Abstract P-34: Cryo-EM Study of Submicrocapsules with a Shell of Nanoparticle Heteroaggregates and Polyelectrolyte Layers

Background: Currently, different approaches of active and passive targeted drug delivery are being developed. One of the most promising methods of targeted drug delivery is the use of capsules. For instance, colloidosomes—capsules consisting of the shell formed by colloidal particles at the interface of the emulsion—can be used for targeted delivery of antitumor drugs or any other drugs in liquid form. Here we present results of cryo-EM study of submicrocapsules with the soybean oil core and with the shell consisting of SiO2 nanoparticles and detonation nanodiamonds (DNDs) stabilized with chitosan and alginate. Methods: Сryo-electron tomography (Cryo-ET) was used to identify the morphological features of the submicrocapsules. Preliminary screening of samples and cryo-ET data collection were performed using Titan Krios cryo-EM (ThermoFisher Scientific, US) equipped with Falcon 2 direct electron detector. The restoration of the tomographic series was carried out using IMOD software. Eman2 was used for segmentation and UCSF Chimera was used for visualization of the 3D model. Submicron capsules were obtained by stabilizing oil droplets with a mixture of SiO2 nanoparticles and DNDs. To form a stable shell, an additional layer of silica particles and polyelectrolyte layers of alginate/chitosan were applied to the droplets of the dispersed phase of the emulsion by physical adsorption. Results: Cryo-EM data showed the presence of submicrocapsules with a diameter in the range of 200-900 nm. Although a significant fraction of submicrocapsules was found to be partially destroyed, results of cryo-ET study of intact capsules demonstrated that silicon dioxide nanoparticles form a net, while DNDs form clusters. Conclusion: Here we demonstrate the results of the study of submicron capsules with a shell of silica nanoparticles and DNDs. It was found that a uniform distribution of DNDs is not a prerequisite for the creation of submicron capsules that contradicts the theoretical model

Abstract OR-2: The Formation of Dps-DNA Complexes under Different Conditions According to Cryo-EM and SAXS

Background: The effect of Dps-DNA co-crystals formation, which occurs in stressed Escherichia coli cells exposed to extreme conditions, is well described in the literature. However, the exact mechanisms of co-crystals formation are yet to be postulated remaining largely unknown. Here we summarize the results obtained by our group over the last few years using cryo-Electron Microscopy (cryo-EM) and Small Angle X-ray Scattering (SAXS). Methods: Samples for cryo-EM were plunge frozen in liquid ethane with Vitrobot Mark IV and studied with Titan Krios (ThermoFisher Scientific, US) cryo-EM, equipped with Falcon 2 direct electron detector, Image corrector (CEOS, Germany), and Volta phase plate. Single Particle Analysis (SPA) and cryo-Electron Tomography (cryo-ET) studies were conducted with 300 kV accelerating voltage in low dose mode using EPU and Tomography software (ThermoFisher Scientific, US). Cryo-EM data processing was conducted using Warp, CryoSPARC, IMOD, EMAN, and Relion software packages. SAXS measurements were performed at the EMBL on the P12 BioSAXS beam line at the PETRAIII storage ring (DESY, Hamburg). Results: In this work, Dps-DNA complex formation is thoroughly studied using complementary cryo-EM (including SPA, cryo-ET, and subtomogram averaging) and SAXS methods. The formation of individual complexes of Dps with small linear DNA fragments and the Dps-Dps interaction was visualized using cryo-EM. It was found that Dps-DNA complex remains stable under various conditions and while the addition of different ions leads to the disruption of co-crystals, the process is completely or partially reversible. Conclusion: Recent studies conducted by our group showed that Dps-DNA co-crystals adopt triclinic or cubic crystal lattice (FEBS Lett., 2019; Biomolecules, 2020). Here we present the results on the studies of Dps interaction with small linear DNA fragments, demonstrate the effects of MgCl2, FeSO4, and EDTA on the Dps-DNA complex and individual Dps protein structure, discuss the influence of the temperature and time on the co-crystals

Abstract P-43: Effect of Glucocerebrosidase Dysfunction on the Pool of Plasma Exosomes of Patients with Gaucher Disease

Background: Extracellular vesicles (EVs) are small membrane vesicles released from different types of cells. EVs are found in many human biological fluids. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies with the plasma membrane. This type of vesicles is characterized by specific exosomal markers. Exosomes extracted from peripheral body liquids could have specific properties associated with different physiological conditions as well as human disorders, including neurodegenerative diseases. Gaucher disease (GD) – is the most common form of lysosomal storage disorders caused by mutations in the glucocerebrosidase (GBA) gene. Lysosome functionality is critical for the regulation of extracellular vesicle secretion and content. In model animals, the inhibition of glucocerebrosidase has been shown to increase the secretion of extracellular vesicles in brain tissues. Amount evaluation of EVs and their size in the biological fluids of patients with GD has not been early performed; therefore, it is unknown whether lysosomal dysfunction found in GD patients influences the plasma pool of EVs. The aim of this study was to evaluate the amount of blood plasma EVs in patients with GD and their characterization for morphology and size. Methods: EVs were isolated from the blood plasma of 8 GD patients and 8 controls by ultracentrifugation, and were characterized using cryo-electron microscopy (cryo-EM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS). Also, the presence of exosomal markers CD9, CD63, CD81, and HSP70 was analyzed by flow cytometry and western blot. Results: Here, it was first shown an increased proportion of exosome fraction in EVs from plasma of GD patients compared to controls by DLS and cryo-EM (p<0.001) that was confirmed by mode size detected by NTA (p<0.02). Moreover, an increased number of double and multilayer vesicles in plasma EVs from GD patients was demonstrated by cryo-EM. We also detected an increase in the expression of exosomal markers on the surface of vesicles from the blood plasma of patients with GD compared to controls. Conclusion: Here, we firstly report that the exosomes obtained from the blood plasma of GD patients have a larger size and altered morphology. Thus, we have shown that lysosomal dysfunction in GD patients leads to a striking alteration of blood plasma extracellular vesicle pool

Structural Insights into Iron Ions Accumulation in Dps Nanocage

Dps (DNA-binding protein from starved cells) is well known for the structural protection of bacterial DNA by the formation of highly ordered intracellular assemblies under stress conditions. Moreover, this ferritin-like protein can perform fast oxidation of ferrous ions and subsequently accumulate clusters of ferric ions in its nanocages, thus providing the bacterium with physical and chemical protection. Here, cryo-electron microscopy was used to study the accumulation of iron ions in the nanocage of a Dps protein from Escherichia coli. We demonstrate that Fe2+ concentration in the solution and incubation time have an insignificant effect on the volume and the morphology of iron minerals formed in Dps nanocages. However, an increase in the Fe2+ level leads to an increase in the proportion of larger clusters and the clusters themselves are composed of discrete ~1–1.5 nm subunits

Abstract OR-2: The Formation of Dps-DNA Complexes under Different Conditions According to Cryo-EM and SAXS

Background: The effect of Dps-DNA co-crystals formation, which occurs in stressed Escherichia coli cells exposed to extreme conditions, is well described in the literature. However, the exact mechanisms of co-crystals formation are yet to be postulated remaining largely unknown. Here we summarize the results obtained by our group over the last few years using cryo-Electron Microscopy (cryo-EM) and Small Angle X-ray Scattering (SAXS). Methods: Samples for cryo-EM were plunge frozen in liquid ethane with Vitrobot Mark IV and studied with Titan Krios (ThermoFisher Scientific, US) cryo-EM, equipped with Falcon 2 direct electron detector, Image corrector (CEOS, Germany), and Volta phase plate. Single Particle Analysis (SPA) and cryo-Electron Tomography (cryo-ET) studies were conducted with 300 kV accelerating voltage in low dose mode using EPU and Tomography software (ThermoFisher Scientific, US). Cryo-EM data processing was conducted using Warp, CryoSPARC, IMOD, EMAN, and Relion software packages..

Structural Rearrangement of Dps-DNA Complex Caused by Divalent Mg and Fe Cations

Two independent, complementary methods of structural analysis were used to elucidate the effect of divalent magnesium and iron cations on the structure of the protective Dps-DNA complex. Small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) demonstrate that Mg$^{2+}$ ions block the N-terminals of the Dps protein preventing its interaction with DNA. Non-interacting macromolecules of Dps and DNA remain in the solution in this case. The subsequent addition of the chelating agent (EDTA) leads to a complete restoration of the structure of the complex. Different effect was observed when Fe cations were added to the Dps-DNA complex; the presence of Fe$^{2+}$ in solution leads to the total complex destruction and aggregation without possibility of the complex restoration with the chelating agent. Here, we discuss these different responses of the Dps-DNA complex on the presence of additional free metal cations, investigating the structure of the Dps protein with and without cations using SAXS and cryo-EM. Additionally, the single particle analysis of Dps with accumulated iron performed by cryo-EM shows localization of iron nanoparticles inside the Dps cavity next to the acidic (hydrophobic) pore, near three glutamate residues

Composite materials based on Ag nanoparticles in situ synthesized on the vaterite porous matrices

We have designed sensors based on Ag nanoparticles synthesized in situ on the vaterite beads. In this article we demonstrate an approach to produce size controllable spherical and elliptical vaterite particles and discuss time-dependent in situ Ag nanoparticles synthesis and its potential effect on surface-enhanced Raman scattering. The time dependent silver reduction synthesis in inorganic porous particles allows to regulate the number and size of Ag nanoparticles. It is shown that the irregular surface and high porosity of vaterite particles and large amount (surface filling factor) of the Ag nanoparticles are the critical parameters to increase the SERS signal to 104 times. Such inorganic composites have a huge potential in medical applications; soon they provide an opportunity to study intracellular processes in vivo. The detailed characterization of the microstructure of these composites was studied by scanning and transmission electron microscopy, including 3D visualization and energy dispersive x-ray microanalysis

Proteome of Glioblastoma-Derived Exosomes as a Source of Biomarkers

Extracellular vesicles (EV) are involved in important processes of glioblastoma multiforme (GBM), including malignancy and invasion. EV secreted by glioblastoma cells may cross the hematoencephalic barrier and carry molecular cargo derived from the tumor into the peripheral circulation. Therefore, the determination of the molecular composition of exosomes released by glioblastoma cells seems to be a promising approach for the development of non-invasive methods of the detection of the specific exosomal protein markers in the peripheral blood. The present study aimed to determine the common exosomal proteins presented in preparations from different cell lines and search potential glioblastoma biomarkers in exosomes. We have performed proteomics analysis of exosomes obtained from the conditioned culture medium of five glioblastoma cell lines. A list of 133 proteins common for all these samples was generated. Based on the data obtained, virtual two-dimensional electrophoresis (2DE) maps of proteins presented in exosomes of glioblastoma cells were constructed and the gene ontology (GO) analysis of exosome proteins was performed. A correlation between overexpressed in glial cell proteins and their presence in exosomes have been found. Thus, the existence of many potential glioblastoma biomarkers in exosomes was confirmed

Polymorphic Protective Dps–DNA Co-Crystals by Cryo Electron Tomography and Small Angle X-Ray Scattering

Rapid increase of intracellular synthesis of specific histone-like Dps protein that binds DNA to protect the genome against deleterious factors leads to in cellulo crystallization—one of the most curious processes in the area of life science at the moment. However, the actual structure of the Dps–DNA co-crystals remained uncertain in the details for more than two decades. Cryo-electron tomography and small-angle X-ray scattering revealed polymorphous modifications of the co-crystals depending on the buffer parameters. Two different types of the Dps–DNA co-crystals are formed in vitro: triclinic and cubic. Three-dimensional reconstruction revealed DNA and Dps molecules in cubic co-crystals, and the unit cell parameters of cubic lattice were determined consistently by both methods