Epitaxially guided assembly of collagen layers on mica surfaces

Ordered assembly of collagen molecules on flat substrates has potential for various applications and serves as a model system for studying the assembly process. While previous studies demonstrated self-assembly of collagen on muscovite mica into highly ordered layers, the mechanism by which different conditions affect the resulting morphology remains to be elucidated. Using atomic force microscopy, we follow the assembly of collagen on muscovite mica at a concentration lower than the critical fibrillogenesis concentration in bulk. Initially, individual collagen molecules adsorb to mica and subsequently nucleate into fibrils possessing the 67 nm D-periodic bands. Emergence of fibrils aligned in parallel despite large interfibril distances agrees with an alignment mechanism guided by the underlying mica. The epitaxial growth was further confirmed by the formation of novel triangular networks of collagen fibrils on phlogopite mica, whose surface lattice is known to have a hexagonal symmetry, whereas the more widely used muscovite does not. Comparing collagen assembly on the two types of mica at different potassium concentrations revealed that potassium binds to the negatively charged mica surface and neutralizes it, thereby reducing the binding affinity of collagen and enhancing surface diffusion. These results suggest that collagen assembly on mica follows the surface adsorption, diffusion, nucleation, and growth pathway, where the growth direction is determined at the nucleation step. Comparison with other molecules that assemble similarly on mica supports generality of the proposed assembly mechanism, the knowledge of which will be useful for controlling the resulting surface morphologies.     

Protofilaments, filaments, ribbons, and fibrils from peptidomimetic self-assembly: implications for amyloid fibril formation and materials science

Deciphering the mechanism(s) of β-sheet mediated self-assembly is essential for understanding amyloid fibril formation and for the fabrication of polypeptide materials. Herein, we report a simple peptidomimetic that self-assembles into polymorphic β-sheet quaternary structures including protofilaments, filaments, fibrils, and ribbons that are reminiscent of the highly ordered structures displayed by the amyloidogenic peptides Aβ, calcitonin, and amylin. The distribution of quaternary structures can be controlled by and in some cases specified by manipulating the pH, buffer composition, and the ionic strength. The ability to control β-sheet-mediated assembly takes advantage of quaternary structure dependent pK(a) perturbations. Biophysical methods including analytical ultracentrifugation studies as well as far-UV circular dichroism and FT-IR spectroscopy demonstrate that linked secondary and quaternary structural changes mediate peptidomimetic self-assembly. Electron and atomic force microscopy reveal that peptidomimetic assembly involves numerous quaternary structural intermediates that appear to self-assemble in a convergent fashion affording quaternary structures of increasing complexity. The ability to control the assembly pathway(s) and the final quaternary structure(s) afforded should prove to be particularly useful in deciphering the quaternary structural requirements for amyloid fibril formation and for the construction of noncovalent macromolecular structures.     

Nanoscale characterization of zein self-assembly

Zein, a major protein of corn, is rich in α-helical structure. It has an amphiphilic character and is capable of self-assembly. Zein can self-assemble into various mesostructures that may find applications in food, agricultural, and biomedical engineering. Understanding the mechanism of zein self-assembly at the nanoscale is important for further development of zein structures. In this work, high-resolution transmission electron microscopy (TEM) images revealed nanosize zein stripes, rings, and discs containing a 0.35 nm periodicity, which is characteristic of β-sheet. TEM images were interpreted in terms of the transformation of original α-helices into β-sheet conformation after evaporation-induced self-assembly (EISA). The presence of β-sheet was also detected by circular dichroism (CD) spectroscopy. Zein β-sheets self-assembled into stripes, which curled into rings. Rings formed discs and eventually spheres. The formation of zein nanostructures was believed to be the result of β-sheet orientation, alignment, and packing.     

Assembly of Alzheimer's Aβ peptide into nanostructured amyloid fibrils

The self-assembly of β-amyloid (Aβ) peptide into highly ordered amyloid fibril structures represents one of the pathological hallmarks of Alzheimer's disease. This process leads to the transient stabilization of ordered or disordered intermediates, which are thought to act as the main pathogenic culprits in neurodegenerative amyloid disease. This review describes recent results from different biophysical techniques, ranging from structure determination to single-particle methods by which the outgrowth of individual fibrils can be followed, and it explains their contributions towards understanding the mechanism of assembly. Finally, we will outline emerging methods and molecules to specifically interfere with the assembly and pathogenic impact of Aβ peptide.     

Programming supramolecular peptide materials by modulating the intermediate steps in the complex assembly pathway: Implications for biomedical applications

Self-assembling peptides form a prominent class of supramolecular materials with in general good biocompatibility. To afford better control over the material properties, tremendous progress has been made in studying the supramolecular organization of the peptide assemblies. This knowledge has helped us to understand the correlation between the molecular structure of the peptide building blocks and the properties of the supramolecular products. However, peptide self-assembly consists of a complex pathway rather than a spontaneous thermodynamic process. This implies that the outcome of the self-assembly is critically governed by the assembly pathway. Here, we are going to discuss how peptide self-assembly can be modulated at the intermediate steps in the self-assembly pathway. The focus will be to demonstrate this engineering approach on the example of zero-dimensional/one-dimensional nanostructure selectivity over the β-sheet assembly pathway. In addition, we provide examples of biomedical applications of such steered peptide assemblies in the field of drug delivery and tissue engineering.     

Combination self-assembly of β-sheet peptides and carbon nanotubes: functionalizing carbon nanotubes with bioactive β-sheet block copolypeptides

Understanding the self-assembly behavior of β-sheet peptides is important, not only in constructing bioactive peptide nanostructures, but also in inhibiting uncontrollable protein aggregation in protein-misfolding diseases. Here, the first systematic investigation of combination self-assembly between β-sheet block copolypeptides and CNTs is presented, demonstrating the presence of several different association modes during the combination self-assembly process. Bioactive β-sheet block copolypeptides can self-assemble by themselves, or can be used to functionalize CNT hybrids depending on the situation. This behavior may be important both for fabricating bioactive peptide/CNT hybrids and for controlling/inhibiting protein-misfolding diseases.     

Structural Insight into IAPP-Derived Amyloid Inhibitors and Their Mechanism of Action

Designed peptides derived from the islet amyloid polypeptide (IAPP) cross-amyloid interaction surface with Aβ (termed interaction surface mimics or ISMs) have been shown to be highly potent inhibitors of Aβ amyloid self-assembly. However, the molecular mechanism of their function is not well understood. Using solution-state and solid-state NMR spectroscopy in combination with ensemble-averaged dynamics simulations and other biophysical methods including TEM, fluorescence spectroscopy and microscopy, and DLS, we characterize ISM structural preferences and interactions. We find that the ISM peptide R3-GI is highly dynamic, can adopt a β-like structure, and oligomerizes into colloid-like assemblies in a process that is reminiscent of liquid–liquid phase separation (LLPS). Our results suggest that such assemblies yield multivalent surfaces for interactions with Aβ40. Sequestration of substrates into these colloid-like structures provides a mechanistic basis for ISM function and the design of novel potent anti-amyloid molecules.     

The characterization of high generation poly(amidoamine) G9 dendrimers by atomic force microscopy (AFM)

Scanning force microscopy (AFM) has been employed to characterize the generation‐9 (G9) poly(amidoamine) (PAMAM) dendrimer packing on a mica surface under various conditions. Well ordered 2‐D arrays from hexagonally packed particles of PAMAM (G9) dendrimers (11.4nm in diameter) were deposited on the mica surface. This may be one of the smallest regular monolayer arrays ever observed. The mechanism considered to be responsible for this 2‐D array packing is the interaction of forces between the dendrimer and the mica surface and between dendrimer molecules as well. Other factors such as molecular interpenetrating and the rigidity of the branch structure obviously play an important role in the 2‐D array formation.     

Parallel-oriented fibrogenesis of a beta-sheet forming peptide on supported lipid bilayers

Peptide self-assembly on substrates is currently an intensively studied topic that provides a promising strategy for fabrication of soft materials and is also important for revealing the surface chemistry of amyloidogenic proteins that aggregate on cell membranes. We investigated the fibrogenesis of a beta-sheet forming peptide Abeta(26-35) on supported lipid bilayers (SLBs) by in situ atomic force microscopy (AFM), circular dichroism (CD), and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The results show that the Abeta(26-35) nanofilaments' growth is oriented to a specific direction and formed a highly ordered, large-scale, parallel-oriented surface pattern on membranes. The parallel-oriented fibrogenesis of Abeta(26-35) was able to occur on different lipid membranes rather than on solid substrates. It implies that the parallel-oriented fibrogenesis was associated with the distinct properties of lipid membranes, such as the fluid nature of lipid molecules on membranes. The membrane fluidity may allow the peptide assemblies to float at the water-membrane interface and easily orient to an energetically favorable state. These results provide an insight into the surface chemistry of peptide self-assembly on lipid membranes and highlight a possible way to fabricate supramolecular architectures on the surface of soft materials.     

Highly-ordered selective self-assembly of a trimeric cationic surfactant on a mica surface

Novel trimeric cationic surfactant tri(dodecyldimethylammonioacetoxy)diethyltriamine trichloride (DTAD) has been synthesized, and its self-assembly morphology on a mineral surface has been studied. From its micelle solution, highly ordered bilayer patterns are obtained on a mica surface, whereas randomly distributed bilayer patches are formed on a silica substrate. The highly ordered bilayer patterns on mica are first caused by the matching of the special structure of DTAD headgroups with the negative charge sites on mica, which leads to the specific nucleation of DTAD on the mica surface via electrostatic interaction. Furthermore, hydrophobic interaction among the DTAD hydrocarbon chains results in the formation of the bilayer structure, and intermolecular hydrogen-bonding among the DTAD headgroups promotes the directional growth of such bilayer structures.     

Dependence of Self-Assembled Peptide Hydrogel Network Structure on Local Fibril Nanostructure

Physically cross-linked, fibrillar hydrogel networks are formed by the self-assembly of β-hairpin peptide molecules with varying degrees of strand asymmetry. The peptide registry in the self-assembled state can be used as a design element to generate fibrils with twisting, nontwisting, or laminated morphology. The mass density of the networks varies significantly, and can be directly related to the local fibrillar morphology as evidenced by small angle neutron scattering (SANS) and in situ substantiation using cryogenic transmission electron microscopy (cryo-TEM) under identical concentrations and conditions. Similarly, the density of the network is dependent on changes in the peptide concentration. Bulk rheological properties of the hydrogels can be correlated to the fibrillar nanostructure, with the stiffer, laminated fibrils forming networks with a higher G' as compared to the flexible, singular fibrillar networks.     

Surface-tuned assembly of porphyrin coordination oligomers

Two self-complementary phenanthroline-strapped porphyrins bearing imidazole arms and C 12 or C 18 alkyl chains were synthesized, and their surface self-assembly was investigated by atomic force microscopy (AFM) on mica and highly ordered pyrrolitic graphite (HOPG). Upon zinc(II) complexation, stable porphyrin dimers formed, as confirmed by DOSY (1)H NMR and UV-visible spectroscopy. In solution, the dimers formed J-aggregates. AFM studies of the solutions dip-coated onto mica or drop-casted onto HOPG revealed that the morphologies of the assemblies formed were surface-tuned. On mica, fiber-like assemblies of short stacks of J-aggregates were observed. The strong influence of the mica's epitaxy on the orientation of the fibers suggested a surface-assisted assembly process. On HOPG, interactions between the alkyl chains and the graphite surface resulted in the stabilization and trapping of monomer species followed by their subsequent association into coordination polymers on the surface. Interdigitation of the alkyl chains of separate polymer strands induced lateral association of wires to form islands that grew preferentially upon drop-casting and slow evaporation. Clusters of laterally assembled wires were observed for the more mobile functionalized porphyrins bearing C 12 chains.     

Multistep growth mechanism of calcium phosphate in the earliest stage of morphology-controlled biomineralization

We studied the effect of surface-functional-group position on precipitate morphology in the earliest stage of calcium phosphate biomineralization and determined the detailed mechanism of precipitation starting from nucleation to precipitate growth. The biomineralization template was a β-sheet peptide scaffold prepared by adsorption with carboxyl groups arranged at strict 7 ? intervals. Phosphate was then introduced. Within 10 s, highly ordered embryos of calcium phosphate were formed and confined by a peptide nanofiber pattern. They repeatedly nucleated and dissolved, with the larger embryos absorbing the smaller ones in a clear demonstration of an Ostwald-ripening-like phenomenon, then aggregated in a line pattern, and finally formed highly ordered nanofibers of amorphous calcium phosphate. This multistep growth process constitutes the earliest stage of biomineralization.     

Mechanistic studies of peptide self-assembly: transient α-helices to stable β-sheets

The pathologic self-assembly of proteins is associated with typically late-onset disorders such as Alzheimer's disease, Parkinson's disease, and type 2 diabetes. Important mechanistic details of the self-assembly are unknown, but there is increasing evidence supporting the role of transient α-helices in the early events. Islet amyloid polypeptide (IAPP) is a 37-residue polypeptide that self-assembles into aggregates that are toxic to the insulin-producing β cells. To elucidate early events in the self-assembly of IAPP, we used limited proteolysis to identify an exposed and flexible region in IAPP monomer. This region includes position 20 where a serine-to-glycine substitution (S20G) is associated with enhanced formation of amyloid fibrils and early onset type 2 diabetes. To perform detailed biophysical studies of the exposed and flexible region, we synthesized three peptides including IAPP(11-25)WT (wild type), IAPP(11-25)S20G, and IAPP(11-25)S20P. Solution-state NMR shows that all three peptides transiently populate the α-helical conformational space, but the S20P peptide, which does not self-assemble, transiently samples a broken helix. Under similar sample conditions, the WT and S20G peptides populate the α-helical intermediate state and β-sheet end state, respectively, of fibril formation. Our results suggest a mechanism for self-assembly that includes the stabilization of transient α-helices through the formation of NMR-invisible helical intermediates followed by an α-helix to β-sheet conformational rearrangement. Furthermore, our results suggest that reducing intermolecular helix-helix contacts as in the S20P peptide is an attractive strategy for the design of blockers of peptide self-assembly.     

Controlled rearrangement of adsorbed undecanol films on mica surfaces induced by an atomic force microscopy tip

An undecanol film adsorbed on a mica surface was found to rearrange and spread in a position-controlled way induced by a tapping mode atomic force microscopy (AFM) probe. AFM images of varying scanning times showed that before forming an ordered monolayer the undecanol molecules were adsorbed on the mica surface in the disordered and disorganized status. With the proceeding of scanning, these undecanol molecules gradually formed an ordered and flat film. Such behavior was caused by the formation of a stable film and had never been reported for other alcohols.     

Synthesis of thermally stable highly ordered nanoporous tin oxide thin films with a 3D face-centered orthorhombic nanostructure

Thin films of nanoporous tin oxide with a 3D face-centered orthorhombic nanostructure have been synthesized by self-assembly that is controlled by post-coating thermal treatment under controlled humidity. In contrast to the conventional evaporation-induced self-assembly (EISA), the films here have no ordered nanostructure after dip-coating. However, the initial coatings are formed under conditions that inhibit significant hydrolysis and condensation for extended periods. This allows the use of postsynthesis thermal vapor treatments to completely control the formation of the nanostructure. With EO106-PO70-EO106 (Pluronic F127) triblock copolymer as the template, highly ordered nanostructures were generated by exposing the disordered films to a stream of water vapor at elevated temperature, which rehydrates the films and allows the formation of the thermodynamically favored phase. Further exposure to water vapor drives the condensation reaction through the elimination of HCl. The X-ray diffraction pattern from the nanostructure was indexed in the space group Fmmm as determined by analysis of 2D small-angle X-ray scattering patterns at various angles of incidence. The nanostructure is then stabilized and made nanoporous by extended controlled thermal treatments. After self-assembly and template removal, the films are thermally stable up to 600 degrees C and retain an ordered, face-centered orthorhombic nanostructure.     

Two-dimensional densely packed DNA nanostructure derived from DNA complexation with a low-generation poly(amidoamine) dendrimer

One of the keys for using deoxyribonucleic acid (DNA) as a nanomaterial relies on how the individual DNA chain can be aligned and how a multitude of DNA chains can be packed into ordered nanostructures. Here we present a simple method for constructing a 2-D densely packed DNA nanostructure using the electrostatic complex of DNA with a poly(amidoamine) (PAMAM) dendrimer of generation two. Ordered DNA arrays are formed by drop-casting an aqueous solution containing positively overcharged complexes onto mica followed by a prolonged incubation. During the incubation, the complexes tend to adsorb onto the negatively charged mica surface through electrostatic attraction. The rodlike complexes organize to form ordered arrays to increase the surface density of the adsorbed complexes and hence the attractive free energy of adsorption. The densely packed nanostructure obtained here is distinguished from the previously reported spheroid or toroid structure derived from DNA complexations with the higher-generation dendrimers.     

Oriented monolayers prepared from lyotropic chromonic liquid crystal

We use a layer-by layer electrostatic self-assembly technique to obtain in-plane oriented aggregates of mesogenic dye molecules cast from lyotropic chromonic liquid crystals (LCLCs) on mica substrates. The aqueous solutions of dye used for deposition are in the nematic phase. Atomic force microscopy and X-ray photoelectron spectroscopy of the dried film reveal that the LCLC molecules adsorb at the charged substrate preserving ordered aggregates of elongated shape characteristic of the nematic phase in the aqueous solution. These elongated aggregates of LCLC molecules form films with in-plane orientational order and are compositionally distinct from the substrate.     

A Rapid Self-Assembly Peptide Hydrogel for Recruitment and Activation of Immune Cells

Self-assembly peptide nanotechnology has attracted much attention due to its regular and orderly structure and diverse functions. Most of the existing self-assembly peptides can form aggregates with specific structures only under specific conditions and their assembly time is relatively long. They have good biocompatibility but no immunogenicity. To optimize it, a self-assembly peptide named DRF3 was designed. It contains a hydrophilic and hydrophobic surface, using two N-terminal arginines, leucine, and two c-terminal aspartate and glutamic acid. Meanwhile, the c-terminal of the peptide was amidated, so that peptide segments were interconnected to increase diversity. Its characterization, biocompatibility, controlled release effect on antigen, immune cell recruitment ability, and antitumor properties were examined here. Congo red/aniline blue staining revealed that peptide hydrogel DRF3 could be immediately gelled in PBS. The stable β-sheet secondary structure of DRF3 was confirmed by circular dichroism spectrum and IR spectra. The observation results of cryo-scanning electron microscopy, transmission electron microscopy, and atomic force microscopy demonstrated that DRF3 formed nanotubule-like and vesicular structures in PBS, and these structures interlaced with each other to form ordered three-dimensional nanofiber structures. Meanwhile, DRF3 showed excellent biocompatibility, could sustainably and slowly release antigens, recruit dendritic cells and promote the maturation of dendritic cells (DCs) in vitro. In addition, DRF3 has a strong inhibitory effect on clear renal cell carcinoma (786-0). These results provide a reliable basis for the application of peptide hydrogels in biomedical and preclinical trials.     

Guided self-assembly of diblock copolymer thin films on chemically patterned substrates

We study the guided self-assembly of symmetric/asymmetric diblock copolymer (BCP) films on heterogeneous substrates with chemically patterned surface by using a coarse-grained phase-separation model. During the procedure, the free energy employed for the BCP films was modeled by the Ginzburg-Landau free energy with nonlocal interaction, and the flat, chemically patterned surface was considered as a heterogeneous surface with short-range interaction with the BCP molecules. The resulting Cahn-Hilliard equation was solved by means of an efficient semi-implicit Fourier-spectral algorithm. Effects of pattern scale, surface chemical potential, and BCP asymmetry on the self-assembly process were explored in detail and compared with those without chemically patterned substrate surfaces. It was found that the morphology of both symmetric and asymmetric BCP films is strongly influenced by the commensurability between the unconstrained natural period lambda* of the bulk BCP and the artificial pattern period. Simulation shows that patterned surface with period close to lambda* leads to highly ordered morphology after self-assembly for both symmetric and asymmetric BCP films, and it also dramatically accelerates the guided self-assembly process. The present simulation is in a very good agreement with the recent experimental observation in BCP nanolithography. Finally, the present study also expects an innovative nanomanufacturing method to produce highly ordered nanodots based on the guided self-assembly of asymmetric BCP films on chemically patterned substrates.