Supramolecular Double Helices from Small C3-Symmetrical Molecules Aggregated in Water.

Supramolecular fibers in water, micrometers long and several nanometers in width, are among the most studied nanostructures for biomedical applications. These supramolecular polymers are formed through a spontaneous self-assembly process of small amphiphilic molecules by specific secondary interactions. Although many compounds do not possess a stereocenter, recent studies suggest the (co)existence of helical structures, albeit in racemic form. Here, we disclose a series of supramolecular (co)polymers based on water-soluble benzene-1,3,5-tricarboxamides (BTAs) that form double helices, fibers that were long thought to be chains of single molecules stacked in one dimension (1D). Detailed cryogenic transmission electron microscopy (cryo-TEM) studies and subsequent three-dimensional-volume reconstructions unveiled helical repeats, ranging from 15 to 30 nm. Most remarkable, the pitch can be tuned through the composition of the copolymers, where two different monomers with the same core but different peripheries are mixed in various ratios. Like in lipid bilayers, the hydrophobic shielding in the aggregates of these disc-shaped molecules is proposed to be best obtained by dimer formation, promoting supramolecular double helices. It is anticipated that many of the supramolecular polymers in water will have a thermodynamic stable structure, such as a double helix, although small structural changes can yield single stacks as well. Hence, it is essential to perform detailed analyses prior to sketching a molecular picture of these 1D fibers.

Disordered Filaments Mediate the Fibrillogenesis of Type I Collagen in Solution.

Collagen type I is one of the major structural proteins in mammals, providing tissues such as cornea, tendon, bone, skin, and dentin with mechanical stability, strength, and toughness. Collagen fibrils are composed of collagen molecules arranged in a quarter-stagger array that gives rise to a periodicity of 67 nm along the fibril axis, with a 30 nm overlap zone and a 37 nm gap zone. The formation of such highly organized fibrils is a self-assembly process where electrostatic and hydrophobic interactions play a critical role in determining the staggering of the molecules with 67 nm periodicity. While collagen self-assembly has been extensively studied, not much is known about the mechanism, and in particular, the nature of the nuclei that initially form, the different stages of the aggregation process, and how the organization of the molecules into fibrils arises. By combining time-resolved cryo-transmission electron microscopy with molecular dynamics simulations, we show that collagen assembly is a multistep process in which the molecules first form filaments which self-organize into fibrils with a disordered structure. The appearance of the D-band periodicity is gradual and starts with the alignment of adjacent filaments at the N-terminal end of the molecules, first leading to bands with a periodicity of 67 nm and then to the formation of gap and overlap regions.

Liquid-Phase Electron Microscopy for Soft Matter Science and Biology.

Innovations in liquid-phase electron microscopy (LP-EM) have made it possible to perform experiments at the optimized conditions needed to examine soft matter. The main obstacle is conducting experiments in such a way that electron beam radiation can be used to obtain answers for scientific questions without changing the structure and (bio)chemical processes in the sample due to the influence of the radiation. By overcoming these experimental difficulties at least partially, LP-EM has evolved into a new microscopy method with nanometer spatial resolution and sub-second temporal resolution for analysis of soft matter in materials science and biology. Both experimental design and applications of LP-EM for soft matter materials science and biological research are reviewed, and a perspective of possible future directions is given.

Crystallization by particle attachment is a colloidal assembly process.

The nucleation of crystals has long been thought to occur through the stochastic association of ions, atoms or molecules to form critical nuclei, which will later grow out to crystals1. Only in the past decade has the awareness grown that crystallization can also proceed through the assembly of different types of building blocks2,3, including amorphous precursors4, primary particles5, prenucleation species6,7, dense liquid droplets8,9 or nanocrystals10. However, the forces that control these alternative pathways are still poorly understood. Here, we investigate the crystallization of magnetite (Fe3O4) through the formation and aggregation of primary particles and show that both the thermodynamics and the kinetics of the process can be described in terms of colloidal assembly. This model allows predicting the average crystal size at a given initial Fe concentration, thereby opening the way to the design of crystals with predefined sizes and properties.

Osteoporotic Bone Recovery by a Highly Bone-Inductive Calcium Phosphate Polymer-Induced Liquid-Precursor.

Osteoporosis is an incurable chronic disease characterized by a lack of mineral mass in the bones. Here, the full recovery of osteoporotic bone is achieved by using a calcium phosphate polymer-induced liquid-precursor (CaP-PILP). This free-flowing CaP-PILP material displays excellent bone inductivity and is able to readily penetrate into collagen fibrils and form intrafibrillar hydroxyapatite crystals oriented along the c-axis. This ability is attributed to the microstructure of the material, which consists of homogeneously distributed ultrasmall (≈1 nm) amorphous calcium phosphate clusters. In vitro study shows the strong affinity of CaP-PILP to osteoporotic bone, which can be uniformly distributed throughout the bone tissue to significantly increase the bone density. In vivo experiments show that the repaired bones exhibit satisfactory mechanical performance comparable with normal ones, following a promising treatment of osteoporosis by using CaP-PILP. The discovery provides insight into the structure and property of biological nanocluster materials and their potential for hard tissue repair.

Photocatalytic activity of exfoliated graphite-TiO2 nanoparticle composites.

We investigate the photocatalytic performance of composites prepared in a one-step process by liquid-phase exfoliation of graphite in the presence of TiO2 nanoparticles (NPs) at atmospheric pressure and in water, without heating or adding any surfactant, and starting from low-cost commercial reagents. These show enhanced photocatalytic activity, degrading up to 40% more pollutants with respect to the starting TiO2-NPs, in the case of a model dye target, and up to 70% more pollutants in the case of nitrogen oxides. In order to understand the photo-physical mechanisms underlying this enhancement, we investigate the photo-generation of reactive species (trapped holes and electrons) by ultrafast transient absorption spectroscopy. We observe an electron transfer process from TiO2 to the graphite flakes within the first picoseconds of the relaxation dynamics, which causes the decrease of the charge recombination rate, and increases the efficiency of the reactive species photo-production.

A Biomimetic Model for Mineralization of Type-I Collagen Fibrils.

The bone and dentin mainly consist of type-I collagen fibrils mineralized by hydroxyapatite (HAP) nanocrystals. In vitro biomimetic models based on self-assembled collagen fibrils have been widely used in studying the mineralization mechanism of type-I collagen. In this chapter, the protocol we used to build a biomimetic model for the mechanistic study of type-I collagen mineralization is described. Type-I collagen extracted from rat tail tendon or horse tendon is self-assembled into fibrils and mineralized by HAP in vitro. The mineralization process is monitored by cryoTEM in combination with two-dimensional (2D) and three-dimensional (3D) stochastic optical reconstruction microscopy (STORM), which enables in situ and high-resolution visualization of the process.

Liquid-liquid phase separation during amphiphilic self-assembly.

The self-assembly of amphiphilic molecules in solution is a ubiquitous process in both natural and synthetic systems. The ability to effectively control the structure and properties of these systems is essential for tuning the quality of their functionality, yet the underlying mechanisms governing the transition from molecules to assemblies have not been fully resolved. Here we describe how amphiphilic self-assembly can be preceded by liquid-liquid phase separation. The assembly of a model block co-polymer system into vesicular structures was probed through a combination of liquid-phase electron microscopy, self-consistent field computations and Gibbs free energy calculations. This analysis shows the formation of polymer-rich liquid droplets that act as a precursor in the bottom-up formation of spherical micelles, which then evolve into vesicles. The liquid-liquid phase separation plays a role in determining the resulting vesicles' structural properties, such as their size and membrane thickness, and the onset of kinetic traps during self-assembly.

Designing stable, hierarchical peptide fibers from block co-polypeptide sequences.

Natural materials, such as collagen, can assemble with multiple levels of organization in solution. Achieving a similar degree of control over morphology, stability and hierarchical organization with equilibrium synthetic materials remains elusive. For the assembly of peptidic materials the process is controlled by a complex interplay between hydrophobic interactions, electrostatics and secondary structure formation. Consequently, fine tuning the thermodynamics and kinetics of assembly remains extremely challenging. Here, we synthesized a set of block co polypeptides with varying hydrophobicity and ability to form secondary structure. From this set we select a sequence with balanced interactions that results in the formation of high-aspect ratio thermodynamically favored nanotubes, stable between pH 2 and 12 and up to 80 °C. This stability permits their hierarchical assembly into bundled nanotube fibers by directing the pH and inducing complementary zwitterionic charge behavior. This block co-polypeptide design strategy, using defined sequences, provides a straightforward approach to creating complex hierarchical peptide-based assemblies with tunable interactions.

Simulation of Calcium Phosphate Prenucleation Clusters in Aqueous Solution: Association beyond Ion Pairing.

Classical molecular dynamics simulations and free energy methods have been used to obtain a better understanding of the molecular processes occurring prior to the first nucleation event for calcium phosphate biominerals. The association constants for the formation of negatively charged complexes containing calcium and phosphate ions in aqueous solution have been computed, and these results suggest that the previously proposed calcium phosphate building unit, [Ca(HPO4)3]4-, should only be present in small amounts under normal experimental conditions. However, the presence of an activation barrier for the removal of an HPO4 2- ion from this complex indicates that this species could be kinetically trapped. Aggregation pathways involving CaHPO4, [Ca(HPO4)2]2-, and [Ca(HPO4)3]4- complexes have been explored with the finding that dimerization is favorable up to a Ca/HPO4 ratio of 1:2.

Native Chemical Ligation for Cross-Linking of Flower-Like Micelles.

In this study, native chemical ligation (NCL) was used as a selective cross-linking method to form core-cross-linked thermosensitive polymeric micelles for drug delivery applications. To this end, two complementary ABA triblock copolymers having polyethylene glycol (PEG) as midblock were synthesized by atom transfer radical polymerization (ATRP). The thermosensitive poly isopropylacrylamide (PNIPAM) outer blocks of the polymers were copolymerized with either N-(2-hydroxypropyl)methacrylamide-cysteine (HPMA-Cys), P(NIPAM- co-HPMA-Cys)-PEG-P(NIPAM- co-HPMA-Cys) (PNC) or N-(2-hydroxypropyl)methacrylamide-ethylthioglycolate succinic acid (HPMA-ETSA), P(NIPAM- co-HPMA-ETSA)-PEG-P(NIPAM- co-HPMA-ETSA) (PNE). Mixing of these polymers in aqueous solution followed by heating to 50 °C resulted in the formation of thermosensitive flower-like micelles. Subsequently, native chemical ligation in the core of micelles resulted in stabilization of the micelles with a Z-average of 65 nm at body temperature. Decreasing the temperature to 10 °C only affected the size of the micelles (increased to 90 nm) but hardly affected the polydispersity index (PDI) and aggregation number ( Nagg) confirming covalent stabilization of the micelles by NCL. CryoTEM images showed micelles with an uniform spherical shape and dark patches close to the corona of micelles were observed in the tomographic view. The dark patches represent more dense areas in the micelles which coincide with the higher content of HPMA-Cys/ETSA close to the PEG chain revealed by the polymerization kinetics study. Notably, this cross-linking method provides the possibility for conjugation of functional molecules either by using the thiol moieties still present after NCL or by simply adjusting the molar ratio between the polymers (resulting in excess cysteine or thioester moieties) during micelle formation. Furthermore, in vitro cell experiments demonstrated that fluorescently labeled micelles were successfully taken up by HeLa cells while cell viability remained high even at high micelle concentrations. These results demonstrate the potential of these micelles for drug delivery applications.

Microscopic structure of the polymer-induced liquid precursor for calcium carbonate.

Many biomineral crystals form complex non-equilibrium shapes, often via transient amorphous precursors. Also in vitro crystals can be grown with non-equilibrium morphologies, such as thin films or nanorods. In many cases this involves charged polymeric additives that form a polymer-induced liquid precursor (PILP). Here, we investigate the CaCO3 based PILP process with a variety of techniques including cryoTEM and NMR. The initial products are 30-50 nm amorphous calcium carbonate (ACC) nanoparticles with ~2 nm nanoparticulate texture. We show the polymers strongly interact with ACC in the early stages, and become excluded during crystallization, with no liquid-liquid phase separation detected during the process. Our results suggest that "PILP" is actually a polymer-driven assembly of ACC clusters, and that its liquid-like behavior at the macroscopic level is due to the small size and surface properties of the assemblies. We propose that a similar biopolymer-stabilized nanogranular phase may be active in biomineralization.

Proteins as supramolecular hosts for C60: a true solution of C60 in water.

Hybrid systems have great potential for a wide range of applications in chemistry, physics and materials science. Conjugation of a biosystem to a molecular material can tune the properties of the components or give rise to new properties. As a workhorse, here we take a C60@lysozyme hybrid. We show that lysozyme recognizes and disperses fullerene in water. AFM, cryo-TEM and high resolution X-ray powder diffraction show that the C60 dispersion is monomolecular. The adduct is biocompatible, stable in physiological and technologically-relevant environments, and easy to store. Hybridization with lysozyme preserves the electrochemical properties of C60. EPR spin-trapping experiments show that the C60@lysozyme hybrid produces ROS following both type I and type II mechanisms. Due to the shielding effect of proteins, the adduct generates significant amounts of 1O2 also in aqueous solution. In the case of type I mechanism, the protein residues provide electrons and the hybrid does not require addition of external electron donors. The preparation process and the properties of C60@lysozyme are general and can be expected to be similar to other C60@protein systems. It is envisaged that the properties of the C60@protein hybrids will pave the way for a host of applications in nanomedicine, nanotechnology, and photocatalysis.

Molecular nucleation mechanisms and control strategies for crystal polymorph selection.

The formation of condensed (compacted) protein phases is associated with a wide range of human disorders, such as eye cataracts, amyotrophic lateral sclerosis, sickle cell anaemia and Alzheimer's disease. However, condensed protein phases have their uses: as crystals, they are harnessed by structural biologists to elucidate protein structures, or are used as delivery vehicles for pharmaceutical applications. The physiochemical properties of crystals can vary substantially between different forms or structures ('polymorphs') of the same macromolecule, and dictate their usability in a scientific or industrial context. To gain control over an emerging polymorph, one needs a molecular-level understanding of the pathways that lead to the various macroscopic states and of the mechanisms that govern pathway selection. However, it is still not clear how the embryonic seeds of a macromolecular phase are formed, or how these nuclei affect polymorph selection. Here we use time-resolved cryo-transmission electron microscopy to image the nucleation of crystals of the protein glucose isomerase, and to uncover at molecular resolution the nucleation pathways that lead to two crystalline states and one gelled state. We show that polymorph selection takes place at the earliest stages of structure formation and is based on specific building blocks for each space group. Moreover, we demonstrate control over the system by selectively forming desired polymorphs through site-directed mutagenesis, specifically tuning intermolecular bonding or gel seeding. Our results differ from the present picture of protein nucleation, in that we do not identify a metastable dense liquid as the precursor to the crystalline state. Rather, we observe nucleation events that are driven by oriented attachments between subcritical clusters that already exhibit a degree of crystallinity. These insights suggest ways of controlling macromolecular phase transitions, aiding the development of protein-based drug-delivery systems and macromolecular crystallography.

A classical view on nonclassical nucleation.

Understanding and controlling nucleation is important for many crystallization applications. Calcium carbonate (CaCO3) is often used as a model system to investigate nucleation mechanisms. Despite its great importance in geology, biology, and many industrial applications, CaCO3 nucleation is still a topic of intense discussion, with new pathways for its growth from ions in solution proposed in recent years. These new pathways include the so-called nonclassical nucleation mechanism via the assembly of thermodynamically stable prenucleation clusters, as well as the formation of a dense liquid precursor phase via liquid-liquid phase separation. Here, we present results from a combined experimental and computational investigation on the precipitation of CaCO3 in dilute aqueous solutions. We propose that a dense liquid phase (containing 4-7 H2O per CaCO3 unit) forms in supersaturated solutions through the association of ions and ion pairs without significant participation of larger ion clusters. This liquid acts as the precursor for the formation of solid CaCO3 in the form of vaterite, which grows via a net transfer of ions from solution according to z Ca2+ + z CO32- → z CaCO3 The results show that all steps in this process can be explained according to classical concepts of crystal nucleation and growth, and that long-standing physical concepts of nucleation can describe multistep, multiphase growth mechanisms.

CryoTEM as an Advanced Analytical Tool for Materials Chemists.

Morphology plays an essential role in chemistry through the segregation of atoms and/or molecules into different phases, delineated by interfaces. This is a general process in materials synthesis and exploited in many fields including colloid chemistry, heterogeneous catalysis, and functional molecular systems. To rationally design complex materials, we must understand and control morphology evolution. Toward this goal, we utilize cryogenic transmission electron microscopy (cryoTEM), which can track the structural evolution of materials in solution with nanometer spatial resolution and a temporal resolution of <1 s. In this Account, we review examples of our own research where direct observations by cryoTEM have been essential to understanding morphology evolution in macromolecular self-assembly, inorganic nucleation and growth, and the cooperative evolution of hybrid materials. These three different research areas are at the heart of our approach to materials chemistry where we take inspiration from the myriad examples of complex materials in Nature. Biological materials are formed using a limited number of chemical components and under ambient conditions, and their formation pathways were refined during biological evolution by enormous trial and error approaches to self-organization and biomineralization. By combining the information on what is possible in nature and by focusing on a limited number of chemical components, we aim to provide an essential insight into the role of structure evolution in materials synthesis. Bone, for example, is a hierarchical and hybrid material which is lightweight, yet strong and hard. It is formed by the hierarchical self-assembly of collagen into a macromolecular template with nano- and microscale structure. This template then directs the nucleation and growth of oriented, nanoscale calcium phosphate crystals to form the composite material. Fundamental insight into controlling these structuring processes will eventually allow us to design such complex materials with predetermined and potentially unique properties.

Silicanin-1 is a conserved diatom membrane protein involved in silica biomineralization.

BACKGROUND: Biological mineral formation (biomineralization) proceeds in specialized compartments often bounded by a lipid bilayer membrane. Currently, the role of membranes in biomineralization is hardly understood. RESULTS: Investigating biomineralization of SiO2 (silica) in diatoms we identified Silicanin-1 (Sin1) as a conserved diatom membrane protein present in silica deposition vesicles (SDVs) of Thalassiosira pseudonana. Fluorescence microscopy of GFP-tagged Sin1 enabled, for the first time, to follow the intracellular locations of a biomineralization protein during silica biogenesis in vivo. The analysis revealed incorporation of the N-terminal domain of Sin1 into the biosilica via association with the organic matrix inside the SDVs. In vitro experiments showed that the recombinant N-terminal domain of Sin1 undergoes pH-triggered assembly into large clusters, and promotes silica formation by synergistic interaction with long-chain polyamines. CONCLUSIONS: Sin1 is the first identified SDV transmembrane protein, and is highly conserved throughout the diatom realm, which suggests a fundamental role in the biomineralization of diatom silica. Through interaction with long-chain polyamines, Sin1 could serve as a molecular link by which the SDV membrane exerts control on the assembly of biosilica-forming organic matrices in the SDV lumen.

Mesoporous Silica Nanoparticle-Coated Microneedle Arrays for Intradermal Antigen Delivery.

PURPOSE: To develop a new intradermal antigen delivery system by coating microneedle arrays with lipid bilayer-coated, antigen-loaded mesoporous silica nanoparticles (LB-MSN-OVA). METHODS: Synthesis of MSNs with 10-nm pores was performed and the nanoparticles were loaded with the model antigen ovalbumin (OVA), and coated with a lipid bilayer (LB-MSN-OVA). The uptake of LB-MSN-OVA by bone marrow-derived dendritic cells (BDMCs) was studied by flow cytometry. The designed LB-MSN-OVA were coated onto pH-sensitive pyridine-modified microneedle arrays and the delivery of LB-MSN-OVA into ex vivo human skin was studied. RESULTS: The synthesized MSNs demonstrated efficient loading of OVA with a maximum loading capacity of about 34% and the lipid bilayer enhanced the colloidal stability of the MSNs. Uptake of OVA loaded in LB-MSN-OVA by BMDCs was higher than that of free OVA, suggesting effective targeting of LB-MSN-OVA to antigen-presenting cells. Microneedles were readily coated with LB-MSN-OVA at pH 5.8, yielding 1.5 µg of encapsulated OVA per microneedle array. Finally, as a result of the pyridine modification, LB-MSN-OVA were effectively released from the microneedles upon piercing the skin. CONCLUSION: Microneedle arrays coated with LB-MSN-OVA were successfully developed and shown to be suitable for intradermal delivery of the encapsulated protein antigen.