-forming polyHis−polyGlu. The formation of complex coacervates by polyHis and polyAsp homopolyelectrolytes in the biologically relevant pH of 4.5 to 5.5, provides support for the complex coacervation of the histidine-rich and aspartate-rich proteins in the naturally occurring squid beak system. With respect to protein secretion and permeation, it can be plausibly claimed that complex coacervation is more favorable than precipitation in introducing macromolecule complexes to the chitin network. The fluidic nature of complex coacervates allows the complexes to spread and permeate the porous network with a dense concentration of proteins. Furthermore, the ability of complex coacervates to modify the interfacial energy provides an avenue for the squid to prepare the hydrophobic chitin surface for subsequent aqueous protein spreading. Controlling the charge density at the genetic level or by local pH changes would allow squid to form different phases from mixtures of similar proteins, allowing it to switch from dilute solutions to dense coacervates to solids. Furthermore, the ability for polyHis and polyAsp to form LbL films that retain liquid characteristics at the interface implies that another way to incrementally process a structure such as a beak is for the His-rich proteins to be laid down onto Asp-rich proteins that were initially secreted with the chitin as the beak matures. This is supported by the observed bias toward aspartate in the untanned region of the beak (in contrast to the rostrum which is histidine rich), n.b., the initial Asp content is still present but its concentration diluted by the His-rich addition. Further investigations using native or recombinant beak proteins are being performed in order to validate this possibility. The ability for the adsorbed polyelectrolytes to maintain a fluidic behavior while preventing precipitation opens interesting avenues that would be useful for various biomaterials and processing applications. This extra degree of control would, for instance, allow control of the flow of coacervates in a spatially controlled fashion in the context of fluidics experiments, to incorporate soluble small molecules such as drugs, dyes, or
4. CONCLUSION This study provides support for the hypothesis that the hard beaks of squids (and cephalopods in general) can be built up by either depositing coacervates of His- and Asp-rich proteins or by adsorbing the proteins one layer at a time in an LbL manner, although a more accurate biological understanding requires the elucidation of squid beak formation in real time with actual precursor proteins. Using polyHis and polyAsp as mimics of squid beak proteins, our data indicate that these polyelectrolytes are able to form complex coacervates, both in solution as well as in adsorbed films such as an LbL assembly. In the latter case, these coacervate-forming polyelectrolytes are shown to form LbL increments that retain characteristics of the fluidic complex coacervates, that is, dehydrated layers that still enable molecular rearrangement. LbLs from coacervate-forming precursors are relevant to biomaterial processing because they possess useful interfacial properties that can be exploited in drug delivery and nanoparticle encapsulation. However, the relationship between complex coacervation in the formation of solution macrophases and as LbL microphases needs to be further explored.
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ASSOCIATED CONTENT
S Supporting Information *
Figures of the following: Proton NMR spectra of polyAsp in D2O; plot of iSPR signal for polyHis−polyAsp LbL and polyHis−polyGlu LbL at 500 mM acetate buffer; pH 5.0, plot of QCM-D frequency and dissipation versus time for both LbLs at 10 mM and 500 mM acetate buffer, pH 5.0; turbidity plot overlaid with zeta potential and size for polyHis−polyAsp complex coacervation at 10 mM acetate buffer, pH 5.0. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (J.H.W.);
[email protected]. sg (A. M.). Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to thank Prof. Bo Liedberg for discussion about iSPR and QCM-D results, and Shahrouz Amini, Yanli Yang, and Matt Menyo for help with micrographs, QCM-D, and NMR respectively. This research is supported by the Singapore National Research Foundation (NRF) through an NRF Fellowship (A.M.) and National Institutes of Health Grant R01-DE018468 (J.H.W.). The project made use of the UCSB MRL Shared Experimental Facilities, which are supported by the MRSEC Program of the NSF under Award No. DMR 1121053, a member of the NSF-funded Materials Research Facilities Network. 1724
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dx.doi.org/10.1021/bm400448w | Biomacromolecules 2013, 14, 1715−1726
