Assembly of partially covered strawberry supracolloids in dilute and concentrate aqueous dispersions.

HYPOTHESIS: Core-corona supracolloids can be assembled in aqueous dispersions by controlling the physical interactions between the corona and core colloidal particles. A raspberry corona configuration with full surface coverage of the core can be reached by inducing strong attractive interactions between the individual particles. A controlled partial surface coverage of the core, i.e. strawberry configuration, is however, more difficult to achieve. Supracolloids with different surface coverage ratio exhibit unique and multifunctional surface properties. EXPERIMENTS: By counterbalancing the multiple physical interactions playing a role during the assembly, the configuration and stability of the assemblies could be fine-tuned over a wide range of concentrations. Supracolloids consisting of polyethylene glycol (PEO)-grafted polymer particles covered by silica nanoparticles were assembled with different configurations, by adjusting the pH and ionic strength of the dispersion, the PEO grafting density and the particles concentration. The self-assembly process and resulting configurations were monitored via cryogenic transmission electron microscopy (Cryo-TEM) and light scattering. FINDINGS: The suitable conditions to assemble supracolloids with partial corona coverage have been established. Stable strawberry supracolloids could be prepared, both for diluted (1 wt%) and concentrated (12 wt%) dispersions. These hybrid supracolloids with well-defined configuration are highly relevant to developing advanced water-borne paints and inks, food dispersions, cosmetic and healthcare products.

Investigating the Morphology and Mechanics of Biogenic Hierarchical Materials at and below Micrometer Scale.

Investigating and understanding the intrinsic material properties of biogenic materials, which have evolved over millions of years into admirable structures with difficult to mimic hierarchical levels, holds the potential of replacing trial-and-error-based materials optimization in our efforts to make synthetic materials of similarly advanced complexity and properties. An excellent example is biogenic silica which is found in the exoskeleton of unicellular photosynthetic algae termed diatoms. Because of the complex micro- and nanostructures found in their exoskeleton, determining the intrinsic mechanical properties of biosilica in diatoms has only partly been accomplished. Here, a general method is presented in which a combination of in situ deformation tests inside an SEM with a realistic 3D model of the frustule of diatom Craspedostauros sp. (C. sp.) obtained by electron tomography, alongside finite element method (FEM) simulations, enables quantification of the Young's modulus (E = 2.3 ± 0.1 GPa) of this biogenic hierarchical silica. The workflow presented can be readily extended to other diatom species, biominerals, or even synthetic hierarchical materials.

Mapping and Controlling Liquid Layer Thickness in Liquid-Phase (Scanning) Transmission Electron Microscopy.

Liquid-Phase (Scanning) Transmission Electron Microscopy (LP-(S)TEM) has become an essential technique to monitor nanoscale materials processes in liquids in real-time. Due to the pressure difference between the liquid and the microscope vacuum, bending of the silicon nitride (SiNx ) membrane windows generally occurs. This causes a spatially varying liquid layer thickness that makes interpretation of LP-(S)TEM results difficult due to a locally varying achievable resolution and diffusion limitations. To mediate these difficulties, it is shown: 1) how to quantitatively map liquid layer thickness for any liquid at less than 0.01 e- Å-2 total dose; 2) how to dynamically modulate the liquid thickness by tuning the internal pressure in the liquid cell, co-determined by the Laplace pressure and the external pressure. It is demonstrated that reproducible inward bulging of the window membranes can be realized, leading to an ultra-thin liquid layer in the central window area for high-resolution imaging. Furthermore, it is shown that the liquid thickness can be dynamically altered in a programmed way, thereby potentially overcoming the diffusion limitations towards achieving bulk solution conditions. The presented approaches provide essential ways to measure and dynamically adjust liquid thickness in LP-(S)TEM experiments, enabling new experiment designs and better control of solution chemistry.

In Situ Manipulation and Micromechanical Characterization of Diatom Frustule Constituents Using Focused Ion Beam Scanning Electron Microscopy.

Biocomposite structures are difficult to characterize by bulk approaches due to their morphological complexity and compositional heterogeneity. Therefore, a versatile method is required to assess, for example, the mechanical properties of geometrically simple parts of biocomposites at the relevant length scales. Here, it is demonstrated how a combination of Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and micromanipulators can be used to isolate, transfer, and determine the mechanical properties of frustule constituents of diatom Thalassiosira pseudonana (T.p.). Specifically, two parts of the diatom frustule, girdle bands and valves, are separated by FIB milling and manipulated using a sharp tungsten tip without compromising their physical or chemical integrity. In situ mechanical studies on isolated girdle bands combined with Finite Element Method (FEM) simulations, enables the quantitative assessment of the Young's modulus of this biosilica; E = 40.0 GPa. In addition, the mechanical strength of isolated valves could be measured by transferring and mounting them on top of premilled holes in the sample support. This approach may be extended to any hierarchical biocomposite material, regardless of its chemical composition, to isolate, transfer, and investigate the mechanical properties of selected constituents or specific regions.

Nucleation of protein mesocrystals via oriented attachment.

Self-assembly of proteins holds great promise for the bottom-up design and production of synthetic biomaterials. In conventional approaches, designer proteins are pre-programmed with specific recognition sites that drive the association process towards a desired organized state. Although proven effective, this approach poses restrictions on the complexity and material properties of the end-state. An alternative, hierarchical approach that has found wide adoption for inorganic systems, relies on the production of crystalline nanoparticles that become the building blocks of a next-level assembly process driven by oriented attachment (OA). As it stands, OA has not yet been observed for protein systems. Here we employ cryo-transmission electron microscopy (cryoEM) in the high nucleation rate limit of protein crystals and map the self-assembly route at molecular resolution. We observe the initial formation of facetted nanocrystals that merge lattices by means of OA alignment well before contact is made, satisfying non-trivial symmetry rules in the process. As these nanocrystalline assemblies grow larger we witness imperfect docking events leading to oriented aggregation into mesocrystalline assemblies. These observations highlight the underappreciated role of the interaction between crystalline nuclei, and the impact of OA on the crystallization process of proteins.

Time-Resolved Cryo-TEM Study on the Formation of Iron Hydroxides in a Collagen Matrix.

The mineralization of collagen via synthetic procedures has been extensively investigated for hydroxyapatite as well as for silica and calcium carbonate. From a fundamental point of view, it is interesting to investigate whether collagen could serve as a generic mineralization template for other minerals, like iron oxides. Here, bio-inspired coprecipitation reaction, generally leading to the formation of magnetite, is used to mineralize collagen with iron hydroxides. Platelet-shaped green rust crystals form outside the collagen matrix, while inside the collagen, nanoparticles with a size of 2.6 nm are formed, which are hypothesized to be iron (III) hydroxide. Mineralization with nanoparticles inside the collagen solely occurs in the presence of poly(aspartic acid) (pAsp). In the absence of pAsp, magnetite particles are formed around the collagen. Time-resolved cryo-TEM shows that during the coprecipitation reaction, initially a beam-sensitive phase is formed, possibly an Fe3+-pAsp complex. This beam-sensitive phase transforms into nanoparticles. In a later stage, sheet-like crystals are also found. After 48 h of mineralization, ordering of the nanoparticles around one of the collagen sub-bands (the a-band) is observed. This is very similar to the collagen-hydroxyapatite system, indicating that mineralization with iron hydroxides inside collagen is possible and proceeds via a similar mechanism as hydroxyapatite mineralization.

Studying Reaction Mechanisms in Solution Using a Distributed Electron Microscopy Method.

Electron microscopy (EM) of materials undergoing chemical reactions provides knowledge of the underlying mechanisms. However, the mechanisms are often complex and cannot be fully resolved using a single method. Here, we present a distributed electron microscopy method for studying complex reactions. The method combines information from multiple stages of the reaction and from multiple EM methods, including liquid phase EM (LP-EM), cryogenic EM (cryo-EM), and cryo-electron tomography (cryo-ET). We demonstrate this method by studying the desilication mechanism of zeolite crystals. Collectively, our data reveal that the reaction proceeds via a two-step anisotropic etching process and that the defects in curved surfaces and between the subunits in the crystal control the desilication kinetics by directing mass transport.

A modular approach toward producing nanotherapeutics targeting the innate immune system.

Immunotherapies controlling the adaptive immune system are firmly established, but regulating the innate immune system remains much less explored. The intrinsic interactions between nanoparticles and phagocytic myeloid cells make these materials especially suited for engaging the innate immune system. However, developing nanotherapeutics is an elaborate process. Here, we demonstrate a modular approach that facilitates efficiently incorporating a broad variety of drugs in a nanobiologic platform. Using a microfluidic formulation strategy, we produced apolipoprotein A1-based nanobiologics with favorable innate immune system-engaging properties as evaluated by in vivo screening. Subsequently, rapamycin and three small-molecule inhibitors were derivatized with lipophilic promoieties, ensuring their seamless incorporation and efficient retention in nanobiologics. A short regimen of intravenously administered rapamycin-loaded nanobiologics (mTORi-NBs) significantly prolonged allograft survival in a heart transplantation mouse model. Last, we studied mTORi-NB biodistribution in nonhuman primates by PET/MR imaging and evaluated its safety, paving the way for clinical translation.

Chain length of bioinspired polyamines affects size and condensation of monodisperse silica particles.

Polyamines play a major role in biosilicification reactions in diatoms and sponges. While the effects of polyamines on silicic acid oligomerization and precipitation are well known, the impact of polyamines chain length on silica particle growth is unclear. We studied the effects of polyamine chain length on silica particle growth and condensation in a known, simple, and salt-free biphasic reaction system; with tetraethyl orthosilicate as organic phase and polyamine dissolved in the aqueous phase. The particles at various growth stages were characterized by Cryo- Transmission Electron Microscopy, Scanning Electron Microscopy, Thermogravimetric Analysis, Zeta Potential, and solid-state NMR analysis. Polyamines were found co-localized within silica particles and the particle diameter increased with an increase in polyamine chain length, whereas silica condensation showed the opposite trend. Particle growth is proposed to progress via a coacervate intermediate while the final particles have a core shell structure with an amine-rich core and silica-rich shell. The results presented in this paper would of interest for researchers working in the field of bioinspired materials.

Hybrid Biodegradable Nanomotors through Compartmentalized Synthesis.

Designer particles that are embued with nanomachinery for autonomous motion have great potential for biomedical applications; however, their development is highly demanding with respect to biodegradability/compatibility. Previously, biodegradable propulsive machinery based on enzymes has been presented. However, enzymes are highly susceptible to proteolysis and deactivation in biological milieu. Biodegradable hybrid nanomotors powered by catalytic inorganic nanoparticles provide a proteolytically stable alternative to those based upon enzymes. Herein we describe the assembly of hybrid biodegradable nanomotors capable of transducing chemical energy into motion. Such nanomotors are constructed through a process of compartmentalized synthesis of inorganic MnO2 nanoparticles (MnPs) within the cavity of organic stomatocytes. We show that the nanomotors remain active in cellular environments and do not compromise cell viability. Effective tumor penetration of hybrid nanomotors is also demonstrated in proof-of-principle experiments. Overall, this work represents a new prospect for engineering of nanomotors that can retain their functionality within biological contexts.

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.

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.