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Electrostatic Self-Assembly of Soft Matter Nanoparticle Cocrystals with Tunable Lattice Parameters Ville Liljestro¨m,†,‡ Jani Seitsonen,‡ and Mauri A. Kostiainen*,† †
Biohybrid Materials, Department of Biotechnology and Chemical Technology, and ‡Molecular Materials, Department of Applied Physics, Aalto University, 00076 Aalto, Finland
ABSTRACT Atomic crystal structure affects the electromagnetic
and thermal properties of common matter. Similarly, the nanoscale structure controls the properties of higher length-scale metamaterials, for example, nanoparticle superlattices and photonic crystals. Electrostatic self-assembly of oppositely charged nanoparticles has recently become a convenient way to produce crystalline nanostructures. However, understanding and controlling the assembly of soft nonmetallic particle crystals with long-range translational order remains a major challenge. Here, we show the electrostatic selfassembly of binary soft particle cocrystals, consisting of apoferritin protein cages and poly(amidoamine) dendrimers (PAMAM), with very large crystal domain sizes. A systematic series of PAMAM dendrimers with generations from two to seven were used to produce the crystals, which showed a dendrimer generation dependency on the crystal structure and lattice constant. The systematic approach presented here offers a transition from trial-and-error experiments to a fundamental understanding and control over the nanostructure. The structure and stability of soft particle cocrystals are of major relevance for applications where a high degree of structural control is required, for example, protein-based mesoporous materials, nanoscale multicompartments, and metamaterials. KEYWORDS: nanoparticle . self-assembly . supramolecular interactions . crystal . dendrimer . ferritin . protein cage
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s described by Pauling's rules, the crystal structure of atoms in a binary ionic crystal is critically dependent on their size ratio, charge valence, and type of bonding.1 The atomic-level organization has far-reaching consequences in bulk, affecting the fundamental electromagnetic, optical, and thermal properties of matter. The phenomenon where properties arise from the crystallographic arrangement spans different material length scales from atoms to large colloidal particles and macroscopic objects.2,3 In order to achieve control over the materials properties, focused research efforts have been made to find similar rules to those for atoms that would govern the assembly of binary crystals consisting of much larger building blocks, such as ligandstabilized nanoparticles and colloidal polymer particles.47 Especially crystals consisting of DNA-functionalized nanoparticles8,9 and patchy particles10,11 have proven to be well controlled, but for other material LILJESTRÖM ET AL.
types the self-assembly of long-range threedimensional crystalline two-component structures remains a major challenge. The crystal structure and unit cell size are dependent primarily on the properties of the nanoparticles, but also the crystallization conditions have been found to affect the final structure.1215 Here we demonstrate a systematic series of binary soft particle cocrystals, where we modulate the particle size ratios and electrostatic binding strengths to yield cocrystals with unprecedented long-range order. This involves using different generations of poly(amidoamine) dendrimers (PAMAM) and apoferritin protein cages as building blocks, which have extremely well-defined dimensions. Biohybrid structures that combine the extreme versatility of synthetic materials and the well-defined structure of biomolecules would in principle offer predictable crystal structures with custom properties.1620 VOL. XXX
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* Address correspondence to mauri.kostiainen@aalto.fi. Received for review August 7, 2015 and accepted October 23, 2015. Published online 10.1021/acsnano.5b04912 C XXXX American Chemical Society
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ARTICLE Figure 1. Schematic presentation of the self-assembling soft nanoparticles. (a) aFT and PAMAM dendrimer generations G2G7 drawn to scale. (b) At low ionic strength the anionic apoferritin forms a cocrystal together with the cationic PAMAM dendrimer. At high ionic strength free counterions screen the electrostatic interaction, yielding a solution with free particles. (c) Dendrimer size can be used to control the lattice constant a. (d) The dendrimer generation (G) and ionic strength [NaCl] present at the assembly affect also the crystal symmetry.
Dendrimers are synthetic polymers with a sequentially branched monodisperse molecular frame, which can be synthesized in different generations with a wide range of molecular masses.21,22 Dendrimers form a specific class of nanoparticles with a soft and flexible structure23 that is responsive to changes in the chemical environment.2426 Due to their highly monodisperse and chemically tunable structure, dendritic molecules have been utilized extensively in hierarchically ordered nanomaterials.27,28 Controlling the primary chemical structure of self-assembling dendrons and dendrimers allows a high control of the resulting crystal structure both in liquid crystalline and solid crystalline form of the dendritic systems.2933 The diameter and the net surface charge of PAMAM dendrimers depend on the dendrimer generation, and it has been demonstrated that they can function efficiently as spacers to control the physical properties of different nanoparticle systems.3436 Due to their wellknown characteristics and spherical shape, we concentrate in this study on cationic PAMAM dendrimers (Figure 1a and Figure S1), which at neutral pH carry a highly protonated primary amine surface. Ferritins are a ubiquitous class of iron storage proteins, commonly consisting of 24 protein subunits that form a hollow spherical protein cage with an outer diameter of 12.5 nm and 432-point-group symmetry.37 Apoferritin (aFT) from Pyrococcus furiosus is known for its ability to sequester inorganic material from solution and form nanoparticles inside its protein cage.38 This biomineralizing property makes aFT an attractive building block when designing higher order LILJESTRÖM ET AL.
nanostructures.39,40 At neutral pH aFT carries a net negative charge (pI ≈ 4.5), which forms a patchy electrostatic potential map on the surface of the protein cage, where especially ion channels carry a negative electrostatic potential.41 aFT has been used as a nanoparticle component to create higher order crystalline structures together with dendrons,42,43 diblock copolymers,44 metal ions,45 cationic metal nanoparticles,46 and fusion proteins.18 However, aFT crystallizes also without the addition of other components (than electrolyte), forming an fcc lattice (a = 183.0184.8 Å, space group F432).4749 In this study we combine these two types of particles, which are monodisperse but still very different in their properties: ferritins are genetically encoded rigid protein particles with anionic patches, and PAMAM dendrimers are synthetic globular and flexible particles with a highly cationic surface, smooth charge distribution, and generation-dependent net charge. The electrostatic interactions on the nanoscale have previously been intensively studied;50 however, we focus on the higher order structures that arise from the self-assembly of charged nanoparticles. PAMAM dendrimers have been used previously as soft spacers to control the distance between nanoparticles,34,35 which suggests that they could also be used to control the lattice constant and crystal symmetry in soft binary crystals. We show that aFT and PAMAM efficiently form cocrystals, where the lattice parameters and crystal stability are controlled by the choice of particles and the electrolyte conditions (Figure 1). VOL. XXX
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ARTICLE Figure 2. Phase separation behavior of aFT-PAMAM assemblies. (a) aFT and PAMAM particles form a white band (scattering from large assemblies) in a vertical electrolyte gradient. (b) Light scattering intensity measured from aFT complexed with PAMAM-G2 and PAMAM-G7 at various NaCl concentrations. High count rate at low ionic strength indicates the presence of large assemblies, and low scattering at high ionic strength indicates the presence of free particles. (c) Volume-average size distributions for aFT (black line) and aFT complexed with PAMAM-G7 at low (red line) and high (blue line) NaCl concentration. (d) Recapitulation of the threshold concentrations (ct) shows that higher PAMAM dendrimer generations form more stable selfassemblies than lower dendrimer generations and disassemble at a higher ionic strength. ct is indicated by the dashed line in b.
RESULTS AND DISCUSSION Free ions in solution screen the electrostatic interactions between charged nanoparticles. Ionic strength is therefore one of the key parameters affecting the electrostatic self-assembly of nanoparticles.46,51,52 The effect of electrolyte concentration was demonstrated in the reversible assemblydisassembly cycle of aFTPAMAM superstructures. Low ionic strength promoted the formation of larger structures that phase separated from the solution and high ionic strength induced solvation of phase-separated nanoparticle assemblies. Even by visual inspection it was clearly observed that at low (∼0 mM) ionic strength combining equal amounts of aFT and PAMAM (generations 27) yielded a rapid formation of large complexes, whereas mixing the particles at high ionic strength (∼300 mM) did not show any formation of visible structures or aggregates. It was also noticed that addition of a NaCl solution into the aFT-PAMAM complex made it dissolve to a clear solution, whereas further dilution with water again caused complexation. A simple demonstration of the ionic-strength-dependent assemblydisassembly behavior was done by placing a nonassembled aFTPAMAM solution with high ionic strength at the bottom of a capillary followed by stepwise addition of sodium chloride solution and finally pure water on top, leading to a vertical gradient of sodium chloride concentration in the capillary. The gradient yielded a sharp white line of large complexes at the vertical position where ionic strength was low enough for assembly to occur (Figure 2a). The line of complexes was stable for hours, until the electrolyte concentration gradient was washed out by diffusion. To acquire a qualitative understanding of the nanoparticle assembly behavior as a function of ionic strength, we carried out a set of dynamic light scattering (DLS) titration experiments. The scattering count rate, which indicates the amount of large assemblies in the solution, was used to monitor the advancing LILJESTRÖM ET AL.
assembly of the aFT-PAMAM structures. When titrating PAMAM into an aFT solution, a clear increase in both the count rate and the mean particle size was observed, showing the formation of large aFT-PAMAM complexes (hydrodynamic diameter, Dh > 1 μm), which did not significantly increase in size after a mPAMAM:maFT ratio of 0.4 was reached (Figure S2). Titrating sodium chloride into the aFT-PAMAM solution caused a distinctive decrease in the count rate at the dendrimer-generation-dependent sodium chloride threshold concentration ct, due to the disassembly of the aFT-PAMAM assemblies (Figure 2b). Here, ct is defined as the concentration where the count rate is 50% of the maximum measured count rate. Finally, at high enough ionic strength, the free aFT particles were identified also from the DLS particle size distribution (Figure 2c), where the measured Dh for aFT is ∼12 nm. On the other hand, when mixing aFT with PAMAM dendrimers at high ionic strength, the count rate was not observed to increase. The size distribution was similar to that of a pure aFT solution showing primarily the presence of single aFT particles; that is, the nanoparticles were present as free particles in the solution as the electrostatic attraction between them was efficiently screened by the electrolyte. However, when the ionic strength of the solution was lowered by titrating with pure water, the particles assembled to larger structures at ct and the scattering count rate increased strongly. The assemblydisassembly process was independent of the dendrimer generation used, except for the lowest generations G0 and G1, which did not efficiently form large secondary complexes together with aFT. However, the ct increased monotonically as a function of the dendrimer generation for G2G7 (Figure 2d) and ranged between 39 mM (G2) and 180 mM NaCl (G7). Considering the dendrimer structure and number of charged groups (see the Supporting Information, Figure S1 and supplementary notes) yields that the VOL. XXX
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ARTICLE Figure 3. SAXS profiles of aFT-PAMAM crystallites. (a) SAXS profiles of crystallites with second- and third-generation PAMAM show only the face-centered structure. Crystallites with the fourth-generation dendrimers show either face-centered or hexagonal structure depending on the ionic strength. Crystallites with G5G7 dendrimers have a hexagonally packed structure. When using higher dendrimer generations, the diffraction peaks are observed to shift to smaller q-values, showing the increase in the lattice constant. (b) Miller indices of assigned reflections for the fcc (left) and hcp (right) structures versus measured q-vector positions for indexed peaks. Solid line presents a linear fit. (c) Interparticle distance dcc for aFT increases with higher dendrimer generations. (d and e) SAXS data compared to the theoretical scattering structure factor S(q) and fitted model (measured data shifted in y-direction for clarity). Space group and unit cell dimensions are Fm3m; a = 20.7 nm in d and P63/mmc; a = 14.9 nm; c = 25.1 nm in e. Detailed SAXS analysis is presented in the Supporting Information.
surface charge concentration of the dendrimers is proportional to 2G/3. This also describes well the ct dependence on dendrimer generation, suggesting that the electrostatic interaction (leading to assembly) can be seen as the mutual attraction of the surface charges that is screened when counterion distribution balances out the surface charge concentration. When the counterion concentration balances out the high surface charge concentration, the electrostatic interaction between the oppositely charged nanoparticles vanishes and the complexes fall apart. This suggests that the assemblydisassembly threshold concentration is more likely proportional to the surface charge concentration than the net charge of the nanoparticles. Periodic nanostructures were achieved by combining aFT with different PAMAM generations in correctly balanced electrolyte concentration. It is known from previous studies on similar systems46,53,54 that electrolyte concentration is the key in achieving well-ordered electrostatic assemblies of oppositely charged nanoparticles, and therefore we tested different electrolyte concentrations in which the superstructures were assembled. Small-angle X-ray scattering (SAXS) was utilized to evaluate the degree of ordering in the aFT-PAMAM superstructures. For all aFT-PAMAM combinations that showed a tendency to self-assemble, the highest degree of crystalline order appeared at moderate electrolyte concentrations compared to the ct, i.e., at sodium chloride concentrations of 1860 mM, LILJESTRÖM ET AL.
which correspond to 1545% of the estimated ct. An exception to this was the combination of aFT and generation 2 PAMAM, which was the lowest dendrimer generation to self-assemble with aFT and showed a lower degree of crystalline order at all of the probed ionic strengths (Figure 3). Importantly, intense diffraction spots, similar to those observed in, for example, protein crystallography, were dominating the 2D scattering pattern of some of the samples, indicating that the size of the crystallites in the solution approached macroscopic length scales (Figure 4). As aFT alone is known to crystallize into close packed fcc structure, the observed aFT-PAMAM crystallites were also assumed to form similar structures, i.e., structures that resemble the close packing of spheres, which are the simple cubic (sc), body-centered cubic (bcc), face-centered cubic (fcc), and hexagonal closepacked (hcp) crystal structures. The relative diffraction peak positions were in good agreement with closepacked sphere models, but the observed crystal symmetries were not the same for all combinations of aFT and PAMAM generations 27 (Figure 3a). That is, apoferritin and PAMAM-G2 and -G3 samples showed exclusively fcc crystal structure (space group Fm3m; number 225, (hkl) = (111), (200), (220), (311), (222); √ √ √ q:q* = 1, (4/3), (8/3), (11/3), 2). Apoferritin-PAMAM (G4) assemblies had fcc crystal symmetry when formed at low (cNaCl = 20 mM) ionic strength. However, assembly at higher (cNaCl = 40 mM) ionic strength yielded a highly crystalline hcp crystal structure (space group VOL. XXX
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√ P63/mmc; number 194; c/a = (8/3), (hkl) = (100), (002), √ (102), (110), (200), (112), (004), (202); q:q* = 1, (9/8), √ √ √ √ √ (27/16), 3, 2, (33/8), (9/2), (123/24)), which is identified by the relative peak positions and the number of identified peaks. All of the first eight allowed reflections were identified from the scattering profile. Further measurements showed that the hcp structure was prevalent also when assembling aFT with PAMAM generations 5, 6, and 7. The lattice constants were determined from SAXS profiles by adapting a linear fit to the identified (hkl) peak positions (Figure 3b). For a cubic lattice the lattice constant a can be calculated from a = (2π/q(hkl)) √ 2 (h þ k2 þ l2) and for a hexagonal close packed √ lattice a = (2π/q(hkl)) ((4/3)(h2 þ hk þ k2) þ 3l2/8). The experimental scattering patterns for fcc and hcp structures (blue and green traces) match well with the respective theoretical scattering patterns (orange trace for a fitted model and black trace for the scattering structure factor S(q)) (Figure 3c,d). The center-to-center distance (dcc) of aFT particles was calculated by assuming that aFT is positioned at the lattice points of the fcc √ or hcp crystals (for an fcc structure dcc = a/ 2 and for LILJESTRÖM ET AL.
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Figure 4. Single-crystallite characteristics observed by SAXS and cryo-TEM. (a) 2D detector view of SAXS intensity measured from large aFTþG3 (18 mM) crystallites with fcc crystal symmetry showing clear diffraction spots. (b and c) cryo-TEM images of large crystalline regions of aFTþG6 60 mM NaCl (b) and aFTþG3 32 mM NaCl (c) showing faceted assemblies. Scale bars are 200 nm. Inset in b: Crystals observed with an optical microscope with sizes approaching 10 μm. Scale bar is 10 μm. Inset in c: Magnification showing the ordered structure.
an hcp structure dcc = a). As expected, the data showed that increasing the dendrimer size increased the lattice constant in the crystalline assemblies. For fcc crystals (PAMAM-G2 to -G4) the lattice constant increased from 14.7 nm to 15.9 nm, and for hcp crystals (PAMAM-G4 to -G7) the lattice constant increased from 14.5 nm to 16.5 nm. The calculated dcc of aFT particles increases linearly with the dendrimer generation from 14.5 nm to 16.5 nm (Figure 3c). Taking into account the dendrimer size, the hcp crystal structure observed for generations 4 to 7 had a significantly higher packing fraction of aFT particles than would be expected when comparing to the fcc structures obtained with the lower dendrimer generations. This is clearly observed with generation 4, where both fcc and hcp structures were observed, and the hcp structure showed a roughly 10% smaller dcc compared to the fcc crystal structure. The deviation in aFT packing fraction and the change in the crystal symmetry are explained by the higher charge of large dendrimers, which implies that the oppositely charged particles have to adopt a different crystal structure to balance the net electric charge of the unit cell. Under optimized conditions the crystals were observed to grow several micrometers in diameter. The presence of multiple large crystals manifests as scattering spots in the 2D SAXS pattern (Figure 4a). In order to support the interpretation of SAXS data, cryogenic electron microscope (cryo-TEM) imaging of selected highly crystalline samples, for example, aFT complexed with G6 and G3, was carried out. The size of the observed crystallites typically approached micrometer length scales and showed the typical appearance of single crystals with different facets (Figure 4b,c). Even larger structures were observed in the cryo-TEM; however, they are not transparent enough for the electron beam, and these structures were observed as black areas (Figure S7d). The majority of the large, above micrometer sized, crystallites are also effectively removed from the TEM grid in the sample preparation process, where the excess of solution is drained from the grid. However, the large crystals can be clearly observed with an optical microscope, which shows the presence of hexagonal and cubic crystals with diameters up to 10 μm (Figure S8). The typical crystallographic features, such as dcc and various crystal projections, for the different crystal structures were identified from the cryo-TEM images (Figure 5). Assemblies in the aFT-PAMAM-G2 sample, which according to SAXS had an intermediate average crystallite size, were observed to have a sheet-like structure morphology with hexagonal symmetry. This matches with the appearance of the [111] direction of an fcc structure (Figure 5a). The observed aFT-PAMAMG2 structures consist of mono- and multilayers, where the structures form one crystal plane at a time shown by the partly formed second layer on top of a wide
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ARTICLE Figure 5. Structural details obtained from the cryo-TEM images. (a) Top: aFTþG2 shows single and multilayer hexagonal sheet structures. Middle: Schematic view along fcc [111] zone axis (left) and inverse Fourier transform from selected Fourier components of selected single (1) and double (2) layer areas. Bottom: Integrated profile along the indicated blue line yielding an aFT center-to-center distance of 15.0 nm. (b) Top: Large cubic lattices consisting of aFTþG3. Inset: Schematic presentation of an fcc lattice faceted by {111} planes viewed along the [100] zone axis. Middle: Magnified view along the fcc [100] zone axis with the unit cell highlighted in blue. Inset shows the fast Fourier transform. Bottom: Schematic view along the [100] zone axis (left) and a filtered inverse Fourier transform from selected Fourier components. (c) Top: Large hexagonal lattice consisting of aFTþG6. Middle: Magnified view along the hcp [110] zone axis with the unit cell highlighted in blue. Inset shows the fast Fourier transform. Bottom: Schematic view along the hcp [110] zone axis (left) and a filtered inverse Fourier transform from selected Fourier components. Scale bars are 200 nm.
monolayer. The average period measured from the TEM image is 15.0 ( 1.0 nm, which corresponds well with aFT dcc = 14.7 nm measured by SAXS. It has been reported that crystal growth of pure aFT crystals also occurs in the Æ111æ directions, forming hexagonal {111} planes,55 which suggests that also the assembly of the aFT-PAMAM cocrystals is similarly directed by the structure of aFT. Larger aFT-PAMAM crystallites formed with the higher generation dendrimers (G3 and G6) show clear crystal facets (Figure 5b,c), which also result from anisotropic crystal growth where the crystal formation is the fastest in defined directions. Also typical fcc [100] and hcp [110] projections could be clearly identified, which aided the identification of the crystal structure. CONCLUSION We have shown that electrostatic self-assembly of aFT and PAMAM can be used in a simple manner to create two-component soft nanoparticle crystals with excellent structural order in aqueous solutions. The resulting crystals are sensitive to the ionic strength and LILJESTRÖM ET AL.
can be reversibly assembled and disassembled. Depending on the dendrimer generation, the electrolyte resistance can be either above or below 150 mM, which is the typical physiological ionic strength. Using building blocks with varying size ratios allows the production of cocrystals with different symmetries, which depend on the particle size and charge ratio. Importantly, two different crystallographic symmetries (fcc and hcp) are allowed (not simultaneously) for the same set of particles, if the ionic strength at assembly is varied. Previous works demonstrate pathways to achieve well-defined nanoperiodic structures and independent control of different parameters, such as particle size, lattice parameters, and crystallographic symmetry in synthetic materials.8,9,11,31 Our concept of using welldefined soft nanoparticles opens up new dimensions for tuning the structural properties, such as crystal symmetry, lattice parameters, selective functionalization, and electrolyte resistance, of biologically relevant soft matter. The results shown here provide systematic insight into the coassembly of proteinpolymer nanoparticle VOL. XXX
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MATERIALS AND METHODS Materials. Apoferritin from Pyrococcus furiosus in a doubledistilled aqueous solution was supplied by MoLiRom (www. molirom.com or MoLiRom srl, Rome, Italy). Poly(amidoamine) dendrimer generation 17 stocks were supplied by Dendritech (www.dendritech.com or Dendritech, Inc., Midland, MI, USA). Dynamic Light Scattering. The hydrodynamic diameter and scattering intensity were measured using a table-top DLS device (Zetasizer Nano Series, Malvern Instruments) with a 4 mW HeNe ion laser at a wavelength of 633 nm and an avalanche photodiode detector at an angle of 173. Experiments were carried out at 25 C. For titration series, the initial sample contained aFT as an aqueous solution (0.15 mg mL1) to which the PAMAM solution (0.15 mg mL1) was sequentially added until a mass ratio m(aFT): m(PAMAM) of 1:1 was reached. Samples were thoroughly mixed after each titration step. The measurements were carried out in Plastibrand semimicro PMMA cuvettes. Zetasizer software (Malvern Instruments) was used to obtain the scattering intensity (count rate) and particle size distributions. DLS data were not further treated, except for the normalization of the count rate, which was carried out by multiplying with a normalization constant Cv to compensate for the dilution of the sample during the titration (Cv = volume/initial volume). Small-Angle X-ray Scattering. The samples were prepared by adding 1.25 μL of NaCl water solution in 5.0 μL of aFT solution (10 mg mL1) to adjust the ionic strength, after which 5.0 μL of PAMAM solution (10 mg mL1) was added under stirring. The liquid samples were sealed between two Kapton foils during the SAXS measurements, and the sample environment was evacuated to reduce scattering from air. The SAXS was measured using a rotating anode Bruker Microstar microfocus X-ray source (Cu KR radiation, λ = 1.54 Å). The beam was monochromated and focused by a Montel multilayer focusing monochromator (Incoatec). The X-ray beam was further collimated by a set of four slits (JJ X-ray), resulting in a final spot size of less than 1 mm at the sample position. The scattered intensity was collected using a Hi-Star 2D area detector (Bruker). Sample-to-detector distance was 1.59 m, and a silver behenate standard sample was used for calibration of the length of the scattering vector q. Onedimensional SAXS data were obtained by azimuthally averaging the 2D scattering data. The magnitude of the scattering vector q is given by q = 4π sin θ/λ, where 2θ is the scattering angle. Cryogenic Transmission Electron Microscopy (cryo-TEM). aFT-PAMAM samples were prepared in the same way as for SAXS measurements. The samples were vitrified on a TEM grid and imaged at liquid nitrogen temperature. Vitrification was done using Fei Vitrobot Mk3 in a saturated water vapor environment. Sample volumes of 3 μL were placed on Quantifoil R 3.5/1 grids, and the excess sample was blotted away with filter paper. Blot time and drain time were both 1 s. After blotting, the grids were plunged into a liquid ethane/propane (1:1) solution that was cooled with liquid nitrogen surrounding the ethane/propane vessel. Vitrified samples were cryo-transferred to the microscope. Imaging was performed with JEOL JEM-3200FSC equipment operating at a 300 kV accelerating voltage and specimen temperature of ∼86 K. Optical Microscopy. Transmission mode optical microscopy was used to characterize the macroscopic morphology of liquid aFT-PAMAM samples. Images were acquired using a Leica DM4500 optical microscope (40 magnification) combined with a Canon 60D digital camera. The scale of the images was calibrated by imaging a ruler at the sample position. Samples for optical microscopy were prepared similarly to the SAXS samples. Conflict of Interest: The authors declare no competing financial interest. Supporting Information Available: The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b04912.
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nanomaterials by combing synthetic and biological soft matter.
Additional DLS, SAXS, cryo-TEM, and optical microscopy data (PDF) Acknowledgment. Financial support from the Academy of Finland (Grants 263504, 267497, 273645), Biocentrum Helsinki, and Emil Aaltonen Foundation is gratefully acknowledged. This work was carried out under the Academy of Finland's Centers of Excellence Programme (20142019) and made use of the Aalto University Nanomicroscopy Centre (Aalto NMC).
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crystals. Moreover, they advance in particular the biohybrid interface aiming to create next-generation
REFERENCES AND NOTES 1. Pauling, L. The Principles Determining the Structure of Complex Ionic Crystals. J. Am. Chem. Soc. 1929, 51, 1010– 1026. 2. Whitesides, G. M.; Grzybowski, B. Self-Assembly at All Scales. Science 2002, 295, 2418–2421. 3. Whitesides, G. M.; Boncheva, M. Beyond Molecules: SelfAssembly of Mesoscopic and Macroscopic Components. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 4769–4774. 4. Kalsin, A. M.; Kowalczyk, B.; Smoukov, S. K.; Klajn, R.; Grzybowski, B. A. Ionic-like Behavior of Oppositely Charged Nanoparticles. J. Am. Chem. Soc. 2006, 128, 15046–15047. 5. Shevchenko, E. V.; Talapin, D. V.; Kotov, N. A.; O'Brien, S.; Murray, C. B. Structural Diversity in Binary Nanoparticle Superlattices. Nature 2006, 439, 55–59. 6. Leunissen, M. E.; Christova, C. G.; Hynninen, A.-P.; Royall, C. P.; Campbell, A. I.; Imhof, A.; Dijkstra, M.; van Roij, R.; van Blaaderen, A. Ionic Colloidal Crystals of Oppositely Charged Particles. Nature 2005, 437, 235–240. 7. Singh, G.; Chan, H.; Baskin, A.; Gelman, E.; Repnin, N.; Král, P.; Klajn, R. Self-Assembly of Magnetite Nanocubes into Helical Superstructures. Science 2014, 345, 1149–1153. 8. Macfarlane, R. J.; Lee, B.; Jones, M. R.; Harris, N.; Schatz, G. C.; Mirkin, C. A. Nanoparticle Superlattice Engineering with DNA. Science 2011, 334, 204–208. 9. Zhang, C.; Macfarlane, R. J.; Young, K. L.; Choi, C. H. J.; Hao, L.; Auyeung, E.; Liu, G.; Zhou, X.; Mirkin, C. A. A General Approach to DNA-Programmable Atom Equivalents. Nat. Mater. 2013, 12, 741–746. 10. Zhang, Z.; Glotzer, S. C. Self-Assembly of Patchy Particles. Nano Lett. 2004, 4, 1407–1413. 11. Gröschel, A. H.; Walther, A.; Löbling, T. I.; Schacher, F. H.; Schmalz, H.; Müller, A. H. E. Guided Hierarchical CoAssembly of Soft Patchy Nanoparticles. Nature 2013, 503, 247–251. 12. Kalsin, A. M.; Grzybowski, B. A. Controlling the Growth of “Ionic” Nanoparticle Supracrystals. Nano Lett. 2007, 7, 1018–1021. 13. Podsiadlo, P.; Krylova, G.; Demortière, A.; Shevchenko, E. Multicomponent Periodic Nanoparticle Superlattices. J. Nanopart. Res. 2011, 13, 15–32. 14. Glotzer, S. C.; Solomon, M. J. Anisotropy of Building Blocks and Their Assembly into Complex Structures. Nat. Mater. 2007, 6, 557–562. 15. Fukuto, M.; Nguyen, Q. L.; Vasilyev, O.; Mank, N.; WashingtonHughes, C. L.; Kuzmenko, I.; Checco, A.; Mao, Y.; Wang, Q.; Yang, L. Crystallization, Structural Diversity and Anisotropy Effects in 2D Arrays of Icosahedral Viruses. Soft Matter 2013, 9, 9633–9642. 16. Abe, S.; Ueno, T. Design of Protein Crystals in the Development of Solid Biomaterials. RSC Adv. 2015, 5, 21366–21375. 17. Sakai, F.; Yang, G.; Weiss, M. S.; Liu, Y.; Chen, G.; Jiang, M. Protein Crystalline Frameworks with Controllable Interpenetration Directed by Dual Supramolecular Interactions. Nat. Commun. 2014, 5, 4634. 18. Sinclair, J. C.; Davies, K. M.; Venien-Bryan, C.; Noble, M. E. M. Generation of Protein Lattices by Fusing Proteins with
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21.
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36.
LILJESTRÖM ET AL.
37. Uchida, M.; Kang, S.; Reichhardt, C.; Harlen, K.; Douglas, T. The Ferritin Superfamily: Supramolecular Templates for Materials Synthesis. Biochim. Biophys. Acta, Gen. Subj. 2010, 1800, 834–845. 38. Yamashita, I. Biosupramolecules for Nano-Devices: Biomineralization of Nanoparticles and Their Applications. J. Mater. Chem. 2008, 18, 3813. 39. Jutz, G.; van Rijn, P.; Santos Miranda, B.; Böker, A. Ferritin: A Versatile Building Block for Bionanotechnology. Chem. Rev. 2015, 115, 1653–1701. 40. Srivastava, S.; Samanta, B.; Jordan, B. J.; Hong, R.; Xiao, Q.; Tuominen, M. T.; Rotello, V. M. Integrated Magnetic Bionanocomposites through Nanoparticle-Mediated Assembly of Ferritin. J. Am. Chem. Soc. 2007, 129, 11776–11780. 41. Douglas, T.; Ripoll, D. R. Calculated Electrostatic Gradients in Recombinant Human H-Chain Femtin. Protein Sci. 1998, 7, 1083–1091. 42. Kostiainen, M. A.; Kasyutich, O.; Cornelissen, J. J. L. M.; Nolte, R. J. M. Self-Assembly and Optically Triggered Disassembly of Hierarchical Dendron-Virus Complexes. Nat. Chem. 2010, 2, 394–399. 43. Kostiainen, M. A.; Ceci, P.; Fornara, M.; Hiekkataipale, P.; Kasyutich, O.; Nolte, R. J. M.; Cornelissen, J. J. L. M.; Desautels, R. D.; van Lierop, J. Hierarchical Self-Assembly and Optical Disassembly for Controlled Switching of Magnetoferritin Nanoparticle Magnetism. ACS Nano 2011, 5, 6394–6402. 44. Lin, Y.; Böker, A.; He, J.; Sill, K.; Xiang, H.; Abetz, C.; Li, X.; Wang, J.; Emrick, T.; Long, S.; et al. Self-Directed SelfAssembly of Nanoparticle/copolymer Mixtures. Nature 2005, 434, 55–59. 45. Sontz, P. A.; Bailey, J. B.; Ahn, S.; Tezcan, F. A. A Metal Organic Framework with Spherical Protein Nodes: Rational Chemical Design of 3D Protein Crystals. J. Am. Chem. Soc. 2015, 137, 11598–11601. 46. Kostiainen, M. A.; Hiekkataipale, P.; Laiho, A.; Lemieux, V.; Seitsonen, J.; Ruokolainen, J.; Ceci, P. Electrostatic Assembly of Binary Nanoparticle Superlattices Using Protein Cages. Nat. Nanotechnol. 2012, 8, 52–56. 47. Hempstead, P. D.; Yewdall, S. J.; Fernie, a R.; Lawson, D. M.; Artymiuk, P. J.; Rice, D. W.; Ford, G. C.; Harrison, P. M. Comparison of the Three-Dimensional Structures of Recombinant Human H and Horse L Ferritins at High Resolution. J. Mol. Biol. 1997, 268, 424–448. 48. Lawson, D. M.; Artymiuk, P. J.; Yewdall, S. J.; Smith, J. M. A.; Livingstone, J. C.; Treffry, A.; Luzzago, A.; Levi, S.; Arosio, P.; Cesareni, G.; et al. Solving the Structure of Human H Ferritin by Genetically Engineering Intermolecular Crystal Contacts. Nature 1991, 349, 541–544. 49. Thomas, B. R.; Carter, D.; Rosenberger, F. Effect of Microheterogeneity on Horse Spleen Apoferritin Crystallization. J. Cryst. Growth 1998, 187, 499–510. 50. Walker, D. A.; Kowalczyk, B.; de la Cruz, M. O.; Grzybowski, B. A. Electrostatics at the Nanoscale. Nanoscale 2011, 3, 1316–1344. 51. Radhakrishnan, A. V.; Ghosh, S. K.; Pabst, G.; Raghunathan, V. A.; Sood, A. K. Tuning DNA-Amphiphile Condensate Architecture with Strongly Binding Counterions. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 6394–6398. 52. Kalsin, A. M.; Fialkowski, M.; Paszewski, M.; Smoukov, S. K.; Bishop, K. J. M.; Grzybowski, B. A. Electrostatic SelfAssembly of Binary Nanoparticle Crystals with a Diamondlike Lattice. Science 2006, 312, 420–424. 53. Liljeström, V.; Mikkilä, J.; Kostiainen, M. A. Self-Assembly and Modular Functionalization of Three-Dimensional Crystals from Oppositely Charged Proteins. Nat. Commun. 2014, 5, 4445. 54. Mikkilä, J.; Rosilo, H.; Nummelin, S.; Seitsonen, J.; Ruokolainen, J.; Kostiainen, M. A. Janus-Dendrimer-Mediated Formation of Crystalline Virus Assemblies. ACS Macro Lett. 2013, 2, 720– 724. 55. Yau, S. T.; Vekilov, P. G. Quasi-Planar Nucleus Structure in Apoferritin Crystallization. Nature 2000, 406, 494–497.
VOL. XXX
’
NO. XX
’
000–000
’
ARTICLE
19.
Matching Rotational Symmetry. Nat. Nanotechnol. 2011, 6, 558–562. Miao, L.; Han, J.; Zhang, H.; Zhao, L.; Si, C.; Zhang, X.; Hou, C.; Luo, Q.; Xu, J.; Liu, J. Quantum-Dot-Induced Self-Assembly of Cricoid Protein for Light Harvesting. ACS Nano 2014, 8, 3743–3751. Park, J. I.; Nguyen, T. D.; de Queirós Silveira, G.; Bahng, J. H.; Srivastava, S.; Zhao, G.; Sun, K.; Zhang, P.; Glotzer, S. C.; Kotov, N. A. Terminal Supraparticle Assemblies from Similarly Charged Protein Molecules and Nanoparticles. Nat. Commun. 2014, 5, 3593. Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. A New Class of Polymers: Starburst-Dendritic Macromolecules. Polym. J. 1985, 17, 117–132. Tomalia, D. A. In Quest of a Systematic Framework for Unifying and Defining Nanoscience. J. Nanopart. Res. 2009, 11, 1251–1310. Das, A. K.; Hsiao, P.-Y. Charged Dendrimers in Trivalent Salt Solutions under the Action of DC Electric Fields. J. Phys. Chem. B 2014, 118, 6265–6276. Welch, P.; Muthukumar, M. Tuning the Density Profile of Dendritic Polyelectrolytes. Macromolecules 1998, 31, 5892–5897. Porus, M.; Clerc, F.; Maroni, P.; Borkovec, M. Ion-Specific Responsiveness of Polyamidoamine (PAMAM) Dendrimers Adsorbed on Silica Substrates. Macromolecules 2012, 45, 3919–3927. Jachimska, B.; yapczy nska, M.; Zapotoczny, S. Reversible Swelling Process of Sixth-Generation Poly(amido Amine) Dendrimers Molecule as Determined by Quartz Crystal Microbalance Technique. J. Phys. Chem. C 2013, 117, 1136– 1145. Rosen, B. M.; Wilson, C. J.; Wilson, D. A.; Peterca, M.; Imam, M. R.; Percec, V. Dendron-Mediated Self-Assembly, Disassembly, and Self-Organization of Complex Systems. Chem. Rev. 2009, 109, 6275–6540. Sun, H.-J.; Zhang, S.; Percec, V. From Structure to Function via Complex Supramolecular Dendrimer Systems. Chem. Soc. Rev. 2015, 44, 3900–3923. Balagurusamy, V. S. K.; Ungar, G.; Percec, V.; Johansson, G. Rational Design of the First Spherical Supramolecular Dendrimers Self-Organized in a Novel Thermotropic Cubic Liquid-Crystalline Phase and the Determination of Their Shape by X-Ray Analysis. J. Am. Chem. Soc. 1997, 119, 1539–1555. Zeng, X.; Ungar, G.; Liu, Y.; Percec, V.; Dulcey, A. E.; Hobbs, J. K. Supramolecular Dendritic Liquid Quasicrystals. Nature 2004, 428, 157–160. Ungar, G.; Liu, Y.; Zeng, X.; Percec, V.; Cho, W.-D. Giant Supramolecular Liquid Crystal Lattice. Science 2003, 299, 1208–1211. Percec, V.; Ahn, C.-H.; Bera, T. K.; Ungar, G.; Yeardley, D. J. P. Coassembly of a Hexagonal Columnar Liquid Crystalline Superlattice from Polymer(s) Coated with a Three-Cylindrical Bundle Supramolecular Dendrimer. Chem. - Eur. J. 1999, 5, 1070–1083. Rosen, B. M.; Wilson, D. A.; Wilson, C. J.; Peterca, M.; Won, B. C.; Huang, C.; Lipski, L. R.; Zeng, X.; Ungar, G.; Heiney, P. A.; et al. Predicting the Structure of Supramolecular Dendrimers via the Analysis of Libraries of AB3 and Constitutional Isomeric AB2 Biphenylpropyl Ether Self-Assembling Dendrons. J. Am. Chem. Soc. 2009, 131, 17500–17521. Frankamp, B. L.; Boal, A. K.; Rotello, V. M. Controlled Interparticle Spacing through Self-Assembly of Au Nanoparticles and Poly(amidoamine) Dendrimers. J. Am. Chem. Soc. 2002, 124, 15146–15147. Frankamp, B. L.; Boal, A. K.; Tuominen, M. T.; Rotello, V. M. Direct Control of the Magnetic Interaction between Iron Oxide Nanoparticles through Dendrimer-Mediated SelfAssembly. J. Am. Chem. Soc. 2005, 127, 9731–9735. Srivastava, S.; Frankamp, B. L.; Rotello, V. M. Controlled Plasmon Resonance of Gold Nanoparticles Self-Assembled with PAMAM Dendrimers. Chem. Mater. 2005, 17, 487–490.
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