Trained Immunity-Promoting Nanobiologic Therapy Suppresses Tumor Growth and Potentiates Checkpoint Inhibition.

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.

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.

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.

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.

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.

Writing silica structures in liquid with scanning transmission electron microscopy.

Silica nanoparticles are imaged in solution with scanning transmission electron microscopy (STEM) using a liquid cell with silicon nitride (SiN) membrane windows. The STEM images reveal that silica structures are deposited in well-defined patches on the upper SiN membranes upon electron beam irradiation. The thickness of the deposits is linear with the applied electron dose. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrate that the deposited patches are a result of the merging of the original 20 nm-diameter nanoparticles, and that the related surface roughness depends on the electron dose rate used. Using this approach, sub-micrometer scale structures are written on the SiN in liquid by controlling the electron exposure as function of the lateral position.

Partial oxidation as a rational approach to kinetic control in bioinspired magnetite synthesis.

Biological systems show impressive control over the shape, size and organization of mineral structures, which often leads to advanced physical properties that are tuned to the function of these materials. Such control is also found in magnetotactic bacteria, which produce-in aqueous medium and at room temperature-magnetite nanoparticles with precisely controlled morphologies and sizes that are generally only accessible in synthetic systems with the use of organic solvents and/or the use of high-temperature methods. The synthesis of magnetite under biomimetic conditions, that is, in water and at room temperature and using polymeric additives as control agents, is of interest as a green production method for magnetic nanoparticles. Inspired by the process of magnetite biomineralization, a rational approach is taken by the use of a solid precursor for the synthesis of magnetite nanoparticles. The conversion of a ferrous hydroxide precursor, which we demonstrate with cryo-TEM and low-dose electron diffraction, is used to achieve control over the solution supersaturation such that crystal growth can be regulated through the interaction with poly-(α,ß)-dl-aspartic acid, a soluble, negatively charged polymer. In this way, stable suspensions of nanocrystals are achieved that show remanence and coercivity at the size limit of superparamagnetism, and which are able to align their magnetic moments forming strings in solution as is demonstrated by cryo-electron tomography.

Graphene oxide single sheets as substrates for high resolution cryoTEM.

CryoTEM is an important tool in the analysis of soft matter, where generally defocus conditions are used to enhance the contrast in the images, but this is at the expense of the maximum resolution that can be obtained. Here, we demonstrate the use of graphene oxide single sheets as support for the formation of 10 nm thin films for high resolution cryoTEM imaging, using DNA as an example. With this procedure, the overlap of objects in the vitrified film is avoided. Moreover, in these thin films less background scattering occurs and as a direct result, an increased contrast can be observed in the images. Hence, imaging closer to focus as compared with conventional cryoTEM procedures is achieved, without losing contrast. In addition, we demonstrate an ~1.8 fold increase in resolution, which is crucial for accurate size analysis of nanostructures.

Three-dimensional structure of P3HT assemblies in organic solvents revealed by cryo-TEM.

Poly(3-hexylthiophene) (P3HT) assemblies in vitrified organic solvents were visualized at nanometer scale resolution by cryo-transmission electron microscopy, low dose electron diffraction, and cryo-tomography revealing a three-dimensional lamellar structure formed by the stacking of the conjugated backbones of P3HT with a distance of 1.7 nm and increased order in the bulk of the nanowire. This combination of techniques reveals local structures in dispersion and the condensed state that play a crucial role in the performance of organic electronic devices.

Nucleation and growth of monodisperse silica nanoparticles.

Although monodisperse amorphous silica nanoparticles have been widely investigated, their formation mechanism is still a topic of debate. Here, we demonstrate the formation of monodisperse nanoparticles from colloidally stabilized primary particles, which at a critical concentration undergo a concerted association process, concomitant with a morphological and structural collapse. The formed assemblies grow further by addition of primary particles onto their surface. The presented mechanism, consistent with previously reported observations, reconciles the different theories proposed to date.

Controlling internal pore sizes in bicontinuous polymeric nanospheres.

Complex polymeric nanospheres were formed in water from comb-like amphiphilic block copolymers. Their internal morphology was determined by three-dimensional cryo-electron tomographic analysis. Varying the polymer molecular weight (MW) and the hydrophilic block weight content allowed for fine control over the internal structure. Construction of a partial phase diagram allowed us to determine the criteria for the formation of bicontinuous polymer nanosphere (BPN), namely for copolymers with MW of up to 17 kDa and hydrophilic weight fractions of ≤0.25; and varying the organic solvent to water ratio used in their preparation allowed for control over nanosphere diameters from 70 to 460 nm. Significantly, altering the block copolymer hydrophilic-hydrophobic balance enabled control of the internal pore diameter of the BPNs from 10 to 19 nm.

Gold nanorods with sub-nanometer separation using cucurbit[n]uril for SERS applications.

The capability of cucurbit[n]uril to align gold nanorods, leading to optical coupling into the infrared region, is shown. Cryo-TEM and tomographic imaging confirm the presence of aligned Au nanorods. Full electromagnetic simulations, which support the observed plasmonic modes and predict large enhancements in the inter-particle junction, are performed. This construct is then further utilized for surface enhanced Raman spectroscopy.

Nucleation and growth of magnetite from solution.

The formation of crystalline materials from solution is usually described by the nucleation and growth theory, where atoms or molecules are assumed to assemble directly from solution. For numerous systems, the formation of the thermodynamically stable crystalline phase is additionally preceded by metastable intermediates . More complex pathways have recently been proposed, such as aggregational processes of nanoparticle precursors or pre-nucleation clusters, which seem to contradict the classical theory. Here we show by cryogenic transmission electron microscopy that the nucleation and growth of magnetite-a magnetic iron oxide with numerous bio- and nanotechnological applications-proceed through rapid agglomeration of nanometric primary particles and that in contrast to the nucleation of other minerals, no intermediate amorphous bulk precursor phase is involved. We also demonstrate that these observations can be described within the framework of classical nucleation theory.

Coiled coil driven membrane fusion between cyclodextrin vesicles and liposomes.

Controlled fusion events between natural membranes composed of phospholipids with synthetic unnatural membranes will yield valuable fundamental information on the mechanism of membrane fusion. Here, fusion between vastly different phospholipid liposomes and cyclodextrin amphiphile based vesicles (CDVs) controlled by a pair of coiled coil forming lipidated peptides was investigated. Fusion events were characterized using lipid and content mixing assays and the resulting hybrid assemblies were characterized with cryo-TEM imaging. The secondary/quaternary structure of the lipidated peptides at the membrane interface was studied using circular dichroism spectroscopy. This is the first example of targeted fusion between natural and non-natural bilayer membranes and the in situ formation of hybrid CDV-liposome structures is of interest as it yields fundamental information about the mechanism through which fusion proceeds.

Controlling the growth and shape of chiral supramolecular polymers in water.

A challenging target in the noncovalent synthesis of nanostructured functional materials is the formation of uniform features that exhibit well-defined properties, e.g., precise control over the aggregate shape, size, and stability. In particular, for aqueous-based one-dimensional supramolecular polymers, this is a daunting task. Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing out attractive noncovalent forces within the hydrophobic core of the polymerizing building blocks with electrostatic repulsive interactions on the hydrophilic rim we managed to switch from elongated, rod-like assemblies to small and discrete objects. Intriguingly this rod-to-sphere transition is expressed in a loss of cooperativity in the temperature-dependent self-assembly mechanism. The aggregates were characterized using circular dichroism, UV and 1H-NMR spectroscopy, small angle X-ray scattering, and cryotransmission electron microscopy. In analogy to many systems found in biology, mechanistic details of the self-assembly pathways emphasize the importance of cooperativity as a key feature that dictates the physical properties of the produced supramolecular polymers.

High-magnesian calcite mesocrystals: a coordination chemistry approach.

While biogenic calcites frequently contain appreciable levels of magnesium, the pathways leading to such high concentrations remain unclear. The production of high-magnesian calcites in vitro is highly challenging, because Mg-free aragonite, rather than calcite, is the favored product in the presence of strongly hydrated Mg(2+) ions. While nature may overcome this problem by forming a Mg-rich amorphous precursor, which directly transforms to calcite without dissolution, high Mg(2+)/Ca(2+) ratios are required synthetically to precipitate high-magnesian calcite from solution. Indeed, it is difficult to synthesize amorphous calcium carbonate (ACC) containing high levels of Mg, and the Mg is typically not preserved in the calcite product as the transformation occurs via a dissolution-reprecipitation route. We here present a novel synthetic method, which employs a strategy based on biogenic systems, to generate high-magnesian calcite. Mg-containing ACC is produced in a nonaqueous environment by reacting a mixture of Ca and Mg coordination complexes with CO(2). Control over the Mg incorporation is simply obtained by the ratio of the starting materials. Subsequent crystallization at reduced water activities in an organic solvent/water mixture precludes dissolution and reprecipitation and yields high-magnesian calcite mesocrystals with Mg contents as high as 53 mol %. This is in direct contrast with the polycrystalline materials generally observed when magnesian calcite is formed synthetically. Our findings give insight into the possible mechanisms of formation of biogenic high-magnesian calcites and indicate that precise control over the water activity may be a key element.

A triptycene-based approach to solubilising carbon nanotubes and C60.

We describe herein the synthesis of a triptycene-based surfactant designed with the ability to solubilise single-walled carbon nanotubes (SWNTs) and C(60) in water through non-covalent interactions. Furthermore, an amphiphilic naphthalene-based surfactant with the same ability to solubilise SWNTs and C(60) has also been prepared. The compounds synthesised were designed with either two ionic or non-ionic tails to ensure a large number of supramolecular interactions with the solvent, thereby promoting strong solubilisation. The surfactants produced stable suspensions in which the SWNTs are dispersed and the surfactant/SWNT complexes formed are stable for more than one year. UV/Vis/NIR absorption spectroscopy, TEM and AFM were employed to probe the solubilisation properties of the dispersion of surfactants and SWNTs in water.

The role of the amorphous phase on the biomimetic mineralization of collagen.

Bone is a hierarchically structured composite material whose basic building block is the mineralized collagen fibril, where the collagen is the scaffold into which the hydroxyapatite (HA) crystals nucleate and grow. Understanding the mechanisms of hydroxyapatite formation inside the collagen is key to unravelling osteogenesis. In this work, we employed a biomimetic in vitro mineralization system to investigate the role of the amorphous precursor calcium phosphate phase in the mineralization of collagen. We observed that the rate of collagen mineralization is highly dependent on the concentration of polyaspartic acid, an inhibitor of hydroxyapatite nucleation and inducer of intrafibrillar mineralization. The lower the concentration of the polymer, the faster the mineralization and crystallization. Addition of the non-collagenous protein C-DMP1, a nucleator of hydroxyapatite, substantially accelerates mineral infiltration as well as HA nucleation. We have also demonstrated that Cu ions interfere with the mineralization process first by inhibiting the entry of the calcium phosphate into the collagen, and secondly by stabilizing the ACP, such that it does not convert into HA. Interestingly, under these conditions mineralization happens preferentially in the overlap regions of the collagen fibril. Our results show that the interactions between the amorphous precursor phase and the collagen fibril play an important role in the control over mineralization.

Multiscale characterization of pathological bone tissue.

Bone is a complex natural material with a complex hierarchical multiscale organization, crucial to perform its functions. Ultrastructural analysis of bone is crucial for our understanding of cell to cell communication, the healthy or pathological composition of bone tissue, and its three-dimensional (3D) organization. A variety of techniques has been used to analyze bone tissue. This article describes a combined approach of optical, scanning electron, and transmission electron microscopy for the ultrastructural analysis of bone from the nanoscale to the macroscale, as illustrated by two pathological bone tissues. By following a top-down approach to investigate the multiscale organization of pathological bones, quantitative estimates were made in terms of calcium content, nearest neighbor distances of osteocytes, canaliculi diameter, ordering, and D-spacing of the collagen fibrils, and the orientation of intrafibrillar minerals which enable us to observe the fine structural details. We identify and discuss a series of two-dimensional (2D) and 3D imaging techniques that can be used to characterize bone tissue. By doing so we demonstrate that, while 2D imaging techniques provide comparable information from pathological bone tissues, significantly different structural details are observed upon analyzing the pathological bone tissues in 3D. Finally, particular attention is paid to sample preparation for and quantitative processing of data from electron microscopic analysis.