Cell-induced collagen alignment in a 3D in vitro culture during extracellular matrix production.

The bone extracellular matrix consists of a highly organized collagen matrix that is mineralized with carbonated hydroxyapatite. Even though the structure and composition of bone have been studied extensively, the mechanisms underlying collagen matrix organization remain elusive. In this study, we used a 3D cell culture system in which osteogenic cells deposit and orient the collagen matrix that is subsequently mineralized. Using live fluorescence imaging combined with volume electron microscopy, we visualize the organization of the cells and collagen in the cell culture. We show that the osteogenically induced cells are organizing the collagen matrix during development. Based on the observation of tunnel-like structures surrounded by aligned collagen in the center of the culture, we propose that osteoblasts organize the deposited collagen during migration through the culture. Overall, we show that cell-matrix interactions are involved in collagen alignment during early-stage osteogenic differentiation and that the matrix is organized by the osteoblasts in the absence of osteoclast activity.

Elucidating the role of water in collagen self-assembly by isotopically modulating collagen hydration.

Water is known to play an important role in collagen self-assembly, but it is still largely unclear how water-collagen interactions influence the assembly process and determine the fibril network properties. Here, we use the H[Formula: see text]O/D[Formula: see text]O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self-assembly. We dissolve collagen in H[Formula: see text]O and D[Formula: see text]O and compare the growth kinetics and the structure of the collagen assemblies formed in these water isotopomers. Surprisingly, collagen assembly occurs ten times faster in D[Formula: see text]O than in H[Formula: see text]O, and collagen in D[Formula: see text]O self-assembles into much thinner fibrils, that form a more inhomogeneous and softer network, with a fourfold reduction in elastic modulus when compared to H[Formula: see text]O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D[Formula: see text]O is less hydrated than in H[Formula: see text]O. This partial dehydration lowers the enthalpic penalty for water removal and reorganization at the collagen-water interface, increasing the self-assembly rate and the number of nucleation centers, leading to thinner fibrils and a softer network. Coarse-grained simulations show that the acceleration in the initial nucleation rate can be reproduced by the enhancement of electrostatic interactions. These results show that water acts as a mediator between collagen monomers, by modulating their interactions so as to optimize the assembly process and, thus, the final network properties. We believe that isotopically modulating the hydration of proteins can be a valuable method to investigate the role of water in protein structural dynamics and protein self-assembly.

Phase-Separated Lipid-Based Nanoparticles: Selective Behavior at the Nano-Bio Interface.

The membrane-protein interface on lipid-based nanoparticles influences their in vivo behavior. Better understanding may evolve current drug delivery methods toward effective targeted nanomedicine. Previously, the cell-selective accumulation of a liposome formulation in vivo is demonstrated, through the recognition of lipid phase-separation by triglyceride lipases. This exemplified how liposome morphology and composition can determine nanoparticle-protein interactions. Here, the lipase-induced compositional and morphological changes of phase-separated liposomes-which bear a lipid droplet in their bilayer- are investigated, and the mechanism upon which lipases recognize and bind to the particles is unravelled. The selective lipolytic degradation of the phase-separated lipid droplet is observed, while nanoparticle integrity remains intact. Next, the Tryptophan-rich loop of the lipase is identified as the region with which the enzymes bind to the particles. This preferential binding is due to lipid packing defects induced on the liposome surface by phase separation. In parallel, the existing knowledge that phase separation leads to in vivo selectivity, is utilized to generate phase-separated mRNA-LNPs that target cell-subsets in zebrafish embryos, with subsequent mRNA delivery and protein expression. Together, these findings can expand the current knowledge on selective nanoparticle-protein communications and in vivo behavior, aspects that will assist to gain control of lipid-based nanoparticles.

Unraveling the Formation of Gelatin Nanospheres by Means of Desolvation.

Gelatin nanoparticles (GNPs) have been widely studied for a plethora of biomedical applications, but their formation mechanism remains poorly understood, which precludes precise control over their physicochemical properties. This leads to time-consuming parameter adjustments without a fundamental grasp of the underlying mechanism. Here, we analyze and visualize in a time-resolved manner the mechanism by which GNPs are formed during desolvation of gelatin as a function of gelatin molecular weight and type of desolvating agent. Through various analytical and imaging techniques, we unveil a multistage process that is initiated by the formation of primary particles that are ∼18 nm in diameter (wet state). These primary particles subsequently assemble into colloidally stable GNPs with a raspberry-like structure and a hydrodynamic diameter of ∼300 nm. Our results create a basic understanding of the formation mechanism of gelatin nanoparticles, which opens new opportunities for precisely tuning their physicochemical and biofunctional properties.

Precise targeting for 3D cryo-correlative light and electron microscopy volume imaging of tissues using a FinderTOP.

Cryo-correlative light and electron microscopy (cryoCLEM) is a powerful strategy to high resolution imaging in the unperturbed hydrated state. In this approach fluorescence microscopy aids localizing the area of interest, and cryogenic focused ion beam/scanning electron microscopy (cryoFIB/SEM) allows preparation of thin cryo-lamellae for cryoET. However, the current method cannot be accurately applied on bulky (3D) samples such as tissues and organoids. 3D cryo-correlative imaging of large volumes is needed to close the resolution gap between cryo-light microscopy and cryoET, placing sub-nanometer observations in a larger biological context. Currently technological hurdles render 3D cryoCLEM an unexplored approach. Here we demonstrate a cryoCLEM workflow for tissues, correlating cryo-Airyscan confocal microscopy with 3D cryoFIB/SEM volume imaging. Accurate correlation is achieved by imprinting a FinderTOP pattern in the sample surface during high pressure freezing, and allows precise targeting for cryoFIB/SEM volume imaging.

Extracellular vesicles contribute to early cyst development in autosomal dominant polycystic kidney disease by cell-to-cell communication.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts within the kidney due to mutations in PKD1 or PKD2. Although the disease remains incompletely understood, one of the factors associated with ADPKD progression is the release of nucleotides (including ATP), which can initiate autocrine or paracrine purinergic signaling by binding to their receptors. Recently, we and others have shown that increased extracellular vesicle (EVs) release from PKD1 knockout cells can stimulate cyst growth through effects on recipient cells. Given that EVs are an important communicator between different nephron segments, we hypothesize that EVs released from PKD1 knockout distal convoluted tubule (DCT) cells can stimulate cyst growth in the downstream collecting duct (CD). Here, we show that administration of EVs derived from Pkd1-/- mouse distal convoluted tubule (mDCT15) cells result in a significant increase in extracellular ATP release from Pkd1-/- mouse inner medullary collecting duct (iMCD3) cells. In addition, exposure of Pkd1-/- iMCD3 cells to EVs derived from Pkd1-/- mDCT15 cells led to an increase in the phosphorylation of the serine/threonine-specific protein Akt, suggesting activation of proliferative pathways. Finally, the exposure of iMCD3 Pkd1-/- cells to mDCT15 Pkd1-/- EVs increased cyst size in Matrigel. These findings indicate that EVs could be involved in intersegmental communication between the distal convoluted tubule and the collecting duct and potentially stimulate cyst growth.

Phase-Separated Liposomes Hijack Endogenous Lipoprotein Transport and Metabolism Pathways to Target Subsets of Endothelial Cells In Vivo.

Plasma lipid transport and metabolism are essential to ensure correct cellular function throughout the body. Dynamically regulated in time and space, the well-characterized mechanisms underpinning plasma lipid transport and metabolism offers an enticing, but as yet underexplored, rationale to design synthetic lipid nanoparticles with inherent cell/tissue selectivity. Herein, a systemically administered liposome formulation, composed of just two lipids, that is capable of hijacking a triglyceride lipase-mediated lipid transport pathway resulting in liposome recognition and uptake within specific endothelial cell subsets is described. In the absence of targeting ligands, liposome-lipase interactions are mediated by a unique, phase-separated ("parachute") liposome morphology. Within the embryonic zebrafish, selective liposome accumulation is observed at the developing blood-brain barrier. In mice, extensive liposome accumulation within the liver and spleen - which is reduced, but not eliminated, following small molecule lipase inhibition - supports a role for endothelial lipase but highlights these liposomes are also subject to significant "off-target" by reticuloendothelial system organs. Overall, these compositionally simplistic liposomes offer new insights into the discovery and design of lipid-based nanoparticles that can exploit endogenous lipid transport and metabolism pathways to achieve cell selective targeting in vivo.

Involvement of ceramide biosynthesis in increased extracellular vesicle release in Pkd1 knock out cells.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited disorder characterized by the development of renal cysts, which frequently leads to renal failure. Hypertension and other cardiovascular symptoms contribute to the high morbidity and mortality of the disease. ADPKD is caused by mutations in the PKD1 gene or, less frequently, in the PKD2 gene. The disease onset and progression are highly variable between patients, whereby the underlying mechanisms are not fully elucidated. Recently, a role of extracellular vesicles (EVs) in the progression of ADPKD has been postulated. However, the mechanisms stimulating EV release in ADPKD have not been addressed and the participation of the distal nephron segments is still uninvestigated. Here, we studied the effect of Pkd1 deficiency on EV release in wild type and Pkd1-/- mDCT15 and mIMCD3 cells as models of the distal convoluted tubule (DCT) and inner medullary collecting duct (IMCD), respectively. By using nanoparticle tracking analysis, we observed a significant increase in EV release in Pkd1-/- mDCT15 and mIMCD3 cells, with respect to the wild type cells. The molecular mechanisms leading to the changes in EV release were further investigated in mDCT15 cells through RNA sequencing and qPCR studies. Specifically, we assessed the relevance of purinergic signaling and ceramide biosynthesis enzymes. Pkd1-/- mDCT15 cells showed a clear upregulation of P2rx7 expression compared to wild type cells. Depletion of extracellular ATP by apyrase (ecto-nucleotidase) inhibited EV release only in wild type cells, suggesting an exacerbated signaling of the extracellular ATP/P2X7 pathway in Pkd1-/- cells. In addition, we identified a significant up-regulation of the ceramide biosynthesis enzymes CerS6 and Smpd3 in Pkd1-/- cells. Altogether, our findings suggest the involvement of the DCT in the EV-mediated ADPKD progression and points to the induction of ceramide biosynthesis as an underlying molecular mechanism. Further studies should be performed to investigate whether CerS6 and Smpd3 can be used as biomarkers of ADPKD onset, progression or severity.

Strategy for optimizing experimental settings for studying low atomic number colloidal assemblies using liquid phase scanning transmission electron microscopy.

Observing processes of nanoscale materials of low atomic number is possible using liquid phase electron microscopy (LP-EM). However, the achievable spatial resolution (d) is limited by radiation damage. Here, we examine a strategy for optimizing LP-EM experiments based on an analytical model and experimental measurements, and develop a method for quantifying image quality at ultra low electron dose De using scanning transmission electron microscopy (STEM). As experimental test case we study the formation of a colloidal binary system containing 30 nm diameter SiO2 nanoparticles (SiONPs), and 100 nm diameter polystyrene microspheres (PMs). We show that annular dark field (DF) STEM is preferred over bright field (BF) STEM for practical reasons. Precise knowledge of the material's density is crucial for the calculations in order to match experimental data. To calculate the detectability of nano-objects in an image, the Rose criterion for single pixels is expanded to a model of the signal to noise ratio obtained for multiple pixels spanning the image of an object. Using optimized settings, it is possible to visualize the radiation-sensitive, hierarchical low-Z binary structures, and identify both components.

Anionic Lipid Nanoparticles Preferentially Deliver mRNA to the Hepatic Reticuloendothelial System.

Lipid nanoparticles (LNPs) are the leading nonviral technologies for the delivery of exogenous RNA to target cells in vivo. As systemic delivery platforms, these technologies are exemplified by Onpattro, an approved LNP-based RNA interference therapy, administered intravenously and targeted to parenchymal liver cells. The discovery of systemically administered LNP technologies capable of preferential RNA delivery beyond hepatocytes has, however, proven more challenging. Here, preceded by comprehensive mechanistic understanding of in vivo nanoparticle biodistribution and bodily clearance, an LNP-based messenger RNA (mRNA) delivery platform is rationally designed to preferentially target the hepatic reticuloendothelial system (RES). Evaluated in embryonic zebrafish, validated in mice, and directly compared to LNP-mRNA systems based on the lipid composition of Onpattro, RES-targeted LNPs significantly enhance mRNA expression both globally within the liver and specifically within hepatic RES cell types. Hepatic RES targeting requires just a single lipid change within the formulation of Onpattro to switch LNP surface charge from neutral to anionic. This technology not only provides new opportunities to treat liver-specific and systemic diseases in which RES cell types play a key role but, more importantly, exemplifies that rational design of advanced RNA therapies must be preceded by a robust understanding of the dominant nano-biointeractions involved.

SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids.

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human-induced pluripotent stem-cell-derived kidney organoids with SARS-CoV-2. Single-cell RNA sequencing indicated injury and dedifferentiation of infected cells with activation of profibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in long COVID.

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.

HPM live µ for a full CLEM workflow.

With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information-known as multimodal imaging or correlative microscopy (CM)-has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution. Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by chemical fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artifacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM. To minimize the delay separating live imaging from cryo-arrest, we developed a high-pressure freezing (HPF) system directly coupled to a light microscope. We address the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. To further explore the potential of cryo-fixation related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification, the HPF core technology has been revisited to allow easy modification of the environmental parameters during vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

Spontaneous organization of supracolloids into three-dimensional structured materials.

Periodic nano- or microscale structures are used to control light, energy and mass transportation. Colloidal organization is the most versatile method used to control nano- and microscale order, and employs either the enthalpy-driven self-assembly of particles at a low concentration or the entropy-driven packing of particles at a high concentration. Nonetheless, it cannot yet provide the spontaneous three-dimensional organization of multicomponent particles at a high concentration. Here we combined these two concepts into a single strategy to achieve hierarchical multicomponent materials. We tuned the electrostatic attraction between polymer and silica nanoparticles to create dynamic supracolloids whose components, on drying, reorganize by entropy into three-dimensional structured materials. Cryogenic electron tomography reveals the kinetic pathways, whereas Monte Carlo simulations combined with a kinetic model provide design rules to form the supracolloids and control the kinetic pathways. This approach may be useful to fabricate hierarchical hybrid materials for distinct technological applications.

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.

Intermolecular channels direct crystal orientation in mineralized collagen.

The mineralized collagen fibril is the basic building block of bone, and is commonly pictured as a parallel array of ultrathin carbonated hydroxyapatite (HAp) platelets distributed throughout the collagen. This orientation is often attributed to an epitaxial relationship between the HAp and collagen molecules inside 2D voids within the fibril. Although recent studies have questioned this model, the structural relationship between the collagen matrix and HAp, and the mechanisms by which collagen directs mineralization remain unclear. Here, we use XRD to reveal that the voids in the collagen are in fact cylindrical pores with diameters of ~2 nm, while electron microscopy shows that the HAp crystals in bone are only uniaxially oriented with respect to the collagen. From in vitro mineralization studies with HAp, CaCO3 and γ-FeOOH we conclude that confinement within these pores, together with the anisotropic growth of HAp, dictates the orientation of HAp crystals within the collagen fibril.

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.